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Gregory Knipp

Purdue University, Industrial and Physical Pharmacy, Faculty Member

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My laboratory's research interests include the preclinical development and ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) in vitro and in vivo characterization of novel therapeutic entities aimed at facilitating translation. Our current research projects include the lead candidate selection and preclinical formulation of cyanide counter measures (NIH U54 Project), the characterization and preclinical formulation of novel antiviral agents (NIH R01 Project), and the development of a novel blood-brain barrier direct contact, triculture model for predicting neurotherapeutic and neurotoxicant (EPA OSCAPE Project) exposure in the brain. We have also been interested in investigating the effects of processing induced dosage form variation on clinical performance utilizing in vitro and in vivo (porcine) models. In particular, our recent work has focused on the utilization of the porcine model for testing the pharmacokinetics of adult and novel pediatric formulations developed within the laboratory.

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Papers by Gregory Knipp

Characterization of Two Nonclassical Members of the Placental Prolactin Family from the Rat

Pediatric Research, Apr 1, 1999

Mammalian Oligopeptide Transporters

John Wiley & Sons, Inc. eBooks, Aug 8, 2007

Identification of two new nonclassical members of the rat prolactin family

Journal of Molecular Endocrinology, Feb 1, 2000

The prolactin (PRL) family is comprised of a group of hormones/cytokines that are expressed in th... more The prolactin (PRL) family is comprised of a group of hormones/cytokines that are expressed in the anterior pituitary, uterus, and placenta. These proteins participate in the control of maternal and fetal adaptations to pregnancy. In this report, we have identified two new nonclassical members of the rat PRL family through a search of the National Center for Biotechnology Information dbEST database. The cDNAs were sequenced and their corresponding mRNAs characterized. Overall, the rat cDNAs showed considerable structural similarities with mouse proliferin-related protein (PLF-RP) and prolactin-like protein-F (PLP-F), consistent with their classification as rat homologs for PLF-RP and PLP-F. The expression of both cytokines/hormones was restricted to the placenta. The intraplacental sites of PLF-RP and PLP-F synthesis differed in the rat and the mouse. In the mouse, PLF-RP was expressed in the trophoblast giant cell layer of the midgestation chorioallantoic and choriovitelline placentas and, during later gestation, in the trophoblast giant cell and spongiotrophoblast layers within the junctional zone of the mouse chorioallantoic placenta. In contrast, in the rat, PLF-RP was first expressed in the primordium of the chorioallantoic placenta (ectoplacental cone region) and, later, exclusively within the labyrinth zone of the chorioallantoic placenta. In the mouse, PLP-F is an exclusive product of the spongiotrophoblast layer, whereas in the rat, trophoblast giant cells were found to be the major source of PLP-F, with a lesser contribution from spongiotrophoblast cells late in gestation. In summary, we have established the presence of PLF-RP and PLP-F in the rat.

Investigating the replacement of carboxylates with carboxamides to modulate the safety and efficacy of platinum(II) thioether cyanide scavengers

Toxicological Sciences

Cyanide represents a persistent threat for accidental or malicious misuse due to easy conversion ... more 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 (IM) 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 of nephrotoxicity. Platinum amino acid complexes with the ability to form 5- or 6-membered rings and possessing either carboxylates or carboxamides are evaluated in vitro for cyanide scavenging. In vivo efficacy was evaluated in the zebrafish and mice cyanide...

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

Clinical Pharmacology & Therapeutics, Aug 18, 2015

Recent data suggest that variability in drug transporter activity may be critically important for... more Recent data suggest that variability in drug transporter activity may be critically important for safe and effective anti-leukemia drug therapy in children. A high-dose methotrexate study performed in 1,883 acute lymphoblastic leukemia patients enrolled in a multicenter Children's Oncology Group clinical trial revealed a relationship between multiple SNP variants in the OATP1B1 gene SLCO1B1 and high-dose methotrexate toxicity (8). The study demonstrated that methotrexate clearance was lower in 1,279 patients carrying one of several loss-of-function SNP variants of SLCO1B1. Significant correlations also were established between lower clearance rates after high-dose methotrexate administration in Brouwer et al.

The effects of excipients on transporter mediated absorption

International Journal of Pharmaceutics, Jun 1, 2010

Traditionally most pharmaceutical excipients used for peroral dosage forms have been considered t... more 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.

Establishment of a hPepT1-stably transfected human cell line: characterization of transporter expression and function

info:eu-repo/semantics/nonPublishe

Biotechnology-Derived Drug Product Development

John Wiley & Sons, Inc. eBooks, Mar 11, 2008

The Evaluation of Peptide/Histidine Transporter 1 (PHT1) Function: Uptake Kinetics Utilizing a COS-7 Stably Transfected Cell Line

PubMed, Oct 1, 2011

There have been relatively few studies focused on the proton-dependent oligopeptide transporter (... more 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.

The Effects of Media on Pharmaceutically Relevant Transporters in the Human HT-29 Adenocarcinoma Cell Line: Does Culture Media Need to be Controlled?

Journal of Pharmaceutical Sciences, Apr 1, 2012

The HT-29 cell line forms a confluent monolayer with tight junctions, but displays different phen... more 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 reversetranscriptase 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 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

Journal of Pharmaceutical Sciences, Oct 1, 2012

Expression and function of drug transporters and metabolic enzymes (DMEs) in the gastrointestinal... more Expression and function of drug transporters and metabolic 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 transporters and DMEs from two different sources of Caco-2 cells. Briefly, cells were cultured in either Minimum Essential Medium Alpha (AMEM) or Dulbecco's Modified Eagle's Medium (DMEM). Total RNA was isolated from each experimental group, and mRNA expression was evaluated using Quantitative Reverse Transcriptase-Polymerase Chain Reaction (QRT-PCR) arrays. Principal component analysis (PCA) 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 intra-and interlaboratory data comparisons.

The Pharmacodynamic Effects of Sirolimus andSirolimus-Calcineurin Inhibitor Combinations on Macrophage Scavenger and Nuclear Hormone Receptors

Journal of Pharmaceutical Sciences, 2007

Background: Sirolimus (SIR) alone or in combination with cyclosporine (CsA) or tacrolimus (TAC) a... more Background: Sirolimus (SIR) alone or in combination with cyclosporine (CsA) or tacrolimus (TAC) are used in solid organ transplantation, but uncertainty remains regarding their respective atherogenic potentials. Methods: THP-1 cells were cultured as macrophages and then treated with plasma trough and peak concentration doses of SIR, SIR/CsA or SIR/TAC to assess the time-and dose-dependent mRNA or protein expression of selected atherogenic genes. The selected atherogenic genes included: the macrophage scavenger receptors (MSRs) CD36, CD68, scavenger receptor (SR)-A, SR-BII, and LOX-1; the nuclear hormone receptors peroxisome proliferator activated receptor gamma (PPARg) and liver-X-receptor alpha (LXRa); and the cholesterol efflux transporter (ABCA-1). Results: SIR-mediated changes in mRNA included the upregulation of ABCA1, downregulation of CD68, SR-A and SR-BII, and concentration-and/or time-dependent effects on CD36, LOX-1, PPARg, and LXRa that did not translate into significant protein changes. With SIR/CsA, the protein expressions of PPARg and ABCA-1 were downregulated at 8 h. In contrast, with SIR/TAC, PPARg, and ABCA-1 protein expressions were upregulated at 8 h. Conclusions: Combination results differed from findings with SIR alone, supporting the observed clinical phenotype with calcineurin inhibitors. These findings may provide a rationale for the development of novel drug delivery strategies to mitigate adverse pharmacodynamic responses.

Structural Properties of Polyethylene Glycol-Polysorbate 80 Mixture, a Solid Dispersion Vehicle

Journal of Pharmaceutical Sciences, Dec 1, 1992

Abatract 0 The structural properties of the mixtures of polysorbate 80 wRh van' ous polyethylene ... more Abatract 0 The structural properties of the mixtures of polysorbate 80 wRh van' ous polyethylene glycols (PEG), viz., PEG 1000, PEG 1450, PEG 3350, and PEG 8oO0, have been investigated by powder X-ray diffraction (XRD) and differential scanning calorimetric studies. These mixtures may be used as solid dispersion vehicles to insure complete dissolution of poorly water-soluble drugs. Although polysorbate 80 is a liquid at room temperature, the PEG-polysorbate 80 mixtures with up to 75% (wb) polysorbate 80 were solid. The XRD studies revealed that the crystal structures (d-spacings) of the PEGS (M, 1000, 1450,3350, and 8OOO) did not change with increasing amounts of polysorbate 80 in the mixture. The intensities of the XRD peaks, however, varied approximately in proportion to the concentration of PEG present. Similarly, the differential scanning calorimetric studies showed that the melting behavior of a PEG-polysorbate 80 mixture was similar to that of the PEG used. The lowering of the mp of a particular PEG due to the presence of 50% (wb) polysorbate 80 in the mixture was c6 "C, and the decrease in mp was <12 "C in the presence of 75% (w/w) polysorbate 80. When enthalpies of fusion of the mixtures were normalized for the amounts of PEGs present, they were similar to those of pure PEGs. These results indicate that the crystalline structure of PEG in a PEG-polysorbate 80 mixture is substantially the same as that of the pure PEG, and that polysorbate 80 is incorporated into the amorphous region of PEG solid structure.

Structural Biology and Function of Solute Transporters: Implications for Identifying and Designing Substrates

Drug Metabolism Reviews, 2002

Solute carrier (SLC) proteins have critical physiological roles in nutrient transport and may be ... more Solute carrier (SLC) proteins have critical physiological roles in nutrient transport and may be utilized as a mechanism to increase drug absorption. However, we have little understanding of these proteins at the molecular level due to the absence of high-resolution crystal structures. Numerous efforts have been made in characterizing the peptide transporter (PepT1) and the apical sodium dependent bile acid transporter (ASBT) that are important for both their native transporter function as well as targets to increase absorption and act as therapeutic targets. In vitro and computational approaches have been applied to gain some insight into these transporters with some success. This represents an opportunity for optimizing molecules as substrates for the solute transporters and providing a further screening system for drug discovery. Clearly the future growth in knowledge of SLC function will be led by integrated in vitro and in silico approaches.

Paracellular Diffusion in Caco-2 Cell Monolayers: Effect of Perturbation on the Transport of Hydrophilic Compounds That Vary in Charge and Size

Journal of Pharmaceutical Sciences, Oct 1, 1997

We applied the principles of molecular-size-restricted diffusion within a negative electrostatic ... more We applied the principles of molecular-size-restricted diffusion within a negative electrostatic field of force to follow the changes in the aqueous pore radius of tight junctions (TJs) induced by perturbants and the accompanying influence on the permeation of neutral (urea and mannitol), cationic (methylamine and atenolol), and anionic (formate and lactate) compounds that vary in size. The perturbants included palmitoyl-DL-carnitine (PC), which opens TJs by an unknown Ca ++-independent mechanism, and ethyleneglycol-bis-(-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), a Ca ++ chelator. Mass transfer resistances of the collagen−coated filter support and the aqueous boundary layers were factored out to yield paracellular permeability coefficients (P P). As viewed from the P P values of urea and mannitol, EGTA exhibited insignificant effects on pore size at low concentrations compared with control, and then caused a dramatic opening of the TJs over a narrow concentration range (1.35−1.4 mM). The P P values for urea and mannitol remained constant at >1.4 mM EGTA. However, PC produced dose−dependent responses from 0 to 0.15 mM that plateaued at >0.15 mM. In general, cations permeated the cellular TJs faster and anions slower than their neutral images. The effects of changes in pore size (4.6 to 14.6 Å in effective radius) on the ability of these solutes to permeate the TJs were analyzed by the Renkin molecular sieving function. These studies established an experimental, theoretical, and quantitative template to assess perturbants of the TJ and define the limits, short of detrimental effects, at which the TJs may be sufficiently perturbed for maximal enhancement of permeation of solutes varying in size and charge. Materials and Methods MaterialssMannitol, urea, formic acid, lactic acid, and methylamine were purchased as 14 C-labeled stock solutions from the American Radiolabeled Company (St. Louis, MO). Atenolol, EGTA, and PC were purchased from Sigma Chemical Company (St. Louis, MO). Hanks' balanced salt solution (HBSS), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonate (HEPES), nonessential amino acids, trypsin/EDTA, and Dulbecco's Modified Eagle Medium (DMEM) were purchased from JRH Biosciences (Lenexa, KS). Penicillin and streptomycin were purchased as a mixture from Irvine Scientific (Santa Ana, CA). Fetal bovine serum and type I rat tail collagen were purchased from Atlanta Biologicals (Norcross, GA) and Collaborative Research (Lexington, MA), respectively. Cell CulturesThe Caco-2 cell line, which originated from a human adenocarcinoma, 15 was obtained from the American Type Culture Collection (Rockville, MD) and was cultured as described previously by Hildago et al. 10 The cells were grown in 150-cm 2 culture flasks

Assessing the performance of amorphous solid dispersions

Journal of Pharmaceutical Sciences, Apr 1, 2012

The characterization and performance of stable amorphous solid dispersion systems were evaluated ... more The characterization and performance of stable amorphous solid dispersion systems were evaluated in 40 research papers reporting active pharmaceutical ingredient (API) dissolution and bioavailability from various systems containing polymers. The results from these studies were broadly placed into three categories: amorphous dispersions that improved bioavailability (∼82% of the cases), amorphous dispersions possessing lower bioavailability than the reference material (∼8% of the cases), and amorphous dispersions demonstrating similar bioavailabilities as the reference material (∼10% of the cases). A comparative analysis of these studies revealed several in vitro and in vivo variables that could have influenced the results. The in vitro factors compared primarily centered on dissolution testing and equipment, content and amount of dissolution media, sink or nonsink conditions, agitation rates, media pH, dissolution characteristics of the polymer, and dispersion particle size. The in vivo factors included reference materials used for bioavailability comparisons, animal species utilized, fasting versus fed conditions, and regional differences in gastrointestinal (GI) content and volume. On the basis of these considerations, a number of recommendations were made on issues ranging from the assessment of physical stability of API-polymer dispersions to in vivo GI physiological factors that require consideration in the performance evaluation of these systems.

Regulatory Considerations in Approval of Follow-On Protein Drug Products

Pharmaceutical Sciences Encyclopedia, Mar 15, 2010

Design of experiment based optimization of an in vitro direct contact triculture blood brain barrier model for permeability screening

Pharmacy & pharmacology international journal, Jul 28, 2021

The in vivo restrictive properties of the blood brain barrier (BBB) largely arise from astrocyte ... more The in vivo restrictive properties of the blood brain barrier (BBB) largely arise from astrocyte and pericyte synergistic cell signaling interactions that underlie the brain microvessel endothelial cells (BMEC). In vivo relevant direct contact between astrocytes, pericytes, and BMECS, to our knowledge, has not been established in conventional Transwell ® based in vitro screening models of the BBB. We hypothesize that a design of experiments (DOE) optimized direct contact layered triculture model will offer more in vivo relevance for screening in comparison to indirect models. Plating conditions including the seeding density of all three cell types, matrix protein, and culture time were assessed utilizing a DOE approach. A second set of DOE methods assessed the influence of medium additives on barrier properties. The optimized model was further assessed for p-glycoprotein function using a substrate and inhibitor along with a set of BBB paracellular and transcellular markers at varying permeation rates. The optimization revealed that length of culture post endothelial cell plating correlated highest with paracellular tightness. In addition, seeding density of the endothelial cell layer influenced paracellular tightness at earlier times of culture, and its impact decreased as culture is extended. At optimal conditions, the model revealed P-gp function along with the ability to differentiate between BBB positive and negative permeants. We have demonstrated that the implementation of DOE based optimization for biologically based systems is an expedited method to establish multi-component in vitro cell models. The direct contact BBB triculture model reveals that the physiologically relevant layering of the three cell types is a practical method of culture to establish a screening model compared to indirect plating methods that incorporate physical barriers between cell types. Additionally, the ability of the model to differentiate between BBB positive and negative permeants suggests that this model may be an enhanced screening tool for potential neuroactive compounds.

Do Sex and Ethnicity Influence Drug Pharmacokinetics in Solid Organ Transplantation?

Graft, Jul 1, 2002

A number of reports have identified differences in drug pharmacokinetics and pharmacodynamics bet... more A number of reports have identified differences in drug pharmacokinetics and pharmacodynamics between males and females, and between patients of different ethnicity. These differences are observed in both solid organ transplant recipients and in nontransplant patients. This review summarizes the pharmacokinetic differences thought to be dependent on sex and ethnicity noted with immunosuppressive agents commonly used in transplantation. This manuscript also touches on how these differences may translate into observed drug interactions. Furthermore, potential factors underlying the sex and ethnicity differences are evaluated. Current evidence points to differences in drug-metabolizing enzymes, transporters, cytokines, and environmental influences to account for the observed heterogeneity in drug response. Many of these factors seem to be under genetic control, and thus areas for further research are suggested.

Effects of Dendrimer-Like Biopolymers on Physical Stability of Amorphous Solid Dispersions and Drug Permeability Across Caco-2 Cell Monolayers

Aaps Pharmscitech, Jun 4, 2018

The potential applications of dendrimer-like biopolymers (DLB) as stabilizing excipients for amor... more The potential applications of dendrimer-like biopolymers (DLB) as stabilizing excipients for amorphous solid dispersion (ASD) of niclosamide, celecoxib, and resveratrol were evaluated based on (1) the formation and physical stability of the ASD and (2) the permeability and flux of the agents across Caco-2 cell monolayers. The evaluation was made by comparing the performance of prototype phytoglycogen derivatives (DLB1, DLB2, and DLB3) with commonly used polymers such as HPMCAS, PVPVA, and Soluplus®. PXRD was used to confirm the formation of the dispersions and detect crystallinity peaks formed during 2-and 4-week storage at 40°C/75% RH. At concentrations below 2 g/mL, the viability of Caco-2 cells remained above 80% for all DLB samples compared to untreated cells in the MTT assay. Permeability studies revealed a repeating pattern in which an increase in the initial concentration (C 0) was associated with a concomitant decrease in the apparent permeability (P app) which we theorize is due to differences in drug-polymer interactions. Niclosamide-DLB1 dispersion had the lowest flux due to a significant reduction in P app. The high increase in the C 0 of celecoxib-DLB2, however, made up for the reduction in the P app and produced the highest flux values compared to other polymers. Resveratrol-DLB3 had a 5× reduction in P app , but C 0 increased from 25.8 to 176 μg/mL led to a higher flux compared to the crystalline drug without polymer. Collectively, these results provide a Bproof-of-concept^basis to demonstrate that DLB excipients have the ability to increase apparent solubility (Sol app), most likely due to drug-binding capacity.

Characterization of Two Nonclassical Members of the Placental Prolactin Family from the Rat

Pediatric Research, Apr 1, 1999

Mammalian Oligopeptide Transporters

John Wiley & Sons, Inc. eBooks, Aug 8, 2007

Identification of two new nonclassical members of the rat prolactin family

Journal of Molecular Endocrinology, Feb 1, 2000

The prolactin (PRL) family is comprised of a group of hormones/cytokines that are expressed in th... more The prolactin (PRL) family is comprised of a group of hormones/cytokines that are expressed in the anterior pituitary, uterus, and placenta. These proteins participate in the control of maternal and fetal adaptations to pregnancy. In this report, we have identified two new nonclassical members of the rat PRL family through a search of the National Center for Biotechnology Information dbEST database. The cDNAs were sequenced and their corresponding mRNAs characterized. Overall, the rat cDNAs showed considerable structural similarities with mouse proliferin-related protein (PLF-RP) and prolactin-like protein-F (PLP-F), consistent with their classification as rat homologs for PLF-RP and PLP-F. The expression of both cytokines/hormones was restricted to the placenta. The intraplacental sites of PLF-RP and PLP-F synthesis differed in the rat and the mouse. In the mouse, PLF-RP was expressed in the trophoblast giant cell layer of the midgestation chorioallantoic and choriovitelline placentas and, during later gestation, in the trophoblast giant cell and spongiotrophoblast layers within the junctional zone of the mouse chorioallantoic placenta. In contrast, in the rat, PLF-RP was first expressed in the primordium of the chorioallantoic placenta (ectoplacental cone region) and, later, exclusively within the labyrinth zone of the chorioallantoic placenta. In the mouse, PLP-F is an exclusive product of the spongiotrophoblast layer, whereas in the rat, trophoblast giant cells were found to be the major source of PLP-F, with a lesser contribution from spongiotrophoblast cells late in gestation. In summary, we have established the presence of PLF-RP and PLP-F in the rat.

Investigating the replacement of carboxylates with carboxamides to modulate the safety and efficacy of platinum(II) thioether cyanide scavengers

Toxicological Sciences

Cyanide represents a persistent threat for accidental or malicious misuse due to easy conversion ... more 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 (IM) 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 of nephrotoxicity. Platinum amino acid complexes with the ability to form 5- or 6-membered rings and possessing either carboxylates or carboxamides are evaluated in vitro for cyanide scavenging. In vivo efficacy was evaluated in the zebrafish and mice cyanide...

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

Clinical Pharmacology & Therapeutics, Aug 18, 2015

Recent data suggest that variability in drug transporter activity may be critically important for... more Recent data suggest that variability in drug transporter activity may be critically important for safe and effective anti-leukemia drug therapy in children. A high-dose methotrexate study performed in 1,883 acute lymphoblastic leukemia patients enrolled in a multicenter Children's Oncology Group clinical trial revealed a relationship between multiple SNP variants in the OATP1B1 gene SLCO1B1 and high-dose methotrexate toxicity (8). The study demonstrated that methotrexate clearance was lower in 1,279 patients carrying one of several loss-of-function SNP variants of SLCO1B1. Significant correlations also were established between lower clearance rates after high-dose methotrexate administration in Brouwer et al.

The effects of excipients on transporter mediated absorption

International Journal of Pharmaceutics, Jun 1, 2010

Traditionally most pharmaceutical excipients used for peroral dosage forms have been considered t... more 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.

Establishment of a hPepT1-stably transfected human cell line: characterization of transporter expression and function

info:eu-repo/semantics/nonPublishe

Biotechnology-Derived Drug Product Development

John Wiley & Sons, Inc. eBooks, Mar 11, 2008

The Evaluation of Peptide/Histidine Transporter 1 (PHT1) Function: Uptake Kinetics Utilizing a COS-7 Stably Transfected Cell Line

PubMed, Oct 1, 2011

There have been relatively few studies focused on the proton-dependent oligopeptide transporter (... more 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.

The Effects of Media on Pharmaceutically Relevant Transporters in the Human HT-29 Adenocarcinoma Cell Line: Does Culture Media Need to be Controlled?

Journal of Pharmaceutical Sciences, Apr 1, 2012

The HT-29 cell line forms a confluent monolayer with tight junctions, but displays different phen... more 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 reversetranscriptase 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 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

Journal of Pharmaceutical Sciences, Oct 1, 2012

Expression and function of drug transporters and metabolic enzymes (DMEs) in the gastrointestinal... more Expression and function of drug transporters and metabolic 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 transporters and DMEs from two different sources of Caco-2 cells. Briefly, cells were cultured in either Minimum Essential Medium Alpha (AMEM) or Dulbecco's Modified Eagle's Medium (DMEM). Total RNA was isolated from each experimental group, and mRNA expression was evaluated using Quantitative Reverse Transcriptase-Polymerase Chain Reaction (QRT-PCR) arrays. Principal component analysis (PCA) 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 intra-and interlaboratory data comparisons.

The Pharmacodynamic Effects of Sirolimus andSirolimus-Calcineurin Inhibitor Combinations on Macrophage Scavenger and Nuclear Hormone Receptors

Journal of Pharmaceutical Sciences, 2007

Background: Sirolimus (SIR) alone or in combination with cyclosporine (CsA) or tacrolimus (TAC) a... more Background: Sirolimus (SIR) alone or in combination with cyclosporine (CsA) or tacrolimus (TAC) are used in solid organ transplantation, but uncertainty remains regarding their respective atherogenic potentials. Methods: THP-1 cells were cultured as macrophages and then treated with plasma trough and peak concentration doses of SIR, SIR/CsA or SIR/TAC to assess the time-and dose-dependent mRNA or protein expression of selected atherogenic genes. The selected atherogenic genes included: the macrophage scavenger receptors (MSRs) CD36, CD68, scavenger receptor (SR)-A, SR-BII, and LOX-1; the nuclear hormone receptors peroxisome proliferator activated receptor gamma (PPARg) and liver-X-receptor alpha (LXRa); and the cholesterol efflux transporter (ABCA-1). Results: SIR-mediated changes in mRNA included the upregulation of ABCA1, downregulation of CD68, SR-A and SR-BII, and concentration-and/or time-dependent effects on CD36, LOX-1, PPARg, and LXRa that did not translate into significant protein changes. With SIR/CsA, the protein expressions of PPARg and ABCA-1 were downregulated at 8 h. In contrast, with SIR/TAC, PPARg, and ABCA-1 protein expressions were upregulated at 8 h. Conclusions: Combination results differed from findings with SIR alone, supporting the observed clinical phenotype with calcineurin inhibitors. These findings may provide a rationale for the development of novel drug delivery strategies to mitigate adverse pharmacodynamic responses.

Structural Properties of Polyethylene Glycol-Polysorbate 80 Mixture, a Solid Dispersion Vehicle

Journal of Pharmaceutical Sciences, Dec 1, 1992

Abatract 0 The structural properties of the mixtures of polysorbate 80 wRh van' ous polyethylene ... more Abatract 0 The structural properties of the mixtures of polysorbate 80 wRh van' ous polyethylene glycols (PEG), viz., PEG 1000, PEG 1450, PEG 3350, and PEG 8oO0, have been investigated by powder X-ray diffraction (XRD) and differential scanning calorimetric studies. These mixtures may be used as solid dispersion vehicles to insure complete dissolution of poorly water-soluble drugs. Although polysorbate 80 is a liquid at room temperature, the PEG-polysorbate 80 mixtures with up to 75% (wb) polysorbate 80 were solid. The XRD studies revealed that the crystal structures (d-spacings) of the PEGS (M, 1000, 1450,3350, and 8OOO) did not change with increasing amounts of polysorbate 80 in the mixture. The intensities of the XRD peaks, however, varied approximately in proportion to the concentration of PEG present. Similarly, the differential scanning calorimetric studies showed that the melting behavior of a PEG-polysorbate 80 mixture was similar to that of the PEG used. The lowering of the mp of a particular PEG due to the presence of 50% (wb) polysorbate 80 in the mixture was c6 "C, and the decrease in mp was <12 "C in the presence of 75% (w/w) polysorbate 80. When enthalpies of fusion of the mixtures were normalized for the amounts of PEGs present, they were similar to those of pure PEGs. These results indicate that the crystalline structure of PEG in a PEG-polysorbate 80 mixture is substantially the same as that of the pure PEG, and that polysorbate 80 is incorporated into the amorphous region of PEG solid structure.

Structural Biology and Function of Solute Transporters: Implications for Identifying and Designing Substrates

Drug Metabolism Reviews, 2002

Solute carrier (SLC) proteins have critical physiological roles in nutrient transport and may be ... more Solute carrier (SLC) proteins have critical physiological roles in nutrient transport and may be utilized as a mechanism to increase drug absorption. However, we have little understanding of these proteins at the molecular level due to the absence of high-resolution crystal structures. Numerous efforts have been made in characterizing the peptide transporter (PepT1) and the apical sodium dependent bile acid transporter (ASBT) that are important for both their native transporter function as well as targets to increase absorption and act as therapeutic targets. In vitro and computational approaches have been applied to gain some insight into these transporters with some success. This represents an opportunity for optimizing molecules as substrates for the solute transporters and providing a further screening system for drug discovery. Clearly the future growth in knowledge of SLC function will be led by integrated in vitro and in silico approaches.

Paracellular Diffusion in Caco-2 Cell Monolayers: Effect of Perturbation on the Transport of Hydrophilic Compounds That Vary in Charge and Size

Journal of Pharmaceutical Sciences, Oct 1, 1997

We applied the principles of molecular-size-restricted diffusion within a negative electrostatic ... more We applied the principles of molecular-size-restricted diffusion within a negative electrostatic field of force to follow the changes in the aqueous pore radius of tight junctions (TJs) induced by perturbants and the accompanying influence on the permeation of neutral (urea and mannitol), cationic (methylamine and atenolol), and anionic (formate and lactate) compounds that vary in size. The perturbants included palmitoyl-DL-carnitine (PC), which opens TJs by an unknown Ca ++-independent mechanism, and ethyleneglycol-bis-(-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), a Ca ++ chelator. Mass transfer resistances of the collagen−coated filter support and the aqueous boundary layers were factored out to yield paracellular permeability coefficients (P P). As viewed from the P P values of urea and mannitol, EGTA exhibited insignificant effects on pore size at low concentrations compared with control, and then caused a dramatic opening of the TJs over a narrow concentration range (1.35−1.4 mM). The P P values for urea and mannitol remained constant at >1.4 mM EGTA. However, PC produced dose−dependent responses from 0 to 0.15 mM that plateaued at >0.15 mM. In general, cations permeated the cellular TJs faster and anions slower than their neutral images. The effects of changes in pore size (4.6 to 14.6 Å in effective radius) on the ability of these solutes to permeate the TJs were analyzed by the Renkin molecular sieving function. These studies established an experimental, theoretical, and quantitative template to assess perturbants of the TJ and define the limits, short of detrimental effects, at which the TJs may be sufficiently perturbed for maximal enhancement of permeation of solutes varying in size and charge. Materials and Methods MaterialssMannitol, urea, formic acid, lactic acid, and methylamine were purchased as 14 C-labeled stock solutions from the American Radiolabeled Company (St. Louis, MO). Atenolol, EGTA, and PC were purchased from Sigma Chemical Company (St. Louis, MO). Hanks' balanced salt solution (HBSS), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonate (HEPES), nonessential amino acids, trypsin/EDTA, and Dulbecco's Modified Eagle Medium (DMEM) were purchased from JRH Biosciences (Lenexa, KS). Penicillin and streptomycin were purchased as a mixture from Irvine Scientific (Santa Ana, CA). Fetal bovine serum and type I rat tail collagen were purchased from Atlanta Biologicals (Norcross, GA) and Collaborative Research (Lexington, MA), respectively. Cell CulturesThe Caco-2 cell line, which originated from a human adenocarcinoma, 15 was obtained from the American Type Culture Collection (Rockville, MD) and was cultured as described previously by Hildago et al. 10 The cells were grown in 150-cm 2 culture flasks

Assessing the performance of amorphous solid dispersions

Journal of Pharmaceutical Sciences, Apr 1, 2012

The characterization and performance of stable amorphous solid dispersion systems were evaluated ... more The characterization and performance of stable amorphous solid dispersion systems were evaluated in 40 research papers reporting active pharmaceutical ingredient (API) dissolution and bioavailability from various systems containing polymers. The results from these studies were broadly placed into three categories: amorphous dispersions that improved bioavailability (∼82% of the cases), amorphous dispersions possessing lower bioavailability than the reference material (∼8% of the cases), and amorphous dispersions demonstrating similar bioavailabilities as the reference material (∼10% of the cases). A comparative analysis of these studies revealed several in vitro and in vivo variables that could have influenced the results. The in vitro factors compared primarily centered on dissolution testing and equipment, content and amount of dissolution media, sink or nonsink conditions, agitation rates, media pH, dissolution characteristics of the polymer, and dispersion particle size. The in vivo factors included reference materials used for bioavailability comparisons, animal species utilized, fasting versus fed conditions, and regional differences in gastrointestinal (GI) content and volume. On the basis of these considerations, a number of recommendations were made on issues ranging from the assessment of physical stability of API-polymer dispersions to in vivo GI physiological factors that require consideration in the performance evaluation of these systems.

Regulatory Considerations in Approval of Follow-On Protein Drug Products

Pharmaceutical Sciences Encyclopedia, Mar 15, 2010

Design of experiment based optimization of an in vitro direct contact triculture blood brain barrier model for permeability screening

Pharmacy & pharmacology international journal, Jul 28, 2021

The in vivo restrictive properties of the blood brain barrier (BBB) largely arise from astrocyte ... more The in vivo restrictive properties of the blood brain barrier (BBB) largely arise from astrocyte and pericyte synergistic cell signaling interactions that underlie the brain microvessel endothelial cells (BMEC). In vivo relevant direct contact between astrocytes, pericytes, and BMECS, to our knowledge, has not been established in conventional Transwell ® based in vitro screening models of the BBB. We hypothesize that a design of experiments (DOE) optimized direct contact layered triculture model will offer more in vivo relevance for screening in comparison to indirect models. Plating conditions including the seeding density of all three cell types, matrix protein, and culture time were assessed utilizing a DOE approach. A second set of DOE methods assessed the influence of medium additives on barrier properties. The optimized model was further assessed for p-glycoprotein function using a substrate and inhibitor along with a set of BBB paracellular and transcellular markers at varying permeation rates. The optimization revealed that length of culture post endothelial cell plating correlated highest with paracellular tightness. In addition, seeding density of the endothelial cell layer influenced paracellular tightness at earlier times of culture, and its impact decreased as culture is extended. At optimal conditions, the model revealed P-gp function along with the ability to differentiate between BBB positive and negative permeants. We have demonstrated that the implementation of DOE based optimization for biologically based systems is an expedited method to establish multi-component in vitro cell models. The direct contact BBB triculture model reveals that the physiologically relevant layering of the three cell types is a practical method of culture to establish a screening model compared to indirect plating methods that incorporate physical barriers between cell types. Additionally, the ability of the model to differentiate between BBB positive and negative permeants suggests that this model may be an enhanced screening tool for potential neuroactive compounds.

Do Sex and Ethnicity Influence Drug Pharmacokinetics in Solid Organ Transplantation?

Graft, Jul 1, 2002

A number of reports have identified differences in drug pharmacokinetics and pharmacodynamics bet... more A number of reports have identified differences in drug pharmacokinetics and pharmacodynamics between males and females, and between patients of different ethnicity. These differences are observed in both solid organ transplant recipients and in nontransplant patients. This review summarizes the pharmacokinetic differences thought to be dependent on sex and ethnicity noted with immunosuppressive agents commonly used in transplantation. This manuscript also touches on how these differences may translate into observed drug interactions. Furthermore, potential factors underlying the sex and ethnicity differences are evaluated. Current evidence points to differences in drug-metabolizing enzymes, transporters, cytokines, and environmental influences to account for the observed heterogeneity in drug response. Many of these factors seem to be under genetic control, and thus areas for further research are suggested.

Effects of Dendrimer-Like Biopolymers on Physical Stability of Amorphous Solid Dispersions and Drug Permeability Across Caco-2 Cell Monolayers

Aaps Pharmscitech, Jun 4, 2018

The potential applications of dendrimer-like biopolymers (DLB) as stabilizing excipients for amor... more The potential applications of dendrimer-like biopolymers (DLB) as stabilizing excipients for amorphous solid dispersion (ASD) of niclosamide, celecoxib, and resveratrol were evaluated based on (1) the formation and physical stability of the ASD and (2) the permeability and flux of the agents across Caco-2 cell monolayers. The evaluation was made by comparing the performance of prototype phytoglycogen derivatives (DLB1, DLB2, and DLB3) with commonly used polymers such as HPMCAS, PVPVA, and Soluplus®. PXRD was used to confirm the formation of the dispersions and detect crystallinity peaks formed during 2-and 4-week storage at 40°C/75% RH. At concentrations below 2 g/mL, the viability of Caco-2 cells remained above 80% for all DLB samples compared to untreated cells in the MTT assay. Permeability studies revealed a repeating pattern in which an increase in the initial concentration (C 0) was associated with a concomitant decrease in the apparent permeability (P app) which we theorize is due to differences in drug-polymer interactions. Niclosamide-DLB1 dispersion had the lowest flux due to a significant reduction in P app. The high increase in the C 0 of celecoxib-DLB2, however, made up for the reduction in the P app and produced the highest flux values compared to other polymers. Resveratrol-DLB3 had a 5× reduction in P app , but C 0 increased from 25.8 to 176 μg/mL led to a higher flux compared to the crystalline drug without polymer. Collectively, these results provide a Bproof-of-concept^basis to demonstrate that DLB excipients have the ability to increase apparent solubility (Sol app), most likely due to drug-binding capacity.