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Biomedical subjects

P C Hiestand

Publications and source records attributed to P C Hiestand.

At least 19 recordsLinked to original sources
PubMed1992

Pharmacologic interactions between the resistance-modifying cyclosporine SDZ PSC 833 and etoposide (VP 16-213) enhance in vivo cytostatic activity and toxicity.

Cyclosporin A reverses multidrug resistance (MDR) and increases the in vivo cytostatic activity and toxicity of the anticancer agent etoposide (VP 16-213). SDZ PSC 833 (PSC 833), a non-immunosuppressive, non-toxic cyclosporin and very active modifier of P-gp 170-mediated MDR, elicits similar effects when administered with adriamycin. The underlying mechanisms, however, are not yet understood. The present pharmacological interaction study with PSC 833 and VP 16-213 was carried out to reveal the nature of this enhancement of cytostatic activity and toxicity. Rats pre-treated with either PSC-833 or solvent received a single dose of VP 16-213. Plasma levels of VP 16-213 were measured by high-performance liquid chromatography (HPLC). The resulting increase in cytostatic activity and toxicity of VP 16-213 mediated by PSC 833 was paralleled by marked changes in the pharmacokinetic parameters of VP 16-213 in vivo. Bioavailability and blood levels of VP 16-213 were significantly increased 30 min after administration if PSC 833 had been given before. The disappearance rate of VP 16-213 from the intravascular compartment was considerably slowed down by PSC 833. In drug-sensitive xenografts of human colon carcinoma, the PSC-833-induced pharmacologic changes in vivo could be counteracted by dose reduction of VP 16-213 while a full therapeutic potential was maintained. Doses of VP 16-213, 1.5 to 2 times smaller, combined with PSC 833, were as effective in terms of tumor-growth inhibition as the maximum tolerated dose of VP 16-213 alone. Thus, pharmacologic interactions between PSC 833 or other resistance modifiers and VP 16-213 and other cytostatic agents require careful attention if they are to be used in humans to overcome MDR.

Adenocarcinoma↗
PubMed1992

SDZ PSC 833, a non-immunosuppressive cyclosporine: its potency in overcoming P-glycoprotein-mediated multidrug resistance of murine leukemia.

Cyclosporin A (CsA, Sandimmune) is known to reverse P-glycoprotein (P-gp170)-mediated multidrug resistance as efficiently as other prototype compounds of resistance modifiers. The immunosuppressive activity and nephrotoxicity of CsA, however, may limit its clinical use. PSC-833, a new cyclosporine, exerts a similar resistance-modifying activity but lacks toxicity or immunosuppressive activity. We have tested its potency in vitro and in vivo on the L1210 leukemia cell line transfected with a full-length cDNA copy of the human mdr I gene, which showed a stable 30-fold resistance towards adriamycin as compared to the parental cell line. In vitro growth of the transfected cell was unchanged. In vivo growth was less aggressive; the survival time of inoculated mice was prolonged. In vitro, PSC-833 was at least as potent as CsA or verapamil in reversing multidrug resistance. In vivo, the drug-resistant L1210 leukemia was completely unresponsive to i.v. monotherapy with adriamycin at its maximum tolerated dose (MTD). PSC-833 enhanced the activity and toxicity of adriamycin. The MTD of adriamycin was about 3 times lower than when given alone. On this basis, the MTD of i.v. adriamycin in combination with oral PSC-833 successfully overcame refractoriness to treatment. Survival times of the mice were considerably prolonged and even some cures of leukemic mice occurred.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Identification of several cyclosporine binding proteins in lymphoid and non-lymphoid cells in vivo.

The immunosuppressant cyclosporine A (CSA) has been shown to bind to the ubiquitous cellular protein, cyclophilin, and to inhibit its rotamase activity. In the present study, 3H-cyclosporine diazirine analogue was used to photolabel viable human cells of lymphoid and fibroblast origin in order to identify the intracellular targets for the drug. While cyclophilin was strongly labeled in situ, additional minor cyclosporine-protein complexes of 25, 40, 46 and 60 kDa were identified in the T cell leukemia cell line Jurkat. These proteins bound specifically, since only active CSA but not inactive CSH or FK506 competed for binding. Photolabeling of MRC5 cells, a CSA resistant human fibroblast cell line, revealed a 25 kDa complex as the major product, while the 46 and 60 kDa bands were not detectable and cyclophilin labeling was only faint, even though both MRC5 and Jurkat cells contain similar cyclophilin concentrations. Thus, our data suggest that the intracellular targets of CSA and/or the accessibility to cyclophilin varies considerably in drug sensitive and resistant cell types, which may contribute to explaining the lymphocyte selectivity of the drug.

Amino Acid Isomerases↗
PubMed1991

FK 506-binding protein proline rotamase is a target for the immunosuppressive agent FK 506 in Saccharomyces cerevisiae.

FK 506 and cyclosporin A are potent immunosuppressive compounds that inhibit T-cell activation by interfering with signal transduction. In vitro, FK 506 binds and inhibits the activity of FK 506-binding protein (FKBP), a peptidylprolyl rotamase (cis-trans isomerase). Cyclosporin A acts similarly on a different proline rotamase, cyclophilin. Experiments described here demonstrate genetically that FKBP is a target for FK 506 in vivo. We have isolated the gene encoding the FKBP proline rotamase (FPR1) from Saccharomyces cerevisiae. The encoded yeast protein is highly homologous with bovine and human FKBP and shares no homology with cyclophilin. Disruption of FPR1 and CPR1 (encoding cyclophilin) individually or in combination is not lethal; thus, either enzymatic proline rotamerization is not essential for life or an unknown proline rotamase can substitute for the missing enzymes. Overexpression or disruption of FPR1 confers resistance to growth inhibition by FK 506, suggesting that FKBP is a target for FK 506 in yeast. However, FKBP is only one of at least two targets because strains lacking FKBP are only partially resistant to FK 506.

Amino Acid Sequence↗
PubMed1989

Yeast cyclophilin: isolation and characterization of the protein, cDNA and gene.

Cyclophilin (CPH) has been isolated from the yeast Saccharomyces cerevisiae, purified to homogeneity and partially sequenced. Oligodeoxyribonucleotides deduced from this sequence were used to isolate the corresponding cDNA and gene. An open reading frame coding for a 162-amino acid (aa) protein with a calculated Mr of 17,392, was deduced from the nucleotide sequence. Comparison between yeast and human CPH shows a very high overall sequence conservation (65% aa homology). The binding of yeast CPH to cyclosporin A is identical to that of human and bovine CPH. Unlike in Neurospora crassa, a mitochondrial form of CPH could not be detected in yeast. Southern-blot analysis of yeast DNA shows that only one CPH-related sequence is present per haploid genome, whereas at least 20 genes or pseudogenes were detected in the human and rat genome. Purified yeast CPH exhibits peptidyl-prolyl cis-trans isomerase activity, albeit to a far lesser extent than the mammalian protein.

Amino Acid Sequence↗
PubMed1988

Molecular characteristics of cyclophilin-cyclosporine interaction.

The ability of cyclophilin to react with derivatives of cyclosporine (CsA) was studied. Cyclophilin was found to interact preferentially with CsA-residues 1, 2, 10 and 11, which, together with residue 3, are the residues known to contribute to the immunosuppressive activity of CsA. The recognition of different CsA-derivatives by cyclophilin was correlated with their immunosuppressive activity in vitro. All CsA-derivatives showing a significant activity did bind to cyclophilin, although some of the CsA-derivatives able to bind cyclophilin exhibited only low activities. The results suggest that binding to cyclophilin might be one requirement for immunosuppressive activity of CsA derivatives. When tested with CsA-derivatives showing various conformational changes, the binding of cyclophilin was strongly specific for the peptide-ring conformation of CsA. No binding of calmodulin to CsA could be detected in several formats of solid-phase enzyme- or radioimmunoassay, suggesting that, in contrast to cyclophilin, calmodulin does not possess sufficient affinity for CsA to bind to it when immobilized on the solid phase.

Calmodulin↗
PubMed1988

A comparison of cyclosporine binding by cyclophilin and calmodulin and the identification of a novel 45 KD cyclosporine-binding phosphoprotein in Jurkat cells.

Cyclosporine mediates its immunosuppressive effect by preventing the synthesis of lymphokine mRNA during the process of T lymphocyte activation. Although the detailed molecular mechanism by which CsA achieves this effect is unknown, two proteins have been identified as putative intracellular CsA-receptor proteins. One of these, calmodulin, is an important Ca++-binding protein and enzyme cofactor and the other, cyclophilin, is a novel protein that is reported to have protein kinase activity. In this study the CsA-binding capacity of both these proteins has been assessed using CsA-coated ELISA plates and CsA-affinity gel matrices. CsA binding was shown by cyclophilin whereas no CsA-calmodulin binding could be detected under identical conditions. However, it was not possible to demonstrate any cyclophilin-associated protein kinase activity. Jurkat cells were probed for the presence of CsA-binding proteins using the CsA-affinity gel matrix; a 17 KD protein, most probably cyclophilin, was identified as the major CsA-binding protein. In addition, a previously unidentified CsA-binding 45 KD phosphoprotein was precipitated from 32P-labeled Jurkat cells. These results would support cyclophilin as the major, if not only, intracellular receptor protein for CsA. However, the relationship between binding of CsA to cyclophilin and/or the 45 KD phosphoprotein and the immunosuppressive effects of CsA is still unknown.

Calmodulin↗
PubMed1987

Cyclophilin binds to the region of cyclosporine involved in its immunosuppressive activity.

Although several cytosolic proteins including calmodulin and cyclophilin have been shown to bind cyclosporine, the direct involvement of these proteins in the immunosuppressive activity of cyclosporine remains to be established. In the present study, a quantitative immunoassay for cyclophilin was developed which made it possible to compare its relative affinity for cyclosporine and any of its analogues. The binding of cyclophilin to cyclosporine coated on a solid phase was revealed by anti-cyclophilin rabbit antiserum followed by antiglobulin-enzyme conjugate. This reaction could be inhibited by addition of free cyclosporine or certain cyclosporine analogues. By studying the binding of cyclophilin to more than fifty cyclosporine derivatives modified singly on each of the eleven amino acid residues, it could be shown that cyclophilin binds to the residues of cyclosporine known to be critical for its immunosuppressive activity. These data identify cyclophilin as a highly discriminating stereospecific binding protein for cyclosporine.

Calmodulin↗
PubMed1986

Prolactin as a modulator of lymphocyte responsiveness provides a possible mechanism of action for cyclosporine.

Lymphocyte responsiveness in rats was found to depend on serum prolactin levels. Blocking pituitary prolactin release with bromocriptine severely reduces lymphocyte reactivity in vitro (mixed lymphocyte reaction) as well as in vivo (graft-versus-host reaction). In addition, evidence for a prolactin/growth hormone-related mRNA species produced in mitogen- and antigen-stimulated lymphocytes has been obtained. Prolactin was shown to compete in a dose-dependent fashion with the immunosuppressant cyclosporine (cyclosporin A) for a common binding site on the surface of T lymphocytes. Further, stimulation of prolactin secretion reversed the immunosuppression induced by cyclosporine. We conclude that prolactin is involved in the maintenance of T-cell immunocompetence and that the immunosuppressive effects of cyclosporine may be mediated by the displacement of prolactin from binding sites on lymphocytes.

Animals↗
PubMed1985

Immunomodulating activities of ethylene-2,2'-bis(dithio)bis(ethanol) and related compounds. Effects on murine lymphocyte proliferation and functions in vitro.

HEDS (2-hydroxyethyl disulfide) and structurally related compounds were found to affect lymphocyte proliferation and functions in vitro. HEDS, and ADA 202-718 (ethylene-2-2'-bis(dithio)bis(ethanol], were shown to stimulate the proliferation of murine spleen cells. The growth of populations of murine T-cells (thymocytes) was not stimulated. HEDS and ADA 202-718 enhanced the allogeneic response in the mixed lymphocyte reaction and stimulated the formation of antibody producing cells in a primary humoral immune response. A similar stimulatory effect was observed in a secondary humoral response towards a T-cell specific antigen, DNP-keyhole limpet hemocyanine. Neither ADA 202-718 nor HEDS exhibited gamma-interferon inducing ability, when tested on quiescent Balb/c spleen cell cultures in absence of antigen. However, HEDS, and especially ADA 202-718 potentiated the allogen-induced gamma-interferon production and release in the mixed lymphocyte reaction. Both HEDS and ADA 202-718 seemed to induce or stimulate the release of an interleukin-1-like activity as well as interleukin-2 in Balb/c spleen cells. No obvious effect, however, was seen with either compound on maturation or immune-phagocytic activity of bone-marrow derived macrophages.

Adjuvants, Immunologic↗