CALCITONIN GENE RELATED PEPTIDE FOR THE TREATMENT OF UNDESCENDED TESTICLES

Abstract: Methods for the treatment of undescended testicles in male animals are described which methods comprise administering to a subject in need of such treatment calcitonin gene-related peptide (CGRP) or an analogue thereof having CGRP activity, optionally in association with a carrier and/or excipient, in an amount effective to cause testicular descent. There is also described pharmaceutical compositions for the treatment of undescended testicles in male animals, which comprise CGRP or an analogue thereof having CGRP activity in association with a pharmaceutically acceptable carrier and/or excipient. The use of CGRP in the treatment of undescended testicles is an effective alternative to invasive surgery.

Description: CALCITONIN GENE RELATED PEPTIDE FOR THE TREATMENT OF UNDESCENDED TESTICLES This invention relates to a method for the non- surgical treatment of undescended testicles in male animals. The invention is also concerned with pharmaceutical compositions for the treatment of undescended testicles. The testes are two glandular organs which secrete semen, and are situated in the scrotum, being suspended by the spermatic cords. For the past 40 or 50 years it has been proposed that testicular descent is controlled by male androgenic hormones (testosterone). Androgens were proposed to act on mesenchymal tissue in the groin known as the gubernaculum, which migrates across the pubic region from the groin to the scrotum during inguino-scrotal testicular descent. The gubernaculum was thought to guide the testes into the scrotum. In humans, approximately 5% of male babies are born with undescended testicles. In 1 to 2% of males, the testes do not descend into the scrotum. In the remaining males, the testes may arrive in the scrotum a few weeks after birth as compared with the normal time of 30 to 36 weeks gestation. These "late descenders" are not quite normal and many have testicles which "re-ascend" out of the scrotum later in childhood. The treatment for undescended testes is invasive surgery (orchidopexy) where the undescended testes are physically transferred to the scrotum. This is usually carried out at 1 to 3 years of age because the testes are known to develop progressive histological abnormality thereafter. This surgical procedure is traumatic for the individual and family involved, is costly, and has attendant risks associated with all forms of surgery requiring general anaesthesia. It has also been proposed to treat undescended testicles by administering the human androgen testosterone or by treatment with HCG (human chronic gonadotrophin) or luteinizing hormone releasing hormone (LHRH). These treatments have proved ineffective. A requirement accordingly exists for a non-surgical and convenient treatment for undescended testicles. In accordance with an aspect of this invention, there is provided a method for the treatment of undescended testicles in male animals which comprises administering to a subject in need of such treatment calcitonin gene-related peptide (hereafter CGRP) or an analogue thereof optionally in association with a carrier and/or excipient, in an amount effective to cause testicular descent. In another aspect, the invention relates to a pharmaceutical composition for the treatment of undescended testicles in male animals, which comprises CGRP or an analogue thereof having CGRP activity in association with a pharmaceutically acceptable carrier and/or excipient. In a further aspect, this invention relates to the use of CGRP or an analogue thereof in the manufacture of a medicament for the treatment of undescended testicles. CGRP is a 37 amino acid peptide produced by alternative splicing of calcitonin mRNA (Rosenfeld et al., Nature, Vol. 304, 1983). CGRP is a neuropeptide and has been described in many sensory nerves but few motor nerves. As used herein, CGRP refers to CGRP from any animal species, such as human, horse, sheep, pig, rat, mouse, etc. Principally, but without limitation, CGRP refers to human CGRP. The term CGRP extends to naturally occurring allelic variants of the CGRP peptide sequence. Human CGRP has the following sequence: H-Ser-Cys-Asn-Thr-Ala-Thr-Cys-Val-Thr-His-Arg-Leu-Ala- Gly- eu-Leu-Ser-Arg-Ser-Gly-Gly-Val-Val- ys-Asp-Asn-Phe- Val-Pro-Thr-Asn-Val-Gly-Ser-Lys-Ala-Phe. CGRP (human) is available commercially from a number of suppliers, such as Peninsular Laboratories. CGRP may be purified from tissues containing it according to well known procedures. Preferably, CGRP is produced by peptide synthetic techniques, such as solid phase peptide synthesis, or is produced by recombinant DNA methods. Analogues of CGRP which comprise amino acid sequence variants fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acids. Generally, insertions within the mature - 4 - coding sequence of CGRP will be smaller than those with the amino or carboxyl terminal fusions, of the order of say 1 to 4 residues. Insertional amino acid sequence variants of CGRP are those in which one or more amino acid residues are introduced into a predetermined site in the CGRP peptide. Deletional variants are characterised by the removal of one or more amino acids from the CGRP peptide sequence. Typically, no more than about 2 to 6 residues are deleted at any one site within the CGRP molecule. Amino acid substitutions are typically of single residues; insertions usually will be on the order of about 1 to 10 amino acid residues; and deletions will range from about 1 to 20 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of two residues or insertion of two residues. Substitutional variants are those in which at least one residue in the CGRP sequence has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Table. TABLE 1 Original Residue Exemplary Substitutions Ala Ser Arg Lys Asn Gin; His Asp Glu Cys Ser Gin Asn Glu Asp Gly Pro His Asn; Gin He Leu; Val Leu He; Val Lys Arg; Gin; Glu Met Leu; He Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val He; Leu Generally amino acids are replaced by other amino acids having like properties, such as hydrophobicity, hydropholicity, electronegativity, bulky side chains, etc. The amino acid variants of CGRP referred to above may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis (Merrifield, J. Am. Chem. Soc. , 85, p2149 (1964) and the like, or by recombinant DNA manipulations upon the gene encoding CGRP of any particular animal. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example M13' mutagenesis. The manipulation of DNA sequences to produce variant proteins which manifest as substitutional, insertional or deletional variants are well known in the art and are described for example in Maniatis et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 1982). The above referenced amino acid sequence variants of CGRP may all be regarded as analogues of CGRP, if they possess CGRP activity as is defined hereinafter. Any compound having CGRP activity as defined hereinafter is regarded as an analogue of CGRP. This includes small organic or inorganic drugs which may, for example, be designed to mimic the three dimensional structure of CGRP or a part thereof. Compounds of this type may be produced as a result of X-ray crystallography of CGRP or other three dimensional modelling techniques. "CGRP activity" is defined as the ability to effect testes descent in animals having undescended testes. A convenient in-vivo assay for CGRP activity utilises isolated male gubernaculum tissue. In the presence of effective quantities of compounds having CGRP activity, the isolated male gubernaculum undergo rhythmic contractions and in some instances serpentine movement. Such activity in the presence of CGRP is not observed with female gubernacula, skeletal muscle or umbilical cord tissue. Using this convenient assay, further details of which are provided in the examples of this specification, compounds may be readily tested for CGRP activity without recourse to undue experimentation. CGRP bioactivity may also be tested in-vivo utilising male animals whose testes have not yet descended, such as new born rats, or animals which have congenitally undescended testes, such as the TS strain of rat. When compounds having CGRP activity are applied to the scrotum of such animals, for example by way of injection, testicular descent takes place. Again, in- vivo models such as described provide a ready means for assessing CGRP activity. Examples of commercially available CGRP include chicken CGRP, human CGRP, biotinyl-CGRP (human), [Tyr']- CGRP (human), biotinyl-CGRPII (human), CGRP (rat and biotinyl-CGRP (rat). CGRP should generally be administered under the guidance of a physician, and pharmaceutical compositions would usually contain an effective amount of the peptide or analogues thereof in conjunction with a conventional, pharmaceutically acceptable carrier. The pharmaceutical carrier employed may be, for example, either a liquid or a solid. Examples of liquid carriers include physiologically buffered saline, dextrose, sterile water, olive oil and the like. Similarly, the carrier may include time delay material well known to the art, such as glyceryl monostearate, ethyl cellulose, hydroxypropylmethyl cellulose, methylmethacrylate and the.like. Examples of solid carriers are lactose, sucrose, talc, gelatin, agar, pectin, magnesium stearate, stearic acid and the like. A wide variety of pharmaceutical forms can be employed. I jectable forms of CGRP generally contain CGRP dissolved in a sterile vehicle such as water, saline, dextrose or the like. Injectable solutions of CGRP may contain, for example, 50μg to 500mg per ml. Injectable solutions may be provided in ampule or vial or non-aqueous liquid suspension. If a solid carrier is used, the preparation can be tableted, placed in a hard gelatin capsule or admixed with a slow release polymer to form a dosage form. The amount of solid carrier will vary widely but will preferably be from about O.lmg to lg. CGRP may be formulated into a cream, ointment, paste, salve or the like for topical application to the scrotum. Such forms may include skin penetrating agents to facilitate the passage of CGRP or analogues thereof into the scrotum. Suitable skin penetrating agents include dimethylsulphoxide (DMSO) and the like. Formulations for topical use containing CGRP or analogues thereof may be applied to the scrotum from 1 to 4 times per day for about 1 to 10 days. For topical administration, the active ingredient may contain from about 0.001% to 10% w/w, e.g. from 1 to 2% by weight of the formulation. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site where treatment is required, such as: liniments, lotions, creams, ointments or pastes. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone and/or moisturiser such as glycerol or an oil such as caster oil. Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredients for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution, or in suspension in an aqueous or non-aqueous fluid with the aid of suitable machinery, with a greasy or non-greasy base. The base may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, bees wax, a mucilage, an oil of natural origin such as almond, corn, caster or olive oils, wool fat or its derivatives or a fatty acid such as stearic acid together with an alcohol such as polypropylene glycol. The formulation may incorporate any suitable surface active agent such as an ionic, cationic or non-ionic surfactant such as sorbitan esters or the like. Preferably, each parenteral dose of CGRP containing pharmaceutical forms will contain a reactive ingredient in an amount from about 0.05mg to about 500mg. If oral dosage units are employed, they will contain the active ingredient in an amount of from about 0.05mg to about 1.Omg. CGRP or analogues thereof may be administered from an implantable or skin-adhesive sustained release article. Examples of suitable systems include copolymers of L-glutamic acid and γ-ethyl-L-glutamate (U. Sidman et al., 1983, "Biopolymers" 22, 1: 547-556), poly(2- hydroxyethyl- ethacrylate) (R. Langer et al., 1981, J. Biomed. Matter. RES., 15: 167-277 and R. Langer et al. 1982, "Chem. Tech." 12: 98-105) or the like. Such articles may, for example, be implanted sub-cutaneously in the scrotum or placed in contact with the skin of the - 10 - scrotum. Animals which may be treated according to the present invention include humans, horses, and other domestic animals. Medicaments or compositions may be prepared by admixing, dissolving, blending, grinding or the like, CGRP with a pharmaceutically acceptable carrier or excipient according to methods well known in the art. CGRP is administered to such an animal in an effective amount. The term "effective amount" refers to an amount effective to cause testicular descent. It will be recognised by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well known variables. The route of administration of CGRP or analogues thereof may be parenteral, topical, or oral. The term "parenteral" as used herein includes intravenous, intramuscular or subcutaneous administration. The subcutaneous form of parenteral administration to the scrotum is generally preferred. CGRP or analogues thereof may be injected into the scrotum at birth or shortly after, on the side of the palpable but undescended testis as a way of stimulating normal migration of the gubernaculum into the scrotum. Preferably, the amount of CGRP or analogues thereof injected into the scrotum in a single bolus injection is from about 20μg to lOOOμg per kilogram, and more preferably lμg per 5 to 10 g body weight. A single scrotal injection may be provided for testis descent, or several injections over a limited time period such as from 1 to 10 days. Where an implantable or skin-adhesive sustained release article is applied to the scrotum or adjacent - 11 - areas, CGRP is slowly released to the scrotum to effect migration of gubernaculum and testis into the scrotum. It will be recognised by one skilled in the art that the optimal quantity and spacing of individual dosages of CGRP and analogues thereof will be determined by factors such as the route and site of administration, and the type and age of the particular animal being treated. Dosage and frequency of administration of CGRP or analogues thereof will often depend upon the judgement of a consulting physician or veterinarian in any particular case. The optimal course of treatment, that is, the number of doses of CGRP or analogues thereof given per day for a defined number of days, can be readily ascertained by those skilled in the art using conventional courses of treatment determination tests. Generally, treatment with CGRP or analogues thereof to effect testes descent usually takes place shortly after birth of an animal. In humans, it is preferred that treatment take place at birth or shortly thereafter. However, animals of any age having undescended testicles may be treated according to the present invention. Without limiting the invention, the applicant believes that CGRP is the chemotactic signal released by the genitofemoral nerve within the scrotum. The applicant considers that CGRP, through some as yet unknown mechanism, causes the gubernaculum to migrate to the scrotum pulling with it the testis to which it is physically connected. Again without limiting this invention, it is the applicant's hypothesis that in humans for example, there may be an anatomical problem with the genitofemoral nerve (such that it does not go into the scrotum), or perhaps a physiological problem (failure to secrete CGRP) which may cause undescended - 12 - testicles). The present invention will now be described, by way of non-limiting example only, with reference to the following non-limiting figures and examples. In Figure 1, there is shown a displacement curve derived by computerized densitometry from serial gubernacular sections incubated with increasing concentrations of unlabelled human CGRP in the presence of radiolabelled human CGRP. EXAMPLE 1 Analysis of the Genitofemoral Nerve (GFN): The genitofemoral nerve (GFN) is essential for inguino-scrotal testicular descent as transection of the nerve prevents gubernacular migration from groin to scrotum (Beasley, S.W. & Hutson, J.M. [1987] Aust. NZ J.Surg. 57, 49-51). It has been previously shown that cutting the GFN blocks the action of androgens without preventing testosterone secretion. This experiment examines the course of the GFN nerve in immature rats. Materials and Methods: Colonies of Sprague-Dawley rats were maintained at the Royal Children's Hospital, Melbourne, on Barastoc mouse-breeder cubes and water ad libitum, and kept in a constant 12 hour light-dark cycle. Ten male rats ranging in age from one to eight days old were used. The animals were anaesthetised with oxygen and 2% halothane and a small transverse incision was made across the lower abdomen to open the peritoneum. The bowel was pushed aside with cotton swabs, and the peritoneum was divided inferior to the renal vessels and medial to the ureter on one side. Each genitofemoral nerve was picked up carefully with forceps, and divided. Several crystals of either diamidinophenyl indole (DAPI - Sigma) or Fast Blue (Sigma) were applied to the distal end of the cut nerve, then the peritoneum and bowel were returned to the normal positions. The wound was sutured with one layer of continuous 6/0 Ethicon silk. 48 hours after this operation the rats were re- anaesthetised with 4% halothane. The thorax was opened, and the inferior vena cava divided below a ligature. Transcardiac perfusion of 10 ml of normal saline was followed by 10 ml of Zamboni's fixative. After removal of the tail, hind legs and dorsal skin the pelvis was stored in Zamboni's fixative for a week at 4°C, before being cleared in dimethyl sulphoxide and stored in Phosphate Buffered Saline (PBS)/30% sucrose overnight. The pelvis was embedded in OCT plastic medium cooled by liquid nitrogen and isopentane, then 20μm frozen sections were cut in the sagittal plane onto 1% gelatin- coated slides. The fluorescence, and thus the course of the nerve, could then be followed by studying serial sections under an epifluorescence microscope (Leitz Diaplan). Both the dyes used emit blue radiation under ultra-violet excitation wave lengths. When studying sagittal sections of the pelvis, the area that would develop into the scrotum was defined as that area of skin between the anus caudally and the immature phallus cranially. Results: Fluorescent tracing of the genital branch of the GFN showed that it runs through the inguinal canal posterolateral to the spermatic cord, and a small branch follows the cord to supply the head of the epididymis and tunica albuginea, but not the testis itself. The' main trunk runs distally behind the testis on the surface of the cremaster muscle, supplying this, then turns - 14 - cranially to enter the gubernaculum from its distal attachment, where branches fan out supplying the gubernacular substance and the caudal epididymis. A branch of the nerve continues past the testis to end in a network of fibres in the subcutaneous tissues and dermis of the region that will become the scrotum. Fluorescent anterograde nerve labelling appeared to be a reliable and specific method of tracing the course of the GFN, as very little fluorescent straining was present outside the nerve itself, provided that the interval between labelling the nerve and sacrificing the animal did not exceed three days. After this time some leakage of dye out of the nerve occurred. The above results show that the GFN is in a position from which it could provide directional information for the gubernaculum in its migration to the scrotum. This example shows for the first time that the GFN extends into the scrotum prior to gubernacular descent. Example 2 hereafter provides evidence of a neuropeptide located within the GFN which is believed to act as a chemotactic signal drawing the gubernaculum and associated testis into the scrotum. EXAMPLE 2 Histochemical Analysis of the GFN: The GFN is analysed histochemically in this Example in an attempt to identify neuropeptides distributed therein which may act as possible transmitters mediating gubernacular and testis descent. Such a transmitter is likely to be a peptide, as these are capable of exerting trophic and modulatory effects over extended periods of time, and often function in a neuro-endocrine manner. Materials and Methods: The animals used in this Example were 12 male and 3 - 15 - female rats, aged 6 days at operation, and 5 male, 5 female and 5 Testicular Feminization Syndrome (TFM) mice aged 10 days old at operation. Antibodies against thirty different neuropeptides 5 were used in this example. These included antibodies against vasoactive intestinal peptide, 5- hydroxytryptamine, somatostatin 8, met-enkephalin, substance P, thyrotrophin releasing hormone, neuropeptide Y and calcitonin gene-relating peptide (CGRP). 0 Antibodies were obtained from Prof. J. Furness, Flinders Medical Centre, with the exception of calcitonin gene- related peptide rabbit anti-rat antibody which was purchased from Peninsula Laboratories, U.S.A. This latter antibody was used in a concentration 1:2000. 5 Animals were anaesthetised, surgically prepared, and tissues removed and fixed as for Example 1. Tissues were embedded in OCT plastic medium, cooled with liquid nitrogen and isopentane, then 14 μm frozen serial sections were cut onto 1% gelatin-coated slides, 0 and left to dry. The spinal cords were cut transversely and the pelvises were cut in the sagittal plane. Slides were pre-incubated for 30 minutes with a covering to 10% normal sheep serum (NSS) to block non-specific staining. Excess serum was removed, then the slides were incubated overnight in a humidified box with various primary antibodies raised against different neuropeptides. The antibody mixture was diluted with PBS, and contained 10% NSS in the final solution. Next day the slides were rinsed for 15 minutes in PBS (phosphate buffered saline), then incubated for one hour with a sheep anti-rabbit fluoro-iso-thiocyanate (FITC) labelled secondary antibody diluted to 1:200 with PBS (Silenus Laboratories, Hawthorn, Vic). Following another rinse, coverslips were mounted with buffered - 16 - glycerol, and the pattern of fluorescence observed with an epifluorescence microscope (Leitz Diaplan). The location of immunohistochemical staining was compared with the retrograde labelling on the same 5 sections by simply changing the excitation filter, as different wave lengths are required in order for FITC (520nm) and DAPI (400nm) to emit light. To quantify cells consistently all sections were counted, and only those cells which stained brightly and in which the 0 nucleus could be clearly defined were counted as positive. Negative controls include either PBS alone, or antiserum with excess synthetic rat CGRP, instead of the primary antibody. The dorsal root ganglia served as a 5 positive control for CGRP. Qualitative data were obtained for rats, and quantitative experiments were performed on 10-day-old mice of male, female and TFM mutant litter mates. Statistical analysis was performed using both the 0 Wilcoxon Rank-Sum test and the t-test. In two 2-day old male rats CGRP staining was carried out on the lower lumbar spinal cords following DAPI- labelling of the bulbocavernosus muscle. Results: 5 CGRP immunoreactivity was present in significant amounts in lumbar motor neurons. In addition, there were many CGRP-immunoreactive sensory fibres throughout the dorsal horn of the spinal cord, and a ventro-medial fibre bundle stained brightly. All other neuropeptides tested, 0 including vasoactive intestinal peptide, 5- hydroxytryptamine, somatostatin 8, met-enkephalin, substance P, thyrotrophin releasing hormone and neuropeptide Y, where absent from the lumbar anterior horn. In male rats double labelling showed that the sites of CGRP staining were very similar to, but not identical with, the DAPI fluorescence within the genitofemoral nerve. About half the DAPI-labelled cells stained for CGRP, as did a small number of non-DAPI-labelled motor neurons which were localised in other distinct regions within the ventral horn. In comparison, in female rats the motor neurons appeared smaller and were fewer in number, and a smaller proportion of them were immunoreactive for CGRP, although other groups of motor neurons were still CGRP positive as in the male (Table 1). Quantitative studies on mice confirmed these results (Table 2), showing that the spinal nucleus of the GFN contained significantly more cells in male mice than in female mice, with TFM mutants having an intermediate number of cells. In addition, in the male a significantly greater proportion of these labelled cells were CGRP-immunoreactive compared with female mice. This proportion was also greater than for TFM mutants, however this difference was not statistically significant. Statistical analysis of the results obtained showed that comparisons between male and female mice were significant for all three parameters studied (p < 0.05). Comparisons between TFM mutants and female mice were significant for the number of DAPI-labelled cells (P < 0.01) and the number of CGRP positive cells also labelled with DAPI (p < 0.05), but not for the percentage of DAPI labelled cells containing CGRP. Results obtained for male mice and TFM mutants were not statistically different. Staining of the lower lumbar spinal cord following DAPI-labelling of the bulbocavernosus muscle showed that CGRP was not present in this nucleus, however, it - 18 - appeared to be localised within two other motor nuclei situated more laterally at this level. The immunohistochemistry results showed that calcitonin gene-related peptide immunoreactivity was present in large amounts in the sexually dimorphic spinal nucleus of the genitofemoral nerve. The proportion of these cells containing CGRP immunoreactivity was greatest in male mice, followed by TFM mutants and female mice, suggesting that CGRP accumulation may be androgen dependent. The presence of CGRP immunoreactivity within the genitofemoral nerve and the sexual differences in the amount present, indicate that CGRP is the transmitter which exerts its effect on migration of the gubernaculum into the scrotum. EXAMPLE 3 Rhythmic Contractile Movement of the Gubernaculum During Testicular Descent: This experiment examines gubernacula in-vitro and in-vivo and the effects of CGRP thereon. Gubernacula (n = 182) from male rats (0-5 days) were incubated in organ culture with CGRP, Vasoactive intestinal peptide (VIP) somatostatin, acetylcholine or control medium. The cultures were examined daily with a dissecting microscope connected to a video camera and tape. Some rats were anaesthetised and inguinoscrotal skin excised to record in-vivo gubernacula by video. One half of CGRP-treated gubernacula (2.5-50x 10 -11 M) showed rhythmic contractions (120-200/min), with higher doses showing more rapid contractility. This compared with 9/40 of VIP-treated gubernacula contracting and 2/26 of controls (at 20/min). Female gubernacula, skeletal muscle or umbilical cord showed no CGRP-induced contractions. Gubernacula in vivo showed vigorous contractility and serpentine movements, which were accentuated by increased intra-abdominal pressure and direct application of CGRP. These studies reveal the gubernaculum is highly motile during testicular descent. Rhythmic contractions suggest smooth muscle-like components and that CGRP may mediate the effect of androgens on gubernacula migration. EXAMPLE 4 Intact GFN is Required for Testicular Descent: Male Sprague-Dawley rats (n = 13) between one and two days old were anaesthetised and the peritoneum opened by a small transverse incision across the lower abdomen. Each genitofemoral nerve (GFN) was isolated, a segment removed and the ends diathermied by the application of a heat source to each end of the divided nerve. The rats were inspected weekly for four weeks for testicular descent. Testicular descent was defined as descent of testis into the scrotum on compression of the abdomen. In 54% of animals treated in the above manner undescended testicles were observed. In control animals where the GFN was not cut all testicles descended. This experiment shows that the GFN must be intact for testicular descent and directly infers that where the passage of CGRP along the GFN is blocked, testicular descent is inhibited. Further, CGRP (470 μmole/day for 14 days) was injected into the right groin lateral to the inguinal region. Water injections at the same site acted as a control. Two out of five right testes were undescended (40%) with water injections compared with four out of six (67%) when CGRP was injected nearby. EXAMPLE 5 CGRP Receptors in the Gubernaculum: In this example, the binding of radioactive CGRP in the gubernaculum was examined in vitro, to determine whether the gubernaculum contained receptors for CGRP. For competition and specificity studies, neonatal Sprague-Dawley male rats were sacrificed by decapitation at two days after birth. Following decapitation, each rat was placed in a supine position and its limbs taped on a metal tray on ice. Using a Zeiss operating microscope, a lower abdominal transverse incision about 2 mm below the umbilicus was made and, by excising flap-shaped inguinoscrotal skin, gubernacula were exposed to show a cone-shaped projection. Increased abdominal pressure produced by pressing on the upper abdomen with cotton wool buds aided the resection of gubernaculum-. Fine microsurgical scissors were used to divide the distal attachment of the caudal epididymis with the gubernaculum. The excised gubernacula were rapidly removed and snap frozen by immersion in liquid nitrogen. Sagittal sections of 20-μm thickness were cut on a cryostat at -12°C and thaw mounted on 0.5% gelatin-coated slides. The sections were dried in a vacuum desiccator overnight at 4°C and stored at -70"C. Alternate sections were stained with haematoxylin and eosin in order to identify fine structures of the 125 I-labelled gubernacula at the time of quantitation. Radiolabelled human CGRP {(2-[ 125 I)iodohistidyl 10 )- human CGRP} was obtained from Amersham International pic. (Buckinghamshire, England) (-2000 Cl/mmol). The following synthetic peptides: human CGRP, rat CGRP (8- 37), rat CGRP (Tyr 27 , 28-37), salmon thyrocalcitonin, human vasoactive intestinal peptide (VIP), somatostatin, and substance P were purchased from Auspep Pty. Ltd. (Melbourne, Australia); human thyrocalcitonin and Serotonin (5-hydroxytryptamine) from Sigma Chemical Co. (St. Louis, MO). The following buffer was used for incubating tissue sections with various ligands: 100 mM HEPES, containing 120 mM NaCl, 1.2 mM MgS0 4 , 2.5 mM KC1, 15 mM NaC 2 H 3 0 2 , 10 mM D-glucose, 1 mM EDTA, 0.5% BSA, and 0.35 mM Bacitoracin. Conditions for optimising binding were examined at 4°C, 22°C, 37 β C, for 2, 6, 24, 30, 48 hours incubations with pH 7.0, 7.4, 8.0 and pH 8.4 buffers. Following the optimisation of incubation conditions, tissue sections were thawed and subsequently incubated for 24 hours at 4°C in the foregoing buffer at pH 8.0, containing 35 pM [ 125 I]-human CGRP. To determine non¬ specific binding, some sections were also incubated with 1 μM unlabelled human CGRP. Displacement of [ 15 I]-human CGRP binding for characterisation of binding properties and specificity were derived by incubating a series of adjacent tissue sections with increasing concentrations of unlabelled CGRP or of the following peptide: rat CGRP (8-37), rat CGRP (Tyr 27 , 28-37), salmon thyrocalcitonin, human thyrocalcitonin, human VIP, somatostatin, serotonin, and substance P. After incubation, the sections were passed through four successive 30-seconds changes of buffer at pH 8.0 on ice, to remove nonspecifically-bound ligand. The sections were then dried in a stream of cold air and exposed to X-ray film (Eastman Kodak Company, New York, U.S.A.) for 7 days at - 22 - room temperature. Radioactive standards used autoradiographic [ 125 I] micro-scales (Amersham International pic, Buckinghamshire, England), which consist of 10 layers of [ 125 I]-incorporated polymer arranged in order of increasing specific activity separated by non-radioactive layers and were simultaneously exposed to the X-ray film. The radioactivity standards were corrected for decay using the exponential equation, the decay constant and the elapsed time interval to the middle of the exposure period. These data enabled computer calibration of the optical density of the autoradiographs in terms of dpm/mg protein. The films, to which incubated gubernacula were exposed, were developed and quantified by computerised densito etry using an EyeCom Model 850 image processor (Log E/Spatial Data systems, Springfield, Virginia, U.S.A.), coupled to a DEC 11/23 LSI computer to obtain color-coded images calibrated in terms of radioligand density. Some of incubated sections were processed for emulsion autoradiography as follows. The sections were dipped for 1 to 2 seconds in Amersham LM-1 emulsion (Amersham International pic, Buckinghamshire, England) immersed in a waterbath at 43°C, dried for one hour at room temperature with a relative humidity of 60%, and stored in plastic slide box containing silica gel at 4°C for exposure for 4 weeks. Slides were then developed for 5 minutes with a 1:1 dilution of Kodak D19 developer with distilled water at 20°C, immersed in a stop solution of 1% acetic acid for 30 seconds at 20°C, in a fixative (a mixture of 1:4 dilution of Ilford Hypam Fixer with distilled water, and Ilford Hypam Hardener) (Ilford Pty. Ltd., Melbourne, Australia) for 10 minutes, and in gently running water for 1 hour. Following development the " sections were dried and stained with haematoxylin to enable localisation of grain densities. Binding saturation was achieved by 24 hours incubation at 4°C with the buffer pH at 8.0. Either longer incubation time, higher incubation temperature, or higher buffer pH than the optimal conditions resulted in higher non-specific binding. Definitive binding studies were, therefore, performed at 4°C for 24 hours, when specific binding was seen. Displacement curves were derived by computerised densitometry from serial sections incubated with increasing concentrations of unlabelled human CGRP in the presence of radiolabelled human CGRP (Figure 1). Computer-derived plots of these data also revealed a single class of binding sites. Rat CGRP (8-37) was potent in competing with [ 125 I]- human CGRP with affinity constants similar to or greater than that of unlabelled human CGRP; salmon calcitonin competed at this site with a lower affinity than human CGRP, while the human calcitonin had only a very low potency; rat CGRP (Tyr 27 , 28-37) also competed with the binding sites but at a lower affinity than human CGRP. However the unrelated peptides, human VIP, somatostatin, serotonin, and substance P do not compete at all for concentrations of up to 1 X 10" 6 M. Light microscopic localisation of [ 125 I]-human CGRP receptor binding using emulsion autoradiography revealed a high density of binding over the cremasteric muscle. A comparison of the autoradiograph with the stained adjacent section shows that grain densities are confined to the thick layers of the inner and outer cremasteric muscles. Binding was fairly low in the central mesenchyme. - 24 - EXAMPLE 6 CGRP Antagonists Delay Testicular Descent: In this Example, the effects of the CGRP antagonist CGRP (8-37) on testicular descent were examined in neonatal mice. Newborn ARC and Brindle substrains MOBR/MOBLO, mice were used in the study. The mice were injected with 25 μl of 10" 4 molar CGRP (2.5 n molar) within the first three days of life. The mice were injected through the skin of the left iliac fossa and the developing left scrotum was entered obliquely. Once approximately half of the injected dose was administered, the scrotal sceptum was pierced to enter the right scrotum and the rest of the injection was continued. At one week of age, and thereafter weekly, 50 μl of 10~ 4 molar CGRP (8-37) was injected. CGRP (8-37) is a competitive inhibitor of CGRP and lacks the first seven amino acids which comprise the active site of CGRP. The mice were inspected weekly for four weeks for testicular descent. Any mice who demonstrated descended testis on inspection were not injected again on that side. Testicular descent was defined as descent of testis into the scrotum on compression of the abdomen. Descent of the gubernaculum was not considered significant. All rectractile testes were included under descended testes. At four weeks of age all mice with undescended testis were sacrificed and subjected to histology to determine whether there were mechanical factors inhibiting testicular descent, i.e. fibrous tissue formation as a result of the injection. The control group of mice were injected with phosphate buffered saline (PBS) at the same volume. Results were compared using non-parametric analysis. The experimental group consisted of 138 mice while the control group consisted of 37 mice. At one week of age all testis were undescended. At two weeks, all 362 testes of the 138 experimental mice were undescended in the experimental group, while 42 out of 74 were undescended in the control group. At three weeks of age 44 out of 362 testes were undescended in the experimental group and all 74 testes were descended in the control group. At four weeks 3 out of 362 testes were undescended. On histological examination of the three mice which demonstrated unilateral undescended testis after four weeks, none showed evidence of a mechanical barrier inhibiting migration of the gubernaculum. It is clear from the above experiment that injection of the competitive inhibitor of CGRP (8-37) into the developing scrotum had delayed descent of the testis in mice compared to the control group. Indeed, CGRP (8-37) completely prevented descent of 3 testes. CGRP (8-37) binds to the receptors and competitively inhibits the activity of CGRP released from the genitofemoral nerve. However, this inhibiting effect of CGRP (8-37) was not irreversible, probably because of diffusion and its biological half-life. It is believed that the descent of all but three testes at four weeks after treatment may be due to the endogenous CGRP competitively binding to the receptors rather than the exogenous antagonist, thereby reversing the effect of the latter. This result does, however, demonstrate that CGRP (8- 37) can prevent or delay testicular descent. This is consistent with CGRP causing testicular descent after release from the nerve fibres of the genitofemoral nerve in the inguino-scrotal area. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within its spirit and scope. The invention also includes all the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively , and any and all combinations of any two or more of said steps or features. TABLE 1 QUANTIFICATION OF CELLS IN THE SPINAL CORDS OF A MALE AND A FEMALE RAT No. of DAPI- No. of CGRP % of DAPI-labelled labelled positive cells containing cells cells CGRP MALE 298 148 50 FEMALE 163 35 22 QUANTIFICATION OF CELLS IN MALE. FEMALE AND TFM MICE No. of DAPI- No. Of CGRP- % of DAPI labelled positive labelled cells Sex cells also contain- containing CGRP ing DAPI mean+-SD MALE 46+or-7 p < 0.05 FEMALE NS TFM SUBSTITUTESHEET

Claims: CLAIMS : 1. A method for the treatment of undescended testicles in male animals which comprises administering to a subject in need of such treatment calcitonin gene- related peptide (CGRP) or an analogue thereof optionally in association with a carrier and/or excipient, in an amount effective to cause testicular descent. 2. A method according to claim 1 wherein said CGRP is human CGRP. 3. A method according to claim 1 wherein said CGRP is administered to the scrotum. 4. A method according to claim 3 wherein said CGRP is administered in the form of a topical preparation. 5. A method according to claim 4 wherein said topical preparation contains a skin penetrating agent. 6. A method according to claim 1 wherein said CGRP is injected into the scrotum or abdomen. 7. A method according to claim 1 wherein said CGRP or analogues thereof are administered from an implantable or skin-adhesive sustained released article. 8. A method according to claim 7 wherein said sustained release article is implanted sub-cutaneously in the scrotum or placed in contact with the skin of the scrotum. 9. A method according to claim 1 wherein said male animal is selected from a human, horse, or other domestic animal. 10. A method according to any one of claims 1 to 9 wherein said CGRP or analogues thereof are administered at or shortly after birth. 11. A composition for the treatment of undescended testicles in male animals, which comprise a CGRP or an analogue thereof having CGRP activity in association with a pharmaceutically acceptable carrier and/or excipient. 12. A composition according to claim 11 wherein said CGRP or analogue thereof is human CGRP. 13. A topical formulation which comprises CGRP or an analogue thereof in association with a skin penetrating agent. 14. A composition according to claim 12 comprising CGRP or an analogue thereof in the form of an implantable or skin-adhesive sustained release article. 15. Use of CGRP or an analogue thereof in the preparation of a composition for use in the therapeutic treatment of undescended testicles in male animals.

Inventors: HUTSON JOHN MEDWYN, HUTSON, JOHN, MEDWYN
Applicants: UNIV MELBOURNE, THE UNIVERSITY OF MELBOURNE
Application year: 1991
Main classification: A61K37
IPC codes: A61K38, A61P13, A61P15