The Neuroanatomic Studies of Albert L. Rhoton Jr. in Historical Context: An Analysis of Origin, Evolution, and Application.

The incorporation of perspective into art and science revolutionized the study of the brain. Beginning in about 1504, Leonardo da Vinci began to model the ventricles of the brain in three dimensions. A few years later, Andreus Vesalius illustrated radically novel brain dissections. Thomas Willis' work, Cerebri Anatome (1664), illustrated by Christopher Wren, remarkably showed the brain undersurface. Later, in the early 1800s, Charles Bell's accurate images of neural structures changed surgery. In the 1960s, Albert L. Rhoton Jr. (1932-2016) began to earn his place among the preeminent neuroanatomists by focusing his lens on microanatomy to harness a knowledge of microneurosurgery, master microneurologic anatomy, and use it to improve the care of his patients. Although his biography and works are well known, no analysis has been conducted to identify the progression, impact, and trends in the totality of his publications, and no study has assessed his work in a historical context compared with the contributions of other celebrated anatomists. We analyzed 414 of 508 works authored by Rhoton; these studies were analyzed according to subjects discussed, including anatomic region, surgical approaches, subjects covered, anatomic methods used, forms of multimedia, and subspecialty. Rhoton taught detailed neuroanatomy from a surgical perspective using meticulous techniques that evolved as the technical demands of neurosurgery advanced, inspiring students and contemporaries. His work aligns him with renowned figures in neuroanatomy, arguably establishing him historically as the most influential anatomist of the neurosurgical era.

Detecting and Quantifying Microscale Chemical Reactions in Pharmaceutical Tablets by Stimulated Raman Scattering Microscopy.

It has been estimated that approximately 50% of all marketed drug molecules are manufactured and administered in the form of salts, often with the goal of improving solubility, dissolution rate, and efficacy of the drug. However, salt disproportionation during processing or storage is a common adverse effect in these formulations. Due to the heterogeneous nature of solid drug formulations, it is essential to characterize the drug substances noninvasively at micrometer resolution to understand the molecular mechanism of salt disproportionation. However, there is a lack of such capability with current characterization methods. In this study, we demonstrate that stimulated Raman scattering (SRS) microscopy can be used to provide sensitive and quantitative chemical imaging of the salt disproportionation reaction of pioglitazone hydrochloride (PIO-HCl) at a very low drug loading (1% w/w). Our findings illuminate a water mediated pathway of drug disproportionation and highlight the importance of noninvasive chemical imaging in a mechanistic study of solid-state chemical reactions.

In Situ Stimulated Raman Scattering (SRS) Microscopy Study of the Dissolution of Sustained-Release Implant Formulation.

Localized drug delivery systems (DDSs) provide therapeutic levels of drug agent while mitigating side effects of systemic delivery. These systems offer controlled release over extended periods of time making them attractive therapies. Monitoring drug dissolution is vital for developing safe and effective means of drug delivery. Currently, dissolution characterization methods are limited to bulk analysis and cannot provide dissolution kinetics at high spatial resolution. However, dissolution rates of drug particles can be heterogeneous with influences from many factors. Insights into finer spatiotemporal dynamics of single particle dissolution could potentially improve pharmacokinetic modeling of dissolution for future drug development. In this work, we demonstrate high-resolution chemical mapping of entecavir, a hepatitis B antiviral drug, embedded in a slow release poly(d,l-lactic acid) formulation with stimulated Raman scattering (SRS) microscopy. By tracking the volume change of individual micron-sized drug particles within the polymer matrix, we establish an analytical protocol for quantitatively profiling dissolution of single crystalline particles in implant formulations in an in situ manner.

Expression Profile of Drug and Nutrient Absorption Related Genes in Madin-Darby Canine Kidney (MDCK) Cells Grown under Differentiation Conditions.

The expression levels of genes involved in drug and nutrient absorption were evaluated in the Madin-Darby Canine Kidney (MDCK) in vitro drug absorption model. MDCK cells were grown on plastic surfaces (for 3 days) or on Transwell® membranes (for 3, 5, 7, and 9 days). The expression profile of genes including ABC transporters, SLC transporters, and cytochrome P450 (CYP) enzymes was determined using the Affymetrix® Canine GeneChip®. Expression of genes whose probe sets passed a stringent confirmation process was examined. Expression of a few transporter (MDR1, PEPT1 and PEPT2) genes in MDCK cells was confirmed by RT-PCR. The overall gene expression profile was strongly influenced by the type of support the cells were grown on. After 3 days of growth, expression of 28% of the genes was statistically different (1.5-fold cutoff, p < 0.05) between the cells grown on plastic and Transwell® membranes. When cells were differentiated on Transwell® membranes, large changes in gene expression profile were observed during the early stages, which then stabilized after 5-7 days. Only a small number of genes encoding drug absorption related SLC, ABC, and CYP were detected in MDCK cells, and most of them exhibited low hybridization signals. Results from this study provide valuable reference information on endogenous gene expression in MDCK cells that could assist in design of drug-transporter and/or drug-enzyme interaction studies, and help interpret the contributions of various transporters and metabolic enzymes in studies with MDCK cells.

Sustained release subcutaneous delivery of BMS-686117, a GLP-1 receptor peptide agonist, via a zinc adduct.

BMS-686117 is an 11-mer GLP-1 receptor agonist with a short intrinsic pharmacokinetic half-life (t(1/2)) of approximately 2h. In order to develop an extended release formulation for once-daily (QD) subcutaneous administration, a non-covalently bonded Zn/BMS-686117 adduct with very low aqueous solubility was prepared through mixing zinc acetate and BMS-686117 solutions, followed by filtration or spray drying. At pH 6.8, free BMS-686117 concentration decreased continuously with the increase of Zn:BMS-686117 ratio. Furthermore, free BMS-686117 concentration increases in the presence of ethylenediaminetetraacetic acid (EDTA), indicating the reversibility of the zinc-peptide association. As solids, the glass transition temperature of Zn/BMS-686117 adduct increases with the increase of Zn:BMS-686117 ratio. A Zn/BMS-686117 adduct suspension, with a molar ratio of zinc:BMS-686117 of 3:1, was dosed subcutaneously to dogs along with two other solution formulations. The Zn/BMS-686117 adduct showed a prolonged BMS-686117 terminal t(1/2) of 8.5h, a mean residence time (MRT) of 16h, and a C(max) value 6-8 times lower than the solution formulations. Additionally, the Zn/BMS-686117 was encapsulated into poly(lactide-co-glycolide) (PLGA) microspheres. The Zn/BMS-686117 microspheres showed an almost zero-order release profile in vitro for at least 18 days, with minimal initial burst, indicating the potential of using this approach for long-term sustained release.

Pulmonary delivery of a GLP-1 receptor agonist, BMS-686117.

Alternate delivery route of therapeutic peptides is an attractive non-invasive option to patients who must chronically self-administer their medication through injections. In recent years, much attention has centered on pulmonary peptide delivery of peptide drugs such as insulin and GLP-1 mimetic peptides in the treatment of type II diabetes. In this study, we assessed the feasibility of delivering BMS-686117, an 11-mer GLP-1 receptor peptide agonist, to the lung in rats via intratracheal administration. The pharmacokinetic profiles of three spray-dried, prototype inhaled powder formulations, 80/20 BMS-686117/trehalose (I), 100% BMS-686117 (II), and 20/80 BMS-686117/mannitol (III), as well as a lyophilized BMS-686117 powder, were compared with intravenously and subcutaneously administered peptide. The spray-dried formulations were mostly spherical particles with narrow particle size distribution between 2 to 10 microm, which are better suited for inhalation delivery than the lyophilized, irregular shape powder with a wide particle size distribution between 2 to 100 microm. Prototype III exhibited the best physical characteristics and in vivo performance, with bioavailability of 45% relative to subcutaneous administration. The T(max) for lung delivered peptide formulations were almost twice as fast as subcutaneous injection, suggesting potential for rapid absorption and onset of action. This study demonstrated that pulmonary delivery is a promising, non-invasive route for the administration of BMS-686117.

Estimation of drug solubility in polymers via differential scanning calorimetry and utilization of the fox equation.

The solubility of drugs in polyethylene glycol 400 (PEG 400) was estimated and rank ordered using a differential scanning calorimetry (DSC) method and the Fox Equation. Drug-polymer binary mixtures of six compounds (Ibuprofen, Indomethacin, Naproxen, and three proprietary compounds: PC-1 through PC-3) with PEG 400 were heat treated using a three-cycle DSC method to establish a correlation between equilibrium solubility and temperature. Thermal events such as heat of fusion, heat of recrystallization and glass transition temperature, T(g), were used to calculate the drug solubility at multiple higher temperatures through the Fox Equation. Subsequently, a van't Hoff plot was constructed to estimate the drug solubility at room temperature, and the values were compared with those measured by HPLC. With the exception of Naproxen, room temperature solubilities of the remaining drug compounds in PEG 400 were determined by this thermal method approach, and compared with those measured by HPLC: 26.7% vs. 24.7% for Ibuprofen, 5.8% vs. 9.6% for Indomethacin, 3.1 % vs. 1.5% for PC-1, 2.3% vs. 1.3% for PC-2, and 1.4% vs. 0.2% for PC-3 in PEG 400. There was good concordance in solubility rank order estimates between the two methods. These collective results support the potential utility of the thermal method as an alternative to other methods for estimation of drug solubility in polymers which is an important determinant in the design of physically-stable amorphous systems.

Rat nasal lavage biomarkers to assess preclinical irritation potential of nasal drug formulations and excipients.

The goal of this study was to evaluate biomarkers of nasal mucosal damage for rapid assessment of irritancy potential of formulations in the rat nasal lavage model, a tool to facilitate nasal formulation development prior to histopathology studies. The nasal cavity of anesthetized rats was lavaged with normal saline 20 min pos-tdose. The collected fluid was analyzed for secreted total protein and biomarkers. Solutions tested include: normal saline, buffers, benzalkonium chloride (BAC), lysophosphatidylcholine (LPC), and four marketed nasal products. Total protein, lactate dehydrogenase and interleukin-1alpha biomarkers were secreted to varying degrees. BAC (0.2%) and LPC (0.5%) exhibiting the strongest response with a signal window ranging from 3.4- to 87-fold greater levels than normal saline. Buffer treatments, excipients, and most marketed nasal products yielded levels similar to normal saline. There was a weak correlation between formulation osmolarity and surface tension with any of the biomarkers. Each nasal formulation elicited a unique protein and biomarker profile with total protein secretion correlated with IL-1alpha secretion suggesting the potential for an inflammatory response. Taken together, rapid and potentially mechanistic information on the preclinical acute irritancy potential of formulations was assessed in the rat nasal lavage model by benchmarking treatments relative to controls and marketed nasal products.

Formation of zinc-peptide spherical microparticles during lyophilization from tert-butyl alcohol/water co-solvent system.

PURPOSE: To understand the mechanism of spherical microparticle formation during lyophilizing a tert-Butyl alcohol (TBA)/water solution of a zinc peptide adduct. METHOD: A small peptide, PC-1, as well as zinc PC-1 at (3:2) and (3:1) ratios, were dissolved in 44% (wt.%) of TBA/water, gradually frozen to -50 degrees C over 2 h ("typical freezing step"), annealed at -20 degrees C for 6 h ("annealing step"), and subsequently lyophilized with primary and secondary drying. Zinc peptide (3:1) lyophile was also prepared with quench cooling instead of the typical freezing step, or without the annealing step. Other TBA concentrations, i.e., 25%, 35%, 54% and 65%, were used to make the zinc peptide (3:1) adduct lyophile with the typical freezing and annealing steps. The obtained lyophile was analyzed by Scanning Electron Microscopy (SEM). The zinc peptide solutions in TBA/water were analyzed by Differential Scanning Calorimeter (DSC). The surface tension of the TBA/water co-solvent system was measured by a pendant drop shape method. RESULTS: With typical freezing and annealing steps, the free peptide lyophile showed porous network-like structure that is commonly seen in lyophilized products. However, with increasing the zinc to peptide ratio, uniform particles were gradually evolved. Zinc peptide (3:1) adduct lyophiles obtained from 25%, 35% and 44% TBA exhibit a distinctive morphology of uniform and spherical microparticles with diameters of approximately 3-4 microm, and the spherical zinc peptide particles are more predominant when the TBA level approaches 20%. Adopting quench cooling in the lyophilization cycle leads to irregular shape fine powders, and eliminating the annealing step causes rough particles surface. When TBA concentration increases above 54%, the lyophiles demonstrate primarily irregular shape particles. CONCLUSIONS: A proposed mechanism of spherical particle formation of the 3:1 zinc peptide encompasses the freezing of a TBA/water solution (20-70% TBA) causing the formation of a TBA hydrate phase ("dispersed TBA hydrate"). Decreasing the temperature further causes the formation of a eutectic mixture between TBA hydrate ("eutectic TBA hydrate") and water. Due to its low aqueous solubility, the zinc peptide adduct accumulates in both of the dispersed and eutectic TBA hydrate phases to form a hydrophobic "oil" phase. Since the eutectic TBA hydrate phase is surrounded by ice, a "solid emulsion" forms to lower the interfacial energy, and gives rise to spherical zinc peptide particles upon solvent sublimation. Possibility of liquid-liquid phase separation during freeze-drying was also investigated, and no evidence was found to support this alternative mechanism.

Mechanistic investigation of Pluronic based nano-crystalline drug-polymer solid dispersions.

PURPOSE: To understand the mechanism of nano-crystalline drug formation in Pluronic (i.e., poly(ethylene oxide-block-propylene oxide) triblock copolymers) based drug-polymer solid dispersions. MATERIALS AND METHODS: Four polymers, Pluronic F127, F108, F68 and PEG 8000, which have different poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) ratio and chain length, were co-spray dried with BMS-347070, a COX-2 inhibitor, to form 50/50 (w/w) drug-polymer solid dispersions. The solid dispersions were analyzed by powder X-ray diffraction (PXRD), modulated differential scanning calorimetry (mDSC), and hot-stage microscopy. Average size of drug crystallites in different polymers was calculated by the Scherrer equation based on peak-broadening effect in PXRD. Two other drug compounds, BMS-A and BMS-B, were also spray dried with Pluronic F127, and the solid dispersions were analyzed by PXRD and mDSC. RESULTS: The average size of BMS-347070 crystallites in PEG 8000, F127, F108 and F68 polymers was 69, 80, 98 and 136 nm, respectively, and the degree of BMS-347070 crystallinity is the lowest in PEG 8000. Hot-stage microscopy showed that 50/50 drug-polymer dispersions crystallized in a two-step process: a portion of the polymer crystallizes first (Step 1), followed by crystallization of drug and remaining polymer (Step 2). The T (g) value of the BMS-347070/Pluronic dispersions after Step 1 (i.e., T(g1)) was measured and/or calculated to be 15-26 degrees C, and that of BMS-347070/PEG 8000 was 60 degrees C. Solid dispersions of BMS-A and BMS-B in Pluronic F127 have T(g1) of 72 and 3 degrees C, respectively; and PXRD showed BMS-A remained amorphous after approximately 3 weeks under ambient condition, while BMS-B crystallized in F127 with an average crystallite size of 143 nm. CONCLUSIONS: The size of drug crystallites in the drug-polymer solid dispersions is independent of polymer topology, but is caused kinetically by a combined effect of nucleation rate and crystal growth rate. When drug-Pluronic solid dispersions crystallize at room temperature, that is close to the T(g1) of the systems, a fast nucleation rate and a relatively slow crystal growth rate of the drug synergistically produced small crystallite size. While the much higher T(g1) value of drug-PEG 8000 led to a slower nucleation rate and an even slower crystal growth rate at room temperature, therefore, small crystallite size and low drug crystallinity were observed. Results from BMS-A/Pluronic and BMS-B/Pluronic systems confirmed this kinetic theory.

Predicting effect of food on extent of drug absorption based on physicochemical properties.

PURPOSE: To develop a statistical model for predicting effect of food on the extent of absorption (area under the curve of time-plasma concentration profile, AUC) of drugs based on physicochemical properties. MATERIALS AND METHODS: Logistic regression was applied to establish the relationship between the effect of food (positive, negative or no effect) on AUC of 92 entries and physicochemical parameters, including clinical doses used in the food effect study, solubility (pH 7), dose number (dose/solubility at pH 7), calculated Log D (pH 7), polar surface area, total surface area, percent polar surface area, number of hydrogen bond donor, number of hydrogen bond acceptors, and maximum absorbable dose (MAD). RESULTS: For compounds with MAD >or= clinical dose, the food effect can be predicted from the dose number category and Log D category, while for compounds with MAD < clinical dose, the food effect can be predicted from the dose number category alone. With cross validation, 74 out of 92 entries (80%) were predicted into the correct category. The correct predictions were 97, 79 and 68% for compounds with positive, negative and no food effect, respectively. CONCLUSIONS: A logistic regression model based on dose, solubility, and permeability of compounds is developed to predict the food effect on AUC. Statistically, solubilization effect of food primarily accounted for the positive food effect on absorption while interference of food with absorption caused negative effect on absorption of compounds that are highly hydrophilic and probably with narrow window of absorption.

Human PEPT1 pharmacophore distinguishes between dipeptide transport and binding.

The human intestinal oligopeptide transporter (PEPT1) facilitates the absorption of dipeptides, tripeptides, and many peptidomimetic drugs. In this study, a large number of peptides were selected to investigate the structural features required for PEPT1 transport. Binding affinity was determined in a Gly-Sar uptake inhibition assay, whereas functional transport was ranked in a membrane depolarization assay. Although most of the peptides tested could bind to PEPT1, not all were substrates. As expected, single amino acids and tetrapeptides could not bind to or be transported by PEPT1. Dipeptide transport was influenced by charge, hydrophobicity, size, and side chain flexibility. The extent of transport was variable, and unexpectedly, some dipeptides were not substrates of PEPT1. These included dipeptides with two positive charges or extreme bulk in either position 1 or 2. Our results identify key features required for PEPT1 transport in contrast to most previously described pharmacophores, which are based on the inhibition of transport of a known substrate.

Carbopol-mediated paracellular transport enhancement in Calu-3 cell layers.

The interaction of Carbopol polymers with mucus producing Calu-3 human bronchial epithelial cells was evaluated to test for potential paracellular transport enhancement. Using desmopressin (1-deamino-8-arginine-vasopressin, DDAVP) as the model peptide, apical treatment with Carbopol polymer gel formulations resulted in molecular size-dependent permeability enhancement with a concomitant drop in the transepithelial electrical resistance (TEER). Permeability enhancement of DDAVP was dependent on the formulation vehicle composition and polymer concentration, was noncytotoxic, and completely reversible. Carbopol 971P displayed the greatest permeability enhancement across Calu-3 cells compared to other more viscous Carbopol polymers 934P and 974P, and other mucoadhesive cellulosic polymers. The greatest enhancement was observed when C971P formulation was prepared in water at a concentration of 0.25% w/v. Enhancement was confirmed in rabbit dosed with intranasal fluorescent dextran 4400. The C(max) and absorption rate each increased by 48% in C971P formulations compared to control, while the relative exposure increased 30%. In conclusion, Carbopol polymers are potentially useful excipients to enhance intranasal peptide absorption. We hypothesize that the permeation enhancement is related to the chelation of extracellular or tight-junctional Ca(2+) by charged polymer carboxylate groups that leads to temporary disruption of tight-junctions, thereby facilitating paracellular transport.

A rabbit model for sublingual drug delivery: comparison with human pharmacokinetic studies of propranolol, verapamil and captopril.

A rabbit model for investigating sublingual drug absorption was established yielding results consistent with clinical data reported in the literature. Using propranolol as a model compound the effect of formulation and dosing variables was explored as a means to characterize the limiting parameters of this model. In addition, verapamil and captopril were selected as reference compounds to compare this model to sublingual absorption in humans. Rabbits were dosed sublingually and systemic absorption was measured over time. Sublingual absorption of propranolol was dependent on dosing solution pH and volume. Intra-oral spray device did not affect the overall exposure compared to instillation using a syringe. Despite species and dosing regimen differences the relative bioavailabilities of propranolol and verapamil were very similar in rabbits and humans. In contrast, captopril absorption from the sublingual cavity of rabbits was low and did not agree with that observed in man. Here we report a sublingual rabbit model of drug delivery and its potential utility in preclinical development of intra-oral dosage forms.

pH-dependent dissolution in vitro and absorption in vivo of weakly basic drugs: development of a canine model.

PURPOSE: The aim of this research was to develop a pH-dependent canine absorption model for studying pH effect on both dissolution in vitro and pharmacokinetics in vivo using the weak bases ketoconazole and dipyridamole as model drugs. METHODS: Ketoconazole and dipyridamole pH-dependent dissolution profiles in vitro were determined by dissolution test at different pH values using USP apparatus II and an Opt-Diss Fiber Optic UV System. In vivo absorption studies for ketoconazole and dipyridamole were performed with crossover design in three groups of beagle dogs under control (no treatment), pentagastrin, and famotidine treatments. Ketoconazole and dipyridamole plasma concentrations were quantified by gradient high performance liquid chromatography mass spectroscopy (HPLC MS/MS). Pharmacokinetic parameters were determined from individual plasma concentration vs. time profiles. RESULTS: Ketoconazole and dipyridamole displayed pH-dependent dissolution. Increasing the pH of the dissolution medium from 1.2 to 6.8 reduced the extent of dissolution of ketoconazole and dipyridamole at 1 h by 96% and 92%, respectively. In vivo studies in dogs under control (no treatment), pentagastrin, and famotidine treatments show marked differences in systemic ketoconazole and dipyridamole exposure. Area under the concentration-time curve (AUC) increased more than 4-fold as compared to control group, whereas it increased nearly 30-fold for ketoconazole and 9-fold for dipyridamole with pentagastrin (gastric pH approximately 2-3) as compared to famotidine (gastric pH approximately 5-7.5) treatment. CONCLUSIONS: This work demonstrates a pH-dependent dissolution in vitro and absorption in vivo for the weak bases ketoconazole and dipyridamole independent of food effects. This model is useful to examine pH-dependent effects on oral drug absorption and for screening formulations to overcome the pH dependency.

In vitro testing of drug absorption for drug 'developability' assessment: forming an interface between in vitro preclinical data and clinical outcome.

Drug 'developability' assessment has become an increasingly important addition to traditional drug efficacy and toxicity evaluations, as pharmaceutical scientists strive to accelerate drug discovery and development processes in a time- and cost-effective manner. The fraction of drug absorbed and the maximum absorbable dose (MAD) can be estimated from in vivo clinical pharmacokinetics, mass balance studies or in vivo drug permeability in humans by different calculation methods. Unfortunately, in vivo data are usually unavailable at the early stages of drug discovery and development, and in vitro screening for the permeability, solubility, activity and toxicity of a drug has become a routine measurement in drug discovery and development. These in vitro data could be used to predict drug 'developability' with different calculation methods before selecting candidates for clinical evaluation. The fraction of drug absorbed in human could be predicted by in vivo human permeability or in vitro Caco2 permeability. For example, if drug permeability in Caco2 cells reaches 13.3 to 18.1 x 10(-6) cm/s, its predicted in vivo permeability in humans would reach 2 x 10(-4) cm/s, and its predicted fraction of drug absorbed would be > 90%, which is defined as highly permeable. The MAD could also be predicted with in vitro permeability, or calculated absorption rate constant. In addition, in vitro solubility and permeability data can also be used for the biopharmaceutics classification system (BCS) and, subsequently, to direct formulation optimization strategies. If drug 'developability' becomes an obstacle for drug delivery based on these in vitro data and predictions at the early stages of drug discovery and development, options such as prodrug approaches could be explored to enhance drug 'developability', in addition to different formulation methods. Therefore, in vitro absorption testing is a highly valuable tool in the decision-making process to select candidates for in vivo clinical studies at early-stage drug discovery and development.

A novel high-throughput pepT1 transporter assay differentiates between substrates and antagonists.

PepT1 is a transporter of proven pharmaceutical utility for enhancing oral absorption. A high-throughput, robust functional assay, capable of distinguishing PepT1 binders from substrates, allowing identification and/or prediction of drug candidate activation was developed. An MDCK epithelial cell line was transfected with rPepT1. The high level of stable rPepT1 expression that was achieved enabled development of a miniaturized PepT1 assay in a 96-well format, which could be scaled to 384 wells. The assay is based on measurement of membrane depolarization resulting from the cotransport of protons and PepT1 substrates. Membrane potential changes are tracked with a voltage-sensitive fluorescent indicator. Control (mock-transfected) cells are used to determine nonspecific membrane potential changes. A variety of fluorescent dyes were tested during initial assay design, including intracellular pH and membrane potential indicators. A membrane potential indicator was chosen because of its superior performance. Upon PepT1 activation with glycylsarcosine, dose-dependent membrane depolarization was observed with an EC50 of 0.49 mM. Maximum depolarization was dependent on the level of PepT1 expression. Testing of 38 known PepT1 substrates, binders, and nonbinders demonstrated that this assay accurately distinguished substrates from binders and from nonbinders. Initial validation of this novel assay indicates that it is sensitive and robust, and can distinguish between transporter substrates and antagonists. This important distinction has been previously achieved only with lower-throughput assays. This assay might also be used to determine substrate potency and establish a high-quality data set for PepT1 SAR modeling.

A novel hPepT1 stably transfected cell line: establishing a correlation between expression and function.

Stably transfected MDCK/hPepT1-V5&His clonal cell lines expressing varying levels of epitope-tagged hPepT1 protein were established to quantify the relationship between transgene hPepT1 expression levels and its functional kinetics in facilitating peptide and peptide-like drug uptake and transport in vitro. The hPepT1 sequence was amplified from Caco-2 cell mRNA, inserted into the pcDNA3.1 -V5&His TOPO plasmid, and transfected into MDCK cells. Transgene protein levels were quantified by Western Blot analysis utilizing a standard curve generated with a positive control protein containing a V5&His epitope. Three clones expressing different levels of the hPepT1 fusion protein (low, medium, and high) were selected for the functional characterization with [14C]Gly-Sar and [3H]carnosine. The MDCK/hPepT1 cells expressed a novel hPepT1/epitope tag protein with an apparent molecular mass of 110 kDa. The [14C]Gly-Sar uptake in the transfected cells was sodium-independent and pH-dependent, demonstrating enhanced uptake, the rate of which increased significantly from the weakly to strongly expressing hPepT1 MDCK/hPepT1 -V5&His clones as compared to the mock cell line at pH 6.0. The uptake and permeability of [14C]Gly-Sar and [3H]carnosine demonstrated a direct correlation between the hPepT1 level of expression, uptake, and transport capabilities. Molecular and functional characterization of the MDCK/hPepT1-V5&His cell line confirmed a directly proportional relationship between Vmax and Papp versus the molar levels of hPepT1 transgene expression. This stably transfected hPepT1 cell line may serve as a useful in vitro model for screening and quantifying peptide and peptide-like drug transport as a function of hPepT1 expression in drug discovery.

Membrane topology of the ZntB efflux system of Salmonella enterica serovar Typhimurium.

The membrane topology of the ZntB Zn(2+) transport protein of Salmonella enterica serovar Typhimurium was determined by constructing deletion derivatives of the protein and genetically fusing them to blaM or lacZ cassettes. The enzymatic activities of the hybrid proteins indicate that ZntB is a bitopic integral membrane protein consisting largely of two independent domains. The first 266 amino acids form a large, highly charged domain within the cytoplasm, while the remaining 61 residues form a small membrane domain containing two membrane-spanning segments. The overall orientation towards the cytoplasm is consistent with the ability of ZntB to facilitate zinc efflux.