Evaluation of the Safety of Drugs and Biological Products Used During Lactation: Workshop Summary.

This report serves as a summary of a 2-day public workshop sponsored by the US Food and Drug Administration (FDA) to discuss the safety of drugs and biological products used during lactation. The aim of the workshop was to provide a forum to discuss the collection of data to inform the potential risks to breastfed infants with maternal use of medications during lactation. Discussions included the review of current approaches to collect data on medications used during lactation, and the considerations for future approaches to design and guide clinical lactation studies. This workshop is part of continuing efforts to raise the awareness of the public for women who choose to breastfeed their infants.

The Influence of Race and Common Genetic Variations on Outcomes After Pediatric Heart Transplantation.

Significant racial disparity remains in the incidence of unfavorable outcomes following heart transplantation. We sought to determine which pediatric posttransplantation outcomes differ by race and whether these can be explained by recipient demographic, clinical, and genetic attributes. Data were collected for 80 black and 450 nonblack pediatric recipients transplanted at 1 of 6 centers between 1993 and 2008. Genotyping was performed for 20 candidate genes. Average follow-up was 6.25 years. Unadjusted 5-year rates for death (p = 0.001), graft loss (p = 0.015), acute rejection with severe hemodynamic compromise (p = 0.001), late rejection (p = 0.005), and late rejection with hemodynamic compromise (p = 0.004) were significantly higher among blacks compared with nonblacks. Black recipients were more likely to be older at the time of transplantation (p < 0.001), suffer from cardiomyopathy (p = 0.004), and have public insurance (p < 0.001), and were less likely to undergo induction therapy (p = 0.0039). In multivariate regression models adjusting for age, sex, cardiac diagnosis, insurance status, and genetic variations, black race remained a significant risk factor for all the above outcomes. These clinical and genetic variables explained only 8-19% of the excess risk observed for black recipients. We have confirmed racial differences in survival, graft loss, and several rejection outcomes following heart transplantation in children, which could not be fully explained by differences in recipient attributes.

Pharmacogenomic information in FDA-approved drug labels: Application to pediatric patients.

Pharmacogenomic (PGx) information is increasingly being incorporated into US Food and Drug Administration-approved drug labels. We reviewed the data source (adults vs. pediatrics) of PGx information in approved drug labels and assessed the suitability of applying adult-derived PGx information and related prescribing recommendations to the care of pediatric patients. We identified 65 drugs with labels containing PGx information and that have also been evaluated in children and found that in the majority of cases (56/65, 86%), the PGx information described was derived from adult studies. The application of PGx information from adults to pediatrics was deemed suitable for 71.4% (n = 40) of the drugs and unclear for 28.6% (n = 16). An ontogeny effect, limited or conflicting data regarding ontogeny of the genetic biomarker, or a difference in the pathophysiology or progression of the adult vs. pediatric disease were the primary reasons for deeming direct application from adults to pediatrics unclear.

A Survey of Neonatal Pharmacokinetic and Pharmacodynamic Studies in Pediatric Drug Development.

Conducting clinical trials in neonates is challenging, and knowledge gaps in neonatal clinical pharmacology exist. We surveyed the US Food and Drug Administration databases and identified 43 drugs studied in neonates or referring to neonates between 1998 and 2014. Twenty drugs were approved in neonates. For 10 drugs, approval was based on efficacy data in neonates, supplemented by pharmacokinetic data for four drugs. Approval for neonates was based on full extrapolation from older patients for six drugs, and partial extrapolation was the basis of approval for four drugs. Dosing recommendations differed from older patients for most drugs, and used body-size based adjustment in neonates. Trial failures were associated with various factors including inappropriate dose selection. Successful drug development in neonates could be facilitated by an improved understanding of the natural history and pathophysiology of neonatal diseases and identification and validation of clinically relevant biomarkers.

Failed Pediatric Drug Development Trials.

Pediatric product development initiatives have stimulated the development of therapies for children, resulting in improved product labeling, increased identification of adverse events, and development of new pediatric formulations. However, 42% of recently completed pediatric trials have failed to establish either safety or efficacy, leading to an inability to label the product for use in children.(1) Characterizing these failed trials, including common contributing factors, is imperative to designing better pediatric trials in the future.

Regulatory experience with physiologically based pharmacokinetic modeling for pediatric drug trials.

Physiologically based pharmacokinetic (PBPK) approaches that incorporate the developmental physiology and ontogeny of cytochrome P450 (CYP) enzymes may have value in the design of pediatric trials. Four recent submissions to the US Food and Drug Administration (FDA) incorporated different PBPK applications to pediatric drug development.Further testing of PBPK models for three drugs showed that these models generally under predicted drug clearance. PBPK modeling may have potential for improving pediatric trials through the learn-and-confirm approaches utilized in current regulatory submissions.

Biomarkers in solid organ transplantation.

This workshop was organized by the US Food and Drug Administration (FDA) Office of Clinical Pharmacology Review Team supporting the Division of Special Pathogen and Transplant Products in the Center for Drug Evaluation and Research. The main goal of the workshop was to enhance the knowledge base regarding biomarkers in solid-organ transplantation via presentation and discussion of scientific findings.

Pediatric dose selection.

Selection of a drug dose in pediatrics is generally based on no or incomplete pharmacokinetic data. Traditionally, allometric, or scaling, techniques have been used; however, they have inherent limitations and may not make optimal use of the drug-specific clinical pharmacokinetic information that is available. Modeling is a tool that holds promise. The future challenge is to create a structured approach to determining pediatric doses for new therapeutic agents.

Species-dependent hepatic metabolism of immunosuppressive agent tacrolimus (FK-506).

The current study aims to investigate species-related differences in the in-vitro hepatic metabolism of tacroliums using liver microsomes obtained from rat, hamster, guinea pig, rabbit, pig, dog, baboon and humans. Tacrolimus metabolism was characterized using high-performance liquid chromatography- ultraviolet light (HPLC-UV) and two soft ionization mass spectrometric techniques; matrix-assisted lasers desorption/ionization (MALDI) and time-of-flight-secondary ion mass spectrometry (TOF-SIMS). The extent of tacrolimus metabolism, when normalized to the cytochrome P-450 content, was in the order: rat < hamster < rabbit < pig < guinea pig < dog < human < baboon. Tacrolimus metabolism exhibited significant qualitative and quantitative differences between the animal species tested. Desmethyl- (MI-MIII), didesmethyl- (MIV-MVI), monohydroxy- (MVII), dihydroxy- (MVIII), epoxide- (MIX), dihydrodiol- (MX), monodesmethyl and monohydroxy- (MXI-MXIII), and didesmethyl and monohydroxy- (MXIV-MXVI) tacroliums metabolites were identified in the species tested. MI-MX were identified in all the species tested; MXI-MXVI were identified in all species except rat, rabbit and guinea pig; and MXIV-MXVI were identified only in baboon. The current investigation was unable to detect any phase II metabolites due to the limitations of the test system used. The analytical methods were not able to differentiate optical and positional isomers of metabolites due to the nature of the analytical tools used, therefore groups of metabolites were identified based on their molecular weights and available information. From the current in-vitro metabolism studies, the pattern of tacroliums metabolism in baboons closely resembled that in humans and thus it is ideal for studying tacroliums metabolism-related work of clinical relevance.

Assessment of the impact of renal impairment on systemic exposure of new molecular entities: evaluation of recent new drug applications.

The US Food and Drug Administration (FDA) is currently developing a guidance for industry to replace a previous guidance, "Pharmacokinetics in Patients With Impaired Renal Function--Study Design, Data Analysis, and Impact on Dosing and Labeling" (renal guidance) issued in May 1998. The impact of the 1998 renal guidance was assessed following a survey of 94 new drug applications (NDAs) for small-molecule new molecular entities (NMEs) approved over the past 5 years (2003-2007). The survey results indicate that 57% of these NDAs included renal impairment study data, that 44% of those with renal data included evaluation in patients on hemodialysis, and that 41% of those with renal data resulted in recommendation of dose adjustment in renal impairment. In addition, the survey results provided evidence that renal impairment can affect the pharmacokinetics of drugs that are predominantly eliminated by nonrenal processes such as metabolism and/or active transport. The latter finding supports our updated recommendation to evaluate pharmacokinetic/pharmacodynamic alterations in renal impairment for those drugs that are mainly eliminated by nonrenal processes, in addition to those that are mainly excreted unchanged by the kidney.

IMPDH1 gene polymorphisms and association with acute rejection in renal transplant patients.

Inosine 5'-monophosphate dehydrogenase 1 (IMPDH1) catalyzes the rate-limiting step of the de novo pathway for purine synthesis and is a major target of the immunosuppressive drug mycophenolic acid (MPA). Few variants of the IMPDH1 gene have been reported. The objective of this study was to identify and characterize IMPDH1 variants to determine whether genetic variation contributes to differences in MPA response and toxicity in transplant patients. Seventeen genetic variants were identified in the IMPDH1 gene with allele frequencies ranging from 0.2 to 42.7%. In this study, 191 kidney transplant patients who received mycophenolate mofetil were genotyped for IMPDH1. Two single-nucleotide polymorphisms, rs2278293 and rs2278294, were significantly associated with the incidence of biopsy-proven acute rejection in the first year post-transplantation. Future studies of the multifactorial nature of acute rejection must consider IMPDH1 polymorphisms in MPA-treated patients.

The impact of pharmacogenomic factors on acute persistent rejection in adult lung transplant patients.

Persistent rejection in the face of treatment and multiple episodes of rejection are associated with the development of chronic rejection and graft loss in solid organ transplantation. The factors that create an environment for rejection that persists in the face of treatment are as yet not understood. The objective of this study was to evaluate the risk factors, including human multidrug resistance gene (MDR1), cytochrome P4503A5 (CYP3A5) and cytokine gene polymorphisms, associated with acute persistent rejection (APR) in lung transplant patients. One hundred and twenty-five adult lung transplant patients were studied. MDR1 G2677T, C3435T and CYP3A5 polymorphisms were assessed by direct sequencing of the polymorphic region in patient DNA. Cytokine genotyping for five cytokines was performed using the polymerase chain reaction-sequence specific primers (PCR-SSP) technique. Multivariate regression analysis was used to identify the predictors of acute persistent rejection. The dependent variable was the presence or absence of acute persistent rejection based on lung biopsies during the first postoperative year. The independent variables were MDR1 G2677T and C3435T, CYP4503A5 and cytokine polymorphisms, survival status, age, gender, survival days and HLA mismatches. The MDR1 C3435T polymorphism and age were independently associated with acute persistent rejection (p = 0.025, odds ratio = 0.29, 95% CI 0.1-0.86 and p = 0.016, odds ratio = 0.94, 95% CI 0.89-0.98, respectively). For the MDR1 C3435T polymorphism, 72% of patients with the C allele had acute persistent rejection in comparison to 52% for TT patients (p = 0.04). For age, a significant difference was found between the nonrejection group and the rejection group (mean+/-S.D. 52.1+/-11.2 vs. 44.4+/-12.3, p = 0.01). This is the first report of the association of a drug disposition genotype with drug-resistant acute rejection in organ transplant patients. The major predictor of acute persistent rejection in the first postoperative year for lung transplant patients was the MDR1 C3435T genotype. This association could be due to drug resistance, altered drug disposition or other immunologic effects associated with P-glycoprotein (P-gp) function. Future prospective treatment algorithms should be developed that will incorporate the knowledge of gene polymorphisms into treatment regimens to improve the outcome following lung transplantation.

The impact of pharmacogenomic factors on steroid dependency in pediatric heart transplant patients using logistic regression analysis.

Many pharmacogenomic predictors of drug response are now available, and include both drug metabolism-disposition factors and drug targets. Information on statistical approaches to analyzing large clinical data sets in relation to genetic polymorphisms is limited. The objective of this study was to evaluate whether logistic regression could identify pharmacogenomic predictors of outcome in a large data set in a complex transplant patient population. Seventy pediatric heart transplant patients were studied. Patients were followed for at least 1 yr post-transplantation as outpatients, and weaned from corticosteroids if clinically appropriate. Logistic regression analysis was used to identify the predictors of steroid dependency. The dependent variable was the presence or absence of steroid therapy at 1 yr post-transplantation. The independent variables were the patients' transplant age, gender, MDR1 C3435T and G2677T, CYP3A53B and cytokine polymorphisms. By chi-square test for the MDR1 C3435T polymorphism, 12 of 18 (67%) patients in the CC group were still on prednisone, whereas only 18 of 47 (38%) of the CT/TT group were still receiving prednisone (p = 0.04). For the IL-10 groups, two of 15 patients with the high producer genotype (13.3%) remained on prednisone, in comparison with 16 of 28 patients with the intermediate producer genotype (57.1%) and 15 of 26 patients with the low producer genotype (57.7%, p = 0.01). Logistic regression analysis confirmed MDR1 C3435T (p = 0.021), and IL-10 polymorphisms (intermediate producer genotype p = 0.015; low producer genotype p = 0.013) as independent risk factors for steroid dependency at 1 yr after transplantation. This approach identifies pharmacogenomic factors, which can be studied more extensively in larger data sets, and used in prospective studies to individualize immunosuppressive therapy following solid organ transplantation.

Preservation of post-transplant lung function with aerosol cyclosporin.

Post-lung transplant use of aerosol cyclosporin (ACsA) is considered by examining the relationship between deposited aerosol dose and effect. In a sub-study of placebo controlled trials of ACsA as a rejection prophylaxis, 15 drug subjects received aerosol dose quantification tests to gage their ability to effectively deposit the nebulised drug in their transplanted lung(s). A total of seven placebo subjects received mock deposition tests. The deposited doses and mock doses were compared to changes in the forced expiratory volume in one second, at six time points during the 2-yr trial period (ACsA was started within 6 weeks post-transplant). Linear relationships were demonstrated between deposited dose and improvement in lung function in the drug subjects at all intervals. Mock dose data from placebo subjects did not demonstrate similar correlation. Based on these results, subjects were grouped by dose and compared. Subjects depositing > or = 5 mg of the drug in the periphery of their transplant(s) had improving pulmonary function on average. Low-dose and placebo subjects demonstrated declines, more A2-A4 rejection events in the latter portion of the trial, and more chronic rejection beyond the end of the trial. A dose-to-effect relationship is demonstrated for aerosol cyclosporin in terms of pulmonary function and biopsy proven rejection.

Measurement of basal, substrate induced and total P-glycoprotein activity in bronchoalveolar lavage T-cell subsets.

BACKGROUND: P-glycoprotein (P-gp) is a member of the ABC transporter superfamily. P-gp activity can be detected by measuring efflux of fluorescent substrates such as rhodamine 123 (R123). Our objectives were to evaluate P-gp activity in T cells freshly isolated from bronchoalveolar lavage (BAL) and to develop a strategy to distinguish between basal, in vitro substrate-induced, and total P-gp activities. METHODS: Cells were obtained from blood (n = 44) and BAL (n = 34), stained for expression of CD3, CD4, CD8, and CD14, and incubated with R123 (0.13 microM) +/- cyclosporine (5 microM), a specific P-gp inhibitor. P-gp activity was detected as median fluorescence intensity (MFI) and percentage of cells falling below a pre-established cutoff. RESULTS: BAL T cells displayed significant basal P-gp activity, which was most apparent when measured as percentage below the cutoff. Induced activity (difference between P-gp activity measured after load and efflux) was determined equally well when using the MFI or the percentage below cutoff parameter. Total activity was represented by the efflux parameters (MFI or percentage below cutoff) or by the activity-time area under the curve (AUC) method. The two efflux parameters correlated well but were insensitive to the time-dependent nature of dye efflux. In the AUC method, two samples with identical R123 brightness or percentage below cutoff after dye efflux can have very different total activities, depending on their basal activity. The AUC method was also most sensitive in distinguishing between P-gp activity in peripheral blood and resident lung T cells. Application of this methodology to the comparison of P-gp activity in BAL and peripheral CD8+ T cells best revealed the elevated total P-gp activity in BAL T cells. CONCLUSIONS: We have systematically evaluated several methodologies for analysis of P-gp activity and arrived at a novel and robust strategy amenable to standardization and evaluation of the effects of P-gp modulators in vivo and in vitro.

P-glycoprotein (P-gp) is upregulated in peripheral T-cell subsets from solid organ transplant recipients.

Immunosuppressive agents such as cyclosporine, tacrolimus, sirolimus, and corticosteroids are substrates for the transmembrane multidrug resistance pump P-glycoprotein (P-gp). Experience in oncologyhas suggested that chronic exposure to P-gp substrates induces upregulation of P-gp activity, which could result in resistance to immunosuppressive drugs. The authors investigated P-gp function in CD4+ and CD8+ T cells from the peripheral blood of solid organ transplant recipients (SOTX). Subjects included 14 stable SOTX (10 liver, 4 lung) and 16 healthy controls. Four-color flow cytometry was used to simultaneously measure intracellular concentration of the fluorescent P-gp substrate Rhodamine 123 (Rh123) and surface expression of CD45RO (nominal memory/effector), CD45RA (naive), and either CD4 or CD8. P-glycoprotein function was measured by a dye efflux assay in which activity was inferred from a decrease in Rh123 fluorescence. CD4+ and CD8+ T cells from patients and control subjects eliminated Rh123, and this activity was inhibited by verapamil, a known P-gp substrate. CD8+ T cells had greater P-gp activity than CD4+ cells, and naive and transitional T cells displayed greater activity than memory T cells. Activity was bimodal in CD8+ CD45RO+ T cells, with a subset of these cells expressing the greatest P-gp activity. Patient CD8+ naive and transitional T cells had upregulated P-gp activity compared to control subjects. We conclude that (1) P-gp activityis significantly upregulated in specific T-cell subsets (CD8+/CD45RA+) in the peripheral blood of SOTX, and (2) the bimodal nature of P-gp response in CD8+ T cells complicates analysis of the effect of chronic administration of P-gp substrates to SOTX.

In the lung aerosol cyclosporine provides a regional concentration advantage over intramuscular cyclosporine.

BACKGROUND: Acute rejection remains an almost universal complication among lung transplant recipients. Refractory rejection as well as chronic systemic immunosuppression is associated with significant morbidity and mortality. Recent studies suggest that aerosol cyclosporine may address these issues by effectively preventing acute cellular rejection while maintaining low systemic drug concentrations. This study was designed to evaluate the concentrations of cyclosporine in blood and lung tissue after aerosol and intramuscular administration. METHODS: Lewis rats were divided into 4 experimental groups: Groups A (n = 33) and B (n = 30) received aerosol cyclosporine 3 and 5 mg/kg, respectively; Groups C (n = 33) and D (n = 30) received systemic cyclosporine 5 and 15 mg/kg, respectively. We used high-performance liquid chromatography to quantitate blood and lung tissue cyclosporine levels at timed intervals. We used the trapezoidal rule to approximate area under the concentration vs time curve (AUC). RESULTS: Aerosol delivery of cyclosporine resulted in higher and more rapid peak drug levels in lung tissue samples than did systemic delivery. At an equivalent 5 mg/kg dose, the cyclosporine AUC was 3 times higher with aerosol delivery than with intramuscular delivery in lung tissue (477,965 vs 157,706 ng x hour/g, respectively). The lung tissue: blood AUC ratio was highest in the aerosol groups (27.3:1 and 17.4:1) compared with the intramuscular groups (8.1:1 and 9.4:1). CONCLUSION: Local aerosol inhalation delivery of cyclosporine provides a regional advantage over systemic intramuscular therapy by providing higher peak concentrations and greater lung tissue exposure.