MNT and Muta™Mouse studies to define the in vivo dose response relations of the genotoxicity of EMS and ENU

Although there are a multitude of in vitro and in vivo studies on the genotoxic activity of EMS, no lifetime carcinogenicity studies, repeat dose mutation data or exposure analysis are available to serve as a solid basis for risk assessment for human exposure cases. The present studies were undertaken to investigate whether a threshold for mutagenic and clastogenic activity in vivo could be established, using the bone marrow micronucleus (MNT) and Muta™Mouse test systems, in the hope to provide reassurance to the patients that their accidental exposure to EMS at doses up to 0.055 mg/kg did not carry a toxicological risk. Dose levels ranging from 1.25 to 260 mg/kg/day were applied orally for up to 28 days. As a reference we included ENU at doses of 1.1–22 mg/kg/day. Our studies showed that daily doses of up to 25 mg/kg/day (bone marrow, GI tract) and 50 mg/kg/day (liver) did not induce mutations in the lacZ gene in the three organs tested. Doses up to 80 mg/kg/day (7-day dosing regime) did not induce micronuclei in mouse bone marrow. The genotoxic activity of EMS became apparent only at higher dose levels. Dose fractionation of EMS (28 times 12.5 mg/kg versus a single high dose 350 mg/kg) provided further evidence for the thresholded dose response of EMS and showed that no cumulation of gene mutations below a threshold was occurring. In contrast, for ENU no threshold was apparent and dose fractionation indicated full additivity of individual dose effects.     

ESR Dosimetry of Irradiated Ascorbic Acid

Purpose. As an alternative to heat and gas exposure sterilization, ionizing radiation is gaining interest as a sterilization process for medicinal products. The aim of this work was to develop equations to describe the ESR curves versus dose and storage time after gamma irradiation of ascorbic acid. Several ESR data sets previously acquired in this laboratory were adopted to check the performance of the models. Results. Limit of detection and limit of discrimination are respectively 0.5 kGy and 2 kGy for ascorbic acid. Linear regression is applicable for doses lower than 25 kGy. Since the radiation dose selected must always be based upon the bioburden of the products and the Degree of Sterility required (ANSI/AAMI/ISO 11137), doses in the range 5–25 kGy could be investigated and linear regression would appear to be the least expensive route to follow and gives good results. Quadratic fit, power function, exponential function and bi-exponential functions are of more general applicability to predict irradiation dose. Decay kinetics for radicals versus storage were considered. Nonhomogeneous kinetics with time-dependent rate (diffusion-controlled second-order reaction) and bi-exponential function appeared valid to reproduce the experimental data. Discrimination between irradiated and unirradiated ascorbic acid is possible after a storage of 800 days. Conclusions. It is worth noting that, at present, ESR is the only technique which proves to be suitable for identification and quantification purposes in irradiated pharmaceuticals. Moreover, other features such as sensitivity, precision, ease and non-destructive readout make ESR superior to other proposed analytical techniques.     

Impact of dose selection on parameter estimation using a rapid binding approximation model of target-mediated drug disposition

The purpose of this study was to examine the role of dose selection on population pharmacokinetic (PK) parameter estimation using a rapid binding approximation of a target-mediated drug disposition (TMDD) model previously developed for interferon-β (IFN-β). A total of 50 replicate datasets each containing 100 subjects were created using NONMEM^®. The study design included IV injection of IFN-β followed by the SC route in a crossover manner, with each dose and route of administration separated by a 1,000 h washout period. Serial plasma PK samples were simulated up to 48 h for all subjects following each dose. Population mean PK parameters were re-estimated in NONMEM^® for each simulated dataset using the same TMDD model after including the following doses (MIU/kg): (A) 1, 3 and 10 (original study); (B) 1, 3 and 7; (C) 1, 3 and 5; (D) 1, 3 and 4; (E) 1 and 3; (F) 3 and 10; or (G) 10 MIU/kg only. Bias in the model fit was assessed by calculating the percent prediction error (PE%) for each of the population mean PK parameters relative to the estimates obtained from the fit to the 1, 3, and 10 MIU/kg doses (Case A). Relatively unbiased population mean PK parameter estimates (median PE% <8%) were obtained only when the study design included 1, 3 and a minimum higher dose of 7 MIU/kg. Bias increased for various parameters when the highest dose was less than 7 MIU/kg along with 1 and 3 MIU/kg being the low and intermediate dose levels. An increase in the bias for binding capacity, R_tot, and the equilibrium dissociation constant, K _D, was observed as the highest dose included in the dataset was reduced from 5 to 3 MIU/kg (median PE% ranged from ?4.71 to ?23.9% and ?4.76 to ?34.6%). Similar increases in the range of median PE% were also observed for other model parameters as the highest dose was reduced from 5 to 3 MIU/kg. Severely biased results were obtained from the study design that included only the 10 MIU/kg dose (Case G) suggesting that it is not sufficient to study just a high dose group. This bias was greatly reduced (median PE% <14%) for all parameters except K _D when the 3 and 10 MIU/kg doses were co-modeled (Case F). Plots of the PE% for R_tot and K _D versus the molar ratio of maximum dose to R_tot suggest that study designs should evaluate at least one IFN-β dose 3.5- to 4-fold higher than R_tot along with the 1 and 3 MIU/kg dose levels to obtain unbiased population PK parameter estimates. In summary, for the IFN-β model and study design, dose selection influences the ability to generate relatively unbiased population mean TMDD parameter estimates, which is based on maximum dose levels relative to R_tot. This simulation study highlights the role of dose selection in optimal study design strategies for drugs such as IFN-β that exhibit TMDD properties.     

Toxicokinetics of tabun enantiomers in anaesthetized swine after intravenous tabun administration

In the present study, we report the first in vivo toxicokinetic study of tabun (O-ethyl-N,N-dimethylphosphoramidocyanidate). The toxicokinetics of the enantiomers of tabun were investigated in anesthetized swine after intravenous administration of 3 × LD₅₀ (161.4 μg/kg) tabun. Blood samples were taken for gas chromatographic–mass spectrometric determination of the tabun enantiomers and for measurement of the activity of red blood cell acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BChE). The tabun enantiomers could be quantified in swine blood to a minimum concentration of 3.0 pg/ml (18.5 pM) and could be detected to a minimum concentration of 1.0 pg/ml (6.2 pM). The concentration–time profiles of both tabun enantiomers were best described by a bi-exponential equation. The elimination of (+)-tabun and (−)-tabun were comparable in the initial phase. In the terminal phase a remarkable difference was found, with terminal half lives of 11.5 min for (+)-tabun and 23.1 min for (−)-tabun. (+)-Tabun showed a markedly longer persistence in vivo than (+)-enantiomers of other G-type nerve agents and could be detected in all swine at least up to 30 min post-injection, (−)-tabun at least up to 90 min post-injection. These results demonstrate a rather rapid elimination of tabun enantiomers in vivo and may provide a toxicokinetic basis for the further development and optimization of medical countermeasures against this nerve agent.     

Clinical implications of dose-dependent cytochrome P-450 drug–drug interactions with antidepressants

The magnitude of drug–drug interactions in vitro involving competitive inhibition of cytochrome (CYP) isozymes by newer antidepressants can theoretically be shown to be dependent upon several factors. These include the concentration of both the substrate and inhibitor, the affinity of the inhibitor for the inhibited isozyme, and an inhibition constant. The purpose of this study was to compare the results from three human drug interaction studies with theoretical considerations and the results from in vitro studies. Of special interest was the relationship between dose or concentration of an enzyme inhibitor and the change in the plasma concentration of a co-administered drug. Observed data were fit to equations using linear regression and nonlinear least squares regression analysis. All three human data sets demonstrated a linear dose or concentration dependency in the magnitude of the observed drug–drug interaction. The results draw attention to the dose dependent nature of drug–drug interactions. As the dose of antidepressant is under clinician control, guidelines are suggested to minimize the clinical impact of antidepressant drug interactions. © 1998 John Wiley & Sons, Ltd.     

Pharmacopeial methodologies for determining aerodynamic mass distributions of ultra-high dose inhaler medicines

Three different impactor methodologies, the Andersen cascade impactor (ACI), next-generation impactor (NGI) and multistage-liquid impinger (MSLI) were studied to determine their performance when testing ultra-high dose dry powder formulations. Cumulative doses of spray-dried mannitol (Aridol™) were delivered to each impactor at a flow rate of 60 L min⁻¹ (up to a max dose of 800 mg delivering 20 sequential 40 mg capsules). In general, total drug collected in both the ACI and NGI falls below the range 85–115% of label claim criteria recommended by the United States of America Food and Drug Administration (FDA) at nominal mannitol doses exceeding 20 mg and 200 mg, respectively. In comparison analysis of the MSLI data, over a 5–800 mg cumulative dosing range, indicated that the percentage of nominal dose recovered from the MSLI was within the ±15% limits set in this study. Furthermore all samples, apart from the 5 mg and 10 mg analysis were within 5% of the nominal cumulative dose. While the MSLI is not routinely used for regulatory submission, the use of this impinger when studying ultra-high dose formulations should be considered as a complementary and comparative source of aerosol deposition data.     

Dose-related pharmacokinetics of coumarin, 7-hydroxycoumarin and 7-hydroxycoumarin glucuronide upon intraperitoneal administration of coumarin and 7-hydroxycoumarin in the rat

Blood concentration-time data of coumarin (C), 7-hydroxycoumarin (7-HC) and 7-hydroxycoumarin glucuronide (7-HCG) were obtained in rats receiving intraperitoneal doses of C ranging from 2.5 to 60 mg/kg and of 7-HC ranging from 2.5 to 20 mg/kg. Coumarin blood levels were fit to pharmacokinetic models using computer programs including NONLIN, modified ESTRIP, RESID and AUCRPP. The other molecular species were treated pharmacokinetically by linear least squares regression analysis. C blood concentrations were indicated to be dose-dependent at doses greater than 10 mg/kg. Only trace amounts of 7-HC were found upon C administration, however, 7-HCG was found in measurable quantities at all dose levels. The blood concentration-time data of 7-HC and 7-HCG were very erratic with considerable intersubject variation. 7-HC levels upon 7-HC dosing were found to have extremely short half-lives of elimination for all animals tested. Deviation from linearity was apparent at the 20 mg/kg dose for 7-HC. The 7-HCG metabolite upon 7-HC dosing also showed dose-dependent kinetics at the 20 mg/kg dose. 7-HCG was found to appear at variable rates in the blood with peak times ranging from about 2 to 50 min.     

Simultaneous determination of sulphamethoxazole and trimethoprim in powder mixtures by attenuated total reflection-Fourier transform infrared and multivariate calibration

A partial least-squares calibration (PLS) procedure in combination with infrared spectroscopy has been developed for simultaneous determination of sulphamethoxazole (SMZ) and trimethoprim (TMP) in raw material powder mixtures used for manufacturing commercial pharmaceutical products. Multivariate calibration modeling procedures, interval partial least squares (iPLS) and synergy partial least squares (siPLS), were applied to select a spectral range that provided the lowest prediction error in comparison to the full-spectrum model. The experimental matrix was constructed using 49 synthetic samples and 15 commercial samples. The considered concentration ranges were 400–900 mg g⁻¹ SMZ and 80–240 mg g⁻¹ TMP. Spectral data were recorded between 650 and 4000 cm⁻¹ with a 4 cm⁻¹ resolution by Fourier transform infrared spectroscopy coupled with attenuated total reflectance (ATR-FTIR) accessory. The proposed procedure was compared with conventional procedure by high performance liquid chromatography (HPLC) using 15 commercial samples containing SMZ and TMP. The results showed that PLS regression model combined to ATR-FTIR is a relatively simple, rapid and accurate procedure that could be applied to the simultaneous determination of SMZ and TMP in routine quality control of powder mixtures. A root mean square error of prediction (RMSEP) of 13.18 mg g⁻¹ for SMZ and 6.03 mg g⁻¹ for TMP was obtained after selection of better intervals by siPLS. Using the proposed procedure it is possible to analyze each sample in less than 3 min considering two replicates (excluding the grinding step). Accuracy was checked by comparison to HPLC method and agreement better than 98.8% was achieved.     

Ex vivo delta opioid receptor autoradiography: CNS receptor occupancy of two novel compounds over their antihyperalgesic dose range

Discovered as part of an effort to identify delta opioid (DOPr or DOR) agonist analgesics, JNJ-20788560 and JNJ-39204880 exhibited high DOR affinity, with K_i values of 1.7 and 2.0 nM, respectively, and were selective for DOR over the mu opioid receptor (MOPr or MOR), with 596- and 122-fold selectivity, respectively. Both compounds stimulated DOR but not MOR induced GTPγS binding and were effective antihyperalgesic agents in the complete Freund's adjuvant model of thermal hyperalgesia in the rat, with oral ED₅₀ values of 13.5 and 35 mg/kg, corresponding to plasma levels of 1 and 9 µM, respectively. Autoradiographic analysis of DOR and MOR occupancy in sections of brain (striatum) and lumbar spinal cord (L4-L6) was determined ex vivo, using radiolabeled naltrindole or DAMGO. Quantitative image analysis resulted in striatal DOR ED₅₀ values of 6.9 and 10.7 mg/kg, for JNJ-20788560 and JNJ-39204880 respectively, and spinal cord values of 6.4 and 3.2 mg/kg, respectively. Neither compound dose-dependently occupied MOR within the dose range studied. Thus, this study confirmed the DOR selectively over MOR of both compounds following their oral administration, and further demonstrated dose-dependent DOR occupancy by each compound across its antihyperalgesic dose range. Importantly, these in vitro, in vivo, and ex vivo data revealed that the greater in vitro potency of JNJ-20788560 was paralleled by its greater in vivo potency, although JNJ-39204880 achieved higher plasma levels following its oral administration. The receptor occupancy levels observed at the pharmacologic ED₅₀ doses of these compounds suggest the need for greater target engagement by JNJ-39204880 than by JNJ-20788560 to elicit a similar therapeutic response.     

N-acetyl-glutamic acid: Evaluation of acute and 28-day repeated dose oral toxicity and genotoxicity

N-acetyl-glutamic acid (NAG) is an endogenously produced mammalian substance and minor constituent of commonly consumed foods. This paper reports the outcome of genotoxicity and acute and repeated dose (28-day) oral toxicology studies conducted with NAG. No evidence of genotoxicity was observed with NAG in in vitro or in vivo studies. No mortalities or evidence of adverse effects was observed in Sprague–Dawley rats following acute oral gavage with NAG at a dose of 2000 mg/kg of body weight. No adverse effects were observed in rats following repeated dose dietary exposure to NAG at target concentrations corresponding to doses of 100, 500, or 1000 mg/kg of body weight/day for 28 days. All rats survived until scheduled sacrifice and no biologically significant or test substance related differences were observed in body weights, feed consumption, clinical signs, functional observational battery (FOB), ophthalmology, hematology, coagulation, clinical chemistry, organ weights or histopathology of any of the treatment groups. Based on the observed results it is concluded that NAG is not genotoxic or acutely toxic. The no-observed-adverse-effect-level (NOAEL) for systemic toxicity from repeated dose (28-day) dietary exposure to NAG was 914 mg/kg of body weight/day for male rats and 1007 mg/kg of body weight/day for female rats.     

Dose selection for toxicity studies: a protocol for determining the maximum repeatable dose.

1. A three-stage protocol is described for a dose-ranging study which defines the maximum repeatable dose (MRD) and provides a preview of the toxicology of new, pharmacologically active, substances before commencing the first formal regulatory toxicity studies, usually of 2 or 4 weeks duration. 2. Additionally, a range of toxicokinetic (TK) data relevant to protocol design for formal studies is generated. 3. Stage A is a dose incrementation process in which the MRD is provisionally determined and basic TK values generated. 4. In stage B the animals are dosed daily for at least 7 d, the MRD is substantiated and a wider range of TK data obtained. 5. In stage C, each of the dose levels identified for a formal study is administered once to investigate the relationship of doses to TK data. 6. This protocol can be completed using as few as 24 rats or six dogs (or primates). 7. Selection of dose levels for the first formal studies can be greatly aided by the results of a well-designed dose-ranging study including TK data. 8. For particularly toxic substances, the findings of studies based on this protocol have frequently been sufficiently clear to warrant early termination of their development.     

High dose benzodiazepines prolong reaction times in chronic users who have major depressive and/or anxiety disorders

Aim

Short term administration of benzodiazepines (BZD) was found to prolong reaction time (RT) in experimental studies. However, studies on long term BZD use did not always adjust for important confounders and showed inconsistent results. We aimed to identify a possible relationship between long term BZD use and RT in BZD users in this large cross-sectional, observational study.

Methods

The RTs of non-users (n = 2404) were compared with low (n = 288), intermediate (n = 74), and high dose BZD users (n = 57) in the Netherlands Study of Depression and Anxiety (NESDA). RTs were obtained from the Implicit Association Test. Analyses were adjusted for sociodemographic characteristics, health indicators, severity of psychopathology and antidepressant use.

Results

Of the NESDA participants, 419 subjects (14.8%) used BZDs. A higher dose of BZDs was associated with prolonged RTs (P = 0.01). When comparing the different dose groups, the high dose group, but not the low and medium dose groups, had significantly longer RTs than the non-users.

Conclusions

Tolerance for the RT prolonging effect of relatively high doses of BZDs does not seem to develop. As prolonged RTs can have adverse consequences in daily life, BZDs should be prescribed conservatively at the lowest possible dose.     

Single oral dose proportionality pharmacokinetics of almitrine bismesylate in humans

A single-blind study was conducted in 10 healthy male subjects. Each subject was tested with four single oral doses of capsules containing 25, 50, 100, 200mg almitrine bismesylate and one dose of placebo. Blood samples were drawn as a function of time and the concentration of almitrine in plasma was determined by gas chromatography utilizing nitrogen-phosphorus detection. Linear regression analysis of the data suggested that a deviation from linearity existed between the area under the plasma concentration time curves and the dose (R = 0.96). Linear analysis of the individual data indicates that a slight negative deviation from linearity is apparent for the 200 mg dose. The same trend was observed for the mean maximum almitrine plasma concentration, Cmax, which ranged from 38.9 +/- 11.8 to 286.2 +/- 99.1 ng ml-1 for the 25 and 200 mg dose, respectively. The time to peak was relatively constant regardless of the administered dose and ranged from 2.4 +/- 0.5 h to 2.8 +/- 0.8 h. Good agreement was obtained between the observed bioavailability parameters and those predicted from the nonlinear fit of the data. Further kinetic analysis of the data revealed mean total body clearance over fraction of dose absorbed ranging from 268.2 +/- 132.8 to 436.4 +/- 191.4 ml min-1 for doses 50 and 200mg, respectively.     

Developmental toxicity of copaiba tree (Copaifera reticulata Ducke, Fabaceae) oleoresin in rat

The oleoresin of the copaiba tree (Copaifera sp., Fabaceae) is traditionally used in Brazilian herbal medicine to treat a variety of illnesses and symptoms. This study, conducted according to the OECD Guideline 414, provides data on the developmental toxicity of oleoresin from C. reticulata (COPA-R) in rats. Pregnant Wistar rats (25 per dose group) were treated by gavage with COPA-R (0, 500, 1000 and 1250 mg/kg bw/day) on gestation days (GD) 6-19 and Caesarean sections performed on GD20. Implantations, living and dead fetuses and resorptions were recorded. Half of the fetuses from each litter were examined for visceral abnormalities and the remaining were cleared and stained for skeleton evaluation. COPA-R was maternally toxic (reduced food intake and weight gain) and embryotoxic (lower fetal body weight and increased occurrence of fetal skeleton variations) at the two highest doses, but did not cause embryo deaths or fetal malformations at any dose level. The study derived an oral no-observed-adverse-effect-level (NOAEL) for maternal and developmental toxicity induced by COPA-R of 500 mg/kg bw/day. The results suggest that copaiba oleoresin does not pose a health risk to pregnant women when used according to the recommended doses (up to five drops, three times a day).     

Extended least squares nonlinear regression: A possible solution to the “choice of weights” problem in analysis of individual pharmacokinetic data

It is often difficult to specify weights for weighted least squares nonlinear regression analysis of pharmacokinetic data. Improper choice of weights may lead to inaccurate and/or imprecise estimates of pharmacokinetic parameters. Extended least squares nonlinear regression provides a possible solution to this problem by allowing the incorporation of a general parametric variance model. Weighted least squares and extended least squares analyses of data from a simulated pharmacokinetic experiment were compared. Weighted least squares analysis of the simulated data, using commonly used weighting schemes, yielded estimates of pharmacokinetic parameters that were significantly biased, whereas extended least squares estimates were unbiased. Extended least squares estimates were often significantly more precise than were weighted least squares estimates. It is suggested that extended least squares regression should be further investigated for individual pharmacokinetic data analysis.This work was supported in part by USUHS Grant RO-7516 and NIH Grants GM26676 and GM26691.     

Analysis of pharmacokinetic data using parametric models. II. Point estimates of an individual's parameters

This is the second in a series of tutorial articles discussing the analysis of pharmacokinetic data using parametric models. In this article the basic issue is how to estimate the parameters of such models. Primary emphasis is placed on point estimates of the parameters of the structural (pharmacokinetic) model. All the estimation methods discussed are least squares (LS) methods: ordinary least squares, weighted least squares, iteratively reweighted least squares, and extended least squares. The choice of LS method depends on the variance model. Some discussion is also provided of computer methods used to find the LS estimates, identifiability, and robust LS-based estimation methods.Work supported in part by NIH grants GM26676 and GM 26691.     

Metabolism and pharmacokinetics of ethyl N-lauroyl-L-arginate hydrochloride in human volunteers

The human metabolism and pharmacokinetics of ethyl N^α-lauroyl-L-arginate hydrochloride (LAE), a new antimicrobial agent for use in foods have been investigated using both in vitro and in vivo techniques with ¹⁴C-LAE and ¹³C-LAE respectively. LAE was readily hydrolysed to the corresponding lauroyl arginine (LAS) on incubation with human plasma samples to the extent of about 50% during 4 h. LAE was stable in simulated gastric fluid but in simulated intestinal fluid it was rapidly hydrolysed to LAS and arginine with more than 90% conversion to arginine after 1 h. Oral doses of ¹³C-LAE in propylene glycol were administered to human volunteers at dose levels of 1.5 mg/kg (4 subjects) and 2.5 mg/kg (2 subjects). LAE was only detected in two plasma samples in one individual at the higher dose level close to the limit of quantification (1 ng/ml). Maximum plasma concentrations of LAS generally occurred at 2 h with mean peak levels of 18.2 ng/ml (1.5 mg/kg dose) and 23.9 ng/ml (2.5 mg/kg dose). Maximum concentrations of ¹³C-arginine occurred earlier (0.5 to 1 h) and at much higher levels than LAS with mean peak levels of 124 ng/ml (1.5 mg/kg dose) and 240 ng/ml (2.5 mg/kg dose). The results showed that in humans LAE was rapidly metabolized to the naturally occurring dietary components lauric acid and arginine.     

Therapeutic effect of centchroman alone and in combination with glycine soya on 7,12-dimethylbenz[α]anthracene-induced breast tumor in rat

Centchroman is a non-steroidal oral contraceptive and has been found to be a candidate drug for breast cancer exhibiting partial to complete remission of lesions in 40.5% of breast cancer patients. The therapeutic efficacy of centchroman was monitored alone and together with glycine soya on growth of 7,12-dimethylbenz[α]anthracene-induced breast tumor in rat. The tumor regression was monitored at different doses of centchroman alone ranging from 0 to 10 mg kg⁻¹ and with glycine soya from 1 × 10⁴ to 5 × 10⁴ mg kg⁻¹ per day until 5 weeks treatment. An optimum tumor treatment opus was established with varying treatment parameters including doses of therapeutic agents and treatment period. The tumors were found to be static with a strong anti-estrogenic effect. Overall our study shows that both centchroman and glycine soya alone and jointly combat with breast cancer.