Early retinopathy progression in four randomized trials comparing insulin glargine and NPH [corrected] insulin.

Early worsening of diabetic retinopathy, characterized by cotton wool spots, intraretinal microvascular abnormalities and/or macular edema, can occur following improvement of glycemic control. In four randomized 28- to 52-week clinical trials comparing insulin glargine and NPH insulin in regard to glycemic control and frequency of hypoglycemia, ophthalmologic examinations and fundus photographs were included to assess frequency of early worsening of retinopathy or other early adverse ocular effects. Retinopathy progression rates at 28 weeks were 7-12% by clinical examination and 3-8% by photographic grading; corresponding rates of clinically significant macular edema (CSME) were 1-8% and 1-4%, respectively. Optic disc swelling was not observed clinically or in photographs. Two of the 24 possible comparisons (four trials, three outcomes, two assessment methods), both of which were photographic assessments in type 2 diabetes, were in/near the nominally significant range and favored NPH insulin: 28-week rates of >or=3-step retinopathy progression (insulin glargine: 16/213, 7.5%; NPH insulin: 6/220, 2.7%; p=0.028) and 52-week CSME rates (26/233, 11.2% and 14/214, 6.5%, respectively; p=0.098). Because the between-treatment differences were small and inconsistent across trials and assessment methods, and because overall rates were consistent with the natural course of diabetic retinopathy, we conclude that it is unlikely that insulin glargine carries a higher risk of early worsening or other early adverse effect than NPH insulin. These trials tended to exclude a large early adverse effect, such as optic disc swelling, but cannot assess longer-term effects; a 5-year randomized trial of insulin glargine versus NPH insulin has been initiated. Data from this manuscript have been presented as posters and published in abstract form at the European Association for the Study of Diabetes 2001 ( DIABETOLOGIA 44(Suppl 1):I-IV(A287), 2001) and the Latin American Diabetes Association 2001 (11-15 November 2001, Punta del Este, Uruguay; Poster 180) congresses.

Sex steroids used in hormonal treatment increase vascular procoagulant activity by inducing thrombin receptor (PAR-1) expression: role of the glucocorticoid receptor.

BACKGROUND: The use of sex steroids in oral contraception or hormonal replacement therapy is associated with an increased risk of cardiovascular thromboembolic complications. Although both the estrogen and the progestin components have been involved, the underlying mechanisms responsible are unclear. METHODS AND RESULTS: This study examined whether sex steroids promote hemostasis indirectly by increasing the procoagulant activity of blood vessels. Treatment of vascular smooth muscle cells with several progestins (progesterone, 3-keto-desogestrel, gestodene, and medroxyprogesterone acetate) upregulated proteolytically activatable thrombin receptor (PAR-1) expression, resulting in a potentiated thrombin-induced tissue factor expression and surface procoagulant activity. In contrast, neither the progestins levonorgestrel, norethisterone, and norgestimate nor the synthetic estrogen 17alpha-ethinylestradiol had such effects. The effect of the stimulatory progestins, which induce glucocorticoid-like effects in several cell systems, was mimicked by dexamethasone and inhibited by the progesterone and glucocorticoid receptor antagonist RU-38486. In addition, long-term administration of progesterone, 3-keto-desogestrel, or medroxyprogesterone acetate to ovariectomized rats increased PAR-1 protein level in the arterial wall, resulting in an increased responsiveness of isolated aortic rings to thrombin. CONCLUSIONS: These data demonstrate that several progestins markedly potentiate the vascular procoagulant effects of thrombin by increasing the availability of membrane thrombin receptors in the smooth muscle, an effect that is most likely due to their glucocorticoid-like activity.

Effect of human growth hormone-releasing hormone on the release of dynorphin-like immunoreactivity, luteinizing hormone, and follicle-stimulating hormone from rat adenohypophysis in vitro.

The effect of GH-releasing hormone (GHRH) on the release of the endogenous opioid dynorphin from rat adenohypophysis was investigated in vitro. Rat anterior pituitary quarters were incubated in vitro, and hormone release into the incubation medium was measured by RIAs. Human pancreatic GHRH [hpGHRH-(1-44)] as well as human Leu27,Gly45-GHRH [GHRH-(1-45)] enhanced the secretion of dynorphin A1-13-like immunoreactivity (Dyn A1-13-IR) in a concentration-dependent manner. The concentrations of hpGHRH-(1-44) that stimulated the release of Dyn A1-13-IR were about 100-fold higher than those that enhanced GH secretion. GH release induced by hpGHRH-(1-44) was blocked by somatostatin (IC50, approximately 10 nM) without affecting hpGHRH-(1-44)-induced release of Dyn A1-13-IR. GH release was elicited by prostaglandin E2, while Dyn A1-13-IR secretion remained unchanged. At concentrations that enhanced Dyn A1-13-IR release, hpGHRH-(1-44) also elicited LH and FSH secretion. The LHRH antagonist D-pGlu1, D-Phe2,D-Trp3,6-LHRH blocked the secretion of Dyn A1-13-IR, LH, and FSH induced by hpGHRH-(1-44), whereas the LHRH antagonist did not influence the simultaneous GH release elicited by hpGHRH-(1-44). A possible direct effect of GHRH on the LHRH receptor was examined in radioligand binding studies using iodinated D-Ala6, des-Gly10-LHRH ethylamide (LHRH-A). The binding of [125I]iodo-LHRH-A to rat anterior pituitary membranes was completely displaced by hpGHRH-(1-44) and GHRH-(1-45). The deduced apparent dissociation constants were about 3 orders of magnitude higher than that of LHRH-A, but were close to those concentrations that enhanced Dyn A1-13-IR release. We conclude that GHRH-induced release of Dyn A1-13-IR is unrelated to GH release. High concentrations of GHRH may interact directly with LHRH receptors on gonadotrophs and thereby enhance the release of LH, FSH, and Dyn A1-13-IR.

Release of dynorphin-like immunoreactivity from rat adenohypophysis in vitro during inhibition of anterior pituitary hormone secretion from individual cell types.

LHRH has previously been found to be the only known hypothalamic releasing factor which can specifically stimulate the release of the opioid dynorphin and other proenkephalin B-derived peptides from the rat adenohypophysis in vitro. In the present study the mechanisms that regulate dynorphin release were further characterized. It was examined whether or not dynorphin release from the adenohypophysis in vitro is altered during inhibition of the secretion of various anterior pituitary hormones. Rat anterior pituitary quarters were incubated in vitro and hormone release into the incubation medium was measured by RIAs. Somatostatin, dopamine, T3, dexamethasone, and 5 alpha-dihydrotestosterone were used to inhibit the secretion of GH, PRL, TSH, ACTH/beta-endorphin, or LH/FSH, respectively. GH, PRL, or beta-endorphin release was inhibited without affecting the simultaneous release of dynorphin A-(1-13)-like immunoreactivity (Dyn A1-13-IR). Concentrations of T3, somatostatin, or dopamine which were effective in suppressing the evoked and/or basal release of TSH, GH, or PRL, respectively, produced no effect on Dyn A1-13-IR release caused by high potassium concentration (40 mM) or LHRH (500 pM). The LHRH-induced release of LH and FSH was inhibited by the glucocorticoid dexamethasone or the androgen 5 alpha-dihydrotestosterone. Under these conditions, Dyn A1-13-IR release was also reduced. However, whereas LH release was completely blocked by 5 alpha-dihydrotestosterone, FSH and Dyn A1-13-IR release was reduced only by 50%. The release of FSH and Dyn A1-13-IR in vitro from anterior pituitary glands taken from rats, castrated 3 weeks before, was enhanced to a similar extent (about 2.5-fold); the simultaneous enhancement of LH release was significantly (P less than 0.005) greater (about 5-fold). We conclude that the mechanisms which regulate the release and/or biosynthesis of dynorphin and other proenkephalin B-derived peptides of the adenohypophysis are similar to those of the gonadotropins but different from those of any other anterior pituitary hormone, and may be more closely related with FSH release than LH release. These data support the view that dynorphin of the normal rat adenohypophysis may be localized in at least a subpopulation of gonadotrophs.

Suppression of follicular maturation by infusion of a luteinizing hormone-releasing hormone agonist starting during the late luteal phase in the stumptailed macaque monkey.

The effect of continuous administration of an LHRH agonist for 14 days on follicular estradiol secretion was studied in adult female stumptailed macaque monkeys. The infusion was started either during the early follicular phase or during the late luteal phase. Both treatments resulted in a rise in serum concentrations of LH and FSH for 2-3 days, after which time values returned to basal levels. Infusion during the early follicular phase induced a rapid rise in serum concentrations of estradiol, which declined after 7-10 days. The normal rise in serum progesterone after midcycle failed to occur, indicating that ovulation had been inhibited. Infusion during the late luteal phase of the cycle prevented the normal rise in serum concentrations of FSH and estradiol at the end of the luteal phase, and there was no indication of follicular estradiol production during the subsequent infusion period. Removal of the minipumps after 14 days was followed promptly by normal cycles, regardless of when the infusion had been started. In a second experiment, infusion of LHRH agonist was started during the luteal phase and extended for 90 days. This resulted in marked and sustained suppression of estradiol secretion and total absence of progesterone rises. These findings show that in the monkey, a rapid and sustained suppression of estradiol production can be obtained if a long term infusion of LHRH agonist is started during the luteal phase of the cycle. Such an approach in women using infusion pumps or sustained delivery preparations may be the most effective way of achieving a medical ovariectomy for the treatment of endometriosis, leiomyomata, and other estrogen-dependent pathological conditions.

Serum concentration and urinary excretion of the luteinizing hormone-releasing hormone agonist buserelin in patients with endometriosis.

We studied the pharmacokinetics of iv and intranasally administered buserelin, a LHRH agonist peptide, in 14 women with endometriosis. Serum and urinary buserelin concentrations were determined by specific RIA (buserelin antiserum AS-639). Intact buserelin and the metabolites in urine were separated by reverse phase high performance liquid chromatography and measured by RIA. The mean serum buserelin concentrations were 101 +/- 33 (+/- SD) ng/mL 20 min and 1.12 +/- 0.12 ng/mL 360 min after its iv injection in 6 women, and the mean elimination half-life between 20 and 360 min was 51 min. In serum, intact buserelin was the main constituent (10 min, 90%; 120 min, 74%; 360 min, 52%), and the major metabolite was the buserelin-(5-9) pentapeptide (10 min, 0.6%; 120 min, 19%; 360 min, 12%). In the urine collected 0-1 h after buserelin administration, intact buserelin was 66% and the 5-9 pentapeptide was 28% of the total excretion. In the urine collected between 6-24 h after buserelin administration, intact buserelin accounted for 67% and the 5-9 pentapeptide for 32% of the total excretion. The urinary buserelin concentration was 1345 +/- 156 micrograms/g creatinine 1 h and 25 +/- 5 micrograms/g creatinine 6-24 h after buserelin administration. Serum LH, FSH, and estradiol concentrations increased acutely up to 10-fold above basal values; the mean peak LH, FSH, and estradiol values occurred at 180-240 min, 240 min, and 24 h, respectively. In therapeutic studies with buserelin nasal spray in 5 women, serum concentrations of 0.9-1.4 ng/mL were found 15 min after a single dose of 300 micrograms, intranasally, and the urinary excretion was 2.52-3.68 micrograms/24 h during daily administration of 3 doses of 300 micrograms at intervals of 8 h. These results confirm that buserelin is slowly inactivated and remains available to pituitary receptors for a prolonged period after its iv or intranasal administration.

Suppression of pituitary and testicular function in normal men by constant gonadotropin-releasing hormone agonist infusion.

In a trial for male fertility control the effects of constant GnRH agonist (buserelin) infusion on pituitary and testicular function was investigated. The agonist was administered sc for 12 weeks to two groups of normal young men using extracorporeal osmotic minipumps. Seven men received 118 +/- 24 (SD) micrograms/day from pumps changed biweekly and four men received 230 +/- 27 micrograms/day from pumps changed weekly. After an initial rise serum LH, FSH, and testosterone decreased. The decrease occurred faster in the high dose group and these subjects had no LH response to acute GnRH stimulation after 4 weeks of treatment, whereas the response was drastically reduced in the group receiving the low dose. Androgen substitution with testosterone undecanoate (80-120 mg orally daily) was initiated when the subjects complained of decreased libido and/or potency or when serum testosterone fell below 10 nmol/liter on average in the fifth week. Sperm counts decreased significantly and below the lower normal limit of 20,000,000/ml. The nadir was reached in week 12 of treatment in the high dose group, and in week 4 post treatment in the low dose group. Despite desensitization of the pituitary and impaired testicular function azoospermia did not occur. A higher dose of agonist appears to be required to achieve this goal.

Luteinizing hormone releasing hormone agonist for contraception in breast feeding women.

During the period of lactation there is a need for a reliable method of contraception since the suppressive effects of lactation on ovulation decline as the duration of breastfeeding is decreased. The aim of this study was to establish that chronic treatment with a LHRH agonist would prevent ovulation throughout the period of lactation and to evaluate the effects of the treatment on estrogen production, bleeding patterns, and nursing practice. Starting 6 weeks postpartum, nine mothers took 300 micrograms LHRH agonist (buserelin), intranasally once daily for the remainder of the duration of breastfeeding [216 +/- 18 days (mean +/- SEM)]. Urinary excretion of LH, estrone, and pregnanediol was compared to that of nine control breastfeeding mothers. In the control subjects follicular development, as assessed by rises in estrone, was minimal during the first 90 days of the study. Thereafter, phases of estrogen secretion were observed. Ovulation occurred in seven of the nine mothers on one to six occasions; time to first ovulation varied from 90-296 days. In the women taking buserelin, LH and estrone were initially stimulated for 1 and 2 weeks, respectively, then declined to basal levels. No ovulations occurred in the treated group. In six treated mothers only minor fluctuations in estrone were observed during the remainder of agonist treatment. In three subjects more frequent and sustained episodes of estrogen secretion were observed, but in contrast to the controls the rises in estrone were not followed by a typical LH surge or a rise in pregnanediol. Bleeding occurred in eight of the nine of the control mothers on one to seven occasions during the study period. The first bleed in five of the mothers was anovular, while other menstrual bleeds occurred in response to falling levels of pregnanediol. Of the mothers taking buserelin, one was amenorrhoeic, and five had only one light bleeding associated with the initial stimulation of estrone. Of the three women with continued fluctuations of estrone, one had three light bleeds, one experienced frequent spotting, while one had regular bleeding. No other side-effects, such as hot flashes or changes in nursing practices, were reported. Our results indicate that LHRH agonist treatment has the potential to be developed as an acceptable method of contraception during the postpartum period. The duration of treatment may be long enough to have a significant effect on maternal-infant well-being without encountering significant problems associated with low estrogen output.

Studies with fragments of a highly active analogue of luteinizing hormone releasing hormone.

The minimal structural requirements for gonadotrophin releasing activity were studied with fragments of a highly active analogue of luteinizing hormone releasing hormone (LH-RH), [D-Ser(But)6]LH-RH(1-9)nonapeptide-ethylamide (Hoe 766). All fragments are related to the C-terminal structure of LH-RH and have increased enzyme stability. Ovulation in phenobarbitone-blocked rats was induced with a median effective dose/rat, of 1.9 microgram of the (3-9)-heptapeptide, Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-ethylamide and 6.8, 18.0 and 38.3 microgram for the (4-9), (5-9) and (6-9) fragments respectively. The (3-9)- heptapeptide and (4-9)-hexapeptide induced release of LH and FSH in phenobarbitone-blocked rats with a ratio similar to that of LH-RH. Degradation of LH-RH by enzyme preparations of liver, kidney and hypothalamic or anterior pituitary tissue was not modified by addition of the (3-9)-heptapeptide fragment. The organ distribution of the 125I-labelled (3-9)-heptapeptide fragments was similar to LH-RH, but not to Hoe 766. The peptide accumulated in liver and kidney, but was eliminated from the anterior pituitary gland 15 min after i.v. injection, whereas Hoe 766 showed progressive accumulation in the pituitary gland (tissue:plasma ratio = 6.6 after 60 min). In contrast to C-terminal fragments of LH-RH, the corresponding fragments of nonapeptide analogues retained significant biological activity, and the minimal structural requirements for LH release may be related to the C-terminal sequence of LH-RH.

Gonadotrophin release by a highly active analogue of luteinizing hormone releasing hormone in rats immunized against luteinizing hormone releasing hormone.

Immunization against luteinizing hormone releasing hormone (LH-RH) in adult male rats produced a progressive decline in LH and FSH in the circulation to low or non-detectable levels. D-Serine-tertiary-butyl6,des-glycine-NH210 LH-RH ethylamide is an analogue of LH-RH having highly active LH-RH properties in the normal rat. Because it is also immunologically different from LH-RH it can stimulate gonadotrophin release from the anterior pituitary gland of rats immunized against LH-RH without interference from the antibody. The analogue stimulated LH and FSH release in rats 15 weeks after immunization against LH-RH when antibody titre was highest, and after long-term (35 weeks) immunization against LH-RH. D-Serine-tertiary-butyl6,des-glycine-NH210 LH-RH ethylamide and related analogues are therefore potentially useful for reversing the effects of immunization against LH-RH.

Effect of long-term infusion of an LH-releasing hormone agonist on testicular function in bulls.

Continuous administration of LH-releasing hormone (LHRH) agonists is an effective method of suppressing testosterone secretion in the male. The effect of the LH-releasing hormone (LHRH) agonist, buserelin, administered to bulls by constant infusion from osmotic minipumps was studied. In one experiment with four treated and one control bull, 109 micrograms buserelin/day were administered for 22 days. Immediately after implantation, serum testosterone concentrations rose from below 35 nmol/l to 35-105 nmol/l, and all four buserelin-infused bulls showed increased testosterone secretion during the treatment period. After removal of the minipumps, testosterone concentrations decreased to pretreatment levels. In a second experiment bulls were infused for 42 days (four treated and one control), and identical results were obtained. Testosterone secretion was stimulated (52-87 nmol/l serum) during the entire treatment period. These results demonstrate that conditions for stimulation of the pituitary-testicular axis may vary between species. Infusion of low doses of LHRH-agonists in bulls has an extended stimulatory effect without immediate desensitization of gonadotrophin release.

Effect of treatment with an agonist of luteinizing hormone releasing hormone on early maturational changes in pituitary and testicular function in the rat.

Male rats aged 30 days were injected once daily for between 1 and 7 days with 50 ng (D-serine t-butyl6, des-glycine-NH210) luteinizing hormone releasing hormone ethylamide (LH-RH agonist), and pituitary and testicular function were assessed. Treatment for 7 days significantly (P less than 0.02) inhibited maturational increases in the pituitary content and serum concentration of gonadotrophins, testicular luteinizing hormone (LH)-receptor concentration and the testicular capacity to secrete testosterone; the pituitary content and serum concentration of prolactin, the hypothalamic content of LH-RH and testicular weight were unaffected. In rats treated with LH-RH agonist, the initial (2 to 3 days) reduction in testicular LH-receptors and the capacity to secrete testosterone probably resulted from acutely raised levels of LH in the blood, whilst later effects may have resulted from the apparently chronic reduction in serum gonadotrophin levels. The latter may reflect a decrease in pituitary responsiveness to repeated stimulation with LH-RH agonist. Despite the extensive loss of testicular LH-receptors and diminished responsiveness, the concentration of HCG which significantly (P less than 0.05) increased testosterone secretion by the testis in vitro was the same (2 pmol/l) as that for testes from control rats.

Pituitary-testicular function is suppressed by an LHRH antagonist but not by an LHRH agonist in the marmoset monkey.

The use of pituitary desensitization by an LHRH agonist (buserelin) to examine pituitary-testicular function was investigated in a New World primate. Six adult male marmoset monkeys were injected s.c. with an LHRH agonist implant (1.5 mg in a rod 0.5 cm long). Pharmacokinetics, determined by radioimmunoassay of urinary buserelin, revealed a rapid initial release of the agonist followed by a steady decline during a 200-day period. The LHRH agonist treatment resulted in a rapid initial rise in plasma LH followed by a return to mean values similar to those seen in the control samples by 7 days after implantation. Using the present protocol, no evidence of subsequent pituitary desensitization or suppression of testicular function was observed, plasma concentrations of LH and testosterone remaining within the normal range during the 200-day study period. In contrast, pituitary-testicular function was suppressed in the male marmoset after blockade of pituitary LHRH receptors by an LHRH antagonist. Five adults were treated with a single s.c. injection of the antagonist [Ac-D-Nal(2)1,D-pCl-Phe2,D-Trp3,D-Ser(Rha)6,AzGlyNH2(10)]-LHRH at a dose of 300 micrograms/kg. The LHRH antagonist induced a marked suppression of plasma LH and testosterone by 6-8 h, the low levels being maintained for 24-48 h. These results show that, whereas treatment with an LHRH antagonist can inhibit pituitary-testicular function in the male marmoset, it may be that desensitization cannot be induced by the LHRH agonist used.

Competition of various LH--RH analogs and fragments with 135I-LH--RH for specific binding sites on isolated pituitary plasma membranes.

The displacement by various LH--RH analogs and fragments of 125I-LH--RH specifically bound to plasma membranes isolated from rat anterior pituitaries was investigated. The addition of increasing amounts of unlabelled LH--RH resulted in an increasing displacement of bound 125I-LH--RH. When some fragments of LH--RH or of an analog with little biological activities were incubated, a much weaker affinity for LH--RH binding sites could be observed. No correlation between biological effectiveness and binding affinity was, however, found when several highly active LH--RH analogs were tested. The potent analogs competed much less efficiently for specific binding sites on isolated plasma membranes than LH--RH. Binding experiments with iodine-labelled (D-Ser(But)6-LH--RH(1--9)-nonapeptide-ethylamide indicated that the small binding affinity of the superactive analog may be due to a lower association rate while the dissociation rate is comparable to that of LH--RH. Contrary to LH--RH, no binding equilibrium was reached during 2 h of incubation of the analog with plasma membranes. The physiological role of LH--RH binding sites on isolated pituitary plasma membranes remains to be elucidated.

The regulation of LHRH action at the pituitary and gonadal receptor level: a review.

Luteinizing hormone releasing-hormone (LHRH) and its highly active agonists are under clinical investigation for the control of reproductive function and for suppression of hormone dependent tumours. The regulation of LHRH action by pituitary receptors and expression of the biological LHRH effect by gonadotropin release and activation of steroid biosynthesis are discussed in this context. Pituitary LHRH receptors are controlled by autoregulation via endogenous LHRH secretion. The gonadal response to LHRH stimulation is regulated by LH action on receptors for LH, prolactin and FSH. Pituitary and gonadal inhibition are achieved by different mechanisms. Continuous exposure to LHRH blocks gonadotropin release and reduces pituitary LH/FSH content, whereas inhibition of steroid biosynthesis requires daily LH release to maintain receptor down-regulation. Pituitary enzymes involved in LHRH degradation at the receptor site are required for terminating hormone action, but their role in modulating hormonal responsiveness is secondary to receptor regulation. Direct gonadal effects of LHRH are exerted in the presence of gonadotropins by modulating the gonadotropin effect, e.g. in hypophysectomized animals. The presence of specific receptors for LHRH agonists in ovarian and testicular tissue suggests local control mechanisms for gonadotropin activation of steroid biosynthesis.

Effects of the luteinizing-hormone-releasing hormone (LHRH) antagonist ramorelix (hoe013) and the LHRH agonist buserelin on dimethylbenz[]anthracene-induced mammary carcinoma: studies with slow-release formulations.

Luteinizing-hormone-releasing hormone (LHRH) agonists and antagonists are antigonadotropic agents for reversible ovarian/testicular suppression in gynaecology and in oncology. Pituitary inhibition and suppression of the gonadal steroids can be maintained with continuous release rates from biodegradable implants or microparticles. The effects of curative and preventive treatment with slow-release formulations of the LHRH agonist buserelin (implants and microparticles) and the LHRH antagonist ramorelix (hoe013) (microparticles) on dimethylbenz[a]anthracene(DMBA)-induced mammary tumours in rats and the pharmacokinetics of these formulations are described. In addition, direct effects of the LHRH antagonist ramorelix on tumour growth were studied. The release rates of the implants (polylactide-glycolide 75:25) and the microparticles (polylactide-glycolide 50:50) were calculated from urinary excretion of the peptides. The curative treatment started at the time of full tumour development (76 days after DMBA induction). A single buserelin implant injection (3.3 mg peptide) resulted in a dramatic tumour regression within 14 days, which was comparable to ovariectomy. It prevented tumour progression for 120 days. Previous studies in rats have shown that ramorelix microparticles (3.6 mg peptide) have a shorter duration of action (about 14 days) in suppression of gonadal function when compared to buserelin microparticles (3.6 mg peptide), where the suppression lasted for about 35 days. As expected, a single injection of ramorelix microparticles (3.6 mg peptide) inhibited tumour progression for only 14 days. This short action is due to a different release profile of the ramorelix microparticles and the different specific activities of peptides incorporated. In the preventive experiments animals were treated 17 days after DMBA induction before tumour development. Treatment with buserelin implants (3.3 mg peptide) every 56 days or with buserelin microparticles (3.6 mg peptide) every 28 days and the treatment with ramorelix microparticles (1.8 mg peptide) every 7 days prevented the development of tumours. Six weeks after the last injection of ramorelix microparticles a strong tumour progression was seen. There was a clear correlation between peptide release and tumour inhibition. The implants and the microparticles were well tolerated, no tissue reaction or side-effects of ramorelix were seen. Treatment of ovariectomized oestradiol-substituted DMBA-treated rats resulted in a marginal (not significant) inhibition in tumour development. LHRH antagonists in slow-release formulations (microparticles or implants) represent a new approach in treatment of hormone-dependent tumours because of the immediate onset of gonadal function and the increased drug efficacy due to the controlled release from biodegradable microparticles.

Pharmacokinetics and endocrine effects of slow release formulations of LHRH analogues.

The LHRH agonists are antigonadotropic agents for reversible ovarian suppression in gynaecology and in oncology. In oncology, pituitary inhibition is maintained with high release rates preferably by implant or microcapsule injection. The pharmacokinetics of buserelin after injection, infusion, and during implant treatment (controlled release) are described. The release rate is monitored by urinary buserelin excretion (fractional excretion of 30% of the daily dose). During therapy, LHRH agonists in serum are measured by specific radioimmunoassays, with or without extraction. A more convenient non-invasive procedure is to measure the amount of buserelin in 24-h urine samples (during injections or nasal spray), or the urinary buserelin/creatinine ratio in morning urine samples (during infusions or implants). After high dose injection, buserelin has a half-life of 80 min, therapeutic plasma concentrations are maintained for 8-12 h. In long-term maintenance with buserelin implants (polylactide-glycolide, 75:25), serum concentrations and urinary excretion showed an extended plateau phase indicating a suitable dose interval of 2-3 months. In endometriosis and leiomyoma, the minimum release rate (urinary buserelin) required for maintenance of steroid suppression was established (buserelin excretion of about 0.5 microgram/g creatinine). Buserelin implants in prostate carcinoma are effective for 2 or 3 months, after a single dose of 6.6 or 10 mg buserelin, respectively. A consistent suppression of serum testosterone secretion was confirmed for more than 2 yr. Buserelin microparticles are effective in rhesus monkeys to completely suppress follicular maturation and oestrogen secretion during 4-6 weeks after a single dose of 3.6 mg buserelin. Recent results on the controlled release of an LHRH antagonist (Hoe 013) from biodegradable microparticles in rats with DMBA-induced mammary tumours indicate that tumour suppression by LHRH antagonists is well tolerated and highly effective. The local tolerance at the injection site of antagonist microparticles is excellent as in the case of LHRH agonists like buserelin.

New analogues of secretin.

For the evaluation of structure/activity relationships, some porcine secretin analogues, modified in the N-terminus, have been synthesized by segment condensation in solution. The secretin activity of the analogues was defined as the volume of pancreatic juice secreted in rats and dogs. The exchange of the N-terminal pentapeptide for the N-terminal pentapeptide of human somatotropin releasing factor (h-SRF) resulted in a peptide ([1-Tyr,2,4-Di-Ala,5-Ile]secretin) with practically no SRF-activity (less than 1% SRF-activity up to 100 micrograms/kg in the rat), but surprisingly high secretin activity (almost 100% in the rat, but only 1150 CU/mg (27%) in the dog). [3-L-Cysteic acid]secretin showed 1750 CU/mg (39%) in the dog, but a less activity (23%) in the rat. [6-D-Phe]secretin and [5-D-allo-Thr]secretin are again strongly species specific. They exhibited an activity of less than 1% in the dog, but about 10-15% in the rat. The smallest secretin activity was observed with [1-Cys,6-Cys]secretin in the oxidized form. The activity in the rat with this analogue was only about 0.2%.

A pharmacological evaluation of a new 3-month depot preparation of buserelin for prostatic cancer.

A new slow-release formulation of buserelin given as a 3-month depot injection was evaluated in four patients with advanced prostatic cancer. Treatment was maintained for a mean period of 15 months. Steady-state urinary buserelin concentrations were reached by the beginning of the 3rd week of treatment and maintained until the end of the 3rd month. Serum testosterone remained suppressed in the castrate range for the duration of the study. This preparation offers an advantage for the patient and clinician over existing methods of gonadotrophin-releasing hormone (GnRH) analogue administration and will enter further clinical trial.

The first clinical use of depot buserelin for advanced prostatic carcinoma.

The agonist analogues of the gonadotrophin-releasing hormone now provide an alternate medical treatment of prostatic cancer. For effect repeated administration is required, either by five or six times daily intranasal or once daily subcutaneous treatment. There is an obvious disadvantage to such regimens in elderly patients who may have difficulty complying with therapy. In order to circumvent these difficulties, sustained release formulations of the agonist analogues have been synthesized. We report the first clinical use of a long-acting formulation of D-Ser (TBU)6-LHRH Ethylamide (buserelin) using a novel polymer material. Twelve symptomatic patients with previously untreated carcinoma of the prostate were treated with depot buserelin, administered once monthly. In all patients, depot buserelin suppressed serum testosterone into the range seen in castrate men at a rate equivalent to that provided by five times daily intranasal therapy. No significant increase in serum testosterone, luteinizing hormone or follicle-stimulating hormone concentrations occurred during the period of follow-up. Long-acting formulations of buserelin offer an advance in the management of prostatic cancer with agonist analogues of the gonadotrophin-releasing hormone.