P-glycoprotein efflux at the blood-brain barrier mediates differences in brain disposition and pharmacodynamics between two structurally related neurokinin-1 receptor antagonists.

CP-122721 and CP-141938 are potent and selective neurokinin-1 (NK(1)) receptor antagonists with very different brain disposition and potency in models of centrally mediated activity. These investigations sought to determine whether differences in potency were related to differences in P-glycoprotein (P-gp) transport at the blood-brain barrier. Both compounds stimulated ATPase activity of human recombinant MDR1 with similar kinetic parameters. Cell-associated drug concentrations of CP-141938 were 9.4-fold lower in KBV1 cells expressing P-gp compared with KB3.1 control cells. In Madin-Darby canine kidney (MDCK) cells expressing human MDR1, asymmetric transport of CP-141938 was 5-fold higher than in wild-type MDCK cells, whereas no asymmetry was observed with CP-122721. In agreement with these differences in cellular transport, the differences in brain/plasma ratio between mdr1a/b(-/-) and FVB mice 1 h following a 3 mg/kg s.c. dose were 3- and 50-fold for CP-122721 and CP-141938, respectively. The effect of inhibiting P-gp efflux on the effects of these agents was evaluated using GR73632-induced foot tapping in gerbils as a model to measure centrally mediated NK(1) antagonism. When gerbils were pretreated with the P-gp inhibitor MS-073 (50 mg/kg s.c.), there was no effect on the activity of CP-122721 (0.05 mg/kg), whereas the percent reversal for CP-141938 (10 mg/kg) increased from 60 to 100%. In gerbils, the brain/plasma ratio for CP-122721 was unaffected by MS-073 pretreatment, whereas the brain/plasma ratio for CP-141938 brain concentrations increased 13-fold. This suggested that P-gp efflux influences the brain disposition and pharmacologic activity of CP-141938, but not CP-122721. Complete response curves for CP-141938 were then determined with respect to dose, and drug concentration in the plasma and brain in the presence and absence of MS-073 pretreatment. The dose and plasma concentration-response curves of CP-141938 were shifted to the left in the presence of MS-073, yet brain concentrations associated with the response were unchanged. This suggested that once in the brain the interaction of CP-141938 with the NK(1) receptor was not affected by P-gp transport. In conclusion, these studies show that brain disposition and centrally mediated in vivo activity of NK(1) antagonists can be profoundly affected by P-gp transport and that such transport should be considered during the design of new agents.

Simple purification of highly active biotinylated P-glycoprotein: enantiomer-specific modulation of drug-stimulated ATPase activity.

A simplified method for the expression and purification of P-glycoprotein (Pgp) is presented. This method is based on the in-frame fusion of both a polyhistidine tail and a 100-amino acid residue biotin acceptor domain of oxaloacetate decarboxylase from Klebsiella pneumoniae at the carboxyl terminus end of Pgp (Pgp-H6BD). The expression/purification protocol for Pgp-H6BD involves high-level expression of the fusion protein in the yeast Pichia pastoris, biotinylation in vitro with biotin ligase, solubilization of crude membrane fractions in detergent, and affinity purification by a combination of nickel and avidin chromatography. Biotinylated Pgp binds to immobilized monomeric avidin and can be eluted with free biotin in a high state of purity. This protocol is rapid and efficient and yields purified Pgp which shows robust ATPase activity, as determined by vanadate-induced trapping of photoactive nucleotides and by direct measurement of ATP hydrolysis by Pgp-H6BD. This method should be useful for structural studies of the protein by spectroscopic or crystallographic approaches. This purified Pgp-H6BD preparation has been used to study the enantiomer-specific effects of inhibitors of Pgp-mediated drug transport on the drug-stimulated ATPase activity of the protein. A series of 1, 4-disubstituted piperazine derivatives with a central chiral carbon and modified at the head and tail groups are shown to stimulate Pgp ATPase activity in a dose-dependent fashion. Some of these compounds are also capable of inhibiting either vinblastine or verapamil stimulation of ATPase activity of Pgp in an enantiomer-specific fashion. The enantiomeric specific inhibitory activity of these compounds suggests complex interactions at a single substrate binding site(s) on Pgp.

P-glycoprotein and cytochrome P-450 3A inhibition: dissociation of inhibitory potencies.

Many P-glycoprotein (P-gp) inhibitors studied in vitro and in vivo are also known or suspected to be substrates and/or inhibitors of cytochrome P-450 3A (CYP3A). Such overlap raises the question of whether CYP3A inhibition is an intrinsic characteristic of P-gp inhibitors, a matter of concern in the development and rational use of such agents. Thus, the purpose of the present study was to determine whether the ability to inhibit P-gp and CYP3A is, in fact, linked and whether specific P-gp inhibitors with limited ability to inhibit CYP3A can be identified. Therefore, the potency of a series of 14 P-gp inhibitors was assessed by measuring their inhibition of the transepithelial flux across Caco-2 cells of digoxin, a prototypical P-gp substrate. CYP3A inhibition was determined from the impairment of nifedipine oxidation by human liver microsomes. Determination of the apparent Ki values for CYP3A inhibition and the IC50s for P-gp and CYP3A inhibition allowed comparison of the relative inhibitory potency of the compounds on the two proteins' function. The IC50s for P-gp inhibition ranged from 0.04 to 3.8 microM. All compounds inhibited CYP3A with apparent Ki values of between 0.3 and 76 microM and IC50s between 1.5 and 50 microM. However, no correlation was found between the extent of P-gp inhibition and CYP3A inhibition, and the ratio of the IC50 for CYP3A inhibition to the IC50 for P-gp inhibition varied from 1.1 to 125. These results demonstrate that, although many P-gp inhibitors are potent inhibitors of CYP3A, a varying degree of selectivity is present. The development and use of P-gp inhibitors with minimal or absent CYP3A inhibitory effects should decrease the impact of drug interactions on the therapeutic use of such compounds.

Identification and characterization of baxepsilon, a novel bax variant missing the BH2 and the transmembrane domains.

The Bax gene is a member of the Bcl2 family that functions to regulate the programmed cell death process. A number of Bax isoforms have been previously identified: alpha, beta, gamma, delta, and omega. Here we report the identification and characterization of an additional Bax variant, termed Baxepsilon. The newly identified Bax variant contains a 97-base insertion generated by alternative splicing which includes a previously unidentified exon between exons 4 and 5. The insertion causes the production of a truncated Bax protein, termed Baxepsilon, which encodes a protein of 164 residues with a calculated molecular weight of 18 kDa. The last 69 amino acids of Baxalpha that encompass the BH2 and the TM domains are missing in Baxepsilon. The Baxepsilon protein, when expressed as a GST fusion protein, associated efficiently with Baxalpha, Baxepsilon, Bcl2, and Bcl-xL. In addition, Baxepsilon was active in inducing apoptosis when tested in a transient transfection assay. Furthermore, the presence of antiapoptotic genes including Bcl2, Bcl-xL, and baculovirus p35 abrogated Baxepsilon and Baxalpha function. Although the newly identified Bax variant was detectable by RT-PCR in several normal mouse tissues, the role of this variant in controlling programmed cell death is currently unknown.

Human (MDR1) and mouse (mdr1, mdr3) P-glycoproteins can be distinguished by their respective drug resistance profiles and sensitivity to modulators.

Possible functional differences between P-glycoproteins (P-gps) encoded by the human MDR1 and mouse mdr1 and mdr3 genes with respect to drug resistance profiles and sensitivity to known modulators have been investigated. For this, the three genes were introduced and overexpressed in the same cellular background, that of Chinese hamster LR73 ovary cells, and drug-resistant clones expressing comparable amounts of the corresponding P-gps were selected under the same conditions. Analysis of the specific drug resistance profiles encoded by each P-gp for colchicine, adriamycin, vinblastine, and actinomycin D revealed overlapping but distinct patterns of drug resistance for the three isoforms. While all three P-gps conferred levels of resistance to vinblastine that did not vary by more than 2.5-fold, each isoform could be clearly distinguished by its capacity to confer resistance to colchicine and actinomycin D. Likewise, the study of structurally related and unrelated P-gp modulators indicated strong isoform-specific differences in the capacity of individual modulators to abrogate vinblastine resistance in the corresponding mdr transfectants. The study of several disubstituted piperazine analogs indicated that minor chemical modifications of the linker region of this modulator had strong effects on the sensitivity profile of each isoform to the modulator. Together, these results indicate that the three P-gp isoforms analyzed have specific and distinguishable functional characteristics with respect to interactions with drugs and modulators. These findings also suggest that P-gp positive murine transplantable tumors should be used with caution in the design and in vivo testing of novel P-gp modulators to be used to reverse multidrug resistance to tumor cells expressing human MDR1.

Structurally distinct MDR modulators show specific patterns of reversal against P-glycoproteins bearing unique mutations at serine939/941.

The mechanism by which P-glycoprotein (P-gp) interacts with a number of structurally unrelated substrates or inhibitors remains unknown. We have recently shown that a serine residue within the predicted transmembrane (TM) domain 11 of P-gps encoded by mouse mdr1 (Ser941) and mdr3 (Ser939) plays an important role in the substrate specificity of P-gp. We wished to determine if Ser939/941 is also important for efficient interaction of P-gp with structurally different modulating agents, a cyclic peptide (cyclosporin A, CsA), a diaminoquinazoline (CP100356), and a chiral, tricyclic structure (CP117227). For this, the capacity of these compounds to modulate the vinblastine (VBL) resistance phenotype of transfected cells expressing similar levels of P-gps bearing either the wild-type Ser or a mutant Phe at position 941 (mdr1) or 939 (mdr3) was initially tested. The Ser-->Phe substitution indeed affected the potency and P-gp isoform specificity of some of the modulators, in particular that of CP117227 (racemic mixture and enantiomers), which were active against wild-type but not mutant mdr3. The modulatory effect of the mutation on CP117227-mediated reversal of VBL resistance was parallelled by a comparable modulation of the steady-state levels of VBL accumulation in Ser939- and Phe939-expressing cells, but was not linked to differential cellular accumulation of the modulator, which was identical in both cell types. To further assess the role of this amino acid residue in P-gp interactions with modulators, the effect of additional mutations (Ala, Cys, Thr, Asp, Tyr, Trp) at that site on potencies of CsA, CP117227 enantiomers, and CP100356 was evaluated.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulatory effects on substrate specificity of independent mutations at the serine939/941 position in predicted transmembrane domain 11 of P-glycoproteins.

The serine residue located at position 939 and 941 in the predicted transmembrane segment 11 of P-glycoprotein (P-gp) encoded by mouse mdr3 and mdr1, respectively, appears to be important for interaction of chemotherapeutic drugs and reversal agents with P-gp. To further understand the role of this residue in this process and to identify the structural requirements involved, we have replaced this serine residue by alanine, cysteine, threonine, tyrosine, tryptophan, and aspartic acid and tested the effect of these mutations on the overall activity and substrate specificity of mdr1 and mdr3. All mutant proteins could be expressed at high levels in the membrane fractions of LR73 Chinese hamster cells transfected with the corresponding mutant cDNAs. All introduced mutations had limited effect on the capacity of mdr1 and mdr3 to confer resistance to vinblastine. The modulatory effect of mutations on resistance to colchicine, adriamycin, and actinomycin D was more dramatic. The hydroxyl group of serine did not seem essential for interaction with these drugs since mutant mdr1 and mdr3 bearing alanine or cysteine at that position behaved essentially as wild type, while threonine-bearing mutants showed significantly reduced resistance to these drugs. The insertion at that site of residues with bulkier side chains had more complex effects on P-gp function. While introducing tyrosine, tryptophan, or aspartic acid caused an almost complete loss of colchicine and adriamycin resistance in both mdr1 and mdr3, the replacement to tyrosine or tryptophan had the opposite effect on mdr1 and mdr3 for actinomycin D resistance, causing either a 3-fold increase or a 4-8-fold decrease in resistance to this drug, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional analysis of P-glycoprotein mutants identifies predicted transmembrane domain 11 as a putative drug binding site.

The substitution of a single serine to phenylalanine residue within the predicted transmembrane domain 11 of P-glycoproteins (P-gps) encoded by mouse mdr1 (Ser941, 1S;Phe941, 1F) or mdr3 (Ser939, 3S; Phe939, 3F) strongly modulates both the overall activity and substrate specificity of the two P-gps. In cell clones expressing either wild-type (1S, 3S) or mutant P-gps (1F, 3F), we show that the modulating effect of the mutation on the levels of adriamycin (ADM) resistance detected in drug cytotoxicity assays is paralleled by a similar modulation of the intracellular accumulation and extracellular efflux of radiolabeled adriamycin ([14C]ADM) from preloaded cells. Cytofluorescence studies with ADM on live cells produce similar results and demonstrate strong nuclear ADM accumulation only in drug-sensitive LR cells and in the 1F expressing cells, with little if any accumulation in 1S, 3S, or 3F expressing cells. Drug cytotoxicity and drug transport assays carried out in the presence of verapamil or progesterone suggest that the Ser to Phe substitution also reduces the capacity of these two reversal agents to modulate P-gp activity. Labeling experiments with the photoactivatable P-gp ligands iodoarylazidoprazosin and azidopine indicate a strong reduction in binding of these photoactivatable probes to the mutant P-gps (1F, 3F) as compared to their wild-type counterparts (1S,3S). These results indicate that the studied mutations in TM11 reduce drug transport by decreasing initial drug binding to P-gp. This phenotype is opposite to that of a mutation near TM3 in human MDR1 (pst 185), where decreased drug transport is associated with increased drug binding and decreased drug release from P-gp [Safa, A. R., Stern, R. K., Choi, K., Agresti, M., Tamai, I., Metha, N. D., & Roninson, I. B. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7225-7229].

N-(5-fluorobenzothiazol-2-yl)-2-guanidinothiazole-4-carboxamide. A novel, systemically active antitumor agent effective against 3LL Lewis lung carcinoma.

N-(5-Fluorobenzothiazol-2-yl)-2-guanidinothiazole-4-carboxam ide (1) is a member of a series of amides found to substantially increase lifespan in mice bearing established micrometastatic 3LL Lewis lung carcinoma. Amide 1 is effective after either oral or intraperitoneal dosing in acute, subacute, or chronic regimens. 1 is well tolerated in this model with an excellent therapeutic index relative to the cytotoxic anticancer drug adriamycin.

Polarized integrin mediates human keratinocyte adhesion to basal lamina.

Epithelial cell interactions with matrices are critical to tissue organization. Indirect immunofluorescence and immunoprecipitations of cell lysates prepared from stratified cultures of human epidermal cells showed that the major integrins expressed by keratinocytes are alpha E beta 4 (also called alpha 6 beta 4) and alpha 2 beta 1. The alpha E beta 4 integrin is localized at the surface of basal cells in contact with the basement membrane, whereas alpha 2 beta 1/alpha 3 beta 1 integrins are absent from the basal surface and are localized only on the lateral surface of basal and spinous keratinocytes. Anti-beta 4 antibodies potently inhibited keratinocyte adhesion to matrigel or purified laminin, whereas anti-beta 1 antibodies were ineffective. Only anti-beta 4 antibodies were able to detach established keratinocyte colonies. These data suggest that alpha E beta 4 mediates keratinocyte adhesion to basal lamina, whereas the beta 1 subfamily is involved in cell-cell adhesion of keratinocytes.

A novel integrin (alpha E beta 4) from human epithelial cells suggests a fourth family of integrin adhesion receptors.

A new member of the integrin superfamily of adhesion receptors was isolated from human epithelial cells. Analogously to other integrins, this molecule is a heterodimer comprised of structurally unrelated subunits, both glycosylated. Unequivocal amino-acid sequence homologies were observed between these subunits and integrin alpha and beta chain sequences, indicating that this epithelial heterodimer is a novel integrin. No obvious serologic cross-reactivities were detected with other integrins. The beta chain of the epithelial integrin displayed a mol. wt significantly higher than other integrin beta chains, possibly due to a large sialic acid content. Integrin heterodimers are grouped into three families, based on which of three beta chains (beta 1, beta 2 and beta 3) they contain. Therefore, the epithelial integrin may represent the prototype of a fourth integrin family, because it contains a structurally distinct beta chain. The designation alpha E beta 4 is proposed for this novel human integrin.

Sulfated N-linked oligosaccharides in mammalian cells. III. Characterization of a pancreatic carcinoma cell surface glycoprotein with N- and O-sulfate esters on asparagine-linked glycans.

In the preceding two papers, we described two new classes of sulfated N-linked oligosaccharides isolated from total cellular 35SO4-labeled macromolecules of different mammalian cell lines. The first class carries various combinations of sialic acids and 6-O-sulfate esters on typical complex-type chains, while the second carries heparin and heparan-like sequences. In this study, we have characterized a sulfophosphoglycoprotein of 140 kDa from FG-Met-2 pancreatic cancer cells whose oligosaccharides share some properties of both these classes. The molecule was localized to the cell surface by electron microscopy using a monoclonal antibody (S3-53) and by cell surface 125I-labeling. Metabolic labeling of the cells with radioactive glucosamine, methionine, inorganic sulfate, or phosphate all demonstrated a single 140-kDa molecule. Pulse-chase analysis and tunicamycin treatment indicated the glycosylation of a putative primary translation product of 110 kDa via an intermediate (120 kDa) to the mature form (140 kDa). Digestion with peptide:N-glycosidase F (PNGaseF) indicated a minimum of four N-linked glycosylation sites. PNGaseF released more than 90% of the [6-3H]GlcNH2 label and 40-70% of 35SO4 label from the immunoprecipitated 140-kDa molecule. The isolated oligosaccharides were characterized as described in the preceding two papers. The majority of [6-3H]GlcNH2-labeled molecules were susceptible to neuraminidase. More than 50% of the 35SO4 label was associated with only 5-10% of the 3H-labeled chains. Some of the sulfated chains were partly sialylated molecules with four to five negative charges. Treatment with nitrous acid released about 25% of the 35SO4 label as free sulfate, together with 6% of the [6-3H]GlcNH2 label, indicating the presence of N-sulfated glucosamine residues. Some of these oligosaccharides were degraded by heparinase and heparitinase. Therefore, while they are not as highly charged as typical heparin or heparan chains, they must share structural features that permit recognition by the enzymes. Thus, this 140-kDa glycoprotein contains at least four asparagine-linked chains substituted with a heterogeneous mixture of sulfated sequences. The heterogeneity of these molecules is as extensive as that described for whole-cell sulfated N-linked oligosaccharides in the preceding two papers.

Six monoclonal antibodies to human pancreatic cancer antigens.

Murine monoclonal antibodies were generated against FG, a human pancreatic carcinoma (HPC) cell line. Of the six monoclonal antibodies, five (S3-15, S3-23, S3-41, S3-60, and S3-110) reacted by indirect immunoperoxidase assays with HPC of the ductal type, and another (S3-53) reacted with both ductal and acinar HPC. Strong reactivity was also found with tumors of the stomach, colon, mouth, lung, and cervix, while a large panel of normal human tissues displayed little reactivity. Indirect immunofluorescence staining revealed that, except for S3-23, the antigens recognized by these antibodies are expressed at the cell surface. Immunoprecipitation of metabolically radiolabeled FG cells indicated that the epitopes recognized by these antibodies are carried by distinct proteins or glycoproteins, differentiated on the basis of the apparent molecular weight and/or subunit composition as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This panel of antibodies will be useful to study antigenic variations associated with malignant cell transformation in HPC as well as other tumors.

Binding of monoclonal antibody (4F2) to its cell surface antigen on dispersed adenomatous parathyroid cells raises cytosolic calcium and inhibits parathyroid hormone secretion.

In the course of characterizing monoclonal antibodies (MAbs) recognizing cell surface antigens on dispersed human parathyroid cells (dPTCs), we identified one MAb (4F2) that bound avidly to parathyroid cells and had marked effects on parathyroid function. The binding of MAb 4F2 to human adenomatous dPTCs resulted in a marked [53.8 +/- 7.9% (+/- SEM)] reduction in low calcium (Ca)-stimulated PTH secretion to levels equivalent to those in cell suppressed by high extracellular Ca (1.5 mM). Typically, these functional effects were optimal at antibody dilutions of 1:10(4) to 1:10(5). Cell viability was confirmed at the conclusion of each experiment by trypan blue exclusion (greater than 90-95%) and cell surface immunofluorescence. Parallel studies using the Ca-sensitive dye Quin-2 showed that inhibition of PTH secretion in 4F2-treated cells was associated with a concomitant increase in cytosolic Ca (Cai) of 188% in 0.5 mM Ca; these values also approached Cai levels in control cells incubated in high Ca. Mab controls, P3 X 63, which do not bind to dPTCs, and Mab LC7-2, which recognizes a different epitope of the same antigen as 4F2 on dPTCs, did not alter PTH secretion or Cai. Immunoprecipitation of 125I-labeled parathyroid cell extracts with MAb 4F2 demonstrated proteins with mol wt of approximately 145, 85, and 45 under nonreducing conditions and 85 and 45 kilodaltons after reduction with 5% mercaptoethanol. These studies suggest that 1) Mab-4F2 binding to its cell surface antigen inhibits PTH secretion by human adenomatous parathyroid cells in vitro; 2) the alterations in secretory function could be related to by an attendant increase in Cai; 3) the 4F2 antigen on dPTCs is a heterodimeric protein of (approximately) 85K and 45K; and 4) the 4F2 antigen may be an important component of the Ca-sensing and/or signal-transducing mechanism in this cell.

"COLO 357," a human pancreatic adenosquamous carcinoma: growth in artificial capillary culture and in nude mice.

The human pancreatic cancer cell line COLO 357 has been xenografted s.c. in athymic Swiss mice. The xenografts grew well to form typical adenosquamous carcinomas. The cells were placed in a perfused artificial capillary system where they formed a solid tumor mass which survived for 7 weeks. In this system, the cells consumed glucose and released enzymes and carcinoembryonic antigen into the extracapillary space.