From the bench to clinical practice: understanding the challenges and uncertainties in immunogenicity testing for biopharmaceuticals.

Unlike conventional chemical drugs where immunogenicity typically does not occur, the development of anti-drug antibodies following treatment with biologics has led to concerns about their impact on clinical safety and efficacy. Hence the elucidation of the immunogenicity of biologics is required for drug approval by health regulatory authorities worldwide. Published ADA 'incidence' rates can vary greatly between same-class products and different patient populations. Such differences are due to disparate bioanalytical methods and interpretation approaches, as well as a plethora of product-specific and patient-specific factors that are not fully understood. Therefore, the incidence of ADA and their association with clinical consequences cannot be generalized across products. In this context, the intent of this review article is to discuss the complex nature of ADA and key nuances of the methodologies used for immunogenicity assessments, and to dispel some fallacies and myths.

Evaluation of regional limb perfusion with amikacin using the saphenous, cephalic, and palmar digital veins in standing horses.

Previous studies have shown that regional limb perfusion (RLP) using the palmar digital (PD) vein delivers therapeutic concentration of amikacin to the distal limb. Our hypothesis was that using the cephalic and saphenous veins for RLP will enable delivery of therapeutic concentrations of amikacin to the distal limb. Nineteen healthy horses participated in the study. The cephalic, saphenous, or PD vein was used to perfuse the limb with amikacin. Two grams of amikacin was used for RLP using the saphenous and the cephalic veins, and one gram was used in the PD vein. Synovial samples were collected from the metacarpo-/metatarsophalangeal (MCP/MTP) joint, and blood samples were collected from the jugular vein. Maximum concentration (Cmax) of amikacin in the MCP/MTP joint using the cephalic and the saphenous vein was 277 and 363 mg/L, respectively. The amikacin concentrations achieved in the synovial fluid of the MCP/MTP joint in the current study were between 69 and 91 times the minimally inhibitory concentration of common susceptible bacterial pathogens causing orthopedic infections in horses. To conclude, this study shows that use of the proximal veins for RLP to treat distal limb infections is a viable alternative to using the palmar or plantar digital vein.

Pharmacokinetic profile of spectinomycin in rats.

Following intravenous (IV) administration, the pharmacokinetics of spectinomycin in rats was found to be on par with its profile in other mammalian species including humans with respect to its overall excretion and half-life at effective concentrations. This study, however, indicates that a small fraction of the spectinomycin dose is retained in peripheral tissues for a prolonged period of time at low concentrations.

Transrepression and transactivation potencies of inhaled glucocorticoids.

The anti-inflammatory activity of inhaled glucocorticoids is primarily mediated through transrepression of pro-inflammatory transcription factors such as AP-1 and NF-kappaB, while systemic side effects are largely attributed to transactivation via glucocorticoid response elements (GRE) in the promoter region of responsive genes. The objective of this study is to investigate whether inhaled corticosteroids exhibit differences in their transactivation and transrepression potencies. A549 human alveolar epithelial type II like cells, stably transfected with a reporter plasmid containing an AP-1, NF-kappaB or GRE induced secreted alkaline phosphatase reporter gene (SEAP), were exposed to a panel of concentrations of the six inhaled and three systemic glucocorticoids. Glucocorticoid-induced changes in SEAP expression were quantified by chemiluminescence. For eight glucocorticoids (budesonide, desisobutyryl-cicle-sonide, dexamethasone, flunisolide, fluocortolone, fluticasone propionate, mometasone furoate, prednisolone) the EC50 for NF-kappaB mediated transrepression was significantly larger than that for both transactivation and transrepression via AP-1. For the remaining glucocorticoid (triamcinolone acetonide), it was greater than that for transactivation. It is concluded that, within the studied cell system, inhaled corticosteroids did not exhibit preferential transrepression, but had higher potencies for transactivation than for transrepression via NF-kappaB and had differential potencies for the two transrepression pathways.

The role of pharmacokinetics and pharmacodynamics in phosphodiesterase-5 inhibitor therapy.

Differences in clinical pharmacology of the currently marketed phosphodiesterase (PDE)5 inhibitors sildenafil, vardenafil and tadalafil are largely determined by their pharmacokinetic (PK) properties and their PDE5 inhibitory activity profile. This review outlines the basic concepts of pharmacokinetics and pharmacokinetic pharmacodynamic (PK/PD) relationships and their relevance to dose selection and applied pharmacotherapy. It is followed by a detailed comparative discussion on the pharmacokinetics and exposure-response relationship of the currently available PDE5 inhibitors, including known drug-drug interactions and dosage adjustments in special populations. The review is aimed at providing a critical assessment of the pharmacokinetics of PDE5 inhibitors, which may assist clinicians in tailoring drug and/or treatment regimens to the unique needs of each individual patient with erectile dysfunction.

Human Ontogeny of Drug Transporters: Review and Recommendations of the Pediatric Transporter Working Group.

The critical importance of membrane-bound transporters in pharmacotherapy is widely recognized, but little is known about drug transporter activity in children. In this white paper, the Pediatric Transporter Working Group presents a systematic review of the ontogeny of clinically relevant membrane transporters (e.g., SLC, ABC superfamilies) in intestine, liver, and kidney. Different developmental patterns for individual transporters emerge, but much remains unknown. Recommendations to increase our understanding of membrane transporters in pediatric pharmacotherapy are presented.

Dynamic modeling of cortisol reduction after inhaled administration of fluticasone propionate.

Fluticasone propionate (FP) is a new corticosteroid that has been developed for the treatment of asthma. The compound has a very high receptor affinity, 18 times that of dexamethasone. After inhalation, FP is systemically available because of inhaled bioavailability. In healthy subjects this may lead to measurable systemic effects, such as cortisol reduction. A clinical study was conducted in 12 healthy volunteers to determine the systemic effects after inhaled administration of single 500-micrograms, 1,000-micrograms, and 2,000-micrograms doses of FP. Blood samples were collected over a 24-hour period after administration. Concentrations of FP and cortisol were measured in plasma by immunoassay. Cortisol reduction was chosen as the pharmacodynamic parameter. A novel linear release rate model was used to parameterize the cortisol data. The pharmacokinetics of FP were linear over the dose range studied. The cortisol release parameters were determined from baseline data (before drug administration). Based on these results, the E50 values for cortisol reduction were then determined for each dose of FP. The average E50 was 0.134 ng/mL for total FP concentrations and 0.013 ng/mL for unbound FP concentrations; these results were not dose dependent. These in vivo pharmacodynamic values measured in healthy subjects are in good agreement with the relatively high receptor affinity of FP.

Dependency of cortisol suppression on the administration time of inhaled corticosteroids.

Endogenous cortisol suppression is one of the major systemic side effects of inhaled corticosteroids in the treatment of asthma. A previously developed pharmacokinetic/ pharmacodynamic approach was used to evaluate the influence of administration time on the cumulative cortisol suppression (CCS) after single doses of the inhaled corticosteroids flunisolide and fluticasone propionate. Administration time-dependent simulations of CCS were performed with drug-specific pharmacokinetic and pharmacodynamic parameters obtained from previous clinical trials. Both drugs showed similar diurnal variation in CCS, dependent on the administration time, with maximum suppression when administered in the early morning at approximately 3 AM. The optimum administration time for minimized CCS was in the afternoon but was shifted from 3 PM for fluticasone propionate to later time points around 7 PM for flunisolide, probably because of the shorter terminal elimination half-life of flunisolide. Regarding peak to trough fluctuation, however, CCS after fluticasone propionate showed only half the administration time dependency as after flunisolide. Therefore, the ratio between CCS after flunisolide and after fluticasone propionate also followed administration time-dependent variations. This led to the conclusion that administration time has to be considered as a pivotal influential factor in clinical studies comparing CCS among different inhaled corticosteroids.

Pharmacokinetic/pharmacodynamic evaluation of systemic effects of flunisolide after inhalation.

The pharmacokinetics and pharmacodynamics of flunisolide were studied in healthy volunteers after inhalation. In the morning on the day the study began, volunteers inhaled 0.5 mg of flunisolide with and without oral administration of charcoal, or 1 mg, 2 mg, and 3 mg of flunisolide with concomitant administration of charcoal. A placebo group was used to assess the endogenous cortisol, granulocyte, and lymphocyte baseline levels. Flunisolide plasma levels were determined by high-performance liquid chromatography using a tandem mass spectrometer as detector (HPLC/MS/MS). Cortisol plasma levels and differential white blood cell counts were obtained over 12 hours. An integrated pharmacokinetic/pharmacodynamic (PK/PD) model was applied to link the flunisolide plasma concentrations with the effects on lymphocytes, granulocytes, and cortisol. Maximum concentration levels of 3 to 9 ng/mL of flunisolide were observed after 0.2 to 0.3 hours for all of the investigated doses. The terminal half-life ranged from 1.3 to 1.7 hours. There was no statistical difference between treatments in the presence or absence of orally administered charcoal. The pharmacokinetic/pharmacodynamic (PK/PD) models satisfactorily described the time-courses of the effects on granulocytes, lymphocytes, and cortisol suppression. The resulting E50-values (concentrations to induce 50% of the maximum effect) concurred with the reported values of in vitro receptor binding affinities. The duration of the systemic effects were short because of the short half-life of the drug. Cumulative cortisol suppression increased with dose administration and ranged from 20% to 36%. The PK/PD simulations resulted in a smaller degree of cortisol suppression for the drug administered at 10 PM. The cumulative change from baseline was slightly smaller for the effects on granulocytes and lymphocytes than those on cortisol. This information promotes the comparison with other inhaled glucocorticoids.

Basic concepts of pharmacokinetic/pharmacodynamic (PK/PD) modelling.

Pharmacokinetic (PK) and pharmacodynamic (PD) information from the scientific basis of modern pharmacotherapy. Pharmacokinetics describes the drug concentration-time courses in body fluids resulting from administration of a certain drug dose, pharmacodynamics the observed effect resulting from a certain drug concentration. The rationale for PK/PD-modelling is to link pharmacokinetics and pharmacodynamics in order to establish and evaluate dose-concentration-response relationships and subsequently describe and predict the effect-time courses resulting from a drug dose. Under pharmacokinetic steady-state conditions, concentration-effect relationships can be described by several relatively simple pharmacodynamic models, which comprise the fixed effect model, the linear model, the long-linear model, the Emax-model and the sigmoid Emax-model. Under non steady-state conditions, more complex integrated PK/PD-models are necessary to link and account for a possible temporal dissociation between the plasma concentration and the observed effect. Four basic attributes may be used to characterize PK/PD-models: First, the link between measured concentration and the pharmacologic response mechanism that mediates the observed effect, direct vs. indirect link; second, the response mechanism that mediates the observed effect, direct vs. indirect response; third, the information used to establish the link between measured concentration and observed effect, hard vs. soft link; and fourth, the time dependency of the involved pharmacodynamic parameters, time-variant vs. time-invariant. In general, PK/PD-modelling based on the underlying physiological process should be preferred whenever possible. The expanded use of PK/PD-modelling is assumed to be highly beneficial for drug development as well as applied pharmacotherapy and will most likely improve the current state of applied therapeutics.

Gender differences in pharmacokinetics and pharmacodynamics.

Several years ago regulatory authorities requested to include women in all phases of clinical drug development in order to thoroughly investigate potential gender differences in the pharmacokinetics and pharmacodynamics of newly developed therapeutic agents. Since then, numerous reports have been published that evaluate the potential existence and impact of gender differences on all aspects of clinical pharmacology. With regard to pharmacokinetics, differences in absorption rate and duration have been reported for several drugs, but generally lack to have major clinical relevance. Differences in oral bioavailability, however, seem to be more important and are usually caused by sex differences in the activity of major intestinal and hepatic metabolic enzymes. Distributional differences have also been identified for numerous compounds. Although the majority of these findings were merely weight effects as women generally have a lower body weight, some of the gender-specific distribution differences persisted after normalization for weight. Possible explanations are differences in body composition between men and women and/or physiological changes during the menstrual cycle as well as differences in plasma protein binding secondary to hormonal characteristics. Frequent and sometimes clinically relevant gender differences could be identified for drug elimination processes and were predominantly linked to the sex-specific expression of metabolic enzyme systems, e.g. CYP3A4 and CYP1A2. In contrast, gender-related differences in renal elimination are generally only of minor importance. With regard to pharmacodynamics, gender differences have been observed in baseline characteristics as well as in drug response, which might both, at least in part, be the consequence of modulation by sex hormones. Some of the most striking examples identified were in pain therapy and perception, glucose management and arrhythmia susceptibility. Since clinical endpoints of efficacy and toxicity are often difficult to monitor and are frequently substituted by surrogate markers that might increase variability and thus mask gender effects, sex-specific differences in pharmacodynamic characteristics can often remain uncovered and further intensive research in this area seems imperative. For the majority of investigated drugs in the past few years, however, no or only very minor gender differences could be detected in pharmacokinetics and/or pharmacodynamics. In addition, the clinical significance of those gender differences identified seem very limited and was only very rarely linked to treatment success or failure. Hence, it is undoubtedly necessary to include women in the clinical drug development process, but it seems questionable whether women of child-bearing capability should be exposed to potential risks in early phase I clinical trials.

Pharmacokinetics of immunosuppressants: a perspective on ethnic differences.

Despite recent advancements in solid organ transplantation, African-American renal allograft recipients continue to exhibit poorer prognosis in long-term clinical outcome and graft survival compared to Caucasian patients. The role of immunosuppressants in post-transplant outcome is crucial, and associations between exposure-related pharmacokinetic parameters and clinical outcome have been made for several drugs in this class. Thus, ethnic differences in the pharmacokinetics of immunosuppressants are potentially a key factor in the observed differences in post-transplant outcome between African-Americans and Caucasians. Ethnic differences in pharmacokinetics of mycophenolate mofetil and azathioprine based on the current literature are either absent or only of minor relevance. Cyclosporine, tacrolimus, sirolimus and everolimus, however, have all been described to exhibit ethnicity-specific differences in bioavailability and/or dose-adjusted systemic exposure, although currently available reports are controversial for some of these drugs. Oral bioavailability of these drugs in African-Americans was between 20 and 50% lower than in Caucasians or Non-African-Americans, leading to higher dose requirements in African-Americans to maintain similar average concentrations of the respective immunosuppressant. Since all four drugs undergo extensive metabolism and are substrates for CYP3A isoenzymes as well as the drug transporter P-glycoprotein, interethnic variability in activity of these enzymes/transporter may provide a common mechanism for the observed ethnic differences. These ethnic differences are most likely mediated via several non-genetic as well as genetic factors, including known genetic variations that impair transporter/enzyme activity in genes such as CYP3A4, CYP3A5 and ABCB1 (MDR1). Appreciation of differences in immunosuppressant pharmacokinetics and dose requirements between African-Americans and Caucasians in clinical practice is expected to improve post-transplant immunosuppressive pharmacotherapy and may thus contribute to equalize prognostic outcome for all transplant patients.

Clinical PK/PD modelling as a tool in drug development of corticosteroids.

Corticosteroids are used for the treatment of a variety of different diseases both locally and systemically. Most therapeutic effects result from glucocorticoid receptor-mediated events, and there seems to be no substance-specific difference in the post-receptor reaction cascade. Therefore, the extent and duration of glucocorticoid effects depend only on the availability of the respective steroid at the receptor site and its affinity to the receptor. This makes glucocorticoids an ideal candidate for PK/PD modelling. Availability at the receptor site is governed by pharmacokinetic parameters such as bioavailability, clearance, protein binding, and volume of distribution. The receptor affinity can easily be measured in vitro. A suitable indirect-response PK/PD model is presented that allows description of the receptor-mediated drug effects such as endogenous cortisol suppression as a function of time. Furthermore, this model allows prediction of the systemic activity of newly developed corticosteroids based on their pharmacokinetics and their respective receptor-binding affinity. The model can also be applied in order to study systemic steroid effects after topical administration or to investigate the effect of the time of dosing on cortisol suppression. Comparison of predictions based on this model and results from large clinical studies are in excellent agreement. Corticosteroids may represent an ideal class of drugs for the successful use of PK/PD modelling during drug development allowing to save time and expenses.

Mechanism-based PK/PD model for the lymphocytopenia induced by endogenous and exogenous corticosteroids.

OBJECTIVE: Lymphocytopenia is a sensitive surrogate marker for the immunological effects of corticosteroids. This pharmacokinetic/pharmacodynamic (PK/PD) study investigated whether the circadian variation of blood lymphocytes observed after placebo is secondary to the circadian rhythm of endogenous cortisol, and developed based on this relationship an improved PK/PD model for a more sensitive description of the effect of low-dose corticosteroid therapy on blood lymphocytes considering the net activity of the exogenous corticosteroid budesonide and endogenous cortisol. METHODS: In an open, parallel study design, 3 mg oral budesonide or placebo were given at 8.00 a.m., 4.00 p.m. and midnight to two groups of 12 volunteers. Lymphocyte counts and serum concentrations of budesonide and cortisol were monitored for 24 hours. A mechanism-based PK/PD model which considered the non-linear protein binding of cortisol and the budesonide-induced cortisol suppression was employed to relate changes in blood lymphocytes to free cortisol levels after placebo and to the net activity of free budesonide and free endogenous cortisol after active treatment. RESULTS: The circadian rhythm of blood lymphocytes observed after placebo could inversely be related to the circadian rhythm of serum cortisol. After budesonide administration, lymphocyte counts could accurately be linked to the net activity of budesonide and endogenous cortisol. The resulting EC50 values for the effect of budesonide on cortisol, budesonide on lymphocytes and cortisol on lymphocytes were 0.063 +/- 0.034, 0.22 +/- 0.13 and 26.3 +/- 15.0 ng/ml (placebo group 15.4 +/- 3.4 ng/ml), respectively. CONCLUSIONS: The presented mechanism-based PK/PD model suggests that blood lymphocytes are under physiological control of cortisol. It further indicates that endogenous and exogenous corticosteroids and their pharmacological interaction need to be considered for modeling the effects of low doses of exogenous corticosteroids on the immune system.

Optimized therapeutic ratio of inhaled corticosteroids using retrometabolism.

During recent years, the treatment of pulmonary diseases could be significantly improved due to the introduction of modern retrometabolism-based corticosteroids with improved therapeutic ratio. It is the goal of all inhaled corticosteroids to produce long lasting therapeutic effects at the pulmonary target site and to minimize systemic side effects by rapid clearance of the absorbed drug and low oral bioavailability. The development of PK/PD models allows predictions of drug effects based on the administered dose. For example, the cumulative suppression of endogenous cortisol release (CCS) as one of the major systemic side effects of inhaled corticosteroid therapy can be described with an integrated Emax based PK/PD model. In order to assess the predictive power of this model, a study was conducted to compare the PK/PD-based predictions with CCS data obtained from actual clinical trials for flunisolide, fluticasone propionate, budesonide and triamcinolone acetonide. CCS was predicted for different single doses from different inhaler devices for each drug and a good correlation was observed. Thus, the presented PK/PD model proved to be a valid tool for predicting CCS of inhaled corticosteroids. By fully understanding the underlying mechanisms it will be possible to further improve their therapeutic index.

Physiologically based pharmacokinetic (PBPK) modeling in children.

This review summarizes the present status of physiologically based pharmacokinetic (PBPK) modeling and simulation (M&S) and its application in support of pediatric drug research. We address the reasons that PBPK is suited to the current needs of pediatric drug development and pharmacotherapy in light of the evolution in pediatric PBPK methodologies and approaches, which were originally developed for the purpose of toxicologic evaluation. Also discussed is the current degree of confidence in using PBPK to support pediatric drug development and registration and the key factors essential for robust results and broader adoption of pediatric PBPK M&S.

A modern view of excipient effects on bioequivalence: case study of sorbitol.

PURPOSE: To examine the effect of common excipients such as sugars (sorbitol versus sucrose) on bioequivalence between pharmaceutical formulations, using ranitidine and metoprolol as model drugs. METHODS: Two single-dose, replicated, crossover studies were first conducted in healthy volunteers (N=20 each) to compare the effect of 5 Gm of sorbitol and sucrose on bioequivalence of 150 mg ranitidine or 50 mg metoprolol in aqueous solution, followed by a single-dose, nonreplicated, crossover study (N=24) to determine the threshold of sorbitol effect on bioequivalence of 150 mg ranitidine in solution. RESULTS: Ranitidine Cmax and AUC0-infinity were decreased by approximately 50% and 45%, respectively, in the presence of sorbitol versus sucrose. Similarly, sorbitol reduced metoprolol Cmax by 23% but had no significant effect on AUC0-infinity. An appreciable subject-by-formulation interaction was found for ranitidine Cmax and AUC0-infinity, as well as metoprolol Cmax. Sorbitol decreased the systemic exposure of ranitidine in a dose-dependent manner and affected bioequivalence at a level of 1.25 Gm or greater. CONCLUSIONS: As exemplified by sorbitol, some common excipients have unexpected effect on bioavailability/bioequivalence, depending on the pharmacokinetic characteristics of the drug, as well as the type and amount of the excipient present in the formulation. More research is warranted to examine other 'common' excipients that may have unintended influence on bioavailability/bioequivalence.

Modeling of pharmacokinetic/pharmacodynamic (PK/PD) relationships: concepts and perspectives.

Pharmacokinetic/pharmacodynamic (PK/PD)-modeling links dose-concentration relationships (PK) and concentration-effect relationships (PD), thereby facilitating the description and prediction of the time course of drug effects resulting from a certain dosing regimen. PK/PD-modeling approaches can basically be distinguished by four major attributes. The first characterizes the link between measured drug concentration and the response system, direct link versus indirect link. The second considers how the response system relates effect site concentration to the observed outcome, direct versus indirect response. The third regards what clinically or experimentally assessed information is used to establish the link between concentration and effect, hard link versus soft link. And the fourth considers the time dependency of pharmacodynamic model parameters, distinguishing between time-variant versus time-invariant. Application of PK/PD-modeling concepts has been identified as potentially beneficial in all phases of preclinical and clinical drug development. Although today predominantly limited to research, broader application of PK/PD-concepts in clinical therapy will provide a more rational basis for patient-specific dosage individualization and may thus guide applied pharmacotherapy to a higher level of performance.