Investigating the Replacement of Carboxylates with Carboxamides to Modulate the Safety and Efficacy of Platinum(II) Thioether Cyanide Scavengers.

Cyanide represents a persistent threat for accidental or malicious misuse due to easy conversion into a toxic gas and access to large quantities through several industries. The high safety index of hydroxocobalamin is a cornerstone quality as a cyanide scavenger. Unfortunately, intravenous infusion of hydroxocobalamin limits the utility in a mass casualty setting. We previously reported platinum(II) [Pt(II)] complexes with trans-directing sulfur ligands as an efficacious alternative to hydroxocobalamin when delivered by a bolus intramuscular injection in mice and rabbits. Thus, to enable Pt(II) as an alternative to hydroxocobalamin, a high safety factor is needed. The objective is to maintain efficacy and mitigate the risk for nephrotoxicity. Platinum amino acid complexes with the ability to form five- or six-membered rings and possessing either carboxylates or carboxamides are evaluated in vitro for cyanide scavenging. In vivo efficacy was evaulated in the zebrafish and mice cyanide exposure models. In addition, Pt(II) complex toxicity and pharmacokinetics were evaluated in a cyanide naive Sprague-Dawley model. Doses for toxicity are escalated to 5x from the efficacious dose in mice using a body surface area adjustment. The results show the carboxamide ligands display a time and pH dependence on cyanide scavenging in vitro and efficacy in vivo. Additionally, exchanging the carboxylate for carboxamide showed reduced indications of renal injury. A pharmacokinetic analysis of the larger bidentate complexes displayed rapid absorption by intramuscular administration and having similar plasma exposure. These findings point to the importance of pH and ligand structures for methionine carboxamide complexes with Pt(II).

Intramuscular administration of glyoxylate rescues swine from lethal cyanide poisoning and ameliorates the biochemical sequalae of cyanide intoxication.

Cyanide-a fast-acting poison-is easy to obtain given its widespread use in manufacturing industries. It is a high-threat chemical agent that poses a risk of occupational exposure in addition to being a terrorist agent. FDA-approved cyanide antidotes must be given intravenously, which is not practical in a mass casualty setting due to the time and skill required to obtain intravenous access. Glyoxylate is an endogenous metabolite that binds cyanide and reverses cyanide-induced redox imbalances independent of chelation. Efficacy and biochemical mechanistic studies in an FDA-approved preclinical animal model have not been reported. Therefore, in a swine model of cyanide poisoning, we evaluated the efficacy of intramuscular glyoxylate on clinical, metabolic, and biochemical endpoints. Animals were instrumented for continuous hemodynamic monitoring and infused with potassium cyanide. Following cyanide-induced apnea, saline control or glyoxylate was administered intramuscularly. Throughout the study, serial blood samples were collected for pharmacokinetic, metabolite, and biochemical studies, in addition, vital signs, hemodynamic parameters, and laboratory values were measured. Survival in glyoxylate-treated animals was 83% compared with 12% in saline-treated control animals (p < .01). Glyoxylate treatment improved physiological parameters including pulse oximetry, arterial oxygenation, respiration, and pH. In addition, levels of citric acid cycle metabolites returned to baseline levels by the end of the study. Moreover, glyoxylate exerted distinct effects on redox balance as compared with a cyanide-chelating countermeasure. In our preclinical swine model of lethal cyanide poisoning, intramuscular administration of the endogenous metabolite glyoxylate improved survival and clinical outcomes, and ameliorated the biochemical effects of cyanide.

Identification of Platinum(II) Sulfide Complexes Suitable as Intramuscular Cyanide Countermeasures.

The development of rapidly acting cyanide countermeasures using intramuscular injection (IM) represents an unmet medical need to mitigate toxicant exposures in mass casualty settings. Previous work established that cisplatin and other platinum(II) or platinum(IV)-based agents effectively mitigate cyanide toxicity in zebrafish. Cyanide's in vivo reaction with platinum-containing materials was proposed to reduce the risk of acute toxicities. However, cyanide antidote activity depended on a formulation of platinum-chloride salts with dimethyl sulfoxide (DMSO) followed by dilution in phosphate-buffered saline (PBS). A working hypothesis to explain the DMSO requirement is that the formation of platinum-sulfoxide complexes activates the cyanide scavenging properties of platinum. Preparations of isolated NaPtCl5-DMSO and Na (NH3)2PtCl-DMSO complexes in the absence of excess DMSO provided agents with enhanced reactivity toward cyanide in vitro and fully recapitulated in vivo cyanide rescue in zebrafish and mouse models. The enhancement of the cyanide scavenging effects of the DMSO ligand could be attributed to the activation of platinum(IV) and (II) with a sulfur ligand. Unfortunately, the efficacy of DMSO complexes was not robust when administered IM. Alternative Pt(II) materials containing sulfide and amine ligands in bidentate complexes show enhanced reactivity toward cyanide addition. The cyanide addition products yielded tetracyanoplatinate(II), translating to a stoichiometry of 1:4 Pt to each cyanide scavenger. These new agents demonstrate a robust and enhanced potency over the DMSO-containing complexes using IM administration in mouse and rabbit models of cyanide toxicity. Using the zebrafish model with these Pt(II) complexes, no acute cardiotoxicity was detected, and dose levels required to reach lethality exceeded 100 times the effective dose. Data are presented to support a general chemical design approach that can expand a new lead candidate series for developing next-generation cyanide countermeasures.

Multispecific targeting of glioblastoma with tumor microenvironment-responsive multifunctional engineered NK cells.

Tumor antigen heterogeneity, a severely immunosuppressive tumor microenvironment (TME) and lymphopenia resulting in inadequate immune intratumoral trafficking, have rendered glioblastoma (GBM) highly resistant to therapy. To address these obstacles, here we describe a unique, sophisticated combinatorial platform for GBM: a cooperative multifunctional immunotherapy based on genetically engineered human natural killer (NK) cells bearing multiple antitumor functions including local tumor responsiveness that addresses key drivers of GBM resistance to therapy: antigen escape, immunometabolic reprogramming of immune responses, and poor immune cell homing. We engineered dual-specific chimeric antigen receptor (CAR) NK cells to bear a third functional moiety that is activated in the GBM TME and addresses immunometabolic suppression of NK cell function: a tumor-specific, locally released antibody fragment which can inhibit the activity of CD73 independently of CAR signaling and decrease the local concentration of adenosine. The multifunctional human NK cells targeted patient-derived GBM xenografts, demonstrated local tumor site-specific activity in the tissue, and potently suppressed adenosine production. We also unveil a complex reorganization of the immunological profile of GBM induced by inhibiting autophagy. Pharmacologic impairment of the autophagic process not only sensitized GBM to antigenic targeting by NK cells but promoted a chemotactic profile favorable to NK infiltration. Taken together, our study demonstrates a promising NK cell-based combinatorial strategy that can target multiple clinically recognized mechanisms of GBM progression simultaneously.

The CUL3/neddylation inhibitor MLN4924 reduces ethanol-induced locomotor sensitization and inflammatory pain allodynia in mice.

Heterologous sensitization of adenylyl cyclase (AC) is defined by an enhanced cAMP response following persistent activation of Gαi/o-coupled receptors. This phenomenon was first observed in cellular models, and later reported in animal models of inflammatory pain or following chronic exposure to drugs of abuse including opioids and cocaine. Recently, we used genome-wide siRNA screening to identify Cullin3 signaling as a mediator of AC sensitization in cellular models. We also showed that pharmacological inhibition of Cullin3 with the neddylation inhibitor, MLN4924, abolished heterologous sensitization of several AC isoforms, including AC1, AC2, AC5, and AC6. Because ACs, especially AC1, have been implicated in alcohol-induced locomotor sensitization and inflammatory pain, we assessed the potential activity of MLN4924 in both murine models. We found that MLN4924 (30 mg/kg, i.p.) accumulated in the brain and reduced both locomotor sensitization induced by repeated alcohol administration and allodynia in an inflammatory pain model. Based on our previous findings that MLN4924 potently blocks AC sensitization in cellular models, we propose that the activity of MLN4924 in both animal models potentially occurs through blocking AC sensitization. Our findings provide the basis for understanding the molecular mechanism and yield a new pathway for drug development for pathological disorders associated with AC sensitization.

Evaluation of 25% Poloxamer As a Slow Release Carrier for Morphine in a Rat Model.

The objectives of this study were to evaluate poloxamer as a slow release carrier for morphine (M) and potential tissue irritation after subcutaneous poloxamer-morphine (PM) injection in a rat model. Based on the result of a previous in vitro work, 25% poloxamer, with and without morphine, and saline were administered in 14 rats' flanks. Blood for morphine concentrations was automatically sampled at multiple preprogrammed time points using the Culex™ unit for 48 h. Skin tissues from the injection sites were harvested and evaluated for histopathological changes. Following M or PM administration, it was determined that the half-life (t1/2) was significantly longer in the PM (5.5 ± 7.2 h) than M (0.7 ± 0.8 h) indicated a slow dissolution of poloxamer with morphine. The tmax was within 15 min and Cmax was approximately three times higher with M than with PM, reaching 716.8 (±153.7 ng/ml) of plasma morphine concentrations. There was no significant difference in total area under the curve and clearance of M versus PM. Histology inflammatory scores were similar between M, PM, and poloxamer but were significantly higher than saline control. We concluded that 25% poloxamer was capable of increasing the t1/2 of morphine, without a significant tissue irritation.

Development of a direct contact astrocyte-human cerebral microvessel endothelial cells blood-brain barrier coculture model.

OBJECTIVES: In conventional in-vitro blood-brain barrier (BBB) models, primary and immortalized brain microvessel endothelial cell (BMEC) lines are often cultured in a monolayer or indirect coculture or triculture configurations with astrocytes or pericytes, for screening permeation of therapeutic or potentially neurotoxic compounds. In each of these cases, the physiological relevancy associated with the direct contact between the BMECs, pericytes and astrocytes that form the BBB and resulting synergistic interactions are lost. We look to overcome this limitation with a direct contact coculture model. METHODS: We established and optimized a direct interaction coculture system where primary human astrocytes are cultured on the apical surface of a Transwell® filter support and then human cerebral microvessel endothelial cells (hCMEC/D3) seeded directly on the astrocyte lawn. KEY FINDINGS: The studies suggest the direct coculture model may provide a more restrictive and physiologically relevant model through a significant reduction in paracellular transport of model compounds in comparison with monoculture and indirect coculture. In comparison with existing methods, the indirect coculture and monoculture models utilized may limit cell-cell signaling between human astrocytes and BMECs that are possible with direct configurations. CONCLUSIONS: Paracellular permeability reductions with the direct coculture system may enhance therapeutic agent and potential neurotoxicant screening for BBB permeability better than the currently available monoculture and indirect coculture in-vitro models.

Absorption of Transdermal Fluoxetine Compounded in a Lipoderm Base Compared to Oral Fluoxetine in Client-owned Cats.

The objective of this study was to compare serum concentrations of transdermal fluoxetine compounded in Lipoderm base versus commercially available oral fluoxetine tablets. Sixteen clinically healthy, client-owned cats that were at least one year of age were enrolled. Cats weighed between three and seven kilograms, had no comorbidities, and were behavior medication naïve. Cats were recruited from January 2016 through April 2016. Eight cats were assigned to each medication group based on owner preference. The cats received either oral (1 mg/kg) or transdermal (5 mg/kg; maximum 25 mg daily) fluoxetine compounded in a transdermal base (PCCA Lipoderm), administered daily for 60 days. Serum levels of fluoxetine and norfluoxetine were assessed as a surrogate for relative efficacy. Serum was collected and analyzed by high-performance liquid chromatography-mass spectrometry/mass spectrometry at baseline and days 5, 10, 30, 45, and 60 post-drug start. Adverse effects were monitored during physical exams, speaking with owners, and laboratory analysis of liver function tests at baseline and days 5, 30, and 60 post-drug start. Serum fluoxetine concentrations significantly differed between the treatment groups at days 45 and 60 post-drug start. Norfluoxetine concentrations significantly differed at days 30, 45, and 60 post-drug start. Blood concentrations of fluoxetine and norfluoxetine significantly differed between oral and transdermal routes after 30 days of treatment. Oral fluoxetine concentrations were consistently higher. Transdermal fluoxetine appeared to be well-tolerated, but a lack of knowledge regarding effective blood levels makes it unclear if a clinical effective response would be obtained at the blood concentrations achieved.

Physicochemical and Preclinical Evaluation of a Novel Buccal Measles Vaccine.

The aim of this study is to develop an orally disintegrating film (ODF) containing a microparticulate measles vaccine formulation for buccal delivery. The measles vaccine microparticles were made with biocompatible and biodegradable bovine serum albumin (BSA) and processed by spray drying. These vaccine microparticles were incorporated in the ODF, consisting of Lycoat RS720®, Neosorb P60W® and Tween 80. The yield of the microparticles was approximately 85-95%, w/w. The mean size of the vaccine microparticles was 3.65 ± 1.89 µm and had a slightly negative surface charge of 32.65 ± 2.4 mV. The vaccine particles were nontoxic to normal cells at high concentrations (500 µg/2.5 × 105 cells) of vaccine particles. There was a significant induction of innate immune response by vaccine microparticles which was observed in vitro when compared to blank microparticles (P < 0.05). The vaccine microparticles also significantly increased the antigen presentation and co-stimulatory molecules expression on antigen presenting cells, which is a prerequisite for Th1 and Th2 immune responses. When the ODF vaccine formulation was dosed in juvenile pigs, significantly higher antibody titers were observed after week 2, with a significant increase at week 4 and plateauing through week 6 comparative to naïve predose titers. The results suggest that the ODF measles vaccine formulation is a viable dosage form alternative to noninvasive immunization that may increase patient compliance and commercial distribution.

Pharmacogenomics of acetaminophen in pediatric populations: a moving target.

Acetaminophen (APAP) is widely used as an over-the-counter fever reducer and pain reliever. However, the current therapeutic use of APAP is not optimal. The inter-patient variability in both efficacy and toxicity limits the use of this drug. This is particularly an issue in pediatric populations, where tools for predicting drug efficacy and developmental toxicity are not well established. Variability in toxicity between age groups may be accounted for by differences in metabolism, transport, and the genetics behind those differences. While pharmacogenomics has been revolutionizing the paradigm of pharmacotherapy for many drugs, its application in pediatric populations faces significant challenges given the dynamic ontogenic changes in cellular and systems physiology. In this review we focused on the ontogenesis of the regulatory pathways involved in the disposition of APAP and on the variability between pediatric, adolescent, and adult patients. We also summarize important polymorphisms of the pharmacogenes associated with APAP metabolism. Pharmacogenetic studies in pediatric APAP treatment are also reviewed. We conclude that while a consensus in pharmacogenetic management of APAP in pediatric populations has not been achieved, a systems biology based strategy for comprehensively understanding the ontogenic regulatory pathway as well as the interaction between age and genetic variations are particularly necessary in order to address this question.

Persistent pharmacokinetic challenges to pediatric drug development.

The development of new therapeutic agents for the mitigation of pediatric disorders is largely hindered by the inability for investigators to assess pediatric pharmacokinetics (PK) in healthy patients due to substantial safety concerns. Pediatric patients are a clinical moving target for drug delivery due to changes in absorption, distribution, metabolism and excretion (ADME) and the potential for PK related toxicological (T) events to occur throughout development. These changes in ADMET can have profound effects on drug delivery, and may lead to toxic or sub-therapeutic outcomes. Ethical, economical, logistical, and technical barriers have resulted in insufficient investigation of these changes by industrial, regulatory, and academic bodies, leading to the classification of pediatric patients as therapeutic orphans. In response to these concerns, regulatory agencies have incentivized investigation into these ontogenic changes and their effects on drug delivery in pediatric populations. The intent of this review is to briefly present a synopsis of the development changes that occur in pediatric patients, discuss the effects of these changes on ADME and drug delivery strategies, highlight the hurdles that are still being faced, and present some opportunities to overcome these challenges.

Association between hTERT rs2736100 polymorphism and sensitivity to anti-cancer agents.

BACKGROUND: The rs2736100 single nucleotide polymorphism (SNP) is located in the intron 2 of human telomerase reverse transcriptase (hTERT) gene. Recent genome-wide association studies (GWAS) have consistently supported the strong association between this SNP and risk for multiple cancers. Given the important role of the hTERT gene and this SNP in cancer biology, we hypothesize that rs2736100 may also confer susceptibility to anti-cancer drug sensitivity. In this study we aim to investigate the correlation between the rs2736100 genotype and the responsiveness to anti-cancer agents in the NCI-60 cancer cell panel. METHODS AND MATERIALS: The hTERT rs2736100 was genotyped in the NCI-60 cancer cell lines. The relative telomere length (RTL) of each cell line was quantified using real-time PCR. The genotype was then correlated with publically available drug sensitivity data of two agents with telomerase-inhibition activity: Geldanamycin (HSP90 inhibitor) and RHPS4/BRACO19 (G-quadruplex stabilizer) as well as additional 110 commonly used agents with established mechanism of action. The association between rs2736100 and mutation status of TP53 gene was also tested. RESULTS: The C allele of the SNP was significantly correlated with increased sensitivity to RHPS4/BRACO19 with an additive effect (r = -0.35, p = 0.009) but not with Geldanamycin. The same allele was also significantly associated with sensitivity to antimitotic agents compared to other agents (p = 0.003). The highest correlation was observed between the SNP and paclitaxel (r = -0.36, p = 0.005). The telomere length was neither associated with rs2736100 nor with sensitivity to anti-cancer agents. The C allele of rs2736100 was significantly associated with increased mutation rate in TP53 gene (p = 0.004). CONCLUSION: Our data suggested that the cancer risk allele of hTERT rs2736100 polymorphism may also affect the cancer cell response to both TERT inhibitor and anti-mitotic agents, which might be attributed to the elevated telomerase-independent activity of hTERT, as well as the increased risk for TP53 gene mutagenesis conferred by the polymorphism. Detailed mechanisms need to be further investigated.

Assessment of juvenile pigs to serve as human pediatric surrogates for preclinical formulation pharmacokinetic testing.

Pediatric drug development is hampered by biological, clinical, and formulation challenges associated with age-based populations. A primary cause for this lack of development is the inability to accurately predict ontogenic changes that affect pharmacokinetics (PK) in children using traditional preclinical animal models. In response to this issue, our laboratory has conducted a proof-of-concept study to investigate the potential utility of juvenile pigs to serve as surrogates for children during preclinical PK testing of selected rifampin dosage forms. Pigs were surgically modified with jugular vein catheters that were externalized in the dorsal scapular region and connected to an automated blood sampling system (PigTurn-Culex-L). Commercially available rifampin capsules were administered to both 20 and 40 kg pigs to determine relevant PK parameters. Orally disintegrating tablet formulations of rifampin were also developed and administered to 20 kg pigs. Plasma samples were prepared from whole blood by centrifugation and analyzed for rifampin content by liquid chromatography-tandem mass spectrometry. Porcine PK parameters were determined from the resultant plasma-concentration time profiles and contrasted with published rifampin PK data in human adults and children. Results indicated significant similarities in dose-normalized absorption and elimination parameters between pigs and humans. Moreover, ontogenic changes observed in porcine PK parameters were consistent with ontogenic changes reported for human PK. These results demonstrate the potential utility of the juvenile porcine model for predicting human pediatric PK for rifampin. Furthermore, utilization of juvenile pigs during formulation testing may provide an alternative approach to expedite reformulation efforts during pediatric drug development.

Summary of the National Institute of Child Health and Human Development-best pharmaceuticals for Children Act Pediatric Formulation Initiatives Workshop-Pediatric Biopharmaceutics Classification System Working Group.

BACKGROUND: The Biopharmaceutics Classification System (BCS) allows compounds to be classified based on their in vitro solubility and intestinal permeability. The BCS has found widespread use in the pharmaceutical community to be an enabling guide for the rational selection of compounds, formulation for clinical advancement, and generic biowaivers. The Pediatric Biopharmaceutics Classification System (PBCS) Working Group was convened to consider the possibility of developing an analogous pediatric-based classification system. Because there are distinct developmental differences that can alter intestinal contents, volumes, permeability, and potentially biorelevant solubilities at different ages, the PBCS Working Group focused on identifying age-specific issues that need to be considered in establishing a flexible, yet rigorous PBCS. OBJECTIVE: We summarized the findings of the PBCS Working Group and provided insights into considerations required for the development of a PBCS. METHODS: Through several meetings conducted both at The Eunice Kennedy Shriver National Institute of Child Health, Human Development-US Pediatric Formulation Initiative Workshop (November 2011) and via teleconferences, the PBCS Working Group considered several high-level questions that were raised to frame the classification system. In addition, the PBCS Working Group identified a number of knowledge gaps that need to be addressed to develop a rigorous PBCS. RESULTS: It was determined that for a PBCS to be truly meaningful, it needs to be broken down into several different age groups that account for developmental changes in intestinal permeability, luminal contents, and gastrointestinal (GI) transit. Several critical knowledge gaps were identified, including (1) a lack of fully understanding the ontogeny of drug metabolizing enzymes and transporters along the GI tract, in the liver, and in the kidney; (2) an incomplete understanding of age-based changes in the GI, liver, and kidney physiology; (3) a clear need to better understand age-based intestinal permeability and fraction absorbed required to develop the PBCS; (4) a clear need for the development and organization of pediatric tissue biobanks to serve as a source for ontogenic research; and (5) a lack of literature published in age-based pediatric pharmacokinetics to build physiologically- and population-based pharmacokinetic (PBPK) databases. CONCLUSIONS: To begin the process of establishing a PBPK model, 10 pediatric therapeutic agents were selected (based on their adult BCS classifications). These agents should be targeted for additional research in the future. The PBCS Working Group also identified several areas where greater emphasis on research was needed to enable the development of a PBCS.

Comparative pharmacokinetics studies of immediate- and modified-release formulations of glipizide in pigs and dogs.

The utility of pigs as preclinical animals for pharmaceutical development was assessed by evaluating the pharmacokinetics and pharmacodynamics of glipizide (Glucotrol®) following oral administration of immediate-release (IR) and modified-release (MR) formulations. Doses of 10 and 30 mg were administered to six male pigs in a crossover design. Blood samples were collected at selected time-points up to 48 h after dose. Relative to the IR formulation, the time to reach the maximum concentration (t(max) ) was delayed with the MR formulation from 1.3 to 8.7 h with the 10 mg dose and to 6.2 h with the 30 mg dose. The relative bioavailability (BA) was approximately 92% at 10 mg and 79% at 30 mg dose. The area under the curve of the plasma concentration versus time curve (AUC) increased nearly proportionally with the dose. Interanimal coefficient of variation (CV) in AUC ranged from approximately 40% to 60%. Blood glucose results suggest that pigs demonstrate formulation-dependent response to glipizide. Compared with the pigs, the 10 mg MR formulation in dogs showed a higher AUC CV of approximately 80%, a t(max) of 5.5 h, and a lower relative BA of 18%. These data indicate that the MR formulation performed less consistently in dogs as compared with humans, whereas the porcine absorption kinetics and BA were consistent with published clinical data.

The effects of intralaboratory modifications to media composition and cell source on the expression of pharmaceutically relevant transporters and metabolizing genes in the Caco-2 cell line.

Expression and function of drug transporters and drug-metabolizing enzymes (DMEs) in the gastrointestinal tract are critical attributes of intestinal physiology that influence the absorption of orally administered compounds. The purpose of this study was to examine the effects of media composition and cell source on mRNA expression and function of pharmaceutically relevant drug transporters and DMEs from two different sources of Caco-2 cells. Briefly, cells were cultured in either minimum essential medium alpha or Dulbecco's modified Eagle's medium. Total RNA was isolated from each experimental group, and mRNA expression was evaluated using quantitative reverse-transcriptase polymerase chain reaction arrays. Principal component analysis was used to analyze results, which indicated variable transporter and metabolic expression attributable to differences in media composition and cell source. In addition, transport properties of paracellular markers and proton-dependent oligopeptide transporter-mediated substrates across Caco-2 cell monolayers were assessed. Transport experiments demonstrated significant differences in both paracellular and transcellular permeation resultant from differences in media composition and cell source. These studies support previous findings that media composition and cell source may significantly impact expressional and functional characteristics of Caco-2 cells. Standardization of culture-related methodology may reduce variability associated with Caco-2 cells, enabling more meaningful intralaboratory and interlaboratory data comparisons.

The effects of media on pharmaceutically relevant transporters in the human HT-29 adenocarcinoma cell line: does culture media need to be controlled?

The HT-29 cell line forms a confluent monolayer with tight junctions, but displays different phenotypes when cultured for 21 days in galactose-supplemented media (differentiated) versus glucose-supplemented media (dedifferentiated). This study is aimed at elucidating how media differences might affect selected drug transporter expression and peptide-based substrate transport toward reducing this variability. A vial of HT-29 cells was amplified and cultured over several passages in four different mediums (American Type Culture Collection recommended McCoy's 5A versus Dulbecco's modified Eagle's media containing glucose, galactose, or neither carbohydrate) with normal supplementation. Transporter mRNA expression was characterized at days 5 and 21 postseeding utilizing SABiosciences quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) drug transporter arrays. Transport studies using [H]histidine, [(3) H]glycylsarcosine, [(3) H]valacyclovir, and [(3) H]carnosine were performed to assess the functional effects of oligopeptide transporter expression changes in HT-29 cells grown in each media. qRT-PCR arrays illustrated variable, media-dependent transporter expression between both the initial and differentiated time points. Permeability studies illustrated considerable media-dependent differences in both paracellular and transcellular substrate fluxes. The results demonstrate that these cells exhibit differing monolayer characteristics and genotypic/phenotypic profile properties when cultured under different media. The results suggest a need for standardization of culture methodologies for reducing inter- and intralaboratory variability.

THE EVALUATION OF PEPTIDE/HISTIDINE TRANSPORTER 1 (PHT1) FUNCTION: UPTAKE KINETICS UTILIZING A COS-7 STABLY TRANSFECTED CELL LINE.

There have been relatively few studies focused on the proton-dependent oligopeptide transporter (POT) superfamily member, Peptide/Histidine Transporter 1 (PHT1), with respect to its contribution to the ADME of peptides and peptide-based drugs. These studies were conducted to determine hPHT1-mediated, H+-dependent uptake kinetics of histidine, carnosine, Gly-Sar and valacyclovir in stably transfected hPHT1-COS-7 cells comparative to kinetics determined in an empty vector (Mock) stably transfected cell line. The results suggest that Gly-Sar appears to be a substrate for PHT1 based on efflux from the stably transfected hPHT1 COS-7 cells. Histidine and Gly-Sar concentration- and time-dependent studies suggest mixed-uptake kinetics. These studies suggest that stably transfected hPHT1-COS-7 cells exhibit different uptake kinetics than those observed in our previous studies and illustrate the requirement for experiments to delineate the physiological role of hPHT1.

ABC and SLC transporter expression and proton oligopeptide transporter (POT) mediated permeation across the human blood--brain barrier cell line, hCMEC/D3 [corrected].

Initial studies indicate that the newly developed hCMEC/D3 cell line may prove to be a useful model for studying the physiology of the human blood-brain barrier (BBB) endothelium. The purpose of this study was to assess the mRNA expression of several ABC and SLC transporters, with an emphasis on the proton-coupled oligopeptide transporter superfamily (POT) transporters in this immortalized BBB cell model. The transport kinetics of POT-substrates was also evaluated. The hCMEC/D3 cell line was maintained in a modified EGM-2 medium in collagenated culture flasks and passaged every 3-4 days at approximately 85%-95% confluence. Messenger RNA (mRNA) expression of a variety of ABC and SLC transporters was evaluated using qRT-PCR arrays, while additional qRT-PCR primers were designed to assess the expression of POT members. The transport kinetics of mannitol and urea were utilized to quantitatively estimate the intercellular pore radius, while POT substrate transport was also determined to assess the suitability of the cell model from a drug screening perspective. Optimization of the cell line was attempted by culturing with on laminin and fibronectin enhanced collagen and in the presence of excess Ca(2+). hCMEC/D3 cells express both hPHT1 and hPHT2, while little to no expression of either hPepT1 or hPepT2 was observed. The relative expression of other ABC and SLC transporters is discussed. While POT substrate transport does suggest suitability for BBB drug permeation screening, the relative intercellular pore radius was estimated at 19 A, significantly larger than that approximated in vivo. Culturing with extracellular matrix proteins did not alter mannitol permeability. These studies characterized this relevant human hCMEC/D3 BBB cell line with respect to both the relative mRNA expression of various ABC and SLC transporters and its potential utility as an in vitro screening tool for brain permeation. Additional studies are required to adequately determine the potential to establish an in vivo correlation.

The effects of excipients on transporter mediated absorption.

Traditionally most pharmaceutical excipients used for peroral dosage forms have been considered to be inert, although they have been known to play an important role in governing the release of the active pharmaceutical ingredient (API) required for the desired therapeutic effect. Of considerable interest is the emerging data demonstrating that many of these "inert" excipients may produce subtle changes that could directly or indirectly alter the activity of membrane-spanning proteins such as transporters. In this way, excipients could be altering the overall ADMET properties of an incorporated drug thereby affecting its intended therapeutic efficacy and/or enhancing adverse side effects. Therefore, given this recent evidence, it seems necessary to review what has been reported in the literature on interactions of excipients with human physiological entities, particularly transporters. As of today, safety/toxicity evaluations are typically based on the appearance of gross morphological changes rather than the effects on a cellular level, the ability of excipients in modifying the pharmacological activity of an active drug could lead to toxicity evaluation in routine for each additive used in oral formulations. Further knowledge on this subject will enable formulators to make more rational decisions in dosage form design and will help answer the question of whether certain excipients should be considered active pharmaceutical components of formulations.