Systemic proliferative changes and clinical signs in cynomolgus monkeys administered a recombinant derivative of human epidermal growth factor.

Epidermal growth factor (EGF) effects have been explored extensively in vivo in rodents, but little is known about trophic responses in nonhuman primates. A previous publication reports the hyperplastic epithelial/parenchymal changes noted in the digestive tract (tongue, esophagus, stomach, intestine, liver, gallbladder, pancreas, and salivary glands) of adult cynomolgus monkeys treated with recombinant human EGF(1-48) (rhEGF(1-48)). This report documents clinical findings and structural effects in the remaining epithelium-containing tissues of these animals. Two monkeys/sex/dose received rhEGF(1-48) by intravenous bolus at 0 (vehicle), 10, 100, 500 (females only), or 1,000 microg/kg/day (males only) daily for up to 2 weeks. Treatment- and dose-related clinical findings included emesis, fecal alterations (soft feces and diarrhea), lacrimation, nasal discharge, hypoactivity, transient hypotension, and salivation after dosing. Male monkeys administered 1,000 microg/kg became moribund after 5 days of treatment and were necropsied. All other monkeys completed the 2-week treatment period. Necropsy findings in nongastrointestinal tissues were: enlarged, pale kidneys at 100 microg/kg and greater; small thymuses seen sporadically at all doses; and enlarged adrenals and small thyroids in males at 1,000 microqg/kg. Respective organ-to-brain weight ratios at 500 and 1,000 microg/kg for kidneys were 1.5- and 2.6-fold greater and for heart were 1.7- and 1.3-fold greater than controls. Microscopically, pronounced dose-related epithelial hypertrophy and hyperplasia were evident in kidney, urinary bladder, skin (epidermis and adnexa), mammary gland, prostate, seminal vesicles, epididymis, uterus, cervix, vagina, thyroid, thymus, tonsillar crypts, cornea, trachea, and pulmonary airways. Epitheliotrophic effects were conspicuous in many tissues at 100 to 1,000 microg/kg. Changes to renal collecting ducts were present at 10 microg/kg, suggesting that kidneys were a relatively sensitive target. Proliferative alterations were not apparent in testes, intraocular structures, brain ependyma and choroid plexus at any dose. Aside from the noted exceptions, rhEGF(1-48) was a pantrophic epithelial mitogen in cynomolgus monkeys when used intravenously at suprapharmacologic doses.

Retinal degeneration in rats induced by CI-1010, a 2-nitroimidazole radiosensitizer.

The anti-cancer compound CI-1010, designated as (R)-alpha-([(2-bromoethyl)amino]methyl)-2-nitro-1H-imidazole-1-ethanol monohydrobromide, has a proposed dual mechanism of action due to alkylating and radiosensitizing activities. To assess potential toxicity, adult Wistar rats were treated with a single intravenous injection (0, 50, 100, 150, 225, or 350 mg/kg) and necropsied at 4 or 29 days following treatment. In a repeated dose experiment, rats were injected daily (0, 10, 40, or 80 mg/kg; 5 doses/wk) for 3 wk and necropsied at the end of week 3 or 7. CI-1010 induced retinal degeneration by 4 days after a single injection of > or = 225 mg/kg or by 3 wk of repeated injections of > or = 40 mg/kg. The locally extensive to diffuse retinal degeneration involved the photoreceptor and outer nuclear layer. The photoreceptor layer was vacuolated and compressed corresponding to ultrastructural evidence of inner segment swelling and outer segment fragmentation. The outer nuclear layer was thinned due to loss of nuclei and contained numerous pyknotic or karyorrhectic nuclei. These nuclear changes were morphologically consistent with apoptosis and many outer nuclear layer nuclei labeled with in situ TdT-mediated dUTP-digoxigenin nick end labeling (Apoptag). The retinal degeneration was nonreversible, evidenced by increased lesion severity and incidence after CI-1010 was withdrawn for either 25 or 28 days.

Preclinical toxicity of a new oral anticancer drug, CI-994 (acetyldinaline), in rats and dogs.

CI-994 (acetyldinaline) is an orally active anticancer drug currently in Phase 1 clinical trials. To assess its preclinical toxicity, CI-994 was administered orally as suspensions to Wistar rats (10/sex/dose) and in capsules to beagle dogs (3/sex/dose) once daily for two weeks. Doses were 1.5, 5, and 15 mg/kg for rats (9, 30, and 90 mg/m2, respectively), and 0.5, 2, and 5 mg/kg for dogs (10, 40, and 100 mg/m2, respectively). Systemic exposure was dose-proportional based on toxicokinetic analysis in dogs. Severe clinical signs and mortality occurred at the highest dose in both species beginning on Day 10. Neutropenia, lymphocytopenia, thrombocytopenia, lymphoid depletion, bone marrow hypocellularity, and testicular degeneration were observed in both species, primarily at the mid- and high-doses. Despite continued treatment, neutrophil counts in dogs returned to control levels in Week 2. Other microscopic findings in rats included splenic hematopoietic depletion at all doses and epithelial cell necrosis in various tissues at 15 mg/kg. Additional bone marrow changes in dogs involved myeloid and megakaryocyte hyperplasia at 2 mg/kg and abnormal myeloid and megakaryocyte maturation at 2 and 5 mg/kg. Except for the testicular effects in both species, all changes were reversible within a 4-week (rat) or 9-week (dog) recovery period. The results of these studies show that target organ effects of CI-994 principally involve tissues with rapidly dividing cell populations and that bone marrow suppression is the dose-limiting toxicity. CI-994 also seems to interfere with the release and/or maturation of cells in the bone marrow.

Recombinant human epidermal growth factor1-48-induced structural changes in the digestive tract of cynomolgus monkeys (Macaca fascicularis).

To determine the cellular effects and potential toxicity of exogenously administered recombinant human epidermal growth factor1-48 (EGF1-48) in primates, intravenous bolus injections were given to 2 cynomolgus monkeys per sex at 0 (vehicle control). 10, 100, 500 (females only), and 1,000 micrograms/kg/day (males only) for up to 2 wk. Males given the suprapharmacologic dose of 1,000 micrograms/kg did not tolerate treatment and were necropsied after 5 days of dosing. All other monkeys completed the 2-wk study. Necropsy findings included enlarged, discolored, pale tan livers at 500 micrograms/kg and greater, firm, thickened pancreata in 500-micrograms/kg females, and enlarged salivary glands at all doses. Relative liver weights were increased at 500 and 1,000 micrograms/kg: mean salivary gland weights in all dose groups were greater than in controls. Histopathologic changes were primarily those of diffuse epithelial cell hypertrophy and hyperplasia in liver (hepatocytes and biliary tract), pancreas, salivary glands, tongue, esophagus, stomach, small and large intestine, and gallbladder. Alterations were dose-related in intensity and occurred in at last some tissues at the lowest dose. In gastric glands, colon crypts, pancreatic ducts, biliary tract, and salivary glands, differentiated epithelial cells were replaced by cells of less differentiated phenotype. These morphologic alterations were consistent with exuberant proliferation induced by this epithelial mitogen. The extent of the proliferative response in tissues of the digestive tract attests to the potency of this fragment of human EGF1-53 in primates. Furthermore, the epithelial proliferation was significantly greater than that reported previously in EGF-treated rodents.

Hepatic microsomal induction profile of carbamic acid [[2,6-bis(1- methylethyl)phenoxy] sulfonyl]-2,6-bis(1-methylethyl) phenyl ester, monosodium salt (PD138142-15), a novel lipid regulating agent.

Induction of hepatic microsomal cytochrome P450 produced by carbamic acid [2,6-bis(1-methylethyl)phenoxy]sulfonyl]-2,6-bis(1-methylethyl) phenyl ester, monosodium salt (PD138142-15), a novel water-soluble inhibitor of acyl-CoA: cholesterol acyltransferase, was examined in male and female rats, dogs, and monkeys, and in male guinea pigs. Relative to control, PD138142-15 increased hepatic microsomal total spectral P450 in all species examined. Hepatic microsomal ethoxyresorufin-O-deethylase, pentoxyresorufin-O-dealkylase, and peroxisomal carnitine acetyltransferase activities and cyanide-insensitive Beta-oxidation were affected only marginally. Erythromycin-N-demethylase activity was increased (2- to 6-fold) in all three species in which it was examined (rat, dog and pig). Marked increases in immunoreactive P450 3A were noted in the rats and dogs, while slight increases were seen in monkeys. Pharmacokinetic studies of PD138142-15 in rats and dogs revealed pronounced decreases (80-90%) in plasma Cmax and AUC within 2 weeks of initiation of daily dosing. In spite of the marked decline in plasma drug levels, efficacy in dogs, as determined by serum cholesterol levels, was maintained for up to 6 weeks with continued dosing. Potential acid (gastric) breakdown products of PD 138142-15 were examined for their hepatic cytochrome P450 induction profiles in rats adn were found to differ both quantitatively and qualitatively from profiles produced by the parent compound. This suggested that induction observed in rats was due to parent PD138142-15 and not to any of the known potential acid breakdown products. The cumulative data establish that PD 138142-15 is an inducer of P450 3A in rats and dogs. The results also suggest that P450 3A is induced in monkeys and pigs as well, although the data are less definitive. Decreases in plasma drug levels imply that the compound may be an autoinducer in dogs and rats. The maintenance of efficacy in spite of decreased drugs levels in dogs suggests that the effects on serum cholesterol are due to a metabolite or that cholesterol lowering effects occur before the compound is metabolized by the liver.

Mechanism of the serum thyroid hormone lowering effect of perfluoro-n-decanoic acid (PFDA) in rats.

The mechanism and consequences of the serum thyroid hormone lowering effect of perfluorodecanoic acid (PFDA) were examined. Thyroid and pituitary gland functions in PFDA-treated rats were assessed by measuring radioiodine uptake from the circulation and the ability of the thyroid gland to secrete thyroxine (T4) and triiodothyronine (T3) in response to thyrotropin-releasing hormone (TRH) stimulation. Serum levels of reverse triiodothyronine (rT3) were measured to test for possible conversion of T4 to a biologically inactive product and the displacement of radiolabeled T4 from rat albumin in vitro by PFDA was examined. Finally, changes in activity of the thyroid hormone-sensitive liver enzymes glycerophosphate dehydrogenase (GPD) and malic enzyme (ME) in response to PFDA were analyzed. Functional activities of the thyroid and/or pituitary glands appear to be somewhat depressed by PFDA treatment. There was no increased conversion of T4 to rT3. PFDA displaced radiolabeled T4 from rat albumin with an affinity similar to thyroxine. The activities of both GPD and ME were significantly increased in livers from PFDA-treated rats. These results suggest that decreased serum levels of thyroid hormones may be due to (1) reduced responsiveness of the thyroid and/or pituitary glands to hormonal stimulation and (2) a displacement of circulating hormones from plasma protein binding sites by PFDA. Increased activity of the liver enzymes GPD and ME does not reflect the reduction in circulating thyroid hormones and indicates that PFDA-treated rats are apparently not functionally hypothyroid at the tissue level.

Effect of thyroxine supplementation on the response to perfluoro-n-decanoic acid (PFDA) in rats.

The effects of thyroxine (T4) supplementation on perfluoro-n-decanoic acid- (PFDA) induced decreases in food consumption, body weight, and body temperature were examined. A dose-response study was carried out with 50-, 100-, 200-, or 250-micrograms/kg ip doses of T4 for 7 d prior to PFDA administration, and daily dosing with T4 was continued for an additional 30 d. From this study a T4 dose of 200 micrograms/kg was chosen, and subsequent experiments were conducted with this dose. Supplementation with T4 at 200 micrograms/kg daily alleviated the hypophagia but not the severe weight loss and hypothermia produced by PFDA treatment. Our results suggest that some component of the thyroid axis plays a role in feeding behavior. In addition, the PFDA-induced wasting syndrome and hypothermia appear to be unrelated to changes in serum thyroid hormones. The unexpected observation that severe weight loss occurred in the presence of essentially normal food intake suggests that PFDA alters basic cellular metabolic processes.

The effects of perfluoro-n-decanoic acid (PFDA) on rat heart beta-receptors, adenylate cyclase, and fatty acid composition.

Perfluoro-n-decanoic acid (PFDA) is a member of a family of surfactants with numerous industrial applications. The acute toxicity of PFDA is characterized by body wasting and delayed lethality. Recent reports have indicated that the effects of PFDA may involve an action on the structure of biological membranes which results in an alteration of function. In the present study we extend our work on the membrane actions of PFDA by examining its effects on myocardial beta-adrenoceptor binding characteristics and adenylate cyclase. Following a single injection of PFDA the apparent number of beta-receptor binding sites was reduced compared to pair-fed controls. This change in beta-receptor binding capacity was reflected in a reduced ability of norepinephrine to activate adenylate cyclase. No alterations were observed in basal adenylate cyclase activity or in the ability of NaF or guanylyl imidodiphosphate to stimulate the enzyme. The fatty acid composition of the heart was changed by PFDA treatment. Our results suggest that the toxic effects of PFDA may be due to an alteration of the membrane lipid bilayer leading to changes in the functional activity of myocardial membranes.

The effects of perfluoro-n-decanoic acid in the rat heart.

Perfluoro-n-decanoic acid (PFDA) is a synthetic chemical resembling a 10-carbon fatty acid. Several studies have suggested that the toxic mechanism of PFDA may involve impaired lipid metabolism and/or altered cell membrane function. We examined the possibility that altered cell membrane structure in the heart might lead to changes in the functional activity of the organ. Functional characteristics were determined in the isolated perfused rat heart by measuring the ability of the heart to respond to either sympathetic nerve stimulation or infused norepinephrine. PFDA reduced the intrinsic resting heart rate and the inotropic response to a stimulus with maximal effects occurring 8 days after dosing. In addition, resting heart rate measured in vivo was found to be reduced in PFDA-treated rats 6 to 8 days after dosing. beta-Receptor binding studies conducted 8 days after a single dose of PFDA showed that the maximum binding capacity was reduced by PFDA treatment without significant changes in receptor affinity. It is concluded that the reduction in the inotropic response to catecholamines following PFDA treatment may be explained in part by lower beta-receptor density in the myocardial cell membrane. These effects may be related to the early fall in serum thyroid hormone levels as previously reported.

Thyroid, bradycardic and hypothermic effects of perfluoro-n-decanoic acid in rats.

A single ip injection of perfluoro-n-decanoic acid (PFDA) to male Wistar rats resulted in an initially rapid, then gradual decrease in food consumption and a parallel loss of body weight. Body temperatures and resting heart rates were significantly depressed by PFDA treatment. As early as 12 h following a single dose of PFDA, serum thyroxine (T4) levels were significantly reduced and remained depressed throughout the 8 day study. Serum triiodothyronine (T3) was reduced by 35% 12 h following PFDA treatment and remained at that level throughout the study. These preliminary data suggest that an action on the thyroid axis may be an early primary response to PFDA and that some of the observed subsequent effects may in part be secondary to the change in thyroid hormone levels.