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WO2025075499A1 - Reducing dopamine feed-back inhibition in dopaminergic cells by tadalafil. - Google Patents

Reducing dopamine feed-back inhibition in dopaminergic cells by tadalafil. Download PDF

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Publication number
WO2025075499A1
WO2025075499A1 PCT/NL2024/050536 NL2024050536W WO2025075499A1 WO 2025075499 A1 WO2025075499 A1 WO 2025075499A1 NL 2024050536 W NL2024050536 W NL 2024050536W WO 2025075499 A1 WO2025075499 A1 WO 2025075499A1
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Prior art keywords
dopamine
dopa
prodrug
agonist
tadalafil
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French (fr)
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Lars Philip VAN DER HEIDE
Marten Piet Smidt
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Universiteit Van Amsterdam
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Universiteit Van Amsterdam
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs

Definitions

  • the invention relates to means and methods for treating dopamine deficiencies in subjects.
  • the invention also relates to means and methods for the treatment of an individual that is receiving levodopa (L-DOPA) as a medicament.
  • the invention also relates to means and methods for the treatment of an individual that has Parkinson’s disease or is at risk of developing this disease.
  • Parkinson’s disease (PD) is the most prevalent neurological disorder after Alzheimer’s pathology. Its primary hallmark is the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) (Kalia and Lang, 2015. Lancet 386: 896-912).
  • This anatomical region arises from the mesencephalon during development and eventually creates projections towards the dorsal striatum, one of the forebrain areas by which the basal ganglia are comprised of (Smidt and Burbach, 2007. Nat Rev Neurosci 8: 21–32; Baladron and Hamker, 2015. Neural Netw 67: 1-13).
  • the functional connectivity between both structures is broadly referred as the nigrostriatal pathway and depends on the release of dopamine (Ikemoto et al., 2015. Behav Brain Res 290: 1731). This neurotransmitter interacts with the striatal postsynaptic receptors after being released by the nigral efferents, ultimately regulating motor control.
  • levodopa-induced dyskinesias standing out the most.
  • Orally- administered levodopa can cross the BBB and, once in the brain, be incorporated into the dopaminergic terminals of the dorsal striatum. There, it is further converted to dopamine by AADC, imported into vesicles by the transporter vMAT2 and ultimately released to the synaptic cleft.
  • This outcome of levodopa treatment accounts for its beneficial effects in patients with PD.
  • levodopa can also be incorporated into the striatal serotonergic terminals, in which AADC is equally present.
  • tadalafil a phosphodiesterase (PDE) 5 and PDE 11 inhibitor, or a variant thereof, is able to increase the endogenous production of dopamine in dopaminergic cells in the presence of an external source of dopamine by means of increasing Tyrosine Hydroxylase (TH) Ser40 phosphorylation.
  • TH Tyrosine Hydroxylase
  • Individuals that receive dopamine or a precursor thereof are subject to negative feedback inhibition caused by an inhibitory effect of dopamine on the endogenous production of L-DOPA.
  • tadalafil removes the inhibition from the endogenous production of L-DOPA in the relevant cells.
  • Tadalafil or a variant thereof is able to increase the endogenous production of dopamine in dopaminergic cells even in the presence of an external source of dopamine.
  • Tadalafil can be administered, at a dose of 0,1 – 40 mg/day.
  • a suitable dose is in the range of 1 mg/day to 40 mg/day.
  • Tadalafil is marketed in dosages of 5, 10 and 20 mg. For the present invention it is possible to use such formulations arriving at a suitable dose of 5 mg/day, 10 mg/day and 20 mg/day.
  • a suitable minimum administered dose of tadalafil or variant thereof is 20 mg/day (0.4 mg/kg), when provided as sole PDE inhibitor.
  • Tadalafil or variant thereof can thereby at least in part reverse the negative feedback inhibition induced by added dopamine, a dopamine agonist, L- DOPA or a prodrug thereof in dopaminergic cells.
  • the selective presence of especially PDE11 in the target cells is at least part of the reason for the increased specificity and/or reduction of side effects.
  • Human PDEs are divided into 11 families. Some families (PDE1, PDE3, PDE4, PDE7 and PDE8) are products of multiple genes (multigene), whereas others derive from a single gene.
  • PDEs share a highly conserved catalytic domain near the carboxyl-terminus, including 11 amino acids in the active site. Minor variations in the catalytic domain are believed to be responsible for the different selectivities towards cAMP or cGMP substrate and various inhibitors. More substantial variation exists in the N-terminus part, where most of the regulatory domains reside (Makhlouf et al., 2006. Int J Impot Res 18: 501–509).
  • PDE11 there is a single gene in the family (PDE11A according to standard nomenclature) with four splicing variants (PDE11A1, PDE11A2, PDE11A3 and PDE11A4).
  • PDE11 or phosphodiesterase 11 is known under a number of different names such as Dual 3',5'-Cyclic-AMP And -GMP Phosphodiesterase 11A; EC 3.1.4.35; CAMP And CGMP Cyclic Nucleotide Phosphodiesterase 11A and PPNAD2.
  • External Ids for PDE11A Gene are: HGNC: 8773; NCBI Gene: 50940; Ensembl: ENSG00000128655; OMIM®: 604961; and UniProtKB/Swiss-Prot: Q9HCR9.
  • PDE11A is a dual-substrate PDE, acting on both cAMP and cGMP. Most studies report similar affinities (Km) for both substrates (Makhlouf, A et al., 2006. Int J Impot Res 18, 501–509).
  • the invention provides dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in the treatment of dopamine deficiency in a subject, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is co-administered in combination with tadalafil or a variant thereof, characterized in that dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered at an effective reduced dose compared to the subject's standard therapeutic dose.
  • the invention also provides dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in the treatment of dopamine deficiency in a subject, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered in combination with tadalafil or a variant thereof, characterized in that the co-administration of tadalafil or a variant thereof enables a reduction in the dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, compared to the standard therapeutic dose required for treating the dopamine deficiency in the individual, while maintaining therapeutic efficacy and reducing the incidence or severity of side effects associated with dopamine, a dopamine agonist, L-DOPA or a prodrug thereof.
  • the reduction of the dose is at least 10%, at least 20%, or preferably at least 50% compared to the individuals standard therapeutic dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof.
  • the treatment of the dopamine deficiency preferably does not comprise the administration of a calcimimetic that lowers parathyroid hormone (PTH) levels such as cinacalcet, baclofen, acamprosate, mexiletine, torasemide, or sulfisoxazole.
  • PTH parathyroid hormone
  • Tadalafil or a variant thereof can be administered, at a dose of 0,1 – 40 mg/day.
  • a suitable dose is in the range of 1 mg/day to 40 mg/day.
  • Tadalafil is marketed in dosages of 5, 10 and 20 mg.
  • a suitable minimum administered dose of tadalafil or variant thereof is 20 mg/day (0.4 mg/kg) when provided as sole PDE inhibitor.
  • the minimum administered dose of tadalafil or variant thereof is preferably 20 mg/day (0.4 mg/kg).
  • the dopamine deficiency is preferably a central nervous system dopamine deficiency.
  • the disease associated with the dopamine deficiency in the individual is preferably Parkinson's disease.
  • the further medicament preferably comprises a PDE11 inhibitor, a PDE2 inhibitor, a guanylate cyclase agonist; a dopa-decarboxylase (DDC) inhibitor; a monoamine oxidase (MAO-B) inhibitor; a catechol-O- methyltransferase (COMT) inhibitor; or a combination thereof.
  • the PDE11 inhibitor is preferably BC11-15; BC11-19; BC11-28; BC11-38; BC11-38-1; BC11-38- 2; BC11-38-3; or BC11-38-4.
  • the PDE2 inhibitor is preferably EHNA (erythro-9-(2-hydroxy-3- nonyl)adenine); BAY 60-7550 (2-[(3,4-dimethoxyphenyl)methyl]-7-[(2R,3R)-2- hydroxy-6-phenylhexan-3-yl]-5-methyl-1H-imidazo[5,1-f][1,2,4]triazin-4-one); PDP (9-(6-phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one; Hcyb1, PF- 051809999 (4-(1-azetidinyl)-7-methyl-5-[1-methyl-5-[5-(trifluoromethyl)-2- pyridinyl]-1H-pyrazol-4-yl]-imidazo[5,1-f][1,2,4]triazine); IC933, or oxindole (2,3- dihydroind
  • the guanylate cyclase agonist is preferably a GUCY-2C agonist.
  • the guanylate cyclase 2C receptor (GUCY2C) agonist is preferably guanylin, lymphoguanylin, enterotoxin or uroguanylin. or a functional derivative of guanylin, lymphoguanylin, enterotoxin or uroguanylin.
  • the dopamine, a dopamine agonist, L-DOPA or a prodrug thereof and tadalafil are preferably administered simultaneously.
  • the Parkinson’s disease is Parkinson’s disease stage 1-5.
  • the invention also provides a method for treating dopamine deficiency in a subject, comprising: - administering to the subject a therapeutically effective dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof; and co-administering to the subject a therapeutically effective dose of tadalafil or a variant thereof; wherein the dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered at an effective reduced dose compared to the subjects standard therapeutic dose.
  • Also provided is a method for treating dopamine deficiency in a subject comprising: - administering to the subject a therapeutically effective dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is known to cause one or more side effects at standard therapeutic doses; and - co-administering to the subject a therapeutically effective dose of tadalafil or a variant thereof, wherein the co-administration of the tadalafil or a variant thereof enables a reduction in the dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof compared to the standard therapeutic dose required for treating the dopamine deficiency in the individual, while maintaining therapeutic efficacy and reducing the incidence or severity of a side effect associated with dopamine, a dopamine agonist, L-DOPA or a prodrug thereof.
  • the treatment preferably does not comprise a calcimimetic that lowers parathyroid hormone (PTH) levels such as cinacalcet, baclofen, acamprosate, mexiletine, torasemide, or sulfisoxazole.
  • PTH parathyroid hormone
  • Tadalafil or a variant thereof can be administered, at a dose of 0,1 – 40 mg/day.
  • a suitable dose is in the range of 1 mg/day to 40 mg/day.
  • Tadalafil is marketed in dosages of 5, 10 and 20 mg.
  • a suitable minimum administered dose of tadalafil or variant thereof is 20 mg/day (0.4 mg/kg) when provided as sole PDE inhibitor.
  • the minimum administered dose of tadalafil or variant thereof is preferably 20 mg/day (0.4 mg/kg).
  • The is preferably a central nervous system dopamine deficiency.
  • the disease associated with dopamine deficiency in the individual is preferably Parkinson's disease.
  • dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in the treatment of dopamine deficiency in a subject, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is co-administered in combination with tadalafil or a variant thereof, characterized in that dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered at a reduced dose compared to the subjects standard therapeutic dose of dopamine, a dopamine agonist, L- DOPA or a prodrug thereof.
  • dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in the treatment of dopamine deficiency in a subject, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered in combination with tadalafil or a variant thereof, characterized in that the co- administration of tadalafil or a variant thereof allows a reduction in the dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, compared to the standard therapeutic dose required for treating the dopamine deficiency, while maintaining therapeutic efficacy and reducing the incidence or severity of side effects associated with dopamine, a dopamine agonist, L-DOPA or a prodrug thereof.
  • the invention provides dopamine, a dopamine agonist, L- DOPA or a prodrug thereof for use in the treatment of Parkinson’s disease in a subject, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is co- administered in combination with tadalafil or a variant thereof, characterized in that dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered at an effective reduced dose compared to the subject's standard therapeutic dose.
  • the invention also provides dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in the treatment of Parkinson’s disease in a subject, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered in combination with tadalafil or a variant thereof, characterized in that the co-administration of tadalafil or a variant thereof enables a reduction in the dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, compared to the standard therapeutic dose required for treating the Parkinson’s disease in the individual, while maintaining therapeutic efficacy and reducing the incidence or severity of side effects associated with dopamine, a dopamine agonist, L-DOPA or a prodrug thereof.
  • the reduction of the dose is at least 10%, at least 20%, or preferably at least 50% compared to the individuals standard therapeutic dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof.
  • the treatment of the Parkinson’s disease preferably does not comprise the administration of a calcimimetic that lowers parathyroid hormone (PTH) levels such as cinacalcet, baclofen, acamprosate, mexiletine, torasemide, or sulfisoxazole.
  • PTH parathyroid hormone
  • the invention provides a method for treating Parkinson’s disease in a subject, comprising: - administering to the subject a therapeutically effective dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof; and co-administering to the subject a therapeutically effective dose of tadalafil or a variant thereof; wherein the dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered at a reduced dose compared to the subjects standard therapeutic dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof.
  • dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in the treatment of Parkinson’s disease in a subject, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is co-administered in combination with tadalafil or a variant thereof, characterized in that dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered at a reduced dose compared to the subjects standard therapeutic dose of dopamine, a dopamine agonist, L- DOPA or a prodrug thereof.
  • dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in the treatment of Parkinson’s disease, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered in combination with tadalafil or a variant thereof, characterized in that the co-administration of tadalafil or a variant thereof allows a reduction in the dose of dopamine, a dopamine agonist, L- DOPA or a prodrug thereof, compared to the standard therapeutic dose required for treating Parkinson’s disease, while maintaining therapeutic efficacy and reducing the incidence or severity of side effects associated with dopamine, a dopamine agonist, L-DOPA or a prodrug thereof.
  • the invention provides a method for treating Parkinson’s disease in a subject, comprising: - administering to the subject a therapeutically effective dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is known to cause one or more side effects at standard therapeutic doses; and - co-administering to the subject a therapeutically effective dose of tadalafil or a variant thereof, wherein the co-administration of the tadalafil or a variant thereof allows for a reduction in the dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof compared to the standard therapeutic dose required for treating Parkinson’s disease in the individual, while maintaining therapeutic efficacy and reducing the incidence or severity of a side effect associated with dopamine, a dopamine agonist, L-DOPA or a prodrug thereof.
  • the reduction in the dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is preferably at least 10%, 20%, or preferably 50% compared to the standard therapeutic dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof.
  • the side effects of standard dopamine, a dopamine agonist, L-DOPA or a prodrug thereof treatment include dyskinesias (involuntary movements that can take the form of chorea, dystonia, ballism, tics, and myoclonus); nausea and vomiting; low blood pressure (hypotension); loss of appetite; anxiety and depression; hallucinations (seeing, hearing, feeling and smelling things that aren't there) and sleep problems.
  • Reducing the incidence or severity of a side-effect involves the reduction of the incidence or severity of at least one of: levodopa- induced dyskinesias (involuntary movements that can take the form of chorea, dystonia, ballism, tics, and myoclonus); nausea and vomiting; low blood pressure (hypotension); loss of appetite; anxiety and depression; hallucinations (seeing, hearing, feeling and smelling things that aren't there) and sleep problems.
  • a drug that can be dopamine, a dopamine agonist, L-DOPA or a prodrug thereof it is preferred that the drug is L- DOPA or a prodrug thereof. It is preferred that the drug is L-DOPA.
  • the invention provides L-DOPA for use in the treatment of Parkinson’s disease in a subject, wherein L-DOPA is co-administered in combination with tadalafil or a variant thereof, characterized in that L-DOPA is administered at an effective reduced dose compared to the subject's standard therapeutic dose.
  • the invention also provides L-DOPA for use in the treatment of Parkinson’s disease in a subject, wherein L-DOPA is administered in combination with tadalafil or a variant thereof, characterized in that the co-administration of tadalafil or a variant thereof enables a reduction in the dose of L-DOPA, compared to the standard therapeutic dose required for treating the Parkinson’s disease in the individual, while maintaining therapeutic efficacy and reducing the incidence or severity of side effects associated with L-DOPA.
  • the reduction of the dose is at least 10%, at least 20%, or preferably at least 50% compared to the individuals standard therapeutic dose of L-DOPA.
  • the treatment of the Parkinson’s disease preferably does not comprise the administration of a calcimimetic that lowers parathyroid hormone (PTH) levels such as cinacalcet, baclofen, acamprosate, mexiletine, torasemide, or sulfisoxazole.
  • PTH parathyroid hormone
  • the invention provides a method for treating Parkinson’s disease in a subject, comprising: - administering to the subject a therapeutically effective dose of L-DOPA; and co-administering to the subject a therapeutically effective dose of tadalafil or a variant thereof; wherein the L-DOPA is administered at a reduced dose compared to the subjects standard therapeutic dose of L-DOPA.
  • L-DOPA for use in the treatment of Parkinson’s disease in a subject, wherein L-DOPA is co-administered in combination with tadalafil or a variant thereof, characterized in that L-DOPA is administered at a reduced dose compared to the subjects standard therapeutic dose of L-DOPA.
  • the reduction in the dose of L-DOPA is preferably at least 10%, 20%, or preferably 50% compared to the patients standard therapeutic dose of L-DOPA.
  • the co-administration is effective in treating Parkinson’s disease in the subject while reducing the incidence and/or severity of side effects associated with L-DOPA treatment of Parkinson patients.
  • L-DOPA for use in the treatment of Parkinson’s disease, wherein L-DOPA is administered in combination with tadalafil or a variant thereof, characterized in that the co-administration of tadalafil or a variant thereof allows a reduction in the dose of L-DOPA, compared to the standard therapeutic dose required for treating Parkinson’s disease, while maintaining therapeutic efficacy and reducing the incidence or severity of side effects associated with L- DOPA.
  • the reduction in the dose of L-DOPA is preferably at least 10%, 20%, or preferably 50% compared to the standard therapeutic dose of L-DOPA.
  • the side effects of standard L-DOPA treatment include levodopa-induced dyskinesias (involuntary movements that can take the form of chorea, dystonia, ballism, tics, and myoclonus); nausea and vomiting; low blood pressure (hypotension); loss of appetite; anxiety and depression; hallucinations (seeing, hearing, feeling and smelling things that aren't there) and sleep problems.
  • Reducing the incidence or severity of a side-effect involves the reduction of the incidence or severity of at least one of: levodopa-induced dyskinesias (involuntary movements that can take the form of chorea, dystonia, ballism, tics, and myoclonus); nausea and vomiting; low blood pressure (hypotension); loss of appetite; anxiety and depression; hallucinations (seeing, hearing, feeling and smelling things that aren't there) and sleep problems.
  • levodopa-induced dyskinesias involuntary movements that can take the form of chorea, dystonia, ballism, tics, and myoclonus
  • nausea and vomiting low blood pressure (hypotension); loss of appetite
  • anxiety and depression hallucinations (seeing, hearing, feeling and smelling things that aren't there) and sleep problems.
  • the invention also provides dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in a method of treating a Parkinson’s disease in a subject characterized in that the treatment of the disease comprises the administration of tadalafil or a variant thereof to the subject. Further provided is dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in a method of treating a Parkinson’s disease in a subject characterized in that the treatment of the disease comprises the administration of tadalafil or a variant thereof to the subject, whereby tadalafil or variant thereof is administered at a minimum dose of 20 mg/day (0.4 mg/kg).
  • the physician adjusts the dose every few days or weeks depending on the patient’s tolerance and control of symptoms.
  • the goal is to increase the dose until a desired balance between symptom relief and side effects is achieved.
  • increments are typically 100 mg/day or smaller, with frequent reassessment.
  • the starting dose for L-DOPA in a Parkinson’s patient that receives the drug for the first time is typically 100 mg 2 or 3-times daily.
  • the dose is gradually increased in number per day and/or amount per dose until a desired balance between symptom relief and side effects is achieved.
  • the patient starts with a low dose and increases it after some time, doses are regularly increased until a suitable balance between effects and side-effects is achieved. When this is achieved this dose is referred to as the standard therapeutic dose.
  • the comparison with and without tadalafil is preferably done under otherwise the same medical treatment conditions.
  • the effective reduced dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is the therapeutically effective dose of said dopamine, a dopamine agonist, L-DOPA or a prodrug thereof when given as treatment of the patient with tadalafil. This effective reduced dose is also typically determined by titration of the medicament in the individual.
  • the term “effective reduced dose” is the same as the term “reduced therapeutic dose” and can be replaced by it. It is not necessary to first find the optimal dose or standard therapeutic dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof to find the suboptimal dose or effective reduced dose of dopamine, a dopamine agonist, L- DOPA or a prodrug thereof for use in combination with tadalafil. A physician typically starts treatment with a set effective dose of tadalafil and a normal low starting dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof which is subsequently titrated until a desired balance between symptom relief and side effects is achieved.
  • Said PDE2 inhibitor preferably is EHNA (erythro-9-(2-hydroxy-3- nonyl)adenine); BAY 60-7550 (2-[(3,4-dimethoxyphenyl)methyl]-7-[(2R,3R)-2- hydroxy-6-phenylhexan-3-yl]-5-methyl-1H-imidazo[5,1-f][1,2,4]triazin-4-one); PDP (9-(6-phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one; Hcyb1; PF- 051809999(4-(1-azetidinyl)-7-methyl-5-[1-methyl-5-[5-(trifluoromethyl)-2- pyridinyl]-1H-pyrazol-4-yl]-imidazo[5,1-f][1,2,4]triazine); IC933; oxindole (2,3- dihydr
  • the invention further provides a method for treating dopamine deficiency in a subject comprising administering dopamine, a dopamine agonist, L-DOPA or a prodrug thereof to a subject in need thereof, characterized in that the treatment of the deficiency further comprises the administration of tadalafil or variant thereof to the subject, whereby the treatment does not comprise a calcimimetic that lowers parathyroid hormone (PTH) levels such as cinacalcet, baclofen, acamprosate, mexiletine, torasemide, or sulfisoxazole.
  • PTH parathyroid hormone
  • Said administration of a guanyl cyclase agonist, a PDE11 inhibitor and/or a PDE2 inhibitor may be before, simultaneous to, or after the administration of dopamine, a dopamine agonist, L- DOPA or a prodrug thereof, and tadalafil or variant thereof.
  • a medicament comprising a sub-optimal dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, and tadalafil or variant thereof.
  • Said administration of a guanyl cyclase agonist may be before, simultaneous to, or after the administration of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, and tadalafil or variant thereof.
  • Said PDE11 inhibitor preferably is selected from BC11-15; BC11-19; BC11- 28; BC11-38; BC11-38-1; BC11-38-2; BC11-38-3; or BC11-38-4.
  • Said administration of a PDE2 inhibitor may be before, simultaneous to, or after the administration of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, and tadalafil or variant thereof.
  • the invention provides dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in a method for the treatment of dopamine deficiency in a subject, characterized in that the treatment of the deficiency further comprises the administration of a PDE11 inhibitor to the subject.
  • L-DOPA which can pass the blood-brain barrier, is usually administered peripherally in combination with a peripheral AADC inhibitor. It reaches the brain via cerebral arteries and passage of the blood- brain barrier. Then, in combination with AADC, L-DOPA is synthesized to dopamine in the brain.
  • Regions of interest are micro-dissected from the collected coronal slices and transferred into a tube for ex vivo pharmacological treatment. Ex vivo pharmacological experiments are performed on micro-dissected brain tissue, for which each slice first is divided into two hemispheres, followed by micro-dissection of their regions of interest (indicated in gray).
  • the regions of interest are the midbrain (MB), the striatum (CS), or the areas connecting these regions.
  • Tyrosine hydroxylase Ser40 phosphorylation levels are upregulated though activation of Gucy2C A-B
  • C Immunohistochemistry for TH (green) and Gucy2C (red) on human postmortum midbrain material. Melanin is represented by a blue signal. Merge of these signals show that Gucy2C is present axons of in human midbrain dopaminergic neurons (white arrows). In a blow up of a TH/melanin positive neurons the presence of Gucy2C is represented in axonal fibers. Figure 6.
  • A Schematic images are shown of each examined brain slice over the rostral- caudal axis with representative western blot images for protein levels of tyrosine hydroxylase (Th) and phospho-Th (Ser40).
  • C-D The effect of 60 minutes exposure to L-DOPA on tyrosine hydroxylase phosphorylation.
  • Micro-dissected mouse striatal slices were incubated with either 100 ⁇ M L-DOPA or vehicle for 30 minutes, followed by exposure to either 10 ⁇ M forskolin or vehicle for another 30 minutes.
  • F Schematic images are shown of each examined brain slice over the rostral- caudal axis with representative western blot images for protein levels of Th and phospho-Th (Ser40).
  • G Quantitative analysis of the effect of 10 ⁇ M forskolin (FSK) on total Th- corrected phospho-Th (Ser40) levels in micro-dissected mouse brain slices pre- incubated with either 100 ⁇ M L-DOPA or vehicle for 30 minutes.
  • Forskolin up- regulates and L-DOPA down-regulates phospho-Th (Ser40) levels. If slices are exposed to forskolin subsequently to 30 minutes of L-DOPA incubation, phospho- Th (Ser40) levels are recovered to baseline levels. For each animal, the area under the curve was calculated per condition (two-tailed paired student’s t-test statistical analysis with Bonferroni correction, n 5 animals). Protein levels were detected by automated western blot analysis using the WesTM (ProteinSimple). The micro- dissected part of one hemisphere (randomly chosen) is used as internal control to the micro-dissected part of the other hemisphere.
  • the amount of phospho-protein levels are corrected for the total amount of protein and data is normalized to the average level of protein of the vehicle condition.
  • Data are minimum to maximum boxplots showing the first quartile, third quartile, and the mean (B, D) or bar charts showing the mean ⁇ SEM (G), *p ⁇ 0.05 and **p ⁇ 0.01.
  • Figure 8 The effect of Pitx3-deficiency on tyrosine hydroxylase levels in the mouse striatum Deficiency in the homeobox gene Pitx3 leads to selective neuronal cell loss of the same group of dopamine neurons that are affected in Parkinson’s disease. As a consequence of this selective loss of neurons, dopaminergic connections to the striatum are affected as well.
  • B Quantitative analysis of total tyrosine hydroxylase (Th) levels. Tyrosine hydroxylase levels are drastically lower in the phenotypically defective Pitx3GFP/GFP mice.
  • L-DOPA treatment results in a reduced phosphorylation of TH and can be rescued by BAY 60-7550 treatment
  • Pharmacological time-line of L-DOPA and BAY 60-7550 combinatorial treatment One hemisphere was treated with vehicle for one hour. The corresponding hemisphere with L-DOPA for 60 minutes. The following striatal slice was incubated BAY 60-7550 for one hour (100 ⁇ M). The corresponding hemisphere was treated with BAY 60-7550 (10 ⁇ M) and L-DOPA (100 ⁇ M).
  • Neuro2A cells were exposed to sildenafil or tadalafil for 60 minutes.
  • Introduction of PDE11A4 down-regulates Ser40 phosphorylation under basal conditions. Without PDE11A4, sildenafil downregulates Ser40 phosphorylation, while tadalafil had no effect. However, when PDE11A4 is introduced, relative Ser40 phosphorylation levels are up-regulated by tadalafil.
  • Figure 16 Dose response curve Tadalafil Neuro2A cells were transfected with DNA plasmid encoding tyrosine hydroxylase (Th) and either polyhistidine-tagged PDE11A4 or pcDNA3.1(+) backbone.
  • Neuro2A cells were exposed to different concentrations of Tadalafil for 60 minutes.
  • A Increasing concentrations of Tadalafil attenuate the initial downregulation of TH-40 Phosphorylation.
  • the present invention relates to the utilization of the endogenous dopamine biosynthesis pathway as a means to address the significantly diminished levels of dopamine in the nigrostriatal dopamine projections in Parkinson’s patients that are on L-DOPA treatment.
  • L-DOPA endogenous dopamine synthesis
  • the extent to which the L-DOPA dose can be reduced depends on the degree of Ser40 phosphorylation and the residual population of dopamine neurons, typically correlating with the stage and phenotype of the disease in individual patients.
  • Off-target effects resulting from L-DOPA treatment can occur due to dopamine synthesis in cells outside the striatum (extrastriatal cells) that do not normally produce dopamine. These extrastriatal cells can synthesize dopamine when exposed to L-DOPA, contributing to the adverse side effects observed in Parkinson’s patients treated with L-DOPA.
  • this invention mitigates both the off- target effects caused by extrastriatal dopamine production in non-dopaminergic, AADC-expressing neurons and the uncontrolled overstimulation of post-synaptic signaling pathways.
  • the endogenous dopamine biosynthesis pathway remains functional, preserving crucial mechanisms that regulate dopamine neurotransmission. This allows for some degree of natural dopamine regulation, including processes such as vesicular packaging and the initiation of dopamine synthesis, in which the subcellular distribution of tyrosine hydroxylase and other proteins involved in dopamine production play a pivotal role.
  • Stage 1 is typically characterized by mild symptoms that generally do not interfere with daily activities. Tremor and other movement symptoms typically occur on one side of the body only. Friends and family may notice changes in posture, walking and facial expressions. In stage 2 the symptoms start getting worse. Tremor, rigidity and other movement symptoms affect both sides of the body. Walking problems and poor posture may become apparent. In this stage, the person is still able to live alone, but completing day-to-day tasks becomes more difficult and may take longer. Stage 3 is considered mid-stage in the progression of the disease. Loss of balance and slowness of movements are hallmarks of this phase. Falls are more common.
  • Parkinson’s disease or at least the motor symptoms part thereof, is commonly treated with dopamine replacement therapy.
  • the dopamine precursor levodopa (L-DOPA), dopamine agonists (DAs), monoamine oxidase B inhibitors and catechol-O-methyltransferase inhibitors are commonly used.
  • dopamine, the prodrug levodopa (L-DOPA) and other dopamine or levodopa prodrugs are commonly used.
  • Prodrugs of dopamine and L-DOPA have been described (see for example Karaman, 2011 (Karaman, 2011. Chem Biol Drug Des 78: 853–863); Fernandez et al., 2003 (Fernandez et al., 2003.
  • L-DOPA is a prodrug of dopamine
  • the prodrugs of L-DOPA are also prodrugs of dopamine.
  • Levodopa (L-DOPA) a precursor (or prodrug) to dopamine, is presently considered an effective and well tolerated dopamine replacing agent.
  • L-DOPA dopa-decarboxylase
  • L-DOPA is effective in the early stages of PD or Parkinsonism and remains effective as the disease progresses. Intolerance of L-DOPA is not known to develop over time.
  • the main drawbacks of L-DOPA are dyskinesias and response fluctuations, which are partly related to its short half-life (Salat et al., 2013. J Parkinson's Disease 3: 255-269).
  • the long-term treatment of PD or Parkinsonism with L-DOPA is complicated by the development of motor complications, including response fluctuations, dyskinesia, and psychiatric abnormalities.
  • L-DOPA induced dyskinesia is particularly troubling as the abnormal, involuntary movements can be disabling and interfere with activities of daily living.
  • an additional drug to the L- DOPA regimen is added from one of three other classes of treatments: dopamine agonists, catechol- O-methyl transferase inhibitors (COMTIs), or monoamine oxidase type B inhibitors (MAOBIs).
  • COMTIs catechol- O-methyl transferase inhibitors
  • MAOBIs monoamine oxidase type B inhibitors
  • combination therapy include ropinirole with L-DOPA (WO1997/048394).
  • materials which can serve as pharmaceutically acceptable carriers or vehicles include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as butylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ring
  • treat or “treating” includes stopping the progression, reversing the progression, preventing and/or reducing or ameliorating a symptom of a central nervous system disorder involving a dopamine deficiency such as Parkinson's disease or Parkinsonism, for example, improving motor function.
  • a dopamine deficiency such as Parkinson's disease or Parkinsonism
  • the words subject and individual are used inter-changeably herein.
  • Reference to a compound described herein is understood to include reference to salts thereof, unless otherwise indicated.
  • salt(s) denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases.
  • a compound described herein contains both a basic moiety, such as, but not limited to, amine, pyridine or imidazole and an acidic moiety, such as, but not limited to, a carboxylic acid, zwitterions ("inner salts") may be formed and are included within the term "salt(s)" as used herein.
  • Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful, e.g. , in isolation or purification steps which may be employed during preparation.
  • Salts of compounds described herein may be formed, for example, by reacting a compound with an amount of acid or base, such as an equivalent amount, in a medium, such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.
  • co-administration refers to the practice of administering two or more therapeutic agents, such as drugs or medicaments, to a subject at or around the same time.
  • the drugs can be given simultaneously, sequentially, or in a staggered manner, depending on the treatment regimen or how the drugs interact.
  • the goal of the co-administration of L-DOPA and tadalafil or a variant thereof is to enhance therapeutic efficacy and reduce side effects of the medications.
  • Co- administration of L-DOPA and tadalafil or a variant thereof involves giving both to the patient, where tadalafil or a variant thereof is used to allow for a lower dose of L-DOPA, thereby reducing the side effects typically caused by L-DOPA.
  • the drugs can be taken together in one combined formulation or separately but during the same treatment period.
  • Compounds The use of simple ester prodrugs of levodopa to improve the pharmacokinetics of the drug has been proposed (U.S. Pat. Nos. 5,017,607; 4,826,875; 4,873,263; 4,771,073; 4,663,349; 4,311,706; Japanese Patent No. JP58024547; Juncos et al., 1987.
  • levodopa methyl ester (Levomet®, CHF 1301) has been described (Chiesi Pharmaceuticals).
  • the ethyl ester of levodopa (TV-1203) is under clinical investigation as a potential therapy for Parkinsonism when co- administered with carbidopa (U.S. Pat. No.5,607,969).
  • a sustained release cellulose formulation of levodopa ethyl ester in a mixture of hydroxypropylmethyl cellulose, hydroxypropyl cellulose, and a carboxyvinyl polymer has been described (U.S. Pat. No.
  • dopamine and levodopa prodrugs are esters of dopamine or levodopa.
  • the invention provides dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in a method for the treatment of dopamine deficiency in a subject characterized in that the treatment of the deficiency further comprises the administration of tadalafil or variant thereof to the subject.
  • the cyclic nucleotide PDEs comprise a group of enzymes that degrade the phosphodiester bond in the second messenger molecules cAMP, cGMP or both. PDEs are commonly known as regulators of signal transduction mediated by these second messenger molecules.
  • the PDE nomenclature signifies the PDE family with an Arabic numeral, then a capital letter denotes the gene in that family, and a second and final Arabic numeral then indicates the splice variant derived from a single gene (e.g., PDE1C3: family 1, gene C, splicing variant 3).
  • PDE1C3 family 1, gene C, splicing variant 3
  • a PDE inhibitor is a drug that blocks one or more of the subtypes of the enzyme phosphodiesterase, thereby preventing the inactivation of the intracellular second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) by the respective PDE subtype(s).
  • cAMP cyclic adenosine monophosphate
  • cGMP cyclic guanosine monophosphate
  • non-selective PDE inhibitors are caffeine; aminophylline; IBMX (3-isobutyl-1-methylxanthine); paraxanthine; pentoxifylline; and theophylline. These compounds are said to be non-selective, however, there activity may still vary. This can be the result of differences in preference; in affinity; and/or in pharmacokinetics, among others.
  • the concentration of a particular PDE inhibitor in vitro that inhibits PDE activity by 50% is known as the IC50 value.
  • Selective PDE inhibitors are often referred to by the PDE type that is most specifically inhibited.
  • a PDE5 inhibitor has an IC50 for PDE5 that is lower than the IC50 for any of the other PDEs.
  • An inhibitor is a PDE inhibitor if it has an IC50 that is in the micro(u) or nano(n) molar range for at least one of the human PDEs.
  • An inhibitor is a selective PDE inhibitor if it has an IC50 for the respective PDE that is at least 10x lower than the IC50 of more than 5 other human PDEs, preferably more than 8, preferably all other human PDEs.
  • a PDE2 inhibitor has an IC50 that is at least 10x lower than the IC50 of the compound for more than 5 other human PDEs, preferably more than 8, preferably all other human PDEs.
  • a PDE11 inhibitor has an IC50 that is at least 10x lower than the IC50 of the compound for more than 5 other human PDEs, preferably more than 8, preferably all other human PDEs.
  • Tadalafil and variants thereof as described herein have the general formula: , wherein R1 is selected from the group consisting of H, halo, and C1-6alkyl; R2 represents an optionally substituted monocyclic aromatic ring selected from the group consisting of benzene, thiophene, furan, and pyridine, or an optionally substituted bicyclic ring attached to the rest of the molecule via one of the benzene ring carbon atoms, wherein the fused ring A is a 5- or 6-membered ring, saturated or partially or fully unsaturated, and comprises carbon atoms and optionally one or two heteroatoms selected from oxygen, sulphur, and nitrogen; R3 represents H or C1-3alkyl, or R3 and R4 together represent a 3- or 4-membered alkyl or alkenyl chain component of a 5- or 6-membered ring; and R4 is selected from the group consisting of H, C1-6alkyl, C
  • a variant of tadalafil preferably has the structural formula indicated above wherein R1 is H, R4 is H or C1-6alkyl; R2 is the bicyclic ring optionally substituted with one or more groups independently selected from halo and C1-3alkyl; and R3 is H or C1-3alkyl. It is preferred that tadalafil or the variant thereof has the structural formula indicated above wherein R1 is H; R2 is the bicyclic ring ; and R3 is H; and R4 is CH3.
  • a guanyl cyclase agonist is preferably a GUCY2C agonist.
  • GUCY2C or Guanylate Cyclase 2C is known under a number of names such as STAR; STA Receptor; GUC2C; Heat-Stable Enterotoxin Receptor; Intestinal Guanylate Cyclase; Guanylyl Cyclase C; EC 4.6.1.2; GC-C; Heat Stable Enterotoxin Receptor; EC 4.6.1; DIAR6 and others.
  • External Ids for GUCY2C Gene are HGNC: 4688 NCBI Entrez Gene: 2984 Ensembl: ENSG00000070019 OMIM®: 601330 UniProtKB/Swiss-Prot: P25092.
  • the guanylin family of peptides has 3 subclasses of peptides containing either 3 intramolecular disulfide bonds found in bacterial heat-stable enterotoxins (ST), or 2 disulfides observed in guanylin and uroguanylin, or a single disulfide exemplified by lymphoguanylin. These peptides bind to and activate cell-surface receptors that have intrinsic guanylate cyclase (GC) activity such as GUCY2C. The peptides are also referred to as GUCY2C agonists. Guanylin is a natural agonistic ligand of GUCY2C. It is a 15 amino acid polypeptide.
  • Guanylin acts as an agonist of the guanylyl cyclase receptor GC-C and among others regulates electrolyte and water transport in intestinal and renal epithelia.
  • the uroguanylin peptide is encoded by the GUCA2B gene.
  • the gene and protein are known under a number of different names Guanylate Cyclase Activator 2B; Prepro-Uroguanylin; GCAP-II; and UGN.
  • peptide shuttles have received growing attention because of their lower cost, reduced immunogenicity, and higher chemical versatility than traditional Trojan horse antibodies and other proteins.
  • Suitable BBB shuttles are described in Oller- Salvia et al., 2016 (Oller-Salvia et al., 2016. Chem Soc Rev 45: 4690-4707), which is incorporated by reference herein for this purpose.
  • the functional guanylin, lymphoguanylin, enterotoxin or uroguanylin derivative is guanylin, lymphoguanylin, enterotoxin or uroguanylin fused to a peptide BBB shuttle of table 1 of Oller-Salvia et al., 2016.
  • the PDE2 inhibitor is EHNA. In embodiments, the PDE2 inhibitor is BAY 60-7550. In embodiments, the PDE2 inhibitor is PDP. In embodiments, the PDE2 inhibitor is PF-05180999.
  • Dopa-decarboxylase (DDC) inhibitors are known in the art, including benserazide (2-amino-3-hydroxy-N'-[(2,3,4-trihydroxyphenyl)methyl] propanehydrazide), carbidopa (2S)-3-(3,4-dihydroxyphenyl)-2-hydrazinyl-2- methylpropanoic acid), methyldopa (L- ⁇ -Methyl-3,4-dihydroxyphenylalanine), alpha-difluoromethyl-DOPA (DFMD; (2S)-2-Amino-2-[(3,4- dihydroxyphenyl)methyl]-3,3-difluoropropanoic acid), 3',4',5,7-tetrahydroxy-8- methoxyisoflavone (3-(3,4-dihydroxyphenyl)-5,7-dihydroxy-8-methoxychromen-4- one), epigallocatechin (EGC; (2R,3R)-2-(3,4,5
  • Catechol-O-methyltransferase (COMT) inhibitors are known in the art, including entacapone ((E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N- diethylprop-2-enamide), tolcapone ((3,4-dihydroxy-5-nitrophenyl)-(4- methylphenyl)methanone), opicapone (5-[3-(2,5-dichloro-4,6-dimethyl-1- oxidopyridin-1-ium-3-yl)-1,2,4-oxadiazol-5-yl]-3-nitrobenzene-1,2-diol), and nitecapone (3-(3,4-dihydroxy-5-nitrobenzyliden)-2,4-pentandion).
  • entacapone (E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N- diethylprop-2-enamide)
  • tolcapone (
  • Dopamine agonists are known in the art, including bromocriptine ((6aR,9R)- 5-bromo-N-[(1S,2S,4R,7S)-2-hydroxy-7-(2-methylpropyl)-5,8-dioxo-4-propan-2-yl-3- oxa-6,9-diazatricyclo[7.3.0.02,6]dodecan-4-yl]-7-methyl-6,6a,8,9-tetrahydro-4H- indolo[4,3-fg]quinoline-9-carboxamide), amantadine (adamantan-1-amine), cabergoline ((6aR,9R,10aR)-N-[3-(dimethylamino)propyl]-N-(ethylcarbamoyl)-7- prop-2-enyl-6,6a,8,9,10,10a-hexahydro-4H-indolo[4,3-fg]
  • Dopamine, a dopamine agonist, levodopa and prodrugs thereof are preferably administered orally.
  • the composition comprising dopamine, levodopa and/or a prodrug thereof is suitable for oral administration.
  • the composition is in a solid oral dose form.
  • the solid oral dose form is a tablet, a capsule, or a softgel.
  • Various other delivery methods can be used.
  • One such other methods is intranasal delivery.
  • Tadalafil is presently marketed as a tablet for oral administration (for instance under the tradename Cialis). It can also be administered in other ways. For instance, as an intravenous injection, or inhalant.
  • Various other delivery methods can be used.
  • the effective amount of dopamine, a dopamine agonist, L- DOPA or a prodrug thereof is a suboptimal dose when compared to the optimal dose of said dopamine, a dopamine agonist, L-DOPA or a prodrug thereof when given as a single treatment.
  • the sub-optimal dose is determined by titration in a patient.
  • dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered in an amount that is lower than a therapeutically effective amount of the compound when administered as a sole therapeutic agent for the treatment of Parkinson's disease or Parkinsonism.
  • dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered in an amount that is lower than a therapeutically effective amount of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof when administered with benserazide for the treatment of Parkinson's disease or Parkinsonism.
  • dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered in an amount that is lower than a therapeutically effective amount of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof when administered with carbidopa for the treatment of PD or Parkinsonism.
  • the suboptimal dose is preferably determined by titration in the patient.
  • the suboptimal dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is between 10% and 75% of the optimal dose of said dopamine, a dopamine agonist, L- DOPA or a prodrug thereof when given as a single treatment.
  • the suboptimal dose is preferably between 10-60%, preferably 20-50%, preferably between 30-50% of the optimal dose when given as a single treatment.
  • the suboptimal dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof in a combination treatment with a further PD medicament is between 10% and 75% of the optimal dose of said dopamine, a dopamine agonist, L-DOPA or a prodrug thereof when given as said combination treatment.
  • the amount of levodopa (L-DOPA) that is effective for treating Parkinson's disease or Parkinsonism is in the range of about 150-1000 mg/day. Suitable lower doses of L-DOPA are in the range of about 25-200 mg/day.
  • the effective amount of tadalafil or variant thereof is at least 0,1 mg/day, such as from about 0,1 mg to about 200 mg per day. In embodiments, the effective amount of tadalafil or variant thereof is a daily dose of about 1 to about 50 mg/day. In embodiments, the effective amount of tadalafil or variant thereof is 5 to about 40 mg/day. In embodiments, the effective amount of tadalafil or variant thereof is about 5 to about 40 mg per day. In some embodiments, the effective amount of tadalafil or variant thereof is about 5, 10 or 20 mg per day. In embodiments, the effective amount of a PDE11 inhibitor is from about 0.1 mg to about 1000 mg per day.
  • the effective amount of the PDE2 inhibitor is about 15 to about 20 mg per day. In embodiments, the effective amount of the PDE2 inhibitor is about 5 to about 20 mg per day. In embodiments, the effective amount of the PDE2 inhibitor is about 25 to about 100 mg per day. In embodiments, the effective amount of a PDE11 inhibitor is from about 0.1 mg to about 1000 mg per day. In embodiments, the effective amount of the PDE11 inhibitor is about 1 to about 500 mg per day. In embodiments, the effective amount of the PDE11 inhibitor is from about 10 to about 100 mg per day. In embodiments, the effective amount of the PDE11 inhibitor is about 20 to about 50 mg per day. In embodiments, the effective amount of the PDE11 inhibitor is about 25 to about 40 mg per day.
  • tadalafil or variant thereof When tadalafil or variant thereof is combined with a PDE2 inhibitor such as EHNA, PF-05180999 (CAS 1394033-54-5) and/or BAY 60-7550, and/or a PDE11 inhibitor such as BC11-38, said tadalafil or variant thereof may be provided at less than 20 mg/day, such as from about 1 mg to about 15 mg per day, including 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg or 14 mg/day.
  • a guanylate cyclase agonist such as a GUCY2C agonist can be administered directly to a subject.
  • tadalafil or variant thereof may be provided 10 mg/day or less, such as from about 0.1 mg to about 9 mg per day, including 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, and 0.9 mg/day.
  • Exemplary effective daily doses of benserazide include, but are not limited to, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 50 mg, 60 mg, 70 mg, 80 mg and 100 mg.
  • adalafil or variant thereof When tadalafil or variant thereof is combined with a dopa-decarboxylase (DDC) inhibitor, e.g., benserazide or carbidopa, said tadalafil or variant thereof may be provided at less than 20 mg/day, such as from about 1 mg to about 15 mg per day, including 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg or 14 mg/day.
  • DDC dopa-decarboxylase
  • the effective amount of a monoamine oxidase (MAO-B) inhibitor e.g., rasagiline or selegiline
  • a monoamine oxidase (MAO-B) inhibitor is in the range of 1 to 500 mg, 2 to 150 mg, 2 to 120 mg, 2 to 80 mg, 2 to 40 mg, 5 to 150 mg, 5 to 120 mg, 5 to 80 mg, 10 to 150 mg, 10 to 120 mg, 10 to 80 mg, 10 to 40 mg, 20 to 150 mg, 20 to 120 mg, 20 to 80 mg, 20 to 40 mg, 40 to 150 mg, 40 to 120 mg or 40 to 80 mg.
  • a typical dose of rasagiline is 1 mg/day which is preferred in the present invention.
  • a typical dose of selegiline is 10 to 20 mg/day which is preferred in the present invention.
  • the dose is typically administered over several time points over the day.
  • the administration is typically done with a selegiline solution containing 5 mg/ml of selegiline.
  • Another MAO-B inhibitor is safinamide. Safinamide can be given in a range of 10-500 mg. It is typically administered with a starting dose of 50 mg per day which can be increased to 100 mg per day or decreased as desired.
  • the effective daily dose of a monoamine oxidase (MAO-B) inhibitor is 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.5 mg, 2 mg, 4 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 750 mg or 1,000 mg.
  • the effective amount of the catechol-O-methyltransferase (COMT) inhibitor is 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.5 mg, 2 mg, 4 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 500 mg.
  • tadalafil or variant thereof When tadalafil or variant thereof is combined with a catechol-O-methyltransferase (COMT) inhibitor, e.g., tolcapone or entacapone, said tadalafil or variant thereof may be provided at less than 20 mg/day, such as from about 1 mg to about 15 mg per day, including 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg or 14 mg/day.
  • the effective amount of a dopamine agonist e.g., pramipexole, ropinirole, rotigotine, is in the range of 0,1 mg – 30 mg per day.
  • the starting dose is typically lower in the first week, which is then increased in the second week. If needed the dose can be further increased in the third week and even again in the fourth week.
  • a typical dose escalation is: In the first week, take 3 times 0.25 mg per day. In the second week, take 3 times 0.5 mg per day. In the third week, take 3 times 0.75 mg per day. In the fourth week, take 3 times 1 mg per day. If necessary, gradually increase to a maximum of 24 mg per day after the fourth week.
  • Rotigotine is typically administered with a skin patch. When used as monotherapy: Increase the initial dose from 2 mg per day to a maximum of 8 mg per day. The maintenance dose is typically 6-8 mg per day.
  • the individual may be administered at the same time.
  • the individual does not have a completely advanced form of PD. In such cases the substantia nigra is (almost) completely destroyed.
  • the individual has Parkinson’s disease stage 1, 2, 3 or 4.
  • the individual has Parkinson’s disease stage 1, 2, or 3.
  • stage 1 or stage 2 Preferably stage 1 or stage 2.
  • Dopaminergic neurons of the substantia nigra are stimulated to produce more dopamine to compensate for the loss of dopaminergic cells in the substantia nigra while at the same time not over-producing dopamine, or having excessive dopamine production in non-target cells, i.e. dopaminergic neurons that are not within the substantia nigra.
  • the possibility of reducing the dose of dopamine, a dopamine agonist, L-DOPA or prodrug thereof reduces the propensity and/or the intensity of side effects of the externally provided dopamine, a dopamine agonist, L-DOPA or prodrug thereof.
  • TH has an N-terminal regulatory domain, a central catalytic domain and a C-terminal tetramerization domain (Tekin et al., 2014. J Neural Transmiss 121: 1451–1481).
  • the most appealing domain to increase TH activity is located in the N-terminal region, which includes three serine residues susceptible to phosphorylation (i.e. Ser19, Ser31 and Ser40). The regulatory importance of these residues is reflected in their broad evolutionary conservation (see WO/2018/160067).
  • Ser31 and Ser40 Independent phosphorylation of Ser31 and Ser40 is known to increase TH activity in vitro and in situ.
  • Ser31 phosphorylation exclusively operates by raising the affinity of TH for one of its cofactors (i.e. BH4), whereas Ser40 also impedes the negative feedback loop by blocking the interaction of dopamine with the catalytic domain of Th (Dunkley et al., 2004. J Neurochem 91: 1025-1043).
  • Ser40 is thus a promising target, and we believe that boosting its phosphorylation solves an important limitation of levodopa treatment. It is an object of the invention to increase TH activity by inducing Ser40 phosphorylation specifically in the (presynaptic) striatum.
  • PKA and PKG The main kinases known to phosphorylate this residue are cAMP- and cGMP-dependent protein kinases (PKA and PKG, respectively) (Campbell et al., 1986. J Biol Chem 261: 10489-10492; Roskoski et al., 1987. J Neurochem 48: 840-845). Both enzymes are basally inactive due to the interaction of the regulatory region with the catalytic center. While the regulatory and catalytic counterparts of PKA correspond to separate polypeptides, PKG is a single amino acidic chain including both domains.
  • the further medicament preferably comprises a PDE11 inhibitor, a PDE2 inhibitor, a dopa-decarboxylase (DDC) inhibitor; a monoamine oxidase (MAO-B) inhibitor; a catechol-O-methyltransferase (COMT) inhibitor; or a combination thereof.
  • the PDE11 inhibitor preferably is BC11-15; BC11-19; BC11-28; BC11-38; BC11-38-1; BC11-38-2; BC11-38-3; or BC11-38-4, preferably BC11-38; BC11-38-1; BC11-38-2; BC11-38-3 or BC11-38-4.
  • the PDE11 inhibitor is BC11-38.
  • the PDE2 inhibitor is preferably EHNA, BAY 60-7550, PDP, PF-05180999, or oxindole.
  • the invention further provides a method for treating dopamine deficiency in a subject comprising administering dopamine, a dopamine agonist, L-DOPA or a prodrug thereof to a subject in need thereof characterized in that the treatment of the deficiency further comprises the administration of tadalafil or a variant thereof to the subject, whereby the administration of tadalafil or variant thereof is at a minimum dosage of 20 mg/day (0.4 mg/kg).
  • Said dopamine deficiency preferably is a central nervous system dopamine deficiency.
  • the invention also provides a method of treating a Parkinson’s disease in a subject comprising administering dopamine, a dopamine agonist, L-DOPA or a prodrug thereof to a subject in need thereof characterized in that the treatment of the disease comprises the administration of tadalafil or variant thereof to the subject, whereby the administration of tadalafil or variant thereof is at a minimum dosage of 20 mg/day (0.4 mg/kg).
  • the treatment preferably comprises treating symptoms of Parkinson’s disease related to central nervous system dopamine deficiency. Said treatment preferably is of patients having PD stage 1-5.
  • a medicament or kit of parts comprising a sub-optimal dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof and tadalafil or variant thereof, for use in the treatment of PD, preferably PD stage 1-5.
  • the invention further provides a method for at least in part decreasing feedback inhibition by dopamine, a dopamine agonist, L-DOPA or a prodrug thereof on dopamine production comprising contacting a dopaminergic cell, preferably a substantia nigra dopaminergic cell, with said dopamine, a dopamine agonist, L-DOPA or prodrug thereof and tadalafil or variant thereof.
  • the dose of externally provided dopamine, a dopamine agonist, L-DOPA or prodrug thereof can be reduced to levels wherein the treatment has less side-effects than a treatment with dopamine, a dopamine agonist, L-DOPA or prodrug at a routine dose.
  • the treatment is particularly effective in early stages of the disease, where the limitation of dopamine production in the individual just becomes apparent in the expression of mild symptoms. In such cases the individual does not have enough dopamine production typically due to the partial disappearance of dopaminergic cells. Particularly in the early stages, however, the individual typically has ample cells to provide adequate production of dopamine upon the treatment of the invention.
  • the invention further provides dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in a method for the treatment of dopamine deficiency in a subject characterized in that the treatment further comprises the administration of tadalafil or variant thereof to the subject.
  • Pharmaceutical Compositions Comprising L-DOPA and tadalafil or variant thereof.
  • An effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular agent being administered, the size of the subject, or the severity of the disease or condition, as is known to a skilled person.
  • a therapeutically effective amount is any amount that is necessary or sufficient for treating Parkinson's disease or Parkinsonism in a subject in combination with a sub-optimal dose of L-DOPA.
  • the effective amount of tadalafil or variant thereof can also vary depending on whether tadalafil or variant thereof is administered with L-DOPA in combination with benserazide, whether tadalafil or variant thereof is administered with L- dopa in combination with carbidopa, whether tadalafil or variant thereof is administered with L-DOPA in combination with a monoamine oxidase type B inhibitor, or whether tadalafil or variant thereof is administered with L-DOPA in combination with a catechol-O- methyl transferase inhibitor.
  • the effective amount of tadalafil or variant thereof may further vary depending on whether the inhibitor is administered with L- DOPA in combination with carbidopa and a monoamine oxidase type B inhibitor, with L-DOPA in combination with carbidopa and a catechol-O- methyl transferase inhibitor, with L-DOPA in combination with benserazide and a monoamine oxidase type B inhibitor, or with L-DOPA in combination with benserazide and a catechol- O- methyl transferase inhibitor.
  • Tadalafil or variant thereof and L-DOPA can be administered at different times or at the same time. In embodiments, L-DOPA and tadalafil or variant thereof are administered simultaneously.
  • L-DOPA and tadalafil or variant thereof may independently be administered to a patient once, twice, three times, four times, five times, six times, 8 times or 12 times a day, as is known to a person skilled in the art.
  • the methods described herein can further comprise administering an effective amount of another therapeutic agent, that is, the pharmaceutical compositions described herein can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures.
  • the particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved.
  • the methods described herein can further comprise administering an effective amount of a PDE11 inhibitor such as BC11-38.
  • the methods described herein can further comprise administering an effective amount of a PDE2 inhibitor such as EHNA, PF-05180999 or BAY 60-7550.
  • the methods described herein can further comprise administering an effective amount of a guanylate cyclase agonist, preferably a guanylate cyclase 2C receptor (GUCY2C) agonist, such as guanylin, lymphoguanylin, enterotoxin or uroguanylin or a functional derivative of guanylin, lymphoguanylin, enterotoxin or uroguanylin.
  • a PDE2 inhibitor such as EHNA, PF-05180999 or BAY 60-7550.
  • the methods described herein can further comprise administering an effective amount of a guanylate cyclase agonist, preferably a guanylate cyclase 2C receptor (GUCY2C) agonist, such as guanylin, lymphoguanylin, entero
  • the methods described herein further comprise administering effective amounts of benserazide and a guanylate cyclase agonist, preferably a guanylate cyclase 2C receptor (GUCY2C) agonist.
  • the methods described herein further comprise administering effective amounts of benserazide and a monoamine oxidase (MAO-B) inhibitor.
  • the methods described herein further comprise administering effective amounts of benserazide and a catechol-O- methyltransferase (COMT) inhibitor.
  • the methods described herein further comprise administering effective amounts of carbidopa and a monoamine oxidase (MAO-B) inhibitor.
  • the methods described herein further comprise administering effective amounts of carbidopa and a catechol-O-methyltransferase (COMT) inhibitor.
  • a monoamine oxidase (MAO-B) inhibitor or a catechol-O- methyltransferase (COMT) inhibitor is administered in addition tadalafil or variant thereof and dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, it can be administered before, after or concurrently with tadalafil or variant thereof and/or dopamine, a dopamine agonist, L-DOPA or a prodrug thereof.
  • the kit comprises: (a) L-DOPA, benserazide, and a pharmaceutically acceptable carrier or vehicle in a first container; and (b) tadalafil or variant thereof and a pharmaceutically acceptable carrier or vehicle in a second container.
  • the kit can further comprise a label or printed instructions instructing the use of tadalafil or variant thereof and L-DOPA to treat Parkinson's disease.
  • the kit can further comprise a label or printed instructions instructing the use of tadalafil or variant thereof and L-DOPA to treat PD or Parkinsonism.
  • the kit can also further comprise a unit dose form of a dopa-decarboxylase (DDC) inhibitor.
  • DDC dopa-decarboxylase
  • the kit comprises: (a) L-DOPA and a pharmaceutically acceptable carrier or vehicle in a first container; (b) tadalafil or variant thereof and a pharmaceutically acceptable carrier or vehicle in a second container, and (c) monoamine oxidase (MAO-B) inhibitor and a pharmaceutically acceptable carrier or vehicle in a third container.
  • the kit comprises: (a) L-DOPA and a pharmaceutically acceptable carrier or vehicle in a first container; (b) tadalafil or variant thereof and a pharmaceutically acceptable carrier or vehicle in a second container, and (c) catechol-O-methyltransferase (COMT) inhibitor and a pharmaceutically acceptable carrier or vehicle in a third container.
  • the kit comprises: (a) L-DOPA, benserazide, and a pharmaceutically acceptable carrier or vehicle in a first container; (b) tadalafil or variant thereof and a pharmaceutically acceptable carrier or vehicle in a second container, and (c) monoamine oxidase (MAO-B) inhibitor and a pharmaceutically acceptable carrier or vehicle in a third container.
  • the kit comprises: (a) L-DOPA, benserazide, and a pharmaceutically acceptable carrier or vehicle in a first container; (b) tadalafil or variant thereof and a pharmaceutically acceptable carrier or vehicle in a second container, and (c) catechol-O- methyltransferase (COMT) inhibitor and a pharmaceutically acceptable carrier or vehicle in a third container.
  • the kit comprises: (a) L-DOPA, carbidopa, and a pharmaceutically acceptable carrier or vehicle in a first container; (b) tadalafil or variant thereof and a pharmaceutically acceptable carrier or vehicle in a second container, and (c) monoamine oxidase (MAO-B) inhibitor and a pharmaceutically acceptable carrier or vehicle in a third container.
  • the kit comprises L-DOPA, tadalafil or variant thereof, a catechol-O- methyltransferase (COMT) inhibitor, and a pharmaceutically acceptable carrier or vehicle in a single container.
  • the kit comprises L-DOPA, tadalafil or variant thereof, benserazide, a monoamine oxidase (MAO-B) inhibitor, and a pharmaceutically acceptable carrier or vehicle in a single container.
  • the kit comprises L-DOPA, tadalafil or variant thereof, benserazide, a catechol-O-methyltransferase (COMT) inhibitor, and a pharmaceutically acceptable carrier or vehicle in a single container.
  • the kit further comprises a catechol-O-methyltransferase (COMT) inhibitor in the single container.
  • a catechol-O-methyltransferase (COMT) inhibitor in the single container.
  • the heterozygous animals are described to have a normal development of the midbrain dopaminergic system, while the homozygous animals are known to have a dramatic loss of neurons of the substantia nigra and its projections to the dorsal striatum (Maxwell et al., 2005. Dev Biol 282: 467–479; Zhao et al., 2004. Eur J Neurosci 19: 1133–1140). Animals were housed on a 12 hour light-dark cycle, with food and water provided ad libitum. All animal experimentation was supported and granted by the Animals Experimentation Committee of the University of Amsterdam (UvA) according to national and international legislation.
  • mice Animals are cared for on a daily basis and sacrificed according to rules and regulations of the Dutch and European law. Ex vivo slicing and chemical treatment Mice were sacrificed by cervical dislocation in compliance with the law. Brains were immediately isolated and sliced on a Leica VT100S vibratome in ice- cold slicing buffer (120 mM Choline Chloride, 3.5 mM KCl, 0.5 mM CaCl2, 6 mM MgSO4, 1.25 mM NaH2PO4, 27.5 mM D-Glucose, 25 mM NaHCO3) under constant oxygenation (95% O2, 5% CO2). Coronal Midbrain (MB) or corpus striatum (CS) slices with a thickness of 250 ⁇ m were collected.
  • MB Choline Chloride
  • CS corpus striatum
  • Proteins were probed using the following antibodies: rabbit Anti-Tyrosine Hydroxylase (P40101, Pel-Freez); rabbit anti-phospho-tyrosine hydroxylase (Ser40) (2791S, CST); rabbit anti- phospho-tyrosine hydroxylase (Ser31) (13041S, CST); rabbit anti- ⁇ -actin (4907S, CST); anti-TOM20 (42406, CST).
  • Antibodies were diluted in antibody diluent (1:10 or 1:50; 042-203, ProteinSimple). Statistical analysis The amounts of phospho-protein levels were corrected for the total amount of the same protein, phosphorylated or not, and normalized to the vehicle condition.
  • Coronal slices were collected as described above, resulting in 12 striatal (CS) slices, five midbrain (MB) slices, and three slices connecting the striatum and the midbrain. Each slice was divided per hemisphere and the regions of interest (depicted in gray in schematic images of Fig. 3A) were micro-dissected. The micro-dissected parts of each coronal slice (one per hemisphere) were pooled and probed for baseline protein levels of total tyrosine hydroxylase (independent of phosphorylation status), phospho-tyrosine hydroxylase (Ser40), phospho-tyrosine hydroxylase (Ser31), and ⁇ -actin (Fig. 3A).
  • each slice per hemisphere and micro-dissect the regions of interest of each slice (see Figure 4, gray part of the schematic images).
  • the micro-dissected part of one hemisphere as internal control for the experimental, treated condition, which is the micro-dissected part of the other hemisphere.
  • the control and experimental conditions are randomly chosen, we can only use this setup if the relative tyrosine hydroxylase levels are similar between hemispheres.
  • Ser40 phosphorylation The counterpart of tyrosine hydroxylase activation via Ser40 phosphorylation is the extensively described mechanism of end-product feedback inhibition via dopamine and other catecholamines (Almas et al., 1992. Eur. J. Biochem 209: 249– 255; Andersson et al., 1992. Biochem J.284: 687–695; Andersson et al., 1988. J Biol Chem 263: 18621–18626; Briggs et al., 2014. J Biochem 155: 183–193; Briggs et al., 2011.
  • mice that are deficient in Pitx3 are used as model for the selective loss of nigrostriatal dopamine neurons and show L-DOPA-reversible impaired performance on select behavioral tests (Ardayfio et al., 2008. Neurobiol Dis 31: 406–412; Hwang et al., 2005. J Neurosci 25: 2132–2137).
  • mice To examine the effects of Pitx3-deficiency on tyrosine hydroxylase levels and regulation in the striatum, we examined and compared tyrosine hydroxylase protein levels between phenotypically normal heterozygous Pitx3GFP/+ and defective homozygous Pitx3GFP/GFP mice. These mice either have a heterozygous (Pitx3GFP/+) or homozygous (Pitx3GFP/GFP) mutation of Pitx3 that carries a green fluorescent protein (GFP) under control of the endogenous Pitx3 promotor.
  • the heterozygous mutation of Pitx3 does not affect nigrostriatal dopamine neuron development and these mice show similar tyrosine hydroxylase levels to Pitx3 wild- type littermates (Maxwell et al., 2005. Dev Biol 282: 467–479; Zhao et al., 2004. Eur J Neurosci 19: 1133–1140). Therefore, the Pitx3 heterozygous mice are used as controls for the homozygous Pitx3-deficient, nigrostriatal dopamine neuron deficient mice. To start, we want to inspect if some sort of compensation on the level of tyrosine hydroxylase phosphorylation develops in a dopamine deficient situation (Fig. 8A-D).
  • Ser31 phosphorylation levels are down-regulated.
  • Ser40 phosphorylation levels can still be up-regulated by forskolin, and to a similar extend as the phenotypically normal situation.
  • Phosphorylation of Ser40 is the main regulatory mechanism to increase the enzymatic activity of tyrosine hydroxylase, thereby boosting the synthesis of L-DOPA from L-tyrosine and thereby increasing dopaminergic biosynthesis.
  • exogenous L-DOPA down-regulates tyrosine hydroxylase Ser40 phosphorylation
  • the induction of tyrosine hydroxylase Ser40 phosphorylation can be achieved also when a substantial proportion of nigrostriatal dopamine neurons is lost.
  • the progressive loss in nigrostriatal dopaminergic projections may actually be a factor to prevent the eventual durability of L-DOPA therapy.
  • the exogenous L-DOPA supports striatal dopamine release while excessive L-DOPA and dopamine can be buffered by pre-synaptic mechanisms (Carta and Bezard, 2011.
  • tyrosine hydroxylase Ser40 phosphorylation a suitable target in the treatment of Parkinson’s disease: either as a complement to L-DOPA therapy or as an alternative to completely circumvent the well-described off-target effects of exogenous L-DOPA.
  • the promotion of tyrosine hydroxylase Ser40 phosphorylation will allow lower L-DOPA dosage and dosages. This is because Ser40 phosphorylation re-activates endogenous dopamine production, which automatically allows lower L-DOPA dosages, as the L-DOPA concentration used is now on-top of the endogenous production.
  • L-DOPA dose can be lowered depending on the extent of Ser40 phosphorylation and the amount of dopamine neurons still present. This is typically reflected in the stage of the disease and the disease phenotype particular to the patient.
  • Off target effects caused by L-DOPA treatment can be the result of dopamine production by cells that are not situated in the striatum (extrastriatal cells) and that are not normally producing dopamine. Such cells can, however, produce dopamine when L-DOPA when is provided to them. This extrastriatal off target dopamine production can seriously contribute to the side effects experienced by the Parkinson patient that is treated with L-DOPA.
  • the present invention insures, that both the off- target effects induced by extrastriatal dopamine production by non-dopaminergic AADC-containing neurons and the uncontrolled overstimulation of post-synaptic signaling routes is reduced, while the endogenous dopamine biosynthesis pathway is not completely shut down.
  • important mechanisms that affect and regulate dopamine neurotransmission will not be circumvented allowing at least some form of natural regulation of dopamine levels.
  • the signaling circuitry that initiates dopamine synthesis or post-synthesis processes such as vesicular packaging.

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Abstract

The invention relates to dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in a method for the treatment of dopamine deficiency in a subject characterized in that the treatment of the deficiency further comprises the administration of tadalafil or variant thereof to the subject. The combination with tadalafil or variant thereof allows a reduction in the amount of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof resulting in reduced extrastriatal side effects. The disclosure further relates to compositions, medicaments and kits of parts comprising dopamine, a dopamine agonist, L-DOPA or a prodrug thereof and tadalafil, optionally further comprising a PDE11 inhibitor, a PDE2 inhibitor, a guanyl cyclase agonist, or a combination thereof.

Description

P135718PC00 Title: Reducing dopamine feed-back inhibition in dopaminergic cells by tadalafil. The invention relates to means and methods for treating dopamine deficiencies in subjects. The invention also relates to means and methods for the treatment of an individual that is receiving levodopa (L-DOPA) as a medicament. The invention also relates to means and methods for the treatment of an individual that has Parkinson’s disease or is at risk of developing this disease. Parkinson’s disease (PD) is the most prevalent neurological disorder after Alzheimer’s pathology. Its primary hallmark is the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) (Kalia and Lang, 2015. Lancet 386: 896-912). This anatomical region arises from the mesencephalon during development and eventually creates projections towards the dorsal striatum, one of the forebrain areas by which the basal ganglia are comprised of (Smidt and Burbach, 2007. Nat Rev Neurosci 8: 21–32; Baladron and Hamker, 2015. Neural Netw 67: 1-13). The functional connectivity between both structures is broadly referred as the nigrostriatal pathway and depends on the release of dopamine (Ikemoto et al., 2015. Behav Brain Res 290: 1731). This neurotransmitter interacts with the striatal postsynaptic receptors after being released by the nigral efferents, ultimately regulating motor control. Accordingly, in PD patients in which there is a prominent loss of neurons in the SNpc, the resultant lack of dopamine release into the striatum triggers the onset of motor symptoms. Classically, these Parkinsonian manifestations can be classified into bradykinesia or slow body movement, tremor at rest, muscular stiffness and gait abnormalities (Kalia and Lang, 2015. Lancet 386: 896-912). Nonetheless, motor impairments only appear when the degeneration in the SNpc occurs in an advanced stage, and they are preceded by a set of non-motor symptoms which often include olfactory dysfunction, depression or constipation (Mahlknecht et al., 2015. J Parkinsons Dis 5: 68197). The physiological cause of these early symptoms is not completely understood and, at least partially, is thought to be mediated by the malfunctioning of networks outside the nigrostriatal pathway and the imbalance of neurotransmitters apart from dopamine (Kalia and Lang, 2015. Lancet 386: 896-912). Interestingly, the latency between the outbreak of the prodromal phase and motor manifestations might be more than a decade (Postuma et al., 2012. Mov Disord 27: 617-626). Therefore, the premotor period could offer physicians a temporal window which might be exploited to prevent the further development of the disease. Unfortunately, two major drawbacks arise when trying to employ this strategy to tackle PD: (1) non-motor symptoms are subtle to the clinical eye and not necessarily linked to this particular disease, and (2) the current comprehension of the molecular basis of sporadic PD is far from complete, rarely considers the underlying etiology of the disorder and often concedes a complex interplay of environmental and genetic factors (Kalia and Lang, 2015. Lancet 386: 896-912; Mahlknecht et al., 2015. J Parkinsons Dis 5: 68197). The most common drugs for Parkinson’s disease are: levodopa (L-DOPA); levodopa combined with peripheral AADC inhibitors (e.g. carbidopa); MAOB inhibitors, COMT inhibitors (Kalia LV and Lang AE, 2015) and dopamine agonist (Armstrong and Okun, 2020, JAMA 323, 548-560). These medicines can be taken orally and are able to cross the blood-brain barrier (BBB) with the exception of carbidopa, which cannot reach the central nervous system (CNS) (Ahlskog et al., 1989. Exp Neurol 105: 152-61; Gershanik, 2015. Mov Disord 30: 103-13). A drawback of these treatments is that they exhibit (severe) side effects. These are thought to be caused among others by the fact that the drugs are not specific enough. The administration of available drugs targeting PD triggers inescapable side effects, with levodopa-induced dyskinesias standing out the most. Orally- administered levodopa can cross the BBB and, once in the brain, be incorporated into the dopaminergic terminals of the dorsal striatum. There, it is further converted to dopamine by AADC, imported into vesicles by the transporter vMAT2 and ultimately released to the synaptic cleft. This outcome of levodopa treatment accounts for its beneficial effects in patients with PD. However, levodopa can also be incorporated into the striatal serotonergic terminals, in which AADC is equally present. It has been reported that dopamine, or ‘false serotonin’, can be synthesized and further released from serotonergic terminals in an activity-dependent manner upon levodopa administration. In elegant experiments performed in 6-OHDA- induced parkinsonian rats, levodopa-derived dyskinesias could be abolished by removing the serotonergic terminals in the dorsal striatum, or by silencing the neurotransmitter release with agonists of serotonin auto-receptors (Carta et al., 2007. Brain 130: 1819-1833). The drugs also affect dopaminergic neurons outside the SNpc such as those in the ventral tegmental area or some hypothalamic nuclei (Upadhya et al., 2016. Behav Brain Res 301: 262-272). Stimulation of dopamine production in these off- target cells can lead to side-effects of the treatment. Furthermore, the dopamine biosynthesis enzymes also participate in the production of compounds other than dopamine. Levodopa treatment can interfere with the synthesis of these other compounds and exert off-target effects in noradrenergic and serotonergic neurons (Carta et al., 2007. Brain 130: 1819-1833; Navailles et al., 2014. CNS Neurosci Ther 20: 671-678). Yet a further drawback of the use of dopamine, levodopa and other dopamine prodrugs is that they inhibit the production of dopamine by the dopaminergic cells themselves. This increases the dependency on medication and increases the side effects associated with unnatural dosing schedules, irregular supply of dopamine and reduces on-target effects still potentially possible in the absence of the inhibition. There is thus a need to reduce or even diminish at least some of the drawbacks associated with the administration of dopamine or prodrugs thereof. BRIEF DESCRIPTION OF THE INVENTION The present invention provides a solution to some of the drawbacks associated with the administration of dopamine or prodrugs thereof. In the present invention it was found that tadalafil, a phosphodiesterase (PDE) 5 and PDE 11 inhibitor, or a variant thereof, is able to increase the endogenous production of dopamine in dopaminergic cells in the presence of an external source of dopamine by means of increasing Tyrosine Hydroxylase (TH) Ser40 phosphorylation. Individuals that receive dopamine or a precursor thereof are subject to negative feedback inhibition caused by an inhibitory effect of dopamine on the endogenous production of L-DOPA. In the examples it is shown that tadalafil removes the inhibition from the endogenous production of L-DOPA in the relevant cells. Re- activation of the pathway is shown by increased TH Ser40 phosphorylation thereby inducing the endogenous production of dopamine by the cells. Tadalafil or a variant thereof is able to increase the endogenous production of dopamine in dopaminergic cells even in the presence of an external source of dopamine. Tadalafil can be administered, at a dose of 0,1 – 40 mg/day. A suitable dose is in the range of 1 mg/day to 40 mg/day. Tadalafil is marketed in dosages of 5, 10 and 20 mg. For the present invention it is possible to use such formulations arriving at a suitable dose of 5 mg/day, 10 mg/day and 20 mg/day. A suitable minimum administered dose of tadalafil or variant thereof is 20 mg/day (0.4 mg/kg), when provided as sole PDE inhibitor. Tadalafil or variant thereof can thereby at least in part reverse the negative feedback inhibition induced by added dopamine, a dopamine agonist, L- DOPA or a prodrug thereof in dopaminergic cells. The selective presence of especially PDE11 in the target cells is at least part of the reason for the increased specificity and/or reduction of side effects. Human PDEs are divided into 11 families. Some families (PDE1, PDE3, PDE4, PDE7 and PDE8) are products of multiple genes (multigene), whereas others derive from a single gene. All PDEs share a highly conserved catalytic domain near the carboxyl-terminus, including 11 amino acids in the active site. Minor variations in the catalytic domain are believed to be responsible for the different selectivities towards cAMP or cGMP substrate and various inhibitors. More substantial variation exists in the N-terminus part, where most of the regulatory domains reside (Makhlouf et al., 2006. Int J Impot Res 18: 501–509). In the case of PDE11, there is a single gene in the family (PDE11A according to standard nomenclature) with four splicing variants (PDE11A1, PDE11A2, PDE11A3 and PDE11A4). This appears to be the case in all mammalian species studied so far (human, rat and mouse). PDE11 or phosphodiesterase 11 is known under a number of different names such as Dual 3',5'-Cyclic-AMP And -GMP Phosphodiesterase 11A; EC 3.1.4.35; CAMP And CGMP Cyclic Nucleotide Phosphodiesterase 11A and PPNAD2. External Ids for PDE11A Gene are: HGNC: 8773; NCBI Gene: 50940; Ensembl: ENSG00000128655; OMIM®: 604961; and UniProtKB/Swiss-Prot: Q9HCR9. PDE11A is a dual-substrate PDE, acting on both cAMP and cGMP. Most studies report similar affinities (Km) for both substrates (Makhlouf, A et al., 2006. Int J Impot Res 18, 501–509). The invention provides dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in the treatment of dopamine deficiency in a subject, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is co-administered in combination with tadalafil or a variant thereof, characterized in that dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered at an effective reduced dose compared to the subject's standard therapeutic dose. The invention also provides dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in the treatment of dopamine deficiency in a subject, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered in combination with tadalafil or a variant thereof, characterized in that the co-administration of tadalafil or a variant thereof enables a reduction in the dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, compared to the standard therapeutic dose required for treating the dopamine deficiency in the individual, while maintaining therapeutic efficacy and reducing the incidence or severity of side effects associated with dopamine, a dopamine agonist, L-DOPA or a prodrug thereof. The reduction of the dose is at least 10%, at least 20%, or preferably at least 50% compared to the individuals standard therapeutic dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof. The treatment of the dopamine deficiency preferably does not comprise the administration of a calcimimetic that lowers parathyroid hormone (PTH) levels such as cinacalcet, baclofen, acamprosate, mexiletine, torasemide, or sulfisoxazole. Tadalafil or a variant thereof can be administered, at a dose of 0,1 – 40 mg/day. A suitable dose is in the range of 1 mg/day to 40 mg/day. Tadalafil is marketed in dosages of 5, 10 and 20 mg. For the present invention it is possible to use such formulations arriving at a suitable dose of 5 mg/day, 10 mg/day and 20 mg/day. A suitable minimum administered dose of tadalafil or variant thereof is 20 mg/day (0.4 mg/kg) when provided as sole PDE inhibitor. The minimum administered dose of tadalafil or variant thereof is preferably 20 mg/day (0.4 mg/kg). The dopamine deficiency is preferably a central nervous system dopamine deficiency. The disease associated with the dopamine deficiency in the individual is preferably Parkinson's disease. The dopamine, a dopamine agonist, L-DOPA or a prodrug and the tadalafil or variant thereof are preferably administered for treating symptoms of Parkinson’s disease related to central nervous system dopamine deficiency. The effective reduced dose of dopamine, a dopamine agonist, L-DOPA, or a prodrug thereof is suboptimal for the individual when administered as a single treatment to the individual. The effective reduced dose is preferably determined by titration in the patient. The effective reduced dose is preferably between 10% and 75% of the standard therapeutic dose when administered as a single treatment to the individual. The treatment of the disease or deficiency is preferably combined with a further medicament. The further medicament preferably comprises a PDE11 inhibitor, a PDE2 inhibitor, a guanylate cyclase agonist; a dopa-decarboxylase (DDC) inhibitor; a monoamine oxidase (MAO-B) inhibitor; a catechol-O- methyltransferase (COMT) inhibitor; or a combination thereof. The PDE11 inhibitor is preferably BC11-15; BC11-19; BC11-28; BC11-38; BC11-38-1; BC11-38- 2; BC11-38-3; or BC11-38-4. The PDE2 inhibitor is preferably EHNA (erythro-9-(2-hydroxy-3- nonyl)adenine); BAY 60-7550 (2-[(3,4-dimethoxyphenyl)methyl]-7-[(2R,3R)-2- hydroxy-6-phenylhexan-3-yl]-5-methyl-1H-imidazo[5,1-f][1,2,4]triazin-4-one); PDP (9-(6-phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one; Hcyb1, PF- 051809999 (4-(1-azetidinyl)-7-methyl-5-[1-methyl-5-[5-(trifluoromethyl)-2- pyridinyl]-1H-pyrazol-4-yl]-imidazo[5,1-f][1,2,4]triazine); IC933, or oxindole (2,3- dihydroindol-2-on). The guanylate cyclase agonist is preferably a GUCY-2C agonist. The guanylate cyclase 2C receptor (GUCY2C) agonist is preferably guanylin, lymphoguanylin, enterotoxin or uroguanylin. or a functional derivative of guanylin, lymphoguanylin, enterotoxin or uroguanylin. The dopamine, a dopamine agonist, L-DOPA or a prodrug thereof and tadalafil are preferably administered simultaneously. The Parkinson’s disease is Parkinson’s disease stage 1-5. The invention also provides a method for treating dopamine deficiency in a subject, comprising: - administering to the subject a therapeutically effective dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof; and co-administering to the subject a therapeutically effective dose of tadalafil or a variant thereof; wherein the dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered at an effective reduced dose compared to the subjects standard therapeutic dose. Also provided is a method for treating dopamine deficiency in a subject, comprising: - administering to the subject a therapeutically effective dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is known to cause one or more side effects at standard therapeutic doses; and - co-administering to the subject a therapeutically effective dose of tadalafil or a variant thereof, wherein the co-administration of the tadalafil or a variant thereof enables a reduction in the dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof compared to the standard therapeutic dose required for treating the dopamine deficiency in the individual, while maintaining therapeutic efficacy and reducing the incidence or severity of a side effect associated with dopamine, a dopamine agonist, L-DOPA or a prodrug thereof. The treatment preferably does not comprise a calcimimetic that lowers parathyroid hormone (PTH) levels such as cinacalcet, baclofen, acamprosate, mexiletine, torasemide, or sulfisoxazole. Tadalafil or a variant thereof can be administered, at a dose of 0,1 – 40 mg/day. A suitable dose is in the range of 1 mg/day to 40 mg/day. Tadalafil is marketed in dosages of 5, 10 and 20 mg. For the present invention it is possible to use such formulations arriving at a suitable dose of 5 mg/day, 10 mg/day and 20 mg/day. A suitable minimum administered dose of tadalafil or variant thereof is 20 mg/day (0.4 mg/kg) when provided as sole PDE inhibitor. The minimum administered dose of tadalafil or variant thereof is preferably 20 mg/day (0.4 mg/kg). The is preferably a central nervous system dopamine deficiency. The disease associated with dopamine deficiency in the individual is preferably Parkinson's disease. Also provided is dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in the treatment of dopamine deficiency in a subject, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is co-administered in combination with tadalafil or a variant thereof, characterized in that dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered at a reduced dose compared to the subjects standard therapeutic dose of dopamine, a dopamine agonist, L- DOPA or a prodrug thereof. Further provided is dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in the treatment of dopamine deficiency in a subject, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered in combination with tadalafil or a variant thereof, characterized in that the co- administration of tadalafil or a variant thereof allows a reduction in the dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, compared to the standard therapeutic dose required for treating the dopamine deficiency, while maintaining therapeutic efficacy and reducing the incidence or severity of side effects associated with dopamine, a dopamine agonist, L-DOPA or a prodrug thereof. In one embodiment the invention provides dopamine, a dopamine agonist, L- DOPA or a prodrug thereof for use in the treatment of Parkinson’s disease in a subject, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is co- administered in combination with tadalafil or a variant thereof, characterized in that dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered at an effective reduced dose compared to the subject's standard therapeutic dose. The invention also provides dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in the treatment of Parkinson’s disease in a subject, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered in combination with tadalafil or a variant thereof, characterized in that the co-administration of tadalafil or a variant thereof enables a reduction in the dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, compared to the standard therapeutic dose required for treating the Parkinson’s disease in the individual, while maintaining therapeutic efficacy and reducing the incidence or severity of side effects associated with dopamine, a dopamine agonist, L-DOPA or a prodrug thereof. The reduction of the dose is at least 10%, at least 20%, or preferably at least 50% compared to the individuals standard therapeutic dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof. The treatment of the Parkinson’s disease preferably does not comprise the administration of a calcimimetic that lowers parathyroid hormone (PTH) levels such as cinacalcet, baclofen, acamprosate, mexiletine, torasemide, or sulfisoxazole. In one embodiment the invention provides a method for treating Parkinson’s disease in a subject, comprising: - administering to the subject a therapeutically effective dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof; and co-administering to the subject a therapeutically effective dose of tadalafil or a variant thereof; wherein the dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered at a reduced dose compared to the subjects standard therapeutic dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof. Also provided is dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in the treatment of Parkinson’s disease in a subject, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is co-administered in combination with tadalafil or a variant thereof, characterized in that dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered at a reduced dose compared to the subjects standard therapeutic dose of dopamine, a dopamine agonist, L- DOPA or a prodrug thereof. The reduction in the dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is preferably at least 10%, 20%, or preferably 50% compared to the patients standard therapeutic dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof. The co-administration is effective in treating Parkinson’s disease in the subject while reducing the incidence and/or severity of side effects associated with dopamine, a dopamine agonist, L-DOPA or a prodrug thereof treatment of Parkinson patients. Also provided is dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in the treatment of Parkinson’s disease, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered in combination with tadalafil or a variant thereof, characterized in that the co-administration of tadalafil or a variant thereof allows a reduction in the dose of dopamine, a dopamine agonist, L- DOPA or a prodrug thereof, compared to the standard therapeutic dose required for treating Parkinson’s disease, while maintaining therapeutic efficacy and reducing the incidence or severity of side effects associated with dopamine, a dopamine agonist, L-DOPA or a prodrug thereof. In one embodiment the invention provides a method for treating Parkinson’s disease in a subject, comprising: - administering to the subject a therapeutically effective dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is known to cause one or more side effects at standard therapeutic doses; and - co-administering to the subject a therapeutically effective dose of tadalafil or a variant thereof, wherein the co-administration of the tadalafil or a variant thereof allows for a reduction in the dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof compared to the standard therapeutic dose required for treating Parkinson’s disease in the individual, while maintaining therapeutic efficacy and reducing the incidence or severity of a side effect associated with dopamine, a dopamine agonist, L-DOPA or a prodrug thereof. The reduction in the dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is preferably at least 10%, 20%, or preferably 50% compared to the standard therapeutic dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof. The side effects of standard dopamine, a dopamine agonist, L-DOPA or a prodrug thereof treatment include dyskinesias (involuntary movements that can take the form of chorea, dystonia, ballism, tics, and myoclonus); nausea and vomiting; low blood pressure (hypotension); loss of appetite; anxiety and depression; hallucinations (seeing, hearing, feeling and smelling things that aren't there) and sleep problems. Reducing the incidence or severity of a side-effect involves the reduction of the incidence or severity of at least one of: levodopa- induced dyskinesias (involuntary movements that can take the form of chorea, dystonia, ballism, tics, and myoclonus); nausea and vomiting; low blood pressure (hypotension); loss of appetite; anxiety and depression; hallucinations (seeing, hearing, feeling and smelling things that aren't there) and sleep problems. When in this document reference is made to a drug that can be dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, it is preferred that the drug is L- DOPA or a prodrug thereof. It is preferred that the drug is L-DOPA. In one embodiment the invention provides L-DOPA for use in the treatment of Parkinson’s disease in a subject, wherein L-DOPA is co-administered in combination with tadalafil or a variant thereof, characterized in that L-DOPA is administered at an effective reduced dose compared to the subject's standard therapeutic dose. The invention also provides L-DOPA for use in the treatment of Parkinson’s disease in a subject, wherein L-DOPA is administered in combination with tadalafil or a variant thereof, characterized in that the co-administration of tadalafil or a variant thereof enables a reduction in the dose of L-DOPA, compared to the standard therapeutic dose required for treating the Parkinson’s disease in the individual, while maintaining therapeutic efficacy and reducing the incidence or severity of side effects associated with L-DOPA. The reduction of the dose is at least 10%, at least 20%, or preferably at least 50% compared to the individuals standard therapeutic dose of L-DOPA. The treatment of the Parkinson’s disease preferably does not comprise the administration of a calcimimetic that lowers parathyroid hormone (PTH) levels such as cinacalcet, baclofen, acamprosate, mexiletine, torasemide, or sulfisoxazole. In one embodiment the invention provides a method for treating Parkinson’s disease in a subject, comprising: - administering to the subject a therapeutically effective dose of L-DOPA; and co-administering to the subject a therapeutically effective dose of tadalafil or a variant thereof; wherein the L-DOPA is administered at a reduced dose compared to the subjects standard therapeutic dose of L-DOPA. Also provided is L-DOPA for use in the treatment of Parkinson’s disease in a subject, wherein L-DOPA is co-administered in combination with tadalafil or a variant thereof, characterized in that L-DOPA is administered at a reduced dose compared to the subjects standard therapeutic dose of L-DOPA. The reduction in the dose of L-DOPA is preferably at least 10%, 20%, or preferably 50% compared to the patients standard therapeutic dose of L-DOPA. The co-administration is effective in treating Parkinson’s disease in the subject while reducing the incidence and/or severity of side effects associated with L-DOPA treatment of Parkinson patients. Also provided is L-DOPA for use in the treatment of Parkinson’s disease, wherein L-DOPA is administered in combination with tadalafil or a variant thereof, characterized in that the co-administration of tadalafil or a variant thereof allows a reduction in the dose of L-DOPA, compared to the standard therapeutic dose required for treating Parkinson’s disease, while maintaining therapeutic efficacy and reducing the incidence or severity of side effects associated with L- DOPA. In one embodiment the invention provides a method for treating Parkinson’s disease in a subject, comprising: - administering to the subject a therapeutically effective dose of L-DOPA, wherein L-DOPA is known to cause one or more side effects at standard therapeutic doses; and - co-administering to the subject a therapeutically effective dose of tadalafil or a variant thereof, wherein the co-administration of the tadalafil or a variant thereof allows for a reduction in the dose of L-DOPA compared to the standard therapeutic dose required for treating Parkinson’s disease in the individual, while maintaining therapeutic efficacy and reducing the incidence or severity of a side effect associated with L-DOPA. The reduction in the dose of L-DOPA is preferably at least 10%, 20%, or preferably 50% compared to the standard therapeutic dose of L-DOPA. The side effects of standard L-DOPA treatment include levodopa-induced dyskinesias (involuntary movements that can take the form of chorea, dystonia, ballism, tics, and myoclonus); nausea and vomiting; low blood pressure (hypotension); loss of appetite; anxiety and depression; hallucinations (seeing, hearing, feeling and smelling things that aren't there) and sleep problems. Reducing the incidence or severity of a side-effect involves the reduction of the incidence or severity of at least one of: levodopa-induced dyskinesias (involuntary movements that can take the form of chorea, dystonia, ballism, tics, and myoclonus); nausea and vomiting; low blood pressure (hypotension); loss of appetite; anxiety and depression; hallucinations (seeing, hearing, feeling and smelling things that aren't there) and sleep problems. The invention also provides dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in a method of treating a Parkinson’s disease in a subject characterized in that the treatment of the disease comprises the administration of tadalafil or a variant thereof to the subject, whereby the treatment does not comprise a calcimimetic that lowers parathyroid hormone (PTH) levels such as cinacalcet, baclofen, acamprosate, mexiletine, torasemide, or sulfisoxazole. The invention also provides dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in a method of treating a Parkinson’s disease in a subject characterized in that the treatment of the disease comprises the administration of tadalafil or a variant thereof to the subject. Further provided is dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in a method of treating a Parkinson’s disease in a subject characterized in that the treatment of the disease comprises the administration of tadalafil or a variant thereof to the subject, whereby tadalafil or variant thereof is administered at a minimum dose of 20 mg/day (0.4 mg/kg). The treatment is preferably a treatment of symptoms of Parkinson’s disease related to central nervous system dopamine deficiency. The dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is preferably a suboptimal dose when compared to the optimal dose of said dopamine, a dopamine agonist, L-DOPA or a prodrug thereof when given as a single treatment or when given without tadalafil. The suboptimal dose is preferably determined by titration in the patient. Titration of dopamine, a dopamine agonist, L-DOPA or a prodrug comprises gradually increasing the dose from a low starting dose based on the patient’s response. The physician adjusts the dose every few days or weeks depending on the patient’s tolerance and control of symptoms. The goal is to increase the dose until a desired balance between symptom relief and side effects is achieved. In the case of L-DOPA, increments are typically 100 mg/day or smaller, with frequent reassessment. The starting dose for L-DOPA in a Parkinson’s patient that receives the drug for the first time is typically 100 mg 2 or 3-times daily. The dose is gradually increased in number per day and/or amount per dose until a desired balance between symptom relief and side effects is achieved. In embodiments, the suboptimal dose of dopamine, a dopamine agonist, L- DOPA or a prodrug thereof is between 10% and 75% of the optimal dose of said dopamine, a dopamine agonist, L-DOPA or a prodrug thereof when given as a single treatment. Said suboptimal dose preferably is less than 1500 mg/day, such as 20-750 mg/day. The standard therapeutic dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is the therapeutically effective dose of said dopamine, a dopamine agonist, L-DOPA or a prodrug thereof when given as treatment of the patient without tadalafil. It is typically determined by titration of the medicament in the individual. The patient starts with a low dose and increases it after some time, doses are regularly increased until a suitable balance between effects and side-effects is achieved. When this is achieved this dose is referred to as the standard therapeutic dose. The comparison with and without tadalafil is preferably done under otherwise the same medical treatment conditions. The effective reduced dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is the therapeutically effective dose of said dopamine, a dopamine agonist, L-DOPA or a prodrug thereof when given as treatment of the patient with tadalafil. This effective reduced dose is also typically determined by titration of the medicament in the individual. The patient starts with tadalafil and a low dose dopamine, a dopamine agonist, L-DOPA or a prodrug thereof and increases it after some time, doses are regularly increased until a suitable balance between effects and side- effects is achieved. When this is achieved this dose is referred to as the effective reduced dose. The effective reduced dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is between 10% and 75% of the standard therapeutic dose of said dopamine, a dopamine agonist, L-DOPA or a prodrug thereof when given as a single treatment. Said effective reduced dose is preferably less than 1500 mg/day, such as 20-750 mg/day. The term “effective reduced dose” is the same as the term “reduced therapeutic dose” and can be replaced by it. It is not necessary to first find the optimal dose or standard therapeutic dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof to find the suboptimal dose or effective reduced dose of dopamine, a dopamine agonist, L- DOPA or a prodrug thereof for use in combination with tadalafil. A physician typically starts treatment with a set effective dose of tadalafil and a normal low starting dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof which is subsequently titrated until a desired balance between symptom relief and side effects is achieved. When administration of tadalafil is interrupted for some reason, the dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof needs to be increased until a new balance between symptom relief and side effects is achieved. At this increased dose the balance between symptom relief and side effects is typically worse than at the titrated dose used in in co-administration with tadalafil. This often results in more side effects at the new therapeutically effective dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof and/or a lower than desired effectivity of new dose because of undesired side effect(s). In embodiments, dopamine, a dopamine agonist, L-DOPA or a prodrug thereof and tadalafil may be administered simultaneously. The dopamine, a dopamine agonist, L-DOPA or a prodrug thereof and tadalafil or variant thereof may be combined with yet a further medicament for the treatment of the disease or the deficiency. Said further medicament preferably comprises a guanylate cyclase agonist; a PDE11 inhibitor, a PDE2 inhibitor, a dopa-decarboxylase (DDC) inhibitor; a monoamine oxidase (MAO-B) inhibitor; a catechol-O-methyltransferase (COMT) inhibitor, or a combination thereof. The dopamine, a dopamine agonist, L-DOPA or a prodrug thereof and tadalafil or variant thereof can be administered simultaneously with the guanylate cyclase agonist; a PDE11 inhibitor, a PDE2 inhibitor, a dopa-decarboxylase (DDC) inhibitor; a monoamine oxidase (MAO-B) inhibitor; a catechol-O-methyltransferase (COMT) inhibitor, or a combination thereof. Said guanyl cyclase agonist preferably is a guanylate cyclase 2C receptor (GUCY2C) agonist such as guanylin, lymphoguanylin , enterotoxin or uroguanylin. or a functional derivative of guanylin, lymphoguanylin, enterotoxin or uroguanylin. Said PDE11 inhibitor is preferably BC11-15; BC11-19; BC11-28; BC11-38; BC11-38-1; BC11-38-2; BC11-38-3; or BC11-38-4. Said PDE2 inhibitor preferably is EHNA (erythro-9-(2-hydroxy-3- nonyl)adenine); BAY 60-7550 (2-[(3,4-dimethoxyphenyl)methyl]-7-[(2R,3R)-2- hydroxy-6-phenylhexan-3-yl]-5-methyl-1H-imidazo[5,1-f][1,2,4]triazin-4-one); PDP (9-(6-phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one; Hcyb1; PF- 051809999(4-(1-azetidinyl)-7-methyl-5-[1-methyl-5-[5-(trifluoromethyl)-2- pyridinyl]-1H-pyrazol-4-yl]-imidazo[5,1-f][1,2,4]triazine); IC933; oxindole (2,3- dihydroindol-2-on) or PF-05180999 (CAS 1394033-54-5). The invention works with substantia nigra cells. The Parkinson’s disease is preferably Parkinson’s disease stage 1-5. Subjects with early to mid-late Parkinson’s disease have remaining substantia nigra cells. These cells can respond to the treatments provided by the invention. The invention further provides a method for treating dopamine deficiency in a subject comprising administering dopamine, a dopamine agonist, L-DOPA or a prodrug thereof to a subject in need thereof, characterized in that the treatment of the deficiency further comprises the administration of tadalafil or variant thereof to the subject, whereby the treatment does not comprise a calcimimetic that lowers parathyroid hormone (PTH) levels such as cinacalcet, baclofen, acamprosate, mexiletine, torasemide, or sulfisoxazole. The invention further provides a method for treating dopamine deficiency in a subject comprising administering dopamine, a dopamine agonist, L-DOPA or a prodrug thereof to a subject in need thereof, characterized in that the treatment of the deficiency further comprises the administration of tadalafil or variant thereof to the subject. The invention further provides a method for treating dopamine deficiency in a subject comprising administering dopamine, a dopamine agonist, L-DOPA or a prodrug thereof to a subject in need thereof, characterized in that the treatment of the deficiency further comprises the administration of tadalafil or variant thereof to the subject, whereby the administration of tadalafil or variant thereof is at a minimum dose of 20 mg/day (0.4 mg/kg). The dopamine deficiency is preferably a central nervous system dopamine deficiency. Further provided is a method of treating Parkinson’s disease in a subject comprising administering dopamine, a dopamine agonist, L-DOPA or a prodrug thereof to a subject in need thereof characterized in that the treatment of the disease further comprises the administration of tadalafil or variant thereof to the subject, whereby the administration of tadalafil or variant thereof is at a minimum dose of 20 mg/day (0.4 mg/kg). The treatment may comprise treating symptoms of Parkinson’s disease related to central nervous system dopamine deficiency. The method of treating Parkinson’s disease may further be combined with the administration of a guanyl cyclase agonist, preferably a guanylate cyclase 2C receptor (GUCY2C) agonist such as guanylin, lymphoguanylin , enterotoxin or uroguanylin. or a functional derivative of guanylin, lymphoguanylin, enterotoxin or uroguanylin, a PDE11 inhibitor and/or a PDE2 inhibitor. Said administration of a guanyl cyclase agonist, a PDE11 inhibitor and/or a PDE2 inhibitor may be before, simultaneous to, or after the administration of dopamine, a dopamine agonist, L- DOPA or a prodrug thereof, and tadalafil or variant thereof. Further provided is a medicament comprising a sub-optimal dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, and tadalafil or variant thereof. Said medicament may further comprise a guanyl cyclase agonist, preferably a guanylate cyclase 2C receptor (GUCY2C) agonist such as guanylin, lymphoguanylin , enterotoxin or uroguanylin. or a functional derivative of guanylin, lymphoguanylin, enterotoxin or uroguanylin, a PDE11 inhibitor and/or a PDE2 inhibitor. Said GUCY2C agonist may be selected from guanylin, lymphoguanylin , enterotoxin or uroguanylin. or a functional derivative of guanylin, lymphoguanylin, enterotoxin or uroguanylin. Said administration of a guanyl cyclase agonist may be before, simultaneous to, or after the administration of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, and tadalafil or variant thereof. Said PDE11 inhibitor preferably is selected from BC11-15; BC11-19; BC11- 28; BC11-38; BC11-38-1; BC11-38-2; BC11-38-3; or BC11-38-4. Said administration of a PDE11 inhibitor may be before, simultaneous to, or after the administration of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, and tadalafil or variant thereof. Said PDE2 inhibitor preferably is selected from EHNA (erythro-9-(2-hydroxy- 3-nonyl)adenine); BAY 60-7550 (2-[(3,4-dimethoxyphenyl)methyl]-7-[(2R,3R)-2- hydroxy-6-phenylhexan-3-yl]-5-methyl-1H-imidazo[5,1-f][1,2,4]triazin-4-one); PDP (9-(6-phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one; Hcyb1; PF- 051809999 (4-(1-azetidinyl)-7-methyl-5-[1-methyl-5-[5-(trifluoromethyl)-2- pyridinyl]-1H-pyrazol-4-yl]-imidazo[5,1-f][1,2,4]triazine) (CAS 1394033-54-5); IC933, oxindole (2,3-dihydroindol-2-on). Said administration of a PDE2 inhibitor may be before, simultaneous to, or after the administration of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, and tadalafil or variant thereof. The invention provides dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in a method for the treatment of dopamine deficiency in a subject, characterized in that the treatment of the deficiency further comprises the administration of a PDE11 inhibitor to the subject. The invention also provides a COMT inhibitor, a MAO-B inhibitor for use in a method for the treatment of dopamine deficiency in a subject, characterized in that the treatment of the deficiency further comprises the administration of a PDE11 inhibitor or tadalafil to the subject. The invention also provides a COMT inhibitor, a MAO-B inhibitor for use in a method for the treatment of dopamine deficiency in a subject, characterized in that the treatment of the deficiency further comprises the administration of a PDE2 inhibitor to the subject. In embodiments, said medicament is for use in the treatment of Parkinson’s disease, preferably Parkinson’s disease stage 1-5. Further provided is a kit of parts comprising the medicament. Said kit of parts may optionally further comprise a PDE11 inhibitor, a PDE2 inhibitor and/or a guanyl cyclase agonist. The medicament or a kit of parts preferably comprises levodopa and tadalafil or variant thereof, for administration of tadalafil or a variant thereof at a dose of 0,1 – 40 mg/day. A suitable dose is in the range of 1 mg/day to 40 mg/day. Tadalafil is marketed in dosages of 5, 10 and 20 mg. For the present invention it is possible to use such formulations arriving at a suitable dose of 5 mg/day, 10 mg/day and 20 mg/day. A suitable minimum administered dose of tadalafil or variant thereof is 20 mg/day (0.4 mg/kg). The medicament or a kit of parts preferably comprises tadalafil in dosage forms comprising 5, 10 and 20 mg tadalafil or variant thereof. The medicament or a kit of parts preferably comprises levodopa and tadalafil or variant thereof, for administration of 20 mg/day of tadalafil or a variant thereof. The dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is preferably packaged to facilitate providing a suboptimal dose or an effective reduced dose, such as 20-750 mg/day. The medicament or a kit of parts preferably does not comprise a calcimimetic that lowers parathyroid hormone (PTH) levels such as cinacalcet, baclofen, acamprosate, mexiletine, torasemide, or sulfisoxazole. Further provided is a medicament or kit of parts comprising a sub-optimal dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof and tadalafil or variant thereof for use in the treatment of Parkinson’s disease, preferably Parkinson’s disease stage 1-5. An embodiment or preference described herein for dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in a method for the treatment of dopamine deficiency in a subject is equally described for methods of treatment of dopamine deficiency in a subject or methods of treating a Parkinson’s disease in a subject, and vice versa. There are treatments and medicaments described in the art for the treatment of dopamine deficiency in a subject according to the invention, which are all suitable for the treatment of Parkinson’s disease. FIGURE LEGENDS Figure 1. Tyrosine hydroxylase phosphorylation as therapeutic target A | The biosynthetic pathway for dopamine. Tyrosine hydroxylase has a predominant role in the biosynthesis pathway of dopamine. Tyrosine hydroxylase uses oxygen (O2), the cofactor tetrahydrobiopterin (BH4), and a ferrous iron (Fe2+) atom in the active site to facilitate the catalytic reaction of L-tyrosine to L-3,4- dihydroxyphenylalanine (L-DOPA). In turn, aromatic amino acid decarboxylase (AADC) decarboxylates L-DOPA to dopamine. B | Schematic diagram of the multi-domain monomer structure of the homo- tetrameric tyrosine hydroxylase protein, indicating the N-terminal regulatory domain, the catalytic domain, and the C-terminal tetramerization domain. C | Schematic diagram of the N-terminal regulatory domain of tyrosine hydroxylase. The regulatory domain bears the main phosphorylation sites, which are the serine (Ser) residues Ser40, Ser31, and the less relevant Ser19. Phosphorylation of tyrosine hydroxylase at Ser40 increases the enzyme’s activity. D | Illustration describing dopamine supplementation therapy by L-DOPA for Parkinson’s disease (PD) patients. L-DOPA, which can pass the blood-brain barrier, is usually administered peripherally in combination with a peripheral AADC inhibitor. It reaches the brain via cerebral arteries and passage of the blood- brain barrier. Then, in combination with AADC, L-DOPA is synthesized to dopamine in the brain. The presence of AADC is, however, not limited to dopamine- synthesizing neurons. Therefore, in addition to the original treatment target area, L-DOPA reaches various AADC-containing neuronal cell types in diverse brain regions. As a consequence, dopamine is synthesized in AADC-containing neurons that normally synthesize neurotransmitters other than dopamine, for example serotonin (5-HT) neurons of the raphe nuclei. E | As L-DOPA production in unspecific brain regions can lead to unwanted and unendurable side effects, an approach to increase L-DOPA and dopamine synthesis limited to tyrosine hydroxylase-containing neurons will be a requisite for a long- term therapeutic treatment. The approach we propose is to increase tyrosine hydroxylase (Th) phosphorylation at Ser40, and therefore to increase the enzyme’s activity, by targeting the intra-cellular signaling mechanisms specific to tyrosine hydroxylase-containing neurons. Figure 2. Ex vivo approach to investigate tyrosine hydroxylase protein expression in mouse acute brain slices A | Preparation of mouse coronal slices along the rostral-caudal axis. Brains from adult mice were removed and sectioned in 250 μm coronal slices using a vibratome. The slices containing the regions of interest were collected for further experimental use. B | Overview of the mouse coronal slices along the rostral-caudal axis that contain dopaminergic projections, including cell-bodies in the midbrain (MB), axons to the corpus striatum (CS), and axon terminals in the CS. Light microscopy pictures of each slice are shown together with corresponding in situ blots (see Okabe et al., 1993 (Okabe et al., 1993. J Histochem Cytochem 41: 927–934) and methods section) as a proxy indicating the distribution of tyrosine hydroxylase (Th) protein expression within each brain slice. A schematic representation of each of the coronal slices is depicted below the in situ blots. The regions of interest per slice, the regions that include the dopaminergic projections as indicated by tyrosine hydroxylase expression, are marked in gray. C | Regions of interest are micro-dissected from the collected coronal slices and transferred into a tube for ex vivo pharmacological treatment. Ex vivo pharmacological experiments are performed on micro-dissected brain tissue, for which each slice first is divided into two hemispheres, followed by micro-dissection of their regions of interest (indicated in gray). The regions of interest are the midbrain (MB), the striatum (CS), or the areas connecting these regions. Per slice, one of the two corresponding micro-dissected regions of interest (randomly chosen) is treated with a compound, while the other is treated with a vehicle. Next, samples were lysed and protein levels were detected by automated western blot analysis using a Wes™ (ProteinSimple). Figure 3. Tyrosine hydroxylase protein distribution in mouse brain slices A | Schematic images of each examined brain slice over the rostral-caudal axis with representative western blot images for baseline protein levels of tyrosine hydroxylase (Th), phospho-Th (Ser40), phospho-Th (Ser31), and β-actin. Protein levels of the micro-dissected parts (marked in gray of the schematic images) are quantified. The slices include twelve striatal (CS) slices, five midbrain (MB) slices, and three slices connecting these brain regions, together comprising dopaminergic projections from the midbrain to the striatum. B-E | Quantitative analysis of (B) total Th, (C) Th-corrected phospho-Th (Ser40), (D) Th-corrected phospho-Th (Ser31), and (E) β-actin-corrected Th protein levels in micro-dissected mouse brain slices. Compared to the midbrain, Th protein levels are 3.55 times higher in the striatum, striatal total Th-corrected phospho-Th (Ser40) levels are 1.77 times higher, and striatal total Th-corrected phospho-Th (Ser31) levels are 1.67 times higher. β-Actin-corrected Th protein levels demonstrate a striatum (CS) to midbrain (MB) ratio of 0.63. Protein levels were detected by automated western blot analysis using the Wes™ (ProteinSimple). Two micro-dissected parts (one from each hemisphere) of each coronal slice are pooled, the amount of phospho-protein levels are corrected for the total amount of protein, and data is normalized to the average level of the protein in all striatal slices. Data are mean ± SEM, *p < 0.05 and **p < 0.01 (two-tailed paired student’s t-test), n = 3. Figure 4. Secondary messenger signaling is involved in the phosphorylation of tyrosine hydroxylase in mouse striatal slices A | There are no differences in relative striatal phosphorylation levels between the right and left hemisphere. To examine possible hemisphere-related effects, we carefully separated micro-dissected regions of interest from the left hemisphere (L) and compared them with the micro-dissected parts from the right (R) hemisphere. Schematic images are shown of each examined brain slice over the rostral-caudal axis with representative western blot images for baseline protein levels of tyrosine hydroxylase (Th), phospho-Th (Ser40), phospho-Th (Ser31), and β-actin. B | Quantitative analysis of total Th-corrected phospho-Th (Ser40) and phospho- Th (Ser31) levels in micro-dissected mouse brain slices. Although there is variance between hemispheres, there is no hemisphere-dependent effect (n = 10). C | Forskolin up-regulates phospho-Th (Ser40) levels and down-regulates phospho- Th (Ser31) levels. To examine the involvement of secondary messengers in the phosphorylation of tyrosine hydroxylase, micro-dissected mouse striatal slices were exposed for 60 minutes to either vehicle or 10 μM forskolin, an adenylyl cyclase activator that increases intracellular levels of the second messenger cyclic adenosine monophosphate (cAMP). Schematic images are shown of each examined brain slice over the rostral-caudal axis with representative western blot images for protein levels of Th, phospho-Th (Ser40), and phospho-Th (Ser31). D | Quantitative analysis of total Th-corrected phospho-Th (Ser40) and phospho- Th (Ser31) levels in micro-dissected mouse brain slices. Exposure to 10 μM forskolin for 60 minutes induces phosphorylation of Th at Ser40 and down- regulates phosphorylation of Th at Ser31 (n = 10). E | pCPT-cAMP up-regulates phospho-Th (Ser40) levels. To examine the direct involvement of cAMP in the phosphorylation of Th, micro-dissected mouse striatal slices were exposed for 60 minutes to either vehicle or 500 μM pCPT-cAMP, a cell- permeable analogue of cAMP. Schematic images are shown of each examined brain slice over the rostral-caudal axis with representative western blot images for protein levels of Th, phospho-Th (Ser40), and phospho-Th (Ser31). F | Quantitative analysis of total Th-corrected phospho-Th (Ser40) and phospho- Th (Ser31) levels in micro-dissected mouse brain slices. Exposure to 500 μM pCPT- cAMP for 60 minutes increases phospho-Th (Ser40) levels, while phospho-Th (Ser31) levels are left unchanged (n = 6). Protein levels were detected by automated western blot analysis using the Wes™ (ProteinSimple). The amount of phospho- protein levels are corrected for the total amount of protein and data is normalized to the average level of protein of the vehicle condition. Data are minimum to maximum boxplots showing the first quartile, third quartile, and the mean, *p < 0.05 and **p < 0.01 (two-tailed paired student’s t-test). Figure 5. Tyrosine hydroxylase Ser40 phosphorylation levels are upregulated though activation of Gucy2C A-B | Guanylin treatment of mouse striatal slices is able to elevate TH-Ser40 phosphorylation. C | Immunohistochemistry for TH (green) and Gucy2C (red) on human postmortum midbrain material. Melanin is represented by a blue signal. Merge of these signals show that Gucy2C is present axons of in human midbrain dopaminergic neurons (white arrows). In a blow up of a TH/melanin positive neurons the presence of Gucy2C is represented in axonal fibers. Figure 6. Tyrosine hydroxylase Ser40 phosphorylation levels are down- regulated by dopamine A-B | The effect of 30 minutes exposure to dopamine on tyrosine hydroxylase phosphorylation. Micro-dissected mouse striatal slices were exposed to either 10 μM dopamine or vehicle for 30 minutes. A| Schematic images are shown of each examined brain slice over the rostral- caudal axis with representative western blot images for protein levels of tyrosine hydroxylase (Th), phospho-Th (Ser40), and phospho-Th (Ser31). B | Quantitative analysis of the effect of 10 μM dopamine on total Th-corrected phospho-Th (Ser40) and phospho-Th (Ser31) levels in micro-dissected mouse brain slices. 30 Minutes exposure to 10 μM dopamine decreases phospho-Th (Ser40) levels (n = 11). C-D | The effect of 60 minutes exposure to dopamine on tyrosine hydroxylase phosphorylation. Micro-dissected mouse striatal slices were incubated with either 10 μM dopamine or vehicle for 60 minutes. C | Schematic images are shown of each examined brain slice over the rostral- caudal axis with representative western blot images for protein levels of Th, phospho-Th (Ser40), and phospho-Th (Ser31). D | Quantitative analysis of the effect of 10 μM dopamine on total Th-corrected phospho-Th (Ser40) and phospho-Th (Ser31) levels in micro-dissected mouse brain slices. 60 Minutes exposure to 10 μM dopamine decreases phospho-Th (Ser40) levels (n = 10). E-G | The effect of forskolin on the down-regulating effects of dopamine. E | Ex vivo pharmacology protocol to test if 10 μM forskolin can reverse the down- regulating effects of 10 μM dopamine on phospho-Th (Ser40) levels. Mouse brain slices are collected, divided by hemisphere, and micro-dissected. Micro-dissected mouse striatal slices were incubated with 10 μM dopamine for 30 minutes, followed by exposure to either 10 μM forskolin or vehicle. F | Schematic images are shown of each examined brain slice over the rostral- caudal axis with representative western blot images for protein levels of Th, phospho-Th (Ser40), and phospho-Th (Ser31). G | Quantitative analysis of the effect of 10 μM forskolin (FSK) on total Th- corrected phospho-Th (Ser40) and phospho-Th (Ser31) levels in 30 minute dopamine pre-incubated micro-dissected mouse brain slices. If striatal slices are exposed to forskolin subsequently to 30 minutes of dopamine incubation, phospho- Th (Ser40) levels are increased while phospho-Th (Ser31) levels remain unaffected (n = 11). Protein levels were detected by automated western blot analysis using the Wes™ (ProteinSimple). The micro-dissected part of one hemisphere (randomly chosen) is used as internal control to the micro-dissected part of the other hemisphere. The amount of phospho-protein levels are corrected for the total amount of protein and data is normalized to the average level of protein of the vehicle condition. Data are minimum to maximum boxplots showing the first quartile, third quartile, and the mean, **p < 0.01 (two-tailed paired student’s t-test). Figure 7. Tyrosine hydroxylase Ser40 phosphorylation levels are down- regulated by L-DOPA but can be rescued in a cAMP-dependent manner via forskolin A-B | The effect of 30 minutes exposure to L-DOPA on tyrosine hydroxylase Ser40 phosphorylation. Micro-dissected mouse striatal slices were exposed to either 100 μM L-DOPA or vehicle for 30 minutes. A | Schematic images are shown of each examined brain slice over the rostral- caudal axis with representative western blot images for protein levels of tyrosine hydroxylase (Th) and phospho-Th (Ser40). B | Quantitative analysis of the effect of 100 μM L-DOPA on total Th-corrected phospho-Th (Ser40) levels in micro-dissected mouse brain slices. 30 Minutes exposure to 100 μM L-DOPA down-regulates phospho-Th (Ser40) levels (two-tailed paired student’s t-test, n = 10). C-D | The effect of 60 minutes exposure to L-DOPA on tyrosine hydroxylase phosphorylation. C | Micro-dissected mouse striatal slices were exposed to either 100 μM L-DOPA or vehicle for 60 minutes. Schematic images are shown of each examined brain slice over the rostral-caudal axis with representative western blot images for protein levels of Th, phospho-Th (Ser40), and phospho-Th (Ser31). D | Quantitative analysis of the effect of 100 μM L-DOPA on total Th-corrected phospho-Th (Ser40) and phospho-Th (Ser31) levels in micro-dissected mouse brain slices. 60 Minutes exposure to 100 μM L-DOPA down-regulates phospho-Th (Ser40) levels, while there is no effect on phospho-Th (Ser31) levels (two-tailed paired student’s t-test, n = 11). E-G | The effect of forskolin on the down-regulating effects of L-DOPA. E | Ex vivo pharmacology protocol to test if 10 μM forskolin can reverse the down- regulating effects of 100 μM L-DOPA on phospho-Th (Ser40) levels. Mouse brain slices are collected, divided by hemisphere, and micro-dissected. Micro-dissected mouse striatal slices were incubated with either 100 μM L-DOPA or vehicle for 30 minutes, followed by exposure to either 10 μM forskolin or vehicle for another 30 minutes. F | Schematic images are shown of each examined brain slice over the rostral- caudal axis with representative western blot images for protein levels of Th and phospho-Th (Ser40). G | Quantitative analysis of the effect of 10 μM forskolin (FSK) on total Th- corrected phospho-Th (Ser40) levels in micro-dissected mouse brain slices pre- incubated with either 100 μM L-DOPA or vehicle for 30 minutes. Forskolin up- regulates and L-DOPA down-regulates phospho-Th (Ser40) levels. If slices are exposed to forskolin subsequently to 30 minutes of L-DOPA incubation, phospho- Th (Ser40) levels are recovered to baseline levels. For each animal, the area under the curve was calculated per condition (two-tailed paired student’s t-test statistical analysis with Bonferroni correction, n = 5 animals). Protein levels were detected by automated western blot analysis using the Wes™ (ProteinSimple). The micro- dissected part of one hemisphere (randomly chosen) is used as internal control to the micro-dissected part of the other hemisphere. The amount of phospho-protein levels are corrected for the total amount of protein and data is normalized to the average level of protein of the vehicle condition. Data are minimum to maximum boxplots showing the first quartile, third quartile, and the mean (B, D) or bar charts showing the mean ± SEM (G), *p < 0.05 and **p < 0.01. Figure 8. The effect of Pitx3-deficiency on tyrosine hydroxylase levels in the mouse striatum Deficiency in the homeobox gene Pitx3 leads to selective neuronal cell loss of the same group of dopamine neurons that are affected in Parkinson’s disease. As a consequence of this selective loss of neurons, dopaminergic connections to the striatum are affected as well. To examine the effects of Pitx3-deficiency on tyrosine hydroxylase (Th) levels (A-D) and regulation (E-H) in the striatum, we examined and compared total Th, phospho-Th (Ser40), and phospho-Th (Ser31) protein levels between phenotypically normal Pitx3GFP/+ and defective Pitx3GFP/GFP mice. A-D | The effect of Pitx3-deficiency on Th levels. A | Representative western blot images for protein levels of Th, phospho-Th (Ser40), and phospho-Th (Ser31) of four untreated micro-dissected striatal mouse brain slices over the rostral-caudal axis, comparing striatal Th levels of Pitx3GFP/+ and Pitx3GFP/GFP mice. Per slice, protein levels were averaged over animals (n = 3) and the averages of Pitx3GFP/+ mice were compared with the averages of Pitx3GFP/GFP mice. B | Quantitative analysis of total tyrosine hydroxylase (Th) levels. Tyrosine hydroxylase levels are drastically lower in the phenotypically defective Pitx3GFP/GFP mice. C | Quantitative analysis of relative tyrosine hydroxylase Ser40 phosphorylation levels. Relative Ser40 phosphorylation levels are comparable between the two genotypes. D | Quantitative analysis of relative tyrosine hydroxylase Ser40 phosphorylation levels. Relative Ser31 phosphorylation levels are lower in the phenotypically defective Pitx3GFP/GFP mice. E-H | The effect of Pitx3-deficiency on tyrosine hydroxylase regulation by forskolin. E-F | Twelve micro-dissected mouse striatal slices of (E) Pitx3GFP/+ mice (n = 3) or (F) Pitx3GFP/GFP (n = 3) mice were exposed to either 10 μM forskolin or vehicle for 60 minutes. The micro-dissected part of one hemisphere (randomly chosen) is used as internal control to the micro-dissected part of the other hemisphere. Per slice, protein levels were averaged over three animals and the averages of the vehicle condition was compared with the averages of the forskolin treatment group. Schematic images are shown of each examined brain slice over the rostral-caudal axis with representative western blot images for protein levels of Th, phospho-Th (Ser40), and phospho-Th (Ser31). G | Quantitative analysis of relative tyrosine hydroxylase Ser40 phosphorylation levels, comparing Pitx3GFP/+ mice (left) and Pitx3GFP/GFP mice (right). Forskolin up-regulates tyrosine hydroxylase Ser40 phosphorylation in both Pitx3GFP/+ mice and Pitx3GFP/GFP mice, and to a similar extent. H | Quantitative analysis of relative tyrosine hydroxylase Ser31 phosphorylation levels, comparing Pitx3GFP/+ mice (left) and Pitx3GFP/GFP mice (right). Forskolin down-regulates Ser31 phosphorylation in the Pitx3GFP/GFP mice. The amount of phospho-protein levels are corrected for the total amount of protein and data is normalized to the average level of protein of the Pitx3GFP/+ mice (B-D) or to the vehicle condition (G-H). Data are minimum to maximum boxplots showing the first quartile, third quartile, and the mean, *p < 0.05 and **p < 0.01 (two-tailed paired student’s t-test). Figure 9. L-DOPA down-regulates the endogenous biosynthesis pathway of dopamine via tyrosine hydroxylase Ser40 phosphorylation Hypothesized model of L-DOPA-induced “silencing” of tyrosine hydroxylase activity, an effect which can be negated — or “rescued” — due to manipulations in cyclic nucleotide levels. Tyrosine hydroxylase is activated by forskolin, an adenylyl cyclase activator that increases intracellular levels of the second messenger cyclic adenosine monophosphate (cAMP) and thereby tyrosine hydroxylase Ser40 phosphorylation levels (upper section). When neurons are exposed to L-DOPA, Th activity is “silenced” or down-regulated (middle section) due to its effect on pSer40 phosphorylation. This effect, however, can be “rescued” via the cyclic nucleotide- dependent manipulation of p Ser40 phosphorylation levels, as exemplified again by the exposure to forskolin (lower section). Figure 10. Theoretical pathways by which a PDE inhibitor can lead to an increase in Th activity PDEs convert the transition from cAMP and/or cGMP to AMP and GMP. cAMP and or cGMP have been described to induce phosphorylation of TH on Ser40 which is directly linked to its enzymatic activity. Figure 11. L-DOPA treatment results in a reduced phosphorylation of TH and can be rescued by IBMX treatment Experiments were performed as in Figure 6E, 6F, and 6G. A | Pharmacological time-line of L-DOPA and IBMX combinatorial treatment. One hemisphere was treated with vehicle for one hour. The corresponding hemisphere with L-DOPA for 60 minutes. The following striatal slice was incubated with IBMX for one hour (100 μM). The corresponding hemisphere was treated IBMX (100 μM) and L-DOPA (100 μM) for one hour. B | Blot image of L-DOPA and IBMX combinatorial treated striatal slices. Clearly an increase of p-Th (Ser40) protein can be observed in the IBMX treated groups. C | Slices treated with L-DOPA for one hour significantly downregulates the levels of p-Th (Ser40) protein (p = 0.01/ p = 0.032 after Bonferroni correction). Incubation with IBMX (100 μM) for 30 minutes significantly increases the levels of p-Th (Ser40) protein (p = 0.002/ p= 0.007 after Bonferroni correction). Combinatorial treatment of L-DOPA together with IBMX significantly rescues the levels of p-Th (Ser40) protein (p = 0.016/ p = 0.047 after Bonferroni correction) compared to L- DOPA treatment (100 μM) for one hour. Figure 12. L-DOPA treatment results in a reduced phosphorylation of TH and can be rescued by BAY 60-7550 treatment Experiments were performed as in Figure 6E, 6F, and 6G. A | Pharmacological time-line of L-DOPA and BAY 60-7550 combinatorial treatment. One hemisphere was treated with vehicle for one hour. The corresponding hemisphere with L-DOPA for 60 minutes. The following striatal slice was incubated BAY 60-7550 for one hour (100 μM). The corresponding hemisphere was treated with BAY 60-7550 (10 μM) and L-DOPA (100 μM). B | Blot image of L-DOPA and BAY 60-7550 combinatorial treated striatal slices. Clearly an increase of p-Th (Ser40) protein can be observed in the BAY 60-7550 treated groups. C | Slices treated with L-DOPA for one hour significantly downregulates the levels of p-Th (Ser40) protein (p = 0.001/ p = 0.007 after Bonferroni correction). Incubation with BAY 60-7550 (10 μM) for 60 minutes significantly increases the levels of p-Th (Ser40) protein (p = 0.003/ p = 0.008 after Bonferroni correction). Combinatorial treatment of L-DOPA together with BAY 60-7550 significantly rescues the levels of p-Th (Ser40) protein (p = 0.013 p = 0.4 after Bonferroni correction) compared to L-DOPA treatment (100 μM) for one hour. Figure 13. PDE11A reduces TH phosphorylation on Ser40 Cells were transfected with WT TH as well as WT isoforms of PDE11A or containing a translational fusion to a HIS tag. A | Neuro2a cells were transfected with TH in combination with empty vector or TH in combination with the WT isoform of PDE11A1, PDE11A2, PDE11A3, PDE11A4. Subsequently, cells were harvested and analyzed for TH expression as well as TH phosphorylation on Ser40. Results indicate that PDE11A4 reduces the phosphorylation of Ser40 most potently. B | Neuro2a cells were transfected with TH in combination with empty vector or TH in combination with the PDE11A translational fusion to HIS isoform of PDE11A1, PDE11A2, PDE11A3, PDE11A4. Subsequently, cells were harvested and analyzed for TH expression as well as TH phosphorylation on Ser40. HIS blot shows the expression levels of PDE11A after transfection. PDE11A4 is expressed best. In addition results indicate that PDE11A4 reduces the phosphorylation of Ser40 most potently. Figure 14. Forskolin does but IMBX does not restore attenuating effects of PDE11 on TH Ser40 Cells were transfected with WT TH as well as the long isoforms of PDE11A: PDE11A4 ( as also used in fig 13). A | Neuro2a cells were transfected with TH in combination with empty vector or TH in combination with the long isoform: PDE11A4 and samples were treated with Forskolin and/or IBMX. Subsequently, cells were harvested and analyzed for TH expression as well as TH phosphorylation on Ser40. Only Forskolin is able to attenuate the decrease in TH-40 phosphorylation in these conditions. B | Quantification of the data (N=4 per condition) in A. ** p < =0.01. Figure 15. Tadalafil enhances tyrosine hydroxylase Ser40 phosphorylation in Neuro2A cells. A | The first-generation phosphodiesterase V (PDE5) inhibitors sildenafil (10 µM) and tadalafil (10 µM) affect tyrosine hydroxylase Ser40 phosphorylation. Neuro2A cells were transfected with DNA plasmid encoding tyrosine hydroxylase (Th) and either polyhistidine-tagged PDE11A4 or pcDNA3.1(+) backbone. Neuro2A cells were exposed to sildenafil or tadalafil for 60 minutes. B | Introduction of PDE11A4 down-regulates Ser40 phosphorylation under basal conditions. Without PDE11A4, sildenafil downregulates Ser40 phosphorylation, while tadalafil had no effect. However, when PDE11A4 is introduced, relative Ser40 phosphorylation levels are up-regulated by tadalafil. Figure 16. Dose response curve Tadalafil Neuro2A cells were transfected with DNA plasmid encoding tyrosine hydroxylase (Th) and either polyhistidine-tagged PDE11A4 or pcDNA3.1(+) backbone. Neuro2A cells were exposed to different concentrations of Tadalafil for 60 minutes. A | Increasing concentrations of Tadalafil attenuate the initial downregulation of TH-40 Phosphorylation. B | Quantification of the data (N=4 per condition) in A. ** P < =0.01. Figure 17. Structure of some PDE2a and some PDE11a inhibitors Figure 18. IC50 values of some PDE2a inhibitors DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the utilization of the endogenous dopamine biosynthesis pathway as a means to address the significantly diminished levels of dopamine in the nigrostriatal dopamine projections in Parkinson’s patients that are on L-DOPA treatment. By specifically targeting dopamine neuron-associated upstream signaling pathways involved in the phosphorylation of tyrosine hydroxylase at Ser40, it has become possible to enhance endogenous dopamine production. This targeted approach focuses on neurons where increased dopamine synthesis is required. Consequently, phosphorylation of tyrosine hydroxylase at Ser40 emerges as an effective therapeutic target in the management of Parkinson's disease, either as a supplement to L-DOPA therapy or as an alternative to bypass the known off-target effects associated with exogenous L-DOPA administration. When used in conjunction with L-DOPA therapy, promoting tyrosine hydroxylase Ser40 phosphorylation allows for a reduction in L-DOPA dosages. This reduction is facilitated by the reactivation of endogenous dopamine synthesis, which works in combination with the exogenous L-DOPA. The extent to which the L-DOPA dose can be reduced depends on the degree of Ser40 phosphorylation and the residual population of dopamine neurons, typically correlating with the stage and phenotype of the disease in individual patients. Off-target effects resulting from L-DOPA treatment can occur due to dopamine synthesis in cells outside the striatum (extrastriatal cells) that do not normally produce dopamine. These extrastriatal cells can synthesize dopamine when exposed to L-DOPA, contributing to the adverse side effects observed in Parkinson’s patients treated with L-DOPA. By facilitating the removal of inhibition on the endogenous dopamine production, this invention mitigates both the off- target effects caused by extrastriatal dopamine production in non-dopaminergic, AADC-expressing neurons and the uncontrolled overstimulation of post-synaptic signaling pathways. At the same time, the endogenous dopamine biosynthesis pathway remains functional, preserving crucial mechanisms that regulate dopamine neurotransmission. This allows for some degree of natural dopamine regulation, including processes such as vesicular packaging and the initiation of dopamine synthesis, in which the subcellular distribution of tyrosine hydroxylase and other proteins involved in dopamine production play a pivotal role. In peripheral tissues, dopamine is released from neuronal cells and is synthesized within specific parenchyma. Dopamine released from sympathetic nerves predominantly contributes to plasma dopamine levels. Despite growing evidence for peripheral source and action of dopamine and the widespread expression of dopamine receptors in peripheral tissues, most studies have focused on functions of dopamine in the central nervous system. Dopamine deficiency is associated with various brain disorders, including schizophrenia, Parkinson’s disease, attention-deficit hyperactivity disorder, and depression. These disorders can be alleviated by pharmacological modulation of dopamine transmission. In the present invention it is preferred that the dopamine deficiency is selected from the group consisting of schizophrenia, Parkinson’s disease, attention-deficit hyperactivity disorder, and depression. Considering that these diseases are all brain or central nervous system diseases it is preferred that the dopamine deficiency is a central nervous system deficiency. A subject with a dopamine deficiency is preferably a subject that has schizophrenia, Parkinson’s disease, attention-deficit hyperactivity disorder, depression or a combination thereof. In a preferred embodiment the dopamine deficiency is Parkinson’s disease. In a particularly preferred embodiment the subject with a dopamine deficiency is a subject that has Parkinson’s disease. Parkinson's Disease (PD) typically start-off slowly with minor symptoms. The disease can progress slowly or fast. During this progression the symptoms grow worse and new symptoms can become apparent. Various stages of disease can be identified that correlate more or less with the progressive loss of dopaminergic neurons from the substantia nigra. Often 5 stages are identified. Stage 1 is typically characterized by mild symptoms that generally do not interfere with daily activities. Tremor and other movement symptoms typically occur on one side of the body only. Friends and family may notice changes in posture, walking and facial expressions. In stage 2 the symptoms start getting worse. Tremor, rigidity and other movement symptoms affect both sides of the body. Walking problems and poor posture may become apparent. In this stage, the person is still able to live alone, but completing day-to-day tasks becomes more difficult and may take longer. Stage 3 is considered mid-stage in the progression of the disease. Loss of balance and slowness of movements are hallmarks of this phase. Falls are more common. Though the person is still fully independent, symptoms significantly impair activities of daily living such as getting dressed and eating. Parkinson’s symptoms are severe and very limiting in stage 4. It’s possible to stand without assistance, but movement may require a walker. The person needs help with activities of daily living and is unable to live alone. Stage 5 is the most advanced and debilitating stage of Parkinson’s disease. Stiffness in the legs may make it impossible to stand or walk. The person requires a wheelchair or is bedridden. Around-the-clock nursing care is required for all activities. The person may experience hallucinations and delusions. While stage five focuses on motor symptoms, the Parkinson’s community acknowledges that there are many important non-motor symptoms as well. Parkinson’s disease, or at least the motor symptoms part thereof, is commonly treated with dopamine replacement therapy. The dopamine precursor levodopa (L-DOPA), dopamine agonists (DAs), monoamine oxidase B inhibitors and catechol-O-methyltransferase inhibitors, are commonly used. Of these, dopamine, the prodrug levodopa (L-DOPA) and other dopamine or levodopa prodrugs are commonly used. Prodrugs of dopamine and L-DOPA have been described (see for example Karaman, 2011 (Karaman, 2011. Chem Biol Drug Des 78: 853–863); Fernandez et al., 2003 (Fernandez et al., 2003. Organic Biomol Chem 1: 767-771); and Di Stefano et al., 2008 (Di Stefano et al., 2008. Molecules 13: 46-68). These references are referred to herein specifically for the dopamine prodrugs mentioned. These references are therefore incorporated by reference herein. As L-DOPA is a prodrug of dopamine the prodrugs of L-DOPA are also prodrugs of dopamine. Levodopa (L-DOPA), a precursor (or prodrug) to dopamine, is presently considered an effective and well tolerated dopamine replacing agent. It is considered the gold standard for symptomatic treatment of PD and contributes significantly to improvements in the quality of life of patients with Parkinson's disease (Salat et al., 2013. J Parkinson's Disease 3: 255-269). When L-DOPA is administered orally, it is quickly decarboxylated and only a small proportion reaches the central nervous system. To counter this, L-DOPA is routinely administered in combination with a dopa-decarboxylase (DDC) inhibitor, such as carbidopa or benserazide. This co-administration of L-DOPA with a dopa- decarboxylase inhibitor extends the half-life of L-DOPA and increases its availability to the brain, thereby prolonging the duration of its symptomatic effect. In the field and for the present invention it is routinely co-administered with L- DOPA when the patient is on an L-DOPA regimen. L-DOPA is effective in the early stages of PD or Parkinsonism and remains effective as the disease progresses. Intolerance of L-DOPA is not known to develop over time. The main drawbacks of L-DOPA are dyskinesias and response fluctuations, which are partly related to its short half-life (Salat et al., 2013. J Parkinson's Disease 3: 255-269). The long-term treatment of PD or Parkinsonism with L-DOPA is complicated by the development of motor complications, including response fluctuations, dyskinesia, and psychiatric abnormalities. L-DOPA induced dyskinesia is particularly troubling as the abnormal, involuntary movements can be disabling and interfere with activities of daily living. Generally, when motor complications develop, an additional drug to the L- DOPA regimen is added from one of three other classes of treatments: dopamine agonists, catechol- O-methyl transferase inhibitors (COMTIs), or monoamine oxidase type B inhibitors (MAOBIs). Although trials have shown that these drugs are beneficial compared to placebo, it remains unclear as to the best way to treat patients experiencing motor complications and whether one class of drug is more effective than another (Stowe et al., 2010. Cochrane Library 7: 1). Examples of combination therapy include ropinirole with L-DOPA (WO1997/048394). Definitions The following are definitions of terms used in this specification. The initial definition provided for a group or term herein applies to that group or term throughout the present specification individually or as part of another group, unless otherwise indicated. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Terms may also be defined in other parts of the description than only in this “Definition” section. The term "optimal dose" as used herein refers to the dose that induces the best "on response", which is usually associated with prominent L-DOPA-induced dyskinesia (LID) in advanced parkinsonian primates and may only reduce the Motor Disability Score (MDS) by 70-75% in some animals. The term "sub-optimal dose" as used herein refers to the dose that produces half of the optimal dose response with significantly reduced, but still ratable, L- DOPA-induced dyskinesia. The term "threshold dose" as used herein refers to the dose that induces a reproducible small improvement of mobility (e.g., 25-30% reduction of Motor Disability Score) without L-DOPA-induced dyskinesia. These doses are determined individually according to the particular motor response of each animal. The term "pharmaceutically acceptable carrier or vehicle" as used herein, refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the tadalafil or variant thereof and/or dopamine, levodopa or prodrug thereof from one organ, or portion of the body, to another organ, or portion of the body. Each carrier or vehicle must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically acceptable carriers or vehicles include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as butylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer' s solution; ethyl alcohol; phosphate buffer solutions; and other non- toxic compatible substances employed in pharmaceutical formulations. Dopamine, a dopamine agonist, levodopa or a prodrug thereof, and tadalafil or variant thereof as referred to herein are administered in an effective amount. The term "effective amount" refers to any amount that is necessary or sufficient for achieving or promoting a desired outcome, e.g., for treating, preventing, or ameliorating a dopamine deficiency, including a symptom of Parkinson's disease or Parkinsonism. In some instances an effective amount is a therapeutically effective amount. A therapeutically effective amount is an amount that is necessary or sufficient for promoting or achieving a desired biological response in a subject, either alone or in combination with another drug. For instance, with a method or composition of the invention it is possible to administer a dose of dopamine, a dopamine agonist, levodopa or prodrug thereof that is sub-optimal when administered alone, but can be optimal when combined with administration of tadalafil or variant thereof as described herein. Such doses are also effective amounts as referred to herein. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular agent being administered, the size of the subject, or the severity of the disease or condition. As used herein, "treat" or "treating" includes stopping the progression, reversing the progression, preventing and/or reducing or ameliorating a symptom of a central nervous system disorder involving a dopamine deficiency such as Parkinson's disease or Parkinsonism, for example, improving motor function. The words subject and individual are used inter-changeably herein. Reference to a compound described herein is understood to include reference to salts thereof, unless otherwise indicated. The term "salt(s)", as employed herein, denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases. In addition, when a compound described herein contains both a basic moiety, such as, but not limited to, amine, pyridine or imidazole and an acidic moiety, such as, but not limited to, a carboxylic acid, zwitterions ("inner salts") may be formed and are included within the term "salt(s)" as used herein. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful, e.g. , in isolation or purification steps which may be employed during preparation. Salts of compounds described herein may be formed, for example, by reacting a compound with an amount of acid or base, such as an equivalent amount, in a medium, such as one in which the salt precipitates or in an aqueous medium followed by lyophilization. As used herein “co-administration” refers to the practice of administering two or more therapeutic agents, such as drugs or medicaments, to a subject at or around the same time. The drugs can be given simultaneously, sequentially, or in a staggered manner, depending on the treatment regimen or how the drugs interact. The goal of the co-administration of L-DOPA and tadalafil or a variant thereof is to enhance therapeutic efficacy and reduce side effects of the medications. Co- administration of L-DOPA and tadalafil or a variant thereof involves giving both to the patient, where tadalafil or a variant thereof is used to allow for a lower dose of L-DOPA, thereby reducing the side effects typically caused by L-DOPA. The drugs can be taken together in one combined formulation or separately but during the same treatment period. Compounds The use of simple ester prodrugs of levodopa to improve the pharmacokinetics of the drug has been proposed (U.S. Pat. Nos. 5,017,607; 4,826,875; 4,873,263; 4,771,073; 4,663,349; 4,311,706; Japanese Patent No. JP58024547; Juncos et al., 1987. Neurology 37: 1242; and Cooper et al., 1987, J Pharm Pharmacol 39: 627- 635). An oral formulation of levodopa methyl ester (Levomet®, CHF 1301) has been described (Chiesi Pharmaceuticals). The ethyl ester of levodopa (TV-1203) is under clinical investigation as a potential therapy for Parkinsonism when co- administered with carbidopa (U.S. Pat. No.5,607,969). A sustained release cellulose formulation of levodopa ethyl ester in a mixture of hydroxypropylmethyl cellulose, hydroxypropyl cellulose, and a carboxyvinyl polymer has been described (U.S. Pat. No. 5,840,756). Oral administration of this formulation to healthy adults pretreated with carbidopa produced a plasma levodopa terminal half-life of 2 hours, comparable to that of Sinemet® CR. A further prodrug of dopamine, a pivaloyl ester of levodopa (NB-355), has been described (European Patent No. 0309827). Following oral administration of NB-355, no rapid increase or elimination of levodopa was observed and duration time was prolonged, while levels of levodopa were low. The potential for using ester prodrugs of levodopa to enhance rectal absorption of the drug has been described (U.S. Pat. Nos.4,663,349; 4,771,073; 4,873,263). Notably, the absorption of simple alkyl esters of levodopa has been shown to be greater following rectal absorption than following oral dosing (Fix et al., 1989. Pharm Res 6: 501-505; Fix et al., 1990. Pharm Res 4: 384-387). This effect is attributed to the decreased abundance of esterases in the large intestine relative to the small intestine. Therefore, selective delivery of a prodrug of levodopa to the large intestine in a sustained release formulation might be expected to provide a greater oral bioavailability and a prolonged exposure to the drug. A series of glycolic acid ester containing prodrugs of dopamine and specifically of levodopa has been described (U.S. Pat. No.4,134,991). Lipid conjugates of levodopa to facilitate the entry of drug into cells and tissues have also been described (U.S. Pat. No.5,827,819). The half-life of levodopa is prolonged and its bioavailability increased by the co-administration of carbidopa. Both drugs have relatively short half-lives of less than about 2 hours. In order to avoid the need for frequent (more than twice per day) dosing of levodopa, it is desirable to deliver levodopa (or prodrug thereof) in a sustained manner. It has been proposed that rectal administration of an ester prodrug of levodopa would be possible as a means to decrease metabolic clearance of levodopa (U.S. Pat. Nos. 4,663,349; 4,771,073; 4,873,263). US7671089 with title “Levodopa prodrugs, and compositions and uses thereof” describes prodrugs of dopamine and levodopa with the structural formula I:
Figure imgf000030_0001
; a stereoisomer thereof, an enantiomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate of any of the foregoing, wherein n is an integer from 1 to 6; each R1 and R2 is independently selected from hydrogen, alkenyl, alkynyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, halo, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; or optionally, when n is 1, then R1 and R2 together with the carbon atom to which R1 and R2 are attached form a cycloalkyl, substituted cycloalkyl, cycloheteroalkyl or substituted cycloheteroalkyl ring; R3 and R4 are independently selected from hydrogen, —C(O)OR7,—C(O)R7, and — (CR8R9)OC(O)R10; R5 is selected from alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, heteroalkyl, substituted heteroalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; R7 is selected from alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; R8 and R9 are independently selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl or optionally, R8 and R9 together with the carbon atom to which R8 and R9 are attached form a cycloalkyl, substituted cycloalkyl, cycloheteroalkyl or substituted cycloheteroalkyl ring; and R10 is selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, heteroalkyl, substituted heteroalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; wherein each substituent group is independently selected from halo, —CN, —NO2, —OH, C1-6 alkyl, and C1-6 alkoxy; with the provisos that when n is 2, and R1, R2, R3 and R4 are hydrogen, then R5 is not methyl or phenyl; when n is 3, and R1, R2, R3 and R4 are hydrogen, then R5 is not methyl; and when n is an integer from 1 to 6, and R1, R2, R3 and R4 are hydrogen, then R5 is not benzyl. These compounds and the preferred compounds which are both described in US7671089 are referred to herein specifically as suitable dopamine and levodopa prodrugs. Preferred dopamine and levodopa prodrugs are esters of dopamine or levodopa. In an aspect, the invention provides dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in a method for the treatment of dopamine deficiency in a subject characterized in that the treatment of the deficiency further comprises the administration of tadalafil or variant thereof to the subject. The cyclic nucleotide PDEs comprise a group of enzymes that degrade the phosphodiester bond in the second messenger molecules cAMP, cGMP or both. PDEs are commonly known as regulators of signal transduction mediated by these second messenger molecules. The PDE nomenclature signifies the PDE family with an Arabic numeral, then a capital letter denotes the gene in that family, and a second and final Arabic numeral then indicates the splice variant derived from a single gene (e.g., PDE1C3: family 1, gene C, splicing variant 3). Different PDEs of the same family are functionally related despite the fact that their amino acid sequences can show considerable divergence. PDEs have different substrate specificities. Some are known cAMP-selective hydrolases (PDE4, 7 and 8); others are known to be cGMP- selective (PDE5, 6, and 9). Others are known to hydrolyse both cAMP and cGMP (PDE1, 2, 3, 10, and 11). A PDE inhibitor is a drug that blocks one or more of the subtypes of the enzyme phosphodiesterase, thereby preventing the inactivation of the intracellular second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) by the respective PDE subtype(s). Various selective and non-selective PDE inhibitors are known. Among the non-selective PDE inhibitors are caffeine; aminophylline; IBMX (3-isobutyl-1-methylxanthine); paraxanthine; pentoxifylline; and theophylline. These compounds are said to be non-selective, however, there activity may still vary. This can be the result of differences in preference; in affinity; and/or in pharmacokinetics, among others. The concentration of a particular PDE inhibitor in vitro that inhibits PDE activity by 50% is known as the IC50 value. Selective PDE inhibitors are often referred to by the PDE type that is most specifically inhibited. A PDE5 inhibitor has an IC50 for PDE5 that is lower than the IC50 for any of the other PDEs. A suitable assay for determining the IC50 value of a compound for a PDE is given in Ceyhan et al., 2012 (Ceyhan et al., 2012. Chemistry Biol 19: 155–163). Assays to determine the IC50 of compounds for specific PDE proteins are also commercially available, see for instance BPS Bioscience (PDE2A = Catalog #: 60321) and PDE11A = Catalog #: 60411). An inhibitor is a PDE inhibitor if it has an IC50 that is in the micro(u) or nano(n) molar range for at least one of the human PDEs. An inhibitor is a selective PDE inhibitor if it has an IC50 for the respective PDE that is at least 10x lower than the IC50 of more than 5 other human PDEs, preferably more than 8, preferably all other human PDEs. A PDE2 inhibitor has an IC50 that is at least 10x lower than the IC50 of the compound for more than 5 other human PDEs, preferably more than 8, preferably all other human PDEs. A PDE11 inhibitor has an IC50 that is at least 10x lower than the IC50 of the compound for more than 5 other human PDEs, preferably more than 8, preferably all other human PDEs. An overview of the PDE proteins and some of their characteristics is provided in Bender and Beavo, 2006 (Bender and Beavo, 2006. Pharmacol Rev 58: 488-520). Tadalafil and variants thereof as described herein have the general formula:
Figure imgf000032_0001
, wherein R1 is selected from the group consisting of H, halo, and C1-6alkyl; R2 represents an optionally substituted monocyclic aromatic ring selected from the group consisting of benzene, thiophene, furan, and pyridine, or an optionally substituted bicyclic ring attached to the rest of the molecule via one of the benzene ring carbon atoms, wherein the fused ring A is a 5- or 6-membered ring, saturated or partially or fully unsaturated, and comprises carbon atoms and optionally one or two heteroatoms selected from oxygen, sulphur, and nitrogen; R3 represents H or C1-3alkyl, or R3 and R4 together represent a 3- or 4-membered alkyl or alkenyl chain component of a 5- or 6-membered ring; and R4 is selected from the group consisting of H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, halo, C3-8cycloalkyl, C3-8cycloalkylC1-3aikyl, and arylC1-3alkyl, wherein aryl is phenyl or phenyl substituted with one to three substituents independently selected from the group consisting of halo, C1-6alkyl, C1-6alkoxy, and methylenedioxy, and heteroarylC1-3alkyl, wherein heteroaryl is thienyl, furyl, or pyridyl, each optionally substituted with one to three substituents independently selected from the group consisting of halo, C1-6alkyl, and C1-6alkoxy. A variant of tadalafil preferably has the structural formula indicated above wherein R1 is H, R4 is H or C1-6alkyl; R2 is the bicyclic ring
Figure imgf000033_0001
optionally substituted with one or more groups independently selected from halo and C1-3alkyl; and R3 is H or C1-3alkyl. It is preferred that tadalafil or the variant thereof has the structural formula indicated above wherein R1 is H; R2 is the bicyclic ring
Figure imgf000033_0002
; and R3 is H; and R4 is CH3. A guanyl cyclase agonist is preferably a GUCY2C agonist. GUCY2C or Guanylate Cyclase 2C is known under a number of names such as STAR; STA Receptor; GUC2C; Heat-Stable Enterotoxin Receptor; Intestinal Guanylate Cyclase; Guanylyl Cyclase C; EC 4.6.1.2; GC-C; Heat Stable Enterotoxin Receptor; EC 4.6.1; DIAR6 and others. External Ids for GUCY2C Gene are HGNC: 4688 NCBI Entrez Gene: 2984 Ensembl: ENSG00000070019 OMIM®: 601330 UniProtKB/Swiss-Prot: P25092. The guanylin family of peptides has 3 subclasses of peptides containing either 3 intramolecular disulfide bonds found in bacterial heat-stable enterotoxins (ST), or 2 disulfides observed in guanylin and uroguanylin, or a single disulfide exemplified by lymphoguanylin. These peptides bind to and activate cell-surface receptors that have intrinsic guanylate cyclase (GC) activity such as GUCY2C. The peptides are also referred to as GUCY2C agonists. Guanylin is a natural agonistic ligand of GUCY2C. It is a 15 amino acid polypeptide. It is secreted by goblet cells in the colon. Guanylin acts as an agonist of the guanylyl cyclase receptor GC-C and regulates electrolyte and water transport in intestinal and renal epithelia. Upon receptor binding, guanylin increases the intracellular concentration of cGMP, induces chloride secretion and decreases intestinal fluid absorption, ultimately causing diarrhea. The peptide stimulates the enzyme through the same receptor binding region as the heat-stable enterotoxins. It is known under a number of names some of which are guanylate cyclase activator 2A; Guanylate Cyclase-Activating Protein 1; Guanylate Cyclase- Activating Protein I; Prepro-Guanylin; GCAP-I; GUCA2; Guanylate Cyclase Activator 2A; and STARA. External Ids for the guanylin gene/protein are HGNC: 4682; Entrez Gene: 2980; Ensembl: ENSG00000197273; OMIM: 139392; and UniProtKB: Q02747. Uroguanylin a natural agonistic ligand of GUCY2C. It is a 16 amino acid peptide that is secreted by cells in the duodenum and proximal small intestine. Guanylin acts as an agonist of the guanylyl cyclase receptor GC-C and among others regulates electrolyte and water transport in intestinal and renal epithelia. In humans, the uroguanylin peptide is encoded by the GUCA2B gene. The gene and protein are known under a number of different names Guanylate Cyclase Activator 2B; Prepro-Uroguanylin; GCAP-II; and UGN. External Ids for the gene and protein are HGNC: 4683; Entrez Gene: 2981; Ensembl: ENSG00000044012; OMIM: 601271; and UniProtKB: Q16661. Lymphoguanylin is a natural agnostic ligand of GUCY2C. It is among others described in Forte et al. Endocrinology. 1999 Apr;140(4):1800-6. Other GUCY2C agonists are functional derivatives of guanylin, lymphoguanylin , enterotoxin or uroguanylin.. A functional derivative of guanylin, lymphoguanylin , enterotoxin or uroguanylin. has the same activity in kind not necessarily in amount. A functional derivative preferably has the same amino acid sequence as guanylin, uroguanylin or STh or has an altered amino acid sequence which is highly similar to guanylin, uroguanylin or STh. One such derivative is linaclotide. Linaclotide is a peptide mimic of endogenous guanylin and uroguanylin. It is a synthetic tetradecapeptide (14 amino acid peptide) with the sequence CCEYCCNPACTGCY (H–Cys–Cys–Glu–Tyr–Cys– Cys–Asn–Pro–Ala–Cys–Thr–Gly–Cys–Tyr–OH). Linaclotide has three disulfide bonds which are between (when numbered from left to right) Cys1 and Cys6, between Cys2 and Cys10, and between Cys5 and Cys13 (Laps et al., 2021. Nature Comm 12: 870). Three similar peptide agonists of GUCY2C are in clinical development: linaclotide (Linzess™, Forest Laboratories and Ironwood Pharmaceuticals, Inc.); SP-304 (plecanatide) and SP-333 (Synergy Pharmaceuticals, Inc.). A functional derivative can have a chemical group attached to the N- and/or C-terminal end. The chemical group can have one or two amino acids in peptide linkage. The functional derivative may be derived from guanylin, lymphoguanylin, enterotoxin or uroguanylin by chemical modification of one or more of the amino acid residue side chains. One such modification or chemical group may be a modification to further facilitate passage of the blood brain barrier. Guanylin that is injected into the blood stream rapidly enters the brain (WO2013016662). Guanylin is capable of passing the blood-brain barrier (BBB). Brain delivery can be a challenge in drug development. Although the blood-brain barrier prevents many drugs from reaching their targets, molecular vectors - known as BBB shuttles - offer great promise to safely overcome this formidable obstacle. In recent years, peptide shuttles have received growing attention because of their lower cost, reduced immunogenicity, and higher chemical versatility than traditional Trojan horse antibodies and other proteins. Suitable BBB shuttles are described in Oller- Salvia et al., 2016 (Oller-Salvia et al., 2016. Chem Soc Rev 45: 4690-4707), which is incorporated by reference herein for this purpose. In a preferred embodiment the functional guanylin, lymphoguanylin, enterotoxin or uroguanylin derivative is guanylin, lymphoguanylin, enterotoxin or uroguanylin fused to a peptide BBB shuttle of table 1 of Oller-Salvia et al., 2016. The fusion is typically done by means of a peptide linkage. A linker can be introduced between the two functional units. The linker is preferably a peptide linker of 1-20, preferably 1-15, preferably 1-10 and more preferably 1-5 amino acid residues. The invention further provides a GUCY2C agonist comprising the sequence H-NDDCELCVNVACTGCLL-OH; H-NDCCELCCNVACTGCL-OH; H- NDDCELCVNVVCTGCL-OH; H-QEECEL[Abu]INMACTGY-OH; H- QEECELCINMACTGCL-OH; H-NTFYCCELCCNPACAGCY-OH; H- NTFYCCELCCAPACTGCY-OH; or H-NTFYCCELCCNPaCAGCY-OH. A GUCY2C agonist may be provided as a peptide, as a conjugate or a protein comprising the peptide. In a preferred embodiment the GUCY2C agonist is a conjugate of the peptide fused to a peptide BBB shuttle of table 1 of Oller-Salvia et al., 2016 (Oller-Salvia et al., 2016. Chem Soc Rev 45: 4690-4707). The fusion is typically done by means of a peptide linkage. A linker can be introduced between the two functional units. The linker is preferably a peptide linker of 1-20, preferably 1-15, preferably 1-10 and more preferably 1-5 amino acid residues. Examples of a PDE11 inhibitor include BC11-15; BC11-19; BC11-28 or BC11- 38. Variants of BC11-38 that can also be used are BC11-38-1; BC11-38-2; BC11-38- 3 and BC11-38-4. The BC11 inhibitors and their activity and method of selection are described in Ceyhan et al., 2012 (Ceyhan et al., 2012. Chemistry Biol 19: 155- 163). In embodiments, the PDE11 inhibitor is BC11-38; BC11-38-1; BC11-38-2; BC11-38-3 or BC11-38-4. In embodiments, the PDE11 inhibitor is BC11-38. Examples of a PDE2 inhibitor include EHNA (erythro-9-(2-hydroxy-3- nonyl)adenine); BAY 60-7550 (2-[(3,4-dimethoxyphenyl)methyl]-7-[(2R,3R)-2- hydroxy-6-phenylhexan-3-yl]-5-methylimidazo[5,1-f][1,2,4]triazin-4(1H)-one); PDP (9-(6-phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one; Hcyb1 (Liu et al., 2018. CNS Neurosci Ther 24: 652–660); PF-05180999 (4-(1-azetidinyl)-7-methyl-5- {1-methyl-5-[5-(trifluoromethyl)-2-pyridinyl]-1H-pyrazol-4-yl}imidazo[5,1- f][1,2,4]triazine); IC933, oxindole (structural formulas are indicated in figure 16, IC50 values of some of the compounds are indicated in figure 17). In embodiments, the PDE2 inhibitor is EHNA. In embodiments, the PDE2 inhibitor is BAY 60-7550. In embodiments, the PDE2 inhibitor is PDP. In embodiments, the PDE2 inhibitor is PF-05180999. Dopa-decarboxylase (DDC) inhibitors are known in the art, including benserazide (2-amino-3-hydroxy-N'-[(2,3,4-trihydroxyphenyl)methyl] propanehydrazide), carbidopa (2S)-3-(3,4-dihydroxyphenyl)-2-hydrazinyl-2- methylpropanoic acid), methyldopa (L-α-Methyl-3,4-dihydroxyphenylalanine), alpha-difluoromethyl-DOPA (DFMD; (2S)-2-Amino-2-[(3,4- dihydroxyphenyl)methyl]-3,3-difluoropropanoic acid), 3',4',5,7-tetrahydroxy-8- methoxyisoflavone (3-(3,4-dihydroxyphenyl)-5,7-dihydroxy-8-methoxychromen-4- one), epigallocatechin (EGC; (2R,3R)-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H- chromene-3,5,7-triol), and epigallocatechin gallate (EGCG; [(2R,3R)-5,7-dihydroxy- 2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-chromen-3-yl] 3,4,5-trihydroxybenzoate). Monoamine oxidase (MAO-B) inhibitors are known in the art, including phenelzine (2-phenylethylhydrazine), tranylcypromine ((1R,2S)-2- phenylcyclopropan-1-amine), selegiline ((2R)-N-methyl-1-phenyl-N-prop-2- ynylpropan-2-amine) and isocarboxazid (N'-benzyl-5-methyl-1,2-oxazole-3- carbohydrazide). Catechol-O-methyltransferase (COMT) inhibitors are known in the art, including entacapone ((E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N- diethylprop-2-enamide), tolcapone ((3,4-dihydroxy-5-nitrophenyl)-(4- methylphenyl)methanone), opicapone (5-[3-(2,5-dichloro-4,6-dimethyl-1- oxidopyridin-1-ium-3-yl)-1,2,4-oxadiazol-5-yl]-3-nitrobenzene-1,2-diol), and nitecapone (3-(3,4-dihydroxy-5-nitrobenzyliden)-2,4-pentandion). Dopamine agonists are known in the art, including bromocriptine ((6aR,9R)- 5-bromo-N-[(1S,2S,4R,7S)-2-hydroxy-7-(2-methylpropyl)-5,8-dioxo-4-propan-2-yl-3- oxa-6,9-diazatricyclo[7.3.0.02,6]dodecan-4-yl]-7-methyl-6,6a,8,9-tetrahydro-4H- indolo[4,3-fg]quinoline-9-carboxamide), amantadine (adamantan-1-amine), cabergoline ((6aR,9R,10aR)-N-[3-(dimethylamino)propyl]-N-(ethylcarbamoyl)-7- prop-2-enyl-6,6a,8,9,10,10a-hexahydro-4H-indolo[4,3-fg]quinoline-9-carboxamide), apomorphine ((6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11- diol), pramipexole ((6S)-6-N-propyl-4,5,6,7-tetrahydro-1,3-benzothiazole-2,6- diamine), ropinirole (4-[2-(dipropylamino)ethyl]-1,3-dihydroindol-2-one), and rotigotine ((6S)-6-[propyl(2-thiophen-2-ylethyl)amino]-5,6,7,8- tetrahydronaphthalen-1-ol). Delivery Methods Dopamine, a dopamine agonist, levodopa and prodrugs thereof are preferably administered orally. In some embodiments, the composition comprising dopamine, levodopa and/or a prodrug thereof is suitable for oral administration. In embodiments, the composition is in a solid oral dose form. In embodiments, the solid oral dose form is a tablet, a capsule, or a softgel. Various other delivery methods can be used. One such other methods is intranasal delivery. Tadalafil is presently marketed as a tablet for oral administration (for instance under the tradename Cialis). It can also be administered in other ways. For instance, as an intravenous injection, or inhalant. Various other delivery methods can be used. One such other methods is intranasal delivery. In embodiments, the effective amount of dopamine, a dopamine agonist, L- DOPA or a prodrug thereof is a suboptimal dose when compared to the optimal dose of said dopamine, a dopamine agonist, L-DOPA or a prodrug thereof when given as a single treatment. In embodiments, the sub-optimal dose is determined by titration in a patient. In embodiments, dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered in an amount that is lower than a therapeutically effective amount of the compound when administered as a sole therapeutic agent for the treatment of Parkinson's disease or Parkinsonism. In embodiments, dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered in an amount that is lower than a therapeutically effective amount of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof when administered with benserazide for the treatment of Parkinson's disease or Parkinsonism. In embodiments, dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered in an amount that is lower than a therapeutically effective amount of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof when administered with carbidopa for the treatment of PD or Parkinsonism. The suboptimal dose is preferably determined by titration in the patient. The suboptimal dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is between 10% and 75% of the optimal dose of said dopamine, a dopamine agonist, L- DOPA or a prodrug thereof when given as a single treatment. The suboptimal dose is preferably between 10-60%, preferably 20-50%, preferably between 30-50% of the optimal dose when given as a single treatment. When dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is provided in a combination treatment with a further medicament such as a monoamine oxidase (MAO-B) inhibitor; a catechol-O-methyltransferase (COMT) inhibitor; or a combination thereof, and as a result of which the optimal dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is reduced, the suboptimal dose is likewise reduced to the same extent. For instance, the suboptimal dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof in a combination treatment with a further PD medicament is between 10% and 75% of the optimal dose of said dopamine, a dopamine agonist, L-DOPA or a prodrug thereof when given as said combination treatment. The amount of levodopa (L-DOPA) that is effective for treating Parkinson's disease or Parkinsonism is in the range of about 150-1000 mg/day. Suitable lower doses of L-DOPA are in the range of about 25-200 mg/day. In embodiments, suitable lower doses of L-DOPA are in the range of about 20-750 mg/day, 25-200 mg/day, about 25-150 mg/day, or about 50-100 mg/day. The effective amount of tadalafil or variant thereof is in the range of 0,1 – 40 mg/day. A suitable dose is in the range of 1 mg/day to 40 mg/day. Tadalafil is marketed in dosages of 5, 10 and 20 mg. For the present invention it is possible to use such formulations arriving at a suitable dose of 5 mg/day, 10 mg/day and 20 mg/day. A suitable minimum administered dose of tadalafil or variant thereof is 20 mg/day (0.4 mg/kg). The effective amount of tadalafil or variant thereof is at least 0,1 mg/day, such as from about 0,1 mg to about 200 mg per day. In embodiments, the effective amount of tadalafil or variant thereof is a daily dose of about 1 to about 50 mg/day. In embodiments, the effective amount of tadalafil or variant thereof is 5 to about 40 mg/day. In embodiments, the effective amount of tadalafil or variant thereof is about 5 to about 40 mg per day. In some embodiments, the effective amount of tadalafil or variant thereof is about 5, 10 or 20 mg per day. In embodiments, the effective amount of a PDE11 inhibitor is from about 0.1 mg to about 1000 mg per day. In embodiments, the effective amount of the PDE11 inhibitor is about 1 to about 500 mg per day. In embodiments, the effective amount of the PDE11 inhibitor is from about 10 to about 100 mg per day. In embodiments, the effective amount of the PDE11 inhibitor is about 20 to about 50 mg per day. In embodiments, the effective amount of the PDE11 inhibitor is about 25 to about 40 mg per day. In embodiments, the effective amount of the PDE2 inhibitor is from about 0.4 mg to about 100 mg per day. In embodiments, the effective amount of the PDE2 inhibitor is about 5 to about 50 mg per day. In embodiments, the effective amount of the PDE2 inhibitor is from about 0.5 to about 10 mg per day. In embodiments, the effective amount of the PDE2 inhibitor is about 10 to about 25 mg per day. In embodiments, the effective amount of the PDE2 inhibitor is about 15 to about 20 mg per day. In embodiments, the effective amount of the PDE2 inhibitor is about 5 to about 20 mg per day. In embodiments, the effective amount of the PDE2 inhibitor is about 25 to about 100 mg per day. In embodiments, the effective amount of a PDE11 inhibitor is from about 0.1 mg to about 1000 mg per day. In embodiments, the effective amount of the PDE11 inhibitor is about 1 to about 500 mg per day. In embodiments, the effective amount of the PDE11 inhibitor is from about 10 to about 100 mg per day. In embodiments, the effective amount of the PDE11 inhibitor is about 20 to about 50 mg per day. In embodiments, the effective amount of the PDE11 inhibitor is about 25 to about 40 mg per day. When tadalafil or variant thereof is combined with a PDE2 inhibitor such as EHNA, PF-05180999 (CAS 1394033-54-5) and/or BAY 60-7550, and/or a PDE11 inhibitor such as BC11-38, said tadalafil or variant thereof may be provided at less than 20 mg/day, such as from about 1 mg to about 15 mg per day, including 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg or 14 mg/day. A guanylate cyclase agonist such as a GUCY2C agonist can be administered directly to a subject. Direct delivery of a GUCY2C agonist to a subject will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. Other modes of administration include topical, oral, catheterized, intranasal and pulmonary administration, suppositories, and transdermal applications, needles, and particle guns or hyposprays. Dose treatment may be a single dose schedule or a multiple dose schedule. Delivery is preferably to the brain of the individual. A preferred route of administration is via the epithelium of the nose. A preferred method for delivery is intranasal delivery, as described (Grassin-Delyle et al., 2012. Pharmacol Therapeutics 134: 366-379). When tadalafil or variant thereof is combined with a guanylate cyclase agonist such as a GUCY2C agonist, said tadalafil or variant thereof may be provided at less than 20 mg/day, such as from about 1 mg to about 15 mg per day, including 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg or 14 mg/day. In case tadalafil or variant thereof is combined with a PDE2 inhibitor and/or a PDE11 inhibitor, and a guanylate cyclase agonist such as a GUCY2C agonist, said tadalafil or variant thereof may be provided 10 mg/day or less, such as from about 0.1 mg to about 9 mg per day, including 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, and 0.9 mg/day. In embodiments, the amount of a dopa-decarboxylase (DDC) inhibitor, e.g., benserazide or carbidopa, that is effective for treating PD or Parkinsonism is in the range of about 1-500 mg/day, such as 2 to 150 mg, 2 to 120 mg, 2 to 80 mg, 2 to 40 mg, 5 to 150 mg, 5 to 120 mg, 5 to 80 mg, 10 to 150 mg, 10 to 120 mg, 10 to 80 mg, 10 to 40 mg, 20 to 150 mg, 20 to 120 mg, 20 to 80 mg, 20 to 40 mg, 40 to 150 mg, 40 to 120 mg or 40 to 80 mg. Exemplary effective daily doses of carbidopa include, but are not limited to, 10 mg, 15 mg, 20 mg, and 25 mg. Exemplary effective daily doses of benserazide include, but are not limited to, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 50 mg, 60 mg, 70 mg, 80 mg and 100 mg. When tadalafil or variant thereof is combined with a dopa-decarboxylase (DDC) inhibitor, e.g., benserazide or carbidopa, said tadalafil or variant thereof may be provided at less than 20 mg/day, such as from about 1 mg to about 15 mg per day, including 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg or 14 mg/day. In embodiments the effective amount of a monoamine oxidase (MAO-B) inhibitor, e.g., rasagiline or selegiline, is in the range of 1 to 500 mg, 2 to 150 mg, 2 to 120 mg, 2 to 80 mg, 2 to 40 mg, 5 to 150 mg, 5 to 120 mg, 5 to 80 mg, 10 to 150 mg, 10 to 120 mg, 10 to 80 mg, 10 to 40 mg, 20 to 150 mg, 20 to 120 mg, 20 to 80 mg, 20 to 40 mg, 40 to 150 mg, 40 to 120 mg or 40 to 80 mg. A typical dose of rasagiline is 1 mg/day which is preferred in the present invention. A typical dose of selegiline is 10 to 20 mg/day which is preferred in the present invention. The dose is typically administered over several time points over the day. The administration is typically done with a selegiline solution containing 5 mg/ml of selegiline. Another MAO-B inhibitor is safinamide. Safinamide can be given in a range of 10-500 mg. It is typically administered with a starting dose of 50 mg per day which can be increased to 100 mg per day or decreased as desired. In embodiments the effective daily dose of a monoamine oxidase (MAO-B) inhibitor is 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.5 mg, 2 mg, 4 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 750 mg or 1,000 mg. When tadalafil or variant thereof is combined with a monoamine oxidase (MAO-B) inhibitor, e.g., rasagiline or selegiline, said tadalafil or variant thereof may be provided at less than 20 mg/day, such as from about 1 mg to about 15 mg per day, including 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg or 14 mg/day. In embodiments the effective amount of a catechol-O-methyltransferase (COMT) inhibitor, e.g., tolcapone or entacapone, is in the range of 1 to 500 mg, 2 to 150 mg, 2 to 120 mg, 2 to 80 mg, 2 to 40 mg, 5 to 150 mg, 5 to 120 mg, 5 to 80 mg, 10 to 150 mg, 10 to 120 mg, 10 to 80 mg, 10 to 40 mg, 20 to 150 mg, 20 to 120 mg, 20 to 80 mg, 20 to 40 mg, 40 to 150 mg, 40 to 120 mg or 40 to 80 mg. Entacapton is typically administered in doses of 200 mg per administration with a maximum of 10 administrations per day. When dopamine, L-dopa or a dopamine agonist is given entacapton is typically administered with each delivery of dopamine, L-dopa or dopamine agonist. Tolcapone is typically administered in doses of 100 mg or 200 mg per administration with a maximum of 3 administrations per day. When dopamine, L-dopa or a dopamine agonist is given tolcapone is typically administered with a delivery of dopamine, L-dopa or dopamine agonist. In embodiments the effective amount of the catechol-O-methyltransferase (COMT) inhibitor is 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.5 mg, 2 mg, 4 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 500 mg. When tadalafil or variant thereof is combined with a catechol-O-methyltransferase (COMT) inhibitor, e.g., tolcapone or entacapone, said tadalafil or variant thereof may be provided at less than 20 mg/day, such as from about 1 mg to about 15 mg per day, including 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg or 14 mg/day. In embodiments the effective amount of a dopamine agonist, e.g., pramipexole, ropinirole, rotigotine, is in the range of 0,1 mg – 30 mg per day. The starting dose is typically lower in the first week, which is then increased in the second week. If needed the dose can be further increased in the third week and even again in the fourth week. A typical dose escalation is: In the first week, take 3 times 0.25 mg per day. In the second week, take 3 times 0.5 mg per day. In the third week, take 3 times 0.75 mg per day. In the fourth week, take 3 times 1 mg per day. If necessary, gradually increase to a maximum of 24 mg per day after the fourth week. Rotigotine is typically administered with a skin patch. When used as monotherapy: Increase the initial dose from 2 mg per day to a maximum of 8 mg per day. The maintenance dose is typically 6-8 mg per day. When the patients has advanced Parkinson's with fluctuations: the typical dose can be 8-16 mg per day, with a maximum of 16 mg per day. Amantadine has an effect on the dopamine receptor and can be seen as a dopamine agonist. It is typically administered 2 times 100 mg per day. Start with 1 time 100 mg per day, and the maximum is 400 mg per day. The dopamine, a dopamine agonist, L-DOPA or prodrug thereof can be administered at the same time as the PDE11 inhibitor, but it can also be given sequentially, either before or after the administration of the PDE11 inhibitor. Best results are obtained when the PDE11 inhibitor is/are administered within one hour of administration of the dopamine, a dopamine agonist, L-DOPA or prodrug thereof. Conveniently they may be administered at the same time. For medical uses in the context of Parkinson’s and the treatment of individuals that have PD, it is preferred that the individual does not have a completely advanced form of PD. In such cases the substantia nigra is (almost) completely destroyed. For the present invention it is preferred that the individual has Parkinson’s disease stage 1, 2, 3 or 4. In a preferred embodiment the individual has Parkinson’s disease stage 1, 2, or 3. Preferably stage 1 or stage 2. An advantage of the present invention is that early stages of the disease can be treated adequately without inducing many of the side-effects associated with the present standard therapies. Dopaminergic neurons of the substantia nigra are stimulated to produce more dopamine to compensate for the loss of dopaminergic cells in the substantia nigra while at the same time not over-producing dopamine, or having excessive dopamine production in non-target cells, i.e. dopaminergic neurons that are not within the substantia nigra. The possibility of reducing the dose of dopamine, a dopamine agonist, L-DOPA or prodrug thereof reduces the propensity and/or the intensity of side effects of the externally provided dopamine, a dopamine agonist, L-DOPA or prodrug thereof. Preventing at least in part, the negative feedback inhibition of dopamine, a dopamine agonist, L-DOPA or prodrug thereof on dopamine production by substantia nigra cells increases the specificity of dopamine action in the brain, when compared to providing a full dose. Preferably, the compounds of the invention are used as a medicine. Medicines of the invention can suitably be used for the treatment of an individual that has PD or is at risk of having PD. Pharmaceutical compositions of the invention are particularly suited for increasing the production of dopamine by dopaminergic cells as described herein. Dopamine biosynthesis is complex and integrated with various other biosynthesis pathways. Dopamine is produced from L-tyrosine in two reactions. The enzyme tyrosine hydroxylase (Th) converts L-tyrosine into levodopa (L-DOPA) and aromatic L-amino acid decarboxylase (AADC) decarboxylates levodopa to form dopamine. Tyrosine Hydroxylase is known under a number of aliases Tyrosine 3- Monooxygenase; Tyrosine 3-Hydroxylase; EC 1.14.16.2; TYH; Dystonia 14; EC 1.14.16; DYT14; and DYT5b. External Ids for TH are HGNC: 11782; Entrez Gene: 7054; Ensembl: ENSG00000180176; OMIM: 191290 and UniProtKB: P07101. TH has an N-terminal regulatory domain, a central catalytic domain and a C-terminal tetramerization domain (Tekin et al., 2014. J Neural Transmiss 121: 1451–1481). The most appealing domain to increase TH activity is located in the N-terminal region, which includes three serine residues susceptible to phosphorylation (i.e. Ser19, Ser31 and Ser40). The regulatory importance of these residues is reflected in their broad evolutionary conservation (see WO/2018/160067). Independent phosphorylation of Ser31 and Ser40 is known to increase TH activity in vitro and in situ. Ser31 phosphorylation exclusively operates by raising the affinity of TH for one of its cofactors (i.e. BH4), whereas Ser40 also impedes the negative feedback loop by blocking the interaction of dopamine with the catalytic domain of Th (Dunkley et al., 2004. J Neurochem 91: 1025-1043). Ser40 is thus a promising target, and we believe that boosting its phosphorylation solves an important limitation of levodopa treatment. It is an object of the invention to increase TH activity by inducing Ser40 phosphorylation specifically in the (presynaptic) striatum. The main kinases known to phosphorylate this residue are cAMP- and cGMP-dependent protein kinases (PKA and PKG, respectively) (Campbell et al., 1986. J Biol Chem 261: 10489-10492; Roskoski et al., 1987. J Neurochem 48: 840-845). Both enzymes are basally inactive due to the interaction of the regulatory region with the catalytic center. While the regulatory and catalytic counterparts of PKA correspond to separate polypeptides, PKG is a single amino acidic chain including both domains. In order to be activated, both kinases depend on the interaction of the corresponding cyclic nucleotide with the regulatory region of the enzyme, so that the catalytic center is released to phosphorylate different substrates (Scholten et al., 2008. Mass Spectrom Rev 27: 331-353). Among their various targets, Ser40 in TH is the preferred target in the present invention.` In embodiments, the treatment may include treatment of the disease with a further medicament. The further medicament preferably comprises a PDE11 inhibitor, a PDE2 inhibitor, a dopa-decarboxylase (DDC) inhibitor; a monoamine oxidase (MAO-B) inhibitor; a catechol-O-methyltransferase (COMT) inhibitor; or a combination thereof. The PDE11 inhibitor preferably is BC11-15; BC11-19; BC11-28; BC11-38; BC11-38-1; BC11-38-2; BC11-38-3; or BC11-38-4, preferably BC11-38; BC11-38-1; BC11-38-2; BC11-38-3 or BC11-38-4. In embodiments, the PDE11 inhibitor is BC11-38. The PDE2 inhibitor is preferably EHNA, BAY 60-7550, PDP, PF-05180999, or oxindole. The invention further provides a method for treating dopamine deficiency in a subject comprising administering dopamine, a dopamine agonist, L-DOPA or a prodrug thereof to a subject in need thereof characterized in that the treatment of the deficiency further comprises the administration of tadalafil or a variant thereof to the subject, whereby the administration of tadalafil or variant thereof is at a minimum dosage of 20 mg/day (0.4 mg/kg). Said dopamine deficiency preferably is a central nervous system dopamine deficiency. The invention also provides a method of treating a Parkinson’s disease in a subject comprising administering dopamine, a dopamine agonist, L-DOPA or a prodrug thereof to a subject in need thereof characterized in that the treatment of the disease comprises the administration of tadalafil or variant thereof to the subject, whereby the administration of tadalafil or variant thereof is at a minimum dosage of 20 mg/day (0.4 mg/kg). The treatment preferably comprises treating symptoms of Parkinson’s disease related to central nervous system dopamine deficiency. Said treatment preferably is of patients having PD stage 1-5. Also provided is a method for reducing feedback inhibition induced by dopamine, a dopamine agonist, L-DOPA or a prodrug thereof on dopamine production by substantia nigra cells comprising stimulating cAMP and/or cGMP production in said substantia nigra cells. Said cAMP and/or cGMP production is preferably stimulated by contacting said cells with dopamine, a dopamine agonist, L-DOPA or a prodrug thereof and tadalafil or variant thereof. The invention also provides a medicament or a kit of parts comprising dopamine, a dopamine agonist, L-DOPA or a prodrug thereof and tadalafil or variant thereof. The invention also provides a medicament or a kit of parts comprising a sub-optimal dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, tadalafil or variant thereof, and a phosphodiesterase (PDE) 11 inhibitor or a PDE2 inhibitor or a combination thereof. Further provided is a medicament or kit of parts comprising dopamine, a dopamine agonist, L-DOPA or a prodrug thereof and tadalafil or variant thereof, for use in the treatment of PD, preferably PD stage 1-5. Further provided is a medicament or kit of parts comprising a sub-optimal dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof and tadalafil or variant thereof, for use in the treatment of PD, preferably PD stage 1-5. The invention further provides a method for at least in part decreasing feedback inhibition by dopamine, a dopamine agonist, L-DOPA or a prodrug thereof on dopamine production comprising contacting a dopaminergic cell, preferably a substantia nigra dopaminergic cell, with said dopamine, a dopamine agonist, L-DOPA or prodrug thereof and tadalafil or variant thereof. A decrease in feedback inhibition is preferably measured by measuring the level of TH S40 phosphorylation in the dopaminergic cell. In embodiments, the cell is also contacted with a PDE11 inhibitor, a PDE2 inhibitor and/or a CUCY2C agonist. The invention further provides a tadalafil or variant thereof, in combination with dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, for use in the treatment of an individual that has PD. The individual preferably has Parkinson’s disease stage 1, 2, 3 or 4. In a preferred embodiments the individual has Parkinson’s disease stage 1, 2 or 3, preferably 1 or 2 and preferably stage 1. It was noted in the present invention that tadalafil or variant thereof, optionally together with a PDE11 inhibitor, a PDE2 inhibitor and/or a guanylin cyclase agonist, stimulates the production of dopamine by dopaminergic cells to a level that at least in part prevents the feedback inhibition of the production of dopamine by dopamine, a dopamine agonist, L-DOPA or prodrug thereof, in dopaminergic cells. As these cells now continue to produce relevant amounts of dopamine, the dose of externally provided dopamine, a dopamine agonist, L-DOPA or prodrug thereof can be reduced to levels wherein the treatment has less side-effects than a treatment with dopamine, a dopamine agonist, L-DOPA or prodrug at a routine dose. The treatment is particularly effective in early stages of the disease, where the limitation of dopamine production in the individual just becomes apparent in the expression of mild symptoms. In such cases the individual does not have enough dopamine production typically due to the partial disappearance of dopaminergic cells. Particularly in the early stages, however, the individual typically has ample cells to provide adequate production of dopamine upon the treatment of the invention. The invention further provides dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in a method for the treatment of dopamine deficiency in a subject characterized in that the treatment further comprises the administration of tadalafil or variant thereof to the subject. Pharmaceutical Compositions Comprising L-DOPA and tadalafil or variant thereof. An effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular agent being administered, the size of the subject, or the severity of the disease or condition, as is known to a skilled person. A therapeutically effective amount is any amount that is necessary or sufficient for treating Parkinson's disease or Parkinsonism in a subject in combination with a sub-optimal dose of L-DOPA. The effective amount of tadalafil or variant thereof can also vary depending on whether tadalafil or variant thereof is administered with L-DOPA in combination with benserazide, whether tadalafil or variant thereof is administered with L- dopa in combination with carbidopa, whether tadalafil or variant thereof is administered with L-DOPA in combination with a monoamine oxidase type B inhibitor, or whether tadalafil or variant thereof is administered with L-DOPA in combination with a catechol-O- methyl transferase inhibitor. The effective amount of tadalafil or variant thereof may further vary depending on whether the inhibitor is administered with L- DOPA in combination with carbidopa and a monoamine oxidase type B inhibitor, with L-DOPA in combination with carbidopa and a catechol-O- methyl transferase inhibitor, with L-DOPA in combination with benserazide and a monoamine oxidase type B inhibitor, or with L-DOPA in combination with benserazide and a catechol- O- methyl transferase inhibitor. Tadalafil or variant thereof and L-DOPA can be administered at different times or at the same time. In embodiments, L-DOPA and tadalafil or variant thereof are administered simultaneously. In embodiments, L-DOPA and tadalafil or variant thereof are administered sequentially. In embodiments, L-DOPA is administered prior to administration of tadalafil or variant thereof. In embodiments, tadalafil or variant thereof is administered prior to administration of L-DOPA. When a dopa-decarboxylase (DDC) inhibitor is administered in addition to tadalafil or variant thereof and L-DOPA, it can be administered before, after or concurrently with tadalafil or variant thereof and/or L-DOPA. L-DOPA and tadalafil or variant thereof may independently be administered to a patient once, twice, three times, four times, five times, six times, 8 times or 12 times a day, as is known to a person skilled in the art. The methods described herein can further comprise administering an effective amount of another therapeutic agent, that is, the pharmaceutical compositions described herein can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. In embodiments, the methods described herein can further comprise administering an effective amount of a PDE11 inhibitor such as BC11-38. In embodiments, the methods described herein can further comprise administering an effective amount of a PDE2 inhibitor such as EHNA, PF-05180999 or BAY 60-7550. In embodiments, the methods described herein can further comprise administering an effective amount of a guanylate cyclase agonist, preferably a guanylate cyclase 2C receptor (GUCY2C) agonist, such as guanylin, lymphoguanylin, enterotoxin or uroguanylin or a functional derivative of guanylin, lymphoguanylin, enterotoxin or uroguanylin. In embodiments, the methods described herein can further comprise administering an effective amount of a dopa-decarboxylase (DDC) inhibitor such as benserazide or carbidopa. In embodiments, the methods described herein further comprise administering an effective amount of a monoamine oxidase (MAO-B) inhibitor. In embodiments, the methods described herein further comprise administering an effective amount of a catechol-O-methyltransferase (COMT) inhibitor. In embodiments, the methods described herein further comprise administering effective amounts of benserazide and a PDE2 inhibitor. In embodiments, the methods described herein further comprise administering effective amounts of benserazide and a guanylate cyclase agonist, preferably a guanylate cyclase 2C receptor (GUCY2C) agonist. In embodiments, the methods described herein further comprise administering effective amounts of benserazide and a monoamine oxidase (MAO-B) inhibitor. In embodiments, the methods described herein further comprise administering effective amounts of benserazide and a catechol-O- methyltransferase (COMT) inhibitor. In embodiments, the methods described herein further comprise administering effective amounts of carbidopa and a monoamine oxidase (MAO-B) inhibitor. In embodiments, the methods described herein further comprise administering effective amounts of carbidopa and a catechol-O-methyltransferase (COMT) inhibitor. When a monoamine oxidase (MAO-B) inhibitor or a catechol-O- methyltransferase (COMT) inhibitor is administered in addition tadalafil or variant thereof and dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, it can be administered before, after or concurrently with tadalafil or variant thereof and/or dopamine, a dopamine agonist, L-DOPA or a prodrug thereof. When a monoamine oxidase (MAO-B) inhibitor or a catechol-O- methyltransferase (COMT) inhibitor is administered in addition to tadalafil or variant thereof, dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, and a dopa-decarboxylase (DDC) inhibitor, it can be administered before, after or concurrently with tadalafil or variant thereof, dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, and/or a dopa-decarboxylase (DDC) inhibitor. Provided herein are kits that can simplify the administration of tadalafil or variant thereof and dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, to a patient. The kit can comprise one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. An exemplary kit comprises a unit dose form of tadalafil or variant thereof and a unit dose form of L-DOPA. In embodiments, the kit comprises: (a) L-DOPA and a pharmaceutically acceptable carrier or vehicle in a first container; (b) a tadalafil or variant thereof and a pharmaceutically acceptable carrier or vehicle in a second container. In embodiments, the kit comprises: (a) L-DOPA, carbidopa, and a pharmaceutically acceptable carrier or vehicle in a first container; and (b) tadalafil or variant thereof and a pharmaceutically acceptable carrier or vehicle in a second container. In embodiments, the kit comprises: (a) L-DOPA, benserazide, and a pharmaceutically acceptable carrier or vehicle in a first container; and (b) tadalafil or variant thereof and a pharmaceutically acceptable carrier or vehicle in a second container. The kit can further comprise a label or printed instructions instructing the use of tadalafil or variant thereof and L-DOPA to treat Parkinson's disease. The kit can further comprise a label or printed instructions instructing the use of tadalafil or variant thereof and L-DOPA to treat PD or Parkinsonism. The kit can also further comprise a unit dose form of a dopa-decarboxylase (DDC) inhibitor. Such a dopa-decarboxylase (DDC) inhibitor can be present in a third container. Thus, in one embodiment, the kit comprises: (a) L-DOPA and a pharmaceutically acceptable carrier or vehicle in a first container; (b) tadalafil or variant thereof and a pharmaceutically acceptable carrier or vehicle in a second container, and (c) a dopa-decarboxylase (DDC) inhibitor and a pharmaceutically acceptable carrier or vehicle in a third container. Example of a dopa-decarboxylase (DDC) inhibitor include, but are not limited to, benserazide and carbidopa. In embodiments, the kit comprises: (a) L-DOPA and a pharmaceutically acceptable carrier or vehicle in a first container; (b) tadalafil or variant thereof and a pharmaceutically acceptable carrier or vehicle in a second container, and (c) monoamine oxidase (MAO-B) inhibitor and a pharmaceutically acceptable carrier or vehicle in a third container. In embodiments, the kit comprises: (a) L-DOPA and a pharmaceutically acceptable carrier or vehicle in a first container; (b) tadalafil or variant thereof and a pharmaceutically acceptable carrier or vehicle in a second container, and (c) catechol-O-methyltransferase (COMT) inhibitor and a pharmaceutically acceptable carrier or vehicle in a third container. In embodiments, the kit comprises: (a) L-DOPA, benserazide, and a pharmaceutically acceptable carrier or vehicle in a first container; (b) tadalafil or variant thereof and a pharmaceutically acceptable carrier or vehicle in a second container, and (c) monoamine oxidase (MAO-B) inhibitor and a pharmaceutically acceptable carrier or vehicle in a third container. In embodiments, the kit comprises: (a) L-DOPA, benserazide, and a pharmaceutically acceptable carrier or vehicle in a first container; (b) tadalafil or variant thereof and a pharmaceutically acceptable carrier or vehicle in a second container, and (c) catechol-O- methyltransferase (COMT) inhibitor and a pharmaceutically acceptable carrier or vehicle in a third container. In embodiments, the kit comprises: (a) L-DOPA, carbidopa, and a pharmaceutically acceptable carrier or vehicle in a first container; (b) tadalafil or variant thereof and a pharmaceutically acceptable carrier or vehicle in a second container, and (c) monoamine oxidase (MAO-B) inhibitor and a pharmaceutically acceptable carrier or vehicle in a third container. In embodiments, the kit comprises: (a) L-DOPA, carbidopa, and a pharmaceutically acceptable carrier or vehicle in a first container; (b) tadalafil or variant thereof and a pharmaceutically acceptable carrier or vehicle in a second container, and (c) catechol-O- methyltransferase (COMT) inhibitor and a pharmaceutically acceptable carrier or vehicle in a third container. The kit may comprise a unit dose form of tadalafil or variant thereof and L- DOPA. In one embodiment, the kit comprises L-DOPA, tadalafil or variant thereof, and a pharmaceutically acceptable carrier or vehicle in a single container. The kit may further comprise a label or printed instructions instructing the use of tadalafil or variant thereof and L-DOPA to treat PD or Parkinsonism. In embodiments, the kit comprises L-DOPA, tadalafil or variant thereof, a dopa-decarboxylase (DDC) inhibitor, and a pharmaceutically acceptable carrier or vehicle in a single container. In embodiments, the kit comprises L-DOPA, tadalafil or variant thereof, a monoamine oxidase (MAO-B) inhibitor, and a pharmaceutically acceptable carrier or vehicle in a single container. In embodiments, the kit comprises L-DOPA, tadalafil or variant thereof, a catechol-O- methyltransferase (COMT) inhibitor, and a pharmaceutically acceptable carrier or vehicle in a single container. In embodiments, the kit comprises L-DOPA, tadalafil or variant thereof, benserazide, a monoamine oxidase (MAO-B) inhibitor, and a pharmaceutically acceptable carrier or vehicle in a single container. In embodiments, the kit comprises L-DOPA, tadalafil or variant thereof, benserazide, a catechol-O-methyltransferase (COMT) inhibitor, and a pharmaceutically acceptable carrier or vehicle in a single container. In embodiments, the kit comprises L-DOPA, tadalafil or variant thereof, carbidopa, a monoamine oxidase (MAO-B) inhibitor, and a pharmaceutically acceptable carrier or vehicle in a single container. In embodiments, the kit comprises L-DOPA, tadalafil or variant thereof, carbidopa, a catechol-O- methyltransferase (COMT) inhibitor, and a pharmaceutically acceptable carrier or vehicle in a single container. In embodiments, the kit further comprises a monoamine oxidase (MAO-B) inhibitor and a pharmaceutically acceptable carrier or vehicle in a third container. In embodiments, the kit further comprises a catechol-O-methyltransferase (COMT) inhibitor and a pharmaceutically acceptable carrier or vehicle in a third container. The invention provides a kit comprising: (a) L-DOPA and tadalafil or variant thereof, and a pharmaceutically acceptable carrier or vehicle in a single container; and (b) instructions for use. In embodiments, the kit further comprises a dopa-decarboxylase (DDC) inhibitor in the single container. In embodiments, the dopa-decarboxylase (DDC) inhibitor is carbidopa or benserazide. In embodiments, the kit further comprises a monoamine oxidase (MAO-B) inhibitor in the single container. In embodiments, the kit further comprises a catechol-O-methyltransferase (COMT) inhibitor in the single container. For the purpose of clarity and a concise description features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described. Examples Example 1 Material & methods Chemicals Calyculin A (9902), forskolin (3828S), and okadaic acid (5934S) were purchased from Cell Signaling Technology. Dopamine (H8502), L-DOPA (D9628) and pCPT-cAMP (C3912) were purchased from Sigma-Aldrich. Except for dopamine (in H2O) and L-DOPA (in 0.5M HCl) are all chemicals were dissolved in dimethyl sulfoxide (DMSO). Animals All in vivo experiments were performed on adult (~3 months old) C57/Bl6/J wild-type or Pitx3-deficient mice. The Pitx3-deficient mice are either heterozygous for wild-type Pitx3 and green fluorescent protein (GFP) that is knocked-in on the Pitx3 locus (Pitx3GFP/+) or homozygous for GFP on the Pitx3 locus (Pitx3GFP/GFP). The heterozygous animals are described to have a normal development of the midbrain dopaminergic system, while the homozygous animals are known to have a dramatic loss of neurons of the substantia nigra and its projections to the dorsal striatum (Maxwell et al., 2005. Dev Biol 282: 467–479; Zhao et al., 2004. Eur J Neurosci 19: 1133–1140). Animals were housed on a 12 hour light-dark cycle, with food and water provided ad libitum. All animal experimentation was supported and granted by the Animals Experimentation Committee of the University of Amsterdam (UvA) according to national and international legislation. Animals are cared for on a daily basis and sacrificed according to rules and regulations of the Dutch and European law. Ex vivo slicing and chemical treatment Mice were sacrificed by cervical dislocation in compliance with the law. Brains were immediately isolated and sliced on a Leica VT100S vibratome in ice- cold slicing buffer (120 mM Choline Chloride, 3.5 mM KCl, 0.5 mM CaCl2, 6 mM MgSO4, 1.25 mM NaH2PO4, 27.5 mM D-Glucose, 25 mM NaHCO3) under constant oxygenation (95% O2, 5% CO2). Coronal Midbrain (MB) or corpus striatum (CS) slices with a thickness of 250 µm were collected. Subsequently, the slices were micro-dissected and divided in two hemispheres to have an internal control. After micro-dissecting the brain areas of interest, the slices were transferred to 32°C constantly oxygenized (95% O2, 5% CO2) artificial cerebrospinal fluid (aCSF; 120 mM NaCl, 3.5 mM KCl, 2.5 mM CaCl2, 1.3 mM MgSO4, 1.25 mM NaH2PO4, 27.5 mM D-Glucose, 25 mM NaHCO3) for 30 minutes. Subsequently, the slices were put at room temperature (RT) for another 30 minutes. In Eppendorf tubes, slices were incubated with – in each experiment specified – compounds that were further diluted in RT aCSF. After pharmacological treatment, aCSF was removed and slices were lysed in warm 1X Laemmli sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS; [Merck Millipore], 10% glycerol [Sigma-Aldrich] and 0.01% w/v bromophenol blue [Sigma-Aldrich] in MilliQ supplemented with 50 mM dithiotretol (DTT; [Merck Millipore]). Samples were sonicated for 3 min in a Bioruptor sonicator (Diagenode) at maximum potency, boiled at 95 °C for 5-10 min, and spun down briefly. Capillary-based western blot analysis (Wes by ProteinSimple) Evaluation of protein expression was performed using the Wes™ automated capillary western blot system (Protein Simple, San Jose, CA, USA) according to manufacturer’s instructions and under default settings. Briefly, prepared cell lysate samples were diluted with MilliQ, combined with the fluorescent master mix (PS-ST01EZ-8, ProteinSimple), and heated at 95°C for 5 min. The samples, biotinylated ladder (PS-ST01EZ-8, ProteinSimple), reagents (including the secondary antibody) from the anti-rabbit detection module (DM-001, ProteinSimple), and primary antibodies were loaded into designated wells in the 12-230 kDa separation module assay plate (PS-PP03, ProteinSimple). The assay plate and a 25-capillary cartridge are inserted into the Wes™ machine. The machine automatically separates the proteins by size, and performs the immunoprobing, incubations, washing steps, and detection. Digital images were analyzed using the Compass software (ProteinSimple). Proteins were probed using the following antibodies: rabbit Anti-Tyrosine Hydroxylase (P40101, Pel-Freez); rabbit anti-phospho-tyrosine hydroxylase (Ser40) (2791S, CST); rabbit anti- phospho-tyrosine hydroxylase (Ser31) (13041S, CST); rabbit anti-β-actin (4907S, CST); anti-TOM20 (42406, CST). Antibodies were diluted in antibody diluent (1:10 or 1:50; 042-203, ProteinSimple). Statistical analysis The amounts of phospho-protein levels were corrected for the total amount of the same protein, phosphorylated or not, and normalized to the vehicle condition. To determine statistical significance for the mouse ex vivo experiments comparing two groups, two-tailed paired student’s t-tests were used. For the ex vivo experiments comparing four groups, area under the curve (AUC) values were determined for each condition per animal for which outer values (the most rostral and most caudal slices) were determined by interpolation. Subsequently, significance was determined by two-tailed paired student’s t-tests followed by Bonferroni’s multiple comparisons post hoc testing. For the in vitro experiments, one way analysis of variance (ANOVA) was performed, followed by Bonferroni’s multiple comparisons post hoc test. The data are expressed as fold change relative to the control condition and presented as bar charts (showing the mean and with or w/o ± SEM) or minimum to maximum boxplots (showing the first quartile, third quartile, and the mean). Differences were considered to be significant at a p-value < 0.05. Asterisks indicate significance (*p < 0.05 and **p < 0.01). Results and discussion Acute slice preparation to investigate tyrosine hydroxylase phosphorylation To investigate and manipulate tyrosine hydroxylase phosphorylation in specific target areas as a whole and with as less contaminating nuclei as possible, we used and developed a novel ex vivo pharmacology approach using acute brain slices. Currently, two main techniques are used to investigate tyrosine hydroxylase levels in mouse brain tissue (Dunkley and Dickson, 2019. J Neurochem 149: 706– 728). The first is a relatively simple setup where non-frozen tissue is sliced coronally in a brain matrix and, subsequently, the nuclei of interest are dissected freehand or with a punch (Ong et al., 2014. J Neurochem 128: 547–560; Pruett and Salvatore, 2013. Mol Neurobiol 47: 988–999; Pruett and Salvatore, 2010. J Neurochem 115: 707–715; Salvatore, 2014. J Neurochem 129: 548–558; Salvatore et al., 2012. J Vis Exp 66: e4171; Salvatore et al., 2009. Exp Neurol 219: 197–207; Salvatore et al., 2009. PLoS One 4: e8466; Salvatore et al., 2004. J Neurochem 90: 245–254; Salvatore et al., 2000. J Neurochem 75: 225–232; Salvatore and Pruett, 2012. PLoS One 7: e29867). In this technique the simplicity may come at the expense of more contaminating nuclei. The other technique uses frozen brain tissue and the collection of specific nuclei is done with a punch (Ong et al., 2011. Neurochem Res 36: 27–33). In this technique multiple brain slices are required to collect a specific brain area, but with less contaminating nuclei. We want to manipulate intra-cellular signaling mechanisms in a physiologically realistic environment, with as little contamination of other brain structures as possible. Due to the possible contamination of other brain structures and the detrimental effects that freeze-thaw cycles can have on cell signaling, the techniques described above are inadequate. Therefore, we developed and used an alternative approach. In our setup we prepare and investigate living acute brain slices, an approach predominantly used in the field of electrophysiology (Ting et al., 2014. In: Patch- Clamp Methods and Protocols, Methods in Molecular Biology. Martina and Taverna (Eds). Humana Press, New York. Vol 1183: 221–242) and comparable to the setup of Sugiyama and colleagues (Sugiyama et al., 2021. J Neurosci 41: 6388– 6414). As illustrated in Figure 2, the brains from adult mice were rapidly removed from the skull, the non-frozen brains sectioned in 250 μm coronal slices with a vibrating blade microtome, and the acute slices containing the brain regions we are interested in were collected (Fig. 2A). As we want to manipulate tyrosine hydroxylase phosphorylation of nigrostriatal dopaminergic neurons, we either collected acute slices containing the corpus striatum (CS), thus where the neurons project to, and slices containing the substantia nigra in the midbrain (MB), where the neurons originate. For adult mice, we can collect roughly 12 slices to include the whole dorsal striatum and five to include the midbrain (Fig. 2B). To minimize the contaminating effects by other brain structures, we micro-dissected the regions of interest of each collected slice (depicted in gray in the schematic images of each slice in figure 2B) freehand using a light microscope and under ice-cold but not freezing conditions (Fig. 2C). On these micro-dissected acute brain slices we performed our ex vivo pharmacology experiments in a polypropylene incubation tube with drugs as specified in each experiment (Fig. 2C). Finally, using the Wes™ (ProteinSimple), we detected and analyzed the protein levels for each micro- dissected slice by automated western blot analysis (Fig. 2C). Unlike cultures or cell homogenates, acute brain slices largely maintain their in vivo cytoarchitecture and synaptic circuitry. This allows the study of specific brain regions in isolation. Therefore, with this approach, we can investigate and manipulate tyrosine hydroxylase phosphorylation of specific brain regions with minimal contamination of other brain structures while maintaining optimal cellular, molecular, and circuitry of the structure of interest (Buskila et al., 2015. Sci Rep 4: 5309; Ting et al., 2014. In: Patch-Clamp Methods and Protocols, Methods in Molecular Biology. Martina and Taverna (Eds). Humana Press, New York. Vol 1183: 221–242). As such, we examined tyrosine hydroxylase levels in the mouse brain (n = 3) over the rostro-caudal axis (Figure 3). Coronal slices were collected as described above, resulting in 12 striatal (CS) slices, five midbrain (MB) slices, and three slices connecting the striatum and the midbrain. Each slice was divided per hemisphere and the regions of interest (depicted in gray in schematic images of Fig. 3A) were micro-dissected. The micro-dissected parts of each coronal slice (one per hemisphere) were pooled and probed for baseline protein levels of total tyrosine hydroxylase (independent of phosphorylation status), phospho-tyrosine hydroxylase (Ser40), phospho-tyrosine hydroxylase (Ser31), and β-actin (Fig. 3A). Quantification of total tyrosine hydroxylase protein levels (Fig. 3B) exposed a specific pattern over the rostro-caudal axis, with a right-skewed peak in tyrosine hydroxylase levels in the more rostral part of the corpus striatum (CS). To compare the average tyrosine hydroxylase levels in the striatum and in the midbrain, a paired-samples t-test was conducted. Average tyrosine hydroxylase protein levels are 3.55 times higher (p < 0.01) in the striatum as compared to the midbrain. Interestingly, if we quantify the phospho-tyrosine hydroxylase levels and correct them for the total tyrosine hydroxylase levels, a more even distribution emerges in both the striatum and midbrain (Fig. 3C-D). This suggests that the relative phosphorylation levels of both Ser40 (Fig. 3C) and Ser31 (Fig. 3D) is evenly distributed in each brain region and that there are no major differences in relative phosphorylation levels in each brain region. The relative phosphorylation levels of Ser40 (p < 0.05; M = 1.77) and Ser31 (p < 0.01; M = 1.67) are, however, on average higher in the striatum as compared to the midbrain. In spite of this, the average level of tyrosine hydroxylase is almost twice as low in the striatum as compared to the midbrain if we correct the total tyrosine hydroxylase protein levels for the levels of the housekeeping protein β-actin (Fig. 3E; p < 0.05; M = 0.63). This indicates that the density of tyrosine hydroxylase is almost twice as low in the striatum as compared to the midbrain. Next, we wanted to manipulate tyrosine hydroxylase phosphorylation in our acute brain slices, with the main focus on the dopaminergic terminals in the striatum as Parkinson’s disease is characterized by a dopamine deficiency in the region of these striatal terminals. To do so, we divided each slice per hemisphere and micro-dissect the regions of interest of each slice (see Figure 4, gray part of the schematic images). For each slice, we use the micro-dissected part of one hemisphere as internal control for the experimental, treated condition, which is the micro-dissected part of the other hemisphere. Although the control and experimental conditions are randomly chosen, we can only use this setup if the relative tyrosine hydroxylase levels are similar between hemispheres. Therefore, we strictly separated the slices from the left hemisphere from the slices from the right hemisphere and examined protein levels of tyrosine hydroxylase, phospho- tyrosine hydroxylase (Ser40), phospho-tyrosine hydroxylase (Ser31), and β-actin (Fig. 4A-B). Indeed, there are no differences in relative phosphorylation levels between the left and right hemisphere, for both Ser40 phosphorylation (Fig. 4B, left panel; t(9) = 0.38, p > 0.05, MLeft = 1.00, MRight = 1.03) and Ser31 phosphorylation (Fig. 4B, right panel; t(9) = 0.65, p > 0.05, MLeft = 1.00, MRight = 1.03). In sum, there are no differences in relative phosphorylation levels between the left and right hemisphere, and these relative phosphorylation levels are similar throughout the striatum. Therefore, we developed a novel approach that can be used to investigate and manipulate tyrosine hydroxylase in non-frozen micro- dissected brain tissue. Mouse striatal tyrosine hydroxylase phosphorylation is regulated via the cyclic AMP second messenger system A wealth of data described that tyrosine hydroxylase is affected by the cAMP second messenger system (Dunkley et al., 2004. J Neurochem 91: 1025–1043; Dunkley and Dickson, 2019. J Neurochem 149: 706–728; Harris et al., 1974. Nature 252: 156–158; Joh et al., 1978. Proc Natl Acad Sci USA 75: 4744–4748; Lovenberg et al., 1975. Proc Natl Acad Sci USA 72: 2955–2958; Morgenroth et al., 1975. J Biol Chem 250: 1946–1948; Roskoski and Roskoski, 1987. J Neurochem 48: 236–242). Therefore, we investigated the influence of cAMP signaling on tyrosine hydroxylase phosphorylation using our ex vivo approach (Fig.4C-F), either via forskolin (Fig. 4C-D) or via pCPT-cAMP (Fig. 4E-F), a cell-permeable analogue of cAMP. Forskolin is used to increase cAMP levels via the activation of adenylyl cycles (Alasbahi and Melzig, 2012. Pharmazie 67: 5–13) and is known to affect tyrosine hydroxylase phosphorylation (Bowyer et al., 1992. Brain Res 591: 261–270; Cheah et al., 1999. J Neurosci Methods 87: 167–174). We collected and micro-dissected coronal striatal slices as described earlier. Per collected slice, one micro-dissected region of interest was exposed to 10 µM forskolin for 60 minutes while the matching part was treated with DMSO vehicle. Subsequently, tyrosine hydroxylase protein levels were examined (Fig. 4C-D). Relative Ser40 phosphorylation levels are significantly increased due to forskolin exposure (Fig. 4D, left panel; t(9) = 6.48, p < 0.01, MForskolin = 1.94). In contrast, relative Ser31 levels are decreased by forskolin (Fig. 4D, right panel; t(9) = 6.81, p < 0.01, MForskolin = 0.81). In a similar approach we tested the effects of 60 minute exposure to 500 µM pCPT- cAMP (Best et al., 1995. J Neurochem 65: 1934–1943; Bobrovskaya et al., 2007. Cell Signal 19,: 1141–1149; Chen et al., 2008. Mol Pharmacol 73: 1816–1828; Tank et al., 2008. Ann New York Acad Sci 1148: 238–248) on tyrosine hydroxylase phosphorylation (Fig. 4E-F). pCPT-cAMP elevated relative Ser40 phosphorylation levels (Fig. 4F, left panel; t(5) = 2.83, p < 0.5, MpCPT-cAMP = 1.30), while there was no effect on Ser31 phosphorylation levels (Fig. 4F, right panel; t(5) = 1.18, p > 0.5, MpCPT-cAMP = 1.05). Ser40 phosphorylation can be activated through Gucy2C receptor activation. In an ex-vivo setup we stimulated mouse striatal slices with the endogenous ligand guanylin which led to an increase in TH-Ser40 phosphorylation (Fig. 5A/B). In order to substantiate the presence of Gucy2C in human midbrain dopamine neurons we performed IMHC for TH and GUCY2C on postmortum human tissue (Fig. 5C). Th data clearly show that Gucy2C is present in TH positive, neuromelanin containing neurons. Thus, in the mouse striatum is Ser40 phosphorylation up-regulated by the activation of the cAMP second messenger system, via forskolin, cAMP analogues, and Guanylin. Ser31 phosphorylation is down-regulated by forskolin, while pCPT- cAMP had no effect. Dopamine down-regulates tyrosine hydroxylase Ser40 phosphorylation The counterpart of tyrosine hydroxylase activation via Ser40 phosphorylation is the extensively described mechanism of end-product feedback inhibition via dopamine and other catecholamines (Almas et al., 1992. Eur. J. Biochem 209: 249– 255; Andersson et al., 1992. Biochem J.284: 687–695; Andersson et al., 1988. J Biol Chem 263: 18621–18626; Briggs et al., 2014. J Biochem 155: 183–193; Briggs et al., 2011. Biochem 50: 1545–1555; Daubner et al., 1992. J Biol Chem 267: 12639– 12646; Daubner et al., 2011. Arch Biochem Biophys 508: 1–12; Dickson and Briggs, 2013. Advances Pharmacol 68: 13-21; Fitzpatrick, 1988. J Biol Chem 263: 16058– 16062; Fujisawa and Okuno, 2005. Biochem Biophys Res Commun 338: 271–276; Gordon et al., 2008. J Neurochem 106: 1614–1623; Gordon et al., 2009. Neurochem Res 34: 1830–1837; Haavik et al., 1991. Eur J Biochem 199: 371–378; Kaushik et al., 2007. J Comput Neurosci 22: 147–160; Maass et al., 2003. Eur J Biochem 270: 1065–1075; Nakashima et al., 2013. Advanc Pharmacol 68: 3-11; Nakashima et al., 2002. J Neurochem 82: 202–206; Nakashima et al., 1999. J Neurochem 72: 2145– 53; Okuno and Fujisawa, 1991. J Neurochem 57: 53–60; Okuno and Fujisawa, 1985. J Biol Chem 260: 2633–2635; Ramsey and Fitzpatrick, 2000. Biochemistry 39: 773–778; Ramsey and Fitzpatrick, 1998. Biochemistry 37: 8980–8986; Ribeiro et al., 1992. Proc Natl Acad Sci USA 89: 9593–9597; Royo and Daubner, 2006. Biochim Biophys Acta - Proteins Proteomics 1764, 786–792; Sura et al., 2004. J Neurochem 90: 970–978; Tekin et al., 2014. J Neural Transmiss 121: 1451–1481). Dopamine interferes with tyrosine hydroxylase activity by binding almost irreversibly and with high affinity to the ferric iron in the catalytic domain of tyrosine hydroxylase, thereby competing with BH4. The result of this strict inhibitory control is that most tyrosine hydroxylase in dopaminergic neurons is in a low activity state, with low Ser40 phosphorylation stoichiometry (Salvatore et al., 2001. J Neurochem 79: 349–360, 2000; Salvatore et al., 2000. J Neurochem 75: 225–232). Therefore, the main regulatory mechanism to increase tyrosine hydroxylase activity is via Ser40 phosphorylation, as it allows catecholamines to dissociate and thereby lifting the inhibitory feedback by increasing the Ki for catecholamines (Ramsey and Fitzpatrick, 1998. Biochemistry 39: 773–778). Here we investigated the effects of exogenous dopamine on tyrosine hydroxylase phosphorylation. We exposed mouse striatal slices to 10 µM dopamine for either 30 minutes (Fig. 6A-B) or 60 minutes (Fig. 6C-D). Relative Ser40 phosphorylation levels are decreased by dopamine, after exposure to dopamine for both 30 minutes (Fig. 6B, left panel; t(10) = 3.97, p < 0.01, M = 0.76) and 60 minutes (Fig. 6D, left panel; t(8) = 3.78, p < 0.01, M = 0.64). There were no effects of dopamine on Ser31 phosphorylation, after both 30 minutes (Fig. 6B, right panel; t(10) = 0.26, p > 0.05, M = 1.01) and 60 minutes (Fig. 6D, right panel; t(9) = 1.14, p > 0.05, M = 0.95). Furthermore, we investigated the rigidity of the dopamine-induced decrease in Ser40 phosphorylation. To do so, we incubated mouse striatal slices with 10 µM dopamine for 30 minutes and subsequently exposed them to either 10 µM forskolin or DMSO vehicle for another 30 minutes (Fig.6E-G). Exposure to forskolin subsequently to dopamine incubation up-regulated Ser40 phosphorylation levels (Fig. 6G, left panel; t(10) = 5.01, p < 0.01, M = 1.94), while there was no effect on Ser31 phosphorylation (Fig. 6G, right panel; t(9) = 0.30, p > 0.05, M = 0.99). In conclusion, dopamine down-regulates Ser40 phosphorylation, while leaving Ser31 phosphorylation unaffected. However, the down-regulating effect of dopamine on Ser40 phosphorylation is not fixed as, subsequently, Ser40 phosphorylation can be up-regulated by activation of the cAMP second messenger system via forskolin. L-DOPA down-regulates Ser40 phosphorylation, but this effect can be rescued with forskolin As precursor to dopamine (Fig. 1A), L-DOPA is currently the gold standard treatment for Parkinson’s disease. The exogenously administered L-DOPA is synthesized to dopamine by AADC, which is present throughout the brain. Given our above described findings that dopamine down-regulates relative tyrosine hydroxylase Ser40 phosphorylation levels, we investigated the effects of L-DOPA on tyrosine hydroxylase phosphorylation as well. First of all, we exposed mouse striatal slices to 100 µM L-DOPA for 30 minutes (Fig. 7A-B). Relative Ser40 phosphorylation levels are significantly decreased after 30 minutes of L-DOPA incubation (Fig. 7B; t(9) = 5.18, p < 0.01, M = 0.66). Secondly, we incubated mouse striatal slices to 100 µM L-DOPA for 60 minutes (Fig. 7C-D). In this setup, relative Ser40 phosphorylation levels were again down-regulated (Fig. 7D, left panel; t(10) = 5.55, p < 0.01, M = 0.60). Here, we additionally quantified relative Ser31 phosphorylation levels, but found no effect of 60 minutes of L-DOPA exposure on Ser31 phosphorylation (Fig.7D, right panel; t(10) = 0.96, p > 0.05, M = 0.98). Next, we checked if the down-regulating effects of L-DOPA on Ser40 phosphorylation can be rescued with forskolin treatment (Fig. 7E-G). To do so, we incubated mouse striatal slices with either 100 μM L-DOPA (LD) or vehicle (VEH) for 30 minutes, followed by exposure to either 10 μM forskolin (FSK) or vehicle (Fig. 7E). Again, L-DOPA significantly decreased (Fig. 7G; t(4) = 6.27, p < 0.01, MVEH = 1.00, MLD = 0.61) and forskolin increases (Fig. 7G; t(4) = 7.13, p < 0.01, MVEH = 1.00, MFSK = 1.74) the relative levels of Ser40 phosphorylation. If we expose mouse striatal slices to forskolin subsequently to L-DOPA treatment, we can counteract and rescue the down-regulating effects of L-DOPA (Fig. 7G; t(4) = 5.68, p < 0.05, MLD = 0.61, MLD+FSK = 1.18). Thus, L-DOPA treatment down-regulates Ser40 phosphorylation. Therefore, exogenous L-DOPA administration leads to considerable consequences for the endogenous dopamine synthesis pathway. However, in a cyclic nucleotide- dependent manner Ser40 phosphorylation levels can be rescued and up-regulated once again, despite the previously initiated down-regulation via L-DOPA. In the Pitx3-deficiency mouse model for selective loss of nigrostriatal dopamine neurons, tyrosine hydroxylase levels are substantially lower but relative Ser40 phosphorylation levels are unaffected From a therapeutic perspective, we want to be able to manipulate Ser40 phosphorylation in a dopamine deficient setting similar to the situation of Parkinson’s disease patients. To mimic such a situation, we used the Pitx3- deficiency mouse model. Pitx3 is a transcription factor that is essential for normal substantia nigra dopamine neuron development (Ardayfio et al., 2008. Neurobiol Dis 31: 406–412; Smidt and Burbach, 2007. Nat Rev Neurosci 8: 21–32; Smidt et al., 2004. Development 131: 1145–1155; Smidt et al., 2004. Cell Tissue Res 318: 35– 43). Mice that are deficient in Pitx3 are used as model for the selective loss of nigrostriatal dopamine neurons and show L-DOPA-reversible impaired performance on select behavioral tests (Ardayfio et al., 2008. Neurobiol Dis 31: 406–412; Hwang et al., 2005. J Neurosci 25: 2132–2137). To examine the effects of Pitx3-deficiency on tyrosine hydroxylase levels and regulation in the striatum, we examined and compared tyrosine hydroxylase protein levels between phenotypically normal heterozygous Pitx3GFP/+ and defective homozygous Pitx3GFP/GFP mice. These mice either have a heterozygous (Pitx3GFP/+) or homozygous (Pitx3GFP/GFP) mutation of Pitx3 that carries a green fluorescent protein (GFP) under control of the endogenous Pitx3 promotor. The heterozygous mutation of Pitx3 does not affect nigrostriatal dopamine neuron development and these mice show similar tyrosine hydroxylase levels to Pitx3 wild- type littermates (Maxwell et al., 2005. Dev Biol 282: 467–479; Zhao et al., 2004. Eur J Neurosci 19: 1133–1140). Therefore, the Pitx3 heterozygous mice are used as controls for the homozygous Pitx3-deficient, nigrostriatal dopamine neuron deficient mice. To start, we want to inspect if some sort of compensation on the level of tyrosine hydroxylase phosphorylation develops in a dopamine deficient situation (Fig. 8A-D). To do so, we compared tyrosine hydroxylase levels of the phenotypically normal Pitx3GFP/+ mice and the defective Pitx3GFP/GFP mice. As expected, tyrosine hydroxylase levels are significantly lower in the striatum of the Pitx3GFP/GFP mice (Fig. 8B; t(3) = 5.85, p < 0.01, M = 0.19), developing tyrosine hydroxylase protein levels that are about 20 percent of the levels of the phenotypically normal Pitx3GFP/+ mice. There is no difference between the two genotypes, however, if we look at the relative striatal Ser40 phosphorylation levels (Fig. 8C; t(3) = 0.12, p > 0.05, M = 0.99). Interestingly, relative striatal Ser31 phosphorylation levels are lower in the Pitx3GFP/GFP mice (Fig. 8D; t(3) = 4.12, p < 0.05, M = 0.63). Next, we tested if there is still room to manipulate tyrosine hydroxylase phosphorylation – and thus if we can intervene – in a tyrosine hydroxylase- deficient situation (Fig. 8E-H). Striatal slices of both the phenotypically normal Pitx3GFP/+ mice and defective, tyrosine hydroxylase-deficient Pitx3GFP/GFP mice were exposed to 10 µM forskolin for 60 minutes (Fig.8E-F). After quantification, we find that for both the Pitx3GFP/+ mice (Fig. 8G, left panel; t(11) = 11.25, p < 0.01, MFSK = 2.32) and Pitx3GFP/GFP mice (Fig. 8G, right panel; t(11) = 23.51, p < 0.01, MFSK = 2.77) relative Ser40 phosphorylation levels are increased by forskolin and to a similar extent as we observed before in the C57/Bl6/J mice (Fig. 4C-D). Interestingly, if we look at relative Ser31 phosphorylation levels we find no effect of forskolin in Pitx3GFP/+ mice (Fig. 8H, left panel; t(11) = 0.63, p > 0.05, MFSK = 1.02), but, although the difference is small, we find a significant down- regulating effect in the Pitx3GFP/GFP mice (Fig. 8H, right panel; t(11) = 2.29, p < 0.05, MFSK = 0.95). In sum, the Pitx3-deficiency mouse model is used as a model for selective nigrostriatal dopamine neuron loss. In these mice, the homozygous mutation of Pitx3 is accompanied with substantial lower levels of tyrosine hydroxylase. Interestingly, the relative Ser40 phosphorylation levels are, however, no different than the phenotypically normal situation. This, while Ser31 phosphorylation levels are down-regulated. Despite the lower levels of tyrosine hydroxylase, relative Ser40 phosphorylation levels can still be up-regulated by forskolin, and to a similar extend as the phenotypically normal situation. We used and developed a novel ex vivo pharmacology approach using acute brain slices to investigate tyrosine hydroxylase phosphorylation. Phosphorylation of Ser40 is the main regulatory mechanism to increase the enzymatic activity of tyrosine hydroxylase, thereby boosting the synthesis of L-DOPA from L-tyrosine and thereby increasing dopaminergic biosynthesis. By manipulating cAMP second messenger signaling and phosphatase activity, we confirmed the influence of these well-described cell signaling routes that affect tyrosine hydroxylase phosphorylation. Furthermore, from a therapeutic perspective, we investigated the effects of exogenous dopamine and L-DOPA on tyrosine hydroxylase phosphorylation. Both dopamine and its precursor L-DOPA, which is used as gold standard treatment for Parkinson’s disease, have detrimental effects on the endogenous striatal dopamine synthesis pathway as they both down-regulate tyrosine hydroxylase Ser40 phosphorylation. Finally, we investigated tyrosine hydroxylase and its manipulation in a mouse model for selective loss of nigrostriatal dopamine neurons. Although the loss in dopaminergic neurons is accompanied by a proportional loss in tyrosine hydroxylase protein levels, relative Ser40 phosphorylation levels remain unchanged and are prone to manipulation via the cAMP second messenger signaling route. These findings highlight the therapeutic opportunities of tyrosine hydroxylase regulation in the treatment of striatal dopamine-deficiency, which is one of the main characteristics of Parkinson’s disease. In our setup, we confirmed the involvement of the cyclic nucleotide second messenger system in the phosphorylation of tyrosine hydroxylase. Both direct and indirect increases in cAMP levels resulted in an up-regulation of tyrosine hydroxylase Ser40 phosphorylation. Then, we investigated the effects of exogenous L-DOPA and dopamine on tyrosine hydroxylase phosphorylation. Especially L-DOPA is of great therapeutic relevance, as L-DOPA therapy is well-established as treatment for the motor deficits of patients with Parkinson’s disease. Our data shows, however, that exogenous increases in both L-DOPA and dopamine have repressing influence on the main activity regulating mechanism of the endogenous striatal dopamine biosynthesis pathway. Thereby, our data supports an earlier report that exogenous L-DOPA decreases tyrosine hydroxylase activity (Gordon et al., 2009. Neurochem Res 34: 1830–1837). Therefore, it could be speculated that long-term L-DOPA therapy leads to a continuous suppression of the endogenous dopamine biosynthesis pathway. Moreover, this would suggest that nigrostriatal dopaminergic terminals of patients that undergo L-DOPA therapy are depending on exogenous L-DOPA as the endogenous L-DOPA production is halted via down- regulation in Ser40 phosphorylation. We did show, however, that the down- regulating effects of L-DOPA on tyrosine hydroxylase Ser40 phosphorylation can be counteracted and rescued in a cAMP-dependent manner (Fig. 9), thereby providing a unique opportunity for therapeutic intervention. However, some studies suggest that there is already compensation for the extensive loss of dopaminergic neuron via increased dopamine synthesis (Perez et al., 2008. J Neurochem 105: 1861–1872; Salvatore, 2012. In: Mechanisms in Parkinson’s Disease - Models and Treatments. Dushanova (Ed), Intech. Chapter 10: 189-212; Snyder et al., 1990. J Pharmacol Exp Ther 253: 867–76; Zigmond et al., 1990. Trends Neurosci 13: 290–296). Therefore, we investigated when a substantial amount of nigrostriatal dopamine neurons is gone if such compensatory mechanisms can be explained via compensation on the level of tyrosine hydroxylase Ser40 phosphorylation. To do so, we used the Pitx3-deficiency mouse model (Ardayfio et al., 2008. Neurobiol Dis 31: 406–412; Hwang et al., 2005. J Neurosci 25: 2132–2137; Smidt and Burbach, 2007. Nat Rev Neurosci 8: 21–32; Smidt et al., 2004. Development 131: 1145–1155; Smidt et al., 2004. Cell Tissue Res 318: 35–43). Striatal tyrosine hydroxylase levels in the defective Pitx3GFP/GFP mice were at about 20 percent of the phenotypically normal Pitx3GFP/+ mice. Even though these mice have substantially lower levels of tyrosine hydroxylase, there is no compensation in relative tyrosine hydroxylase Ser40 phosphorylation levels. Moreover, Ser40 phosphorylation can be manipulated with forskolin in a similar manner as we have seen before. Interestingly, relative Ser31 phosphorylation levels were down-regulated in the defective Pitx3GFP/GFP mice. The function of Ser31 phosphorylation is, however, less clear as compared to Ser40 phosphorylation. Therefore, the function of this down-regulation in Ser31 phosphorylation remains elusive. Nevertheless, these data show that there are no major compensatory mechanisms that result in adjusted levels of relative tyrosine hydroxylase Ser40 phosphorylation and, importantly, repression of dopamine synthesis can be prevented as shown by the fact that Ser40 phosphorylation can be up-regulated in a cAMP-dependent manner, even in the presence of amounts of dopamine or levodopa that would otherwise repress endogenous dopamine production. Hence, these data show that tyrosine hydroxylase regulation via phosphorylation is an interesting therapeutic target to overcome striatal dopamine deficiency, even in a setting where a substantial proportion of dopaminergic neurons is lost. Especially with regard to our finding that exogenous L-DOPA down-regulates tyrosine hydroxylase Ser40 phosphorylation, it is of importance that the induction of tyrosine hydroxylase Ser40 phosphorylation can be achieved also when a substantial proportion of nigrostriatal dopamine neurons is lost. In fact, the progressive loss in nigrostriatal dopaminergic projections may actually be a factor to prevent the eventual durability of L-DOPA therapy. At first, when the striatal terminals are preserved (or largely preserved), the exogenous L-DOPA supports striatal dopamine release while excessive L-DOPA and dopamine can be buffered by pre-synaptic mechanisms (Carta and Bezard, 2011. Neuroscience 198: 245–251; Chang and Webster, 1995. Br J Pharmacol 116: 2637–2640; Misu et al., 1986. Neurosci Lett 72: 194–198). The dopamine transporter (DAT) and dopamine D2 auto-receptors (D2Rs) can prevent that post-synaptic dopamine receptors are excessively stimulated and maintain synaptic dopamine levels within a physiological range (Ford, 2014. Neuroscience 282: 13–22; Gainetdinov et al., 1998. Brain Res Rev 26: 148–153; Leviel, 2011. J Neurochem 118: 475–489; Liu and Kaeser, 2019. Curr Opin Neurobiol 57: 46–53). However, when the degradation of nigrostriatal dopamine neurons progresses, this buffering capacity and thus the ability to maintain physiological normal synaptic dopamine levels is progressively lost and thereby the ability to prevent overstimulation of postsynaptic dopamine receptors (Carta and Bezard, 2011. Neuroscience 198: 245–251; Cenci, 2014. Front Neurol 5: 1–15; Cenci, 2007. Trends Neurosci 30: 236–243; Cenci and Lundblad, 2006. J Neurochem 99: 381–392). Furthermore, prolonged exogenous L-DOPA exposure can have other adverse effects as well, in other brain regions and affecting other neurotransmitter systems (Fig. 1D). To illustrate, it is well-described that L-DOPA can induce off-target effects via the release of extrastriatal dopamine by non-dopaminergic AADC- containing neurons (Carta and Bezard, 2011. Neuroscience 198: 245–251; Cenci et al., 2011. CNS Neurol Disord - Drug Targets 10: 670–684; Cenci and Konradi, 2010. In: Björklund, A., Cenci, M.A. (Eds.), Progress in Brain Research. Elsevier B.V., pp. 209–233; Cenci, 2007. Trends Neurosci 30: 236–243; Cenci, 2014. Front Neurol 5: 1–15; Cenci and Lundblad, 2006. J Neurochem 99: 381–392; Chagraoui et al., 2020. Int J Mol Sci 21: 294; De Deurwaerdère et al., 2017. Prog Neurobiol 151: 57–100; Navailles et al., 2013. ACS Chem Neurosci 4: 680–692; Nutt and Holford, 1996. Ann Neurol 39: 561–573; Stansley and Yamamoto, 2015. Toxics 3: 75–88). These off-target effects will only get worse as both the dose and the number of L- DOPA doses per day are usually increased a few years after initiation of L-DOPA therapy (Cenci et al., 2011. CNS Neurol Disord - Drug Targets 10: 670–684; Nutt and Holford, 1996. Ann Neurol 39: 561–573). Thus, the progressive loss of dopaminergic neurons, the increasing dose(s) of progressive intermittent oral administration of L-DOPA, and the dysregulated release of dopamine from non- dopaminergic AADC-containing neurons will give rise to swings in extracellular levels of dopamine and leads to pulsatile stimulation of dopamine receptors. Altogether, we can conclude that the effects of L-DOPA are paradoxical: L-DOPA administration elevates striatal dopamine neurotransmission but on the other hand can lead to off-target effects, can desensitize the post-synaptic dopamine receptor-induced effects, and down-regulates the endogenous dopamine biosynthesis system. Hence, the present invention provides the use of the endogenous dopamine biosynthesis pathway to overcome the greatly reduced levels of dopamine in the nigrostriatal dopamine projections. By targeting dopamine neuron-specific upstream signaling pathway routes of tyrosine hydroxylase Ser40 phosphorylation, it is possible to boost the endogenous dopamine biosynthesis pathway. In this way the neurons are targeted where elevations in dopamine synthesis is wanted. This makes tyrosine hydroxylase Ser40 phosphorylation a suitable target in the treatment of Parkinson’s disease: either as a complement to L-DOPA therapy or as an alternative to completely circumvent the well-described off-target effects of exogenous L-DOPA. As a complement to L-DOPA therapy, the promotion of tyrosine hydroxylase Ser40 phosphorylation will allow lower L-DOPA dosage and dosages. This is because Ser40 phosphorylation re-activates endogenous dopamine production, which automatically allows lower L-DOPA dosages, as the L-DOPA concentration used is now on-top of the endogenous production. How much the L- DOPA dose can be lowered depends on the extent of Ser40 phosphorylation and the amount of dopamine neurons still present. This is typically reflected in the stage of the disease and the disease phenotype particular to the patient. Off target effects caused by L-DOPA treatment can be the result of dopamine production by cells that are not situated in the striatum (extrastriatal cells) and that are not normally producing dopamine. Such cells can, however, produce dopamine when L-DOPA when is provided to them. This extrastriatal off target dopamine production can seriously contribute to the side effects experienced by the Parkinson patient that is treated with L-DOPA. By removing the inhibition of the endogenous dopamine production the present invention insures, that both the off- target effects induced by extrastriatal dopamine production by non-dopaminergic AADC-containing neurons and the uncontrolled overstimulation of post-synaptic signaling routes is reduced, while the endogenous dopamine biosynthesis pathway is not completely shut down. Moreover, by invoking the endogenous biosynthesis route, important mechanisms that affect and regulate dopamine neurotransmission will not be circumvented allowing at least some form of natural regulation of dopamine levels. For example, the signaling circuitry that initiates dopamine synthesis or post-synthesis processes such as vesicular packaging. In such processes the subcellular distribution of tyrosine hydroxylase and other proteins involved in dopamine synthesis play an important part. As we identified the cAMP signaling pathway as one of the main routes that can be addressed to modulate tyrosine hydroxylase Ser40 phosphorylation in the striatum, we here provide the targeting of signaling pathways that can increase the concentrations of cyclic nucleotides in a way that is specific for the nigrostriatal dopaminergic terminals. We can either promote the synthesis of cyclic nucleotides or inhibit the degradation of cyclic nucleotides in a nigrostriatal dopamine neuron- specific way. This boosts the endogenous dopamine biosynthesis pathway in a nigrostriatal dopamine neuron-specific way as well. This can be achieved via the activation of adenylyl cyclases or guanylyl cyclases that can increase the production of cyclic nucleotides, or via the inhibition of phosphodiesterases (PDEs, Fig. 10) to inhibit the degradation of cyclic nucleotides. Boosting the endogenous dopamine synthesis pathway via tyrosine hydroxylase Ser40 phosphorylation can be a promising non-invasive target to overcome the locomotor deficits seen in Parkinson’s disease in an endogenous manner. L-DOPA down-regulates Ser40 phosphorylation, but this effect can be rescued with IBMX Next, we checked if the down-regulating effects of L-DOPA on Ser40 phosphorylation can be rescued by 3-isobutyl-1-methylXBnthine (IBMX), which is generally used as non-selective phosphodiesterase inhibitor (Fig. 11 A-C). To do so, we incubated mouse striatal slices with either 100 μM L-DOPA (LD) or vehicle (VEH) for 60 minutes and/or 100 μM IBMX or vehicle (Fig. 11A, 11B). Again, L- DOPA significantly decreased (Fig. 11C; MVEH = 1.00, MLD = 0.74) and IBMX increases (Fig. 11C; MVEH = 1.00, MIBMX = 2.71) the relative levels of Ser40 phosphorylation. If we expose mouse striatal slices to IBMX simultaneously to L- DOPA treatment, we can counteract and rescue the down-regulating effects of L- DOPA (Fig. 11C; MLD = 0.74, MLD+IBMX = 1.28). In a similar setup the type 2 phosphodiesterase inhibitor Bay 60-7550 was analyzed (Fig. 12) in order to establish whether this compound is able to attenuate L-DOPA induced inhibition of Ser-40 phosphorylation. After application of L-DOPA (100 µM) to mouse striatal slices the level of Ser-40 phosphorylation is lowered to 0.69 (p = 0.002/ p = 0.007 after Bonferroni correction). Application of Bay 60-7550 is able to restore this level to 1.08 (p = 0.013/ p = 0.04 after Bonferroni correction), whereas application of Bay 60-7550 to naive slices elevates the level of TH-40 Phosphorylation to1,6 (p = 0.003/ p = 0.008 after Bonferroni correction). This shows that the inhibition of Ser-40 phosphorylation by L-DOPA can be attenuated by inhibiting phosphodiesterase activity in dopaminergic terminals. We explored the effects of several effectors on tyrosine hydroxylase Ser40 phosphorylation when in the presence of phosphodiesterase 11A (PDE11A). To do so, we introduced either tyrosine hydroxylase and polyhistidine-tagged PDE11A4 or tyrosine hydroxylase only to Neuro2A cells, and exposed both conditions to the same effectors. Endogenously, Neuro2A cells neither contain detectable levels of tyrosine hydroxylase, nor PDE11A. In order to asses which isoform of PDE11A is causal related to the lowering of TH-40 Phosphorylation we transfected Neuro2A cells with an expression plasmid for TH and PDE11A1-A4 (transcript variant 1-4; geneID 50940) (Fig. 13). All transcript variants were able to lower the amount of TH-Ser40 phosphorylation, with the strongest effect from variant PDE11A4 (Fig. 13A). In order to be able to correct for expression levels of the transfected PDE11A variants we created a similar setup were we transfected HIS fusion proteins (His-6x) of PDE11A1-A4 (Fig. 13B). The corrected data indicated that PDE11A4 is still the most active variant and will be used in subsequent experiments. To investigate whether general mechanisms driving Ser40 phosphorylation are still functional in the presence of PDE11A, we tested the effects of 60 minutes exposure to the adenylyl cyclase activator forskolin and IBMX (Fig. 14). Without PDE11A4, both 10 µM forskolin (Fig. 14B; M = 2.22) and 100 µM IBMX (Fig. 14B; M = 1.49) induced elevations in Ser40 phosphorylation. The introduction of PDE11A4 down-regulated Ser40 phosphorylation (Fig. 14B; M = 0.66), but can be up-regulated by forskolin (Fig. 14B; M = 2.21) to a similar extent by forskolin without the presence of PDE11A4. Surprisingly, exposure to IBMX was not able to significantly alter the relative levels of Ser40 phosphorylation anymore (Fig. 14B; M = 0.52). In order to establish that whether we can attenuate the PDE11A4 induced lowering of TH-40 phosphorylation we treated cells transfected with TH and PDE11A4-HIS(6x) with Forskolin and the general PDE inhibitor IBMX (Fig. 14). Forskolin was able to attenuate the lower level of TH-40 phosphorylation, but IBMX was not (Fig. 14B). PDE5 should be one of the most expressed PDEs within Neuro2A cells. Given the sequence similarity between PDE11 and PDE5, several PDE5 inhibitors such as sildenafil (Viagra) and tadalafil (Cialis) cross-react with PDE11 (Cichero et al., 2013. Chem Biol Drug Design 82: 718-731). Therefore, we tested the influence of 60 minute exposure of these PDE5 inhibitors on tyrosine hydroxylase Ser40 phosphorylation, also in the presence of PDE11A4 (Fig. 15). In the absence of PDE11A4 10 µM sildenafil, surprisingly, down-regulated Ser40 phosphorylation (Fig. 15B,; M = 0.75) while 10 µM tadalafil had no effect (Fig. 15B; M = 1.13). When we additionally introduced PDE11A4, basal Ser40 phosphorylation levels were again down-regulated (Fig.15B; M = 0.54), but sildenafil had no significant effect on Ser40 phosphorylation anymore (Fig. 15B; M = 0.36). However, while in the absence of PDE11A4 it exerted no effect, tadalafil significantly increased tyrosine hydroxylase Ser40 phosphorylation levels in the presence of PDE11A4 (Fig. 15B; M = 0.88). To further explore the effects of tadalafil on tyrosine hydroxylase Ser40 phosphorylation, we performed a dose-response curve and exposed tyrosine hydroxylase-transfected Neuro2A cells to tadalafil at different concentrations (1, 3, 10, and 30 µM) for 60 minutes (Fig. 16). Firstly, the introduction of PDE11A4 down-regulated basal Ser40 phosphorylation (Fig. 16; M = 0.71). However, when exposed to tadalafil Ser40 phosphorylation levels were significantly upregulated at all concentrations (Fig. 16; M > 0.81). In conclusion, tyrosine hydroxylase Ser40 phosphorylation is regulated by PDE11A, as its introduction alone down-regulates phosphorylation levels. Forskolin can still overrule these effects as it elevates Ser40 phosphorylation levels to similar extent in the absence of PDE11A. The non-selective PDE inhibitor IBMX, however, lost its effectiveness in elevating Ser40 phosphorylation in the presence of PDE11A. Importantly, the PDE5 inhibitor tadalafil had no effect without the introduction PDE11A, but significantly increased Ser40 phosphorylation levels in the presence of PDE11A, indicating that selective inhibition of PDE11A4 is possible and enhances TH-Ser40 phosphorylation.

Claims

Claims 1. Dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in the treatment of dopamine deficiency in a subject, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is co-administered in combination with tadalafil or a variant thereof, characterized in that dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered at an effective reduced dose compared to the subject's standard therapeutic dose. 2. Dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use in the treatment of dopamine deficiency in a subject, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered in combination with tadalafil or a variant thereof, characterized in that the co-administration of tadalafil or a variant thereof enables a reduction in the dose of dopamine, a dopamine agonist, L- DOPA or a prodrug thereof, compared to the standard therapeutic dose required for treating the dopamine deficiency in the individual, while maintaining therapeutic efficacy and reducing the incidence or severity of side effects associated with dopamine, a dopamine agonist, L-DOPA or a prodrug thereof. 3. Dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use of claim 1 or claim 2, wherein the reduction of the dose is at least 10%, at least 20%, or preferably at least 50% compared to the patients standard therapeutic dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof. 4. Dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use according to any of claims 1-3, characterized in that the treatment of the dopamine deficiency does not comprise the administration of a calcimimetic that lowers parathyroid hormone (PTH) levels such as cinacalcet, baclofen, acamprosate, mexiletine, torasemide, or sulfisoxazole. 5. Dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use according to any of claims 1-4, wherein the administered dose of tadalafil or variant thereof is 5 mg/day, 10 mg/day or 20 mg/day (0.4 mg/kg). 6. Dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use according to any of claims 1-5, wherein the dopamine deficiency is a central nervous system dopamine deficiency. 7. Dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use of claims 1-6, wherein the disease associated with the dopamine deficiency in the individual is Parkinson's disease. 8. Dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for according to any of claims 1-7, wherein the dopamine, a dopamine agonist, L-DOPA or a prodrug and the tadalafil or variant thereof are administered for treating symptoms of Parkinson’s disease related to central nervous system dopamine deficiency. 9. Dopamine, a dopamine agonist, L-DOPA, or a prodrug thereof for use according to any of claims 1-8, wherein the effective reduced dose of dopamine, a dopamine agonist, L-DOPA, or a prodrug thereof is suboptimal for the individual when administered as a single treatment to the individual. 10. Dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use of claim 9, wherein the effective reduced dose is determined by titration in the patient. 11. Dopamine, a dopamine agonist, L-DOPA, or a prodrug thereof for use according to claim 10, wherein the effective reduced dose is between 10% and 75% of the standard therapeutic dose when administered as a single treatment to the individual. 12. Dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use according to any of claims 1-11, further comprising treatment of the disease or deficiency with a further medicament. 13. Dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use of claim 12, wherein the further medicament comprises a PDE11 inhibitor, a PDE2 inhibitor, a guanylate cyclase agonist; a dopa-decarboxylase (DDC) inhibitor; a monoamine oxidase (MAO-B) inhibitor; a catechol-O-methyltransferase (COMT) inhibitor; or a combination thereof. 14. Dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use of claim 13, wherein the PDE11 inhibitor is BC11-15; BC11-19; BC11-28; BC11-38; BC11- 38-1; BC11-38-2; BC11-38-3; or BC11-38-4. 15. Dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use of claim 13 or 14, wherein the PDE2 inhibitor is EHNA (erythro-9-(2-hydroxy-3- nonyl)adenine); BAY 60-7550 (2-[(3,4-dimethoxyphenyl)methyl]-7-[(2R,3R)-2- hydroxy-6-phenylhexan-3-yl]-5-methyl-1H-imidazo[5,1-f][1,2,4]triazin-4-one); PDP (9-(6-phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one; Hcyb1, PF- 051809999 (4-(1-azetidinyl)-7-methyl-5-[1-methyl-5-[5-(trifluoromethyl)-2- pyridinyl]-1H-pyrazol-4-yl]-imidazo[5,1-f][1,2,4]triazine); IC933, or oxindole (2,3- dihydroindol-2-on). 16. Dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use according to any of claims 13-15, wherein said guanylate cyclase agonist is a GUCY-2C agonist. 17. Dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use of claim 16, wherein the guanylate cyclase 2C receptor (GUCY2C) agonist is guanylin, lymphoguanylin, enterotoxin or uroguanylin. or a functional derivative of guanylin, lymphoguanylin, enterotoxin or uroguanylin. 18. Dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use according to any of claims 1-17, wherein the dopamine, a dopamine agonist, L- DOPA or a prodrug thereof and tadalafil are administered simultaneously. 19. Dopamine, a dopamine agonist, L-DOPA or a prodrug thereof for use according to any of 7-18, wherein Parkinson’s disease is Parkinson’s disease stage 1-5. 20. A method for treating dopamine deficiency in a subject, comprising: - administering to the subject a therapeutically effective dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof; and co-administering to the subject a therapeutically effective dose of tadalafil or a variant thereof; wherein the dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is administered at an effective reduced dose compared to the subjects standard therapeutic dose. 21. A method for treating dopamine deficiency in a subject, comprising: - administering to the subject a therapeutically effective dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof, wherein dopamine, a dopamine agonist, L-DOPA or a prodrug thereof is known to cause one or more side effects at standard therapeutic doses; and - co-administering to the subject a therapeutically effective dose of tadalafil or a variant thereof, wherein the co-administration of the tadalafil or a variant thereof enables a reduction in the dose of dopamine, a dopamine agonist, L-DOPA or a prodrug thereof compared to the standard therapeutic dose required for treating the dopamine deficiency in the individual, while maintaining therapeutic efficacy and reducing the incidence or severity of a side effect associated with dopamine, a dopamine agonist, L-DOPA or a prodrug thereof. 22. The method of claim 20 or claim 21, wherein the treatment does not comprise a calcimimetic that lowers parathyroid hormone (PTH) levels such as cinacalcet, baclofen, acamprosate, mexiletine, torasemide, or sulfisoxazole. 23. The method according to any of claims 20-22, characterized in that the administered dose of tadalafil or variant thereof is 5 mg/day, 10 mg/day or 20 mg/day (0.4 mg/kg). 24. The method according to any of claims 20-23, wherein the dopamine deficiency is a central nervous system dopamine deficiency. 25. The method according to any of claims 20-24, wherein the disease associated with dopamine deficiency in the individual is Parkinson's disease.
PCT/NL2024/050536 2023-10-02 2024-10-02 Reducing dopamine feed-back inhibition in dopaminergic cells by tadalafil. Pending WO2025075499A1 (en)

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