Inhibition of prolyl hydroxylation and procollagen processing in chick-embryo calvaria by a derivative of pyridine-2,4-dicarboxylate. Characterization of the diethyl ester as a proinhibitor.

The biochemical and morphological consequences of procollagen prolyl 4-hydroxylase inhibition by pyridine-2,4-dicarboxylic acid (2,4-PDCA) and its diethyl ester (diethyl-2,4-PDC) were studied in chick-embryo calvaria, which predominantly synthesize type I collagen. Half-maximal inhibition of tissue hydroxyproline formation required 650 microM-2,4-PDCA, whereas the Ki with respect to chicken prolyl 4-hydroxylase in vitro was 2 microM. In contrast, half-maximal inhibition was caused by 10 microM-diethyl-2,4-PDC in the intact calvaria, although chicken prolyl 4-hydroxylase in vitro was not inhibited even at 1 mM. The collagenous material produced in the presence of diethyl-2,4-PDC showed an altered 'melting' profile and a lowering of the transition temperature by 10 degrees C, indicating misalignment and thermal instability of its triple-helical structure. Amount and electrophoretic mobility of procollagen type I chains were increased in a dose-dependent manner. The amounts of partially processed species and alpha-chains were decreased, without change in mobility. This marked effect on procollagen-collagen conversion in the intact calvaria suggests that the underhydroxylated collagenous material generated in the presence of diethyl-2,4-PDC is resistant to or acts as endogenous secondary inhibitor of type I procollagen N-proteinase. Electron microscopy of treated calvaria cells showed dilated rough endoplasmic reticulum and numerous phagolysosomes, indicating intracellular retention and lysosomal degradation of the newly synthesized underhydroxylated collagenous material. In summary, these results identify 2,4-PDCA and diethyl-2,4-PDC as the first prolyl 4-hydroxylase-directed inhibitor/proinhibitor pair that affects intra- and extra-cellular events during collagen formation.

Beneficial effects of inhibitors of prolyl 4-hydroxylase in CCl4-induced fibrosis of the liver in rats.

S 0885 and HOE 077 inhibit CCl4-induced liver fibrosis in rats, as shown by significantly reduced hydroxyproline content of the liver and improved liver histology. Mortality of drug-treated animals is significantly diminished. Serum collagen parameters correlate well with the hydroxyproline content of the liver and can be used as noninvasive markers for the fibrotic process. HOE 077 is a proinhibitor, which by itself does not inhibit prolyl 4-hydroxylase. HOE 077 is well absorbed from the gastrointestinal tract. It is taken up by rat liver and is converted to the active metabolites. At a concentration of 1 mM, HOE 077 does not affect collagen synthesis in human fibroblasts, bovine chondrocytes and chicken calvaria. At therapeutic doses the compound does not reduce collagen content of kidney, lung, aorta, femur epiphysis, skin and tendon of the rat, validating the high specifity of the liver selective prodrug/inhibitor conversion. From animal experiments, a human daily dose of 0.5-1 g can be extrapolated.

Chronic intravenous toxicity of the new antibiotic cefpirome in monkeys.

Chronic intravenous toxicity studies in monkeys were carried out with 3-[(2,3-cyclopenteno-1-pyridinium)-methyl]-7-[2-syn-methoximino - 2-(2-aminothiazol-4-yl)-acetamido]-ceph-3-em-4-carboxylate (cefpirome, HR 810; CAS 84957-29-9) a new cephalosporin derivative. In a 90-day study in rhesus monkeys (4 males/4 females per group) dosages of 0, 50, 160 and 500 mg/kg/day were administered. In a 6-month study 5 groups of 6 male and 6 female cynomolgus monkeys received NaCl-solution (0.9%), the vehicle, and 50, 200 or 800/400 mg/kg/d (the highest dosage had to be lowered after the first week due to acute drug intolerance). For clarification of the dose relationship to the findings in the 800/400 mg/kg group, a supplementary 6-month study with 500 mg/kg cefpirome including a vehicle control was also performed. 50 mg cefpirome/kg/d was well tolerated; so too were 160 and 200 mg/kg apart from a slight beta 2-microglobulinuria and/or enzymuria. Almost exclusively at the high dosages retching and vomiting, and exclusively at the high dosages diarrhea, inappetence and physical weakness were sporadically seen in the first phase of the studies. 500 and 400 mg/kg led to increasing signs of discrete renal tubular changes (enzymuria, beta 2-microglobulinuria, cylindruria and minimal histological changes in 2 animals of the 400 mg/kg group). In one rhesus monkey (500 mg/kg) and two cynomolgus monkeys (800 mg/kg) severe kidney damage had developed within the first week. In all dosage groups of the 90-day study special histological methods revealed a dose-dependent increase and enlargement of lysosomes in the epithelia of the proximal renal tubules. Increased cytolysis was, however, not observed. In all the studies there was a dose-dependent increase in the kidney weights of the intermediate and highest dosage groups. The females of the 400 mg/kg group showed slight anemia accompanied by a slight increase in the reticulocyte count. One animal of this group died prematurely probably due to pulmonary embolism. The signs of slight renal impairment including lysosome enlargement, and the slight anemia proved to be reversible.

Subchronic and chronic toxicity of the new antibiotic cefpirome in rats.

Subchronic and chronic toxicity studies in rats were performed with 3-[(2,3-Cyclopenteno-1-pyridinium)-methyl]- 7-[2-syn-methoximino-2-(2-aminothiazol-4-yl)-acetamido] -ceph-3-em-4- carboxylate (cefpirome, HR 810), a new cephalosporin derivative. In subchronic (14 day) studies cefpirome was intravenously administered in dose levels up to 1500 mg/kg/d with good kidney tolerance. Signs of renal functional impairment were observed (800 and 1500 mg/kg) but histologically no morphological changes could be detected. The chronic intraperitoneal administration (90 day) of cefpirome at dose levels of 400 or 1600 mg/kg/d resulted in some reversible changes in hematology (slight anemia), serum-chemistry parameters (liver), urinalysis (proteinuria), and histopathology (increased numbers and enlargement of lysosomes in proximal tubular epithelia of the kidneys and pigment deposits in follicle epithelia of the thyroids), predominantly in high-dose animals. The "no effect level" is considered to be 100 mg/kg/d in this study.

Pharmacological studies on androgen suppression in therapy of prostate carcinoma.

In hormone-dependent prostate carcinoma, androgens can be suppressed into the castrate range by LHRH agonists. Testosterone secretion is blocked at two levels: testicular androgens and adrenal androgens. In humans, the contribution of testicular androgens is about 95%, whereas in the rat, the adrenal androgen secretion is negligible. Pharmacological studies were performed on the suppressive effect of the LHRH agonist, buserelin on androgen-dependent organs in adult rats. The reduction in pituitary and testicular binding capacity was monitored during treatment by injection, or by long-term infusion. Marked differences in suppressive mechanisms activated by the different regimens were observed. Changes in testicular steroid biosynthesis were analysed by incubation of testes after treatment with HCG, measuring the spectrum of C21/C19-steroids in incubation media. In particular, the levels of intraprostatic androgens were determined during treatment with daily buserelin injections, or with sustained release formulations of buserelin. The tissue content of testosterone and 5-alpha-dihydrotestosterone (DHT) were both markedly lowered. In castrate rats, stimulation of adrenal function by ACTH infusion had no effect on the prostate weight or intratesticular T/DHT content. Combination therapy during the initial phase of treatment by an androgen receptor blocker (cyproterone acetate) and buserelin (infusion or implants) was more effective to suppress prostate weight and intra-prostatic T/DHT content than therapy with the single compounds alone. Spermatogenesis and fertility were suppressed after prolonged treatment periods of 6-12 months; the testicular atrophy was not reversible in these long-term injection studies. Similar studies in dogs and monkeys have shown a different result: inhibition of spermatogenesis was fully reversible. It is concluded that studies on the mechanism of androgen suppression by LHRH agonists and the effects on androgen dependent organs provide useful information for the improvement in therapy of hormone-dependent prostate carcinoma.

Type IV collagen antigens in serum of rats with experimental fibrosis of the liver.

Specific and sensitive radioimmunoassays for the N-terminal (7S-collagen) and C-terminal (NC 1) crosslinking domains of type IV collagen were used to study the effects of prolonged treatment of rats with CCl4 on the concentrations of these antigens in serum. After four to six weeks of CCl4 treatment a highly significant increase of type IV collagen antigen levels in serum was to be seen. These elevated levels exhibited a significant correlation to the increased amounts of hydroxyproline in liver samples, when the samples were taken during CCl4 treatment. Analysis of the molecular weight distribution of serum antigens by gelfiltration revealed for 7S-collagen a bimodal distribution, whereas NC 1 eluted mainly as one peak. This peak coeluted with the high molecular weight peak of 7S-collagen, suggesting that this peak might represent intact type IV collagen. The lower molecular weight peak of 7S-collagen antigen eluted at a position which was comparable to standard 7S-collagen. Determination of type IV collagen antigens in serum might be of diagnostic usefulness for the follow up and treatment control of hepatic fibrosis.

The different mechanisms for suppression of pituitary and testicular function.

The differential mechanisms reducing androgen secretion by LHRH agonists are discussed with relevance to clinical therapy. LH secretion can be desensitised by exposure to agonists using high doses, frequent injections or sustained release/constant infusion. The desensitized pituitary is refractory to hypothalamic stimulation. Pituitary receptor suppression is associated with depletion of pituitary gonadotrophin content, and a decline of LH and FSH secretion to a basal rate. Recovery of LH responsiveness to endogenous LHRH stimulation requires restitution of gonadotrophin content (about 7 days in rats). After long-term infusions in normal men, testosterone secretion recovers within 7-10 days. The binding capacity of testicular LH/hCG receptors is reduced in rats after supraphysiological gonadotrophin stimulation, by agonists or directly by hCG, concomitantly the steroidogenic capacity of the testis in vitro is impaired. Qualitative changes in androgen biosynthesis are a marked fall in testosterone production and dose-dependent enhancement of progesterone production. After 12 months of buserelin injections, the changes in hCG-stimulated rat testes are an increased ratio of progesterone/17-OH-progesterone (inhibition of 17-hydroxylase), a reduced capacity for secretion of androstenedione and testosterone (block of 17,20-desmolase), and increased 5 alpha-pregnane-3,20-dione (this steroid inhibits the 17,20-desmolase, similarly to progesterone). After treatment, Leydig cell function recovers completely. Leydig cell hyperplasia is observed as a result of the steroidogenic changes. These findings in rats have not been observed in dogs, monkeys or in humans.(ABSTRACT TRUNCATED AT 250 WORDS)

Habekacin: nephrotoxicity, pharmacokinetics and prophylactic efficacy in rats.

1-N[(S)-4-amino-2-hydroxybutyryl]-kanamycin B (habekacin), a new aminoglycoside antibiotic found in 1973 was tested for its nephrotoxicity, pharmacokinetics and prophylactic efficacy in 351 female rats. Increased urinary elimination of tubule cells and malate dehydrogenase (MDH) demonstrated tubulotoxicity even at the minimal dosage of 2.5 mg/kg/d. At high dosages (100 or 50 mg/kg/d) habekacin produced more tubule damage than dibekacin. At lower dosages (20, 10 or 5 mg/kg/d) both aminoglycosides showed similar effects. Additionally, possible glomerular lesions were found at high dosages (100 mg/kg/d) as indicated by proteinuria, CAF (cellulose acetate foil)-electrophoresis of the urinary protein and raised albumin/globulin ratio. - Pharmacological studies revealed serum concentrations similar to dibekacin, in renal tissue, however, the concentrations of habekacin were much higher than those of dibekacin. - In experimental E. coli pyelonephritis, 9 single doses of habekacin or dibekacin (5 mg/kg) given prophylactically reduced the bacterial counts significantly; a single dose of the antibiotics (5 mg/kg) was slightly effective.

Antifertility effects of an LH-RH analogue in male rats and dogs.

The antifertility effects of a highly active LH-RH analogue, D-Ser(Bu)6-LH-RH(1-9)nonapeptide-ethylamide (buserelin) were studied in male rats and dogs. Pituitary-testicular function was not impaired by a "physiological" dose of 5 ng/rat; this dose gave reproducible LH release during chronic administration. At higher dose testicular LH receptors and responsiveness to HCG were diminished in intact prepubertal and adult rats. Pituitary inhibition was independent of gonadal or adrenal steroid feedback, and hypothalamic LH-RH as well as pituitary LH and FSH were reduced by 4 weeks treatment of castrate/adrenalectomized rats with 50 ng buserelin. In male dogs, a dose of 2.5 micrograms/kg sc reduced serum testosterone to 6% of controls within 8 weeks of treatment. Treatment was continued for 6 months and testicular involution was found to be reversible within 8 weeks of stopping treatment. LH-RH analogues at "supraphysiological" doses can be used as antifertility agents, but suppression of sexual activity in male dogs under treatment indicates that loss of libido will be a problem.