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WO2025224050A1 - Méthodes de traitement de patients souffrant d'hypomélanose de ito - Google Patents

Méthodes de traitement de patients souffrant d'hypomélanose de ito

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Publication number
WO2025224050A1
WO2025224050A1 PCT/EP2025/060846 EP2025060846W WO2025224050A1 WO 2025224050 A1 WO2025224050 A1 WO 2025224050A1 EP 2025060846 W EP2025060846 W EP 2025060846W WO 2025224050 A1 WO2025224050 A1 WO 2025224050A1
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WIPO (PCT)
Prior art keywords
hypomelanosis
ito
gene
rhoa
rock
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PCT/EP2025/060846
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English (en)
Inventor
Jérôme DELON
Rana EL MASRI
Paul KUENTZ
Pierre VABRES
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universite Bourgogne Europe
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Centre Hospitalier Universitaire de Bordeaux
Universite Paris Cite
Original Assignee
Universite Bourgogne Europe
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Centre Hospitalier Universitaire de Bordeaux
Universite Paris Cite
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Publication of WO2025224050A1 publication Critical patent/WO2025224050A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • 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
    • A61K31/01—Hydrocarbons
    • A61K31/015—Hydrocarbons carbocyclic
    • 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
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4155—1,2-Diazoles non condensed and containing further heterocyclic rings
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • 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
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders

Definitions

  • the present invention is in the field of medicine, in particular rare neurocutaneous disease.
  • Somatic mosaicism anomalies are rare diseases that manifest on the skin as linear hypopigmentation zones along the Blaschko lines and are classified using broad, undefined terms such as "Hypomelanosis of Ito" 1 .
  • orphan diseases i.e. those with no known therapeutic resources to date
  • the genetic origins of these rare diseases remain unclear, hindering diagnosis and patient care.
  • the study of these genetic bases has only recently become possible, thanks to the development of high throughput sequencing. This approach led us to the discovery of mutations in the MTOR and RHOA genes that cause the "Hypomelanosis of Ito" syndrome 2,3 .
  • Gal3 is a subunit of a heterotrimeric G protein (aPy) coupled to specific transmembrane receptors known as G-protein coupled receptors (GPCR) 4,5 .
  • GPCR G-protein coupled receptors
  • the Gal3 subunit loads with GTP in exchange for GDP, causing it to change conformation and dissociate from both the GPCR and the P and y subunits.
  • the Gal3-GTP active form can then interact with downstream effectors and kinases to regulate a variety of essential cellular functions such as cytoskeletal modifications, gene transcription, cell migration, and cell cycle division.
  • the signalling process ends with the hydrolysis of GTP, which is mediated by the intrinsic GTPase activity of the Ga subunit.
  • the inventors have identified the functional consequences of this mutation and propose a molecular and cellular mechanism by which the clinical characteristics of those patients manifest and pave the path for new therapeutic approaches.
  • the present invention is defined by the claims.
  • the present invention relates to methods of treatment of patients suffering from hypomelanosis of Ito, and in particular suffering from hypomelanosis of Ito caused by a mutation in a gene coding for a G protein alpha subunit.
  • Hypomelanosis of Ito is a clinical term for patients with mosaic syndromes characterized by skin hypopigmentation and developmental disorders. The genetic causes of these rare diseases remain largely unclear.
  • GNA13 is a new gene that causes Hypomelanosis of Ito. They identified an identical mutation in this gene in four unrelated patients exhibiting pigmentary mosaicism. In depth functional investigations revealed that this is an activatory mutation that alters the cytoskeleton and morphology of melanocytes via a hyperactivation of the RHOA/ROCK signalling pathway.
  • the invention refers to a method for treating hypomelanosis of Ito in a subject in need thereof comprising administering a therapeutically effective amount of a ROCK inhibitor and/or RHOA inhibitor.
  • the term “subject” or “patient” refers to any mammal, such as a rodent, a feline, a canine, and a primate.
  • the subject is a human.
  • the subject according to the invention has or is susceptible to have hypomelanosis of Ito, and particularly an hypomelanosis of Ito caused by at least one mutation in a gene coding for a G-alpha subunits, and more particularly an hypomelanosis of Ito caused by at least one mutation in the gene coding for the G subunits alpha 13 (GNA13 gene).
  • the subject according to the invention has or is susceptible to have a skin linear hypopigmentation along Blaschko lines, asymmetric facial dysmorphism, limb asymmetry and malformation, wound healing issues, teeth and ocular anomalies, gastroenterological and nephrological abnormalities, and neurological defects due to malformative hydrocephalus.
  • HI Hypomelanosis of Ito
  • pigmentary mosaicism refers to a group of very rare neurocutaneous orphan-disease that causes unusual patches of light-colored (hypopigmented) skin and may be associated with eye, nervous system, and skeletal problems. Hypomelanosis of Ito can be caused by mutations in the MTOR and RHOA genes as previously disclosed 2 ' 3 .
  • the inventors discovered a novel mutation in the GNA13 gene that encodes for Gal 3 in four unrelated patients with Hypomelanosis of Ito who all have cutaneous and developmental anomalies but no major neurological disorders.
  • hypomelanosis of Ito is caused by at least one mutation in a gene coding for a G alpha subunit.
  • G alpha subunits has its general meaning in the art and refers to one of the three types of subunit of guanine nucleotide binding proteins, which are membrane-associated heterotrimeric G proteins.
  • Guanine nucleotide binding proteins are membrane-associated, heterotrimeric proteins composed of three subunits: alpha, beta and gamma (IPR001770).
  • G proteins act as signal transducers, relaying a signal from a ligand-activated GPCR (G protein-coupled receptor) to an enzyme or ion channel effector.
  • the heterotrimeric G protein alpha subunit is composed of two domains: a GTP -binding domain and a helical insertion domain.
  • the GTP -binding domain is homologous to Ras-like small GTPases, and includes switch regions I and II, which change conformation during activation. There are several isoforms of G protein alpha subunit, many of which have splice variants.
  • G alpha subunits includes but are not limited to : Gs alpha subunit (or Gas) which is encoded by the gene GNAS gene (Its Entrez reference is 2778, and its Uniprot reference is P63092); G12 alpha subunits (or Gal2) which is encoded by the gene GNA12 gene (Its Entrez reference is 2768 and its Uniprot reference is Q03113); G13 alpha subunits (or Gal3) which is encoded by the gene GNA13 gene (Its Entrez reference is 10672 and its Uniprot reference is Q14344); G q alpha subunits (also known as Gaq) which is encoded by the gene GNAQ gene (Its Entrez reference is 2776 and its Uniprot reference is P50148); Gn alpha subunits (also known as Gal 1) which is encoded by the gene GNA11 gene (Its Entrez reference is 2767 and its Uniprot reference is P29992); G14 alpha subunits (also known as Gal4) which is encoded by
  • the hypomelanosis of Ito is caused by at least one mutation in a gene coding a G alpha subunit, said mutation causes a substitution of an arginine residue (R) by another amino acid residue in the Gal3, Gas, Gaq and/or Gal 1 subunits.
  • the Gal 3 subunits comprises or consists of the amino acid sequence of SEQ ID NO: 1
  • the Gas subunits comprises or consists of the amino acid sequence of SEQ ID NO: 2
  • the Gaq subunits comprises or consists of the amino acid sequence of SEQ ID NO: 3
  • the Gal l subunits comprises or consists of the amino acid sequence of SEQ ID NO: 4
  • hypomelanosis of Ito is caused by at least one mutation in the Gal3, Gas, Gaq and/or Gal 1 subunits.
  • the at least one mutation consists of a substitution of an arginine residue (R) by another amino acid residue, such as a lysine residue (K), a cysteine residue (C), a histidine residue (H), a glutamine residue (Q).
  • R an arginine residue
  • K a lysine residue
  • C cysteine residue
  • H histidine residue
  • Q glutamine residue
  • hypomelanosis of Ito is caused by a substitution of an arginine residue (R) in a G alpha subunit selected from the group consisting of Gal 3 subunit, Gaq subunit, Gal 1 subunit or Gas subunit.
  • hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 200 in the Gal 3 subunit.
  • hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 200 by a lysine residue (K) in the Gal 3 subunit.
  • hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 200 in the SEQ ID NO: 1.
  • hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 200 by a lysine residue (K) in the SEQ ID: 1.
  • the inventors identify the mutation R200K in the Gal 3 subunit in four unrelated patients exhibiting pigmentary mosaicism.
  • This hypomelanosis of Ito is characterized by a skin linear hypopigmentation along Blaschko lines, asymmetric facial dysmorphism, limb asymmetry and malformation, wound healing issues, teeth and ocular anomalies, gastroenterological and nephrological abnormalities, and neurological defects due to malformative hydrocephalus
  • hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 183 in the Gaq and/or Gal l subunits.
  • hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 183 in the SEQ ID NO:3 and/or SEQ ID NO:4.
  • the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 183 by a glutamine residue (Q) in the Gaq and/or Gal l subunits.
  • the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 183 by a glutamine residue (Q) in the SEQ ID NO:3 and/or SEQ ID NO:4.
  • the hypomelanosis of Ito is associated with a Sturge-Weber syndrome.
  • Sturge-Weber syndrome is a rare congenital neurological and skin disorder. It is often associated with port-wine stains of the face, glaucoma, seizures, intellectual disability, and ipsilateral leptomeningeal angioma (cerebral malformations and tumors). It is a mosaic disease arising from somatic activating mutations in GNAQ, which encodes the G protein subunit alpha q or from somatic activating mutations in GNA17, which encodes the G protein subunit alpha 11.
  • the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 201 in the Gas subunit.
  • the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 201 by a cysteine residue (C) or a histidine residue (H) in the Gas subunit.
  • the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 201 in SEQ ID NO:2.
  • the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 201 by a cysteine residue (C) or a histidine residue (H) in SEQ ID NO:2.
  • the hypomelanosis of Ito is associated with a McCune- Albright syndrome.
  • McCune-Albright syndrome is a rare and complex genetic disorder, with estimated prevalence between 1/100,000 and 1/1,000,000, affecting the bone, skin and endocrine systems. It is a mosaic disease arising from somatic activating mutations in GNAS, which encodes the alpha-subunit of the Gs heterotrimeric G protein.
  • treating refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
  • Rho-associated kinase also known as or “Rho-kinase” or “ROCK” or “ROK” has its general meaning in the art and refers to an effector of the small GTPases RhoA, RhoB and RhoC, and belongs to the serine-threonine protein kinase family.
  • Rho kinases which were the first downstream effectors of RhoA to be discovered were found to mediate RhoA-induced actin cytoskeletal changes through effects on myosin light chain phosphorylation.
  • Rho-kinase has pleiotropic functions including the regulation of cellular contraction, motility, morphology, polarity, cell division, and gene expression.
  • ROCK contains several domains including a kinase; a coiled-coil region which contains a RhoA- binding domain and a pleckstrin homology and cysteine-rich domain at the C-terminal.
  • ROCK exists in two isoforms, ROCK1 and ROCK2 (T. Ishizaki et al, EMBO J., 1996, 15, 1885-1893).
  • ROCK has been identified as an effector molecule of RhoA, a small GTP -binding protein (G protein) that plays a key role in multiple cellular signaling pathways.
  • ROCK inhibitor refers to a natural or synthetic compound capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with the activities of ROCK including, for example, reduction or blocking the interaction between ROCK (i.e ROCK1 and/or ROCK2) and RhoA.
  • the inhibitor is selective.
  • the selective ROCK inhibiting compounds are not limited to a particular manner of selective ROCK inhibition.
  • one or more of the selective ROCK inhibiting compounds selectively inhibit ROCK1 activity over ROCK2 activity.
  • one or more of the selective ROCK inhibiting compounds selectively inhibit ROCK2 activity over ROCK1 activity.
  • one or more of the selective ROCK inhibiting compounds selectively inhibit both ROCK1 activity and ROCK2 activity with similar capability.
  • biological activity of ROCK is meant regulating actin organization by phosphorylation and activation of LIM kinase and/or myosin light chains (MLC) as well as regulating cell migration by promoting cellular contraction.
  • the inhibitor specifically binds to ROCK (i.e. ROCK1 and/or ROCK2) in a sufficient manner to inhibit the biological activity of ROCK, i.e. the ROCK pathway. Binding to ROCK and inhibition of the biological activity of ROCK may be determined by any competing assays well known in the art.
  • the assay may consist in determining the ability of the agent to be tested as ROCK inhibitor to bind to ROCK. The binding ability is reflected by the Kd measurement.
  • Kd is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e.
  • Kd/Ka Kd/Ka and is expressed as a molar concentration (M).
  • Kd values for binding biomolecules can be determined using methods well established in the art.
  • an inhibitor that "specifically binds to ROCK is intended to refer to an inhibitor that binds to human R0CK1 and/or ROCK2 with a Kd of IpM or less, lOOnM or less, lOnM or less, or 3nM or less. Then a competitive assay may be settled to determine the ability of the agent to inhibit biological activity of ROCK.
  • the functional assays may be envisaged such as evaluating the ability to inhibit a) F-actin polymerization and/or b) MLC phosphorylation and/or c) reverse the morphological effects (cell shape, motility) induced by Gal 3 R200K mutant as described in example (see Figure 4).
  • the ROCK inhibitor may be a compound selected from the group consisting of nucleic acid (e.g., a short interfering ribonucleic acid (siRNA)), antibodies, aptamers, polypeptides (including peptide or peptidometics) and small molecules.
  • nucleic acid e.g., a short interfering ribonucleic acid (siRNA)
  • siRNA short interfering ribonucleic acid
  • aptamers e.g., a short interfering ribonucleic acid (siRNA)
  • polypeptides including peptide or peptidometics
  • the ROCK inhibitor is a small organic molecule.
  • small organic molecule refers to a molecule of size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g.; proteins, nucleic acids, etc.); preferred small organic molecules range in size up to 2000 Da, and most preferably up to about 1000 Da.
  • ROCK inhibitors include but are not limited to; H-1152; AT-13148; 0- Elemene; Chroman 1; DJ4; GSK-576371; GSK429286A; LX-7101; RKI-1447; TCS-7001; isoquinoline derivatives such as Fasudil, Hydroxyfasudil, Belumosudil, Ripasudil, Verosudil, Netarsudil, Thiazovivin, Y-27632, Y-30141, Y-33075, Y-39983 and their derivatives.
  • Fasudil (hexahydro-l-(5-isoquinolylsulfonyl)-lH-l,4-di-azepime), also named as HA- 1077, is an isoquinoline sulfonamide derivative and a clinically available ROCK inhibitor codeveloped by Asahi Kasei of Japan and Department of Pharmacology of Nagoya University. Its Cas Number is 103745-39-7. A series of fasudil analogs were synthesized and their selectivity and inhibitory activity against ROCK were evaluated 10 ' 17 .
  • Thiazovivin is a drug which acts as a potent and selective ROCK inhibitor. Its CAS Number is 1226056-71-8.
  • Rhosin is a cell-permeable compound that directly targets Rho GEF binding domain. Its CAS Number is 1173671-63-0.
  • Hydroxyfasudil (l-(l-Hydroxy-5-isoquinolinesulfonyl)homopiperazine hydrochloride hydrate) is an active metabolite of Fasudil in vivo, which has higher affinity to ROCK than Fasudil. Its Cas Number is 155558-32-0. Belumosudil, also known as SLx-2119 binds to and inhibits the serine/threonine kinase activity of ROCK2 and is indicated for the treatment of chronic graft-versus host disease (chronic GvHD). Its CAS Number is 911417-87-3.
  • Ripasudil also known as K-l 15 is a derivative of fasudil which is used for the treatment of glaucoma and ocular hypertension. Its CAS Number is 223645-67-8.
  • Verosudil also known as AR-12286 is another potent and selective ROCK Inhibitor investigated for the treatment of glaucoma. Its CAS Number is 1414854-42-4.
  • Netarsudil also known as AR-13324 is another potent and selective ROCK Inhibitor clinically available for the treatment of glaucoma. Its CAS Number is 254032-66-0.
  • H-l 152 (4-methyl-5-[[(2S)-2-methyl-l,4-diazepan-l-yl]sulfonyl]isoquinoline), is another isoquinoline derivative which has been optimized on the basis of fasudil. Its CAS Number is 451462-58-1.
  • Y-27632 ((+)-(R)-trans-4-(l -Aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide) is another type of ROCK inhibitor which inhibits both ROCK1 and ROCK2 through competitively binding to the ATP binding pocket. Its CAS Number is 146986-50-7.
  • AT13148 is a novel oral ROCK inhibitor which has been described in Rath N et al. 18
  • Chroman l is a highly potent and selective ROCK inhibitor which is more potent against ROCK2 than ROCK1. Its Cas number is 1273579-40-0.
  • DJ4 also known as EX-A7863 ((2E,5Z)-5-((3a,7a-dihydro-lH-pyrrolo[2,3-b]pyridin- 3-yl)methylene)-2-(phenethylimino)thiazolidin-4-one), is a selective multi-specific ATP competitive inhibitor of activity of ROCK 1, ROCK2, MRCKa and MRCKP kinases.
  • GSK-576371 is a selective ROCK inhibitor which has been described in Phrommintikul A. et al 19 .
  • GSK429286A N-(6-fluoro-lH-indazol-5-yl)-6-methyl-2-oxo-4-[4-
  • the ROCK inhibitor is Fasudil or Y-27632.
  • ROCK inhibitors include those described in the international patent publications WO98/06433, WO00/09162, WO00/78351, WOOl/17562, WO02/076976, EP1256574, W002/100833, W003/082808, W02004/009555, W02004/024717, W02004/108724, W02005/003101, W020Q5/035501, W02005/035503, W02005/035506, W02005/058891 , W02005/074642, W02005/074643, W02005/Q80934, W02005/082367, W02005/082890, W02005/097790, W02005/100342, W02005/103050, W02005/105780, W02005/108397, W02006/044753, W02006/051311, W02006/057270, W02006/058120 , W02006/072792, WO201 1107608 Al, W0
  • the ROCK inhibitor of the invention is a peptide or a peptidometic.
  • peptidomimetic refers to a small protein-like chain designed to mimic a peptide.
  • the ROCK inhibitor of the invention is an aptamer.
  • “Aptamers” are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999.
  • Peptide aptamers consist of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two-hybrid methods (Colas et al., 1996). Then, after raising aptamers directed against ROCK as described above, the skilled man in the art can easily select those inhibiting ROCK.
  • a platform protein such as E. coli Thioredoxin A
  • the ROCK inhibitor of the invention is an antibody (the term including “antibody portion”).
  • the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody.
  • the portion of the antibody comprises a F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody.
  • antibody includes both naturally occurring and non-naturally occurring antibodies. Specifically, “antibody” includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, “antibody” includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.
  • Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with antigenic forms of ROCK. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization.
  • Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides.
  • Other suitable adjuvants are well-known in the field.
  • the animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.
  • the antigen may be provided as synthetic peptides corresponding to antigenic regions of interest in ROCK.
  • lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hybridoma.
  • cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996).
  • cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen.
  • Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.
  • an antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule.
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
  • the Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
  • CDRs complementarity determining regions
  • FRs framework regions
  • CDR1 through CDR3 complementarity determining regions
  • the second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected.
  • the third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected.
  • the fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3 A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs.
  • the above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies.
  • humanized forms of the antibodies some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen.
  • Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules.
  • a "humanized" antibody retains a similar antigenic specificity as the original antibody.
  • Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference.
  • mice have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies.
  • the animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest.
  • monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.
  • KAMA human anti-mouse antibody
  • the present invention also provides for F(ab') 2, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences.
  • the present invention also includes so-called single chain antibodies.
  • the various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM.
  • IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4.
  • the antibody according to the invention is a single domain antibody.
  • the term “single domain antibody” (sdAb) or “VHH” refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.
  • VHH refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3.
  • CDRs complementarity determining region
  • CDR complementarity determining region
  • VHHs can readily be prepared by an ordinarily skilled artisan using routine experimentation.
  • the VHH variants and modified form thereof may be produced under any known technique in the art such as in vitro maturation.
  • VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2.
  • Antigen-specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells.
  • VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations.
  • the high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals.
  • the affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations).
  • VHHs derived from camelid are readily expressed in and purified from the E.
  • VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells.
  • the “Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695).
  • the “Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E.
  • coli see for example US 6,765,087 and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).
  • moulds for example Aspergillus or Trichoderma
  • yeast for example Saccharomyces, Kluyveromyces, Hansenula or Pichia
  • Anti-sense oligonucleotides including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of mRNA of interest (here ROCK or RHOA mRNA) by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of proteins of interest (i.e. ROCK1, ROCK2 or RHOA), and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding the protein of interest can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion.
  • biological activity of RHOA is meant regulating actin organization by phosphorylation and activation of LIM kinase and/or myosin light chains (MLC) as well as regulating cell migration by promoting cellular contraction.
  • the inhibitor specifically binds to RHOA in a sufficient manner to inhibit the biological activity of RHOA, i.e. the RHOA/ROCJ pathway. Binding to RHOA and inhibition of the biological activity of RHOA may be determined by any competing assays well known in the art.
  • the assay may consist in determining the ability of the agent to be tested as RHOA inhibitor to bind to RHOA. Then a competitive assay may be settled to determine the ability of the agent to inhibit biological activity of RHOA.
  • the functional assays may be envisaged such as evaluating the ability to inhibit a) F-actin polymerization and/or b) MLC phosphorylation and/or c) reverse the morphological effects (cell shape, motility) induced by Gal 3 R200K mutant as described in example (see Figure 4 or 5).
  • the RHOA inhibitor may be a compound selected from the group consisting of nucleic acid (e.g., antisense oligonucleotide, siRNA or shRNA), antibodies, aptamers, polypeptides (including peptide or peptidomimetic) and small molecules.
  • the RHOA inhibitor is a small organic molecule.
  • RHOA inhibitors include but are not limited to Rhosin, G04, CT04, CCG- 1423 and ScafflO-8.
  • G04 is an active metabolite of Rhosin (D-Tryptophan (2E)-2-(6- quinoxalinylmethylene)hydrazide hydrochloride). Its Cas Number is 1281870-42-5.
  • RhoA Scaffl0-8 bound to RhoA, inhibits the AKAP-Lbc-mediated RhoA activation. Its Cas Number is 777857-56-4.
  • the ROCK inhibitor and/or RHOA inhibitor of the present invention is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.
  • pharmaceutically acceptable excipients such as a carboxylate, a carboxylate, a carboxylate, a carboxylate, a carboxylate, a carboxylate, a carboxylate, a carboxylate, sulfate, a pharmaceutically acceptable.
  • pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a ROCK inhibitor and/or RHOA inhibitor according to the invention for use in the treatment of hypomelanosis of Ito.
  • hypomelanosis of Ito is associated with at least one mutation in a gene coding for a G alpha subunit.
  • hypomelanosis of Ito is associated by at least one mutation in the Gal3, Gas, Gaq and/or Gal 1 subunits.
  • the ROCK inhibitor is Y-27632 or Fasudil.
  • FIGURE
  • Figure 1 Effect of R200K mutation identified in patients on actin organization and cell morphology. Quantification of F-actin content (A.) and cell morphology parameters including perimeter (B.) circularity (C.) and solidity (D.) of Gal 3 WT-YFP, Gal 3 R200K-YFP and Gal 3 Q226L-YFP (an artificial mutant generated to mimic a constitutively active form of Gal3, used here as a positive control) overexpressed in B16-F0 cells. The data are represented as the mean +/- SEM. **: P ⁇ 0.01, ***: P ⁇ 0.001, ****: p ⁇ 0.0001.
  • FIG. 1 Effect of R200K mutation identified in patients on cytoskeletal proteins.
  • B16-F0 cells expressing Gal 3 WT-YFP, Gal 3 R200K-YFP and Gal 3 Q226L-YFP were fixed and labelled with anti-pMLC or anti-vinculin antibodies, and Hoechst for nuclei 48 h after being transfected.
  • Figure 5 Effect of Fasudil, another ROCK inhibitor, on pMLC, actin content and cell morphology.
  • B16-F0 cells expressing Gal3 WT-YFP, Gal3 R200K-YFP or Gal3 Q226L-YFP were stimulated or not by MSH (Melanocyte Stimulating Hormone) for 48 hours.
  • MSH Melanocyte Stimulating Hormone
  • This example includes four unrelated affected children and their unaffected parents. Individuals were phenotyped and recruited by dermatologists and geneticists in Dijon and other French cities thanks to a nationwide collaborative effort to identify genes involved in skin mosaic syndromes.
  • index case sequencing was performed as a skin/blood pair.
  • Melanoma (B16-F0 or SK-mel28) and keratinocyte (HaCaT) cell lines were grown in DMEM (Thermo Fisher Scientific) with 10% FCS. Seventy percent confluent cultures were transfected according to the manufacturer’s protocol with the lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) and cultured for 48 h including an overnight cell starvation step. The cells were then fixed, labeled and examined under a fluorescence microscope.
  • RHOA and ROCK inhibition were performed in serum-free medium incubation for 4 hours and overnight using Rho Inhibitor I (CT04, Cytoskeleton Inc.) and Y27632 (Calbiochem) at final concentrations of 2 pg/ml and 5 pM, respectively.
  • HaCaT cells were seeded at 2.5 xl05 cells/well in six-well plates. The next day, B16-F0 cells were added to each well containing the keratinocytes at a keratinocytes to melanocytes seeding ratio of 2.5: 1. Following transfection, a serum-free medium containing 100 nM MSH was added and left for two days. Cultures were then fixed and labelled for immunofluorescence microscopy analysis.
  • Melanocyte Stimulating Hormone (MSH, Merck) was administered in serum-free medium for 48 hours following transfection at a final concentration of 100 nM.
  • MSH Melanocyte Stimulating Hormone
  • cells were harvested with Trypsin/EDTA and the amount of melanin was quantified.
  • a comparable number of cells were lysed with 100 pl 1 N NaOH, 10% DMSO, heated at 80 °C for lh30, and vortexed repeatedly to homogenize.
  • Cell extracts were placed in 96-well plates in duplicate. The relative melanin content was determined by measuring absorbance at 490 nm with a Clariostar reader, and a wide range of pure synthetic melanin (Merck).
  • Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 10 min at room temperature (RT) and permeabilized with 0.1% Saponin (Fluka), 0.2% BSA (Sigma) for 15 min at RT. The latter buffer is used as a washing buffer all along the labeling.
  • Cells were incubated with primary antibodies anti-pMLC (Cell Signaling Technology), anti- vinculin (Life Technologies) or anti-RHOA-GTP (CurieCoreTech) for Ih at RT.
  • the primary antibodies used were anti-TRPl and anti-cytokeratin (abeam).
  • Lentivirus packaging was performed in HEK293T cells by co-transfection of the lentiviral plasmid encoding Gal3-pLVX with the packaging plasmids pVSVG, p8.9 and pREV. The media was changed the next day, and collected after 24 hours for centrifugation to collect the lentiviruses.
  • Sk-mel28 cells were seeded in flat bottom 96-well plates (Corning, Falcon, 80 000 cells/well) and incubated with the lentiviruses for 2 days until confluence.
  • the Essen Bioscience WoundMaker was used to create scratch-wounds of a standardized width (-600 pm) on the cell monolayer. Cells were tracked using the automated live-cell Essen IncuCyte Zoom live-cell microscopy system, taking an image every two hours. The Incucyte S3 software was used for image analysis and wound confluence determination.
  • the mutated Arg200 is highly conserved
  • Arg200 that is mutated into a Lys in the patients we study here, is found in the switch I region (data not shown), which is a flexible region that can bind to GTP or GDP, and change its conformation depending on the protein's activation state, allowing Gal3 to interact with various downstream signalling effectors.
  • Gal 3 R200K variant alters cellular morphology and increases actin polymerisation
  • Non-muscle myosins are proteins that interact with actin to control cell morphology.
  • pMLC myosin light chains
  • vinculin which is a component of focal adhesions that links the cytoskeleton to extracellular matrix proteins.
  • Vinculin staining and quantification of the vinculin dots revealed that cells expressing the Gal 3 R200K mutant have fewer focal adhesions and are more likely to be localized at the cell periphery than cells expressing the WT form (Fig. 2B).
  • ROCK1 and ROCK2 are RHOA effectors that have been shown to phosphorylate MLC.
  • the inhibition of both RHOA and ROCK had no effect on the F-actin content of cells expressing WT Gal3, but it largely blocked the increased F-actin polymerization caused by the Gal3 mutants (Fig. 3D, 3F).
  • Gal 3 R200K inhibits melanosomes transfer to keratinocytes
  • the skin pigmentation process occurs through two important steps: (i) the production of melanin by the melanocytes in vesicles called melanosomes which undergo maturation steps, and (ii) the transfer of melanosomes from melanocytes to keratinocytes. Because the latter step is dependent on the cytoskeleton and the morphology of the melanocytes, we hypothesized that the R200K-induced changes in melanocytes shape could thus disrupt the transfer of melanosomes to keratinocytes.
  • Melanin Stimulating Hormone (MSH) is a hormone that induces the maturation of melanosomes and their transfer to keratinocytes through some morphological changes in the melanocytes.
  • GNA13 as a new gene at the root of dermal mosaic syndromes, could reveal common pathogenic pathways, improving our understanding of their causes and, ultimately, opening up new therapeutic avenues such as the use of RHOA or ROCK inhibition that restored some of the cell alterations caused by the mutation, specifically the cell morphology and cytoskeleton.

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Abstract

L'hypomélanose d'Ito est un terme clinique désignant les patients atteints de syndromes mosaïques caractérisés par une hypopigmentation cutanée et des troubles du développement. Les causes génétiques de ces maladies rares restent en grande partie inconnues. Nous décrivons dans la description que GNA13 est un nouveau gène qui provoque l'hypomélanose de Ito. Nous avons identifié une mutation identique dans ce gène chez quatre patients sans liens de parenté présentant un mosaïcisme pigmentaire. Des études fonctionnelles approfondies ont révélé qu'il s'agit d'une mutation activatrice qui modifie le cytosquelette et la morphologie de mélanocytes via une hyperactivation de la voie de signalisation RHOA/ROCK. Nos résultats indiquent en outre que cette pathologie n'est pas nécessairement causée par une production réduite de mélanine, mais peut être due à un défaut dans le transfert de mélanosomes vers les kératinocytes en raison d'altérations de forme cellulaire. Par conséquent, nos résultats suggèrent pour la première fois un mécanisme par lequel les symptômes cliniques de patients atteints de l'hypomélanose de Ito, et ouvrent la voie à de nouvelles approches thérapeutiques. Globalement, la présente invention concerne une méthode de traitement d'un patient atteint d'hypomélanose d'Ito par administration d'un inhibiteur de ROCK et/ou d'un inhibiteur de RHOA.
PCT/EP2025/060846 2024-04-22 2025-04-22 Méthodes de traitement de patients souffrant d'hypomélanose de ito Pending WO2025224050A1 (fr)

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