The effects of prolactin receptor blockade in a murine endometriosis interna model.

Endometriosis in an estrogen-dependent disease that is characterized by the presence of endometrial tissue outside the uterine cavity leading to pain and infertility in many affected women. Highly efficient treatment options which create a hypo-estrogenic environment can cause side effects such as hot flushes and bone mass loss that are not favorable for premenopausal women. Previous work has demonstrated that increased local or systemic prolactin seems to be involved in the pathogenesis of endometriosis. Here we examined two prolactin receptor (PRLR) blocking antibodies in a murine endometriosis interna model which relies on the induction of systemic hyperprolactinemia in female SHN mice. The severity of the disease is determined by the degree of endometrial invasion into the myometrium. In this model, endometriosis was inhibited by clinical gold standards such as progestins and anti-estrogenic approaches. PRLR blockade completely inhibited endometriosis in this mouse model to the same extent as the anti-estrogen faslodex or the GnRH antagonist cetrorelix. In contrast to cetrorelix and faslodex, the PRLR antibodies did not decrease relative uterine weights and were thus devoid of anti-estrogenic effects. We therefore hypothesize that PRLR antibodies may present a novel and highly efficient treatment option for endometriosis with a good safety and tolerability profile. Clinical studies are on the way to test this hypothesis.

Improved in vivo antitumor effect of a daunorubicin - GnRH-III bioconjugate modified by apoptosis inducing agent butyric acid on colorectal carcinoma bearing mice.

Compared to classical chemotherapy, peptide-based drug targeting is a promising therapeutic approach for cancer, which can provide increased selectivity and decreased side effects to anticancer drugs. Among various homing devices, gonadotropin-releasing hormone-III (GnRH-III) peptide represents a suitable targeting moiety, in particular in the treatment of hormone independent tumors that highly express GnRH receptors (e.g. colon carcinoma). We have previously shown that GnRH-III[(4)Lys(Ac),(8)Lys(Dau = Aoa)] bioconjugate, in which daunorubicin was attached via oxime linkage to the (8)Lys of a GnRH-III derivative, exerted significant in vivo antitumor effect on subcutaneously developed HT-29 colon tumor. In contrast, results of the study reported here indicated that this compound was not active on an orthotopically developed tumor. However, if Lys in position 4 was acylated with butyric acid instead of acetic acid, the resulting bioconjugate GnRH-III[(4)Lys(Bu),(8)Lys(Dau = Aoa)] had significant tumor growth inhibitory effect. Furthermore, it prevented tumor neovascularization, without detectable side effects. Nevertheless, the development of metastases could not be inhibited by the bioconjugate; therefore, its application in combination with a metastasis preventive agent might be necessary in order to achieve complete tumor remission. In spite of this result, the treatment with GnRH-III[(4)Lys(Bu),(8)Lys(Dau = Aoa)] bioconjugate proved to have significant benefits over the administration of free daunorubicin, which was used at the maximum tolerated dose.

Assessment of inhibin B as a biomarker of testicular injury following administration of carbendazim, cetrorelix, or 1,2-dibromo-3-chloropropane in Wistar han rats.

Although histopathology is considered the gold standard for assessing testicular toxicity in the nonclinical setting, identification of noninvasive biomarkers for testicular injury are desirable to improve safety monitoring capabilities for clinical trials. Inhibin B has been investigated as a noninvasive biomarker for testicular toxicity. This study investigates the correlation of Inhibin B in Wistar Han rats with the onset and reversibility of testicular histopathology from classical testicular toxicants carbendazim, cetrorelix acetate (CTX), and 1,2-dibromo-3-chloropropane (DBCP). The dose regimen included Interim (day 8), Drug (day 29), and nondosing Recovery (day 58) Phases. Inhibin B was not effective at predicting the onset of carbendazim- or CTX-mediated testicular pathology in rats. Inhibin B was reduced by DBCP administration at the end of the Drug Phase only, acting as a leading indicator of the onset of testicular toxicity before the onset of germ cell depletion. However, since Inhibin B was only decreased at the end of the Dosing Phase and not at the Recovery Phase, when the onset of testicular pathology occurred, it is unclear if monitoring Inhibin B would provide sufficient advanced warning for the onset of testicular pathology. Furthermore, follicle stimulating hormone was decreased following CTX and DBCP administration in the Interim Phase and CTX in the Drug Phase. Inhibin B has limited predictive capacity as a leading testicular biomarker in rats.

Targeted therapy of breast and gynecological cancers with cytotoxic analogues of peptide hormones.

Gynecological cancers such as breast, ovarian, and endometrial carcinoma express receptors for luteinizing hormone-releasing hormone (LHRH), bombesin/gastrin-releasing peptide (BN/GRP), and somatostatin (SST). These tumors are therefore suitable candidates for targeted therapy with cytotoxic hybrid molecules consisting of a cytotoxic radical and a peptide hormone analogue as a carrier. These compounds have been shown to be more active and less toxic in vivo than nontargeted chemotherapy in models of various human cancers which express the respective receptors. The current review summarizes experimental and clinical findings with cytotoxic peptide hormone analogues of LHRH (AN-152 [AEZS 108], AN-207), BN/GRP (AN-215), and SST (AN-238) in breast, ovarian, and endometrial cancers.

A recombinant protein LHRH-PE40 for tumour therapy: preclinical safety studies.

LHRH-PE40, a recombinant DNA-derived protein composed of LHRH and Pseudomonas aeruginosa exotoxin A, is being developed for the treatment of malignant tumours. This experiment was designed to assess its preclinical safety. Reproductive toxicity studies, pharmacokinetic studies, single- and repeat-dose intraperitoneal or intravenous toxicity studies in mice, rats and monkeys were conducted to assess the toxicity of LHRH-PE40. In intravenous single-dose studies in mice, the LD50 was 731.26 microg/kg and 676.03 microg/kg in male and female mice respectively. In single-dose studies and repeat-dose range-finding studies in rats, dose-limited severe vascular leakage syndromes occurred. In repeat-dose long-term studies, except drug-related vascular leakage syndromes, other drug-related changes included decreased testis weight and testis atrophy. In single-dose and repeat-dose studies in monkeys, dose-limited acute tubular necrosis of the kidneys was the chief finding. In reproductive studies, drug-related changes were decreased food intakes, decreased testis weight and uterus weight, decreased foetus weight and increased foetus mortality, increased maternal and F1 offspring mortality and decreased maternal and F1 offspring body weight. Pharmacokinetic studies showed a similar half-time of distribution and clearance in mice and monkeys. Tissue distribution showed a high concentration in the kidneys and a low concentration in the brain. LHRH-PE40 induced vascular leak syndromes in rats and acute tubular necrosis in monkeys. It also led to testicle atrophy in rats and overt productive toxicity to parents and F1 generations in mice. Because of these findings, it should be monitored carefully in human clinical trials for things such as respiratory, urinary and reproductive toxicities.

Immunological hormone atrophy by gonadotropin-based drug.

Luteinizing hormone releasing hormone-Pseudomonas aeruginosa exotoxin A (LHRH-PE40) and sequence-modified LHRH-PE40 (mLHRH-PE40) are two anti-tumour drugs developed by fusing either native or modified LHRH with the same PE40. This study was designed to evaluate their toxicity on the male genital tract. Male rats were treated intravenously or intraperitoneally with either LHRH-PE40 or mLHRH-PE40 on alternate days for 12 weeks. Serum and testes were examined to detect anti-LHRH, anti-LHRH-PE40 or anti-mLHRH-PE40 using an ELISA assay, testosterone level was measured by radioimmunoassay, and testicular morphology was evaluated. Rats receiving LHRH-PE40 intraperitoneal injections had higher anti-LHRH antibody titres, lower serum testosterone concentration and remarkable testicular atrophy, whereas those administrated with intravenous injections of either LHRH-PE40 or mLHRH-PE40 demonstrated no such changes. We concluded that the testicular atrophy can be attributed to higher titre of anti-LHRH antibodies, which was affected by both drug delivery and the LHRH motif of the chimeric protein.

Effect of gonadotropin-releasing-hormone-induced hypogonadism on insulin action as assessed by euglycemic clamp studies in men.

OBJECTIVE: In view of the relationship between hyperandrogenism and impaired insulin action in women with polycystic ovarian syndrome, we sought to investigate the effects of decreasing androgen levels on insulin action in men. DESIGN: Prospective clinical study. SETTING: University hospital. PATIENT(S): Ten normal healthy men. INTERVENTION(S): GnRH agonist, 3.75 mg monthly for 3 months. MAIN OUTCOME MEASURE(S): Rate of glucose uptake, serum insulin, and glucose levels. RESULT(S): GnRH treatment caused a significant fall in circulating testosterone levels, from 572 +/- 58 ng/dL to 178 +/- 66 ng/dL (P<.05). However, as assessed by euglycemic hyperinsulinemic clamp studies, there was no significant change in glucose uptake. CONCLUSION(S): In normal men, administration of a GnRH analogue was associated with a decrease in testosterone levels but not in insulin action.

Targeting cytotoxic conjugates of somatostatin, luteinizing hormone-releasing hormone and bombesin to cancers expressing their receptors: a "smarter" chemotherapy.

Chemotherapy is one of the main modalities in the therapy of cancer. However, an improvement in the efficacy and a reduction in the toxicity of chemotherapeutic agents remains a great challenge to oncologists. A specific delivery of cytotoxic drugs to cancerous cells may help improving both aspects. Peptide hormones, for which receptors have been found in various human cancers, can serve as carriers for a local delivery of cytotoxic agents or radiopharmaceuticals to the tumors, as demonstrated by the successful clinical use of radiolabeled somatostatin analog Octreoscan for the detection and treatment of some somatostatin receptor-positive tumors. Thus, in recent years we developed a series of cytotoxic peptide hormone conjugates based on derivatives of hypothalamic hormones such as somatostatin and luteinizing hormone-releasing hormone (LHRH), and the brain-gut hormone bombesin. To create targeted conjugates with high cytotoxic activity, a derivative of doxorubicin (DOX), 2-pyrrolino-DOX (AN-201), which is 500-1, 000 times more active than its parent compound, was developed. This agent was coupled to somatostatin octapeptide RC-121 to form cytotoxic conjugate AN-238, and to [D-Lys6]LHRH carrier to produce analog AN-207. Cytotoxic bombesin hybrid AN-215 also contains AN-201. DOX was likewise linked to [D-Lys6]LHRH to form AN-152. A comprehensive testing of these cytotoxic conjugates in experimental models of various human and rodent cancers led to their selection as candidates for clinical trials.

Effects of medical or surgical castration on erectile function in an animal model.

The goal of this study was to investigate the effects of medical castration (luteinizing hormone-receptor hormone [LH-RH] agonist treatment) or surgical castration on erectile function in an animal model. New Zealand White male rabbits were either kept intact (control); surgically orchiectomized; or treated for 2, 4, or 8 weeks with the LH-RH agonist leuprolide acetate (107 microg/kg/mo). At 2 weeks, plasma testosterone levels of orchiectomized and leuprolide acetate-treated animals were 12.8% and 57.4% of intact control animals, respectively. Erectile function was assessed by continuously recording systemic arterial pressure (SAP) and intracavernosal blood pressure (ICP) and determining the ICP:SAP ratios in response to electrical stimulation of the pelvic nerve at varying frequencies (2.5-32 Hz). Androgen deprivation by surgical (orchiectomy) or medical (leuprolide acetate) castration reduced ICP at all frequencies tested but did not alter SAP. Administration of the phosphodiesterase type 5 inhibitor vardenafil (10 microg/kg) did not enhance ICP in surgically orchiectomized or leuprolide acetate-treated animals. Nitric oxide synthase and arginase activities in the corpus cavernosum were not significantly altered by surgical or medical castration. Further, Masson trichrome staining of erectile tissue from androgen-ablated animals showed a reduction in smooth muscle content. These data demonstrate that androgen deprivation achieved by surgical or medical castration adversely affects penile hemodynamics and erectile function without producing significant changes in the activities of nitric oxide synthase or arginase. We conclude that androgen deprivation produces structural alterations in the corpus cavernosum leading to corporal veno-occlusive dysfunction.

Cytotoxic activity of gonadotropin-releasing hormone (GnRH)-pokeweed antiviral protein conjugates in cell lines expressing GnRH receptors.

Pokeweed antiviral protein (PAP), a 29-kDa ribosome-inactivating protein isolated from the leaves of Phytolacca americana, has potent cytotoxic activity once it enters the cytoplasm of a cell. It is incapable of entering cells by itself. Therefore, our objective was to determine whether a GnRH analog could be used to deliver PAP specifically to cells expressing GnRH receptors. D-Lys(6)-GnRH-Pro(9)-ethylamide was conjugated to PAP (GnRH-PAP). Chinese hamster ovary cells stably transfected with cDNA for the murine GnRH receptor and a mouse gonadotroph tumor cell line that expresses endogenous GnRH receptors (alphaT3-1 cells) were used to evaluate the cytotoxic effects of GnRH-PAP. We also examined cytotoxicity of GnRH-PAP using human endometrial, breast, and prostate cancer cell lines. Treatment of GnRH receptor-positive cells with GnRH-PAP resulted in dose-dependent cytotoxicity. Cytotoxicity of GnRH-PAP was dependent on number of GnRH receptors (r(2) = 0.871, P < 0.05) and duration of exposure of GnRH-PAP to the cells. In contrast, GnRH-PAP was not cytotoxic to Chinese hamster ovary cells not harboring GnRH receptors. Moreover, the cytotoxic activity of GnRH-PAP could be inhibited by addition of excess GnRH analog. Neither PAP nor GnRH analog alone was cytotoxic. These results suggest that GnRH analogs can be used to specifically deliver toxin molecules to cells that express GnRH receptors. Thus, a new class of biomedicines that act as hormonotoxins against cells expressing GnRH receptors provides a novel approach for inhibiting reproduction and treating cancers that are dependent on reproductive hormones.

Regulation of targeted chemotherapy with cytotoxic lutenizing hormone-releasing hormone analogue by epidermal growth factor.

Targeting chemotherapy selectively to cancers can reduce the toxic side effects. AN-152, a conjugate of doxorubicin and [D-Lys6]-luteinizing hormone-releasing hormone (LH-RH), is more potent against LH-RH receptor-bearing cancers and produces less peripheral toxicity than doxorubicin. Many cancers, e.g., 50% of breast cancers, but few normal tissues express these receptors, providing a selective target for this cytotoxic conjugate. In this study, the effectiveness of AN-152 was heightened by receptor up-regulation. The cytotoxic effect of AN-152 can be regulated by the number of active LH-RH receptors on cancer cells. LH-RH receptor-positive (MCF-7) and -negative (UCI-107) cancer cells were treated with epidermal growth factor (EGF) or the somatostatin analogue, RC-160. EGF and RC-160 have been shown previously to regulate LH-RH receptors through phosphorylation. The effect of receptor regulation, by hormone exposure, on the cytotoxicity of AN-152 and doxorubicin and on the cellular uptake of AN-152, [D-Lys6]LH-RH, or doxorubicin was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and by two-photon laser scanning microscopy. The results demonstrated that the cellular entry of the conjugate was: (a) specific for cancers with LH-RH receptors; (b) up-regulated by EGF; (c) down-regulated by RC-160; and (d) the cytotoxicity of the AN-152 paralleled the efficiency of entry. This study illustrates the potential use of receptor regulation for increasing the efficacy of chemotherapeutic approaches that are directed to cell surface receptors.

Chronic toxicity and reversibility of antifertility effect of immunization against gonadotropin-releasing hormone in male rats and rabbits.

The chronic systemic toxicity of immunization with gonadotropin-releasing hormone, conjugated to tetanus toxoid (GnRH-TT), was investigated in male rats and rabbits in order to start Phase I clinical trials. Groups of rats and rabbits were immunized with GnRH-TT dissolved in aqueous adjuvant. The antigen was administered at weeks 0, 4, and 8, followed by boosters to maintain high antibody titers. At termination (8-9 months after first immunization), twenty rats and ten rabbits exhibiting the highest mean anti-GnRH titers and all the controls were selected for complete toxicological evaluation. In the rat study, a castrated control group was included for comparison with the immunized group. The hematological and serum chemistry parameters of immunized rats and rabbits were not affected in a significant manner. Most of the changes in serum chemistry of immunized rats were also found in castrated rats, indicating that the changes are most likely due to the withdrawal of androgenic support. The weights of the testes, epididymides, and sex accessory glands were lower in all immunized animals. There was significant atrophy of the germinal epithelium, which, however, sustained a population of Sertoli cells, spermatogonia, and pachytene spermatocytes. Other morphological changes in the prostate, seminal vesicles, pituitary, and mammary gland reflected the effect of androgen withdrawal. The decrease in the weight of liver, kidney, and heart seen in the immunized rats was also present in castrated rats and was not associated with any histopathological changes. The reversibility of immunization-induced infertility was investigated by mating the rats with normal females. Four months after the start of immunization, 9 out of 10 immunized rats were infertile whereas by nine months, all rats had regained fertility. Thus, it is concluded that immunization with GnRH-TT had no systemic toxicological effects in the adult male rats and rabbits for the period studied. The results also indicated that the GnRH-TT immunization had an antifertility effect in male rats. Fertility was restored following cessation of immunization and decline in anti-GnRH antibody titers.

Stability of cytotoxic luteinizing hormone-releasing hormone conjugate (AN-152) containing doxorubicin 14-O-hemiglutarate in mouse and human serum in vitro: implications for the design of preclinical studies.

Recently, we developed a series of cytotoxic peptide conjugates containing 14-O-glutaryl esters of doxorubicin (DOX) or 2-pyrrolino-DOX (AN-201). Serum carboxylesterase enzymes (CE) can partially hydrolyze these conjugates in the circulation, releasing the cytotoxic radical, before the targeting is complete. CE activity in serum of nude mice is about 10 times higher than in human serum. Thus, we found that the t(1/2) of AN-152, an analog of luteinizing hormone-releasing hormone (LH-RH) containing DOX, at 0.3 mg/ml is 19. 49 +/- 0.74 min in mouse serum and 126.06 +/- 3.03 min in human serum in vitro. The addition of a CE inhibitor, diisopropyl fluorophosphate (DFP), to mouse serum in vitro significantly (P < 0. 01) prolongs the t(1/2) of AN-152 to 69.63 +/- 4.44 min. When DFP is used in vivo, 400 nmol/kg cytotoxic somatostatin analog AN-238 containing AN-201 is well tolerated by mice, whereas all animals die after the same dose without DFP. In contrast, DFP has no effect on the tolerance of AN-201. A better tolerance to AN-238 after DFP treatment is due to the selective uptake of AN-238 by somatostatin receptor-positive tissues. Our results demonstrate that the suppression of the CE activity in nude mice greatly decreases the toxicity of cytotoxic hybrids containing 2-pyrrolino-DOX 14-O-hemiglutarate and brings this animal model closer to the conditions that exist in humans. The use of DFP together with these peptide conjugates in nude mice permits a better understanding of their mechanism of action and improves the clinical predictability of the oncological and toxicological results.

Effective treatment of advanced estrogen-independent MXT mouse mammary cancers with targeted cytotoxic LH-RH analogs.

Cytotoxic agents linked to hormonal carriers provide new approaches to tumor therapy, and LH-RH receptors expressed by breast cancers can be used for targeting chemotherapeutic compounds. In the present study, large, advanced estrogen-independent MXT mouse mammary cancers were treated with cytotoxic LH-RH analog AN-152 containing doxorubicin (DOX) or AN-207 incorporating superactive derivative 2-pyrrolino-DOX (AN-201). These cytotoxic hybrid molecules were administered once i.v., close to their maximum tolerated doses, at various time intervals after transplantation of tumors. The cytotoxic LH-RH analogs and the radicals alone, given at earlier stages of tumor development, inhibited growth of MXT cancers. Cytotoxic LH-RH conjugate AN-207 had significantly stronger effect than its respective cytotoxic radical, particularly when larger tumors were treated, causing 95%, 89%, 100% and 96% tumor growth reduction when administered on days 1, 7, 10 or 14, respectively. AN-152, AN-201, and DOX, given on day 14, were virtually ineffective. Histological characteristics of tumor cell proliferation and cell death were analyzed in large MXT cancers 1-4 days after treatment with AN-207 and AN-201. AgNOR scores were decreased and apoptotic indices increased after treatment of tumors with AN-207 or AN-201, but enhanced apoptosis and decreased AgNOR numbers persisted longer in the case of AN-207. In contrast to AN-201, AN-207 also increased the extent of necrosis in tumors. In conclusion, on the basis of its powerful inhibitory effect on the aggressive MXT mouse mammary tumor, the cytotoxic LH-RH analog AN-207 could be considered for treatment of advanced human mammary carcinomas that express LH-RH receptors.

Direct luteinizing hormone action triggers adrenocortical tumorigenesis in castrated mice transgenic for the murine inhibin alpha-subunit promoter/simian virus 40 T-antigen fusion gene.

Transgenic (TG) mice, expressing the Simian Virus 40 T-antigen (Tag) under a 6-kb fragment of the murine inhibin alpha-subunit promoter (inh alpha p), develop gonadal tumors of granulosa/theca or Leydig cell origin. We showed previously that adrenocortical tumors develop if the TG mice are gonadectomized but never develop in intact animals. However, if functional gonadectomy was induced by GnRH antagonist treatment or by cross-breeding the TG mice into the hypogonadotropic hpg genetic background, neither gonadal nor adrenal tumors appeared. Since the most obvious difference between the gonadectomized and GnRH-antagonist-treated or Tag/hpg double mutant mice is the elevated gonadotropin secretion in the first group, we examined whether the adrenal tumorigenesis would be gonadotropin-dependent. Surprisingly, both the adrenal tumors and a cell line (C alpha 1) derived from one of them expressed highly functional LH receptors (LHR), as assessed by Northern hybridization, immunocytochemistry, ligand binding, and human CG (hCG)-stimulated cAMP and steroid production. No FSH receptor expression was found in the adrenal tumors by RT-PCR. hCG treatment of the C alpha 1 cells stimulated their proliferation, as measured by [3H]thymidine incorporation. This effect was related to hCG-stimulated steroidogenesis since progesterone, testosterone, and estradiol, at physiological concentrations, also stimulated the C alpha 1 cell proliferation. Different adrenocortical cells expressed initially LHR and Tag, whereas both were highly expressed in the tumor cells. In conclusion, the high level of functional LHR in the adrenal tumors indicates that this receptor can function as tumor promoter when ectopically expressed and stimulated by the ligand hormone.

Cytotoxic analogs of luteinizing hormone-releasing hormone containing doxorubicin or 2-pyrrolinodoxorubicin, a derivative 500-1000 times more potent.

Doxorubicin (DOX) and its daunosamine-modified derivative, 2-pyrrolino-DOX, which is 500-1000 times more active than DOX, were incorporated into agonistic and antagonistic analogs of luteinizing hormone-releasing hormone (LH-RH). The conjugation of DOX with LH-RH analogs was performed by using N-(9-fluorenylmethoxycarbonyl)-DOX-14-O-hemiglutarate, a dicarboxylic acid ester derivative of DOX. Coupling this derivative covalently to the epsilon-amino group of the D-Lys side chain of agonist [D-Lys6]LH-RH or antagonistic analog AC-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys-Leu-Arg-Pro-D-Ala-NH 2 [where Nal(2) = 3-(2-naphthyl)alanine, Pal(3) = 3-(3-pyridyl)alanine, and Phe(4CI) = 4-chlorophenylalanine] was followed by the removal of the 9-fluorenylmethoxycarbonyl protective group to yield cytotoxic derivatives of LH-RH analogs containing DOX. From these DOX containing LH-RH hybrids, intensely potent analogs with daunosamine-modified derivatives of DOX can be readily formed. Thus, cytotoxic LH-RH agonist containing DOX (AN-152) can be converted in a 66% yield by a reaction with a 30-fold excess of 4-iodobutyraldehyde in N,N-dimethylformamide into a derivative having 2-pyrrolino-DOX (AN-207). Hybrid molecules AN-152 and AN-207 fully preserve the cytotoxic activity of their radicals, DOX or 2-pyrrolino-DOX, respectively, in vitro, and also retain the high binding affinity of the peptide hormone portion of the conjugates to rat pituitary receptors for LH-RH. These highly potent cytotoxic analogs of LH-RH were designed as targeted anti-cancer agents for the treatment of various tumors that possess receptors for the carrier peptide. Initial in vivo studies show that the hybrid molecules are much less toxic than the respective cytotoxic radicals incorporated and significantly more active in inhibiting tumor growth.

Antitumour effect of a gonadotropin-releasing-hormone antagonist (MI-1544) and its conjugate on human breast cancer cells and their xenografts.

Our gonadotropin-releasing hormone (GnRH) antagonist analogue MI-1544 ([Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10]GnRH) was developed as a potential contraceptive material, because it decreased the luteinizing hormone level without unfavourable side-effects. The antagonist was covalently bound to poly[Lys-(Ac-Glu0.96-DL-Ala3.1)] (AcEAK)-a branched polypeptide having a polylysine backbone--resulting in a MI-1544-AcEAK conjugate. According to our in vitro experiments the MI-1544 induced a 33%-35% decrease in cell numbers of MCF-7 and MDA-MB-231 human breast cancer cell lines at a dose of 30 microM. The biodegradable polymeric carrier, AcEAK, alone inhibited cell proliferation by only 13%-15%, while the MI-1544-AcEAK conjugate, applied at the same dose, was capable of producing 45%-50% inhibition of cell proliferation. Our in vivo experiments using immunosuppressed mice showed that MI-1544, applied twice daily s.c., inhibited the growth of oestrogensensitive and -insensitive xenografts by 65% and 30% respectively. This effect was potentiated (70%) in both types of xenografts by the presence of the polymeric carrier in the conjugate; however, the carrier by itself did not cause tumour growth inhibition. The polymeric polypeptide carrier is supposed to increase the stability of the GnRH antagonist and to prevent the rapid excretion of the covalently bound peptide molecule. The antagonist and its conjugate may have various direct and indirect effects on breast cancer cells and, as a consequence, the new GnRH antagonist conjugates are suitable for treating an extended range of breast cancers.

Acute and subchronic toxicity studies with detirelix, a luteinizing hormone-releasing hormone antagonist, in the rat and monkey.

Acute (single dose), 2-week, and 3-month toxicology studies were conducted with detirelix, a luteinizing hormone-releasing hormone (LHRH) antagonist, in rats and cynomolgus monkeys. Acute studies were conducted by intravenous and subcutaneous injection. Subchronic studies were conducted by daily subcutaneous injection. Clinical signs after a single intravenous dose included lethargy, edema, cyanosis, pallor, and red ears in rats at greater than or equal to 0.3 mg/kg and lethargy and facial flushing in monkeys at greater than or equal to 0.5 mg/kg. In subchronic studies, detirelix at greater than or equal to 0.4 mg/kg/day (rats) and at greater than or equal to 0.2 mg/kg/day (monkeys) produced atrophy of the reproductive organs, inhibition of ovulation and spermatogenesis, decreased body weight gain in male rats and monkeys, and increased body weight gain in female rats. In the rat, morbidity and/or mortality occurred throughout the treatment phase at a subcutaneous dose of greater than or equal to 2.0 mg/kg/day. In both species, the time to recovery of normal reproductive organ morphology and function was directly related to dose. Exogenous testosterone decreased the severity of reproductive and body weight effects in male rats. In conclusion, the acute effects of detirelix were consistent with peripheral vasodilation. Subchronic effects were associated with inhibition of pituitary gonadotropic and gonadal hormone secretion.

False positive result for a peptide drug in the gene conversion assay with Saccharomyces cerevisiae strain D7.

A battery of mutagenicity tests was performed with nafarelin, an agonist analogue of luteinizing hormone releasing hormone (LHRH) containing tryptophan (Trp) and histidine (His). Included were the Ames assay and the gene conversion assay with yeast strain D7. Both tests were negative without S9 activation, and the Ames test was negative with S9, but the yeast test was positive with S9 activation. Since the yeast test is based on conversion of cells to Trp independence, release of Trp by metabolism of the drug could account for the positive result. The test was repeated using Trp instead of the drug. The result was positive even at the lowest Trp concentration. In another experiment with the drug, amino acid analysis of the incubation mixture revealed the presence of Trp but no detectable His. Since the Ames test is based on mutation to His-independent cells, these data are completely consistent with the negative result in the Ames test and the false positive result in the yeast test. These data suggest the need for caution in interpreting the results from mutagenicity assays with peptide drugs.