WO1996006634A1

WO1996006634A1 - Method for the treatment of indirect inguinal hernias and related conditions

Info

Publication number: WO1996006634A1
Application number: PCT/AU1995/000531
Authority: WO
Inventors: John Medwyn Hutson; Georgia Armat Paxton
Original assignee: The University Of Melbourne; Royal Children's Hospital Research Foundation
Priority date: 1994-08-29
Filing date: 1995-08-24
Publication date: 1996-03-07
Also published as: AUPM771894A0

Abstract

A method for the treatment of an indirect inguinal hernia or related condition in a male or female animal, including a human, comprises administering to a subject in need of such treatment human or other calcitonin gene-related peptide (CGRP) or an analogue thereof having CGRP activity, optionally in association with a pharmaceutically acceptable carrier and/or excipient, in an amount effective to cause closure of the hernial sac in the subject.

Description

METHOD FOR THE TREATMENT OF INDIRECT INGUINAL HERNIAS

AND RELATED CONDITIONS

FIELD OF THE INVENTION

This invention relates to a method for the treatment of indirect inguinal hernias and related conditions such as scrotal hydroceles and hydroceles of the spermatic cord, and in particular to the use of calcitonin gene-related peptide or an analogue thereof having calcitonin gene-related peptide activity in this treatment.

BACKGROUND OF THE INVENTION

Inguinal hernia is the most common congenital abnormality requiring surgical operation. In mid-gestation, the processus vaginalis develops as a diverticulum of the peritoneum within the gubernaculum, which is the mesenchymatous ligament attaching the testis to the anterior abdominal wall. At the start of the last trimester of pregnancy, in males the gubernaculum migrates from the inguinal abdominal wall to the scrotum and the processus vaginalis enlarges as a tubular diverticulum inside the gubernaculum. The testis descends inside this tubular extension of the abdominal cavity and arrives in the scrotum usually just before birth. Prior to birth in both males and females the processus vaginalis begins to obliterate, completely occluding any potential hernia in the female, and in the male separating the distal end of the processus vaginalis, now called the tunica vaginalis and containing the testis, from the abdominal cavity. At birth, approximately 85% of infants have a patent processus vaginalis, but this decreases to approximately 60% at one year. In adults approximately 15-35% still have a patent processus vaginalis.

By convention, an "indirect" inguinal hernia occurs when an intraabdominal structure, such as the bowel, protrudes through the patent processus vaginalis to create a lump in the groin. About 40% of inguinal hernias in children occur in the first six months after birth, and hernias are 8-10 times more common in boys than girls. Ninety percent of hernias in infants and children are unilateral and 10% bilateral. In very young patients who develop a clinical hernia, the chance of developing another hernia on the opposite side is greater than in older children. Hence, surgery is often performed on both sides in small infants.

Patency of the processus vaginalis may also lead to two other related clinical problems or conditions, scrotal hydrocele and hydrocele of the spermatic cord. A scrotal hydrocele occurs when the patent processus vaginalis is very narrow, preventing the entry of bowel loops, but allowing intraperitoneal fluid to trickle down to the scrotum, where it accumulates around the testis. Hydrocele of the spermatic cord is a dilatation of the processus vaginalis separate from and above the testis but connected by a narrow patent channel to the peritoneum.

Both these conditions would be simply observed in infancy in the hope that spontaneous obliteration of the processus vaginalis will occur and the fluid will be resorbed. Surgical treatment is offered (with inguinal herniotomy, as for a normal indirect inguinal hernia) after two years of age. No treatment (medical or surgical) is performed currently in babies, but a suitable non-surgical treatment of hydroceles in infancy would ensure speedy and complete obliteration.

Further description of the clinical conditions of inguinal hernias scrotal hydroceles and hydroceles of the spermatic cord is contained, for example, in Jones' Clinical Paediatnc Surgery, 4th Edition, Blackwell Scientific Publications (Eds. J.M.Hutson, S.W.Beasley and A.A. Woodward), pages 170-174.

The clinical diagnosis of an inguinal hernia in a child is always an indication for surgery because of the risk of incarceration, where the bowel becomes stuck within the hernial sac. This may proceed to strangulation, where the blood supply to the bowel is compromised and a bowel obstruction or death of the bowel may ensue. In addition, there is a significant risk of atrophy of the testis, because the testicular vessels passing to the testis are compressed where they exit the abdominal cavity through the inguinal canal. Strangulated hernias carry an approximate 10% risk of subsequent testicular atrophy.

At present, the standard treatment for an inguinal hernia is an operation called herniotomy. Under general anaesthesia, the inguinal canal is surgically opened and the patent processus vaginalis, which is a thin membranous tube connecting the abdominal cavity to the scrotum, is separated from the vas deferens and the testicular artery and veins. The processus vaginalis is then divided surgically and closed with a suture at the upper end of the inguinal canal, which is known as the internal inguinal ring.

Surgical treatment of an inguinal hernia is the most common procedure for congenital abnormality and the most common operation performed by paediatric surgeons around the world. Since this treatment is performed under general anaesthesia there is a risk of attendant complications. While children may be given a full general anaesthetic, have the hernia or hernias repaired and be discharged on hour or two after operation; in very premature infants or newborn babies, it may be necessary to observe the patient overnight to avoid respiratory complications following general anaesthetic.

To date, no non-surgical treatment of inguinal hernias has been sufficiently successful to be widely adopted in practice, and in particular, no hormone therapy has been successful in the past.

The only hormonal treatment which may have any effect on the patency of the processus vaginalis is the use of human chorionic gonadotrophin (hCG). This has never been used directly to treat inguinal hernias but is in current use for the putative treatment of undescended testes. There have been reports showing that the incidence of patency of the processus vaginalis in children with undescended testis is significantly lower following hCG treatment. Elder (Journal of Urology Vol.148:624-626, 1992) and Herzog et al. (Journal of Urology Vol.148:622-623, 1992) found a close correlation between patency of the processus vaginalis and the degree of epididymal development. The development of the epididymis is controlled by testosterone, which is stimulated to be produced from the testis by administration of hCG. Treatment with hCG in children with undescended testes reduced the frequency of a patent processus vaginalis from 69% to 31%. The degree of epididymal development was conversely increased with hormone treatment. These studies suggest a link between stimulation of androgens and the patency of inguinal hernias, although this has never been used as a method of treating inguinal hernias.

The effect of androgens on closure of the processus vaginalis does not appear to be a direct one, as the human processus vaginalis does not contain androgen receptors, as measured from 45 biopsies of the processus taken in children having orchidopexy for undescended testes (Johansen, European Urology 23:466-468, 1993).

In work leading to the present invention, it has been discovered that calcitonin gene-related peptide (hereafter CGRP) and analogues having CGRP activity are effective in causing closure of the hernial sac without surgery. This non-surgical treatment has significant advantages over operative treatment as no general anaesthesia would be required and probably no admission to hospital would be needed as the treatment could be performed simply as an outpatient, for example, by topical application or by injection. In addition, it has significant theoretical advantages over the use of hCG treatment which would produce systemic side effects including precocious puberty. CGRP could be administered locally to the groin, thereby avoiding many systemic side effects and in addition, not causing precocious puberty since it has no direct androgenic effect.

The use of CGRP or an analogue having CGRP activity in the treatment of undescended testes in male animals is disclosed in International Patent

Application No. PCT/AU91/00135 (WO 91/15246) and by Hutson et al. Curr. Opin. Invest. Drugs 2(6):635-643 (1993), however these compounds have not been previously proposed for use in the treatment of inguinal hernias.

SUMMARY OF THE INVENTION According to one aspect of the present invention, there is provided a method for the treatment of an indirect inguinal hernia or related condition in a male or female animal, which comprises 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 pharmaceutically acceptable carrier and/or excipient, in an amount effective to cause closure of the hernial sac in the subject.

In another aspect, the present invention relates to the use of CGRP or an analogue thereof having CGRP activity, optionally in association with a pharmaceutically acceptable carrier and/or excipient, in the treatment of an indirect inguinal hernia or related condition in a male or female animal.

In yet another aspect, the invention relates to a pharmaceutical composition for the treatment of an indirect inguinal hernia or related condition in a male or female animal, 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 having CGRP activity in the manufacture of a medicament for the treatment of an indirect inguinal hernia or related condition in a male or female animal.

Animals which may be treated in accordance with the present invention include humans, as well as domestic animals. - 6 -

The references herein to "related condition" include other clinical conditions which are characterised by patency of the processus vaginalis, including scrotal hydrocele and hydrocele of the spermatic cord. The present invention also extends to the treatment of both complete and incomplete indirect inguinal hernia.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as

"comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

DETAILED DESCRIPTION OF THE INVENTION

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. The term CGRP extends to naturally occurring allelic variants of the CGRP peptide sequence.

Principally, but without limitation, CGRP refers to human σ-CGRP and β-

CGRP.

The sequences of human σ-CGRP, human /?-CGRP, rat CGRP, chicken CGRP and truncated CGRP analogues are set out in Hutson et al. (1993) supra.

CGRP (human) is available commercially from a number of suppliers, such as Celltech, USA and 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 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.

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.

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 or truncated 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. Known deletional or truncated variants of CGRP include CGRP (8-37) and CGRP

(21-37) disclosed in Hutson et al. (1993), supra.

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

Oriαinal Residue Exemplary Substitutions

Ala Ser

Arg Lys

Asn Gin; His

Asp Glu

Cys Ser

Gin Asn

Glu Asp

Gly Pro

His Asn; Gin

lie Leu; Val

Leu lie; 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, hydrophilicity, 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.

In International Patent Application No. PCT/AU91/00135, "CGRP activity" is defined as the ability to effect testes descent in animals having undescended testes. A convenient in-vivo assay for CGRP activity is disclosed which utilises isolated male gubernaculum tissue. In the presence of effective quantities of compounds having CGRP activity, the isolated male gubernacula 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 International Application No. PCT/AU91/00135, 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.

In the context of the present invention, "CGRP activity" is also defined as the ability to effect closure of the hernial sac in a subject having an inguinal hernia, and experimental procedures for determining such activity are disclosed hereinafter.

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 conventional, pharmaceutically acceptable carriers and/or excipients.

The formulation of such therapeutic compositions is well known to persons skilled in this field. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The pharmaceutical carriers employed may be, for example, either liquid or 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.

It is especially advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the human subjects to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and/or diluent. The specifications for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active ingredient for the particular treatment.

A wide variety of pharmaceutical forms can be employed. Injectable 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, 50/ g to 500mg per ml. Injectable solutions may be provided in ampoule 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 0.1mg to 1g. CGRP may be formulated into a cream, ointment, paste, salve or the like for topical application to the groin. Such forms may include skin penetrating agents to facilitate the passage of CGRP or analogues thereof through the skin. Suitable skin penetrating agents include dimethylsulphoxide (DMSO) and the like. Formulations for topical use containing CGRP or analogues thereof may be applied to the groin 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 1.0mg. 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 -ethyl-L-glutamate (U. Sidman et al., 1983, Biopolymers 22, 1 : 547-556), poly(2-hydroxyethyl-methacrylate) (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 subcutaneously in the groin or placed in contact with the skin of the groin.

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 an animal having an inguinal hernia in an effective amount. The term "effective amount" refers to an amount effective to cause closure of the hernial sac. 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. Administration by injection or topical application to the groin is generally preferred.

Preferably, the amount of CGRP or analogues thereof injected in a single bolus injection is from about 20//g to 1000//g per kilogram, and more preferably 1 /g per 5 to 10 g body weight. A single injection may be provided for treatment of a hernia, 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 groin or adjacent areas, CGRP is slowly released to effect closure of the hernial sac.

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.

Further features of the present invention are more fully described in the following Example(s). It is to be understood, however, that this detailed description is included solely for the purposes of exemplifying the present invention, and should not be understood in any way as a restriction on the broad description of the invention as set out above.

In the accompanying figures:

Figure 1 is a graph showing in vitro fusion of hernial sac specimens from males;

Figure 2 is a graph of age-adjusted data showing in vitro fusion of hernial sac specimens from males;

Figure 3 is a graph showing in vitro fusion of hernial sac specimens from females; Figure 4 is a graph showing in vitro fusion of hernial sac specimens in serum-free media; Figure 5 is a graph showing in vitro fusion of hernial sac specimens in serum-containing media; and

Figure 6 is a graph showing in vitro fusion of hernial sac specimens over an incubation period of 2-4 days.

EXAMPLE 1

This Example demonstrates that CGRP induces fusion of the processus vaginalis in the human, on exposure of patent human hernial sacs to exogenous CGRP in vitro.

A. METHODS

(1) Preparation of Hernial Sacs.

Hernial sacs were collected from male and female infants during the surgical repair of inguinal hernias at the Royal Children's Hospital, Melbourne, Australia. The hernial sac consists of the patent processus vaginalis and the surrounding cremaster muscle, and may contain abdominal viscera. During herniotomy, the hernial sac is lifted up and the vas and vessels, which lie posteriorly, are separated from it. The caudal end of the sac is cut and the free end twisted to the level of the internal inguinal ring to push anything contained in the sac back into the abdominal cavity. This end is then transfixed and ligated, and the remainder of the tissue excised. The resulting "tube" of tissue was collected for experimental work.

Tissue was transported in modified Hank's Balanced Salt Solution (HBSS)

[Flow Laboratories, Irvine, Scotland]. Using a Zeiss operating microscope, the hernial sac was opened longitudinally to expose the endothelial lining. Small strips of tissue were cut carefully with as little trauma as possible to surrounding tissue. External muscle and connective tissue layers were left intact. If the endothelial surface appeared to be ripped or damaged, that part of the tissue was discarded. The end of the hernial sac which had been twisted by forceps during hemiotomy was evident by the imprint of the forceps' teeth. This tissue was also discarded.

(2) Preparation of the Organ-Culture System. Stainless steel grids [Becton-Dickinson, Lincoln Park, New Jersey] were placed in the centre well of Falcon organ-culture plates [Becton-Dickinson, Lincoln Park, New Jersey]. Several drops of a heated solution of 2% Agar [BDH Chemicals Ltd., Poole, England] were placed on the grids and levelled with a scalpel blade.

The outer well of the culture plate was filled with 2 ml of Tyrodes salt solution. The centre well was filled with 650 μ\ of serum-free Dulbecco's Modification of Eagle's Medium (DMEM) containing antibiotics (100 lU/ml penicillin and 100 //g/ml streptomycin) and 1 mg/ml L-Glutamine.

Experimental plates contained a further 50 μ\ of a 10^"5M solution of human Calcitonin Gene-Related Peptide (hCGRP) [Celltech, USA] diluted in PBS [Oxoid, Unipath Laboratories, Basingstoke, Hampshire] in the centre well. Control plates contained an equal volume of PBS alone.

(3) Assembling the Organ-Culture System.

Strips of hernial sac tissue were placed on the grids in the organ-culture plates. Using forceps, and with the aid of the dissecting microscope, the tissue was gently folded in half so the peritoneal surfaces were opposed. Plates were incubated for 48 hours at 37°C in an humidified atmosphere of 5% carbon dioxide and 95% air.

(4) Processing Tissue Specimens.

After the incubation period, tissue specimens were held in place with a drop of hot agar, and fixed in Bouin's fixative overnight. Tissues were processed through graded alcohols and xylene to paraffin wax. Specimens were rotated through an angle of 90° when embedded in paraffin. This was in order to show the two layers of hernial sac tissue, (i.e. the opposing peritoneal surfaces), in the same section. Blocks were sectioned at 10 / m, stained with Haematoxylin and Eosin, and examined under light microscopy at low, medium and high power.

(4.1) Appearance of sections.

Sections appeared as either two rectangles of tissue lying one on top of the other, or as a folded strip of tissue, depending on which angle of 90° they were rotated through when they were embedded. The cellularity of the tissue varied between hernial sac specimens. In all specimens, the peritoneal surface was seen as a layer of cells above amorphous pink staining (collagenous) tissue. In some cases, several layers of cells were noted below the endothelial lining. The walls of the hernial sac varied in appearance between specimens. The inner part was usually collagenous tissue with scattered dark staining nuclei. Bundles of striated muscle were seen in the outer wall of some specimens, other specimens had an outer layer of nondescript cellular tissue, and some showed the collagenous tissue through the full thickness. Blood vessels were seen in many sections; if these ran longitudinally, the two closely opposing endothelial surfaces (filled with blood) looked very similar to tissue "fusion" under low power magnification.

B. COMPILATION OF RESULTS.

Results were compiled from tissue specimens cultured using serum-free media, under the standardised conditions described above, and were separated by sex. A total of 80 tissue specimens were cut from 19 hernial sacs collected from 12 males aged between 42 weeks gestational age and 5 years. A total of 29 tissue specimens were cut from 6 hernial sacs collected from 4 females aged between 3 months and 7 years.

Tissue specimens were not used in the compilation of results if two layers of tissue could not be seen in section, if they had unfolded during incubation, or if it was evident that the endothelial surface had been disrupted prior to, or during, organ-culture preparation.

(1) Criteria Used in Classification of Results. If two layers of hernial sac (with opposing peritoneal surfaces) were not seen in section (i.e. tissue unfolded or sectioned through the wrong plane), or if the peritoneal surface was absent for more than half the total length on one side, tissue specimens were classified as invalid for the compilation of results.

Tissue was classified as unfused if two distinct peritoneal surfaces could be seen under high power magnification [x400]. In nearly all cases, a gap could be seen between the two layers at low power magnification [x40]. If 20% or less of the total length of opposing surfaces looked like fusion (see below), tissue specimens were still classified as unfused. A small zone of fusion in an otherwise obviously unfused specimen was taken as clumsy handling of the tissue with forceps.

Tissue was classified as fused if no gap could be seen between opposing peritoneal surfaces for at least half of the total length (i.e. the epithelial cell layer was closely opposed and the connective tissue below this layer was indistinguishable between the two sides). Some of the tissues classified as fused appeared to be actively fusing; several layers of cells were observed along the line where the peritoneal surfaces should have been in opposition, and tissue surfaces appeared to be growing together. No distinction was made between different appearances of fused specimens, as the cellularity of hernial sacs varied. As fusion of the processus vaginalis is followed by obliteration, a non- cellular state may represent a later point in the fusion process.

C. RESULTS The following data refer to tissue specimens cultured in serum-free conditions with the addition of PBS (control) or CGRP (experimental) as specified. TABLE 1 Totals : Fusion of Hernial Sac Specimens from Males.

Control CGRP

Number Fused 6 17

Number Not Fused 16 10

Rate of Fusion in Total 6/22 17/27 Countable Sample 27.3% 63.0%

TABLE 2 Totals : Fusion in Hernial Sac Specimens from Females.

Control CGRP

Rate of Fusion in Total 5/11 3/5 Countable Sample 45.5% 60%

These results are shown graphically in Figures 1 and 3.

Results for males were adjusted according to the age of the child.

Children older than two years of age were not included in the sample population.

This was done because no cases of fusion were observed in either control or experimental culture conditions in tissue specimens from males of this age and above.

TABLE 3 Totals : Fusion of Hernial Sac Specimens from Males (Age-Adjusted Data).

Control CGRP

Rate of Fusion in Total 6/20 17/22 Countable Sample 30.0% 77.3%

These results are shown graphically in Figure 2.

D. DISCUSSION

Using serum-free media for organ-culture, a difference was apparent in the rate of fusion of tissue specimens between control and experimental conditions.

In specimens collected from male infants and children, and cultured with CGRP, fusion was observed in 17/27 (63%) of cases, compared to fusion in 6/22 (27.3%) specimens cultured under controlled conditions. The p-value from these data gave a 98% confidence interval with standard chi-square testing. These results are taken from the entire male sample.

It was found that there were no cases of fusion in the small number of older children (age > 2 years), and the distribution of fused hernial sac specimens varies markedly between different ages. Most cases of fusion occurred in tissue specimens from infants aged 3 months or under.

The age of the (male) child was then taken into account in the compilation of results: children ages > 2 years were not included in the sample population. The remaining data refer to hernial sac tissue specimens collected from infants aged < 11/12, 7 (from a total of 8) of these infants were aged < 3/12. In hernial sac tissue from these children, fusion was observed in 17/22 (77.3%) of specimens cultured with CGRP, and in 6/20 (30%) of specimens cultured under control conditions. A p-value <0.01 was derived from these figures using chi- square testing. Hernial sacs collected from female children also demonstrate fusion in an organ-culture system. The difference in rate of fusion between control and experimental conditions was smaller than in the male, however CGRP did cause fusion in a greater percentage of samples. Any figures derived from these data are limited by the extremely small sample size, and are insignificant with chi- square testing.

Although preliminary, the results obtained in this study show a difference in the rate of fusion of hernial sac tissue cultured with and without CGRP. Specimens exposed to CGRP in organ-culture were more commonly observed to be fused at the end of the culture period. This applied to tissue collected from both male and female infants and children.

Human hernial sacs represent a patent processus vaginalis. These results indicate that CGRP may function to cause the fusion and obliteration of the processus seen as a part of normal gubemacular development in the male and the female, and that local administration of CGRP could cause fusion of an abnormally patent processus postnatally. This would alleviate the need for surgery and its associated complications in infants and children.

EXAMPLE 2

This Example relates to work which involves a number of technical changes to the procedures of Example 1, including optimised culture conditions and improved quantitative analysis of the results.

A. METHODS

The methods as previously described in Example 1 were used in this Example with the changes set out below.

In Example 1 , the concentration of CGRP was 714nM whereas the concentration used in this Example is 7/ M, that is, the concentration has been increased by a factor of ten. The orientation of the specimen has also been improved. The specimen has to be rotated through 90° so that both the bottom and top layer of tissue, as well as the fold, could be seen in the section. After processing, the fold is quite difficult to distinguish therefore the possibility of incorrect orientation of the tissue played a large factor. This problem has now been rectified by placing a piece of prolene thread along the side or edge of the specimen so that the specimen could be embedded in the correct orientation without discrepancy.

In addition, the culture conditions used differ from those of Example 1 where the media used were CMRL-1066 media containing 10% foetal calf serum and Dubecco's Modification of Eagle's Medium alone for serum-free conditions. In this Example, Dubecco's was used for the serum containing experiments, and for the serum-free conditions Iscove's medium was used with the necessary serum-free additives; insulin (2//g/ml), transferrin (10//g/ml), BSA (0.75%) and lipids (Gibco, Grand Island, USA). These additives were used to optimise cell viability in the organ culture system.

The Hank's Balanced Salt Solution was changed once a week so that the solution did not turn alkaline over time and become suboptimal for viability of the specimen.

In Example 1, specimens were placed in Bouin's Fixative overnight and processed. In this Example, the specimens are fixed in formalin for 2 hours and processed, so that the histological material can be used for a variety of other methods, e.g. immunocytochemistry and in situ hybridisation.

B. COMPILATION OF RESULTS.

The criteria for assessing the degree of fusion was previously subjective and determined by observing fusion as "the epithelial cell layer being closely opposed and connective tissue below this layer being indistinguishable between the two sides". An unfused specimen was categorised as two opposing epithelial layers that were ordered, linear and usually a gap between the two layers. In the present Example, fusion has been quantitated as follows: every sixth section is drawn with the aid of a drawing tube and measured on the Sigma Plotter. The length of fusion is defined as Length of Fusion/fLength of Non-fusion + Length of Fusion). All the results from the Sigma Plot were compiled and mean and standard error were calculated.

C. RESULTS

The results obtained in the Example using optimised culture conditions and improved quantitative analysis of the results are shown graphically in Figures 4, 5 and 6. Figure 4, which sets out the results of experiments carried out in serum- free media, shows a very significant difference between the use of CGRP and phosphate-buffered saline (PBS). By contrast in Figure 5, which sets out the results of experiments in serum-containing media, the difference is less. It is proposed that this is because serum contains CGRP and therefore the controls have endogenous CGRP present in the test system. When this is removed in the serum-free conditions, the difference between the treated and the control samples is significantly enhanced.

Persons skilled in this art will appreciate that variations and modifications may be made to the invention as broadly described herein, other than those specifically described without departing from the spirit and scope of the invention. It is to be understood that this invention extends to include all such variations and modifications.

Claims

CLAIMS:

1. A method for the treatment of an indirect inguinal hernia or related condition in a male or female animal, which comprises 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 pharmaceutically acceptable carrier and/or excipient, in an amount effective to cause closure of the hernial sac in the subject.

2. A method according to claim 1 , wherein said subject is a human.

3. A method according to claim 1 , wherein said CGRP or analogue is human CGRP or an analogue thereof.

4. A method according to claim 1 , wherein said CGRP or analogue is applied topically to the groin region of the subject.

5. A method according to claim 4, wherein said CGRP or analogue is administered in the form of a topical preparation.

6. A method according to claim 5, wherein said topical preparation contains a skin penetrating agent.

7. A method according to claim 1 , wherein said CGRP or analogue is injected into the groin region of the subject.

8. A method according to claim 1, wherein said CGRP or analogue is administered from an implantable or skin-adhesive sustained release article.

9. A method according to claim 8, wherein said sustained release article is implanted subcutaneously in or placed in contact with the skin of the groin region of the subject.

10. Use of CGRP or an analogue thereof having CGRP activity, optionally in association with a pharmaceutically acceptable carrier and/or excipient in the treatment of an indirect inguinal hernia or related condition in a male or female animal.

11. Use of CGRP or an analogue thereof, having CGRP activity in the manufacture of a medicament for the treatment of an indirect inguinal hernia or related condition in a male or female animal.

12. A pharmaceutical composition for the treatment of an indirect inguinal hernia or related condition in a male or female animal, which comprises CGRP or an analogue thereof having CGRP activity in association with a pharmaceutically acceptable carrier and/or excipient.

13. A composition according to claim 12, wherein said CGRP or analogue is human CGRP or an analogue thereof.

14. A composition according to claim 12, in the form of a topical preparation.

15. A composition according to claim 14, wherein said topical preparation contains a skin penetrating agent.

16. A composition according to claim 12 in the form of an injectable preparation.

17. A composition according to claim 12, in the form of an implantable or skin- adhesive sustained release article.