Evaluation of apomorphine HCl effects on reproductive endpoints: studies in male rats and dogs.

Apomorphine HCl is currently in phase III clinical trials for the treatment of erectile dysfunction. The potential for reproductive toxicity in males was assessed based on a 13-week rat study--a fertility study in male rats--and a 6-month study in dogs. The subcutaneous (s.c.) route was selected to simulate the sublingual route in humans. Dosages of apomorphine were 0.0 (vehicle), 0.8, 2, and 8 mg/kg/day in the 13-week study in rats (20/group), with 8 mg/kg/day used for only 9 weeks. In the fertility study, 24 males/group were cohabited with females, and doses were 0.0, 0.2, 0.8, and 2 mg/kg/day. Males were treated for 4 weeks prior to cohabitation and for 5 weeks throughout cohabitation. Organ weights, including testis and left epididymis, sperm count and morphology in the epididymis, and sperm motility in the vas deferens were evaluated. Male fertility index, and in females, the numbers of fetuses, implantation sites, and corpora lutea were counted. Male dogs (five/group) were dosed with 0.04, 0.1, or 0.4 mg/kg/day for 6 months. Epididymal and prostate weight, and testicular and epididymal histology were evaluated. Daily morbidity/mortality, weekly clinical observations, body weight, and food consumption were evaluated in all studies. No adverse effect was observed in any of the reproductive parameters in the studies. The NOEL for reproductive toxicity was approximately equal to 0.4 mg/kg/day in dogs and > or = 2 mg/kg/day in rats. These doses in rats and dogs correlated with plasma levels of approximately 240 and 130 ng/mL, and AUCs of 200 and 100 ng.h/mL, respectively. These levels suggest a safety margin for the evaluated male reproductive endpoints of at least 104 times based on the Cmax, and 44 times based on AUC of the clinical dose.

Evaluation of the toxicity of ISIS 2302, a phosphorothioate oligonucleotide, in a four-week study in cynomolgus monkeys.

The toxicity of ISIS 2302, a phosphorothioate oligonucleotide with antisense activity against human ICAM-1 mRNA, was investigated in cynomolgus monkeys (young adult). The oligonucleotide was administered by slow bolus injection every other day for 28 days (14 doses) at dose levels of 0, 2, 10, and 50 mg/kg/injection. The basic group size consisted of three male and three female monkeys which were sacrificed 2 days after the last dose. An additional 2 monkeys/sex in the vehicle control and 50 mg/kg dose groups remained on study for a 28-day treatment-free period. No treatment-related deaths occurred during this study, however, one monkey in the 10 mg/kg dose group was markedly lethargic after the first dose. Other clinical observations included periocular swelling (> or = 10 mg/kg) on the first day of the study, and bruising in all dose groups throughout the study. Bruising was associated with a dose-dependent prolongation of clotting times, particularly activated partial thromboplastin times (APTT), that was transient in nature. Bruises occurred around site of intravenous dosing or blood collection, and were manifested as subcutaneous hemorrhages upon microscopic evaluation. There were no corresponding alterations in hematology parameters including RBC or platelet counts. Other treatment-related microscopic alterations noted were intracytoplasmic eosinophilic granules and vacuolation in proximal tubular epithelial cells at 10 and 50 mg/kg, with free RBC in renal proximal tubular lumens at 50 mg/kg. Serum chemistry parameters including BUN and creatinine levels were normal in all dose groups and there were no notable alterations in urinalysis parameters. Granules and vacuolations in kidneys were reversed following a 4-week treatment free period. In general, 10 and 50 mg/kg ISIS 2302 produced dose-dependent changes in clotting times and the kidney that were reversible, while 2 mg/kg ISIS 2302 produced no remarkable alterations.

A dietary toxicity/oncogenicity study of tributyl phosphate in the rat.

Tri-n-butyl phosphate (TBP, CAS No. 126-73-8), an industrial chemical, was administered in the diet at concentrations of 0, 200, 700 and 3000 ppm to groups of 50 male and 50 female Sprague-Dawley rats for 2 years. Body weights and food consumption were measured weekly for the first 13 weeks and monthly thereafter. Hematology was performed at 12, 18 and 24 months; urinalyses were performed at 3 weeks and 3, 6, 12 and 18 months. All surviving animals were euthanized after 24 months of treatment. Macroscopic postmortem examinations were performed on all animals; complete histopathological evaluation was performed on control and high dose animals; target organs were examined in all dose groups. Significant decreases in body weight gain occurred in males and females receiving the 3000 ppm concentration and a slight decrease in weight gain occurred in females receiving the 700 ppm concentration. The only clinical sign attributed to TBP was an increased incidence of red discoloration of the urine in some high-dose males. Survival, hematology and urinalysis parameters were unaffected by treatment at any concentration. A dose-related increase in the incidence and severity of urinary bladder hyperplasia and the incidence of urinary bladder papillomas was evident in male and female rats receiving the 700 and 3000 ppm concentrations. Transitional cell carcinomas were present in six of 49 males and two of 50 females and a squamous cell carcinoma was present in one of 49 males in the group which received 3000 ppm. The oncogenic effects showed a clear threshold of 700 ppm in the diet. The NOEL (no observable effect level) for chronic toxicity was 200 ppm. Mean intake of TBP was 9 and 12 mg/kg/day for males and females, respectively, receiving 200 ppm; 33 and 42 mg/kg/day for males and females, respectively, receiving 700 ppm, and 143 and 182 mg/kg/day for males and females, respectively, receiving 3000 ppm. TBP was negative in genotoxicity tests, suggesting that the tumors are induced by nongenotoxic mechanisms.

A dietary oncogenicity study of tributyl phosphate in the CD-1 mouse.

Tri-n-butyl phosphate (TBP), an industrial chemical, was administered in the diet at concentrations of 0, 150, 1000 or 3500 ppm to groups of 50 male and 50 female CD-1 mice for 18 months. Survival, clinical signs and hematology parameters were unaffected by treatment at any concentration. Initial weight losses and significant decreases in body weight gain occurred in males and females receiving the high dose (3500 ppm) of TBP in diet. A significant dose-related increase in absolute and relative liver weights was seen in male and female mice which received the two highest dietary concentrations of TBP (1000 and 3500 ppm). The incidence of hepatocellular adenomas was significantly increased in male mice treated with 3500 ppm TBP in diet. No other tumors were associated with TBP administration in this study. The NOEL for chronic toxicity was 150 ppm, or 28.9 mg/kg/day for females and 24.1 mg/kg/day for males. Although rats treated chronically with TBP have exhibited urinary bladder hyperplasia and urinary bladder papillomas and transitional cell carcinomas, no urinary bladder alterations attributed to TBP administration occurred in this study.

Subchronic oral toxicity of cellulose acetate in rats.

Cellulose acetate was administered by way of a dietary admixture to Sprague-Dawley rats (20/sex/group) at dose levels of 0, 500, 2500 and 5000 mg/kg body weight/day for 94-96 days. Physical observations, body weight and food consumption measurements were made before testing and throughout the study. Ophthalmoscopic examinations were conducted on all animals before testing and just prior to study termination. Haematology, clinical chemistry and urinalysis were performed at 1.5 and 3 months on 10 animals/sex/group. After 3 months of treatment the animals were killed, terminal body weights and organ weights were measured and ratios calculated. Histopathological examination of tissues from the control and high-dose groups was conducted. The evaluation of physical observations, ophthalmology, body weight, food consumption, haematology, clinical chemistry, organ-to-body-weight ratios, gross pathology and histopathology revealed no evidence of an adverse effect related to treatment with cellulose acetate.

[A six-month intraperitoneal repeated dose toxicity study of tazobactam/piperacillin and tazobactam in rats].

Tazobactam (TAZ) is a newly developed beta-lactamase inhibitor. Tazobactam/Piperacillin (TAZ/PIPC) is a formulation consisting of TAZ and PIPC in a ratio of 1:4. A six-month intraperitoneal repeated dose toxicity study of TAZ/PIPC and TAZ including a one-month recovery period were carried out using male and female rats. The doses were 200, 400 and 800 mg/kg/day for TAZ/PIPC, and 40, 80 and 160 mg/kg/day for TAZ. The results were as follows. 1. No test article-related deaths occurred during the study period. No effect on clinical finding of survival rats was evident. 2. There was no dose-related increases of food consumption in both the males and females given TAZ/PIPC and PIPC. Slight reductions in body weight gain occurred in males given 800 mg/kg/day of TAZ/PIPC. 3. Decreases in erythrocyte, hemoglobin and hematocrit, and increases in reticulocytes were seen only at study termination in the group given 800 mg/kg/day of TAZ/PIPC. Increases in reticulocytes were seen only at study termination in the females given 80 or 160 mg/kg/day of TAZ. 4. A decrease in triglyceride levels was observed in the males given 800 mg/kg/day of TAZ/PIPC or 160 mg/kg/day of TAZ. 5. The ophthalmoscopic examination or urinalysis show no test article-related changes. 6. Enlarged ceca in all groups of animals given TAZ/PIPC and in the females given 160 mg/kg/day of TAZ were observed. 7. An increase of relative organ weight in liver was noted in the males and females given 800 mg/kg/day of TAZ/PIPC, in the males given 80 or 160 mg/kg/day of TAZ and in the females given 160 mg/kg/day of TAZ. 8. In the hepatocytes, accumulation of PAS-positive materials which was identified histochemically and ultrastructurally as glycogen, was present in the males given 800 mg/kg/day of TAZ/PIPC and in the males given 80 or 160 mg/kg/day of TAZ. 9. After a one-month recovery period, the changes of liver had generally disappeared, suggesting that they were reversible. 10. From the histopathological changes of liver, the no-toxic dose level in both the males and females was 400 mg/kg/day and 40 mg/kg/day for TAZ/PIPC and TAZ, respectively.

Oxcarbazepine monotherapy in postherpetic neuralgia unresponsive to carbamazepine and gabapentin.

OBJECTIVES: We present the results of a preliminary, open-label trial to evaluate the efficacy and tolerability of oxcarbazepine in postherpetic neuralgia (PHN) unresponsive to treatment with antiepileptic drugs (carbamazepine and gabapentin) and local anesthetic blocks. MATERIALS AND METHODS: Twenty-four patients were treated with oxcarbazepine monotherapy for 8 weeks. Starting dose was 150 mg/day, subsequently increased by 150 mg/day every 2 days until a maintenance dose of 900 mg/day. Pain was assessed using a visual analog scale (VAS). RESULTS: There was a significant decrease in the mean VAS score following 8 weeks of treatment (Delta=5.33; paired t-test: P <0.0001) compared with baseline. Oxcarbazepine was effective from the first week of treatment. There was a significant reduction in allodynia, leading to improvements in patients' functioning and quality of life. Oxcarbazepine was generally well tolerated. CONCLUSION: Oxcarbazepine appears to be a promising alternative monotherapeutic approach for patients affected by PHN.

The sensitizing potential of primary amyl acetate in the guinea pig.

Primary amyl acetate is a liquid mixture of the isomeric forms of pentyl acetate, which is used as a volatile organic solvent. Because of the possibility for skin contact, primary amyl acetate was investigated for its potential to cause allergic contact dermatitis. Using a guinea pig maximization procedure, primary amyl acetate was found to be a possible marginal skin sensitizer.

Chronic toxicity, oncogenic potential, and reproductive toxicity of p-nitroaniline in rats.

Dose levels for these studies were selected mainly on the basis of subchronic studies, although consideration was also given to workplace exposure levels and proposed mechanism of tumor formation with structurally similar compounds. For the chronic study, groups of 60 male and 60 female Sprague-Dawley CD (Registered Trademark of Charles River Breeding Laboratories, Portage, MI) rats were given 0, 0.25, 1.5, or 9.0 mg/kg/day paranitroaniline (PNA) by gavage in corn oil for a period of 2 years. Parameters monitored included clinical observations, ophthalmoscopic exams, body weights, food consumption, hematology, clinical chemistry, and urinalysis at regular intervals throughout the study. All gross lesions and over 40 tissues were examined histologically for all control and high-dosage-level animals. Gross lesions, spleens, and livers of low- and mid-dosage groups were also examined histologically. For the reproduction study, groups of 15 male and 30 female rats, designated as F0 generation, were given PNA at the same levels as the chronic study for 14 weeks prior to mating and during mating, gestation, and lactation. Selected groups of 15 male and 30 female rats of the F1 generation received the same dose of PNA for 18 weeks prior to mating and during mating, gestation, and lactation. F2 pups were observed through weaning at which time they were euthanized. Observations made during the study included body weights, food consumption, mating and fertility indices, pup and litter survival indices, and histopathology of selected tissues. In the chronic study, except for a slight decrease in survival of high-dose male rats late in the study, survival in all treated groups was comparable to controls. Blood methemoglobin levels were elevated in the mid- and high-dosage groups, while slight anemia was observed in the high-dosage group also. Spleen weights were significantly increased in the high-dosage groups. An accumulation of brown pigment was observed in the cytoplasm of the sinusoidal macrophages or littoral cells of the liver and in the reticuloendothelial cells of the spleen. No treatment-related increase in tumor incidence was observed. In the reproduction study, no consistent pattern of effect from treatment between the F0 and F1 generation was seen in mating, pregnancy, or fertility indices. Thus, administration of PNA at levels which produced significant methemoglobinemia and low-level anemia in the rat and histological changes in the spleen produced no tumors or reproducible effects on reproductive performance.

The effects of long-term dietary administration of the rubber accelerator, N-oxydiethylene thiocarbamyl-N-oxydiethylene sulfenamide, to rats.

Continuous administration of diets containing 0, 20, 60, 200, or 600 ppm of N-oxydiethylene thiocarbamyl-N-oxydiethylene sulfenamide to groups of male and female Sprague-Dawley rats (60/sex/group) for over 2 years resulted in oncogenic and toxic effects which generally were limited to the high-dose (600 ppm) group. The most significant findings of this study were in the urinary system of the high-dose males and females. Postmortem examinations revealed a compound-related increase in both benign and malignant urothelial tumors in these animals but not in animals at lower doses. These observations in the high-dose animals paralleled an increase in kidney weights and non-neoplastic urinary tract abnormalities. In both sexes of the high-dose group, body weights were significantly lower and the incidence of rales was significantly higher than those of control animals throughout the course of the study. While some statistically significant changes in hematology, chemical chemistry, and urinalysis values were noted, no compound-related effect on these parameters was evident.

Inhibition of coagulation by a phosphorothioate oligonucleotide.

In the development of antisense therapeutics, there have been a number of hybridization-independent effects characterized for phosphorothioate oligodeoxynucleotides. One such effect is the transient prolongation of clotting times following intravenous infusion of high doses. In this study, inhibition of clotting times was characterized by determining the time course of both APTT and plasma oligonucleotide following intravenous infusion of ISIS 2302 in cynomolgus monkeys. Prolongation of APTT was also achieved by addition of ISIS 2302 to citrated blood from untreated monkeys, allowing the investigation of the mechanism of inhibition in vitro. Results from this study clearly indicate that the intrinsic pathway (APTT) was more sensitive to inhibition than the extrinsic pathway (PT). The prolongation of APTT was also shown to be transient and closely correlated with plasma oligonucleotide concentrations. The extent of APTT prolongation can be controlled by minimizing peak plasma oligonucleotide concentrations through lowering the dose or prolonging infusion duration. Direct addition of ISIS 2302 to blood produced quantitatively similar inhibition of clotting times. This effect was similar for a number of different phosphorothioate oligodeoxynucleotides, but oligonucleotides containing phosphodiester linkages and 2'-propoxy linkages were much less inhibitory. Additional in vitro studies indicated that the mechanism of inhibition was independent of that of heparin and possibly involved selective inhibition of the intrinsic pathway as well as the common clotting pathway. Investigation of selective clotting factors indicated that there was no direct inhibition of the enzymatic activity of factor Xa, XIa, or thrombin using chromogenic substrates. However, ISIS 2302 did produce a concentration-dependent increase in clotting time when fibrinogen was used as the substrate for thrombin.

Activation of the alternative pathway of complement by a phosphorothioate oligonucleotide: potential mechanism of action.

Intravenous infusion of high doses of phosphorothioate oligonucleotides in monkeys has been associated with transient alterations in hematologic and hemodynamic parameters, which appear to be secondary to complement activation. ISIS 2302, a phosphorothioate oligonucleotide specific for human intracellular adhesion molecule-1, was used to further characterize complement activation in monkeys. Complement activation occurred selectively through the alternative pathway resulting in increased plasma concentrations of the complement split products Bb, C3a and C5a. Marked fluctuations in circulating neutrophil counts and reductions in cardiac output were closely associated with peak production of anaphylatoxins C3a and C5a. Changing both dose and infusion duration revealed that complement activation is related to plasma levels of oligonucleotide, and that there is a minimum threshold concentration of approximately 50 micrograms/ml of ISIS 2302 that is required to activate complement. Dose regimens in which plasma concentrations do not exceed this threshold do not result in complement activation. Further investigation reveals that plasma concentrations of a key regulatory component of the alternative pathway, Factor H, were also decreased after administration of ISIS 2302. Decreases in Factor H levels are suggestive of a possible mechanism of complement activation. Direct interaction between ISIS 2302 and Factor H was demonstrated in a competition assay, where increasing concentrations of ISIS 2302 eluted Factor H from a heparin-sepharose column. These data demonstrate a clear correlation between plasma oligonucleotide concentrations and complement activation. Interactions between ISIS 2302 and Factor H may lead to activation of the alternative complement pathway.