Assessment of drug-drug interactions between daclatasvir and methadone or buprenorphine-naloxone.

Hepatitis C virus (HCV) infection is common among people who inject drugs, including those managed with maintenance opioids. Pharmacokinetic interactions between opioids and emerging oral HCV antivirals merit evaluation. Daclatasvir is a potent pangenotypic inhibitor of the HCV NS5A replication complex recently approved for HCV treatment in Europe and Japan in combination with other antivirals. The effect of steady-state daclatasvir (60 mg daily) on stable plasma exposure to oral opioids was assessed in non-HCV-infected subjects receiving methadone (40 to 120 mg; n = 14) or buprenorphine plus naloxone (8 to 24 mg plus 2 to 6 mg; n = 11). No relevant interaction was inferred if the 90% confidence interval (CI) of the geometric mean ratio (GMR) of opioid area under the plasma concentration-time curve over the dosing interval (AUCτ) or maximum concentration in plasma (C max) with versus without daclatasvir was within literature-derived ranges of 0.7 to 1.43 (R- and S-methadone) or 0.5 to 2.0 (buprenorphine and norbuprenorphine). Dose-normalized AUCτ for R-methadone (GMR, 1.08; 90% CI, 0.94 to 1.24), S-methadone (1.13; 0.99 to 1.30), and buprenorphine (GMR, 1.37; 90% CI, 1.24 to 1.52) were within the no-effect range. The norbuprenorphine AUCτ was slightly elevated in the primary analysis (GMR, 1.62; 90% CI, 1.30 to 2.02) but within the no-effect range in a supplementary analysis of all evaluable subjects. Dose-normalized C max for both methadone enantiomers, buprenorphine and norbuprenorphine, were within the no-effect range. Standardized assessments of opioid pharmacodynamics were unchanged throughout daclatasvir administration with methadone or buprenorphine. Daclatasvir pharmacokinetics were similar to historical data. Coadministration of daclatasvir and opioids was generally well tolerated. In conclusion, these data suggest that daclatasvir can be administered with buprenorphine or methadone without dose adjustments.

Active case finding for tuberculosis among people who inject drugs on methadone treatment in Dar es Salaam, Tanzania.

SETTING: Active case finding is a World Health Organization (WHO) endorsed strategy for improving tuberculosis (TB) case detection. Despite WHO recommendations for active case finding among people who inject drugs (PWID), few studies have been published. The historical focus of case finding has been in populations that are human immunodeficiency virus-positive, incarcerated or at higher occupational risk. OBJECTIVE: We sought to examine the yield of active case finding among PWID newly started on methadone in Tanzania. DESIGN: Of 222 methadone clients, 156 (70%) met with study administrators; 150 consented to participate, 139 (93%) of whom were male. The median age was 34 years. A symptom-based questionnaire was developed by the investigators and administered to every consenting patient by a native Swahili speaker. RESULTS: Of the 150 patients surveyed, 16 (11%) had one or more TB symptoms and were referred for laboratory testing. Six new TB cases were identified in this active case finding program, with a prevalence of 4%. CONCLUSION: This study presents the first data on TB prevalence in a population of PWID in Tanzania. This prevalence is 23 times that of the general Tanzanian TB prevalence of 0.2%. These results have significant implications for TB control.

Genetic variation in OPRD1 and the response to treatment for opioid dependence with buprenorphine in European-American females.

Two commonly prescribed treatments for opioid addiction are methadone and buprenorphine. Although these drugs show some efficacy in treating opioid dependence, treatment response varies among individuals. It is likely that genetic factors have a role in determining treatment outcome. This study analyses the pharmacogenetic association of six polymorphisms in OPRD1, the gene encoding the delta-opioid receptor, on treatment outcome in 582 opioid addicted European Americans randomized to either methadone or buprenorphine/naloxone (Suboxone) over the course of a 24-week open-label clinical trial. Treatment outcome was assessed as the number of missed or opioid-positive urine drug screens over the 24 weeks. In the total sample, no single-nucleotide polymorphisms (SNPs) in OPRD1 were significantly associated with treatment outcome in either treatment arm. However, sex-specific analyses revealed two intronic SNPs (rs581111 and rs529520) that predicted treatment outcome in females treated with buprenorphine. Females with the AA or AG genotypes at rs581111 had significantly worse outcomes than those with the GG genotype when treated with buprenorphine (P=0.03, relative risk (RR)=1.67, 95% confidence interval (CI) 1.06-2.1). For rs529520, females with the AA genotype had a significantly worse outcome than those with the CC genotype when (P=0.006, RR=2.15, 95% CI 1.3-2.29). No significant associations were detected in males. These findings suggest that rs581111 and rs52920 may be useful when considering treatment options for female opioid addicts, however, confirmation in an independent sample is warranted.

Determination of the intra- and interlaboratory reproducibility of the low volume eye test and its statistical relationship to the Draize eye test.

The reproducibility of toxicologic test methods, including alternative tests, is a key scientific and regulatory concern. In the present work, historic rabbit eye irritation data were used to determine the intra- and interlaboratory reproducibility of the low volume eye test (LVET). The standard Draize eye irritation test was used as the basis for comparison. The LVET and Draize tests had similar degrees of intra- and interlaboratory reproducibility as determined by examination of their coefficients of variation, although the variability in LVET results was directionally lower. Results from 70 parallel Draize and LVET tests indicated a strong positive association between results from the two tests, for corneal, iridial, conjunctival, and maximum average scores (MAS). Correlation coefficients were 0.60, 0.73, 0.69, and 0.73, respectively (P

Comparison of the up-and-down, conventional LD50, and fixed-dose acute toxicity procedures.

The up-and-down procedure (UDP), fixed-dose procedure (FDP) and conventional LD50 tests were compared to determine their consistency in chemical hazard classification for acute oral toxicity according to the European Economic Community (EEC) system. There was consistent classification for 23 out of 25 cases between the UDP and the conventional LD50 results, in 16 out of 20 cases between the FDP and the conventional LD50, and in seven out of 10 cases between the UDP and the FDP. The UDP needed only between six and 10 animals of one sex (fewer than either the LD50 or the FDP). Available literature indicates that the sexes are usually similar in their acute toxicity responses and that of females are often more sensitive than males when acute toxicity differences do exist, thus obviating the need for both sexes to be tested in most cases. Unlike the FDP, the UDP also estimates an LD50, thus providing data directly applicable to all current hazard classification systems based on acute oral toxicity.

Respiratory and immunological responses of guinea pigs to enzyme-containing detergents: a comparison of intratracheal and inhalation modes of exposure.

Guinea pigs were exposed once a week for 10 weeks by intratracheal exposure to solutions of 3, 1, 0.3, or 0.1 micrograms of the enzyme protein, Subtilisn Carlsberg (Alcalase), in 250 micrograms of a detergent base. Other groups of guinea pigs were exposed by inhalation (6 hr per day, 4 days a week) to 1 mg/m3 of the aerosolized detergent base containing either 3.5, 1.1, 0.3, or 0.1% Alcalase protein. Evaluations of gross respiratory responses immediately following each intratracheal exposure revealed a significant dose response in respiratory symptoms measurable after the fourth exposure and continuing throughout the study. In the inhalation experiment, during Weeks 4 through 10, animals were observed to have respiratory symptoms which were dependent upon both the dose of enzyme and on total exposure to the enzyme/detergent atmosphere. For both intratracheal and inhalation routes of exposure, the initial appearance of respiratory symptoms coincided with the first appearance of measurable serum allergic antibodies specific to Alcalase. The allergic antibody levels increased with time and dosage by both routes of exposure, and the antibody titers generated by the intratracheal administration of antigen were comparable to those generated by the inhalation route of exposure. These results indicate that the intratracheal technique is appropriate for the evaluation of the respiratory allergic response to a protein.

Reducing the number of rabbits in the low-volume eye test.

Although the Draize eye irritation test has provided important and useful information for eye safety assessments, considerable effort has been directed toward refining the assay procedure, reducing the number of animals used, and replacing this assay with alternative methods. The low-volume eye test (LVET) is a refinement of the Draize eye irritation test that uses 1/10 the volume of test substance placed directly on the cornea. The level and duration of eye irritation in the LVET are less than those in the Draize procedure, which means that it is a less stressful test. Furthermore, LVETs are more predictive of human response. Statistical studies have been conducted to determine the effects of reducing the number of animals used in the Draize test. These results suggested that a three-animal test would provide essentially the same information as the six-animal test. A similar analysis has not been performed on results from the LVET. Accordingly, the present study was undertaken to evaluate previously existing LVET data to determine if the number of animals used in a LVET can be decreased as has been shown for the Draize test. The results of the analysis are consistent with the findings of earlier evaluations of classical Draize data. Three-animal subsets from 119 six-animal LVETs provided the correct classification greater than 92% of the time for three different classification schemes. Furthermore, the discrepancies between the three-animal subsets and the six-animal maximum average score tended to be smaller than those observed for the Draize test.(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous corneal dystrophy and generalized basement membrane changes in Fischer-344 rats.

Groups of young, sexually mature Fischer-344 rats (n = 25/sex) obtained from commercial breeders were examined ophthalmologically and histopathologically to determine the prevalence and severity of corneal basement membrane lesions (corneal dystrophy) and basement membrane changes in select nonocular tissues. Results disclosed a high incidence of corneal basement membrane dystrophy in rats of both sexes from all breeders; however, severity levels were significantly increased in rats obtained from one breeder when compared to others. Furthermore, rats that displayed the most advanced corneal lesions also exhibited more severe basement membrane changes in other organs, especially renal tubules and vascular internal laminae. These findings suggest that both ocular and nonocular dystrophic changes may have been linked through common physiologic (or genetic) mechanisms. Animals that displayed basement membrane lesions were not considered to represent compromised biologic test systems.

Chronic toxicity and carcinogenicity studies of olestra in Fischer 344 rats.

Two 2-year feeding studies were carried out in Fischer 344 rats with olestra, a mixture of the hexa-, hepta- and octaesters of sucrose formed with long-chain fatty acids. Olestra was fed at 0, 0.99, 4.76 or 9.09% (w/w) of the diet in the first study, and at 0 or 9.09% (w/w) of the diet in the second. Daily observations, feed consumption and body weights, ophthalmoscopic examinations, organ weights, serum chemistry, haematology, urinalysis and histopathological evaluations revealed no evidence of any adverse effects associated with olestra ingestion. Relative to controls, there was a higher incidence of basophilic liver foci in olestra-fed female rats at 12 months. At 24 months, foci were observed in most animals in all groups but were more numerous in olestra-fed females. The foci were not associated with hepatic tumours, alterations in liver function, or increases in liver weight and therefore not considered to represent a toxic response to olestra. Isolated statistically significant differences in mortality, mononuclear cell leukaemia, and pituitary adenomas were observed but were not considered to be related to olestra ingestion since they were not reproducible across the two studies, generally not dose responsive, not consistent between sexes, and the incidences were within the ranges for historical and contemporary laboratory controls. The results of the two studies show that olestra was not toxic or carcinogenic when fed to rats at up to 9% of the diet for 24 months.

Comparison of the up-and-down method and the fixed-dose procedure for acute oral toxicity testing.

The acute oral toxicity data for 10 compounds, generated by using two alternative methods in rats, the up-and-down method and the fixed-dose procedure, were compared with those obtained from the classical LD50 test. In this evaluation, both alternative methods offered a reduction in animal use, while providing adequate information to rank the compounds according to the EEC classification for acute oral toxicity. In addition to the ranking, these alternative methods also provided useful information on signs of toxicity and gross autopsy findings, although the results varied depending on the method used. Of the three methods, the up-and-down method required the fewest animals. Although the up-and-down method used only females, the LD50 values obtained were in good agreement with those obtained by the classical method, which used both sexes. It is concluded that the up-and-down method and the fixed-dose procedure are acceptable alternative methods to the classical LD50 test, and the choice of method depends on the type of toxicity information required.

Characterization of a method for quantitating food consumption for mutation assays in Drosophila.

Quantitation of food consumption is necessary when determining mutation responses to multiple chemical exposures in the sex-linked recessive lethal assay in Drosophila. One method proposed for quantitating food consumption by Drosophila is to measure the incorporation of 14C-leucine into the flies during the feeding period (Thompson and Reeder: Environmental Mutagenesis 10:357-365, 1987). Three sources of variation in the technique of Thompson and Reeder have been identified and characterized. First, the amount of food consumed by individual flies differed by almost 30% in a 24 hr feeding period. Second, the variability from vial to vial (each containing multiple flies) was around 15%. Finally, the amount of food consumed in identical feeding experiments performed over the course of 1 year varied nearly 2-fold. The use of chemical consumption values in place of exposure levels provided a better means of expressing the combined mutagenic response. In addition, the kinetics of food consumption over a 3 day feeding period for exposures to cyclophosphamide which produce lethality were compared to non-lethal exposures. Extensive characterization of lethality induced by exposures to cyclophosphamide demonstrate that the lethality is most likely due to starvation, not chemical toxicity.

Risk assessment for aflatoxin: II. Implications of human epidemiology data.

A review of epidemiology literature revealed that only studies conducted in Africa and Asia included data adequate to permit quantitative assessment of the dose-response relationship between aflatoxin exposure levels and liver cancer rates. Although these studies were judged adequate, their direct use to predict risks in U.S. populations may be questioned since hepatitis B virus (HBV) infections are far more common in the studied areas than in the U.S. Recent research indicates that, if aflatoxin contributes to the development of liver cancer, it almost always does so in the presence of HBV infection. The African/Asian data do not permit us to estimate the potency of aflatoxin in the absence of HBV. Recognizing this, these data can only be used to establish upper limits for the predicted excess lifetime risk for liver cancer in the U.S. When used in conjunction with aflatoxin exposure estimates for the Southeast U.S., these data predict a liver cancer rate, due to aflatoxin alone, far above that actually observed due to all causes; this provides an indication of the conservatism of this approach. Data from the Southeast U.S. may be used to estimate an excess lifetime risk for liver cancer of 2.17 x 10(-6) x (aflatoxin intake, ng/kg/day).

Morphometric assessment of thymic size variation in laboratory rabbits.

Sections of thymus from New Zealand white rabbits used as controls in 28-day and 91-day percutaneous toxicity studies conducted at different laboratories were morphometrically assessed. Measurements of total thymic area, medullary area, and cortical area were greater for 28-day vs 91-day studies conducted at a given laboratory, but varied from one laboratory to another. Relative thymic measurements (percent medulla, percent cortex, and medulla:cortex ratio) were similar for studies conducted at each laboratory and from one laboratory to another regardless of study duration. A decrease in thymic size occurred between approximately 16 and 25 weeks of age (i.e., 28-day studies vs 91-day studies) due to proportionally equivalent decreases in both the cortical and medullary areas. The consistency of the relative measurements can be used to assist in distinguishing changes in thymic size due to aging from changes in size due to stress or toxicity which would be expected to differentially affect the cortical and medullary areas. Appreciation of the normal variation in thymic size is needed when evaluating results of toxicity testing in rabbits. Data are provided as to the degree of normal variation of rabbit thymic size expected within and across percutaneous toxicity studies, with considerations for interpreting such data.

Hepatocellular vacuolization in rabbits: effects of feed restriction, orchiectomy and ovariectomy.

Previous studies have reported that striking variations in hepatocellular vacuolization occurs in rabbits and the magnitude of vacuolization correlated independently with weight and sex. The current study evaluated the effects of feed restriction and gonadectomy on this hepatocellular vacuolization. For 28 days, rabbits were fed either ad libitum (ad lib group), 100% of the National Research Council's recommended feed intake required for growth (100% group), or 50% of the NRC recommended feed intake required for growth (50% group). Feed consumption, weight gain, final body weight, absolute liver weight, and relative liver weight were not significantly different between the ad lib and 100% groups. Values for these parameters for both groups were significantly greater than for the 50% group. Rabbits in the 50% group had significantly less hepatocellular vacuolization than rabbits in the 100% group. Hepatocellular vacuolization in the 100% group did not differ from rabbits fed ad libitum. Hepatocellular vacuolization in the ad lib group was greater than in the 50% group but this difference was not significant. Ovariectomy and orchiectomy did not significantly alter hepatocellular vacuolization in either female or male rabbits, respectively, that were fed ad libitum for 28 days. However, intact females had significantly greater hepatocellular vacuolization than either intact or orchiectomized males. Conversely, hepatocellular vacuolization in ovariectomized females was not significantly different from that in intact and orchiectomized males. There were no significant differences in feed consumption, weight gain, final body weight, and absolute and relative liver weights among these intact and gonadectomized groups. Results of these studies indicate feed consumption can affect the degree of hepatocellular vacuolization in rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethylenediaminetetra(methylenephosphonic acid): genotoxicity, biodistribution, and subchronic and chronic toxicity in rats.

Ethylenediaminetetra(methylenephosphonic acid), EDITEMPA, was tested for oral toxicity in rats in a 13-wk feeding study (at doses of 0, 5, 50 and 500 mg/kg/day) and in a chronic feeding study (at doses of 0, 4, 20 and 100 mg/kg/day). EDITEMPA was also tested for genotoxicity in the Ames, mouse lymphoma, unscheduled DNA synthesis, and in vivo cytogenetics assays. Additionally, absorption, distribution and excretion (ADE) studies were conducted following administration of [14C]EDITEMPA to rats by gavage and via the feed and drinking-water. The principal finding in the 13-wk study was mild anaemia in male and female rats given 500 mg/kg/day, which was resolved during a 9-wk recovery period. In the chronic study, there was no substantial evidence of any treatment-related toxicity or carcinogenicity. Differences in survival of control and treated females (noted late in the study) were interpreted to represent unusually good survival in controls; however, a compound-related increase in mortality could not be completely ruled out. Tests for genotoxicity were all negative. ADE studies revealed that [14C]EDITEMPA was poorly absorbed from the gastro-intestinal tract and that most of the absorbed dose was rapidly excreted by the kidneys or sequestered in bone. The gavage route of administration led to four- to six-fold increases in bone EDITEMPA levels as compared with administration in the feed and drinking-water, respectively. These results suggest that no significant toxicity or carcinogenicity concerns arise from EDITEMPA when it is administered in the feed at the concentrations tested. Reversible anaemia was seen only at very high doses and was interpreted as being secondary to EDITEMPA's ability to interfere with iron absorption and utilization. Localization of EDITEMPA in bone indicated a high degree of affinity for mineralizing tissues, consistent with its chelating properties. There was, however, no effect on bone resorption or mineralization. A comparison of human drinking-water levels of 3500 ppm EDITEMPA (based on a no-effect level of 100 mg/kg/day in rats) with the estimated worst-case exposure in humans of 0.01 ppm suggested a safety margin greater than 1 x 10(5).

Variable hepatocellular vacuolization associated with glycogen in rabbits.

Marked variation in hepatocellular vacuolization was present in New Zealand white rabbits used as controls in 28-day and 91-day percutaneous studies conducted at 5 different laboratories. Vacuoles in hepatocytes of alcohol-fixed and formalin-fixed livers contained periodic acid-Schiff (PAS)-positive material which was removed with diastase digestion, indicating the presence of glycogen. The magnitude of hepatocyte vacuolization was subjectively assessed by light microscopy using 5 histologic grades. Quantitative measurements of hepatocyte perimeter and lobule radius for representative liver sections of each histologic grade corroborated the different grades used. Factors associated significantly with the degree of vacuolization were sex (females were affected more severely than males), body weight, relative liver weight, and the laboratory conducting the study. Also apparent were variations in mean severity of hepatocyte vacuolization between studies conducted at the same laboratory, and variation in severity of vacuolization within individual studies. Duration of the study and season had no significant association with the degree of vacuolization. Marked variation of hepatocellular vacuolization due to glycogen accumulation must be recognized when evaluating results of toxicity testing in rabbits.

Effects of adenosine and xanthine derivatives on breathing during acute hypoxia in the anesthetized newborn piglet.

Neonates of animals and humans exhibit a paradoxical ventilatory response to hypoxia characterized by an initial increase in minute ventilation followed by a late, sustained decrease. Exogenous adenosine analogues cause respiratory depression, and the xanthine derivative aminophylline, a competitive inhibitor of adenosine receptors, decreases the amount of hypoxic ventilatory depression in the newborn piglet. Other xanthine derivative such as enprofylline are weak adenosine antagonists. The purpose of this report is to test the hypothesis that enprofylline would not reverse ventilatory depression caused by hypoxia, supporting the suggestion that adenosine contributes to hypoxic ventilatory depression. To confirm the weak adenosine antagonism of enprofylline, L-N6-(phenylisopropyl)adenosine (PIA) was administered to six newborn piglets until respiratory depression was achieved. Either aminophylline or enprofylline was then administered. Aminophylline, but not enprofylline, reversed the respiratory depression caused by PIA. In seven additional piglets, respiratory depression was first produced by 10% oxygen breathing and the ability of saline, aminophylline, and enprofylline to reverse the decrease in ventilation was evaluated. The administration of either saline or enprofylline produced little change in minute ventilation (9.8% +/- 3.7% and -11.7% +/- 7.7%, respectively), whereas aminophylline consistently produced an increase (43.5% +/- 7.3% [P less than 0.001]). Both aminophylline and enprofylline increased heart rate (P less than 0.01), whereas saline produced no significant change. Blood pressure was increased by enprofylline but not by aminophylline or saline. These findings suggest that, in the anesthetized newborn piglet, adenosine contributes to ventilatory depression caused by hypoxia.

A confirmatory study of the up-and-down method for acute oral toxicity testing.

Ten materials have been tested in parallel both by the "classical" method for acute oral toxicity (LD50) and by the up-and-down method. Materials tested included laundry and dishwashing detergents, a shampoo, a flavor, potassium hydroxide, and caffeine. All testing was done in Sprague-Dawley rats. Excellent agreement was seen between the two methods. The classical method typically used 40 to 50 animals while the up-and-down method required only six to nine animals per material.

Human and rabbit eye responses to chemical insult.

Groups of eight human volunteers and eight albino rabbits, under controlled laboratory conditions, were exposed in one eye without subsequent rinsing to the same concentrations and volumes of four prototype consumer products: fabric softener, shampoo, hand soap, and laundry detergent. Dose volume was 0.10 or 0.01 ml. The dose concentrations were selected to produce moderate effects with recovery within 24 to 48 hr. Two irritation scales were employed with both human and animal subjects: the Draize scale by a technician and a medical scale used with slit lamp examination by an ophthalmologist. Eyes were examined by both graders before and after dosing at specified intervals until recovery. Mean and maximum irritation scores are presented for each grading time, method, and exposure, as are the mean hours to recovery (clearing) for each exposure. Recovery times for human eyes were consistent with those reported previously for accidental human exposures to similar materials. Correlation coefficients for time to clear, comparing human vs rabbit for each dose volume-species combination across the four test products, were 0.72, 0.1 ml-human vs 0.01 ml-rabbit; 0.66, 0.01 ml-human vs 0.01 ml-rabbit; 0.40, 0.01 ml-human vs 0.1 ml-rabbit; 0.35, 0.1 ml-human vs 0.1 ml-rabbit. Thus, recovery time obtained under conditions of the "Low-Volume" test (0.01 ml-rabbit) better correlates with human eye recovery time (either dose volume) than does recovery time under Draize test conditions (0.10 ml-rabbit).(ABSTRACT TRUNCATED AT 250 WORDS)