Effect of tumor promoting contraceptive steroids on growth and drug metabolizing enzymes in rat liver.

Liver tumor formation in rats treated with oral contraceptive steroids for long periods has been associated with the tumor promoting potential of these agents. As other known liver tumor promoters, e.g., phenobarbital and hexachlorocyclohexane, induce liver growth and hepatic monooxygenases, we investigated whether or not estrogens have similar effects. Female rats were treated with a wide range of doses of ethinylestradiol, including human contraceptive doses, which are approximately 1 microgram/kg. The physiological estrogen estradiol was studied for comparison. Also included were norethynodrel and norethisteron and its acetate and enanthate because these human progestins act predominantly estrogenic in rats. Daily s.c. doses of ethinylestradiol (0.5 mg/kg) produced a rapid increase of liver mass, DNA, RNA, and protein which was almost maximal after 7 days. The percentages of parenchymal cells involved in DNA synthesis and mitosis were enhanced up to 20-fold, suggesting parenchymal hyperplasia as the main cause of liver growth. Sinus wall cells showed a proportionate increase of number and DNA synthesis. Likewise, all other steroids tested produced significant increases of liver mass and DNA. For ethinylestradiol and estradiol extrapolated threshold doses were in the range of 1 microgram/kg. These doses are below those used in previous tumor promotion studies in rats. Using 5 different substrates to check monooxygenase activities of isolated liver microsomes, no induction or only very weak induction by estrogens was found. These studies suggest that induction of liver growth may be a property relevant for the tumor promoting activity of estrogens; in contrast, induction of hepatic monooxygenases does not appear to be necessary for liver tumor promotion in the rat.

Value of enzyme determinations in urine for the diagnosis of nephrotoxicity in rats.

Excretion of urinary lactate dehydrogenase (LDH, EC 1.1.1.27), gamma-glutamyltransferase (gamma-GT, EC 2.3.2.2), alkaline phosphatase (ALP, EC 3.1.3.1), alanine aminopeptidase (AAP, EC 3.4.11.-), alanine aminotransferase (GPT, EC 2.6.1.2) and N-acetyl-beta-D-glucosaminidase (NAG, EC 3.2.1.30) was studied following a single i.v. application of 1 mg mercuric chloride/kg body weight or a radio contrast medium (SH H 340 AB) at a dose of 7.5 g iodine/kg body weight in rats. Measurements of urinary enzymes and serum urea nitrogen and creatinine were carried out on the second, third, fourth and ninth days after treatment. Histological examinations of kidneys were performed on day 9. A drastic increase in urinary LDH and moderate increase in gamma-GT, ALP and AAP and a very slight increase in GPT was observed in the first 18-h urine samples after mercuric chloride. This increase in enzymuria was associated with a drastic increase in serum urea nitrogen and creatinine, with a maximum on day 4. The radio contrast medium-treated animals showed a similar but less pronounced pattern of urinary enzymes excretion and only a slight increase of serum urea nitrogen on day 2. A good correlation was found between histological findings and enzymuria as well as serum urea nitrogen and creatinine. Thus, determination of only some urinary enzymes (LDH and gamma-GT) is valuable in predicting early nephrotoxicity and sufficient for the diagnosis of proximal tubule damage in rats.

Isolation and identification of 15-beta-hydroxy cyproterone acetate as a new metabolite of cyproterone acetate in dog, monkey and man.

15-beta-hydroxy cyproterone acetate could be identified by thin layer chromatography, mass spectrum, NMR and IR spectrum as a major unconjugated metabolite of cyproterone acetate in plasma and urine of dog, monkey and man. This metabolite has been found to interferein the Mattingly method for the determination of 11-hydroxy-corticosteroids which suggests, that this method is inadequate in controling the adrenal function of subjects treated with cyproterone acetate.

Proliferative liver lesions and sex steroids in rats.

In standard two year tumorigenicity studies some gestagens, alone or in combination with an estrogen have been shown to produce proliferative liver lesions (PLL) in rats. Estrogens in general have not produced PLLs; for ethinyl estradiol the situation is equivocal. The steroids which increase the incidence of PLLs have not been shown to have the characteristics usually associated with classical hepatocarcinogens such as diethylnitrosamine (e.g. negative mutagenicity and cytoxicity, failure to bind covalently to DNA). Therefore, another mechanism must be assumed. A possible explanation is given by the ability demonstrated for a few steroids (mestranol alone or in combination with norethindrone, ethinylestradiol) to promote preneoplastic islands induced by diethylnitrosamine, resulting in an increase in tumor incidence over rats given only the carcinogen. Spontaneous preneoplastic islands in rats occur in various incidence in different strains and we have recently shown that their reaction to steroids-enhanced proliferation-is the same as the effect on preneoplastic islands produced by classical carcinogens. Sex steroids also enhance the growth or normal hepatocytes. It, therefore, seems reasonable to assume that sex steroids produce PLLs by acting on spontaneous preneoplastic lesions and not by the mechanisms usually associated with classical carcinogens. Extrapolating the incidence of PLLs in rats to a risk in humans may be highly misleading because of different pharmacokinetic patterns. Even in rats treated orally for two years with sex steroids dosed as multiples of the human contraceptive dose, resorption and biovailability may vary by a factor of 10 or more. It is, therefore, inappropriate to state that one sex steroid is more active in the production of PLLs than another, unless the data from pharmacokinetic studies are taken into account. Available evidence shows that sex steroids act as tumor promotors and enhance the growth of normal hepatocytes. It is extremely doubtful whether a sex steroid producing PLLs in the rat after two years of oral treatment can be classified as an initiating tumorigen for the rat. Substantial risk for humans cannot be inferred, because of the high incidence of spontaneous preneoplastic lesions in rats, and because of the very high sensitivity of the rat liver to a variety of chemically unrelated compounds including sex steroids.

The use of urinary N-acetyl-beta-D-glucosaminidase (NAG) for the detection of contrast-media-induced 'osmotic nephrosis' in rats.

Excretion of urinary N-acetyl-beta-glucosaminidase (NAG), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and gamma-glutamyltransferase (gamma-GT) was studied following single intravenous administrations of a non-ionic monomeric contrast medium (iohexol) at doses of 5.0, 7.5, 10.0 and 12.5 g iodine/kg body wt in rats. Measurements of urinary enzymes, serum urea nitrogen and serum creatinine were carried out on the 2nd, 3rd, 4th and 8th days after treatment. Histological examinations of kidneys were performed on days 3 and 8. From 5.0 g iodine/kg onwards urinary NAG showed a dose-dependent and significant (multiple comparison, alpha = 0.05) increase in the first 18-h urine samples after application. A significant increase in urinary LDH could be observed only at the highest dose of 12.5 g iodine/kg. All other biochemical parameters showed no differences when compared to the control group. The dose-dependent increase in lysosomal NAG correlated with the histological findings, i.e. there was dose-dependent vacuolization of proximal tubular cells, so-called 'osmotic nephrosis'.

Diagnostic value of serum creatine kinase and lactate dehydrogenase isoenzyme determinations for monitoring early cardiac damage in rats.

Serum activities of creatine kinase (CK, EC 2.7.3.2) and CK isoenzymes, lactate dehydrogenase (LDH, EC 1.1.1.27) and LDH isoenzymes, alpha-hydroxybutyrate dehydrogenase (alpha-HBDH, no EC) and the LDH/alpha-HBDH ratio were studied following a single s.c. application of 5-250 mg isoproterenol/kg body weight (b.w.) in rats. Measurements of the serum enzymes and histological and enzyme-histochemical examinations of hearts were performed 2, 4, 6, 8 and 24 h after treatment. A drastic increase in serum levels of the isoenzymes CK-MB, LDH1, LDH2 and alpha-HBDH and decrease in the ratio LDH/alpha-HBDH were observed from 2 h onwards after isoproterenol application in all dose groups, the maximum effect being after 4-8 h. Focal cellular injury in the myocardium could also be observed from 2 h onwards after isoproterenol application by an enzyme-histochemical method using nitro blue tetrazolium (NBT) whereas the earliest histological alterations using haematoxylin and eosin (HE) stain could be detected 6 h after treatment. A dose-dependent effect as to enzyme values as well as to myocardial necrosis was observed 24 h after isoproterenol application. No kidney damage could be detected on the basis of serum urea nitrogen and histological examinations. Thus, measurement of serum activities of CK-MB isoenzyme alone or LDH1-2 isoenzymes in combination with other tests to exclude kidney damage are valuable indicators of cardiac lesions in rats.

Animal toxicity studies performed for risk assessment of the once-a-month injectable contraceptive Mesigyna.

Results from toxicity studies performed for risk assessment of the combined injectable hormonal preparation Mesigyna are reviewed. Both components of Mesigyna, i.e., estradiol valerate (E2Val) and norethisterone enanthate (NET-EN), have been thoroughly investigated as individual compounds and some limited toxicity data have been obtained for the combination. Most findings which were gathered in these studies from different animal species occurred in the gonads, accessory genital and endocrine organs and can be related to the known species-specific pharmacological activity of a typical estrogen or progestin, respectively. No additional or unexpected information which might indicate a possible estrogen/progestin interaction was gained from the administration of the combined preparation to animals. Based on the results from toxicity testing, there were no objections to the long-term therapeutic use of Mesigyna for hormonal contraception. The predictive value of the effects (including the tumorigenicity) observed in the common laboratory animals with regard to human safety is critically discussed, taking the vast amount of previous experience with hormonal contraceptives into consideration. The conclusion is drawn that there is no animal model for safety assessment of sex steroids that adequately represents the human situation. Quantitative extrapolations from animal toxicity findings to humans, therefore, are not possible. Especially, the value of long-term studies and of toxicity studies on estrogen/progestin combinations is put into question. Like endocrine pharmacology studies, the toxicity studies with these steroid hormones are useful for the characterization of the possible endocrine pharmacological profile only.

PIP: Considerable research has been conducted on the 2 steroid components of the once-a-month injectable contraceptive, Mesigyna. These steroids are estradiol valerate and norethisterone enanthate. Most findings from the limited toxicity studies of the combined injectable in different animal species were limited to the gonads and accessory genital and endocrine organs. The steroids have a toxicological activity profile in each of the species, which indicate that they act as a typical steroidal estrogen or progestin. They are no different than comparable compounds as used in oral contraceptives. Other than these findings, researchers did not acquire any more or unexpected information that would suggest a possible estrogen/progestin interaction from administering the combined preparation to animals. The findings of the toxicity testing do not suggest problems with the long-term use of Mesigyna for hormonal contraception. Yet, the fundamental species differences in endocrinology, metabolism of compounds, and pharmacokinetics make it virtually impossible to quantitatively extrapolate from findings of animal toxicity studies to human, e.g., those on tumorigenicity. A critical review of in vitro and animal toxicity studies of both compounds individually and combined lead to the conclusion that no animal model for safety assessment of sex steroids exists that correctly replicates the human environment. Toxicologists with Schering AG even question the value of long-term studies and toxicity studies on estrogen/progestin combinations. Animal models can be used, however, to characterize a possible endocrine pharmacological profile of newly developed steroids. Any such animal studies need not last any longer than 6 months.

Effect of norethisterone acetate on serum lipid and lipoprotein parameters as well as on blood coagulation in female monkeys (M. fascicularis).

The effects of daily oral administration of a high dose of 10 mg norethisterone acetate (NET-Ac.)/kg/day over 14 weeks on serum lipid and lipoprotein parameters as well as on blood coagulation were investigated in female monkeys (M. fascicularis). Measurements of lipids and lipoprotein cholesterol were performed in weeks--5 and -1 before treatment and in weeks 4, 8 and 12 after treatment. In addition, various blood coagulation and fibrinolytic parameters were determined in weeks 11-14 after treatment with NET-Ac. Furthermore, the serum levels of norethisterone (NET) were determined in order to monitor the real systemic compound exposure and revealed that Cmax and AUC (0-3 h) values reached for norethisterone in this experiment in monkeys were about 25 times higher than those obtained after an oral contraceptive dose of NET-Ac. in women. The results of lipid and lipoprotein cholesterol determinations showed decreases in serum total lipids, phospholipids, triglycerides and total cholesterol associated with similar decreases in HDL-, LDL- and VLDL-cholesterol fractions after NET-Ac.-treatment in monkeys. These effects were observed from week 4 onwards and maintained their magnitude up to week 12 after treatment. Since both HDL- and LDL-cholesterol fractions decreased, the HDL/LDL-ratio remained almost unchanged. Thus, the results obtained in this study after high-dose treatment with NET-Ac. in monkeys did not indicate any changes of lipid and lipoprotein parameters which in humans are supposed to be associated with an increased risk of cardiovascular lesions, namely a decrease in HDL- and increase in LDL-cholesterol fractions. The results of blood coagulation and fibrinolytic parameters showed increased antithrombin-III and plasminogen levels besides minor changes in other parameters, thus indicating that NET-Ac.-treatment does not contribute to an increased risk of cardiovascular thrombotic events in the cynomolgus monkey.

[Histochemical and histological investigations on the vagina of the beagle she-dog during various functional conditions (author's transl)]. / Histochemische und histologische Untersuchungen an der Vagina der Beagle-Hündin während verschiedener Funktionszustände

Histotopochemistry and histology of vaginal epithelium in female beagles were studied during oestrus, metoestrus-dioestrus, post partum period and at days 20, 30, 40, 50, 60 of pregnancy. During oestrus the epithelium is uniform throughout the whole vagina: it presents itself as a high, uncornified, stratified squamous epithelium with some glycogen and lipid droplets but devoid of leucocytes. The intercellular gaps of the stratum intermedium give strong reactions for ATPase and alkaline phosphatase. The activities of oxidoreductases studied decrease continuously from basal to apical. During gravidity, post partum period and metoestrus-dioestrus distinct morphological and histochemical differences can be stated between the cranial and caudal vaginal portions. Caudal vaginal epithelium outside oestrus remains of stratified squamous type. It exhibits strong mucification during pregnancy. The PAS-positive mucous substances prefer a position in the enlarged intercellular gaps of stratum intermedium and superficiale. During pregnancy the epithelium is relatively rich in acid and completely devoid of alkaline phosphatases. Outside oestrus the epithelium of the cranial vaginal region is a relatively flat, stratified columnar one and contains leucocytes with regularity. Also the cranial vaginal portion undergoes mucification during pregnancy with a maximum about day 33. The mucous material is situated intracellularly and not within the intercellular gaps. Further, larger intraepithelial mucus cysts are observed. Alkaline phosphatase is found during gravidity in the basal region and an adluminal border of the epithelium. The reactions for oxidoreductases are strongest in the columnar cell layer which shows more functional adaptations than the remainder of the epithelium. Histochemical tests for beta-D-glucuronidase and leucine aminopeptidase give negative results in the whole vagina during all different functional stages studied.

Considerations regarding the development of individual nonclinical test strategies in the European Community.

The wish of pharmaceutical companies to be given the most binding possible preclinical test programmes for a new product, and the tendency of authorities to regulate to a great extent the preclinical testing of new drugs (especially at a multinational level), has led to differing requirements and practices in preclinical testing in different areas of the world. These differing requirements and practices have of necessity brought in their wake varying scientific criteria, with particular regard to animal protection, ethical standards, as well as imposing apparently unjustified extra financial costs. In order to improve this situation, the development of drug-specific preclinical test strategies is proposed in a drafted E. C. Note for Guidance, which incorporates already existing drug-testing guidelines and method recommendations. This draft Note for Guidance points to general methods of analysing problems and reaching decisions and thus appears to be worthy of recommendation as practical and desirable. It requires the co-operation of drug producers and supervisory authorities at a high scientific and ethical level. With regard to the state-of-the art and the socio-political background, the fulfillment of these requirements would appear not only to be appropriate but also imperative. To put them into practice would contribute enormously to the improvement of drug development and to de-emotionalization of the public debate. Therefore, comments on these draft guidelines from societies and associations are urgently sought and awaited with keen interest.

Characterization of a new potent heparin. 1 communication: Determinations of anticoagulant activity of a high potency heparin preparation in vivo using dog as an experimental animal and in vitro using dog and human plasma.

The anticoagulant effect of a highly potent heparin preparation was compared with a commercially available heparin in vivo after i.v. application in beagle dogs. The anticoagulant activity was determined using thrombin time, activated partial thromboplastin time and whole blood clotting time 5, 10 and 30 min after application. The relative potency of the heparin preparation (Schering) was found to be 1.62 to 2.52 times higher than heparin used for comparison (150 USP units/mg). The anticoagulant properties of both preparations were also studied in vitro using heparin concentrations from 0.44 to 7.0 microgram/ml dog and human plasma. The relative potency in vitro experiments using dog or human plasma were 1.62 and 1.63, respectively, on the basis of activated partial thromboplastin time. It was further demonstrated that the anticoagulant effect as determined by activated partial thromboplastin time in vitro on human plasma was 2 to 2.4 times more pronounced in both heparin preparations when compared to the effect exerted on dog plasma.

Characterization of a new potent heparin. 2nd communication: Chemical analysis of the carbohydrate content and determination of the biological activity of a new potent heparin preparation in vitro, using protamine neutralization and amidolytic methods for factor Xa and thrombin.

A new potent heparin preparation was further characterized for carbohydrate content and for biological activities in vitro using the protamine neutralization test and amidolytic methods for factor Xa and thrombin. The chemical analysis of the heparin preparations showed similar amounts of glucosamine and glucuronic acid as known for other heparin preparations. In protamine neutralization test the new heparin was 1.32 times more active than commercial heparin. The amidolytic method using chromogenic substrates S-2222 for factor Xa and S-2238 for thrombin demonstrated that the new heparin was also at least two times more effective than commercial heparin in increasing the rate of inactivation of these serin proteases through antithrombin III.

Characterization of a new potent heparin. 5th Communication: Further characterization by affinity chromatography on antithrombin-sepharose and its comparison with a commercially available heparin.

A highly potent heparin (Heparin Schering) was characterized in comparison to commercial heparin by affinity chromatography on antithrombin-III-sepharose. Heparin Schering consists of 83% high affinity and 17% low affinity fractions, whereas commercial heparin contained only 40% high affinity heparin and 60% low affinity heparin. The specific biological activities per microgram uronic acid and elution pattern on antithrombin-sepharose of high affinity heparin fraction were similar for both heparin preparations. It is concluded that the 2 times more potent anticoagulant activity of heparin Schering is mainly due to the enrichment of high affinity fraction (83%), which is also present in commercial heparin but at a remarkably lower percentage (40%).

Characterization of a new potent heparin. 3rd Communication: Determinations of anticoagulant activity of a new potent heparin preparation by thrombelastography in vitro using citrated dog and human blood.

The anticoagulant effect of a new potent heparin preparation was compared with a commercially available heparin by thrombelastography in vitro using citrated dog and human blood. The relative potency of the new heparin was found to be 1.45 to 1.77 with dog citrated blood and 1.77 to 2.41 with human citrated blood on the basis of different thrombelastographic parameters.