FDA recommendations for preclinical testing of gonadotropin releasing hormone (GnRH) analogues.

Gonadotropin-releasing hormone (GnRH) agonists and antagonists are synthetic analogues synthesized by modifications of the naturally occurring hypothalamic decapeptide GnRH. These modifications significantly increase the biological potency and duration of action of GnRH agonists as well as the solubility, potency, and duration of action of GnRH antagonists while decreasing GnRH antagonists toxicity. The field of GnRH analogues has expanded significantly during the past few years in terms of the number of analogues, therapeutic indications, formulations, and mode of administration. This paper provides recommendations for nonclinical testing of GnRH analogues and reflects the type and degree of toxicity testing expected by the Division. However, these recommendations are not formal guidelines in that alternative testing methods will be considered. Furthermore, these recommendations should not be used as guidance for testing of other new drugs.

Heifer fertility and its relationship with cow fertility and production traits in Holstein dairy cattle.

Breeding receipts from three AI units were merged with Ontario Dairy Herd Improvement Corporation and Record of Performance production records. Data comprised 53,705 heifer, 41,253 lactation 1, 14,688 lactation 2, and 3054 lactation 3 records by daughters of 2150 sires represented in 15,877 herd-year-seasons of birth. Three measures of heifer fertility, three measures of cow fertility, and three measures of production were investigated. Measures of heifer fertility were ages at first and last breeding and number of inseminations per conception. Cow fertility traits were days from calving to first breeding, days open, and number of inseminations per conception. Production traits were breed class average milk, breed class average fat, and fat percentage. Relationships among these nine traits for the first three lactations were estimated using a maximum likelihood multiple-trait procedure. The linear mixed model for each trait included fixed effects of herd-year-season of birth and genetic groups of sire and the random effect of sire. Transformations of the data for nonnormality had no influence on the estimates of genetic and phenotypic parameters. The heritability of .12 for age at first insemination, which was higher than other heifer fertility traits, indicated that selection would result in genetic response. Genetic and phenotypic correlations between heifer fertility and cow fertility and production traits in all three lactations were not different from zero. There was no genetic antagonism between fertility and subsequent production traits.

Relationships between fertility and production in Holstein dairy cattle in different lactations.

Record of Performance and Dairy Herd Improvement Corporation production records of Ontario Holstein cows were merged with breeding receipts of three Ontario AI units from September 1981 through December 1985. Relationships between fertility and production in the first three lactations were investigated for 97,368 daughters of 3806 sires in 22,768 herd-hear-seasons of calving. Fertility traits were days from calving to first insemination, number of inseminations per conception, and days open. Production traits were age and month of calving adjusted 305-d milk and fat yields and fat percentage. Multiple-trait maximum likelihood was used to estimate variances and covariances. Heritabilities for the first three lactations were .18, .18, and .19 for milk yield; .20, .19, and .19 for fat yield; and .58, .52, and .48 for fat percentage. Heritabilities of fertility traits ranged from .03 to .06. Genetic and phenotypic correlations between fertility and production traits in all three lactations were essentially 0. Genetic correlations between different lactation production traits ranged from .2 to .65. Repeatabilities of fertility traits ranged from .05 to .16 in different lactations. Repeatabilities for production traits in different lactations ranged from .51 to .77. Genetic and phenotypic correlations between fertility and production in the subsequent lactation and between production and subsequent lactation fertility were also very low or zero.

Relationship of bull fertility with daughter fertility and production traits in Holstein dairy cattle.

The phenotypic and genetic correlations between fertility ratings of AI bulls for conception rate and their estimated breeding values for daughters' fertility and production traits were calculated. Genetic correlations between fertility ratings of bulls for conception and heifer fertility traits (age at first breeding, age at last breeding, and number of insemination per conception) were negative and ranged from -.04 to -.23, indicating daughters of bulls with high fertility ratings were younger at first breeding and required fewer services to conceive. In general, genetic correlations between fertility ratings of bulls for conception rate and cow fertility traits (days from calving to first breeding, days open, and number of inseminations per conception) and production traits (breed class average milk and fat and fat percentage) in the first two lactations were also moderate to high and in the favorable direction. Although heritability of both male and female fertility is low, these data indicate that heavy use of sires with high fertility ratings could have a mild positive effect on both male and female fertility. Evidence is also found to indicate that in this breed, selection for increased milk yield should not impair genetic ability of cows to reproduce.

Food and Drug Administration guidelines for reproductive toxicity testing.

The Food and Drug Administration generally requires reproductive toxicity testing of all new drugs to be used by pregnant women or women or men of reproductive potential. These requirements may vary among the centers within the FDA. Reproductive and developmental toxicity is usually tested in one or two animal species and is divided into three segments to represent treatment throughout the reproductive process. The FDA monitors adverse drug effects on human reproduction through postmarketing surveillance.

Effect of nutritional status on propylthiouracil-induced protection against acetaminophen hepatotoxicity in the rat.

The effect of pair feeding of euthyroid rats compared with propylthiouracil (PTU) treated rats on acetaminophen (APAP)-induced hepatotoxicity was studied. Also, the effect of food deprivation of both the euthyroid and PTU-induced hypothyroid rats for 24 h, as well as forced feeding of the euthyroid rats after a toxic dose of APAP, was determined. Pair feeding decreased both protein and energy intake compared with ad libitum-fed controls and resulted in decreased growth rate similar to that for the PTU treated rats. In contrast to the protective effect of PTU pretreatment, decreased protein energy intake by the euthyroid rats either tended to make them more susceptible to acetaminophen-induced hepatotoxicity or had no effect as assessed by elevation of serum transaminases (SGOT,SGPT) and by hepatic necrotic score. Pair feeding also significantly altered drug disposition with an increase in the molar ratio of urinary APAP-mercapturic acid conjugate, but not the absolute amount, suggesting possible increased cytochrome P-450 dependent drug metabolizing enzyme activity. Compared with PTU-fed, in the pair-fed the molar ratio of glucuronide conjugate decreased and sulfate conjugate increased. Hepatic reduced glutathione (GSH) concentrations before and 4 h after a toxic dose of acetaminophen administration were higher in the PTU pretreated compared with euthyroid rats. Fasting of the PTU pretreated rats for 24 h after acetaminophen administration abolished the PTU-induced protective effect. Forced feeding of the euthyroid rats after a toxic dose of acetaminophen increased rather than decreased the toxicity when compared with euthyroid ad libitum-fed rats. Data suggest that higher concentrations of hepatic glutathione in the PTU pretreated compared with euthyroid rats before and 4 h after acetaminophen administration contribute to PTU-induced protection. Forced feeding of rats when the liver is severely damaged and its function compromised is harmful rather than protective. We conclude that the nutritional state of the animal significantly influences drug toxicity and should be taken into consideration in designing drug therapy and evaluation of drug toxicity.

Possible role of pancreatic insulin and glucagon in the hyperlipidemia and obesity of obese strain of chicken.

Circulating levels of insulin, glucagon, thyroid hormones as well as lipid levels were determined in an obese strain of chicken and their lean controls. Hepatic and muscle glycogen and lipids were also measured. Obese birds had higher plasma lipids accompanied by significantly higher insulin and lower glucagon levels compared to lean controls. Hepatic and muscle triglycerides were also higher in obese birds. Plasma T4 level was significantly higher in obese but T3 was not different in the two groups. Results suggest that genetically obese birds have significantly increased insulin/glucagon ratios as previously reported in the PTU induced hypothyroid-obese chicks (Horm. Metab. Res. 12: 51, 1980) and this could have causal relationship to hyperlipidemia and obesity observed in these birds.

Propylthiouracil-induced hypothyroid hyperlipidemic chick: a model for clofibrate-induced toxicity.

An animal model for clinically observed clofibrate (p-chlorophenoxy isobutyrate, CPIB)-induced toxicity has been tested. It is demonstrated that propylthiouracil-induced hypothyroid-hyperlipidemic chick develops severe toxic manifestations following clofibrate administration. Toxic symptoms are characterized by listlessness, drowziness, and extreme muscular weakness. This is associated with elevation of blood urea nitrogen, creatine phosphokinase, uric acid and glutamic oxaloacetic transaminase. Histological examination of muscle specimen from chicks exhibiting toxic syndrome showed degeneration and vacuolization of muscle fibers. The biochemical and histological changes observed are quite similar to those reported in clinical practice in some patients given clofibrate. It is suggested that this chick model could be used to investigate the biochemical basis of clofibrate toxicity.

Failure of exogenous prostaglandin to afford complete protection against acetaminophen-induced hepatotoxicity in the rat.

The protective effect of 16, 16-dimethylprostaglandin E2 (dm-PGE2) against acetaminophen-induced hepatotoxicity was determined in the rat. The dm-PGE2 was administered at two dose levels both before and after acetaminophen administration. The hepatotoxicity was evaluated by a rise in serum transaminases 24 h after acetaminophen administration and by histological examination of liver preparations. The urinary acetaminophen and its metabolites were determined by high-pressure liquid chromatography. The results suggest that exogenous dm-PGE2 administration had a modest protection against acetaminophen-induced hepatotoxicity, in contrast to its well established cytoprotective effect against many noxious agents in the gastrointestinal tract. Prostaglandin treatment had little effect on acetaminophen metabolites excretion in the urine, suggesting that it did not affect the cytochrome P-450-dependent mixed-function oxidase drug-metabolizing enzyme system. The livers from dm-PGE2-acetaminophen-treated rats showed less advanced necrosis compared to those from saline-acetaminophen-treated rats. Whereas only 2 of 13 rats died in the prostaglandin-treated group, 4 of 13 rats died in the saline-treated group.

Inhibitory effect of propylthiouracil-induced hypothyroidism in rat on oxidative drug metabolism.

The effect of propylthiouracil (PTU) pretreatment on in vivo and in vitro oxidative drug metabolism was determined in the rat. Whereas pentobarbital sleeping time (PBST) and zoxazolamine paralysis time (ZZPT) were used as indices of in vivo drug metabolizing activity, biotransformation of aminopyrine and aniline by hepatic microsomal preparations were used as indices of in vitro drug metabolizing enzymes activities. PTU pretreatment significantly prolonged both PBST and ZZPT. Whereas PTU did not affect microsomal protein concentration or cytochrome P-450 content, it significantly decreased microsomal cytochrome c reductase and aniline hydroxylase activities. These changes in enzymatic activities were observed in microsomal preparations from either non-fasted or 24-hr fasted rats. Our results suggest that PTU-induced hypothyroidism modifies the metabolism and effectiveness or toxicity of concomitantly administered drugs.

Lack of relationship between plasma insulin and glucagon levels and angiographically-documented coronary atherosclerosis.

In 120 consecutive patients undergoing diagnostic coronary arteriography, fasting blood glucose, plasma insulin, glucagon, serum cholesterol and triglyceride concentrations were measured. The insulin-glucose ratio and insulin-glucagon ratio were calculated. Forty-five patients had normal coronary arteries, 19 had single vessel coronary artery disease and 56 patients had multiple vessel disease. Fasting blood glucose was greater than 120 mg/100 ml in 37 patients (group A) and included 9 of the 10 known diabetics, 3 of whom were being treated with insulin. Seventy-seven patients included in group B had fasting blood glucose concentration less than 120 mg/100 ml. Patients with multiple vessel coronary disease in either group had higher blood glucose and cholesterol concentrations than those with normal coronary arteries or the ones with single vessel disease, but they did not have higher plasma insulin or glucagon levels nor increased insulin-glucose or insulin-glucagon ratios. With comparable extent of coronary artery disease patients in group A had higher plasma insulin levels and insulin-glucagon ratios than those in group B, but no correlation exists between the presence or extent of coronary atherosclerosis and these variables in either group. Thus, neither fasting plasma insulin level nor insulin-glucagon ratio predicts the status of underlying coronary atherosclerosis in either diabetics or nondiabetics.

Effect of acetaminophen toxicity on erythrocyte osmotic fragility in the Fisher rat.

The protective effect of propylthiouracil (PTU) pretreatment against acetaminophen-induced erythrocyte osmotic fragility was determined in the male Fisher rat. Hepatotoxicity was assessed for comparative purposes. PTU (0.15%) was fed in chow for a period of 12 days. Acetaminophen (1 g/kg body wt) was then administered orally by a stomach tube after an overnight fast. The rats were killed either 4 or 24 hr later. Erythrocyte osmotic fragility was determined by the extent of hemolysis in various concentrations of NaCl solutions. Hepatotoxicity was assessed by a rise in serum transaminases and by histological examination of hepatic tissue. PTU treatment when compared with control not only protected rats against acetaminophen-induced hepatotoxicity as reported before, but also protected against erythrocyte osmotic fragility. The time course of acetaminophen toxicity seems to be similar for liver and erythrocyte since both showed damage after 24 hr but not after 4 hr of acetaminophen administration. The data show that PTU pretreatment affords protection against acetaminophen-induced increased erythrocyte osmotic fragility even when their glutathione concentrations were not significantly different, suggesting that PTU per se has a protective effect.

Effects of propylthiouracil on urinary metabolites of cyclophosphamide in rats.

Our previous studies have shown a protective effect of propylthiouracil (PTU) pretreatment against the toxicity of cyclophosphamide (CP). The present study was undertaken to investigate the mechanism of the PTU protection. CP is metabolized by the cytochrome P-450 drug-metabolizing enzyme system in the liver to alkylating metabolites, to active antineoplastic agents, and to acrolein, the most toxic and least antineoplastic metabolite. Measurements of CP metabolites in blood and urine during a 4-hr i.v. infusion of CP (50 mg/kg body weight/hr) showed urinary acrolein excretion to be 2.5 times higher in control rats as compared to PTU-treated rats. Since it has been reported that urinary acrolein levels are directly related to the frequency and severity of hemorrhagic cystitis, it is concluded from our observations that prevention of hemorrhagic cystitis is probably mediated by the PTU effect on lowering urinary acrolein concentration and excretion. Serum alkylating activity was significantly higher in the PTU-pretreated rats, which may enhance the antineoplastic potential of CP.

Hepatotoxicity and metabolism of acetaminophen in male and female rats.

This present study was designed to assess the role of metabolic and pharmacokinetic factors in the lower susceptibility of female rats compared to male rats to xenobiotics metabolized by the cytochrome P-450-dependent mixed-function oxidase (MFO) system. Adult intact male and female Sprague-Dawley rats were administered labeled acetaminophen (1 g/kg body weight + 5 microCi [3H]acetaminophen) after an overnight fast. They were bled and killed at 0.5, 1, 2, 3, 6, 12, 24, and 36 h after drug administration. The percentage of [3H]acetaminophen radioactivity remaining in blood, liver, GI tract, and excreted in the urine was determined at all time intervals. Plasma prothrombin time and serum transaminases were determined as indices of hepatotoxicity. Hepatic GSH and glycogen were assayed. Total urinary acetaminophen and its metabolites and the molar percent of various metabolites excreted during the first 6 h were determined. Castrated male and ovariectomized female rats and their respective controls were also given acetaminophen (APAP) and were killed 24 h later to determine hepatotoxicity. The extent of hepatic damage in the intact male rats was greater and appeared sooner than in the female rats. Hepatic GSH and glycogen were depleted earlier in female rats. The percent of the administered dose excreted in the urine during the first 6 h was 17.5 for the male rat versus 24.5 for the female rat. While the APAP glucuronide conjugate concentration was significantly higher, the APAP sulfate conjugate concentration was lower in the female than it was in the male rat. Although peak radioactivity in serum was reached by 30 min in both male and female rats, suggesting quick intestinal absorption, it was significantly higher in female rats and was associated with decreased intestinal and hepatic levels and increased urinary excretion when compared to male rats. While castration of male rats decreased susceptibility to hepatotoxicity, ovariectomy of female rats tended to increase susceptibility to hepatotoxicity in comparison to their respective controls. Our data suggest that aside from the reported sex differences in the cytochrome P-450-dependent MFO enzymes, there are significant differences in GSH utilization. There are also significant changes in glucuronidation and sulfation pathways, as well as in the pharmacokinetics of acetaminophen, which tend to protect female rats against acetaminophen hepatotoxicity.

Effect of riboflavin status on acetaminophen toxicity in the rat.

The effect of riboflavin status on acetaminophen hepatotoxicity was determined in the rat. Groups of rats were fed one of the following diets: "riboflavin-free" (RFF), low riboflavin (LRF), high riboflavin (HRF), or high riboflavin pair-fed (HRF pair-fed) with RFF group. After riboflavin deficiency was established by determining erythrocyte glutathione reductase activity coefficient, rats in all groups were administered a toxic dose of acetaminophen (1 g/kg body weight) orally. Their controls were given the vehicle alone. All animals were killed 24 h later and hepatotoxicity was assessed by the elevation of serum transaminases and by a necrotic score based on histological examination. The RFF diet induced biochemical riboflavin deficiency, decreased food intake and body weight gain, and was associated with almost complete protection against acetaminophen toxicity. Rats on the LRF diet, with less severe riboflavin deficiency and no significant change in weight gain, showed some necrosis, but it was much less than in the HRF ad libitum-fed rats. The HRF pair-fed rats with no biochemical riboflavin deficiency but with considerable growth retardation also showed very little hepatic necrosis. Our results suggest that riboflavin deficiency protects rats against acetaminophen toxicity but it is confounded by decreased food consumption and body weight.

Prevention of acetaminophen hepatotoxicity by propylthiouracil in the glutathione depleted rat.

This study was designed to investigate the protective effect of PTU pretreatment against acetaminophen hepatotoxicity in rats whose hepatic GSH had been depleted by prior diethylmaleate (DEM) administration. A single injection of DEM depleted hepatic GSH showing lowest level after 90 min in both control and PTU pretreated rats. Triple injection schedule kept the hepatic GSH concentrations consistently very low up to 6 hr. Whereas a toxic dose of acetaminophen administration did not effect SGOT and SGPT levels after 30 hr in PTU pretreated rats given either a single or multiple injections of DEM, the same dose of acetaminophen in the control rats raised these transaminases to a very high level. High activity of transaminases was associated with significant histological hepatic damage. Our results suggest that PTU pretreatment affords significant protection against acetaminophen hepatotoxicity even under conditions when hepatic GSH concentrations have been significantly depleted prior to acetaminophen administration.

Comparative effects of soy and casein protein on plasma cholesterol concentrations.

The effects of soy and casein protein on cholesterol metabolism were determined under normocholesterolemic and hypercholesterolemic conditions in male Wistar rats and in Swiss White mice. Hypercholesterolemia was induced by feeding cholesterol and cholic acid in the diet for a period of 2 weeks. Our results demonstrate that there are significant species differences. The hypercholesterolemic effect of exogenous cholesterol was more pronounced in the mouse than in the rat. The rats fed the basal (no cholesterol, no cholic acid) semisynthetic diet containing either soy or casein had plasma cholesterol concentrations similar to those observed in chow-fed controls. However, plasma cholesterol concentrations in the mice fed the basal semisynthetic diet containing either of the two proteins were significantly elevated compared to the control mice fed commercial chow. Rat data demonstrate that the hypercholesterolemia induced by exogenous cholesterol administration is of a lesser magnitude in the presence of soy compared to casein as a dietary protein source. The mice data show that the concentrations of hepatic and biliary cholesterol and plasma triglycerides and in response to exogenous cholesterol are significantly lower in the soy protein diet compared to the casein protein diet.

Hypolipidemic and glycogenolytic effect of clofibrate (CPIB) in hypothyroid mice: role of insulin and glucagon.

1. The role of endogenous glucagon and insulin on the hypolipidemic and glycogenolytic effect of clofibrate was determined in the euthyroid and propylthiouracil (PTU)-induced hypothyroid mice. 2. PTU was fed in diet (0.15%) for 2 weeks and then clofibrate added to diet (0.25%) for 4 weeks. 3. Both PTU and clofibrate significantly increased liver weight but had no effect on kidney weight. PTU significantly decreased plasma triglycerides (TG) and increased cholesterol (Ch). 4. Clofibrate had a significant hypotriglyceridemic effect in both euthyroid and hypothyroid mice but did not affect plasma cholesterol. 5. Clofibrate decreased hepatic glycogen in euthyroid but not in hypothyroid mice. 6. Glucose-6-phosphatase activity was not affected by either PTU or clofibrate. 7. Neither PTU nor clofibrate affected hepatic TG or Ch. 8. Biliary lipid changes due to PTU treatment were reversed by clofibrate administration. 9. Since plasma insulin and glucagon levels were not affected by clofibrate in either euthyroid or hypothyroid mice, our results suggest that the hypotriglyceridemic and glycogenolytic effect of clofibrate is not mediated by changes in circulating insulin and glucagon ratio. 10. Moreover, while the glycogenolytic effect of clofibrate seems to be dependent, the hypotriglyceridemic effect seems to be independent of thyroid hormones.

Elevated insulin/glucagon ratios and decreased cyclic AMP levels accompany the glycogen and triglyceride storage syndrome in the hypothyroid chick.

The role of endogenous glucagon and insulin on the hepatic glycogen and triglyceride storage syndrome in propylthiouracil (PTU)-induced hypothyroidism was investigated in the chick. PTU feeding in the diet resulted in a progressive increase in liver glycogen concentration associated with a concomitant decrease in hepatic glucose-6-phosphatase (G-6-Pase) activity. Plasma glucagon level was significantly decreased and insulin significantly increased after two days of PTU administration. These enzyme and hormone changes were associated with a significant increase in hepatic glucose-6-phosphate (G-6-P) and a decrease in cyclic AMP levels. Although our results do not directly prove, the data does suggest that the hepatic glycogen storage syndrome observed in the PTU-induced hypothyroidism in the chick is mediated through changes in pancreatic glucagon and insulin secretion. The extent of glycogen accumulation was inversely related to G-6-Pase which is a rate limiting glycogenolytic enzyme. A significant increase in the plasma insulin/glucagon ratio, along with a significant decrease in the hepatic cyclic AMP concentration, could most likely also account for the excessive hepatic triglyceride accumulation in the PTU-treated chicks.