Laszlo Endrenyi

Evaluation of the genetic component of variability in CYP3A4 activity: a repeated drug administration method.

The CYP3A4 enzyme contributes to the disposition of more than 60 therapeutically important drugs and displays marked person-to-person variability of the catalytic function. However, the extent of genetic contribution to variability in CYP3A4 activity remains elusive. Recently, we showed that a comparison of between- (SDb2) and within-person (SDW2) variances provides an estimate of the genetic component of variability in drug disposition. The aim of the present analysis was to assess the genetic control of CYP3A4 activity in vivo. A computerized literature search was conducted covering 1966 to September 1999 to identify studies reporting repeated administration of CYP3A4 substrates. The genetic contribution (rGC) to disposition of each CYP3A4 substrate was obtained by the formula (SDb2-SDW2)/SDb2. The rGC values approaching 1.0, point to overwhelming genetic control, whereas those close to zero suggest that environmental factors dominate. A total of 16 studies with 10 different CYP3A4 substrates were identified (n = 161 subjects). The rGC for hepatic CYP3A4 activity as measured by midazolam plasma clearance or the erythromycin breath test was 0.96 (0.92-0.98) (95% Cl) and 0.89 (0.65-0.98), respectively (P < 0.05). The point estimates of rGC for composite (hepatic + intestinal) CYP3A4 activity measured after oral administration of cyclosporine, ethinylestradiol, ethylmorphine, nifedipine and nitrendipine, ranged from 0.66-0.98 (median: 0.83) (P < 0.05). Cyclosporine data suggested a higher genetic control of CYP3A4 at night than during the day. These data indicate that further molecular genetic investigations are warranted to identify genetic variants at CYP3A4 or elsewhere in the genome which contribute to regulation of CYP3A4 activity.

Hypothesis : Comparisons of inter- and intra-individual variations can substitute for twin studies in drug research

Twin studies are useful devices to determine the heritability of persistent but variable characteristics that tend to differ among individuals. Drug responses are not persistent affairs; they are temporary characteristics. One therefore may ask whether twin studies are necessary to assess the genetic element in pharmacological responsiveness. To measure the genetic component contributing to their variability, it seems logical to investigate the response variation by repeated drug administration to given individuals, and to compare the variability of the responses within and between individuals. We attempt here to describe a theoretical background of this venture, and to show some results of the exercise. Potential sources of error or uncertainty are discussed.

Limits for the Scaled Average Bioequivalence of Highly Variable Drugs and Drug Products

AbstractPurpose. To provide a rational procedure for establishing regulatory bioequivalence (BE) limits that can be applied in determinations of scaled average BE for highly-variable (HV) drugs and drug products. Methods. Two-period crossover BE investigations with either 24 or 36 subjects were simulated with assumptions of a coefficient of variation of 10, 20, 30, or 40%. The decline in the fraction of accepted studies was recorded as the ratio of geometric means (GMR) for the two formulations was raised from 1.00 to 1.45. Acceptance of BE was evaluated by scaled average BE, assuming various BE limits, and, for comparison, by unscaled average BE. A procedure for calculating exact confidence limits in two-period studies is presented, and an approximate method, based on the linearization of the regulatory model, is applied. Results. A mixed model is proposed for average BE. Accordingly, at low variabilities, the BE limit is constant, ±BELo, generally log(1.25). Beyond a logarithmic, limiting, “switching” variability (σo), in the region of HV drugs, the approach of scaled average BE is applied with limits of ±(BELo /σo). It is demonstrated that the performance of the mixed model corresponds to these expectations. The effect of σo and of the resulting BE limits is also demonstrated. Scaled average BE, with all reasonable limits for HV drugs, requires fewer subjects than an unscaled average BE. In two-period studies, the exact and approximate methods calculating confidence limits yield very comparable inferences. Conclusions. Scaled average BE can be effectively applied, with the recommended limits, for determining the BE of HV drugs and drug products. The limiting, “switching” variability (σo) will have to be established by regulatory authorities.

Screening of malignant hyperthermia susceptible families by creatine phosphokinase measurement and other clinical investigations

SummaryWe investigated 56 families afflicted with malignant hyperpyrexia. One hundred and twenty-four individuals within these families had had an episode of malignant hyperthermia, of whom we saw seventy-two. Serum creatine phosphokinase (CPK) was statistically higher in affected individuals and in close relatives than in normal volunteers. The magnitude of the serum CPK elevations varied significantly between families. While in some families the serum CPK was clearly elevated in affected individuals, in other families the serum CPK was normal or only moderately or inconsistently raised. In these latter families serum CPK measurement was therefore of little or no value in identifying afflicted members.The incidence of musculoskeletal abnormalities was greater in affected individuals and in close relatives than in the general population. Thus, the concomitant presence of both a high serum CPK and a musculoskeletal abnormality in an individual belonging to a family known to be susceptible to malignant hyperthermia was a better indicator of the MH trait than was the presence of only one of these parameters.For reasons which we do not fully understand, MHS individuals were found to require fewer anaesthetics than normal persons. The incidence of MH crises within each family fell significantly following investigation, counselling, and issuance of Medic-AIert bracelets.RésuméNotre étude a porté sur cinquant-six familles. Cent vingt-quatre membres de ces familles avaient présenté des épisodes d’hyperthermie maligne et nous avons pu investiguer soixante-douze de ceux-ci.Nous avons retrouvé des taux de CPK sériques plus élevés chez des individus affectés et leurs proches parents que chez les volontaires normaux. Le degré d’élévation des CPK variait cependant d’une famille à l’autre. Alors que dans certaines familles les individus affectés présentaient des taux nettement élevés, dans d’autres familles, des taux normaux ou peu élevés étaient retrouvés chez les sujets affectés. Dans ce second groupe de familles, la détermination des CPK sériques n’est donc pas un bon moyen de dépister les individus susceptibles de présenter un épisode d’hyperthermie maligne. L’incidence des anomalies musculo-squelettiques était plus élevés chez les sujets affectés et chez leurs proches parents que l’incidence de ces anomalies au sein de la population générale. Donc, la présence concomitante d’un taux élevé de CPK et d’anomalies musculo-squelettiques retrouvée chez un sujet membre d’une famille présentant une histoire d’hyperthermie maligne est un meilleur indicateur de susceptibilité à l’hyperthermie qu’un seul de ces paramètres isolés.Pour des raisons encore mal comprises, les sujets susceptibles à l’hyperthermie maligne requièrent moins d’anesthésiques que les sujets normaux.L’incidence des épisodes d’hyperthermie maligne a diminué de façon significative dans les familles investiguées, conseillées, et auxquelles on avait remis des bracelets Medic-Alert.

Evaluation of Bioequivalence for Highly Variable Drugs with Scaled Average Bioequivalence

Bioequivalence studies are performed to demonstrate in vivo that two pharmaceutically equivalent products (in the US) or alternative pharmaceutical products (in the EU) are comparable in their rate and extent of absorption. By definition, for highly variable drugs (HVDs), the estimated within-subject variability is >30%. HVDs often fail to meet current regulatory acceptance criteria for average bioequivalence (ABE). The determination of the bioequivalence of HVDs has been a vexing problem since the inception of the current regulations. It is of concern not only to the generic industry but also to the innovator industry. This article reviews the definition of HVDs, the present regulatory recommendations and the approaches proposed in the literature to deal with the bioequivalence problems of HVDs. The approach of scaled ABE (SABE) is proposed as the most adequate procedure to solve the problem. It is demonstrated that SABE has firm theoretical foundations. In fact, statistical tests similar to SABE are used in various fields, such as psychology and quality control. Algorithms and numerical examples are presented to calculate SABE from the data in conventional two-period and replicate-design studies. The most important feature of SABE is that a fixed sample size is adequate to demonstrate bioequivalence regardless of within-subject variability. The conditions for reaching consistent regulatory decisions with SABE are discussed. The required sample size, for a given statistical power, depends on the regulatory criteria. Sample sizes with different criteria are demonstrated and compared with those arising from a recent informal US FDA proposal.Pragmatic considerations lead to modifications of the theoretical concept of SABE. Several modifications are proposed, including reference scaling, restriction on the estimated geometric mean ratios and possibly limiting SABE to only secondary bioequivalence metrics such as the maximum concentration. Each proposal has its own merit but is also a source of new controversy. Overall, the statistical evaluation of SABE is more complex than that of ABE, which means higher regulatory burden. Standardized open software could be very useful in this regard. A small program script is presented to calculate SABE confidence limits.

Evaluation of the bioequivalence of highly-variable drugs and drug products

AbstractPurpose. To establish procedures for the effective evaluation of bioequivalence (BE) for highly-variable drugs and drug products (HVD/P). Methods. 2- and 4-period crossover BE studies with 24 subjects were simulated which generally assumed within-subject coefficients of variation of 40%. The relationship between the fraction of studies in which BE was accepted (the statistical power) and the ratio of geometric means (GMR) of the two formulations was evaluated for various methods of analysis. These included, primarily, scaled average BE (ABE), the corresponding approach of expanding BE limits (BEL), and, for comparison, unscaled ABE and scaled individual BE (IBE). Results. Scaled ABE and expanding BEL showed very similar properties in both 2- and 4-period studies. They had steeper power curves than scaled IBE. Unscaled ABE had very low statistical power. The acceptance of BE by unscaled and scaled ABE and expanding BEL was almost independent of subject-by-formulation interaction and the ratio of within-subject variations of the two formulations. By contrast, the conclusions reached by scaled IBE were strongly affected by these parameters. Conclusions. Scaled ABE and expanding BEL evaluate BE effectively for HVD/P in both 2- and 4-period investigations. However, additional, useful information can be obtained from 4-period studies.

Comparative drug elimination capacity in man-glutethimide, amobarbital, antipyrine, and sulfinpyrazone.

The apparent elimination half‐lifes were determined for 4 drugs that undergo hydroxylation in men. Glutethimide (500 mg), amobarbital sodium (130 mg), antipyrine (1.0 gm), and sulfinpyrazone (400 mg) were each taken orally by 10 healthy men at weekly intervals. When the half‐lifes in these 10 individuals were compared, positive correlations were found among 3 out of 4 drugs: glutethimide‐amobarbital (r 0.69, p < 0.05), glutethimide‐sulfinpyrazone (r 0.64, p < 0.05), and amobarbital‐sulfinpyrazone (r 0.87, p < 0.01). If these data are generally valid, it would mean, for example, that one could, from the sulfinpyrazone half‐life in a given subject, predict the amobarbital half‐life of the same individual within about ± 7 hours (30% of average half‐life, while the interindividual differences could be 300%). Antipyrine half‐life was not correlated with the half‐life of any of the other 3 drugs. The urinary levels of 6β‐hydroxycortisol were also determined in the same sub;ects and compared with the half‐lifes of the 4 drugs. The correlation coefficients taken singly were not statistically significant, but they were all negative and collectively seem to indicate an expected trend.

Design of experiments for the precise estimation of dose-response parameters: the Hill equation

Optimal experimental designs were evaluated for the precise estimation of parameters of the Hill model. The optimally effective designs were obtained by using the criterion of D-optimization. For the Hill model, optimal designs replicate 3 sampling points. These points were shown to be quite sensitive to the behavior of the experimental error. Since an investigator is often uncertain about error conditions in biological studies, a practical approach would use the sampling scheme calculated for an intermediate error condition. Thus, if the behavior of error variances is not known, precise parameters of the Hill model are obtained by choosing concentrations which yield fractional responses (responses divided by their asymptotic, maximum value) of 0.086, 0.581 and 1.0. When experimental constraints limit the maximum attainable concentration and response, all design points are lowered. Appropriate designs can be constructed based on the design which is optimal when constraints result in a maximum attainable fractional response of 0.5. The optimal designs were found to be robust when the parameter values assumed by the investigator did not equal their true values. The estimating efficiencies obtained by using two frequently applied designs were assessed. Uniformly spaced concentrations yielded imprecise parameters. Six-point, geometrically spaced designs gave generally good results. However, their estimating efficiency was generally exceeded by the recommended sampling schemes even in the presence of uncertainty about error conditions. The method exemplified in this paper can be used for other models.

Design of Experiments for Estimating Enzyme and Pharmacokinetic Parameters

Principles are described for optimally estimating precise model parameters. D-optimized designs, which minimize the volumes of joint confidence regions for the parameters, generally replicate p sampling points. (p is the number of parameters.) The observations are repeated at equal frequency when the estimation of all model parameters is essential, but often in unequal proportions if only some of the parameters are of interest. Usually, one of the design points yields the maximal experimentally attainable response. In the presence of constant relative errors another point measures the smallest possible response. The principles of optimization are applied to simple enzyme and pharmacokinetic models, and designs yielding the most precise parameters are described. These designs provide guidelines, and not rules, for experimentation. In practice, a few observations could test the validity of the model while others could be obtained close to the proposed design points. The usefulness of the strategy is demonstrated by the substantially reduced estimating efficiency (larger parameter variance) of frequently applied experimental designs, especially when the number of measurements is increased.

Optimal design of experiments for the estimation of precise hyperbolic kinetic and binding parameters

Abstract Optimal designs were evaluated for the estimation of precise parameters in kinetic and binding experiments in which the concentration dependence of the response (reaction velocity or bound ligand concentration) is characterized by a hyperbola. The designs were evaluated by maximizing the determinant of the appropriate information matrix. It is demonstrated that highest precision is obtained by replicating basic two-point designs. However, the calculated optimal designs should serve only as guidelines and not rules for experimentation. In the presence of constant variance, half of the observations should be obtained at the highest practically attainable concentration. In kinetic studies, the remaining measurements should yield half of the maximally attainable velocity. In binding experiments, the second half of observations for either bound (b) or free (f) ligand concentrations should be made at a total concentration of c = P + K (K is the dissociation constant, and P the binding capacity). With constant coefficient of variation (constant relative error), kinetic experiments should be performed, at equal frequency, at the highest and lowest attainable concentration. Half of the binding experiments should be made at their lowest precision: at the highest possible concentration when b is measured, and at the smallest feasible concentration when f is obtained. The other half of the readings should be taken at c = P − K (if P > K) for the measurement of b, and at c = P + K for observations of f. Deviations from the calculated optimal designs result in diminished efficiency (increased variance) of the estimated parameters. The reduction can be substantial with some frequently used experimental designs, especially when the number of observations is not very small. Therefore, the first few readings could consider the validation of the model, but additional measurements should follow the guidelines presented here.