Porcine Prediction of Pharmacokinetic Parameters in People: A Pig in a Poke?

The minipig has become an animal of considerable interest in preclinical drug development. It has been used in toxicology research and in examining/establishing regulatory guidelines as a nonrodent animal model. We have reviewed some basic issues that one would want to consider in the development and testing of any animal model for humans. The pig is a reasonable alternative to the dog, but there are some clear limitations and unexplained disparities in the literature, which require further study; primary among these is the need for standardization in choice of breed and sex and routine protocols. The minipig offers numerous advantages over other established animal models, and it has similarities to the human with regard to anatomy, physiology, and biochemistry. The gastrointestinal tract is structurally and functionally similar to humans. This appears to be true for enzymes and transporters in the gut as well, but more study is needed. One major concern is assessment of oral drug absorption, especially with regard to potential food effects due to gastric emptying differences, yet this does not appear to be a consistent observation. Hepatic metabolism seems to reflect enzymatic patterns in humans, with some differences. Kidney function seems similar to humans but requires further study. We have analyzed literature data that suggest the pig would offer a reasonable model for human oral bioavailability and for allometric predictions of clearance. The minipig appears to be the model for dermal absorption in humans, and we discuss this in terms of literature data and our own in-house experience.

Controversies in allometric scaling for predicting human drug clearance: an historical problem and reflections on what works and what does not.

This review focuses on a discussion of the controversies in allometric scaling (AS) for predicting human clearance from a mathematical and statistical perspective. First, a history of allometric scaling in comparative biology and its use in pharmacokinetics are reviewed. It is shown that the application of AS in predicting human clearance values based on a limited number of animal species (typically, 3 or 4) contains fundamental statistical errors when AS was first introduced from comparative biology. Second, the mathematical nature of various allometrically-based methods is revealed and the soundness of these methods is assessed. It is demonstrated that any of these methods, which incorporate a correction factor in a traditional allometric approach (varying-exponent allometry), not only reduces the statistical power of the allometric analysis, but are also incorrect with regard to aspects of biology. Finally, it is concluded that allometry remains a valuable tool for predicting human clearance, and should be applied in the context of a fixed exponent. However, fixed-exponent allometry does not provide satisfactory accuracy in predicting human clearance, since it is not able to capture the biological differences among species. Therefore, it is recommended that the overall effort in predicting human pharmacokinetics should be directed to the collection and generation of reliable data (both in vitro and in vivo) along with a better understanding of the DMPK properties of the chemical entity.

Controversy in the allometric application of fixed- versus varying-exponent models: a statistical and mathematical perspective.

This commentary is a reply to a recent article by Mahmood commenting on the authors' article on the use of fixed-exponent allometry in predicting human clearance. The commentary discusses eight issues that are related to criticisms made in Mahmood's article and examines the controversies (fixed-exponent vs. varying-exponent allometry) from the perspective of statistics and mathematics. The key conclusion is that any allometric method, which is to establish a power function based on a limited number of animal species and to extrapolate the resulting power function to human values (varying-exponent allometry), is infused with fundamental statistical errors.

Formulation and in vivo evaluation of chlorpropham (CIPC) oral formulations.

The objective of these studies was to examine the in vivo performance of oral formulations of chlorpropham (CIPC). In order to develop a new oral formulation several different solubilization techniques were evaluated, namely: cosolvents, surfactants, and complexing agents. The solubilization data indicated that a conventional solution formulation was not plausible. Two self-emulsifying drug delivery systems (SEDDS) were developed and evaluated for stability. Both SEDDS formulations were found to be chemically stable. In vivo analysis of a SEDDS formulation, a suspension formulation and an intravenous bolus dose was conducted in F344 rats. Pharmacokinetic analysis of the formulation data indicated that the SEDDS formulation provided only marginally better oral bioavailability compared to a suspension formulation. While SEDDS formulations often result in greater bioavailability this was not observed for CIPC. In vivo analysis indicate that CIPC results in a situation where the dissolution rate of CIPC from the suspension is not rate limiting, rather the absorption rate in the GI tract is rate-limiting. This paradigm is the result of CIPCs low melting point and the relatively small particle size of the suspension which facilitate the dissolution in the GI tract.

Utility of the coefficient of determination (r2) in assessing the accuracy of interspecies allometric predictions: illumination or illusion?

The appropriateness of relying on the coefficient of determination (r(2)) as a statistical metric for judging the predictability of human clearance (CL) based on interspecies animal data was assessed. An explicit mathematical expression was derived for r(2) as a function of species body weight and the corresponding measured value of CL. The derived mathematical function demonstrated that r(2) is numerically large in most instances. Simulations using random CL generated from a common combination of species of mouse, rat, and monkey resulted in an r(2) of 0.75 as the minimum, and 0.95 and 0.98 at 50th and 75th percentiles, respectively, given that total CL values increase with increasing species body weight. Analysis of literature data also indicated that the prediction accuracy of human CL was not correlated with values of r(2). Therefore, it is concluded that r(2) is a limited statistical measure when assessing allometric scaling for the purpose of predicting human CL.

Interspecies prediction of human drug clearance based on scaling data from one or two animal species.

A data-driven approach was adopted to derive new one- and two-species-based methods for predicting human drug clearance (CL) using CL data from rat, dog, or monkey (n = 102). The new one-species methods were developed as CL(human)/kg = 0.152 x CL(rat)/kg, CL(human)/kg = 0.410 x CL(dog)/kg, and CL(human)/kg = 0.407 x CL(monkey)/kg, referred to as the rat, dog, and monkey methods, respectively. The coefficient of the monkey method (0.407) was similar to that of the monkey liver blood flow (LBF) method (0.467), whereas the coefficients of the rat method (0.152) and dog method (0.410) were considerably different from those of the LBF methods (rat, 0.247; dog, 0.700). The new rat and dog methods appeared to perform better than the corresponding LBF methods, whereas the monkey method and the monkey LBF method showed improved predictability compared with the rat and dog one-species-based methods and the allometrically based "rule of exponents" (ROE). The new two-species methods were developed as CL(human) = a(rat-dog) . W (human)(0.628) (referred to as rat-dog method) and CL(human) = a(rat-monkey) . W (human)(0.650) (referred to as rat-monkey method), where a(rat-dog) and a(rat-monkey) are the coefficients obtained allometrically from the corresponding two species. The predictive performance of the two-species methods was comparable with that of the three-species-based ROE. Twenty-six Wyeth compounds having data from mouse, rat, dog, monkey, and human were used to test these methods. The results showed that the rat, dog, monkey, rat-dog, and rat-monkey methods provided improved predictions for the majority of the compounds compared with those for the ROE, suggesting that the use of three or more species in an allometrically based approach may not be necessary for the prediction of human exposure.

A global examination of allometric scaling for predicting human drug clearance and the prediction of large vertical allometry.

Allometrically scaled data sets (138 compounds) used for predicting human clearance were obtained from the literature. Our analyses of these data have led to four observations. (1) The current data do not provide strong evidence that systemic clearance (CL(s); n = 102) is more predictable than apparent oral clearance (CL(po); n = 24), but caution needs to be applied because of potential CL(po) prediction error caused by differences in bioavailability across species. (2) CL(s) of proteins (n = 10) can be more accurately predicted than that of non-protein chemicals (n = 102). (3) CL(s) is more predictable for compounds eliminated by renal or biliary excretion (n = 33) than by metabolism (n = 57). (4) CL(s) predictability for hepatically eliminated compounds followed the order: high CL (n = 11) > intermediate CL (n = 17) > low CL (n = 29). All examples of large vertical allometry (% error of prediction greater than 1000%) occurred only when predicting human CL(s) of drugs having very low CL(s). A qualitative analysis revealed the application of two potential rules for predicting the occurrence of large vertical allometry: (1) ratio of unbound fraction of drug in plasma (f(u)) between rats and humans greater than 5; (2) C logP greater than 2. Metabolic elimination could also serve as an additional indicator for expecting large vertical allometry.

On the observed large interspecies overprediction of human clearance ("vertical allometry") of UCN-01: further support for a proposed model based on plasma protein binding.

The prediction of a human clearance (CL) value for UCN-01, an extreme example of vertical allometry (a large overprediction by allometric scaling), was examined using commonly used simple allometry and the "rule of exponents," as well as a newly proposed model, which quantitatively incorporates plasma protein-binding information from rats and humans. Simple allometry and the rule of exponents were shown to overpredict the human CL value of UCN-01 by about 5000- and 1750-fold, respectively. The new model incorporating the ratio of fraction unbound between rats and humans improved the prediction by about 20-fold compared to the rule of exponents. The model is expected to improve if a more accurate measurement of the unbound fraction in human plasma is obtained. The prediction of volume distribution for UCN-01 by allometric scaling was also shown to be dependent on the difference of fraction unbound between animal species and humans. In summary, plasma protein binding has been demonstrated to be an important measure for interspecies scaling of pharmacokinetics.

A porcine model for fixed drug eruptions in humans: the case of antipyrine in the Yucatan micropig.

To date, there is no acceptable animal model to investigate fixed drug eruptions (FDEs) in humans. We briefly report here observations suggesting that the Yucatan micropig may be a useful animal model for that purpose. During an investigation of antipyrine absorption and disposition, we observed the development of FDEs after intravenous administration of a 1 g dose. Our observations were consistent with those reported in several investigations of humans taking a single dose of antipyrine. To confirm these results, a naïve micropig was challenged. A male uncastrated Yucatan micropig (27.2 kg) was given a 1 g dose of antipyrine intravenously. After 30 days, this pig was rechallenged with the same intravenous dose of antipyrine (1 g). Blood samples were obtained to examine immunological endpoints. During the initial challenge, a fluid plaque (ca. 1-1.5 cm) appeared on the left hip of the pig ca. 6 h after dosing. After the rechallenge, inflamed pink patches were observed at the same sites where the blisters formed initially; however, no blisters re-formed. Changes of neutrophil, lymphocyte and eosinophil levels from baseline were noted 8 h after challenge. The micropig did not seem otherwise affected by the FDEs. These observations suggest that the Yucatan micropig, or swine in general, may be a useful animal model for detecting drugs that may cause FDEs in humans.

A novel model for prediction of human drug clearance by allometric scaling.

Sixty-one sets of clearance (CL) values in animal species were allometrically scaled for predicting human clearance. Unbound fractions (f(u)) of drug in plasma in rats and humans were obtained from the literature. A model was developed to predict human CL: CL=33.35 ml/min x (a/Rf(u))(0.770), where Rf(u) is the f(u) ratio between rats and humans and a is the coefficient obtained from allometric scaling. The new model was compared with simple allometric scaling and the "rule of exponents" (ROE). Results indicated that the new model provided better predictability for human values of CL than did ROE. It is especially significant that for the first time the proposed model improves the prediction of CL for drugs illustrating large vertical allometry.

A mathematical description of the functionality of correction factors used in allometry for predicting human drug clearance.

The functionality of the correction factors, maximum life-span potential (MLP), and brain weight (BrW) used in allometry is mathematically described. Correction by MLP or BrW is equivalent to a multiplication of some constants by the predicted values in humans from simple allometry, but they have no relationship to measured pharmacokinetic parameters in the animal species. The values of these constants (F(MLP) or F(BrW)) were calculated for some commonly used combinations of animal species. For all combinations of animal species, the value of F(BrW) is always greater than that of F(MLP) with a fold-increase of about 1.3 to 1.9. Different combinations of species give different values of F(BrW) and F(MLP). In addition, the role of correction factors (MLP and BrW) or the "rule of exponents" (ROE) was evaluated. An intrinsic defect in using correction factors or ROE was revealed; different study designs will produce significantly different prediction results. However, ROE may still serve as a useful practical approach in predicting human CL since it was derived from real observations and has been applied to many examples.

Accuracy of allometrically predicted pharmacokinetic parameters in humans: role of species selection.

A general equation was derived, which directly describes the mathematical relationship between the allometrically predicted pharmacokinetic (PK) parameters in humans and the body weights of animal species (along with their corresponding measured PK parameters). It was shown, with use of the derived equation, that the predicted values in humans, based on combinations of animal species commonly used in allometry, are heavily dependent on certain species, for example, the dog. In contrast, parameter values from the rat made no contribution to the predicted human values, as long as the rat was not the smallest species used. Monte Carlo simulations were further performed to examine the species or weight dependence. The cost-effective combinations of animal species, in terms of number and species type, were theoretically examined through simulations. Finally, literature data demonstrated the species or weight dependence predicted from the equation and as illustrated through the Monte Carlo simulations. Appreciation of this species or weight dependence should guide researchers in selecting animal species and designing optimal experiments in the application of allometric scaling.

Dermal absorption of camphor, menthol, and methyl salicylate in humans.

Camphor, menthol, and methyl salicylate occur in numerous over-the-counter products. Although extensively used, there have been no estimates of human exposure following administration via dermal application. Furthermore, there is little information about the pharmacokinetics of those compounds. The authors report the plasma concentrations of the intact compounds as a function of dose following dermal patch application. Three groups of 8 subjects (4 male, 4 female) applied a different number of commercial patches (2, 4, or 8) to the skin for 8 hours. Plasma samples were assayed using sensitive and selective gas-chromatographic methods. For the 8-patch group, the average maximum plasma concentrations (Cmax +/- SD) were 41.0 +/- 5.8 ng/mL, 31.9 +/- 8.8 ng/mL, and 29.5 +/- 10.5 ng/mL for camphor, menthol, and methyl salicylate, respectively. The corresponding values for the 4-patch group were 26.8 +/- 7.2 ng/mL, 19.0 +/- 5.4 ng/mL, and 16.8 +/- 6.8 ng/mL. The harmonic mean terminal half-lives were 5.6 +/- 1.3 hours, 4.7 +/- 1.6 hours, and 3.0 +/- 1.2 hours for camphor, menthol, and methyl salicylate, respectively. The 2-patch group had measurable but low plasma concentrations of each compound. Low-dose dermal application for an extended time results in low plasma concentrations of all 3 compounds. Four and 8 patches, when applied for 8 hours, gave measurable and nearly proportional plasma concentrations. Although unable to determine the absolute dermal bioavailability of these compounds, there appears to be relatively low systemic exposure to these potentially toxic compounds, even when an unrealistically large number of patches are applied for an unusually long time.

Protein expression pattern of P-glycoprotein along the gastrointestinal tract of the Yucatan micropig.

The purpose of this study is to characterize the distribution pattern of P-gp protein levels along the entire GI tract in the Yucatan micropig, which is being developed as a model for human drug bioavailability. Small and large intestines were freshly obtained and divided into about 37 segments and 10 segments, respectively (ca., 1 foot/segment). Epithelial cells from the small intestine were obtained by an elution method; whereas, a scraping method was applied to the large intestine. Total cellular protein was isolated from the epithelial cells. Western blot analysis using P-gp antibody showed that the amount of P-gp protein increased distally from the duodenum to the ileum over approximately a 10-fold range. P-gp protein in the large intestine was present at a higher level in the central portion, but the absolute amount was much less than what was found in the small intestine.

Sensitive and rapid isocratic liquid chromatography method for the quantitation of curcumin in plasma.

An HPLC assay was developed using three methods of plasma sample preparation in order to quantitate curcumin, the main constituent in the herbal dietary supplement turmeric. Each method involves simple and rapid processing of samples (either an ethyl acetate or chloroform extraction) with resulting different quantitation limits for curcumin. The assay was developed in an effort to quantify extremely low curcumin plasma concentrations observed in preliminary in vivo studies. The most sensitive assay can reliably detect concentrations down to 2.5 ng/ml. Plasma quantitation was precise and accurate based on both intra- and inter-day validations as indicated by low values for coefficients of variation and bias, respectively (< or =15%). The analytical validation was reproducible between different analysts. The resulting analytical method couples desired sensitivity with the ease of an isocratic system.