EP1148888A2

EP1148888A2 - Treatment of erectile dysfunctions with a c-type natriuretic polypeptide (cnp) as monotherapy or in combination with phosphodiesterase inhibitors

Info

Publication number: EP1148888A2
Application number: EP00909112A
Authority: EP
Inventors: Wolf-Georg Prof. Dr. Forssmann; Hans-Jürgen MÄGERT; Christian Dr. Stief; Andrea Küthe; Stefan Ückert; Armin Becker; Udo Jonas
Original assignee: FORSSMANN WOLF GEORG PROF DR; MAEGERT HANS JUERGEN
Current assignee: FORSSMANN WOLF GEORG PROF DR; MAEGERT HANS JUERGEN
Priority date: 1999-01-27
Filing date: 2000-01-27
Publication date: 2001-10-31
Also published as: CA2361239A1; US20020128176A1; WO2000044399A2; DE19903087A1; WO2000044399A3; JP2002536299A; AU3150800A

Abstract

The invention relates to medicaments containing a combination of CNP or fragments or derivatives with obtained physiological action of the CNP and phosphodiesterase inhibitors.

Description

Treatment of erectile dysfunctions with C-type natriuretic polypeptide (CNP) as monotherapy or in combination with phosphodiesterase inhibitors

The present invention relates to a medicament for the treatment of erectile dysfunctions and the use of the C-type natriuretic polypeptide (CNP) and its variants individually or in combination with phosphodiesterase inhibitors for the treatment of erectile dysfunctions.

The relaxation of the smooth muscles of the corpus cavernosum penis, which leads to penile erection, is triggered by a complex interplay of various factors, whereby the ultimately formed intracellular secondary messenger cyclic adenosine monophosphate (cAMP) plays an essential role (Stief et al., 1997). Its concentration is determined on the one hand by the activity of enzymes catalyzing cAMP, namely the adenylate cyclases, and on the other hand by cAMP-degrading (hydrolyzing) enzymes, the phosphodiesterases (PDEs). To date, more than ten different phosphodiester isoenzymes are known, some of which hydrolyze cAMP, but others also the related secondary messenger cyclic guanosine monophosphate (cGMP) (Beavo and Reifsnyder, 1990; Burns et al., 1996; Taher et al., 1997). Since a cAMP-hydrolyzing phosphodiesterase, PDE3A, is inhibited by cGMP, cGMP-hydrolyzing phosphodiesterase 5 (PDE5) has itself become the focus of research into erectile dysfunctions in recent years. Sildenafil is a PDE5 inhibitor currently used under the Viagra is already successfully used as a therapeutic agent for the treatment of erectile dysfunctions (Boolell et al., 1997). However, undesirable side effects of sildenafil, such as eye flicker, headache or circulatory failure, have been described.

The object of the present invention was to provide an agent which reduces or avoids the side effects mentioned.

This object is achieved by a medicament containing a combination of CNP and phosphodiesterase inhibitors. CNP can also be replaced or supplemented by fragments or derivatives with the physiological effect of the CNP obtained. Partial fragments contained in the CNP prohormone sequence are particularly suitable as fragments. The amino acid sequences of the CNP, proCNP and preproCNP are shown below:

Human preproCNP (prepropeptide)

MHLSQLLACALLLTLLS RPSEAKPGAPPKVPRTPPAEELAEPQAAGGG

QKKGDKAPGGGGANLKGDRSRLLRDLRVDTKSRAAWARLLQEHPNAR

KYKGANKKGLSKGCFGLKLDRIGSMSGLGC

Human proCNP (propeptide)

KPGAPPKVPRTPPAEELAEPQAAGGGQKKGDKAPGGGGANLKGDRSR LLRDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRI GSMSGLGC

Human CNP (mature peptide)

GLSKGCFGLKLDRIGSMSGLGC Suitable derivatives are, in particular, those structures derived from the formula given which arise from the conservative exchange of amino acids in this sequence. Derivatives of the CNP containing amidated, phosphorylated, acylated, alkylated and / or linkages of side chains are also suitable.

Fragments are also obtained by deletion of amino acids which are not involved in the function and can be used as medicaments according to the invention.

The medicament preferably also contains necessary auxiliaries and / or carriers which enable or support the galenic application. When the medicament according to the invention is administered intravenously, it is advisable to dissolve the components in question in appropriate preparations suitable for intravenous administration. Other forms of administration are oral, intracavernous, intraurethral, locally topical, transcutaneous.

Particularly suitable phosphodlesterase inhibitors are those of PDE5 phosphodiesterase. These include, in particular, Zaprinast, Dipyridamol, Milrinon and / or Sildenafil.

According to the invention, the use of CNP or fragments or derivatives of the CNP with preserved physiological effect of the CNP for the treatment of erectile dysfunctions is claimed. In an advantageous embodiment of the use according to the invention, CNP or fragments or derivatives of CNP with a physiological value obtained Effect of CNP used in combination with phosphodiesterase inhibitors.

According to the invention, an increase in the intracellular cAMP level is achieved by stimulating the formation (not the inhibition of hydrolysis) of the PDE3A inhibitor cGMP.

In tests on male patients, both intravenous and intracavernal application of CNP resulted in an immediate erection that lasted for about an hour. Surprisingly, when used in combination with phosphodiesterase inhibitors such as sildenafil, it was found that the doses required to produce an erection could be significantly reduced by the simultaneous application of CNP. In some of the subjects, the side effects such as flare-ups or headaches that occurred with exclusive sildenafil treatment were partially reduced until they completely disappeared.

The figure shows the effect of CNP and ANP / CDD99-126 on smooth muscle preparations of human corpus cavernosum penis in the form of the effect of CNP and ANP / CDD99- ₁ 26 vorkontrahierter on the relaxation of smooth muscle preparations. In contrast to ANP / CDD99-126, CNP already shows a significant relaxing effect at a concentration of 1 nM, which finally leads to a relaxation corresponding to 60% of the tone existing after norepinephrine administration when increased to 1 μM. example 1

In the enzymes that catalyze the synthesis of cGMP, the guanylate cyclases, a distinction must be made between two different subgroups, namely the soluble cytosolic and the membrane-bound guanylate cyclases (for an overview, see Wedel and Garbers, 1997). So far it was assumed that only the soluble cytosolic guanylate cyclases play a role in triggering the erection. In order to check the possible involvement of membrane-bound guanylate cyclases, the expression of the genes for the membrane-bound guanylate cyclases GC-A, GC-B and GC-C as well as for two retinal membrane-bound guanylate cyclases (database identifier: HSRETGC and HSRETCG) was carried out in the corpus cavernosum - tissue checked as follows using the RT-PCR technique:

Total RNA was isolated from biopsy samples from human corpus cavernosum and the subsequent transcription of the messenger RNA (mRNA) contained in 5 μg total RNA into copy DNA first strand (cDNA first strand) as already described (Magert et al. , 1995; Magert et al., 1998). Each 1/300 of the first-strand cDNA batches were then used in standard RT-PCR reactions (Saiki et al., 1988) with the primer pairs derived from cDNA database entries and specific for the different guanylate cyclases. The reaction conditions were as follows:

1 x denaturation 94 ° C 5 min then 35 x denaturation 98 ° C for 1 sec

Primer hybridization 50 ° C 30 sec

Elongation 72 ° C 1 min

finally 1 X elongation 72 ° C 7 min

The products obtained were separated electrophoretically in an agarose gel and, after staining with ethidium bromide, visualized using ultraviolet transmitted light.

In fact, with the primers specific for guanylate cyclase B (GC-B) (sense primer GCB-1S: GGGTGCTGTGGCCTCTGG I I I I I CGG, antisense primer GCB-2AS: CCCTGCATC I I I CCACAATTCGAAG) a homogeneous band could be obtained. The isolation of the corresponding DNA fragment from the agarose gel, the subsequent sequencing with a DNA fluorescence sequencer using the primers used for amplification and the comparison of the sequence obtained with the entries in the EMBL / GenBank databases finally confirmed it as GC-B specific.

Example 2

Since it is known that GC-B is the receptor for the peptide hormone C-type natriuretic polypeptide (CNP) formed in the brain, it was checked as follows whether human CNP has relaxing effects on the smooth muscles of the corpus cavernosum or an increase in intracellular concentration of cyclic nucleoside monophosphates induced:

Functional organ bath studies:

Strip preparations of human cavernous muscles were in the with a modified KREBS-RINGER solution (NaCI 120 mM, NaHC0 ₃ 25.6 mM, KCI 4.7 mM, CaCI ₂ 2.5 mM, NaH ₂ P0 ₄ 1.2 mM, MgCI ₂ 1.2 mM, glucose 22 mM, 2Na ⁺ (Ca ²⁺ ) EDTA 0.1 mM, (pH 7.2-7.4) filled bath chambers of an organ bath system (Isolated Organ Apparatus IOA 5306, Föhr Medical Instruments GmbH, Seeheim, Germany) The bath solution was heated to 37 ° C. and was continuously gassed with a mixture of 95% O2 and 5% CO _2. A passive prestress of 5 mN (0.5 g) followed The muscle strips were then stimulated with 6 μM norepinephrine (NOR) and after reaching stable contraction plateaus, the peptides human Atrial Natriuretic Peptide / Cardiodilatin (ANP / CDD99-126), which had been chemically synthesized in our company, were (see Forssmann et al., 1998) and human C-type natriuretic peptides (CNP) (Ogawa et al., 1992) in cumulative r Dosage (0.01 - 1000 nM) added. Mechanical changes in muscle tension were recorded with an analog-digital measurement data acquisition system (MacLab®, AD Instruments, Castle Hill, Australia).

Determination of tissue concentrations of cyclic nucleotides: To determine the time and dose-dependent effects of ANP / CDD99-126 and CNP on the intracellular concentration of cAMP and cGMP, strips of cavernous musculature were incubated with KREBS-RINGER solution in 2 ml reaction vessels analogous to the organ bath experiments, continuously gassed with carbogen and with 6 μM NOR stimulated. Subsequently, the incubation with ANP / CDD9 ₉ was - ₁₂ 6 and CNP (0.1, 10 and 1000 nM) for 10 min. This was done in the presence of 1 mM isobutylmethylxanthine (IBMX) to prevent the degradation of cyclic nucleotides by the activity of phosphodiesterases. After the end of the incubation period, the tissue strips were transferred to prepared reaction vessels, snap-frozen in liquid nitrogen and then homogenized to a fine powder. Cyclic nucleotides were extracted by resuspending the tissue homogenates in 1 ml of 70% ethanol, sonifying the suspensions for 7 min and then centrifuging (10 min at 1,700 g). The alcoholic supernatants were drawn off and freeze-dried, and the lyophilisates were taken up in 1 ml of RIA buffer (50 mM NaAc pH 6.0). In each case 200 μl of the resuspended samples were placed in reaction vessels and acetylated with 6.7 μl of a mixture of triethyiamine / acetic acid (ratio 2: 1). The quantification of cAMP and cGMP was carried out by radioimmunochemical methods using rabbit antibodies against acetylated cAMP and cGMP.

In the test series with different peptide concentrations, a significant relaxation was achieved from 1 nM CNP, which even increased to 60% of the initial tension when increased to 1 μM. With ANP / CDD99- ₁₂ 6 (the ligand of guanylate cyclase A) showed in corresponding concentrations, no significant relaxation, which confirms the importance of CNP and its receptor, guanylate cyclase B, for the tone regulation of the smooth muscles of the penis (see FIG.).

Credentials:

Beavo, J.A. and Reifsnyder, D.H. (1990). Primary sequence of cyclic nucleotide phosphodiesterase isoenzymes and the design of selective inhibitors. Trends Pharmac. Be. 11: 150-155.

Boolell, M., Allen, M.J., Ballard, S.A., Gepi-Attee, S., Muirhead, G.J., Naylor, A.M., Osterloh, I.H. and Gingell, C. (1997). Sildenafil: an orally active type 5 cyclic GMP-specific phosphodiesterase inhibitor for the treatment of penile erectile dysfunction. Int. J. Impot. Res. 8: 47-52.

Burns, F., Zhao, A.Z. and Beavo, J.A. (1996). Cyclic nucleotide phosphodiesterases: gene complexity, regulation by phosphorylation, and physiological implications. Adv. Pharmacol. 36: 29-48.

Forssmann, W.G., Richter, R. and Meyer, M. (1998). The endoerine heart and natriuretic peptides: histochemistry, cell biology, and functional aspects of the renal urodilatin system. Histochem. Cell. Biol. 110: 335-357.

Magert, HJ., Reinecke, M., David, I., Raab, HR, Adermann, K., Zucht, HD, Hill, O., Hess, R. and Forssmann, WG (1998). Uroguanylin: Gene structure, expression, processing as a peptide hormone, and co-storage with somatostatin in gastrointestinal D-cells. Regul. Pept. 73: 165-176. Magert, HJ., Hadrys, T., Cieslak, A., Gröger, A., Feller, S. and Forssmann, WG (1995). cDNA sequence and expression pattern of the putative pheromone carrier aphrodisin. Proc. Natl. Acad. Be. USA 92: 2091-2095.

Ogawa, Y., Nakao, K., Nakagawa, O., Komatsu, Y., Hosoda, K., Suga, S., Arai, H., Nagata, K., Yoshida, N. and Imura, H. ( 1992). Human C-type natriuretic peptides. Characterization of the gene and peptide. Hypertension 19: 809-813.

Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Hörn, G.T., Mullis, K.B. and Ehrlich, H.A. (1998). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491

Stief, CG., Noack, T. and Anderson, K.E. (1997). Signal transduction in cavernous smooth muscle. World J. Urol. 15: 27-31.

Taher, A., Meyer, M., Stief, CG., Jonas, U. and Forssmann, W.G. (1997). Cyclic nucleotide phosphodiesterase in human cavernous smooth muscle. World J. Urol. 15: 32-35.

Wedel, B.J. and Garbers, D.L. (1997). New insights on the functions of the guanylyl cyclase reeeptors. FEBS Lett. 410: 29-33.

Claims

claims

1. Medicament containing a combination of CNP or fragments or derivatives with preserved physiological effect of the CNP and phosphodiesterase inhibitors.

2. Medicament according to claim 1 further comprising auxiliaries and / or carriers.

3. Medicament according to claim 2, wherein by means of the excipients and / or carriers, the medicament can be administered intravenously, orally, intracavernously, intraurethrally, locally topically, transcutaneously.

4. Medicament according to one of claims 1 to 3, wherein the phosphodiesterase inhibitors are zaprinast, dipyridamole, milrinone and / or sildenafil.

5. Use of CNP or fragments or derivatives of the CNP with preserved physiological effect of the CNP for the treatment of erectile dysfunctions

6. Use according to claim 5 in combination with phosphodiesterase inhibitors.