Longitudinal assessment of liver stiffness using ARFI technique does not support increased risk of fibrosis in rheumatoid arthritis patients on methotrexate.

AIMS: To assess the liver stiffness in patients with rheumatoid arthritis treated with methotrexate monotherapy using non-invasive, ultrasound-based elastography (acoustic radiation force impulse (ARFI) imaging) in a longitudinal approach. METHODS: In total, 23 MTX-naive patients were longitudinally assessed using acoustic radiation force impulse (ARFI) imaging. Baseline assessments were carried out between July 2018 and April 2019, and the follow-up evaluations took place after an average of 2.6 years. The main outcome variable was the mean shear wave velocity as measured by the ARFI method. It was calculated from 10 valid ARFI measurements for each patient. Inferential statistical analyses (within-group comparisons) were performed using t-tests for dependent samples or suitable nonparametric procedures. RESULTS: The main finding was that observed ARFI shear wave velocities did not increase during the observation period. In fact, this parameter decreased over time from 1.07 m/s (SD = 0.23) at baseline without MTX exposure to 0.97 m/s (SD = 0.16) at follow-up after a mean of 2.6 years (P = 0.013). Moreover, the magnitude of the change in shear wave velocity could not be predicted by indicators of inflammation or disease activity, BMI, age, sex or NSAR intake (corresponding regression analysis: corrected R2 = 0.344; P = 0.296). CONCLUSIONS: No increased risk of liver fibrosis was found in RA patients treated with MTX monotherapy during observation period.

TNF inhibitors significantly attenuate the humoral immune response to COVID-19 vaccination in patients with rheumatoid arthritis.

Objective: Several studies on the immunogenicity of vaccination against coronavirus disease 2019 (COVID-19) in patients with immune-mediated inflammatory diseases have evaluated the influence of DMARDs. The aim of the work presented here was to compare the humoral vaccine response after two vaccinations between patients with RA undergoing TNF inhibitor therapy and healthy controls. Methods: We assessed the humoral immune response, as measured by titres of neutralizing antibodies against the S1 antigen of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in patients with RA and anti-TNF treatment vs. controls without immunomodulatory medication. One hundred and seven fully vaccinated individuals were included at 6 ± 1 weeks after the second vaccination [BioNTech/Pfizer (72.9%), AstraZeneca (17.8%) and Moderna (9.3%)]. Immune responses in terms of antibody titres were compared between both subgroups with (n = 45) and without (n = 62) exposure to anti-TNF medication. The comparison was performed as a cross-sectional, single-centre study approach using non-parametric tests for central tendency. Results: Anti-TNF medication produced a significantly impaired humoral immune response to vaccination against COVID-19. The maximum immune response was detected in 77.4% of control patients, whereas this decreased to 62.2% in participants treated with TNF inhibitors (P = 0.045; effect size, d = 0.194). Patients on combination treatment (anti-TNF medication and MTX, 17 of 45 subjects in the treatment group) did not differ significantly regarding humoral immune response compared with patients on monotherapy with TNF inhibitors only (P = 0.214). Conclusion: TNF inhibitors significantly reduce the humoral response following dual vaccination against COVID-19 in patients with RA.

Janus kinase (JAK) inhibitors significantly reduce the humoral vaccination response against SARS-CoV-2 in patients with rheumatoid arthritis.

OBJECTIVES: Recently, a number of studies have explored the possible attenuation of the immune response by disease-modifying antirheumatic drugs (DMARDs) in patients with rheumatoid arthritis (RA). Our study objective was to investigate the presumed attenuated humoral response to vaccination against SARS-CoV-2 in patients with RA treated with Janus kinase (JAK) inhibitors with or without methotrexate (MTX). The immune responses were compared with controls without RA. METHOD: The humoral vaccination response was evaluated by determining titres of neutralising antibodies against the S1 antigen of SARS-CoV-2. One hundred and thirteen fully vaccinated individuals were included at 6 ± 1 weeks after second vaccination (BioNTech/Pfizer (69.9%), AstraZeneca (21.2%), and Moderna (8.9%)). In a cross-sectional and single-centre study design, we compared titres of neutralising antibodies between patients with (n = 51) and without (n = 62) medication with JAK inhibitors. RESULTS: Treatment with JAK inhibitors led to a significantly reduced humoral response to vaccination (P = 0.004). A maximum immune response was seen in 77.4% of control patients, whereas this percentage was reduced to 54.9% in study participants on medication with JAK inhibitors (effect size d = 0.270). Further subanalyses revealed that patients on combination treatment (JAK inhibitors and MTX, 9 of 51 subjects) demonstrated an even significantly impaired immune response as compared to patients on monotherapy with JAK inhibitors (P = 0.028; d = 0.267). CONCLUSIONS: JAK inhibitors significantly reduce the humoral response following dual vaccination against SARS-CoV-2. The combination with MTX causes an additional, significant reduction in neutralising IgG titres. Our data suggest cessation of JAK inhibitors in patients with RA in the context of vaccination against SARS-CoV-2. Key Points • It was shown that DMARD therapy with JAK inhibitors in patients with rheumatoid arthritis leads to an attenuation of the humoral vaccination response against SARS-CoV-2. • The effect under medication with JAK inhibitors was significant compared to the control group and overall moderate. • The combination of JAK inhibitors with MTX led to an additive and significant attenuation of the humoral response.

Methotrexate significantly reduces the humoral vaccination response against SARS-CoV-2 in older but not younger patients with rheumatoid arthritis.

To assess the humoral response to vaccination against SARS-CoV-2 in patients with rheumatoid arthritis treated with methotrexate (MTX). In total, 142 fully vaccinated individuals were included at 6 ± 1 weeks after their second vaccination [BioNTech/Pfizer (70.4%), AstraZeneca (20.4%), and Moderna (9.2%)]. The primary goal was to assess the humoral immune response as measured by titres of neutralising antibodies against the S1 antigen of SARS-CoV-2. In a cross-sectional, single-centre study, titres were compared between patient subgroups with (n = 80) and without (n = 62) methotrexate exposure. MTX patients showed a significantly reduced humoral response to vaccination in the oldest patient subgroup (> 70 years: P = 0.038), whereas titres of neutralising antibodies were not significantly different between MTX and non-MTX patients in patients less than 70 years of age (< 56 years: P = 0.234; 56-70 years: P = 0.446). In patients > 70 years, non-MTX patients showed a maximum immune response in 76.5% of cases, whereas this percentage was reduced to 53.7% in study participants on MTX medication (effect size d = 0.21). Older age in patients with rheumatoid arthritis in combination with methotrexate results in a significantly reduced humoral response after vaccination against SARS-CoV-2. Our data underline the importance of age regarding the humoral response and may support the temporary cessation of methotrexate, particularly in elderly patients in the context of vaccination against SARS-CoV-2.

Synthesis and structure-activity studies on novel analogs of human growth hormone releasing hormone (GHRH) with enhanced inhibitory activities on tumor growth.

The syntheses and biological evaluations of new GHRH analogs of Miami (MIA) series with greatly increased anticancer activity are described. In the design and synthesis of these analogs, the following previous substitutions were conserved: D-Arg2, Har9, Abu15, and Nle27. Most new analogs had Ala at position 8. Since replacements of both Lys12 and Lys21 with Orn increased resistance against enzymatic degradation, these modifications were kept. The substitutions of Arg at both positions 11 and 20 by His were also conserved. We kept D-Arg28, Har29 -NH2 at the C-terminus or inserted Agm or 12-amino dodecanoic acid amide at position 30. We incorporated pentafluoro-Phe (Fpa5), instead of Cpa, at position 6 and Tyr(Me) at position 10 and ω-amino acids at N-terminus of some analogs. These GHRH analogs were prepared by solid-phase methodology and purified by HPLC. The evaluation of the activity of the analogs on GH release was carried out in vitro on rat pituitaries and in vivo in male rats. Receptor binding affinities were measured in vitro by the competitive binding analysis. The inhibitory activity of the analogs on tumor proliferation in vitro was tested in several human cancer cell lines such as HEC-1A endometrial adenocarcinoma, HCT-15 colorectal adenocarcinoma, and LNCaP prostatic carcinoma. For in vivo tests, various cell lines including PC-3 prostate cancer, HEC-1A endometrial adenocarcinoma, HT diffuse mixed ß cell lymphoma, and ACHN renal cell carcinoma cell lines were xenografted into nude mice and treated subcutaneously with GHRH antagonists at doses of 1-5µg/day. Analogs MIA-602, MIA-604, MIA-610, and MIA-640 showed the highest binding affinities, 30, 58, 48, and 73 times higher respectively, than GHRH (1-29) NH2. Treatment of LNCaP and HCT-15 cells with 5µM MIA-602 or MIA-690 decreased proliferation by 40%-80%. In accord with previous tests in various human cancer lines, analog MIA-602 showed high inhibitory activity in vivo on growth of PC-3 prostate cancer, HT-mixed ß cell lymphoma, HEC-1A endometrial adenocarcinoma and ACHN renal cell carcinoma. Thus, GHRH analogs of the Miami series powerfully suppress tumor growth, but have only a weak endocrine GH inhibitory activity. The suppression of tumor growth could be induced in part by the downregulation of GHRH receptors levels.

DO PREVIOUS OFFENCES PREDICT VIOLENT ACTS IN PSYCHIATRIC PATIENTS? A RETROSPECTIVE STUDY IN HUNGARY.

AIM: To investigate the presence of offences in the previous past history of perpetrators of violent acts who have undergone forced medical treatment. METHODS: The documentation of all patients released over a 10-year period from the National Institute of Forensic Psychiatry (IMEI) was reviewed. A comparison was drawn between patients who were convicted of any type of offense before the violent act (patients with previous offences-PPO) and those who were not (patients with no previous offences-PNO). RESULTS: Eighty-six (29%) and 208 (71%) patients formed the PPO and PNO groups, respectively. Prior contact with psychiatric services was significantly higher in the PPO group (p=0.038) and this group was also more likely to offend under the influence of a psychoactive substance (p<0.001). Exceptional brutality and other qualifying factors were more frequent in the PNO group (p=0.019). CONCLUSION: As IMEI is the only forensic institution in Hungary, the picture presented here reflects the situation in the entire country. A recidivism rate of 29% is within the internationally published range.

Comparative in vitro biological evaluation of daunorubicin containing GnRH-I and GnRH-II conjugates developed for tumor targeting.

Hormone based drug targeting is a promising tool for selective tumor therapy. In this study, synthesis and systematic comparative biological evaluation of novel drug containing analogs of gonadotropin-releasing hormone GnRH-I and GnRH-II is reported demonstrating their suitability for tumor targeting. The cytotoxic conjugates were prepared by the attachment of the chemotherapeutical agent daunorubicin (Dau) to GnRH analogs directly or through an enzyme-labile spacer with oxime linkage. All conjugates were found to be proteolytically stable under circumstances applied in biological assays. Both GnRH-I and GnRH-II were able to bind similarly to high-affinity GnRH-I receptors on human pituitary and human prostate cancer cells. The in vitro long-term cytotoxic effect of the conjugates was comparable with that of the free drug in human breast and colon cancer cell lines. Furthermore, a concentration-dependent cellular uptake profile was observed. The in vitro apoptotic effect of the compounds was evaluated by flow cytometry analysis using annexin-V. Our results show that both the GnRH-I and the GnRH-II based analogs might be applied for targeted tumor therapy.

Synthesis of new potent agonistic analogs of growth hormone-releasing hormone (GHRH) and evaluation of their endocrine and cardiac activities.

In view of the recent findings of stimulatory effects of GHRH analogs, JI-34, JI-36 and JI-38, on cardiomyocytes, pancreatic islets and wound healing, three series of new analogs of GHRH(1-29) have been synthesized and evaluated biologically in an endeavor to produce more potent compounds. "Agmatine analogs", MR-356 (N-Me-Tyr(1)-JI-38), MR-361(N-Me-Tyr(1), D-Ala(2)-JI-38) and MR-367(N-Me-Tyr(1), D-Ala(2), Asn(8)-JI-38), in which Dat in JI-38 is replaced by N-Me-Tyr(1), showed improved relative potencies on GH release upon subcutaneous administration in vivo and binding in vitro. Modification with N-Me-Tyr(1) and Arg(29)-NHCH3 as in MR-403 (N-Me-Tyr(1), D-Ala(2), Arg(29)-NHCH3-JI-38), MR-406 (N-Me-Tyr(1), Arg(29)-NHCH3-JI-38) and MR-409 (N-Me-Tyr(1), D-Ala(2), Asn(8), Arg(29)-NHCH3-JI-38), and MR-410 (N-Me-Tyr(1), D-Ala(2), Thr(8), Arg(29)-NHCH3-JI-38) resulted in dramatically increased endocrine activities. These appear to be the most potent GHRH agonistic analogs so far developed. Analogs with Apa(30)-NH2 such as MR-326 (N-Me-Tyr(1), D-Ala(2), Arg(29), Apa(30)-NH2-JI-38), and with Gab(30)-NH2, as MR-502 (D-Ala(2), 5F-Phe(6), Ser(28), Arg(29),Gab(30)-NH2-JI-38) also exhibited much higher potency than JI-38 upon i.v. administration. The relationship between the GH-releasing potency and the analog structure is discussed. Fourteen GHRH agonists with the highest endocrine potencies were subjected to cardiologic tests. MR-409 and MR-356 exhibited higher potency than JI-38 in activating myocardial repair in rats with induced myocardial infarction. As the previous class of analogs, exemplified by JI-38, had shown promising results in multiple fields including cardiology, diabetes and wound healing, our new, more potent, GHRH agonists should manifest additional efficacy for possible medical applications.

Inhibitory effects of GHRH antagonists on human GH-secreting adenoma tissue.

Experimental data indicate that antagonists of growth hormone-releasing hormone (GHRH) could be used clinically in disorders characterized by excessive GHRH/growth hormone (GH) secretion, but direct evidence for the effectiveness of GHRH antagonists on human pituitary tissue is still lacking. In this study, we investigated the inhibitory effect of our GHRH antagonists MZ-4-71 and JV-1-36 and the somatostatin (SST) analog RC-160 on superfused pituitary cells obtained from a human GH-secreting adenoma. Using Western blot analysis and immunohistochemistry, we demonstrated profuse expression of the GHRH receptor and its major splice variant SV1 and an increase in the expression of Gsa protein in the adenoma tissue. Exposure of the tumor cells to exogenous pulses of GHRH induced definite GH responses, causing a 3- to 5-fold elevation of the basal GH level. The antagonists MZ-4-71 and JV-1-36 did not alter basal GH secretion, indicating that the adenoma cells did not secrete GHRH in an autocrine manner. However, both antagonists prevented the stimulatory effect of exogenous GHRH. Similarly to the GHRH antagonists, neither SST-14 nor the SST analog RC-160 had an effect on the basal GH secretion of the tumor cells, but both peptides inhibited the stimulatory effect of exogenous GHRH, with RC-160 being more potent than SST. Our study provides direct evidence for the effectiveness of potent GHRH antagonists such as MZ-4-71 and JV-1-36 on human pituitary GH-secreting adenoma tissue and strongly suggests that these drugs could be used for therapy of GHRH-associated forms of acromegaly, particularly for those patients in whom surgery fails or is not an option.

Combining growth hormone-releasing hormone antagonist with luteinizing hormone-releasing hormone antagonist greatly augments benign prostatic hyperplasia shrinkage.

PURPOSE: Benign prostatic hyperplasia often affects aging men. Antagonists of the neuropeptide growth hormone-releasing hormone reduced prostate weight in an androgen induced benign prostatic hyperplasia model in rats. Luteinizing hormone-releasing hormone antagonists also produce marked, protracted improvement in lower urinary tract symptoms, reduced prostate volume and an increased urinary peak flow rate in men with benign prostatic hyperplasia. We investigated the influence of a combination of antagonists of growth hormone-releasing hormone and luteinizing hormone-releasing hormone on animal models of benign prostatic hyperplasia. MATERIALS AND METHODS: We evaluated the effects of the growth hormone-releasing hormone antagonist JMR-132, given at a dose of 40 µg daily, the luteinizing hormone-releasing hormone antagonist cetrorelix, given at a dose of 0.625 mg/kg, and their combination on testosterone induced benign prostatic hyperplasia in adult male Wistar rats in vivo. Prostate tissue was examined biochemically and histologically. Serum levels of growth hormone, luteinizing hormone, insulin-like growth factor-1, dihydrotestosterone and prostate specific antigen were determined. RESULTS: Marked shrinkage of the rat prostate (30.3%) occurred in response to the combination of growth hormone-releasing hormone and luteinizing hormone-releasing hormone antagonists (p<0.01). The combination strongly decreased prostatic prostate specific antigen, 6-transmembrane epithelial antigen of the prostate, interleukin-1ß, nuclear factor-κß and cyclooxygenase-2, and decreased serum prostate specific antigen. CONCLUSIONS: A combination of growth hormone-releasing hormone antagonist with luteinizing hormone-releasing hormone antagonist potentiated a reduction in prostate weight in an experimental benign prostatic hyperplasia model. Results suggest that this shrinkage in prostate volume was induced by the direct inhibitory effects of growth hormone-releasing hormone and luteinizing hormone-releasing hormone antagonists exerted through their respective prostatic receptors. These findings suggest that growth hormone-releasing hormone antagonists and/or their combination with luteinizing hormone-releasing hormone antagonists should be considered for further development as therapy for benign prostatic hyperplasia.

Regulation of pituitary inhibin/activin subunits and follistatin gene expression by GnRH in female rats.

Pituitary inhibin B, activin B, and follistatin are local regulators of FSH. Activin B is a homodimeric molecule (ß(B)-ß(B)), while inhibin B contains an α and a ß(B) subunit. The regulation of gene expression of α, ß(B), and follistatin by local and endocrine hormones was examined in pituitaries from female rats and in perifused pituitary cells by RT-PCR. Ovariectomy (OVX) induced an elevation in the mRNA level of α and ß(B) subunits and follistatin. Short-term (4 h) treatment of pituitary cells with GnRH decreased both the inhibin α and the inhibin/activin ß(B) subunit mRNA levels, while long-term treatment (20 h) with 100 nM GnRH stimulated the expression of both subunits. In contrast, the mRNA level of follistatin was elevated after the short-term GnRH treatment. Long-term exposure of pituitary cells to estradiol and inhibin B suppressed the mRNA expression of ß(B) and had no effect on the expression of α subunit and follistatin. Our results demonstrate that the increased expressions of inhibin/activin subunits and follistatin in the post-OVX period can be induced by the lack of gonadal negative feedback, resulting in a high GnRH environment in the pituitary. This study reports for the first time that GnRH administered in high doses and for a long period stimulates the gene expression of inhibin/activin subunits and thereby may contribute to the stimulatory effect of OVX on the expression of these genes.

Sexual dimorphism in the effect of concomitant progesterone administration on changes caused by long-term estrogen treatment in pituitary hormone immunoreactivities of rats.

BACKGROUND: Since in clinical practice long-term estrogen (E) treatment is frequently applied, our aim was to study the effect of concomitant progesterone (P) administration on changes caused by long-term estrogen treatment in the secretion of LH, FSH, PRL and GH. MATERIAL/METHODS: Diethylstilbestrol (DES), P or both in silastic capsules were implanted under the skin of prepubertal Sprague-Dawley male and female rats. Animals survived for two or five months. We have also studied whether the changed hormone secretion caused by DES can return to normal level 1 or 2 months after removing DES capsule. RESULTS: 1.) The males more rapidly responded than females with decreasing basal LH release upon treatments. The basal FSH release was decreased only in males. The effect of DES persisted in males; however, in females basal LH and FSH levels were upregulated after removal of DES capsule. 2.) The basal GH levels were low in each group. The body weight and length were depressed by DES in both genders and P little blunted this effect. The body weight and length in males remained low after removal of DES capsule, in females it was nearly similar to intact rats. 3.) There was no sexual dimorphism in the effect of steroids on PRL secretion. In both genders DES extremely enhanced the PRL levels, P prevented the effect of DES. PRL levels returned to intact value after removal of DES influence. 4.) Removal of DES capsule reversed the changes in the immunohistochemical appearance of hormone immunoreactivities. CONCLUSIONS: There was sexual dimorphism in the change of basal gonadotropic hormone and GH secretion but not of PRL upon DES and DES+P treatments. P was basically protective and this role may be mediated by P receptors locally in the pituitary gland.

A correlation of endocrine and anticancer effects of some antagonists of GHRH.

GHRH receptor antagonists inhibit growth and metastasis of a large number of experimental tumors expressing the pituitary GHRH receptor (pGHRH-R) and its major splice variant SV1. In this study, using Western blot, we demonstrated that DBTRG-05 and U-87MG human glioblastoma cell lines express pGHRH-R at levels 6-15 times higher than SV1. To reveal a correlation between the anticancer activity and the endocrine potency on inhibition of GH release, we compared the antitumor effect of GHRH antagonists JV-1-63 and MZJ-7-138 on growth of DBTRG-05 human glioblastomas grafted into athymic nude mice with their inhibitory potency on GH release. JV-1-63 strongly suppressed the stimulated GH secretion induced by clonidine in rats and inhibited the exogenous GHRH-induced GH surge by 88-99% in vivo and in vitro. MZJ-7-138 decreased the stimulated GH secretion by 58% in vitro and showed only a tendency to inhibit GH secretion in vivo. The strong inhibitor of GH release JV-1-63 reduced tumor growth of DBTRG-05 glioblastomas in nude mice by 46%, while the weak GH release suppressor MZJ-7-138 did not have an effect. Exposure of DBTRG-05 cells to the GHRH antagonists in vitro caused an upregulation of mRNA expression for pGHRH-R and a downregulation of SV1 expression, with JV-1-63 having significantly greater effects than MZJ-7-138. Our results demonstrate that a positive correlation exists between the endocrine potency and the antiproliferative efficacy of GHRH antagonists in tumors strongly expressing pGHRH-R.

New derivatives of GnRH as potential anticancer therapeutic agents.

GnRH (gonadotropin-releasing hormone), a decapeptide produced by the hypothalamus, plays an important role in the reproduction by regulating the pituitary-gonadal axis. Continuous high doses of GnRH or its superactive agonists result in desensitization of the pituitary gonadotropes and a suppression of sex steroid production by the gonads (chemical castration). Based on these effects, the treatment with GnRH agonists has become a widely used hormonal therapy of the sex-steroid dependent tumors. It was also demonstrated that most tumor cells contain GnRH receptors, and the direct antiproliferative effect of GnRH analogs on cancer cells might be mediated by these receptors. Development of new GnRH derivatives is focused on the decrease of their hormonal potency resulting in higher selectivity of the antitumor activity. One of the most promising natural GnRH analogs, lamprey (l) lGnRH-III, was isolated from see lamprey. This variant of GnRH binds to GnRH receptors and inhibits proliferation of various cancer cells. However, its endocrine effect is insignificant in mammals. lGnRH-III dimers and conjugates were prepared and were shown to have increased antiproliferative effects on various cancer cells, while their hormonal activity was lower than that of the native hormone. lGnRH-III was applied as targeting moiety to deliver anticancer agents to tumor cells. Research data concerning lGnRH-III and its analogs represent a new outlook for research trends of the application of GnRH compounds in cancer chemotherapy. Studies on the effects of lGnRH-III derivatives including antiproliferative effects, cytotoxicity, hormonal actions, and enzymatic stability are reviewed in this article.

Endocrine and antineoplastic actions of growth hormone-releasing hormone antagonists.

Potent antagonists of growth hormone-releasing hormone (GHRH) have been developed for the treatment of disorders caused by excessive GHRH or growth hormone (GH) production and for therapy of cancers. GHRH antagonists suppressed the release of GH and insulin-like growth factor (IGF)-I in transgenic mice overexpressing human (h) GHRH gene, an animal model of human acromegaly. It was also shown in GH3 rat pituitary tumor cells overexpressing the human pituitary GHRH receptor (pGHRH-R) that GHRH antagonists can inhibit c-AMP production and GH secretion through the human receptor. These observations indicate that GHRH antagonists could be used clinically in disorders characterized by excessive GHRH/GH secretion. Many recent studies demonstrate that GHRH antagonists can inhibit tumor growth by several mechanisms. By indirect action through pGHRH-Rs these antagonists suppress circulating GH/IGF-I level, which results in the inhibition of cancers that depend on GH and/or IGF-I as growth factors. However, GHRH antagonists are also effective inhibitors of tumor IGF-II production, which is a potent mitogen but independent of GH. GHRH antagonists can inhibit tumor cell proliferation by direct action on tumor cell receptors, suppressing the IGF-II and other growth factor production of tumor cells. In addition, various human tumors and tumor cell lines secrete GHRH peptide and respond to GHRH with proliferation. This finding suggests that GHRH functions as an autocrine growth factor and that GHRH antagonists can block its effects on tumor growth. Recently, we demonstrated the expression of hGHRH-R and its splice variants in various human cancers. Antiproliferative action of GHRH antagonists on these cancers indicates that the direct inhibitory effects of GHRH antagonists are mediated by tumoral GHRH receptors.

Structure-activity study on the LH- and FSH-releasing and anticancer effects of gonadotropin-releasing hormone (GnRH)-III analogs.

UNLABELLED: GnRH-III was reported to have selective FSH-releasing activity in rats and significant anticancer potency on human breast cancer cells. To improve either of these effects, 14 analogs were synthesized and investigated for FSH/LH stimulation and breast cancer inhibition. Analogs with single amino acid changes in positions 5-7 or 10 showed small or no difference in the FSH- or LH-releasing activity compared with GnRH-III but their anticancer potency decreased significantly. Modification of the terminal amino acids, side chain cyclization at the 6-8 regions, or combined amino acid changes at positions 4, 6 and/or 8 resulted in the decrease of both effects. Gonadotropin-releasing activity of Arg(8)-GnRH-III was improved 3-11-fold. A copolymer conjugate of GnRH-III showed 2-3-fold anticancer activity while losing endocrine potency. CONCLUSION: The activation of GnRH-receptors on pituitary and breast cancer cells requires a specific structure and/or conformation that makes possible to improve the anticancer selectivity of GnRH analogs.

Structure, enzymatic stability and antitumor activity of sea lamprey GnRH-III and its dimer derivatives.

Direct antitumor activity of sea lamprey (Petromyzon marinus) gonadotropin-releasing hormone III (Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH(2); lGnRH-III) was described on several tumor cells. To improve the selectivity of antitumor effects without increasing the hormone releasing activity and to enhance the enzymatic stability, lGnRH-III dimers were prepared via disulfide bond formation. Our results demonstrate that the lGnRH-III dimer derivatives exhibited higher antiproliferative effect and enzymatic stability in comparison with the native lGnRH-III, while lower LH-releasing potency was determined. In order to find a correlation between the biological and structural features of these compounds, the conformation of lGnRH-III and its dimer derivatives was determined by ECD, VCD, FT-IR and (1)H NMR.

Lipopeptide antagonists of growth hormone-releasing hormone with improved antitumor activities.

Antagonists of growth hormone-releasing hormone (GHRH) synthesized previously inhibit proliferation of various human cancers, but derivatisation with fatty acids could enhance their clinical efficacy. We synthesized a series of antagonists of GHRH(1-29)NH(2) acylated at the N terminus with monocarboxylic or alpha,omega-dicarboxylic acids containing six to sixteen carbon atoms. These peptides are analogs of prior potent antagonists JV-1-36, JV-1-38, and JV-1-65 with phenylacetyl group at their N terminus. Several new analogs, including MZ-J-7-46 and MZ-J-7-30, more effectively inhibited GHRH-induced GH release in vitro in a superfused rat pituitary system than their parent compound JV-1-36 and had increased binding affinities to rat pituitary GHRH receptors, but they showed weaker inhibition of GH release in vivo than JV-1-36. All antagonists acylated with fatty acids containing 8-14 carbon atoms inhibited the proliferation of MiaPaCa-2 human pancreatic cancer cells in vitro better than JV-1-36 or JV-1-65. GHRH antagonist MZ-J-7-114 (5 mug/day) significantly suppressed the growth of PC-3 human androgen-independent prostate cancers xenografted into nude mice and reduced serum IGF-I levels, whereas antagonist JV-1-38 had no effect at the dose of 10 mug/day. GHRH antagonists including MZ-J-7-46 and MZ-J-7-114 acylated with octanoic acid and MZ-J-7-30 and MZ-J-7-110 acylated with 1,12-dodecanedicarboxylic acid represent relevant improvements over earlier antagonists. These and previous results suggest that this class of GHRH antagonists might be effective in the treatment of various cancers.

Increased activity of antagonists of growth hormone-releasing hormone substituted at positions 8, 9, and 10.

Antagonists of human growth hormone-releasing hormone (hGHRH) with increased potency and improved enzymatic and chemical stability are needed for potential clinical applications. We synthesized 21 antagonistic analogs of hGHRH(1-29)NH(2), substituted at positions 8, 9, and 10 of the common core sequence [phenylacetyl-Tyr(1), d-Arg(2,28), para-chloro-phenylalanine 6, Arg(9)/homoarginine 9, Tyr(10)/O-methyltyrosine 10, alpha-aminobutyric acid 15, norleucine 27, Har(29)] hGHRH(1-29)NH(2). Inhibitory effects on hGHRH-induced GH release were evaluated in vitro in a superfused rat pituitary system, as well as in vivo after i.v. injection into rats. The binding affinities of the peptides to pituitary GHRH receptors were also determined. Introduction of para-amidinophenylalanine 10 yielded antagonists JV-1-62 and -63 with the highest activities in vitro and lowest receptor dissociation constants (K(i) = 0.057-0.062 nM). Antagonists JV-1-62 and -63 also exhibited the strongest effect in vivo, significantly (P < 0.05-0.001) inhibiting hGHRH-induced GH release for at least 1 h. Para-aminophenylalanine 10 and O-ethyltyrosine 10 substitutions yielded antagonists potent in vitro, but His(10), 3,3'-diphenylalanine 10, 2-naphthylalanine 10, and cyclohexylalanine 10 modifications were detrimental. Antagonists containing citrulline 9 (in MZ-J-7-72), amidinophenylalanine 9 (in JV-1-65), His(9), d-Arg(9), citrulline 8, Ala(8), d-Ala(8), or alpha-aminobutyric acid 8 substituents also had high activity and receptor affinity in vitro. However, in vitro potencies of analogs with substitution in position 9 correlated poorly with acute endocrine effects in vivo, as exemplified by the weak and/or short inhibitory actions of antagonists JV-1-65 and MZ-J-7-72 on GH release in vivo. Nevertheless, antagonist JV-1-65 was more potent than JV-1-63 in tests on inhibition of the growth of human prostatic and lung cancer lines xenografted into nude mice. This indicates that oncological activity may be based on several mechanisms. hGHRH antagonists with improved efficacy could be useful for treatment of cancers that depend on insulin-like growth factors or GHRH.

Effects of long-term treatment with the luteinizing hormone-releasing hormone (LHRH) agonist Decapeptyl and the LHRH antagonist Cetrorelix on the levels of pituitary LHRH receptors and their mRNA expression in rats.

The effects of depot formulations of the luteinizing hormone-releasing hormone (LHRH) agonist Decapeptyl (25 microg/day) for 30 days or LHRH antagonist Cetrorelix pamoate (100 microg/day) for 30 days and daily injections of 100 microg of Decapeptyl for 10 days on the expression of mRNA for pituitary LHRH receptor (LHRH-R) and the levels of LHRH-R protein were evaluated in rats. Serum sex steroid concentrations and the weights of the reproductive organs were greatly reduced in all groups treated with analogs, demonstrating an efficient blockade of the pituitary-gonadal axis. Decapeptyl microcapsules elevated serum LH in female rats, but decreased it in male rats. LHRH-R mRNA expression in female pituitaries was reduced to 41% and 56-65% on days 10 and 30, respectively, whereas LHRH-R protein was 64% of control on day 10 and returned to pretreatment levels on day 30. Decapeptyl microcapsules reduced LHRH-R mRNA expression in male pituitaries to 58% on day 30 but not LHRH-R protein. Daily injections of Decapeptyl caused a desensitization of LH responses in female rats, while raising LHRH-R mRNA expression in female rats by 23% and LHRH-R protein levels by 119%. Cetrorelix pamoate reduced serum LH in female rats and diminished LHRH-R mRNA to 30% and 26% and LHRH-R protein to 57% and 48% on days 10 and 30, respectively. Elevated LHRH-R protein levels of ovariectomized rats were reduced after 10-day treatment with Cetrorelix or 100 microg/day Decapeptyl. Thus, changes in the mRNA expression after treatment with Cetrorelix, but not always Decapeptyl, paralleled those of LHRH-R protein. The inhibitory effect of Cetrorelix on serum LH, pituitary LHRH-R mRNA, and LHRH-R protein was greater than that of Decapeptyl.