Jerome Hochman - Academia.edu (original) (raw)

Papers by Jerome Hochman

Research paper thumbnail of Drug–Drug Interactions: Regulatory and Theoretical Considerations, and an Industry Perspective

The Royal Society of Chemistry eBooks, Nov 20, 2015

In this review, a number of theoretical bases and regulatory framework are presented for drug–dru... more In this review, a number of theoretical bases and regulatory framework are presented for drug–drug interactions (DDIs), with emphasis on those related to absorption and distribution. Also presented is an industry perspective on how to approach these issues in support of drug development. Overall, holistic integration and understanding of the pharmaceutical (e.g., pH-dependent solubility) and pharmacological (e.g., gastrointestinal physiology and therapeutic margin) profiles, as well as pharmacokinetics and underlying absorption and disposition determinants (e.g., clearance, volume of distribution, permeability and protein binding) of drug candidates in various clinical setting should be considered as this can be valuable in ensuring the safe and effective use of new drugs.

Research paper thumbnail of Influence of P-glycoprotein on the transport and metabolism of indinavir in Caco-2 cells expressing cytochrome P-450 3A4

PubMed, 2000

Caco-2 cells grown in the presence of 1alpha,25-di-OH vitamin D(3) (di-OH vit D(3)) were used as ... more Caco-2 cells grown in the presence of 1alpha,25-di-OH vitamin D(3) (di-OH vit D(3)) were used as a model to evaluate the effects of P-glycoprotein (Pgp) efflux on CYP3A4-mediated metabolism of indinavir during intestinal absorption. Caco-2 cells grown under these conditions demonstrated significant CYP3A4 activity and maintained Pgp-mediated directional transport of indinavir. Metabolism of indinavir in the di-OH vit D(3)-treated cells correlated with the level of CYP3A activity and generated metabolites consistent with CYP3A4-mediated metabolism. During transport experiments, indinavir metabolites are selectively secreted into the apical compartment, consistent with Pgp-mediated efflux. Using formation of the most abundant metabolite, M6, as a marker for indinavir metabolism, we observed that the extent of indinavir metabolism is not significantly affected by the direction of indinavir transport or by inhibition of Pgp with cyclosporin A. However, because Pgp efflux results in higher indinavir transport in the basolateral-to-apical direction than in the apical-to-basolateral direction, the ratio of M6 produced normalized to the amount of drug transported across the monolayer was higher for apical-to-basolateral transport. Thus, Pgp efflux in a direction opposite to absorptive transport results in more metabolite produced per mole of drug that is absorbed. In summary, the results support a role of Pgp in increasing intestinal presystemic metabolism and in removal of CYP3A4-generated metabolites from the intracellular compartment.

Research paper thumbnail of Comparative studies of drug-metabolizing enzymes in dog, monkey, and human small intestines, and in Caco-2 cells

PubMed, Jun 1, 1996

Drug-metabolizing enzymes were studied in subcellular fractions of dog, monkey, and human small i... more Drug-metabolizing enzymes were studied in subcellular fractions of dog, monkey, and human small intestines, and in the human adenocarcinoma cell line Caco-2, a commonly used in vitro absorption model. Immunoblot analysis indicated the presence of enzymes related to cytochrome P450 (CYP) 1A1/CYP1A2, CYP2D6, CYP3A, and carboxylesterases (ESs) in human and monkey intestines, and of CYP3A and ES in dog intestines. Catalytically, human and monkey intestines exhibited significant and comparable testosterone 6 beta-hydroxylase, (+)-bufuralol 1'-hydroxylase, and ES activities. In contrast, dog intestine possessed moderate testosterone 6 beta-hydroxylase, much lower ES, and undetectable bufuralol hydroxylase activities. In addition, low tolbutamide methylhydroxylase activity was observed in human and monkey intestines, but not in dog intestines. Of the phase I enzymes investigated, only ES was detected immunologically and functionally in Caco-2 cells. With respect to phase II enzymes, human and monkey intestines contained relatively high intestinal glucuronyltransferase, N-acetyltransferase (NAT), sulfotransferase, and glutathione S-transferase activities. Except for NAT, all phase II enzymes studied were detectable in dog intestines. In Caco-2 cells, acetaminophen sulfation activity was below the limit of detection, whereas all other conjugating activities were evident. Studies of enzyme kinetics and inhibition by known inhibitors of testosterone 6 beta-hydroxylase activity, the major intestinal mono-oxygenase in all species, revealed some similarities between the responsible enzymes. Comparative studies with human liver microsomes suggested the possible involvement of CYP3A enzymes in the intestinal catalysis of testosterone 6 beta-hydroxylation similar to those observed with human hepatic CYP3A. Further studies on ESs, however, revealed multiplicity and species and/or tissue differences in the microsomal and cytosolic enzymes. Based on kinetic studies, monkey intestines and Caco-2 cells possessed NAT activities, with properties similar to those in human intestine and liver. Overall, the results demonstrated that both the preparations of small intestines and Caco-2 cells exhibited significant drug-metabolizing enzyme activities, although several differences were noted between the intestinal enzymes in the animals or in the Caco-2 cells and those found in humans.

Research paper thumbnail of Mini Review on Molecular Modeling of P-Glycoprotein (Pgp)

Current Topics in Medicinal Chemistry, Aug 1, 2007

Research paper thumbnail of Associations Between Class I MHC Antigens and Viral Glycoproteins Detected by Fluorescence Resonance Energy Transfer

Springer eBooks, 1987

Cytotoxic T lymphocytes, CTL, recognize viral antigens of infected cells in the context of the ce... more Cytotoxic T lymphocytes, CTL, recognize viral antigens of infected cells in the context of the cells’ class I MHC antigens (Zinkernagel and Doherty, 1979). The basis of this MHC restriction is not defined. The weight of the evidence favors recognition of some complex of all or part of viral antigen with the class I antigen. The route of formation of this complex and its stability are still unclear. In some instances, influenza virus infection (Townsend et al., 1984; Yewdell et al., 1985) and SV40 transformation (Gooding and O’Connell, 1983) it is evident that the CTL are primarily directed against internal viral proteins, the nucleocapsid protein of the influenza virus or the T antigen of SV40. This, together with the demonstration that peptides derived from these proteins (Townsend, et al., 1986) can sensitise target cells, strongly implies that these antigens are partly digested, and associated with class I MHC antigens before appearing at the cell surface. Such a mechanism of association parallels that mechanism for the formation of nominal antigen/class II MHC antigen complexes for presentation to helper T cells (Buus et al., 1987; Unanue and Allen, 1987) and unifies our view of antigen presentation to T cells. However, the generality of this scheme is still not clear. Some virus antigens, even the influenza virus nucleocapsid, can be presented to cells under conditions in which processing is blocked (Wraith and Vessey, 1986), suggesting that they associate with class I antigens without internalization and processing.

Research paper thumbnail of Renal elimination of a novel and potent α<sub>v</sub>β<sub>3</sub>integrin antagonist in animals

Xenobiotica, 2004

ABSTRACT

Research paper thumbnail of Diffusion in the mitochondrial inner membrane : implications for the mechanism of electron transfer and hexaammineruthenium, an effective electron donor to cytochrome oxidase

Research paper thumbnail of Mobility of cytochrome c on mitochondrial membranes measured by fluorescence recovery after photo bleaching

Research paper thumbnail of LeCluyse, E. L., Audus, K. L. & Hochman, J. H. Formation of extensive canalicular networks by rat hepatocytes cultured in collagen-sandwich configuration. Am. J. Physiol. 266, C1764-C1774

The American journal of physiology

Rat primary hepatocytes were cultured under different extracellular matrix configurations and eva... more Rat primary hepatocytes were cultured under different extracellular matrix configurations and evaluated for the acquisition and maintenance of structural and functional cell polarity. De novo repolarization of the plasma membrane was variable in rate and extent in hepatocyte cultures maintained on a conventional single layer of either gelled or ungelled collagen. However, cultures maintained in a collagen-sandwich configuration initiated uniform formation of a contiguous anastomosing network of bile canaliculi throughout the entire culture. Localization of apical membrane markers demonstrated normal distribution at the canalicular membrane. A marked rearrangement of the intracellular microfilaments to the cell periphery was observed and coincided with the development of the bile canaliculi. Acquisition of normal bile canalicular function and integrity was observed within 3-4 days postoverlay as indicated by the concentration and retention of carboxyfluorescein within the canalicular...

Research paper thumbnail of Conditionally Immortalized Intestinal Epithelial Cells: A New Model for Studying Intestinal Epithelial Cell Turnover

Annals of the New York Academy of Sciences, 1998

In order to understand intestinal epithelial cell turnover, it is essential to consider the equil... more In order to understand intestinal epithelial cell turnover, it is essential to consider the equilibrium between cell proliferation and cell depletion by apoptosis, taking into account all intermediate steps of intestinal cell differentiation. In spite of a need to obtain a model for studying the processes involved in each step between enterocyte proliferation and death, currently used cell culture models do not resemble natural conditions in full. 1 Adult villus enterocytes in primary culture have a short life span, and also undergo de-differentiation. Attempts to induce differentiation in long-term cultures of intestinal crypt-like cells have been unsatisfactory. Human tumor cell lines, such as Caco-2 and HT-29, are able to differentiate into an enterocyte-like phenotype in culture, but maintain essential characteristics of tumor cells, being unsuitable for studies of regulatory mechanisms in normal intestinal cell differentiation and its termination in apoptotic death. With the identification and characterization of viral oncogenes has come the idea that differentiated cell lines could be established by transformation of primary cultures with cloned immortalizing genes. However, cells transformed with wild-type oncogenes generally acquire properties similar to those of tumor cells, and expression of tissue-specific differentiated characteristics is, in many cases, altered qualitatively or quantitatively by transformation. 2 Conditional immortalization of the intestinal epithelial cells by using temperature-sensitive mutants of SV40 LT-Ag (tsA58 mutant) seems to offer a more acceptable solution. SV40 LT-Ag acts by inactivating negative regulators of cell proliferation that overcome cell senescence. 3 Reduction or removal of the oncogene activity by a simple temperature switch leaves the cells paralyzed at the point in the cycle at which the immortalizing component is essential. In theory at least, SV40 large T-antigenimmortalized cell lines can retain the phenotype of the differentiated lineage of the parent cells at the time of immortalization.

Research paper thumbnail of Specific associations of fluorescent beta-2-microglobulin with cell surfaces. The affinity of different H-2 and HLA antigens for beta-2-microglobulin

Journal of Immunology, Apr 1, 1988

CTL and serologically de ned antigens of B2m,H-3 region.

Research paper thumbnail of with oral administration of a BACE1 inhibitor in nonhuman primates

BACE1 cleavage of Amyloid Precursor Protein is an essential step in the generation of the potenti... more BACE1 cleavage of Amyloid Precursor Protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic Aβ42 peptides in Alzheimer's disease. While previous mouse studies have shown brain Aβ lowering after BACE1 inhibition, extension of such studies to non-human primates or man was precluded by poor potency, brain penetration, and pharmacokinetics, of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor "TC-1" was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of Aβ in CSF and plasma could be evaluated. TC-1, a potent inhibitor (IC 50 ~0.4 nM), has excellent passive membrane permeability, low susceptibility to Pglycoprotein transport, and lowered brain Aβ levels in a mouse model. I.V. infusion of TC-1 led to a significant but transient lowering of CSF and plasma Aβ levels in conscious rhesus monkeys, since it underwent CYP3A4 mediated metabolism. Oral co-dosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys, led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPPβ, Aβ40, Aβ42 and plasma Aβ40 levels. CSF Aβ42 lowering showed an EC 50 of ~20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell based assay. These results demonstrate the first in vivo proof of concept of CSF Aβ lowering following oral administration of a BACE1 inhibitor in a non-human primate.

Research paper thumbnail of Correction to: Liposome Click Membrane Permeability Assay for Identifying Permeable Peptides

Pharmaceutical Research, Apr 1, 2021

Research paper thumbnail of Glycoproteins Detected by Fluorescence Resonance Energy Transfer

Research paper thumbnail of Correction to: Liposome Click Membrane Permeability Assay for Identifying Permeable Peptides

Pharmaceutical Research, 2021

A Correction to this paper has been published: https://doi.org/10.1007/s11095-021-03033-9

Research paper thumbnail of Hochman JH, Chiba M, Nishime J, et al. Influence of P-glycoprotein on the transport and metabolism of indinavir in Caco-2 cells expressing cytochrome P-450 3A4

Journal of Pharmacology and Experimental Therapeutics

ABSTRACT

Research paper thumbnail of Yamazaki M, Neway WE, Ohe T, Chen IW, Rowe JF, Hochman JH et al. In vitro substrate identification studies for P-glycoprotein-mediated transport: species difference and predictability of in vivo results. J Pharmacol Exp Ther 296: 723-735

Journal of Pharmacology and Experimental Therapeutics

ABSTRACT

Research paper thumbnail of Comparative studies of drug-metabolising enzymes in dog, monkey, and human small intestine, and in Caco-2 cells

Drug Metabolism and Disposition

Drug-metabolizing enzymes were studied in subcellular fractions of dog, monkey, and human small i... more Drug-metabolizing enzymes were studied in subcellular fractions of dog, monkey, and human small intestines, and in the human adenocarcinoma cell line Caco-2, a commonly used in vitro absorption model. Immunoblot analysis indicated the presence of enzymes related to cytochrome P450 (CYP) 1A1/CYP1A2, CYP2D6, CYP3A, and carboxylesterases (ESs) in human and monkey intestines, and of CYP3A and ES in dog intestines. Catalytically, human and monkey intestines exhibited significant and comparable testosterone 6 beta-hydroxylase, (+)-bufuralol 1'-hydroxylase, and ES activities. In contrast, dog intestine possessed moderate testosterone 6 beta-hydroxylase, much lower ES, and undetectable bufuralol hydroxylase activities. In addition, low tolbutamide methylhydroxylase activity was observed in human and monkey intestines, but not in dog intestines. Of the phase I enzymes investigated, only ES was detected immunologically and functionally in Caco-2 cells. With respect to phase II enzymes, hu...

Research paper thumbnail of Overview of siRNA Delivery and Mechanisms: In-vitro and In-vivo Applications

siRNAs are RNA duplexes approximately 19-21 nucleotides in length which in the context of endogen... more siRNAs are RNA duplexes approximately 19-21 nucleotides in length which in the context of endogenous eukaryotic proteins catalyze degradation of specific mRNAs in eukaryotic cells. The guide strand which is complementary to the targeted mRNA, incorporates into the RISC complex resulting in catalytic cleavage of the complementary mRNA sequence leading to transient knock down of protein expression. Using delivery vehicles to deliver siRNA into the cytosol of cells, siRNAs have been applied to study protein functions in vitro and in vivo. The ability to design siRNAs targeting specific proteins based on known sequence as well as the potential to knock down gene expression in vivo makes RNAi a particularly attractive technique for evaluation of protein functions in cellular and physiological processes as well as a promising novel therapeutic modality. From a drug metabolism perspective the ability to knock down proteins in liver in vivo and in hepatocytes in vitro makes RNAi a particula...

Research paper thumbnail of Specific associations of fluorescent β-2-microglobulin with cell surfaces. The affinity of different H-2 and HLA antigens for β-2-microglobulin

The Journal of Immunology

We prepared single-labeled FITC derivatives of beta-2-microglobulin (b2m) and examined their inte... more We prepared single-labeled FITC derivatives of beta-2-microglobulin (b2m) and examined their interactions with class I MHC Ag H chains on living cells. Human b2m was reacted with FITC under mild conditions and separated by hydroxylapatite chromatography into three peaks containing single labeled derivatives of b2m peaks A, B, and C, and a peak containing the unmodified protein. The three fluorescent derivatives labeled the surfaces of cells bearing class I MHC Ag. The labeling was specific for class I MHC Ag as indicated by failure to label cells in the presence of excess unlabeled b2m and failure to label the HLA-negative cell lines Daudi and 721.221. Mouse cells labeled with fluorescent human b2m were recognized by mAb to the class I MHC Ag and by virus-restricted cytotoxic T lymphocytes, suggesting that labeling with the fluorescent b2m does not significantly alter the structure of class I MHC Ag or impair their ability to present viral antigens to cytotoxic T lymphocytes. We det...

Research paper thumbnail of Drug–Drug Interactions: Regulatory and Theoretical Considerations, and an Industry Perspective

The Royal Society of Chemistry eBooks, Nov 20, 2015

In this review, a number of theoretical bases and regulatory framework are presented for drug–dru... more In this review, a number of theoretical bases and regulatory framework are presented for drug–drug interactions (DDIs), with emphasis on those related to absorption and distribution. Also presented is an industry perspective on how to approach these issues in support of drug development. Overall, holistic integration and understanding of the pharmaceutical (e.g., pH-dependent solubility) and pharmacological (e.g., gastrointestinal physiology and therapeutic margin) profiles, as well as pharmacokinetics and underlying absorption and disposition determinants (e.g., clearance, volume of distribution, permeability and protein binding) of drug candidates in various clinical setting should be considered as this can be valuable in ensuring the safe and effective use of new drugs.

Research paper thumbnail of Influence of P-glycoprotein on the transport and metabolism of indinavir in Caco-2 cells expressing cytochrome P-450 3A4

PubMed, 2000

Caco-2 cells grown in the presence of 1alpha,25-di-OH vitamin D(3) (di-OH vit D(3)) were used as ... more Caco-2 cells grown in the presence of 1alpha,25-di-OH vitamin D(3) (di-OH vit D(3)) were used as a model to evaluate the effects of P-glycoprotein (Pgp) efflux on CYP3A4-mediated metabolism of indinavir during intestinal absorption. Caco-2 cells grown under these conditions demonstrated significant CYP3A4 activity and maintained Pgp-mediated directional transport of indinavir. Metabolism of indinavir in the di-OH vit D(3)-treated cells correlated with the level of CYP3A activity and generated metabolites consistent with CYP3A4-mediated metabolism. During transport experiments, indinavir metabolites are selectively secreted into the apical compartment, consistent with Pgp-mediated efflux. Using formation of the most abundant metabolite, M6, as a marker for indinavir metabolism, we observed that the extent of indinavir metabolism is not significantly affected by the direction of indinavir transport or by inhibition of Pgp with cyclosporin A. However, because Pgp efflux results in higher indinavir transport in the basolateral-to-apical direction than in the apical-to-basolateral direction, the ratio of M6 produced normalized to the amount of drug transported across the monolayer was higher for apical-to-basolateral transport. Thus, Pgp efflux in a direction opposite to absorptive transport results in more metabolite produced per mole of drug that is absorbed. In summary, the results support a role of Pgp in increasing intestinal presystemic metabolism and in removal of CYP3A4-generated metabolites from the intracellular compartment.

Research paper thumbnail of Comparative studies of drug-metabolizing enzymes in dog, monkey, and human small intestines, and in Caco-2 cells

PubMed, Jun 1, 1996

Drug-metabolizing enzymes were studied in subcellular fractions of dog, monkey, and human small i... more Drug-metabolizing enzymes were studied in subcellular fractions of dog, monkey, and human small intestines, and in the human adenocarcinoma cell line Caco-2, a commonly used in vitro absorption model. Immunoblot analysis indicated the presence of enzymes related to cytochrome P450 (CYP) 1A1/CYP1A2, CYP2D6, CYP3A, and carboxylesterases (ESs) in human and monkey intestines, and of CYP3A and ES in dog intestines. Catalytically, human and monkey intestines exhibited significant and comparable testosterone 6 beta-hydroxylase, (+)-bufuralol 1'-hydroxylase, and ES activities. In contrast, dog intestine possessed moderate testosterone 6 beta-hydroxylase, much lower ES, and undetectable bufuralol hydroxylase activities. In addition, low tolbutamide methylhydroxylase activity was observed in human and monkey intestines, but not in dog intestines. Of the phase I enzymes investigated, only ES was detected immunologically and functionally in Caco-2 cells. With respect to phase II enzymes, human and monkey intestines contained relatively high intestinal glucuronyltransferase, N-acetyltransferase (NAT), sulfotransferase, and glutathione S-transferase activities. Except for NAT, all phase II enzymes studied were detectable in dog intestines. In Caco-2 cells, acetaminophen sulfation activity was below the limit of detection, whereas all other conjugating activities were evident. Studies of enzyme kinetics and inhibition by known inhibitors of testosterone 6 beta-hydroxylase activity, the major intestinal mono-oxygenase in all species, revealed some similarities between the responsible enzymes. Comparative studies with human liver microsomes suggested the possible involvement of CYP3A enzymes in the intestinal catalysis of testosterone 6 beta-hydroxylation similar to those observed with human hepatic CYP3A. Further studies on ESs, however, revealed multiplicity and species and/or tissue differences in the microsomal and cytosolic enzymes. Based on kinetic studies, monkey intestines and Caco-2 cells possessed NAT activities, with properties similar to those in human intestine and liver. Overall, the results demonstrated that both the preparations of small intestines and Caco-2 cells exhibited significant drug-metabolizing enzyme activities, although several differences were noted between the intestinal enzymes in the animals or in the Caco-2 cells and those found in humans.

Research paper thumbnail of Mini Review on Molecular Modeling of P-Glycoprotein (Pgp)

Current Topics in Medicinal Chemistry, Aug 1, 2007

Research paper thumbnail of Associations Between Class I MHC Antigens and Viral Glycoproteins Detected by Fluorescence Resonance Energy Transfer

Springer eBooks, 1987

Cytotoxic T lymphocytes, CTL, recognize viral antigens of infected cells in the context of the ce... more Cytotoxic T lymphocytes, CTL, recognize viral antigens of infected cells in the context of the cells’ class I MHC antigens (Zinkernagel and Doherty, 1979). The basis of this MHC restriction is not defined. The weight of the evidence favors recognition of some complex of all or part of viral antigen with the class I antigen. The route of formation of this complex and its stability are still unclear. In some instances, influenza virus infection (Townsend et al., 1984; Yewdell et al., 1985) and SV40 transformation (Gooding and O’Connell, 1983) it is evident that the CTL are primarily directed against internal viral proteins, the nucleocapsid protein of the influenza virus or the T antigen of SV40. This, together with the demonstration that peptides derived from these proteins (Townsend, et al., 1986) can sensitise target cells, strongly implies that these antigens are partly digested, and associated with class I MHC antigens before appearing at the cell surface. Such a mechanism of association parallels that mechanism for the formation of nominal antigen/class II MHC antigen complexes for presentation to helper T cells (Buus et al., 1987; Unanue and Allen, 1987) and unifies our view of antigen presentation to T cells. However, the generality of this scheme is still not clear. Some virus antigens, even the influenza virus nucleocapsid, can be presented to cells under conditions in which processing is blocked (Wraith and Vessey, 1986), suggesting that they associate with class I antigens without internalization and processing.

Research paper thumbnail of Renal elimination of a novel and potent α<sub>v</sub>β<sub>3</sub>integrin antagonist in animals

Xenobiotica, 2004

ABSTRACT

Research paper thumbnail of Diffusion in the mitochondrial inner membrane : implications for the mechanism of electron transfer and hexaammineruthenium, an effective electron donor to cytochrome oxidase

Research paper thumbnail of Mobility of cytochrome c on mitochondrial membranes measured by fluorescence recovery after photo bleaching

Research paper thumbnail of LeCluyse, E. L., Audus, K. L. & Hochman, J. H. Formation of extensive canalicular networks by rat hepatocytes cultured in collagen-sandwich configuration. Am. J. Physiol. 266, C1764-C1774

The American journal of physiology

Rat primary hepatocytes were cultured under different extracellular matrix configurations and eva... more Rat primary hepatocytes were cultured under different extracellular matrix configurations and evaluated for the acquisition and maintenance of structural and functional cell polarity. De novo repolarization of the plasma membrane was variable in rate and extent in hepatocyte cultures maintained on a conventional single layer of either gelled or ungelled collagen. However, cultures maintained in a collagen-sandwich configuration initiated uniform formation of a contiguous anastomosing network of bile canaliculi throughout the entire culture. Localization of apical membrane markers demonstrated normal distribution at the canalicular membrane. A marked rearrangement of the intracellular microfilaments to the cell periphery was observed and coincided with the development of the bile canaliculi. Acquisition of normal bile canalicular function and integrity was observed within 3-4 days postoverlay as indicated by the concentration and retention of carboxyfluorescein within the canalicular...

Research paper thumbnail of Conditionally Immortalized Intestinal Epithelial Cells: A New Model for Studying Intestinal Epithelial Cell Turnover

Annals of the New York Academy of Sciences, 1998

In order to understand intestinal epithelial cell turnover, it is essential to consider the equil... more In order to understand intestinal epithelial cell turnover, it is essential to consider the equilibrium between cell proliferation and cell depletion by apoptosis, taking into account all intermediate steps of intestinal cell differentiation. In spite of a need to obtain a model for studying the processes involved in each step between enterocyte proliferation and death, currently used cell culture models do not resemble natural conditions in full. 1 Adult villus enterocytes in primary culture have a short life span, and also undergo de-differentiation. Attempts to induce differentiation in long-term cultures of intestinal crypt-like cells have been unsatisfactory. Human tumor cell lines, such as Caco-2 and HT-29, are able to differentiate into an enterocyte-like phenotype in culture, but maintain essential characteristics of tumor cells, being unsuitable for studies of regulatory mechanisms in normal intestinal cell differentiation and its termination in apoptotic death. With the identification and characterization of viral oncogenes has come the idea that differentiated cell lines could be established by transformation of primary cultures with cloned immortalizing genes. However, cells transformed with wild-type oncogenes generally acquire properties similar to those of tumor cells, and expression of tissue-specific differentiated characteristics is, in many cases, altered qualitatively or quantitatively by transformation. 2 Conditional immortalization of the intestinal epithelial cells by using temperature-sensitive mutants of SV40 LT-Ag (tsA58 mutant) seems to offer a more acceptable solution. SV40 LT-Ag acts by inactivating negative regulators of cell proliferation that overcome cell senescence. 3 Reduction or removal of the oncogene activity by a simple temperature switch leaves the cells paralyzed at the point in the cycle at which the immortalizing component is essential. In theory at least, SV40 large T-antigenimmortalized cell lines can retain the phenotype of the differentiated lineage of the parent cells at the time of immortalization.

Research paper thumbnail of Specific associations of fluorescent beta-2-microglobulin with cell surfaces. The affinity of different H-2 and HLA antigens for beta-2-microglobulin

Journal of Immunology, Apr 1, 1988

CTL and serologically de ned antigens of B2m,H-3 region.

Research paper thumbnail of with oral administration of a BACE1 inhibitor in nonhuman primates

BACE1 cleavage of Amyloid Precursor Protein is an essential step in the generation of the potenti... more BACE1 cleavage of Amyloid Precursor Protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic Aβ42 peptides in Alzheimer's disease. While previous mouse studies have shown brain Aβ lowering after BACE1 inhibition, extension of such studies to non-human primates or man was precluded by poor potency, brain penetration, and pharmacokinetics, of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor "TC-1" was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of Aβ in CSF and plasma could be evaluated. TC-1, a potent inhibitor (IC 50 ~0.4 nM), has excellent passive membrane permeability, low susceptibility to Pglycoprotein transport, and lowered brain Aβ levels in a mouse model. I.V. infusion of TC-1 led to a significant but transient lowering of CSF and plasma Aβ levels in conscious rhesus monkeys, since it underwent CYP3A4 mediated metabolism. Oral co-dosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys, led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPPβ, Aβ40, Aβ42 and plasma Aβ40 levels. CSF Aβ42 lowering showed an EC 50 of ~20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell based assay. These results demonstrate the first in vivo proof of concept of CSF Aβ lowering following oral administration of a BACE1 inhibitor in a non-human primate.

Research paper thumbnail of Correction to: Liposome Click Membrane Permeability Assay for Identifying Permeable Peptides

Pharmaceutical Research, Apr 1, 2021

Research paper thumbnail of Glycoproteins Detected by Fluorescence Resonance Energy Transfer

Research paper thumbnail of Correction to: Liposome Click Membrane Permeability Assay for Identifying Permeable Peptides

Pharmaceutical Research, 2021

A Correction to this paper has been published: https://doi.org/10.1007/s11095-021-03033-9

Research paper thumbnail of Hochman JH, Chiba M, Nishime J, et al. Influence of P-glycoprotein on the transport and metabolism of indinavir in Caco-2 cells expressing cytochrome P-450 3A4

Journal of Pharmacology and Experimental Therapeutics

ABSTRACT

Research paper thumbnail of Yamazaki M, Neway WE, Ohe T, Chen IW, Rowe JF, Hochman JH et al. In vitro substrate identification studies for P-glycoprotein-mediated transport: species difference and predictability of in vivo results. J Pharmacol Exp Ther 296: 723-735

Journal of Pharmacology and Experimental Therapeutics

ABSTRACT

Research paper thumbnail of Comparative studies of drug-metabolising enzymes in dog, monkey, and human small intestine, and in Caco-2 cells

Drug Metabolism and Disposition

Drug-metabolizing enzymes were studied in subcellular fractions of dog, monkey, and human small i... more Drug-metabolizing enzymes were studied in subcellular fractions of dog, monkey, and human small intestines, and in the human adenocarcinoma cell line Caco-2, a commonly used in vitro absorption model. Immunoblot analysis indicated the presence of enzymes related to cytochrome P450 (CYP) 1A1/CYP1A2, CYP2D6, CYP3A, and carboxylesterases (ESs) in human and monkey intestines, and of CYP3A and ES in dog intestines. Catalytically, human and monkey intestines exhibited significant and comparable testosterone 6 beta-hydroxylase, (+)-bufuralol 1'-hydroxylase, and ES activities. In contrast, dog intestine possessed moderate testosterone 6 beta-hydroxylase, much lower ES, and undetectable bufuralol hydroxylase activities. In addition, low tolbutamide methylhydroxylase activity was observed in human and monkey intestines, but not in dog intestines. Of the phase I enzymes investigated, only ES was detected immunologically and functionally in Caco-2 cells. With respect to phase II enzymes, hu...

Research paper thumbnail of Overview of siRNA Delivery and Mechanisms: In-vitro and In-vivo Applications

siRNAs are RNA duplexes approximately 19-21 nucleotides in length which in the context of endogen... more siRNAs are RNA duplexes approximately 19-21 nucleotides in length which in the context of endogenous eukaryotic proteins catalyze degradation of specific mRNAs in eukaryotic cells. The guide strand which is complementary to the targeted mRNA, incorporates into the RISC complex resulting in catalytic cleavage of the complementary mRNA sequence leading to transient knock down of protein expression. Using delivery vehicles to deliver siRNA into the cytosol of cells, siRNAs have been applied to study protein functions in vitro and in vivo. The ability to design siRNAs targeting specific proteins based on known sequence as well as the potential to knock down gene expression in vivo makes RNAi a particularly attractive technique for evaluation of protein functions in cellular and physiological processes as well as a promising novel therapeutic modality. From a drug metabolism perspective the ability to knock down proteins in liver in vivo and in hepatocytes in vitro makes RNAi a particula...

Research paper thumbnail of Specific associations of fluorescent β-2-microglobulin with cell surfaces. The affinity of different H-2 and HLA antigens for β-2-microglobulin

The Journal of Immunology

We prepared single-labeled FITC derivatives of beta-2-microglobulin (b2m) and examined their inte... more We prepared single-labeled FITC derivatives of beta-2-microglobulin (b2m) and examined their interactions with class I MHC Ag H chains on living cells. Human b2m was reacted with FITC under mild conditions and separated by hydroxylapatite chromatography into three peaks containing single labeled derivatives of b2m peaks A, B, and C, and a peak containing the unmodified protein. The three fluorescent derivatives labeled the surfaces of cells bearing class I MHC Ag. The labeling was specific for class I MHC Ag as indicated by failure to label cells in the presence of excess unlabeled b2m and failure to label the HLA-negative cell lines Daudi and 721.221. Mouse cells labeled with fluorescent human b2m were recognized by mAb to the class I MHC Ag and by virus-restricted cytotoxic T lymphocytes, suggesting that labeling with the fluorescent b2m does not significantly alter the structure of class I MHC Ag or impair their ability to present viral antigens to cytotoxic T lymphocytes. We det...