Barry S. Levine

Difluoromethylornithine in combination with tamoxifen in female rats: 13-week oral toxicity study.

Purpose: Cancer chemoprevention is the use of pharmacologic or natural agents to inhibit the development of cancer. Difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme in the biosynthesis of polyamines. DFMO has demonstrated chemopreventive efficacy in animal models of tumorigenesis. Tamoxifen (TAM) is currently used for treatment of estrogen receptor-positive breast carcinoma and has demonstrated efficacy in chemoprevention of breast cancer in women at high risk for the disease. The administration of tamoxifen with DFMO is being considered for development by the National Cancer Institute as a potential drug regimen for the chemoprevention of breast carcinoma. Methods: The toxicity of DFMO in combination with TAM was evaluated in female rats following 13 weeks of daily administration by gavage. Dose groups were vehicle control, DFMO (1000 mg/kg per day), low TAM (0.25 mg/kg per day), high TAM (2.5 mg/kg per day), low combination (1000 + 0.25) and high combination (1000 + 2.5). Results: No mortalities occurred in the study. Clinical signs of toxicity were limited to dermal lesions consisting of scab formation and abrasions produced by DFMO. Administration of either DFMO or TAM resulted in decreased body weight gains, with coadministration having an additive effect. Serum albumin, total protein, cholesterol and triglyceride levels were decreased in all drug-treated dose groups, although histologic evidence of liver lesions were not seen. TAM resulted in increased numbers of red blood cells, whereas DFMO produced a slightly anemic response. DFMO produced lesions in the small intestine consisting of necrosis of crypt epithelium and crypt microabscess, which were enhanced by TAM coadministration. Administration of TAM resulted in histologic changes in the ovaries, fallopian tube, vagina, cervix and uterus, indicating that inhibition of ovulation and reproductive cycle arrest in the proestrus stage had occurred. Coadministration with DFMO did not affect the changes to the reproductive system induced by TAM. Conclusions: Coadministration of DFMO with tamoxifen did not result in toxicity unique to the combination drug regimen, but rather toxicity resulted from administration of each drug. Under the conditions of the study, the overall toxicity produced by dual administration of DFMO with tamoxifen was additive with respect to the toxicity associated with each agent alone.

Stress response and drug metabolism in mice

A modified stress model was studied to investigate the relationship between the stress response and drug metabolism in mice. Stress was induced in male CD-1 mice by a daily i.p. injection of hypertonic (1.5 M) saline for up to 3 days, whereas control animals received isotonic (0.15 M) saline. Two hours after receiving the saline injection on the first, second, and third day, animals were euthanized, and serum corticosterone (CORT) and liver aminopyrine N-demethylase (AD) and aniline hydroxylase (AH) activities were determined. To detect any effect of osmotic stimulation, a second control group was given 1.5 M saline as drinking water. There was no difference in CORT levels, AD activity, or AH activity between untreated animals and 0.15 M saline treatment. Intra-peritoneal injection of 1.5 M saline markedly increased serum CORT concentrations compared to 0.15 M saline regardless of the duration of the treatment. Injection of 1.5 M saline also decreased both hepatic enzyme activities each time point. Osmotic stimulation alone by hypertonic drinking water had no significant effect on CORT levels, AD activity, or AH activity. In another series of experiments, intact, sham-operated, and adrenalectomized mice were exposed to the stress model. Injected hypertonic saline decreased AD and AH activities in intact and sham-operated animals compared to isotonic saline-treated animals but both enzyme activities were reduced after adrenalectomy regardless of saline treatment used. In conclusion, a suitable model was established to study the interactions between the stress response and the hepatic drug metabolism in mice.

Regular ArticleStress Response and Drug Metabolism in Mice

A modified stress model was studied to investigate the relationship between the stress response and drug metabolism in mice. Stress was induced in male CD-1 mice by a daily i.p. injection of hypertonic (1.5 M) saline for up to 3 days, whereas control animals received isotonic (0.15 M) saline. Two hours after receiving the saline injection on the first, second, and third day, animals were euthanized, and serum corticosterone (CORT) and liver aminopyrine N-demethylase (AD) and aniline hydroxylase (AH) activities were determined. To detect any effect of osmotic stimulation, a second control group was given 1.5 M saline as drinking water. There was no difference in CORT levels, AD activity, or AH activity between untreated animals and 0.15 M saline treatment. Intra-peritoneal injection of 1.5 M saline markedly increased serum CORT concentrations compared to 0.15 M saline regardless of the duration of the treatment. Injection of 1.5 M saline also decreased both hepatic enzyme activities each time point. Osmotic stimulation alone by hypertonic drinking water had no significant effect on CORT levels, AD activity, or AH activity. In another series of experiments, intact, sham-operated, and adrenalectomized mice were exposed to the stress model. Injected hypertonic saline decreased AD and AH activities in intact and sham-operated animals compared to isotonic saline-treated animals but both enzyme activities were reduced after adrenalectomy regardless of saline treatment used. In conclusion, a suitable model was established to study the interactions between the stress response and the hepatic drug metabolism in mice.

Thirteen-week oral toxicity study of difluoromethylornithine in combination with tamoxifen citrate in female dogs

Purpose: Cancer chemoprevention is the use of pharmacologic or natural agents to inhibit the development of cancer. Difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme in the biosynthesis of polyamines. DFMO has demonstrated chemopreventive efficacy in animal models of tumorigenesis. Tamoxifen (TAM), a nonsteroidal antiestrogen, is approved for use in the treatment of estrogen receptor-positive breast carcinoma and has demonstrated efficacy in chemoprevention of breast cancer in women at high risk for the disease. The administration of TAM with DFMO is being considered for development by the National Cancer Institute as a potential drug regimen for the chemoprevention of breast carcinoma. Methods: The toxicity of DFMO in combination with TAM was evaluated in female Beagle dogs following 13 weeks of daily oral administration by capsule. Dose levels in milligrams per kilogram body weight per day were: 0 (vehicle control), 100 DFMO, 0.1 TAM, 1.0 TAM, 0.1 TAM + 100 DFMO and 1.0 TAM + 100 DFMO. Results: No mortalities occurred. Diarrhea was produced by TAM and vaginal discharge, due to reproductive tract lesions, was produced by both DFMO and TAM, either alone or in combination. DFMO decreased reticulocyte counts and TAM increased counts of mature neutrophils. DFMO alone resulted in lesions to the intestines and ovaries, and cornified epithelium of vagina and cervix. TAM produced cornified epithelium of vagina and cervix, and numerous lesions in the ovaries, fallopian tube, uterus, cervix and vagina which were likely due to an estrogen agonist effect. Coadministration of DFMO increased the incidence and/or severity of these reproductive tract lesions. Each compound alone produced ovarian atrophy, and antral follicles and corpora lutea were completely absent in the 1.0 TAM + 100 DFMO group. Conclusions: Coadministration of DFMO and TAM resulted in additive toxicity involving the female reproductive system.

Oral Antiplatelet Efficacy of the Platelet GPIIb/IIIa Antagonist, DMP754 in Non-Human Primates

Binding kinetic studies with XV459, the active form of DMP754, demonstrated comparable binding kinetics (Kd and Koff) with platelets obtained from either human or baboons which were different from that with platelets obtained from dogs. Therefore, the present study was undertaken to evaluate the antiplatelet efficacy of DMP754 following oral administration in baboons. The dose levels evaluated were 0.1 and 1.0 mg/kg, IV and 0.1, 0.3, 1.0, and 3.0 mg/kg, oral of DMP754. Oral doses of DMP754 resulted in dose- and time-related inhibition of platelet aggregation along with a modest effect on bleeding time prolongation. DMP754 at similar oral doses had 24 hours of antiplatelet effects in baboon as compared to 8-12 hours duration of antiplatelet efficacy in dogs. At maximal antiplatelet doses DMP754 demonstrated no significant effects on platelet count, clinical chemistry or hemodynamic profiles in baboons. These data suggest that DMP754 is a potent orally active antiplatelet agent with extended duration after once a day oral administration in non-human primate.

Determination of phenethylamine, a phenethyl isothiocyanate marker, in dog plasma using solid-phase extraction and gas chromatography-mass spectrometry with chemical ionization

Phenethyl isothiocyanate is unstable in aqueous media and at low pH, and rapidly degrades to phenethylamine. Concentrations of phenethylamine, a phenethyl isothiocyanate marker, in dog plasma, were determined utilizing solid-phase extraction and gas chromatography-mass spectrometry with chemical ionization using acetone as the reagent gas. Deuterated d5-amphetamine was used as an internal standard. After extraction, phenethylamine and d5-amphetamine were derivatized using MBHFBA. Ions monitored for d5-amphetamine were m/z 337 and 338; and for phenethylamine were m/z 318 and 319. Precision and accuracy were studied using control solutions prepared in naive dog plasma (80 and 300 ng/ml). Intra-day variability was determined using six replicates of each control solution analyzed on a single day. The relative standard deviation for the 80 ng/ml control was 12.9% and for the 300 ng/ml it was 12.1%. Relative accuracy was 10.9% for the low control and -4.1% for the high control. Inter-day variability was determined over a 6-day period. For the 80 and 300 ng/ml control solutions, the relative standard deviations were 15.8 and 9.1%, respectively, and relative accuracy values were 10.1 and -5.2%, respectively. Standard curves were prepared in naive dog plasma and were linear over the range of phenethylamine assayed (10-500 ng/ml). The results of this study indicate that the proposed method is simple, precise, accurate and sensitive enough for analysis of large numbers of plasma samples.

Determination of ampicillin in New Zealand white rabbit plasma using column switching technique and HPLC

SummaryThe purpose of this study was to develop a simple and fast analytical method for quantitation of ampicillin in rabbit plasma, suitable for analysis of large numbers of samples collected from experimental animals. The concentration of ampicillin in rabbit plasma was determined utilizing ion-pair reverse-phase high performance liquid chromatography (HPLC) with UV detection and a column switching technique. Plasma samples were treated with a perchloric acid solution to precipitate proteins and centrifuged to pellet the precipitated proteins. Cephalexin was used as an internal standard. The C18 precolumn was placed in the injector loop of the Rheodyne injector. Samples were injected with the injector in the load position and the precolumn was washed free from interfering compounds. When the injector was switched to the inject position, the mobile phase was passed through the precolumn taking relatively pure compounds onto the analytical column. The limit of quantitation was established to be 400 ng mL−1 of plasma. The standard curves were linear over the range of ampicillin concentrations assayed, 400 to 10,000 ng mL−1 of rabbit plasma, and had a mean regression coefficient of 0.9962 (±0.0043). Intra-day variability was determined using six replicates of controls (low and high) analyzed on a single assay. The percent of relative accuracy for low and high controls were 5.67 and 1.12, respectively. Inter-day variability was determined over a four day period analyzing replicates of each control. The percent of relative accuracy for low and high controls were 4.33 and 1.63, respectively.

Oral Toxicity of 1,2-Dithiole-3-Thione, a Potential Cancer Chemopreventive Agent, in the Rat

This study examined the toxicity of 1,2-dithiole-3-thione (D3T) in rats following 14 days of daily oral (gavage) administration. D3T, an extensive inducer of hepatic phase II drug-metabolizing enzymes, has demonstrated cancer preventive efficacy in rodent models of tumorigenesis and is a candidate drug for cancer prevention. Male and female CD rats (5/sex/dose group) received D3T at dose levels of 0 (corn oil vehicle control), 2, 6, 20, and 60 mg/kg/day. Oral administration of D3T for 14 days at 20 mg/kg/day resulted in decreased activity, lethargy, rough coat, and piloerection, and toxicologically significant lesions in the stomach, characterized by apoptotic necrosis and hyperplasia of the glandular mucosa. Administration of D3T at 60 mg/kg/day produced anemia in females, decreased body weight gain in males, and increased vacuolation of adrenal cortical cells. Increased liver weights, vacuolation of hepatocytes, and serum chemistry changes, indicative of altered liver function, were observed at 6 mg/kg/day, which were likely due to a pharmacologic effect of D3T on the liver and not considered to be toxicologically significant. Under the conditions of the study, the no-observed-adverse effect level (NOAEL) was 6 mg/kg/day.

High Performance Liquid Chromatographic Determination of Fumaric Acid in Rat Plasma, Urine, and Fecal Samples

Abstract Fumaric acid, a Krebs cycle intermediate, is a potential cancer chemoprevention agent. A high performance liquid chromatographic procedure with UV detection for determination of fumaric acid in large numbers of rat plasma, urine and fecal samples was developed. Fumaric acid was extracted from plasma, urine, and fecal samples utilizing solid phase extraction using Clean Up® Quaternary Amine 1 mL (plasma, fecal samples) and 3 mL (urine) extraction columns followed by reverse phase high performance liquid chromatography with UV detection at 215 nm. Standard curves for plasma (1 μg/mL - 200 μg/mL), urine (5 μg/mL - 200 μg/mL), and fecal material (25 μg/g - 500 μg/g) were analyzed and replicate analysis of controls were used to determine intra-day and inter-day variability. Chlorofumaric acid was used as an internal standard for plasma and fecal samples; trans-glutaconic acid for urine samples. Precision and accuracy were studied using control solutions (low and high) prepared in naive rat plasma, uri...

Genetic toxicology assessment of HI-6 dichloride.

The oxime HI‐6 dichloride [1‐(2 hydroxyiminomethyl‐1‐pyridino)‐3‐(4‐carbamoyl‐1‐pyridino)‐2‐oxapropane dichloride monohydrate] has shown to be a potent reactivator of cholinesterase activity and may have efficacy for the treatment of organo‐phosphate intoxication [SIPRI, 1976; Schenk et al.; Arch Toxicol 36:71–81, 1976]. As part of a preclinical safety assessment program, the genetic toxicology of HI‐6 dichloride was evaluated in a series of assays designed to measure induction of gene mutations and chromosomal aberrations. HI‐6 dichloride gave negative responses in the Salmonella mutagenicity assay and in the CHO/HGPRT gene mutation assay. Dose‐dependent increases in the frequency of chromosomal aberrations were noted when HI‐6 dichloride was tested in cultured CHO cells and in cultured human peripheral blood lymphocytes. The mouse lymphoma gene mutation assay, reputed to measure both gene mutations and chromosomal deletions, was negative in the absence of metabolic activation. Depending on the criteria employed, a negative or equivocal response was seen in the presence of rat liver‐derived S‐9 mix. An in vivo rat bone marrow metaphase assay performed to further investigate the in vitro clastogenic responses was negative. The results from these studies indicate that HI‐6 dichloride does not induce gene mutations in vitro; however, it is clastogenic in vitro but does not appear to be clastogenic in vivo.