Transport vs. Metabolism: What Determines the Pharmacokinetics and Pharmacodynamics of Drugs? Insights From the Extended Clearance Model.

The well-stirred hepatic clearance model (WSHM) has been expanded to include drug transporters (i.e., extended clearance model [ECM]). However, the consequences of this expansion in understanding when transporters vs. metabolic enzymes will affect the pharmacokinetic (PK) and pharmacodynamic (PD) of drugs remains opaque. Identifying the rate-determining step(s) in systemic or tissue drug PK/PD will allow accurate predictions of drug PK/PD and drug-drug interactions (DDIs). Here, we clarify the implications of the ECM on PK/PD of drugs.

Ontogeny of Hepatic Drug Transporters as Quantified by LC-MS/MS Proteomics.

Protein expression of major hepatic uptake and efflux drug transporters in human pediatric (n = 69) and adult (n = 41) livers was quantified by liquid chromatography / tandem mass spectroscopy (LC-MS/MS). Transporter protein expression of OCT1, OATP1B3, P-gp, and MRP3 was age-dependent. Particularly, significant differences were observed in transporter expression (P < 0.05) between the following age groups: neonates vs. adults (OCT1, OATP1B3, P-gp), neonates or infants vs. adolescents and/or adults (OCT1, OATP1B3, and P-gp), infants vs. children (OATP1B3 and P-gp), and adolescents vs. adults (MRP3). OCT1 showed the largest increase, of almost 5-fold, in protein expression with age. Ontogenic expression of OATP1B1 was confounded by genotype and was revealed only in livers harboring SLCO1B1*1A/*1A. In livers >1 year, tissues harboring SLCO1B1*14/*1A showed 2.5-fold higher (P < 0.05) protein expression than SLCO1B1*15/*1A. Integration of these ontogeny data in physiologically based pharmacokinetic (PBPK) models will be a crucial step in predicting hepatic drug disposition in children.

Interindividual variability of CYP2C19-catalyzed drug metabolism due to differences in gene diplotypes and cytochrome P450 oxidoreductase content.

Large interindividual variability has been observed in the metabolism of CYP2C19 substrates in vivo. The study aimed to evaluate sources of this variability in CYP2C19 activity, focusing on CYP2C19 diplotypes and the cytochrome P450 oxidoreductase (POR). CYP2C19 gene analysis was carried out on 347 human liver samples. CYP2C19 activity assayed using human liver microsomes confirmed a significant a priori predicted rank order for (S)-mephenytoin hydroxylase activity of CYP2C19*17/*17 > *1B/*17 > *1B/*1B > *2A/*17 > *1B/*2A > *2A/*2A diplotypes. In a multivariate analysis, the CYP2C19*2A allele and POR protein content were associated with CYP2C19 activity. Further analysis indicated a strong effect of the CYP2C19*2A, but not the *17, allele on both metabolic steps in the conversion of clopidogrel to its active metabolite. The present study demonstrates that interindividual variability in CYP2C19 activity is due to differences in both CYP2C19 protein content associated with gene diplotypes and the POR concentration.The Pharmacogenomics Journal advance online publication, 1 September 2015; doi:10.1038/tpj.2015.58.

Spontaneous resolution of acute rejection and tolerance induction with IL-2 fusion protein in vascularized composite allotransplantation.

Vascularized composite allotransplantation (VCA) has emerged as a treatment option for treating nonlife-threatening conditions. Therefore, in order to make VCA a safe reconstruction option, there is a need to minimize immunosuppression, develop tolerance-inducing strategies and elucidate the mechanisms of VCA rejection and tolerance. In this study we explored the effects of hIL-2/Fc (a long-lasting human IL-2 fusion protein), in combination with antilymphocyte serum (ALS) and short-term cyclosporine A (CsA), on graft survival, regulatory T cell (Treg) proliferation and tolerance induction in a rat hind-limb transplant model. We demonstrate that hIL-2/Fc therapy tips the immune balance, increasing Treg proliferation and suppressing effector T cells, and permits VCA tolerance as demonstrated by long-term allograft survival and donor-antigen acceptance. Moreover, we observe two distinct types of acute rejection (AR), progressive and reversible, within hIL-2/Fc plus ALS and CsA treated recipients. Our study shows differential gene expression profiles of FoxP3 versus GzmB, Prf1 or interferon-γ in these two types of AR, with reversible rejection demonstrating higher Treg to Teff gene expression. This correlation of gene expression profile at the first clinical sign of AR with VCA outcomes can provide the basis for further inquiry into the mechanistic aspects of VCA rejection and future drug targets.

The concept of fraction of drug transported (ft ) with special emphasis on BBB efflux of CNS and antiretroviral drugs.

The fraction of drug transported (ft) into or out of a tissue is a concept useful to understand the impact of transporters on absorption, distribution, metabolism, and excretion (ADME) and tissue distribution of a drug. Here, ft is utilized to explain the impact of transporters on central nervous system (CNS) distribution of drugs, drug interactions (DDI), and to predict the unbound brain concentration (Cu,b) of the drug. The latter is important to ascertain if Cu,b is sufficient for efficacy.

Evaluation of various static and dynamic modeling methods to predict clinical CYP3A induction using in vitro CYP3A4 mRNA induction data.

Several drug-drug interaction (DDI) prediction models were evaluated for their ability to identify drugs with cytochrome P450 (CYP)3A induction liability based on in vitro mRNA data. The drug interaction magnitudes of CYP3A substrates from 28 clinical trials were predicted using (i) correlation approaches (ratio of the in vivo peak plasma concentration (Cmax) to in vitro half-maximal effective concentration (EC50); and relative induction score), (ii) a basic static model (calculated R3 value), (iii) a mechanistic static model (net effect), and (iv) mechanistic dynamic (physiologically based pharmacokinetic) modeling. All models performed with high fidelity and predicted few false negatives or false positives. The correlation approaches and basic static model resulted in no false negatives when total Cmax was incorporated; these models may be sufficient to conservatively identify clinical CYP3A induction liability. Mechanistic models that include CYP inactivation in addition to induction resulted in DDI predictions with less accuracy, likely due to an overprediction of the inactivation effect.

A PBPK Model to Predict Disposition of CYP3A-Metabolized Drugs in Pregnant Women: Verification and Discerning the Site of CYP3A Induction.

Besides logistical and ethical concerns, evaluation of safety and efficacy of medications in pregnant women is complicated by marked changes in pharmacokinetics (PK) of drugs. For example, CYP3A activity is induced during the third trimester (T3). We explored whether a previously published physiologically based pharmacokinetic (PBPK) model could quantitatively predict PK profiles of CYP3A-metabolized drugs during T3, and discern the site of CYP3A induction (i.e., liver, intestine, or both). The model accounted for gestational age-dependent changes in maternal physiological function and hepatic CYP3A activity. For model verification, mean plasma area under the curve (AUC), peak plasma concentration (Cmax), and trough plasma concentration (Cmin) of midazolam (MDZ), nifedipine (NIF), and indinavir (IDV) were predicted and compared with published studies. The PBPK model successfully predicted MDZ, NIF, and IDV disposition during T3. A sensitivity analysis suggested that CYP3A induction in T3 is most likely hepatic and not intestinal. Our PBPK model is a useful tool to evaluate different dosing regimens during T3 for drugs cleared primarily via CYP3A metabolism.CPT: Pharmacometrics & Systems Pharmacology (2012) 1, e3; doi:10.1038/psp.2012.2; advance online publication 26 September 2012.

Biologics and donor bone marrow cells for targeted immunomodulation in vascularized composite allotransplantation: a translational trial in swine.

INTRODUCTION: Bone marrow (BM) infusion following organ transplantation is a prerequisite for potential donor-antigen-specific tolerance induction. We developed a preclinical swine model to determine the optimal dose of BM cells to achieve microchimerism. Furthermore, induction therapy was optimized by augmenting the BM infusion with biologics in the form of costimulatory blockade: cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4-Ig). MATERIALS AND METHODS: Yucatan miniature swine (n = 12) underwent total body and thymic irradiation for cytodepletion. Animal groups received 15, 30, or 60 million cells per kilogram of whole unmodified BM. The optimal dose of BM cell infusion (BMT) was then applied to subsequent experiments evaluating the addition of CTLA4lg. Group 1 (control) received no treatment. Group 2 received FK506 only; group 3 received irradiation, BMT, and FK506; group 4 received FK506 and CTLA4-lg. RESULTS: Microchimerism was established in all animals after BM cell infusion; at postoperative day 9, it was significantly increased for 60 million cells per kilogram (P = .0001). Transplanted animals in group 1 rejected the allograft 5 to 8 days after transplantation. Group 2 rejected the allograft (skin and muscle) 30 to 32 days after transplantation (2 days after cessation of immunosuppression). Group 3 rejected the skin portion of the allograft at 50, 52, and 53 days posttransplant. Remaining allograft components (muscle, bone, nerve, vessel) survived indefinitely. Group 4 animals demonstrated significantly prolonged muscle survival beyond 150 days posttransplant; the skin component survived past 150 days in two of three animals. Skin and muscle histology in all long-term surviving animals were normal. CONCLUSIONS: BM cell infusion with 60 million cells per kilogram results in stable levels of microchimerism. The addition of costimulatory blockade (CTLA4lg) prolonged allograft skin survival and overall graft survival. Such targeted immunomodulatory protocols might facilitate immune tolerance and eliminate the need for multidrug immunosuppression to maintain graft survival after vascularized composite allotransplantation.

Regional P-glycoprotein activity and inhibition at the human blood-brain barrier as imaged by positron emission tomography.

We used positron emission tomography (PET) to evaluate the contribution of P-glycoprotein (P-gp), present at the human blood-brain barrier (BBB), to regional drug distribution in the brain. Eleven healthy volunteers underwent PET imaging with [(11)C]-verapamil before and during cyclosporine A infusion. Regional P-gp inhibition was expressed as cyclosporine A-induced percentage change in the distributional clearance of verapamil (K(1)) in the brain, normalized to the regional blood flow (rCBF). K(1) estimates were similar across gray-matter regions of the brain and lower in the white matter regions, but all these estimates were considerably lower than rCBF. Normalization of K(1) by rCBF diminished the differences in estimates related to gray matter and white matter. In contrast, the K(1) for the pituitary, which is situated outside the BBB, approximated the rCBF. The magnitude of P-gp inhibition was comparable across BBB-protected brain structures. Our results indicate that P-gp and its inhibition equally affect the distribution of drugs (and therefore their neuro-efficacy and toxicity) in the various brain regions protected by the BBB.

Composite tissue vasculopathy and degeneration following multiple episodes of acute rejection in reconstructive transplantation.

Transplant vasculopathy has not been systematically investigated in composite tissue allotransplantation (CTA). The impact of multiple acute rejections (ARs) on long-term graft outcomes in reconstructive transplantation remains unknown. This study in a rat hind-limb allotransplantation model systematically analyzes vasculopathy and tissue-specific pathological changes secondary to multiple AR episodes. LEW rats were transplanted with BN rat hind limbs and treated as follows: Group 1 (Iso): isografts. Group 2 (CsA): Cyclosporine (CsA) qd; Group 3 (mult AR): CsA and dexamethasone only when AR was observed. No AR was observed in Groups 1 and 2. Multiple AR were observed in Group 3, and each episode was completely reversed (clinically) with pulsed CsA + dexamethasone treatment. Group 3 animals demonstrated significant vascular lesions along with skin and muscle atrophy, upregulation of profibrotic gene expression and fibrosis when compared to Groups 1 and 2. In addition, allograft bone was sclerotic, weak and prone to malunion and nonunion. Interestingly, vasculopathy was a late finding, whereas muscle atrophy with macrophage infiltration was seen early, after only a few AR episodes. Taken together, multiple AR episodes lead to vasculopathy and tissue-specific pathology in CTA. This is the first evidence of 'composite tissue vasculopathy and degeneration (CTVD)' in CTA.