A Phase II trial of a combination herbal supplement for men with biochemically recurrent prostate cancer.

BACKGROUND: Men with biochemical recurrence (BCR) of prostate cancer are typically observed or treated with androgen-deprivation therapy. Non-hormonal, non-toxic treatments to slow the rise of PSA are desirable. We studied a combination herbal supplement, Prostate Health Cocktail (PHC), in prostate cancer cell lines and in a population of men with BCR. METHODS: PC3, LAPC3 and LNCaP cells were incubated with increasing concentrations of PHC suspension. Men previously treated for prostate cancer with surgery, radiation or both with rising PSA but no radiographic metastases were treated with three capsules of PHC daily; the primary end point was 50% PSA decline. Circulating tumor cells (CTCs) were identified using parylene membrane filters. RESULTS: PHC showed a strong dose-dependent anti-proliferative effect in androgen-sensitive and independent cell lines in vitro and suppression of androgen receptor expression. Forty eligible patients were enrolled in the clinical trial. Median baseline PSA was 2.8 ng ml(-1) (1.1-84.1) and 15 men (38%) had a PSA decline on study (1-55% reduction); 25 (62%) had rising PSA on study. The median duration of PSA stability was 6.4 months. Two patients had grade 2/3 transaminitis; the only other grade 2 toxicities were hyperglycemia, hypercalcemia and flatulence. There were no significant changes in testosterone or dihydrotestosterone. CTCs were identified in 19 men (47%). CONCLUSIONS: Although the primary end point was not met, PHC was well tolerated and was associated with PSA declines and stabilization in a significant number of patients. We believe this is the first report of detecting CTCs in men with BCR prostate cancer. Randomized studies are needed to better define the effect of PHC in men with BCR.

Diagnostic role of [F-18]-FDG positron emission tomography in restaging renal cell carcinoma.

AIMS: To retrospectively assess the diagnostic utility of positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) in restaging renal cell carcinoma. MATERIALS AND METHODS: We performed whole-body PET scans (45 minutes after intravenous injection of 10 - 15 mCi FDG) for restaging 25 patients (18 male, 7 female, 42 - 81 years old) with known or suspected metastatic renal cell carcinoma. Prior treatments included immunotherapy (n = 1), nephrectomy (n = 16), nephrectomy followed by chemotherapy (n = 3), by radiation therapy (n = 1), and by combined chemoradiation therapy (n = 4). Contrast-enhanced chest, abdomen and pelvis CT studies were available for all patients. Diagnostic validation was by histological sampling (n = 2) and clinical and imaging follow-up for up to 1 year (n = 23). RESULTS: PET was concordant with the findings of CT in 18 patients (3 TN, 15 TP). PET was discordant with CT in 7 patients (28% of total). PET was falsely negative in 6 of these patients and did not demonstrate hypermetabolism in pulmonary (n = 4), mediastinal (n = 2), adrenal (n = 1) and lytic osseous (n = 2) metastatic lesions. PET was falsely positive in the remaining 1 patient in the discordant group with lumbar facet arthropathy. The diagnostic performance of PET in detection of recurrent and metastatic renal cell carcinoma revealed a sensitivity of 71%, specificity of 75%, accuracy of 72%, negative predictive value of 33% and positive predictive value of 94%. CONCLUSIONS: FDG PET demonstrates modest accuracy in the diagnostic imaging evaluation of patients with suspected or known metastatic renal cell carcinoma. A negative study may not exclude disease while a positive study is suspicious for malignancy.

Therapeutic implications of enhanced G(0)/G(1) checkpoint control induced by coculture of prostate cancer cells with osteoblasts.

Osteoblastic metastases are common in lethal prostate cancer. Effective therapy for bone metastases is lacking. Thus, developing an appropriate in vitro screening system is critical to prioritize which of the newly developed agents should undergo additional expensive and time-consuming in vivo evaluation in bone metastases animal models. In the past, such in vitro screening evaluated the response of prostate cancer cells to chemotherapeutic agents in monoculture without the presence of osteoblasts. In such monoculture, prostate cancer cells have a high (i.e., >90%) proliferative growth fraction. In contrast, the growth fraction (i.e., mean: 7.1 +/- 0.8%; median: 3.1%) in 117 metastatic sites of prostate cancer obtained from 11 androgen ablation failing patients at "warm" autopsy was found to be >10-fold lower. To better mimic the lower growth fraction observed clinically, LNCaP human prostate cancer cells were cocultured with membrane-separated hFOB human osteoblasts. Such coculturing significantly lowered the growth fraction of the LNCaP cells (i.e., from >90 to <30%) without enhancing their low rate (i.e., <5%) of apoptosis. This lowering of the growth fraction was documented using flow cytometry, Ki-67 immunohistochemistry, and 5-bromo-2-deoxyuridine incorporation. Using RNase protection assays, it was documented that coculture with osteoblasts causes enhanced p53, p27, and p21 expression leading to a decrease in the number of LNCaP cells entering the cell cycle (i.e., enhanced number of LNCaP cells in G(0)-G(1) and a decrease in S and G(2)-M and thus the growth fraction). This osteoblast-induced enhanced G(0)-G(1) checkpoint control affected the chemosensitivity of LNCaP cells. This was documented by coculturing LNCaP cells with hFOB cells to condition the medium for 3 days to lower the growth fraction to <30% before exposing the LNCaP cells for 48 h to various concentrations of Taxol, doxorubicin, or thapsigargin (TG). In standard high (i.e., >90%) growth fraction cultures (i.e., cultures in the absence of osteoblast-conditioned medium), there was a dose-dependent and significant (P < 0.05) increase in apoptosis of LNCaP cells exposed to Taxol or doxorubicin. In contrast, even the highest dose of Taxol (1 microM) did not enhance apoptosis of lower growth fraction LNCaP cells cultured in osteoblast-conditioned medium. Similarly, only the highest concentration of doxorubicin (1 microM) enhanced apoptosis in lower growth fraction cells. In contrast, 100 nM TG induced high levels of apoptosis in both lower and high-growth fraction LNCaP cultures. These results demonstrate that the osteoblast/LNCaP coculture system is a better in vitro screen than monoculture to identify proliferation-independent agents for the treatment of prostate cancer bone metastases, and TG is such an agent.

Malformations of angiogenesis in the low differentiated human carcinomas. immunohistochemical study.

Our previous observations showed that the perivascular mesenchyma of the thin-walled vessels (capillaries) in cancers may be the source of organ-specific stem cells. We suggested that the cells forming vascular channels in altered stroma participate in the tumor development. This study was designed to examine the distribution of the vessels and their appearance in the breast, lung and colon cancers. Using immunohistochemical methods, we have shown that in the low differentiated tumors both CD31 and factor VIII antigens may be expressed in capillaries chiefly on the periphery of neoplastic foci. Many of these vessels were discontinuous, with interruptions or unformed tubules. Sporadically, CD31 protein and factor VIII antigens were not expressed in capillaries inside the very low differentiated cancer cases. It is difficult to assess by immunohistochemical means whether the vascular malformations are the primary or secondary phenomena in the malignancy and why these abnormalities were especially visible in some low differentiated cancers.

Stopped-flow and Brownian dynamics studies of electrostatic effects in the kinetics of binding of 7-methyl-GpppG to the protein eIF4E.

The kinetics of binding 7-methyl-GpppG, an analogue of the 5'-mRNA cap, to the cap-binding protein eIF4E, at 20 degrees C, in 50 mM Hepes-KOH buffer, pH 7.2, and 50, 150 and 350 mM KCl, was measured using a stopped-flow spectrofluorometer, and was simulated by means of a Brownian dynamics method. For most of the stopped-flow measurements a single bimolecular step is an inadequate description of the binding mechanism and an additional step is required to accommodate the kinetic data. The rate constants derived from assumed one-step and two-step binding models were determined. The forward rate constants towards the complex formation decrease, and the reverse rate constants increase, with increasing ionic strength. The association rate constants derived from the stopped-flow measurements and the computed diffusional encounter rate constants agree, indicating that the first observed step can be viewed as a diffusionally controlled encounter of the protein and the ligand. Moreover, comparison of experimental and computed bimolecular association rate constants indicate that the experimentally observed decrease of the rate constants with the increasing ionic strength is caused by two factors. The first is less effective steering of the ligand towards the binding site at higher ionic strengths, and the second is that for higher ionic strengths the ligand must be closer to the binding site to induce the fluorescence quenching.

Inhibition of the growth of Caki-I human renal adenocarcinoma in vivo by luteinizing hormone-releasing hormone antagonist Cetrorelix, somatostatin analog RC-160, and bombesin antagonist RC-3940-II.

BACKGROUND: Metastatic or recurrent renal cell carcinoma (RCC) is a therapeutic challenge because it is resistant to chemotherapy and external radiotherapy. No uniformly effective therapeutic agents are available for the management of patients with RCC. Hormones and growth factors may play a role in promoting the transformation and/or proliferation of kidney neoplasms. METHODS: Luteinizing hormone-releasing hormone (LH-RH) antagonist Cetrorelix (SB-75), somatostatin analog RC-160, and bombesin antagonist RC-3940-II were tested for their effects on the growth of the Caki-I renal adenocarcinoma cell line xenografted into nude mice. RESULTS: After 4 weeks of treatment, tumor volume was significantly (P < 0.01) decreased in animals receiving RC-160, to 167.5 +/- 34.2 mm3, compared with the control group (485.7 +/- 77.2 mm3). LH-RH antagonist SB-75 and bombesin antagonist RC-3940-II also significantly reduced the volume of Caki-I tumors, to 159.9 +/- 18.1 and 234.7 +/- 81.8 mm3, respectively. Somatostatin analog RC-160 decreased serum levels for growth hormone (GH) and insulin-like growth factor-I compared with controls. Treatment with RC-160, Cetrorelix, and RC-3940-II significantly reduced the number of high-affinity receptors for epidermal growth factor on Caki-I tumors. CONCLUSIONS: LH-RH antagonist Cetrorelix, somatostatin analog RC-160, and bombesin antagonist RC-3940-II effectively inhibit the growth of human Caki-I renal adenocarcinomas in nude mice. These peptide analogs should be considered for the therapy of patients with metastatic or recurrent RCC.

Luteinizing hormone-releasing hormone antagonist Cetrorelix (SB-75) and bombesin antagonist RC-3940-II inhibit the growth of androgen-independent PC-3 prostate cancer in nude mice.

BACKGROUND: Hormones like bombesin (BN)/gastrin-releasing peptide (GRP) and luteinizing hormone-releasing hormone (LH-RH) and growth factors such as epidermal growth factor (EGF) might be involved in the relapse of prostate cancer under androgen ablation therapy. Interference with receptors for BN/GRP, LH-RH, or EGF might provide a therapeutic approach to inhibit tumor growth of androgen-independent prostate cancer. METHODS: LH-RH antagonist Cetrorelix (SB-75) and the BN/GRP antagonist RC-3940-II were tested for their effects on the growth of the androgen-independent PC-3 human prostate cancer cell line xenografted into nude mice. Tumor growth, serum hormone levels, and receptor concentrations for BN/GRP and EGF were measured. RESULTS: When the treatment was started, tumor volume in all groups was 70-80 mm3. After 4 weeks, tumor volume in the control animals injected with saline was 871 +/- 233 mm3 and that of animals treated with Cetrorelix only 197 +/- 61 mm3. The BN/GRP antagonist RC-3940-II also significantly reduced PC-3 tumor volume in nude mice to 122 +/- 20 mm3. The combination of Cetrorelix and RC-3940-II produced no additional inhibition. High-affinity receptors for EGF were detected in the tumor membranes and their number was significantly decreased after administration of Cetrorelix or RC-3940-II. CONCLUSIONS: These findings demonstrate that LH-RH antagonists and BN/GRP antagonists inhibit the growth of the androgen-independent prostate cancer cell line PC-3 in vivo. Both analogs may exert a direct inhibitory effect on tumor growth through a down-regulation of EGF receptors.

Inhibition of growth of androgen-independent DU-145 prostate cancer in vivo by luteinising hormone-releasing hormone antagonist Cetrorelix and bombesin antagonists RC-3940-II and RC-3950-II.

The aim of this study was to test the antagonist of LH-RH (Cetrorelix), agonist [D-Trp6]LH-RH (triptorelin) and new bombesin antagonists RC-3940-II and RC-3950-II for their effect on the growth of an androgen-independent prostate cancer cell line, DU-145, xenografted into nude mice. Xenografts were grown in male nude mice, and after 4 weeks, the animals were treated either with saline (control) or with one of the analogues. One group of mice was given a combination of Cetrorelix and RC-3950-II. Treatment was given for 4 weeks. Tumour and body weights, and tumour volumes were measured. At sacrifice, tumours were dissected for histological examination and receptor studies. Serum was collected for measurement of hormone levels. The final tumour volume in control animals injected with saline was 577 +/- 155 mm3 and that of animals treated with Cetrorelix only 121.4 +/- 45 mm3 (P < 0.01). Bombesin antagonists RC-3940-II and RC-3950-II also significantly reduced DU-145 tumour volume in nude mice to 84.9 +/- 19.9 and 96.8 +/- 28 mm3, respectively. Agonist [D-Trp6]LH-RH did not significantly inhibit tumour growth. Serum levels of LH were decreased to 0.08 +/- 0.02 ng/ml (P < 0.05) in the Cetrorelix treated group as compared to 1.02 +/- 0.1 ng/ml for the controls, and testosterone levels were reduced to castration levels (0.01 +/- 0.01 ng/ml). Specific receptors for EGF and LH-RH in DU-145 tumours were significantly downregulated after treatment with Cetrorelix, RC-3940-II and RC-3950-II. Although LH-RH could be a local regulator of growth of prostate cancer, the fall in LH-RH receptors is not fully understood and the inhibitory effects of Cetrorelix and bombesin antagonists on DU-145 tumour growth might be attributed at least in part to a downregulation of EHF receptors. Since Cetrorelix and bombesin antagonists inhibit growth of androgen-independent DU-145 prostate cancers, these compounds could be considered for the therapy of advanced prostate cancer in men, especially after relapse.

Growth hormone-releasing hormone antagonist MZ-4-71 inhibits in vivo proliferation of Caki-I renal adenocarcinoma.

In view of evidence that growth hormone (GH) and insulin-like growth factors (IGF) may play a role in the development of renal cell carcinoma (RCC), we investigated the effects of growth hormone-releasing hormone (GH-RH) antagonist MZ-4-71 on the proliferation of the human renal adenocarcinoma cell line Caki-I in vitro and in vivo. Male nude mice bearing xenografts of human Caki-I RCC were treated for 4 weeks with MZ-4-71 injected s.c. twice daily at a dose of 20 microg per animal. Tumor growth, serum, liver, and tumor IGF levels and IGF-I receptor concentrations in Caki-I cell membranes were measured. After 4 weeks of therapy, the final volume of Caki-I tumors in nude mice treated with MZ-4-71 was significantly (P < 0.01) decreased to 52.6 +/- 12.3 mm3 as compared with controls that measured 504.2 +/- 104.1 mm3. Treatment with GH-RH antagonist also significantly reduced tumor weight, serum levels of GH and IGF-I, liver concentrations of IGF-I, and tumor levels of IGF-I and IGF-II. High-affinity binding sites for IGF-I were detected in the cell membranes of Caki-I tumors. IGF-I and IGF-II stimulated the proliferation of Caki-I cells in tissue cultures. Antagonist MZ-4-71 could inhibit in vitro growth of Caki-I cells, but only at high concentrations. Our findings demonstrate that GH-RH antagonist MZ-4-71 can significantly inhibit the growth of Caki-I RCC. MZ-4-71 may exert its suppressive effect on tumor growth through a reduction in GH release from the pituitary and the subsequent decrease in the production of IGF-I in the liver and IGF-I and II by the tumors. The efficacy of MZ-4-71 suggests that this compound could be considered for the therapy of recurrent or metastatic RCC.

Inhibition of in vivo proliferation of androgen-independent prostate cancers by an antagonist of growth hormone-releasing hormone.

Tumour-inhibitory effects of a new antagonist of growth hormone-releasing hormone (GH-RH), MZ-4-71, were evaluated in nude mice bearing androgen-independent human prostate cancer cell lines DU-145 and PC-3 and in Copenhagen rats implanted with Dunning R-3327 AT-1 prostatic adenocarcinoma. After 6 weeks of therapy, the tumour volume in nude mice with DU-145 prostate cancers treated with 40 microg day(-1) MZ-4-71 was significantly decreased to 37 +/- 13 mm3 (P < 0.01) compared with controls that measured 194 +/- 35 mm3. A similar inhibition of tumour growth was obtained in nude mice bearing PC-3 cancers, in which the treatment with MZ-4-71 for 4 weeks diminished the tumour volume to 119 +/- 35 mm3 compared with 397 +/- 115 mm3 for control animals. Therapy with MZ-4-71 also significantly decreased weights of PC-3 and DU-145 tumours and increased tumour doubling time. Serum levels of GH and IGF-I were significantly decreased in animals treated with GH-RH antagonist. In PC-3 tumour tissue, the levels of IGF-I and IGF-II were reduced to non-detectable values after therapy with MZ-4-71. The growth of Dunning R-3327 AT-1 tumours in rats was also significantly inhibited after 3 weeks of treatment with 100 microg of MZ-4-71 day(-1) i.p. as shown by a reduction in tumour volume and weight (both P-values < 0.05). Specific high-affinity binding sites for IGF-I were found on the membranes of DU-145, PC-3 and Dunning R-3327 AT-1 tumours. Our results indicate that GH-RH antagonist MZ-4-71 suppresses growth of PC-3, DU-145 and Dunning AT-1 androgen-independent prostate cancers, through diminution of GH release and the resulting decrease in the secretion of hepatic IGF-I, or through mechanisms involving a lowering of tumour IGF-I levels and possibly an inhibition of tumour IGF-I and IGF-II production. GH-RH antagonists could be considered for further development for the therapy of prostate cancer, especially after the relapse.

Regression of rat Dunning R-3327-H prostate carcinoma by treatment with targeted cytotoxic analog of luteinizing hormone-releasing hormone AN-207 containing 2-pyrrolinodoxorubicin.

The effects of AN-207, a new targeted cytotoxic analog of LH-RH, were evalued in rats bearing hormone-dependent Dunning R-3327-H prostate carcinomas. AN-207 consists of the agonist [D-Lys(6)]LH-RH linked to 2-pyrrolino-doxorubicin, an intensely potent derivative of doxorubicin. In the first experiment, 2-pyrrolinodoxorubicin was administered at a concentration of 50 nmol/kg, as a single drug (AN-201) and as an unconjugated mixture with [D-Lys(6)]LH-RH or conjugated to the carrier [D-Lys(6)]LH-RH (AN-207). Following the second administration of radical AN-201 alone or mixed with the carrier, all rats died with signs of general toxicity, but all animals treated with the conjugate AN-207, survived. After 5 weeks of treatment with a total dose of 150 nmol/kg AN-207, the tumors regressed from an initial volume of 8.35 +/- 1.7 cm(3) to 4.47 +/- 0.8 cm(3), while tumors in the control group measured 17.84 +/- 2.2 cm(3). The therapy with AN-207 also significantly reduced tumor weight and tumor burden. In the second experiment, we compared the efficacy and toxicity of 3 injections of 25 nmol/kg AN-201 or 25 nmol/kg and 50 nmol/kg AN-207. The initial tumor volume in all groups was between 3.9 and 4.5 cm(3). After 5 weeks of therapy, the tumors of rats treated with 50 nmol/kg AN-207 regressed to 2.3 +/- 0.51 cm(3), whereas 25 nmol/kg AN-201 was still toxic in contrast to 25 nmol/kg AN-207, while the reduction in final tumor volume was similar (6.76 +/- 1.4 cm(3) and 6.74 +/- 1 cm(3), respectively), as compared to 15.6 +/- 2.2 cm(3) for untreated animals. High capacity LH-RH receptors were found in the membranes of untreated Dunning tumor specimens, but after treatment with AN-207, they could no longer be detected. This is the first demonstration that the new targeted cytotoxic LH-RH analog AN-207 is an effective antitumor agent. Our work indicates that the cytotoxic analog AN-207 is much less toxic than the antineoplastic radical (AN-201) incorporated, and significantly more active in inhibiting tumor growth. Further development of approaches based on targeted cytotoxic analog AN-207 may lead to major improvements in current palliative therapy of prostate cancer.

Effect of chronic administration of a new potent agonist of GH-RH(1-29)NH2 on linear growth and GH responsiveness in rats.

The effects of a repeated or continuous administration of a potent agonistic analog of growth hormone-releasing hormone (GH-RH), [Dat1, Thr8, Orn12,21, Abu15, Nle27, Asp28, Agm29] hGH-RH(1-29)(JI-36), on the linear growth and the GH responses to bolus injections of GH-RH(1-29)NH2 were investigated in male rats about 7 weeks old. Body weight and tail length were monitored. Basal serum GH and IGF-I concentrations and GH responses to GH-RH(1-29)NH2 were measured by RIA. Chronic administration of GH-RH agonist JI-36 by continuous release from osmotic minipumps at the rate of 0.2 microgram/h or twice daily injections of 0.5 and 5 micrograms/rat for 4 weeks significantly speeded up the growth of rats as measured by the tail length. The acceleration of growth was similar in the 3 groups and was associated with stimulation of IGF-I secretion. The GH response to bolus injection of GH-RH(1-29)NH2 was preserved in all groups and no attentuation of the response occurred in rats treated for 4 weeks with agonist JI-36 as compared with the control group. Our results indicate that chronic administration of GH-RH agonist JI-36 significantly increases the growth rate without affecting somatotroph responsiveness in rats. It is therefore likely that this class of GH-RH agonists may be useful clinically.

Chronic administration of the luteinizing hormone-releasing hormone (LHRH) antagonist cetrorelix decreases gonadotrope responsiveness and pituitary LHRH receptor messenger ribonucleic acid levels in rats.

Continuous exposure to LHRH or its agonistic analogs results in a reduction of LHRH receptor sites and messenger RNA (mRNA) transcripts as well as in desensitization of the pituitary gonadotropes. To determine, whether LHRH antagonists might be similar in this respect to the agonists, we treated male rats for 4 weeks with daily sc injections of LHRH antagonist [Ac-D-Nal2,Phe(4Cl)2,D-Pal(3)3, D-Cit6,D-Ala10]LHRH (Cetrorelix acetate) or LHRH agonist, [D-Trp6]LHRH, in doses of 100 micrograms/animal-day. Another group of rats received a single im injection of 4.5 mg Cetrorelix pamoate depot, a sustained delivery formulation of the LHRH antagonist. An iv stimulation test with LHRH (200 ng/rat) was performed after 4 weeks of treatment. The rats were killed, and pituitary LHRH receptor characteristics were measured by RRA. To examine the effect of LHRH antagonist treatment on the expression of the pituitary LHRH receptor gene, some of the rats injected with Cetrorelix pamoate depot were killed after 2 weeks, and levels of LHRH receptor mRNA were determined by Northern blot and dot blot hybridization to a 32P-labeled rat complementary DNA probe. Our data show that LHRH-stimulated LH secretion at 30 min was suppressed by approximately 33% (P < 0.01) in rats pretreated with [D-Trp6]LHRH compared to that in animals injected with LHRH alone. Pretreatment of the rats with the LHRH antagonist suppressed the LH response to LHRH more markedly, the LH levels at 30 min were decreased by 89.8% and 96% in groups treated with Cetrorelix acetate and Cetrorelix pamoate depot, respectively. The testosterone response was virtually abolished in groups receiving Cetrorelix. The concentration of pituitary receptors for LHRH fell by 69% in the [D-Trp6]LHRH group, whereas the reductions in the Cetrorelix acetate group and in the group that received Cetrorelix pamoate depot were 77% and 82%, respectively. Treatment with Cetrorelix pamoate depot led to a 75-80% decrease in the levels of 5.0- and 4.5-kilobase forms of LHRH receptor mRNA compared to those in the control group. Dot blot analysis also showed 83% reduction in the mRNA for LHRH receptor. In conclusion, these data demonstrate that prolonged administration of LHRH antagonists such as Cetrorelix causes an impairment of gonadotropin secretion and a marked decrease in the levels of LHRH receptors as well as in the expression of the LHRH receptor gene. Thus, the down-regulation of pituitary LHRH receptors produced by LHRH antagonists appears to be similar to that resulting from the agonists.

Down-regulation of pituitary receptors for luteinizing hormone-releasing hormone (LH-RH) in rats by LH-RH antagonist Cetrorelix.

Antagonists of luteinizing hormone-releasing hormone (LH-RH), unlike the LH-RH agonists, suppress gonadotropins and sex steroid secretion immediately after administration, without initial stimulatory effects. [Ac-D-Nal(2)1,D-Ph(4Cl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-R H (SB-75; Cetrorelix) is a modern, potent antagonistic analog of LH-RH. In this study, the binding characteristics of receptors for LH-RH in membrane fractions from rat anterior pituitaries were investigated after a single injection of Cetrorelix at a dose of 100 microg per rat. To determine whether the treatment with Cetrorelix can affect the concentration of measurable LH-RH binding sites, we applied an in vitro method to desaturate LH-RH receptors by chaotropic agents such as manganous chloride (MnCl2) and ammonium thiocyanate (NH4SCN). Our results show that the percentages of occupied LH-RH receptors at 1, 3, and 6 h after administration of Cetrorelix were approximately 28%, 14%, and 10%, respectively, of total receptors. At later time intervals, we could not detect occupied LH-RH binding sites. Ligand competition assays, following in vitro desaturation, demonstrated that rat pituitary LH-RH receptors were significantly (P < 0.01) down-regulated for at least 72 h after administration of Cetrorelix. The lowest receptor concentration was found 3-6 h after Cetrorelix treatment and a recovery in receptor number began within approximately 24 h. The down-regulation of LH-RH binding sites induced by Cetrorelix was accompanied by serum LH and testosterone suppression. Higher LH-RH receptor concentrations coincided with elevated serum hormone levels at later time intervals. Our results indicate that administration of LH-RH antagonist Cetrorelix produces a marked down-regulation of pituitary receptors for LH-RH and not merely an occupancy of binding sites.

Somatostatin analog RC-160 inhibits the growth of human osteosarcomas in nude mice.

We investigated the effects of the potent somatostatin analog RC-160 on the growth of human osteosarcoma cell lines SK-ES-1 and MNNG/HOS, transplanted into nude mice or cultured in vitro. Growth of SK-ES-1 and MNNG/HOS tumors in nude mice was significantly inhibited after 4 weeks of treatment with daily s.c. injections of 100 micrograms RC-160, as measured by a reduction in tumor volume and weight. Histologically, the number of mitotic cells was decreased in the groups treated with RC-160. In mice bearing either tumor model, administration of RC-160 significantly decreased serum growth hormone and insulin-like growth factor I (IGF-I) levels. Specific high-affinity receptors for somatostatin and epidermal growth factor were found on membranes of MNNG/HOS tumors but not on SK-ES-1 tumors. Receptor analyses also demonstrated high-affinity binding sites for IGF-I on membranes of both tumors. In cell cultures, the proliferation rate of MNNG/HOS cells, but not of SK-ES-1, was significantly suppressed by RC-160. Our findings demonstrate that RC-160 can significantly inhibit the growth of SK-ES-1 and MNNG/HOS osteosarcomas in mice.

Inhibition of growth of human small cell and non-small cell lung carcinomas by antagonists of growth hormone-releasing hormone (GH-RH).

Insulin-like growth factors-I and-II (IGF-I and IGF-II) may be involved in the proliferation of human lung carcinomas. The purpose of this study was to investigate the effects of two potent antagonists of growth hormone-releasing hormone (GH-RH), MZ-4-71 and MZ-5-156 on the growth of the H69 human small cell lung cancer (SCLC) and H157 non-SCLC (NSCLC) lines transplanted into nude mice or cultured in vitro. Nude mice bearing H69 and H157 tumors were treated for 3-5 weeks with MZ-4-71 or MZ-5-156 injected s.c. twice a day at a dose of 20 mu g/animal. Growth of H69 and H157 tumors in nude mice was significantly inhibited by MZ-4-71 and MZ-5-156 as shown by a reduction in tumor volume and weight. In animals bearing H157 NSCLC, treatment with MZ-4-71 decreased IGF-I and IGF-II levels in tumor tissue. Levels of IGF-I, but not of IGF-II in serum and liver tissue of H157 tumor-bearing nude mice treated with MZ-4-71 were decreased. High affinity binding sites for ICF-I were demonstrated on membranes of H69 and H157 tumors. In cell cultures, the proliferation rate of H69 SCLC cells was suppressed by 10(-7)-10(-5) M MZ-4-71, but H157 NSCLC line was only inhibited by 10(-5) M antagonist. Our findings demonstrate that the GHRH antagonists MZ-4-71 and MZ-5-156 can inhibit the growth of SCLC and NSCLC. This new approach to the management of lung cancer merits further investigation.

Inhibition of growth of human osteosarcomas by antagonists of growth hormone-releasing hormone.

BACKGROUND: Insulin-like growth factor I (IGF-I) may be involved in the proliferation of human osteosarcomas. Most of the IGF-I found in blood is produced in the liver, where transcription of the IGF-I gene is regulated by growth hormone (GH). Recently, we synthesized various potent antagonists of GH-releasing hormone (GH-RH), including [Ibu0, D-Arg2, Phe(4-Cl)6, Abu15, Nle27]hGH-RH(1-28)Agm, which is also called MZ-4-71. PURPOSE: We investigated the effects of this antagonist on the growth of the human osteosarcoma cell lines SK-ES-1 and MNNG/HOS, transplanted into nude mice or cultured in vitro. METHODS: Nude male mice bearing SK-ES-1 and MNNG/HOS tumors were treated for 4 and 3 weeks, respectively, with MZ-4-71 administered from osmotic minipumps at a dose of 40 micrograms per animal per day. Tumor volume, tumor weight, and levels of receptors for IGF-I were determined. IGF-I levels in serum, tumor, and liver tissue were measured by radioimmunoassay. In other experiments, tumor-bearing nude mice were treated subcutaneously for 3 weeks with the GH-RH agonist hGH-RH(1-29)NH2 or with MZ-4-71 for 13 days at doses of 50 micrograms per animal per day. Effects of MZ-4-71, hGH-RH(1-29)NH2, and human GH (hGH) on cell proliferation and on the production of IGF-I and cyclic adenosine monophosphate were also evaluated in SK-ES-1 and MNNG/HOS cells in vitro. RESULTS: The growth of SK-ES-1 and MNNG/HOS tumors in nude mice was significantly inhibited by MZ-4-71, as measured by a reduction in tumor volume and weight (all P values < .05). MZ-4-71 treatment of either SK-ES-1 or MNNG/HOS tumor-bearing animals decreased tumor tissue IGF-I levels. The growth of MNNG/HOS xenografts was stimulated by hGH-RH(1-29)NH2 (P < .01). IGF-I levels in serum of tumor-bearing nude mice treated subcutaneously for 13 days with MZ-4-71 were decreased (both P values < .01). High-affinity binding sites for IGF-I were demonstrated on cell membranes of SK-ES-1 and MNNG/HOS tumors. In cell cultures of both osteosarcomas, IGF-I production was stimulated by 25 ng/mL hGH but was not changed by 10 ng/mL hGH-RH(1-29)NH2 or 5 microM MZ-4-71. Incorporation of [3H]thymidine into DNA in SK-ES-1 (but not MNNG/HOS) cells was increased by 25 ng/mL IGF-I (P < .01). The proliferation rate of the two cell lines was not affected by 5-50 ng/mL hGH-RH(1-29)NH2 or 1-80 ng/mL hGH but was suppressed by 10(-6)-10(-5) M MZ-4-71. CONCLUSIONS: Our findings demonstrate that the GH-RH antagonist MZ-4-71 can significantly inhibit the growth of SK-ES-1 and MNNG/HOS osteosarcomas in mice.

Synthesis and biological evaluation of superactive agonists of growth hormone-releasing hormone.

Analogs of the 29 amino acid sequence of human growth hormone-releasing hormone (hGH-RH) with agmatine (Agm) in position 29, desaminotyrosine (Dat) in position 1, norleucine (Nle) in position 27, and L-alpha-aminobutyric acid (Abu) in position 15 have been synthesized, and their biological activity was evaluated. Some peptides contained one or two residues of ornithine (Orn) instead of Lys in positions 12 and 21 and additional replacements in positions 8 and 28. All analogs were found to be more potent than hGH-RH-(1-29)-NH2 in the superfused rat pituitary cell system. In tests in vivo in rats after subcutaneous administration, the analogs JI-22, [Dat1, Orn12,21, Abu15, Nle27, Agm29]hGH-RH-(1-29); JI-34, [Dat1, Orn12,21,Abu15,Nle27, Asp28, Agm29]hGH-RH-(1-29); JI-36, [Dat1, Thr8, Orn12,21, Abu15,Nle27,Asp28,Agm29]hGH-RH-(1-29); and JI-38, [Dat1,Gln8, Orn12,21,Abu15,Nle27,Asp28,Agm29]hGH-RH-(1 -29) displayed a potency 44.6,80.9,95.8, and 71.4 times greater, respectively, than that of hGH-RH-(1-29)-NH2 at 15 min and 217.1, 89.7, 87.9, and 116.8 times greater at 30 min. After intravenous administration, JI-22, JI-36, and JI-38 were 3.2-3.8 times more potent than hGH-RH-(1-29)-NH2 at 5 min and 6.1-8.5 times more active at 15 min. All analogs were found to have higher binding affinities for GH-RH receptors on rat pituitary cells than hGH-RH-(1-29)-NH2. Because of high activity and greater stability, these analogs could be considered for therapy of patients with growth hormone deficiency.

Evaluation of the in vitro and in vivo activity of the L-, D,L- and D-Cit6 forms of the LH-RH antagonist Cetrorelix (SB-75).

The objective of this study was to examine the in vivo and in vitro gonadotropin-inhibiting potencies, edematogenic activities and the receptor binding affinities of the D-Cit6, D,L-Cit6 and L-Cit6 forms of the LH-RH antagonist Cetrorelix (SB-75) [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH- RH. In order to demonstrate the suppressive effects of two different diastereomers of SB-75 and their racemic mixture on LH and FSH release, [D-Cit6] SB-75 was injected subcutaneously in doses of 2.5 and 10 micrograms/rat, [D,L-Cit6]-SB-75 in doses of 5 and 20 micrograms/rat and [L-Cit6] SB-75 in doses of 12.5 and 50 micrograms/rat to castrated male rats. Two hours after administration, there was no difference in LH levels between rats injected with the L-form and control animals, indicating a low activity and/or a rapid enzymatic degradation of this peptide. The (1:1) diastereomeric mixture was only about half as potent in suppression of LH release compared to [D-Cit6] SB-75. Serum FSH levels were suppressed significantly (p < 0.01) for more than 48 h after the administration of 10 micrograms [D-Cit6] SB-75 and 20 micrograms of [D,L-Cit6] SB-75, respectively. [D-Cit6] SB-75 administered at a dose of 2 micrograms/rat induced 100% inhibition of ovulation, while 4 micrograms/rat of the D,L-Cit6 peptide were necessary to produce the same effect.(ABSTRACT TRUNCATED AT 250 WORDS)