Rapid suppression of plasma testosterone levels and tumour...

JPET #112326

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Rapid suppression of plasma testosterone levels and tumour growth in the

Dunning rat model treated with degarelix, a new GnRH antagonist

Marc Princivalle, Pierre Broqua, Richard White, Jessica Meyer, Gaell Mayer, Lucy

Elliott, Ketil Bjarnason, Robert Haigh and Christopher Yea

Ferring Research Ltd, Southampton, UK (M.P., P.B., J.M., G.M., L.E., R.H., C.H.)

Ferring Inc, San Diego, USA (R.W.)

Ferring International Center, Copenhagen, Denmark (K.B.)

JPET Fast Forward. Published on December 19, 2006 as DOI:10.1124/jpet.106.112326

Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on December 19, 2006 as DOI: 10.1124/jpet.106.112326

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Running Title: Tumour suppression with degarelix, a new GnRH antagonist

Corresponding author: Marc Princivalle, Ph.D., Ferring Research Ltd, Chilworth

Science Park, 1 Venture Road, SO16 7NP Southampton, UK. Phone: +4423 8076

3480, Fax: +4423 8076 6253, [email protected]

Number of text pages: 22

Number of tables: 2

Number of figures: 4

Number of references: 40

Number of words in the abstract: 230

Number of words in the introduction: 806

Number of words in the discussion: 704

Abbreviations: GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone;

FSH, follicle stimulating hormone; LHRH, Luteinizing hormone releasing hormone;

SPF, Specific pathogen free; MW, Molecular weight; LPL, Leuprolide; RIA,

radioimmunoassay; ANOVA, analysis of variance

Section assignment: Endocrine and Diabetes


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Abstract

Degarelix (FE 200486) is a member of a new class of water-soluble (>50 mg/ml)

GnRH antagonists in clinical development for prostate cancer. Upon subcutaneous

administration degarelix forms a gel resulting in a sustained release of the compound

into the circulation immediately blocking GnRH receptors in the pituitary inducing a

fast and sustained suppression of gonadotrophin secretion in rats and primates. One of

the few animal models of prostate adenocarcinoma is the Dunning R3327H rat

carcinoma transplanted into Copenhagen rats. The growth of the Dunning tumour can

be inhibited by various treatments reported to be effective in the clinic such as GnRH

superagonists, antiandrogens, 5-alphareductase inhibitors, tyrosine kinase inhibitors

and surgical castration. We report in this study that degarelix produces a fast and

sustained suppression of the pituitary gonadal axis in rats and a similar inhibition of

tumour growth compared to surgical castration in the Dunning R3327H rat carcinoma

model. Firstly, degarelix as been compared with D-Trp6-LHRH and surgical

castration on a short-term study (2 months) and secondly, degarelix has been

compared with Leuprolide and surgical castration on a long-term study (12 months).

In both studies, degarelix demonstrated a sustained inhibition of tumour growth at

least comparable to surgical castration. These data provide a convincing profile of

degarelix as a potential candidate for the clinical management of sex steroid-

dependent pathologies, such as prostate cancer, where long-term reversible chemical

castration is required.


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Prostate cancer is a leading cause of mortality and morbidity of men in the

industrialised world, second only to lung cancer. In these countries, where the

incidence is highest, a man has a 10% chance of developing prostate cancer, and a 3-

4% chance of dying from the disease (Cook and Sheridan, 2000). In the European

Community and in the US, where both the prevalence and incidence of prostate

cancer are increasing, more than 500’000 new cases are diagnosed each year, with an

estimated 50’000 deaths (Jemal et al., 2004; Kirkels and Rietbergen, 1997). The

number of patients with prostate cancer is expected to increase steadily over the next

decade because the average age of men is increasing and the incidence of prostate

cancer increases more rapidly with age than the incidence of other cancers (Jemal et

al., 2004).

A widely recognised feature of prostate cancer is its high sensitivity to testosterone

deprivation. The current medical approach in the treatment of androgen-dependent

prostate tumours is to suppress testosterone production using agonist analogues of the

hypothalamic gonadotrophin-releasing hormone (GnRH, also known as LHRH) that

control the secretion of luteinizing hormone (LH) and follicle-stimulating hormone

(FSH) from the anterior pituitary gland. Chronic administration of GnRH

superagonists, eventually leads to a desensitisation of pituitary GnRH receptors and a

corresponding inhibition of gonadotrophin and testosterone secretion (Heber et al.,

1982; Nett et al., 1981; Rivier et al., 1978). However, a major drawback of agonist

therapy is that it initially stimulates gonadotrophin and testosterone secretion,

resulting in a surge in LH and testosterone levels for 2-3 weeks before castration is

completed and a subsequent delay in therapeutic benefit. This temporary increase in

testosterone levels can exacerbate the hormone-sensitive cancer, as well as patient


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symptoms such as sudden paraplegia due to spinal cord compression by epidural

metastases, exacerbation of bone pain and urinary retention (Kahan et al., 1984).

Therefore anti-androgens are often co-administered in the initial phase of GnRH

receptor agonist treatment to minimise the stimulatory effects of the testosterone surge

on the prostate cancer cells (Dupont et al., 1988). Furthermore, “depot” injections

may result in small bursts of androgens (acute on chronic phenomenon), which may

over-stimulate growth of tumours that have adapted (through gene mutations) to low

levels of androgens by over-activity of androgen receptors and associated

transcription factors (Suzuki et al., 2003; Langeler et al., 1993; Montgomery et al.,

2001).

In the search for alternatives to superagonist therapy, researchers have been

developing GnRH antagonists that suppress the release of gonadotrophins by binding

competitively to pituitary GnRH receptors. GnRH antagonists do not induce the LH

and testosterone surge, but instead work directly to reduce the release of

gonadotrophin and testosterone within hours. Despite the advantages of GnRH

antagonists over agonists in suppressing serum gonadal steroids, many of the peptide

GnRH antagonists investigated so far show histamine–releasing properties and/or

solubility limitations that affect their clinical usefulness or even preclude their

development as drugs (Bagatell et al., 1993; Bagatell et al., 1995; Hocart et al., 1987;

Flouret et al., 1992).

Degarelix (FE 200486) is a member of a new class of water-soluble (>50 mg/ml)

GnRH antagonists forming a subcutaneous gel, that has only very weak histamine-

releasing potential compared to other GnRH antagonists (Broqua et al., 2002; Tornoe


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et al., 2004; Schwach et al., 2003; Schwach et al., 2004). When administered

subcutaneously in rats and primates, degarelix immediately blocks GnRH receptors in

the pituitary resulting in fast and sustained suppression of gonadotrophin secretion

with the absence of initial stimulation of the gonadotrophic axis (Broqua et al., 2002).

The present study compares the pharmacological activity of a new GnRH antagonist,

degarelix to two widely and currently used GnRH superagonists (D-Trp6-LHRH and

Leuprolide) in an experimental model of prostate adenocarcinoma in both short and

long-term studies. The Dunning R3327H rat carcinoma transplanted into Copenhagen

rats (Redding and Schally, 1981; Block et al., 1977; Claflin et al., 1977) is of

dorsolateral prostatic origin, developed spontaneously in an aged animal, has

histological and biochemical similarities to the human prostatic carcinoma and is

androgen-dependent (Hierowski et al., 1983; Daehlin et al., 1986; Daehlin and

Damber, 1987). The growth of the Dunning tumour can be inhibited by various

treatments reported to be effective in the clinic such as GnRH superagonists,

antiandrogens, 5-alphareductase inhibitors, tyrosine kinase inhibitors and surgical

castration (Ichikawa et al., 1988; Pinski et al., 1994; George et al., 1999; Presnell et

al., 1998; Tennant et al., 2000).

Blockage of the GnRH receptor by GnRH antagonists produces a fast, profound and

sustained suppression of gonadotrophin release and therefore testosterone secretion in

human males (Jockenhovel et al., 1988; Salameh et al., 1991; Bagatell et al., 1993;

Bagatell et al., 1995; Klingmuller et al., 1993; Pinski et al., 1994). Consequently,

antagonists such as degarelix, have been recognized as potential drugs for the

management of sex steroid-dependent pathologies, such as prostate cancer.


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Material and methods

Animals

In vivo studies were performed by Oncodesign, l’Arbesle, France. Male

Copenhagen/Hsd rats, 7-8 weeks old and weighing 100-125g, were obtained from

Harlan Breeding Center (Indianapolis, USA). Animals were observed for 7 days in a

specific-pathogen-free (SPF) animal care unit before tumour implantation. The animal

care unit (INRA, Dijon, France) is authorized by the French Ministry of Agriculture

and Research (Agreement No. A21100). All animal experiments were performed

according to ethical guidelines of animal experimentation. Animals were maintained

in rooms under controlled conditions of temperature (25°C ± 1°C, humidity (55 ±-

1%), photoperiod (12h light/12h dark) and air exchange. The experimentation room is

maintained at a positive pressure to prevent outside contamination into the rat colony.

Animals were housed in polycarbonate cages (UAR, Epinay sur Orge, France) with

sterile wood shavings (UAR) as bedding, which was replaced twice a week. Diet

(controlled and sterilised granules, AO4) was purchased from UAR. Food and water

were provided ad libitum. Water bottles were cleaned, sterilised and replaced once a

week. The water was sterilised by filtration using 0.2 µm absolute filters (Millipore,

USA)

Tumour preparation

Cryopreserved R-3327H tumour trocar pieces were obtained from John Hopkins

Oncology Centre (Dr J.T. Isaacs, Baltimore, MD) and kept in liquid nitrogen. Before

implantation, the trocar pieces were thawed by immersing the vial in a 37°C water


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bath and tumours washed in RPMI medium. Surgical operations were performed

under isofluran anaesthesia. The hairs from the flank region of rats were shaved and a

skin incision was made. Fresh tumour trocar pieces of R-3327H tumour were serially

implanted subcutaneously into the right flank of the rats (1 tumour/rat). Before

implantation, the mean weight of 10 tumour fragments was 48.5 ± 2.6 mg.

Test substance

Degarelix (N-acetyl-3-(naphtalen-2-yl)-D-alanyl-4-chloro-D-phenylalanyl-3-(pyridin-

3-yl)-D-alanyl-L-seryl-4-[[[(4S)-2,6-dioxohexahydropyrimidin-4-yl]carbonyl]amino]-

L-phenylalanyl-4-(carbamoylamino)-D-phenylalanyl-L-leucyl-N6-(1-methylethyl)-L-

lysyl-L-prolyl-D-alaninamide), FE 200486 acetate salt (Batch No 2010-042-01, MW:

1632.3, peptide content: 88.2%, purity:98.8%) was supplied by Ferring Research

Institute (San Diego, USA) as a white powder. It was stored at -20°C. The

manipulation of compound was performed under laminar flow conditions. The vehicle

of degarelix was 5% Mannitol diluted in sterile distilled water. The degarelix

solutions were prepared just before administration to animals.

D-Trp6-LHRH acetate, (Triptorelin, Batch No 0537085, MW: 1311.5, purity: 89.9%)

was obtained from Bachem (Zurich, Switzerland) as a white powder. It was stored at -

20°C. The manipulation of compound was performed under laminar flow conditions.

Leuprolide-depot (LPL, Enantone, Batch No S306) was supplied by Oncodesign from

Takeda (Puteaux, France). The vehicle for Leuprolide was composed of cellulose and

D-mannitol diluted in distilled water just before administration to rats.

Treatment doses and regimens


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Treatments started at D0 when the tumour size reached approximately 300 mm3.

Before treatment, the mean tumour size of each group at randomisation was not

statistically different. For the short-term study, D-Trp6-LHRH was administered

subcutaneously at 0.5 mg base/kg daily for 62 days (n=10). At this dose, D-Trp6-

LHRH induced a complete suppression of testosterone after the flare-up period to

levels published in the literature (Redding and Schally, 1981; Redding and Schally,

1990; Schally et al., 1986). Degarelix was administered subcutaneously at 1.0 mg

base/kg monthly (n=10). Pharmacological profile of degarelix has been described

recently (Broqua et al., 2002; Agerso et al., 2003). At this dose, degarelix induced

complete and rapid suppression of testosterone. One control group received a monthly

subcutaneous injection of 5% mannitol (n=10) and another control group was

castrated and injected subcutaneously with 5% mannitol once a month (n=10). The

blood samples were collected at D-1, D0, D1, D2, D3, D4, D5, D6, D7, D10, D14,

D21, D28, D,35, D42, D49 and D62. For the group treated with D-Trp6-LHRH, blood

samples were also taken 2 hours after treatment. For the long-term study, Leuprolide-

Depot was administered subcutaneously at 1.5 mg base/kg every 3 weeks until day

288 (n=11). At this dose, Leuprolide-depot induced a complete suppression of

testosterone after the flare-up period to levels published in the literature (Okada et al.,

1991). Degarelix was administered subcutaneously at 1.0 mg base/kg monthly (n=11)

(Broqua et al., 2002; Agerso et al., 2003). One control group received a monthly

subcutaneous injection of 5% mannitol (n=11) and another control group was

castrated and injected subcutaneously with 5% mannitol once a month (n=11). Blood

samples were collected at D0, D2 and every month thereafter until the end of the

study D343.


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Blood sample collection and testosterone assay

Blood samples (approximately 500µl) were taken 2 hours after treatment from the

caudal vein into tubes containing lithium heparin and immediately centrifuged at

1000xg for 10 min at 4°C to obtain plasma samples. The samples were collected in

propylene tubes and stored at -80°C. Plasma testosterone was assayed by

radioimmunosay following the manufacturer’s instructions (Diagnostic System

Laboratories).

Castration

A 0.8 cm incision was made transversally in the abdomen, just above the pubis. The

anterior abdominal muscles and skin were incised and the testes were delivered into

the surgical field, and removed from animals. The incisions were closed separately

using Dexon needle and suture thread (Ref. 58419, Sherwood, Bondoufle, France).

Sacrifice of animals

The animals were sacrificed under anaesthesia with isofluran by cervical dislocation.

The R-3327H tumours, seminal vesicles, prostates, testis of each rat were removed,

cleaned and weighed. Animal reaching the maximal tumour size defined by the

welfare of the animal were sacrificed.

Tumour volume determination

The length and width of the tumour were measured once a week with callipers and the

volume of the tumour was estimated by the formula: (length x width2)/2

Statistical analysis


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The statistical tests were performed using StatView Software (Abacus Concept,

Berkeley, USA). The following parameters were compared: the plasma testosterone

level at each sampling time, the mean body weight change, the weight of tumours,

seminal vesicles, testes and prostates at the end of the experiment, the tumour

volumes on each day of measurement. Statistical analysis of the treatment was

performed using an ANOVA followed by Bonferroni/Dunn multiple comparison

procedures or by unpaired t-tests. A p value < 0.05 was considered significant.

Results

Short-term effects of degarelix, D-Trp6-LHRH and surgical castration on the growth

of the Dunning R-3327H

Effects of degarelix and D-Trp6-LHRH on plasma testosterone levels.

In rats treated with D-Trp6-LHRH 0.5 mg base/kg daily (Figure 1), testosterone levels

were initially raised to more than 20 ng/ml (flare-up effect). Testosterone levels then

decreased to a low level at D5 (80 pg/ml) and thereon slowly to castrate levels, at

D28. Thereafter castration levels (below detection limit (<25 pg/ml)) were maintained

to the end of the study (D62). In rats treated with degarelix 1 mg/kg monthly, the

initial flare-up is not observed; moreover plasma testosterone quickly reached

castrated levels at D3 (below detection limit (<25 pg/ml)) and remained at this level

throughout the time course of the experiment with a pattern similar to castration. In

control animals, testosterone level is within normal range (Figure 1).

Effects of degarelix and D-Trp6-LHRH on tissues weights


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Testes weights at the end of the study (D62) represent an integrated measure of the

pituitary-gonadal axis activity over time. Testes weights was significantly lower

(p<0.01) in rats treated with degarelix (410 ± 40 mg) compared to rats treated with D-

Trp6-LHRH (900 ± 120 mg). Control animals showed normal testes weights (2650 ±

250 mg) compared with the two treated groups (Table 1). However, no statistically

relevant differences were observed for the weight of the seminal vesicles and

prostates of the animals treated with D-Trp6-LHRH or degarelix (40 ± 10 mg and 30 ±

20 mg respectively) (Table 1). Prostates from castrated animals were too small to be

weighted accurately.

Effects of degarelix, D-Trp6-LHRH and surgical castration on tumour volume

Degarelix and surgical castration produced an immediate suppression of tumour

growth that lasted throughout the study period (Figure 2). Tumour volumes in rats

treated with degarelix were significantly smaller than control rats at D21. This

difference lasted until the end of the study at D62. For the D-Trp6-LHRH group, there

is no apparent suppression of the tumour growth until D38 compared to control

animals. A significant difference (p<0.01) between D-Trp6-LHRH treated rats and

controls occurred at D49 and last until the end of the study at D62 (Figure 2, Table 1).

Effects of degarelix, D-Trp6-LHRH and surgical castration on tumour weight

At the end of the study (D62), tumours were excised and weighed. Tumours in the

degarelix treated and surgically castrated groups had similar weight (310 ± 150 mg

and 370 ± 120 mg respectively). Tumours in the degarelix treated group were

significantly smaller (p<0.01) than tumours in the D-Trp6-LHRH treated group (370 ±

120 versus 1340 ± 750 mg) (Table 1).


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Long-term effects of degarelix, leuprolide-depot and surgical castration on the

growth of the Dunning R-3327H

Effects of degarelix, surgical castration and leuprolide-depot on plasma testosterone

levels.

Degarelix suppressed testosterone to castration levels within 2 days of treatment

initiation and maintained castration levels throughout the course of the study. Onset of

testosterone suppression by leuprolide-depot was delayed after the flare-up period,

castration levels being achieved within a month. Thereafter testosterone remained

suppressed until treatment was stopped. Control animals stayed within normal range

(about 2 ng/ml) throughout the study (Figure 3). Data from animals of the control and

leuprolide-depot groups have been discarded after 223 days because of the smaller

group size (animals reaching the maximal size of the tumour have been sacrificed).

However, data from castrated and degarelix-treated groups were continued until the

end of the study (D343).

Effects of degarelix, surgical castration and leuprolide-depot on tumour volume

Degarelix profoundly suppressed tumour growth immediately after administration and

with an efficacy similar to surgical castration. In contrast, similar to the short-term

study, during the first month there is no obvious suppression of tumour growth for the

leuprolide-depot compared to the control group (Figure 4). Moreover, escape from

androgen deprivation occurred sooner in rats treated with leuprolide-depot. Data from

control groups or treated with leuprolide-depot were discarded after D223 due to low

animal numbers remaining (animals reaching the maximal size of the tumour have


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been sacrificed, Table 2). At D223, the mean tumour volume for the leuprolide-depot

group (2489 ± 1063 mm3) was higher than degarelix and castration groups (666 ± 291

mm3 and 820 ± 552 mm3 respectively). Degarelix treatment inhibited tumour growth

throughout the experiment with efficacy at least identical to surgical castration until

D343 (2140 ± 1165 mm3 and 1897 ± 1391 mm3 respectively, Figure 4, Table 2).

Discussion

Our interest in developing a GnRH antagonist was stimulated by the need for

improved therapeutics for the management of sex steroid-dependent pathologies

including uterine leiomyoma, endometriosis, and sex-steroid dependent cancer, such

as prostate cancer. Prostate cancer is currently managed with long-acting (1 or 3

months) formulations of GnRH superagonists that initially stimulate pituitary LH and

FSH release as well as gonadal steroids, and then, after 2 to 4 weeks, desensitise the

gonadotrophe cells, leading to suppression of gonadotrophin and sex steroid secretion.

An alternative pharmacological approach is the use a GnRH antagonist which

immediately blocks GnRH receptors in the pituitary resulting in fast and sustained

suppression of gonadotrophin secretion together with a tight control over the

testosterone level (below castration level) which could translate into significant

clinical advantages. In the first study we investigated the short-term effects of

degarelix on tumour growth over a 2 month period; we have demonstrated that

suppression of the pituitary-gonadal axis occurred sooner in rats treated with degarelix

than in rats treated with D-Trp6-LHRH as evidenced by the shorter time to castration

levels of testosterone. Differences in the mechanisms of pituitary-gonadal suppression

produced by GnRH superagonists and GnRH antagonists can explain this different

onset of action. D-Trp6-LHRH will first stimulate LH release (flare-up effect) from


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the pituitary and then testosterone secretion from the testes before suppressing the

pituitary-gonadal axis by a “down-regulation” mechanism. In contrast, degarelix will

immediately bind to the pituitary GnRH receptor, block it and preclude substantial

occupation and stimulation by endogenous GnRH and therefore release of LH/FSH

from the pituitary. The flare of testosterone initially produced by D-Trp6-LHRH

translates into a significant delay in onset of tumour inhibition compared to degarelix

and surgical castration. The data are consistent with a correlation between the time to

castrate and the time to suppress the tumour growth. Over a 2 month period, this delay

has significant consequences on the overall anti-tumour efficacy as indicated by the

measurement of tumour weight. More importantly, in the second study (long-term

effect) tumour growth started to escape androgen-deprivation after about 5 months of

treatment with leuprolide-depot. In contrast, degarelix induced a rapid suppression of

plasma testosterone levels and sustained inhibition of tumour growth over the whole

study period. Two hypotheses can be proposed to explain the premature tumour

growth from androgen deprivation in rats treated with leuprolide-depot. Firstly, there

are reports suggesting that low concentrations of GnRH superagonists can stimulate

the proliferation of human prostate cancer cell lines by acting through tumour GnRH

receptors (Limonta et al., 1999; Bahk et al., 1998; Qayum et al., 1990). The Dunning

tumour expresses GnRH receptors, it is therefore possible that long-term exposure to

leuprolide may stimulate proliferative pathways or promote adaptation to androgen-

deprivation. Secondly, it has been suggested that escape from androgen deprivation

occurs when the tumour has adapted to low levels of androgens. In tumours escaping

from androgen deprivation, androgen receptors and downstream pathways for control

of androgen-dependent gene expression are still functional. Escape from androgen

deprivation could result from mutation of androgen receptors leading to ligand-


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independent activation, or from super sensitivity of the androgen receptors and/or

activation of alternative transcription factors. In that situation even very low levels of

androgens will be sufficient to maintain tumour survival and expansion.

In conclusion, degarelix is a new GnRH antagonist blocking immediately GnRH

receptors in the pituitary resulting in a fast and sustained suppression of

gonadotrophin secretion and inducing a sustained inhibition of tumour growth at least

comparable to surgical castration. The fast cessation of testosterone production by

degarelix is clearly advantageous, allowing an immediate inhibition of tumour growth

and avoiding other side effects of the flare. Such a rapid anti-tumour response cannot

be achieved by a GnRH superagonist. In the long-term studies, superiority of

degarelix over a GnRH agonist is demonstrated; escape to androgen independence

occurred sooner in animals treated with the GnRH superagonist leuprolide. These data

provide a convincing profile of degarelix (GnRH antagonist) as a more effective

GnRH blocker than the superagonists to inhibit the growth of the Dunning tumour

possibly due a better short-term and long-term testosterone control and provide a

convincing profile of degarelix as a potential candidate for the clinical management of

sex steroid-dependent pathologies where long-term chemical castration is required.

Acknowledgments

The authors thank Dr Wolfgang Koechling for his attentive reading and advice

regarding preparation of this manuscript. The authors also thank Dr David Smart, Dr

Andy Sheppard and Dr Dominic Corkill for helpful discussions and technical

assistance.


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Legends for Figures

Figure 1: Effects of degarelix (1 mg/kg subcutaneous, monthly), D-Trp6-LHRH (0.5

mg/kg subcutaneous, daily) and surgical castration on plasma testosterone levels (D0-

D63) in rats bearing a Dunning R3327H prostate tumour. Results are the mean ± SEM

of 10 rats.

Figure 2: Effects of degarelix (1 mg/kg subcutaneous, monthly), D-Trp6-LHRH (0.5

mg/kg subcutaneous, daily) and surgical castration on tumour weight at the end of the

study (D62) in rats bearing a Dunning R-3327H prostate tumour. Results are the mean

± SEM of 10 rats.

Figure 3: Effects of degarelix (1 mg/kg subcutaneous, monthly), leuprolide-depot (1.5

mg/kg subcutaneous, every 3 weeks) and surgical castration on plasma testosterone

levels (D0-D343) in rats bearing a Dunning R-3327H prostate tumour. Results are the

mean ± SEM of 10-11 rats. # Data from animals of the control and leuprolide-depot

groups have been discarded after 223 days (animals reaching the maximal size of the

tumour have been sacrificed).

Figure 4: Effects of degarelix (1 mg/kg subcutaneous, monthly), leuprolide-depot (1.5

mg/kg subcutaneous, every 3 weeks) and surgical castration on tumour volume (D0-

D343) in rats bearing a Dunning R-3327H prostate tumour. Results are the mean ±

SEM of 10-11 rats. # Data from animals of the control and leuprolide-depot groups

have been discarded after 223 days (animals reaching the maximal size of the tumour

have been sacrificed).


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Tables

Table 1: Short-term study. Effects of degarelix (1 mg/kg subcutaneous, monthly), D-Trp6-LHRH (0.5 mg/kg subcutaneous, daily) and surgical

castration on tumour volume, tumour weight, testis weight, prostate weight and seminal vesicles weights at the end of the short-term study (D62)

in rats bearing a Dunning R-3327H prostate tumour. Results are the mean ± SD of 10 rats.

Treatment Nb of animal D62

Tumour volume D62 [mm3]

Tumour weight D62 [mg]

Testis weight D62 [mg]

Prostate weight D62 [mg]

Seminal vesicles D62 [mg]

5% Mannitol 10 2295 ± 1035 2930 ± 162 2650 ± 260 370 ± 120 960 ± 230

Castration 10 351 ± 174**, †† 310 ± 150**, †† a 80 ± 20**

D-Trp6-LHRH

10 818 ± 413** 1340 ± 750** 900 ± 120** 40 ± 10** 100 ± 20**

Degarelix 10 326 ± 87**, †† 370 ± 120**, †† 410 ± 40**, †† 30 ± 20** 100 ± 30**

a Prostate weight not determined. ** p<0.01 versus 5% Mannitol, †† p<0.01 versus D-Trp6-LHRH

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Table 2: Long-term study. Effects of degarelix (1 mg/kg subcutaneous, monthly), Leuprolide-depot (1.5 mg/kg subcutaneous, every 3 weeks)

and surgical castration on tumour volume in rats bearing a Dunning R-3327H prostate tumour. Results are the mean ± SD of 10-11 rats.

Day 0 Day 223 Day 343 Treatment

No. of animals

Tumour volume [mm3]

No. of animals

Tumour volume [mm3]

No. of animals

Tumour volume [mm3]

5% Mannitol 11 247 ± 61 5b 6262 ± 1696 0 -

Castration 11 260 ± 71 11 666 ± 291** 11 2140 ± 1165

Leuprolide 11 277 ± 87 7b 2489 ± 1063** 0 -

Degarelix 10a 257 ± 76 10 820 ± 552**, †† 9c 1897 ± 1391

a 1 animal died before the start of the treatment. b animals reaching the maximal size of the tumour were sacrificed. c 1 animal has been

sacrificed before the end of the study. ** p<0.01 versus 5% Mannitol, †† p<0.01 versus D-Trp6-LHRH

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