Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
.: WO91/15~6 PCT/AU91/00135
2~3~
:
,~
CALCITONIN GENE RELATED PElrrIDE FOR THE TREATMENT OF VNDESCENDED
TESTICLES
This inventlon relates to a method for the non- ::
surglcal treatment of undescended testicles in male ;~
animals. The lnvention is also concerned~with
pharmaceutical compositions for the treatment o~ :
undescended testicles.
The testes are two glandular organs which secre~e
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 androgenlc
hormones (testosterone). Androgens were proposed to act
on mesenchymal tissue in the groin known as the
gubernaculum, which migrate-q across the pubic region from
the groin to the scrotum during inguino-scrotal
- WO91/15~6 PCT/AU91/0013~
2~3~ 2 - ~
- ~ 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 l 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.
~ WO91/15~6 PCT/AU91/00135
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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. 30~, 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-Leu-Leu Ser-Arg-Ser-Gly-Gly-Val-Val-Lys-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
: : - ,.. :
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WO91/~6 PCT/AU91/00135
% ~ ,'3 ~
coding sequence of CGRP will be smaller than those with
the amino or carboxyl terminal fusions, of the order of
say l 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 l to lO amino acid residues; and deletions will
range from about l 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
dif~erent residue inserted in its place. Such
substitutions generally are made in accordance with the
following Table.
WO 91/15246 PCI~/AU91/S10135
_ 5 _ 2~3~
TABLE l
Oriqinal Residue Exem~lary Substitu-tions
Ala Ser
Arg Lys
Asn Gln; Hls
- Asp Gl u
Cys Ser
Gln Asn
Glu Asp
l O Gly Pro
His . Asn; Gln
Ile Leu; Val
Leu Ile; Val
Lys Arg; Gln; Glu
l 5 Met Leu, Ile
Phe Met; Leu; Tyr
Ser Thr
Thr Ser
Trp Tyr
Tyr Trp; Phe
Val Ile; Leu
". , : ,, ,: ., .. : - ,, :, .. ,. -
W091/15~6 PCT/A1J91tO0135
6 -
Generally amino acids are replaced by other amino
acids having like properties, such as hydrophobicity,
hydropholicity, electronegativity, bulky slde 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 manipulatlons
upon the gene encoding CGRP of any particular animalO
Techniques for making substitution mutations at
predetermined sites in DNA having a known sequence are
well known, for example Ml3' 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, l982).
The above referenced amino acid sequence variants of
CGRP may all be regarded as analogues of CGRP, if they
possess CGRP activity as is deined 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, bs 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 techniquesO
"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
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- , .
WO91/15246 PCT/AU9l/00135
_ 7 _ 2~3~
effective quantities of compounds having CGRP activity,
the lsolated male gubernaculum undergo rhythmic
contractions and in some instances serpentlne movementO
Such activity-in the presence of CGRP is not observed
with female gubernacula, skeletal muscle or u~bilical
cord tissue. Using this convenient assay, further
details of which are provided in the examples of this
specification, compounds may be readily ~ested 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, ln~
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), tTyr']-
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,
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~ WO9l/15~6 PCT/AUgl/00l35
; ~? ; ~
2~3~ - 8 -
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. Injectable forms of CGRP genarally contain
CGRP dissolved in a sterile vehicle such as water,
saline, dextrose or the like. In~ectable solutions of
CGRP may contain, for example, 50~g to 500mg per mlD
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 (DMS0) 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
: ,, ::: . i ~
WO9l/15~6 PCT/A~91~0135
9 2~803~
ace~one 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
l.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 y-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 sub-cutaneously
in the scrotum or placed in contact with the skin of the
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P~ 00~5
W~91/15~6
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scrotum. Animals which may be treated accordlng 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 o CGRP or analogues
thereof may be parenteral, topical, or oral. The term
"parenteral" as used herein includes intravenous,
intramuscular or subcutaneous administration. The
Z0 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.
Pre~erably, the amount of CGRP or analogues thereof
inJected into the scrotum in a single bolus injection is
from about 20,ug to lOOO~g per kilogram, and more
preferably l,ug per 5 to 10 g body weight. A single
~0 scrotal injection may be provided for testis descent, or
several injections over a limited time period such as
~rom 1 to 10 days.
Where an implantable or skin-adhesive sustained
release article is applied to the scrotum or adjacent
':' ' ' ' ~ , ~ ' ", ', ` ~
WO91/15~6 PCT/AU91/00135
'~ 11- 2~35~
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 ~he 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 adminlstration of CGRP or
analogues thereof will often depend upon the judgement of
a consulting physician or veterinarian in any particular
case.
The optimal course o~ 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 ~ests.
Generally, treatment with CGRP or analogues thereof
to effect testes descent usually ta~es 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 chemotac*ic 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
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W091/15~6 PC~/A~91/00~3S
20~35~ 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 l, there is shown a displacement curYe
derived by computerized densitometry from serial
gubernacular sections incubated with increasing
concentrations of unlabelled human ~GRP in the presence
of radiolabelled human CGRP.
.
EXAMP~E 1
Analysis of,the Genito~emoral Nerve (GFN~:
The genitofemoral nerve ( GFN ) is essential ~or
inguino-scrotal testicular descent as transection of the
nerve prevents gubernacular migration from groin to
scrotum (~easley, S.W. & Hutson, J.M. [l987] Aust. NZ
J.Surg. 57, 49-5l). 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 cyrle. Ten male rats ranging
ln age ~rom 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
:, ., . ,: , : - -: ...
- :: : . .... : ~ . .,...... :.
WO91/15~6 PCT/AU91/00135
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of either diamidinophenyl indole (DAPI - Sigma) or Fast
Blue (Si~ma) 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. ~he thorax was opened,
and the inferior vena cava divided below a ligature.
Transcardiac perfusion of lO 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 4C, before
being cleared in dimethyl sulphoxide and storad in
Phosphate Buffered Saline (PBS)/30% sucrose overnight.
The pelvis was embedded in UCT plastic medium cooled
by liquid nitrogen and isopentane, then 20,um 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 epifluore~cence 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
ollows 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 surace of
the cremaster muscle, supplying this, then turns
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PCr/A~D~
WO91~15~6
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cranially to enter the gubernaculum from its distal
attachment, where branches fan out supplying the
gubernacular substance and the caudal epididymis.
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 hecome 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
lS 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
Histoch~mical 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
. , . . . ................ . .,. , ~ . - , :.
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W091/15~6 PCr/AU91/00135
~ - 15 - 2~03~
female rats, aged 6 days at operation, and 5 male, 5
female and 5 Testicular Feminization Syndrome (TFM) mice
aged lO days old at operation.
Antibodies against thirty different neuropeptides
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).
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 l:2000.
Animals were anaesthetised, surgically prepared, and
tissues removed and fixed as for Example l.
Tissues were embedded in OCT plastic medium, cooled
with liquid nitrogen and isopentane, then l4 ,um frozen
serial sections were cut onto 1% gelatin-coated slides,
and left to dry. The spinal cords were cut transversely
and the pelvises were cut in the sayittal plane. ~lides
were pre-incubated for 30 minutes with a covering to lO~
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 lO~
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 l:200 with
P~S (Silenus Laboratories, Hawthorn, Vic.). Following
another rinse, coverslips were mounted with buffered
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: W09l/15~6 PCT/~U9~ 13~
..; . ~, ' ~
2~8~35~ - 16 -
- glycerol, and the pattern of fluorescence observed wlth
an epiflu~rescence microscope (Leitz Diaplan).
The location of immunohistochemical staining was
compared with the retrograde labelliny on the same
sections by simply changing the excitation filter~ as
different wa~e lengths are required in order for FITC
(520nm) and DAPI (400nm) to emit light. To quantify
cells consistentl~ all sections were counted, and only
those cells which stained brightly and in whieh the
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
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 mutan-t litter matesO
Statistical analysis was performed using both the
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:
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,
includin~ vasoactive intestinal peptide, 5-
hydroxytryptamine, somatostatin 8, met-enkephalin,
substance P, thyrotrophin releasing hormone and
neuropeptide Y, where absent from the lumbar anterior
horn.
- . . , ::
-WO9t/15~6 YCT/AU91/00135
~ - 17 - 2~$~3~4
In male rats double labelllng 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 ~or '`
CGRP, as did a small number of non-DAPI-labelled motor
neurons which werei 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 l).
Quantitative studies on micP 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 ~
O.Ol) 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
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W091/15~6 PCT/AU9ltO0135
- 18 -
2 ~ ~ ~ 3 5 ~ 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 e~erts 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 CGR~ thereon.
Gubernacula (n = 182) from male rats (O-S 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
10-11M) 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,
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WO91/15~6 PCT/AU91/00135
9 2 ~
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 migrationO
EXAMPLE 4
Intact GFN is Required fo~ 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, testlcular
descent is inhibited.
Further, CGRP (470 ~mole/day for 14 days) was
in;ected into the right groin lateral to the inguinal
region. Water in;ections at the same site acted as a
control. Two out of five right testes were undescended
,
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WV9l/15~6 PCT/AU91/00135
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(40%) with water in~ections compared with four out of six
(67~ when CGRP was in~ected 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 andiits 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-,um thickness were cut on a cryostat at
-12C and thaw mounted on 0.5~ gelatin-coated slides.
The sections were dried in a vacuum desiccator overnight
at 4C and stored at -70C. Alternate sections were
stained with haematoxylin and eosin in order to identify
ine structures of the 12sI-labelled gubernacula at the
tlme of quantitation.
Radiolabelled human CGRP {(2-[l2sI)iodohistidyl10)-
human CGRP} was obtained from Amersham International plc.
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~ WO91/15~6 PCT/~U91/00135
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(Buckinghamshire, England) (~2000 Cl/mmol). The
following synthetic peptides: human CGRP, rat CGRP ~8-
37), rat CGRP (Tyr27, 28-37), salmon thyrocalcitonin,
human vasoactive intestinal peptide (VIP), soma~ostatin,
and substance P were purchased from Au~pep Pty. Ltd.
(Melbourne, Australia); human thyrocalcitonin and
Serotonin (5-hydroxytryptamine) from Sigma Chemical Co.
(St. Louis, M0).
The following buffer was used for incubating tissue
sections with various ligands: lO0 mM HEPES, containing
120 mM NaCl, 1.2 mM MgS0~, 2.5 mM KCl, 15 mM NaC2H302,
10 mM D-glucose, 1 mM EDTA, 0.5~ BSA, and 0.35 mM
Bacitoracin.
Conditions for optimising binding were examined a~
4C, 22C, 37C, 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 4C in the foregoing buffer at pH 8.0,
containing 35 pM [125I]-human CGRP. To determine non-
specific binding, some sections were also incubated with
l ,uM unlabelled human CGRP. Displacement of [12sI]-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 (Tyr27, 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
- non~pecifically-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
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WO91/15~6 PCT/A W1/00135
~ 3~ - 22 -
room temperature. Radioactive standards used
autoradiographic t125I3 micro-scales (Amersham
International plc., Buckinghamshire, England), which
consist of lO layers of [125I]-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
densitometry using an EyeCom Model 850 image processor
(Log E/Spatial Data systems, Springfield, Virginia,
U.S.A.), coupled to a DEC ll/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 l to 2 seconds in Amersham LM-l emulsion
(Amersham International plc., Buckinghamshire, England)
immersed in a waterbath at 43C, dried for one hour at
room temperature with a relative humidity of 60~, and
stored in plastic slide box containing silica gel at ~C
for exposure for 4 weeks. Slides were then developed for
5 minutes with a l:l dilution of Kodak Dl 9 developer with
distilled water at 20C, immersed in a stop solution of
l~ acetic acid for 30 seconds at 20C, in a fixative (a
mixture of l:4 dilution of Ilford Hypam Fixer with
distilled water, and Ilford Hypam Hardener) (Ilford Pty.
Ltd., Melbourne, Australia) for lO minutes, and in gently
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~ - 23 - 20~a~5~
running water for l hour. Followlng development the
sections were dried and stained with haematoxylin to
enable localisation of grain densitles.
Binding saturation was achieved by 24 hours
incubation at 4C with the buffer pH at 8Ø Either
longer lncubatlon time, higher lncubation temperature, or
higher buffer pH than the optimal condi~ions r2sulted in
higher non-specific binding. Definitive bindin~ studies
were, therefore, performed at 4C for 24 hours, when
speclf~c binding was ~een.
Displacemen~ curves were derived by computeri~ed
densitometry from serial sections incubated with
increasing concentrations of unlabelled human CGRP in the
presence of radiolabelled human CG~P (Figure l).
Computer-derlved plots of these data also revealed a
single class of binding sites.
Rat CGRP (8-37) was potent in competing with [l25I~-
human CGRP with affinity constants similar to or greater
than that of unlabelled human CGRP; salmon calciton~n
competed at this slte wlth a lower affinity than human
CGRP, while the human calcitonin had only a very low
potency, rat CGRP (Tyr27, 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 l X lO-6M.
Light microscopic localisation of ~125I]-human CBRP
receptor binding using emulsion autoradiography revealed
a high density of binding over the cremasteric muscle. A
comparison of the autoradiograph wlth the stained
ad~acent sectlon shows that grain densities are confined
to the thick layers of the inner and outer cremasteric
muscles. ~lnding was fairly low in the central
mesenchyme.
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PCT/AU91/0~135
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EXAMPLE 6
C~P A~tagonists Delay Testicular Descent:
n this Example, the effects of theiCGRP antagonist
CG~P (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 ~l 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 ~l 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.
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WO91/15~6 PCT/AU91/00135
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The control group of mlce were in~ected with
phosphate buffered saline t 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 mlce.
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 in~ection
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, CG~P (8-37)
completely prevented descent of 3 testes.
CGRP (8-37) binds to the receptors and compet~tively
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
WO91/15246 PCT/AU91/00135
2~3~3~ - 26 -
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 ~hat the invention
includes all such variations and modifications which fall
within its spirit and scope. The invention also includes
:O 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.
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TABLE 1
OUANTIFICATION 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 Z98 148 50
FEMALE 163 35 22
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WO 91/15~6 PCT/AU91/0013$
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OUANTIFICATION OF C~L$ I~ MALE, F~MALE AND TFM ~ICE
No. of DAPI- No. of CGRP- % o~ DAPI
labelled positive labelled cells
sex cells also contain- containing CGRP
ing
DAPI
mean+-SD mean+-SD mean~-SD
143 60 42
138 71 5
MALE 281 251 +or- 104 101 114+or-53 36 46+or 7
336 178 53
356 178 46
p < 0.01 p < 0.01 p < 0O05
109 18 17
117 55 47
FEMALE 69 107 +or- 22 27 32 +or-14 39 30 +or- 12
122 2~ 23
1 26
p < O.01 p < 0.05 NS
210 101 48
221 123 56
TFM 168 184 +or- 33 39 70+or-40 23 36 +or- 15
174 46 26
141 39 28
SUBStlTUTE SHEET
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