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S M Stepkowski

Publications and source records attributed to S M Stepkowski.

At least 37 records · Page 2Linked to original sources
PubMed1998

Relative tissue distributions of cyclosporine and sirolimus after concomitant peroral administration to the rat: evidence for pharmacokinetic interactions.

The authors sought to determine the effect of concomitant peroral (PO) administration of cyclosporine (CsA) and sirolimus (SRL, rapamycin) on the tissue distributions of CsA and SRL in the rat. Groups of four adult male Wistar-Furth rats were treated for 14 days with 2.5, 5.0, or 10.0 mg CsA/kg x day. Other groups of four adult male Wistar-Furth rats were treated for 14 days with a 1-to-6.25 weight-to-weight ratio of SRL to CsA at SRL doses of 0.4, 0.8, or 1.6 mg/kg x day. Concentrations of CsA and SRL in homogenates of heart, intestinal, kidney, liver, lung, muscle, spleen, and testes were compared to those in whole blood (WB). There was a large, dose-dependent, distinctive distribution of CsA among rat tissues, as has previously been well documented. At a constant molar dose ratio, concomitant oral administration of SRL produced an approximately two-fold increase in the concentrations of CsA in rat tissues, although SRL did not change the CsA tissue-to-WB partition coefficients. Concomitant oral CsA administration produced dose-dependent increases in SRL tissue concentrations and decreases in the SRL tissue-to-WB partition coefficients. The increases in tissue and WB concentrations on coadministration of both agents may be explained either by an increase in absorption caused by competition between the two agents for binding sites on P-glycoprotein in the gut, a reduced rate of metabolism, or to an as yet unidentified elimination mechanism. The dose-independent and unchanged CsA tissue-to-WB partition coefficients suggest that SRL does not affect the equilibrium of CsA between the central and tissue compartments, namely the tissue uptake or intracellular binding. Altered values of the SRL tissue-to-WB partition coefficients suggest that, under the conditions studied, CsA disturbs the equilibrium of SRL between the central and tissue compartments.

Absorption↗
PubMed1997

Induction of tolerance toward rat cardiac allografts by treatment with allochimeric class I MHC antigen and FTY720.

BACKGROUND: The combination of FTY 720, a novel immunosuppressant, and allochimeric class I MHC proteins bearing donor-type amino acid (aa) epitope substitutions for host-type sequences induces tolerance of Wistar Furth (WF; RT1.Au) heart allografts in ACI (RT1.Aa) recipients. METHODS: Allochimeric alpha(1h)l58-80-RT1.Aa proteins were produced by substituting the allogeneic nucleotide sequence encoding 10 aa residues unique to the alpha1 helical (alpha1h) region of RT1.Al Lewis (Asp58, Arg62, Glu63, Gln65, Lys66, Gly69, Asn70, Asn73, Ser77, and Asn80) for native RT1.Aa residues. The RT1.Au and the RT1.Al haplotypes share four of these aa (Arg62, Glu63, Gln65, and Gly69). A baculovirus/Spodoptera frugiperda insect cell system was used to express the alpha(1h)l58-80-RT1.Aa proteins. RESULTS: The addition of a 3-day oral gavage of 0.05 mg/kg/day FTY720 to a single portal vein injection of 10 microg alpha(1h)l58-80-RT1.Aa protein induced permanent acceptance of WF heart allografts in 16 of 26 ACI recipients (>100 days); the alpha(1h)l58-80-RT1.Aa protein alone only modestly prolonged WF heart survival (13.8+/-0.8 days). The same tolerogenic protocol did not prolong the survival of third-party Brown Norway (RT1.An) heart allografts (14.3+/-2.5 days) compared with FTY720 alone (14.0+/-2.3 days; NS). Tolerant ACI recipients bearing primary WF heart allografts for more than 100 days accepted second WF hearts, but promptly rejected third-party Brown Norway heart grafts (9.3+/-1.5 days). The tolerant state was transferred to irradiated ACI rats (400 rad) with either purified T cells (4-10 x 10[7]) or serum (1-2 ml) from tolerant hosts, and was not broken by daily intraperitoneal injections of interleukin-2 (1000 U/day; 7 days). CONCLUSIONS: The combination of allochimeric protein with FTY720 induces transplantation tolerance, a state that may be associated with the appearance of donor-specific regulatory factors.

Animals↗
PubMed1997

Localization of cryptic tolerogenic epitopes in the alpha1-helical region of the RT1.Au alloantigen.

BACKGROUND: Transplantation tolerance is induced by perioperative administration of host class I major histocompatibility complex proteins bearing donor-type amino acid (a.a.) epitopes substituted for native residues. Herein we demonstrate that two cryptic tolerogenic a.a. epitopes are localized in the alpha1-helical region of the rat RT1.Au class I major histocompatibility complex alloantigen. METHODS: Three allochimeric proteins were produced by superimposing the nucleotides encoding donor-type RT1.Au a.a. onto the host RT1.Aa backbone using the polymerase chain reaction-based method of gene splicing with overlap extension. We substituted nucleotide sequences encoding all nine (Arg62, Glu63, Gln65, Gly66, Gly69, His70, Val73, Asn74, and Asn77; alpha1h u62-77-RT1.Aa), the first four (Arg62, Glu63, Gln65, and Gly69; alpha1h u62-69-RT1.Aa), or the last four (His70, Val73, Asn74, and Asn77; alpha1h u70-77-RT1.Aa) alpha1-helical RT1.Au polymorphic a.a. in RT1.Aa cDNA. A baculovirus/Spodoptera frugiperda (Sf9) expression system was harnessed for production of the proteins. RESULTS: Untreated ACI (RT1a) rats reject Wistar-Furth (WF; RT1u) heart allografts at a mean survival time of 8.9+/-1.0 days. A single portal vein injection of 10 microg of alpha1h u62-77-RT1.Aa protein had no effect on the survival of WF heart allografts (10.5+/-0.6 days) in ACI hosts. Interestingly, portal vein administration of 10 microg of alpha1h u70-77-RT1.Aa induced transplantation tolerance toward WF grafts in four of six untreated ACI recipients (>100 days; P<0.01). In contrast, 10 microg of alpha1h u62-69-RT1.Aa only prolonged the survival of WF heart allografts in ACI hosts (14.0+/-0.8 days; P<0.01). However, when combined with a 7-day course of cyclosporine (4.0 mg/kg; oral gavage), alpha1h u62-69-RT1.Aa induced tolerance toward WF allografts in five of seven ACI recipients (>170 days; P<0.01). Long-term survival of WF grafts was not achieved when a 7-day course of cyclosporine was administered alone (14.3+/-3.0 days) or with 10 microg of alpha1h u62-77-RT1.Aa (14.6+/-0.6 days; NS). CONCLUSIONS: These findings suggest that the use of allochimeric proteins may provide a novel approach to the induction of tolerance.

Animals↗
PubMed1997

Distribution of sirolimus in rat tissue.

OBJECTIVES: To examine the distribution of sirolimus (SRL, rapamycin), an immunosuppressive macrolide antibiotic, in the tissues of adult male Wistar-Furth rats following continuous intravenous infusion (CIVI) and repeated daily peroral gavage (PO). DESIGN AND METHODS: Animals received 14-day courses of SRL by either CIVI (0.04-0.4 mg/kg/day) or PO (0.4-1.6 mg/kg/day) administration. Samples of whole blood and homogenates of five solid organs (heart, kidney, liver, lung and spleen), and portions of intestinal, muscle and testicular tissues were prepared on day 13 of CIVI treatment or 24 hours after administration of the 14th PO dose. SRL concentrations were determined by high performance liquid chromatography with reference to calibration curves produced from SRL-spiked whole blood or tissue homogenates prepared from drug-free animals. RESULTS: Following PO but not CIVI administration, SRL concentrations in whole blood and all tissues increased linearly in relation to dose. SRL was extensively distributed among most tissues tested (tissue partitions coefficients of > 40 were observed in some cases). Comparatively, SRL whole blood concentrations were low. The ratio between the SRL whole blood concentrations after PO versus after CIVI administration (at like doses of 0.4 mg/kg/day) was 0.04. Therefore, we inferred that the oral bioavailability of SRL was low. CONCLUSIONS: The linear relationships between PO dose and SRL concentrations in whole blood and tissues may be attributed to the low oral bioavailability of SRL, which is indicated by the low levels of SRL observed in whole blood and tissues after PO administration. The nonlinear relationships between CIVI dose and SRL concentrations in whole blood and tissues may result because although whole blood depots may be saturated with SRL, the tissues continue to absorb SRL as the dose of SRL increases. Thus, because a high percentage of SRL is widely distributed into tissues stores, caution must be used when administering this drug in humans.

Animals↗
PubMed1997

Cytokine mRNA expression in tolerant heart allografts after immunosuppression with cyclosporine, sirolimus or brequinar.

We sought to examine the impact of the preferential activation of Th2 cells on the induction and maintenance of a tolerant state in heart allograft rat recipients treated with a short course of cyclosporine (CsA), sirolimus (SRL) or brequinar (BQR). A quantitative polymerase chain reaction (PCR) method was used to measure the levels of cytokine mRNAs, namely interferon (IFN)-gamma and interleukin (IL)-2 in T helper 1 (Th1) cells and IL-4, IL-5 and IL-10 in Th2 cells. Our main findings were that on day 5 postgrafting allografts from untreated recipients had increased levels of IFN-gamma (216 +/- 119 fg), IL-2 (449 +/- 75 fg), IL-4 (6.2 +/- 1.3 fg), IL-5 (34.8 +/- 9.3 fg) and IL-10 (1554 +/- 184 fg) mRNAs compared with normal hearts. CsA reduced the levels of IFN-gamma, IL-2, IL-5 and IL-10, but not IL-4, mRNAs. SRL did not affect the expression of cytokine mRNAs. BQR decreased the levels of IFN-gamma, IL-2 and IL-10, but not IL-5 or IL-4 mRNAs. Compared with grafts from untreated recipients, those from CsA- or BQR-treated tolerant hosts (day 100) displayed undetectable IL-2 mRNA levels, and reduced levels of IFN-gamma, IL-4 and IL-10 mRNAs. In fact, the patterns of cytokine mRNA expression in grafts from CsA- and BQR-treated tolerant hosts were similar to those of normal hearts. Grafts from SRL-treated tolerant hosts merely showed slightly increased Th2 cell activity. In conclusion the selective activation of Th2 cells is not absolutely required for induction or maintenance of tolerance.

Animals↗
PubMed1997

Synergistic mechanisms by which sirolimus and cyclosporin inhibit rat heart and kidney allograft rejection.

The studies presented herein examined the mechanism(s) whereby sirolimus (SRL) and cyclosporin (CsA) act synergistically to block allograft rejection. Combination index (CI = 1 reflects additive, CI > 1 antagonistic, and CI < 1 synergistic, effects) analysis documented potent synergism between SRL and CsA to block allograft rejection. Combinations of the two drugs produced synergistic prolongation of heart (CI = 0.001-0.2) or kidney (CI = 0.03-0.5) allograft survival at SRL/CsA ratios ranging from 1:12.5 to 1:200. Pharmacokinetic analysis of the individual drugs showed that CsA does not affect the blood levels of SRL, and SRL mildly increases the levels of CsA in SRL/CsA-treated rats. Quantitative polymerase chain reaction analysis was used to document that both subtherapeutic (1.0 mg/kg) and therapeutic (2.0 or 4.0 mg/kg) CsA doses inhibited the expression of interferon-gamma (IFN-gamma) (P < 0.03) and IL-2 (P < 0.003) mRNA produced by T helper (Th) 1 cells, as well as IL-10 (P < 0.001), but not IL-4 (NS) mRNA produced by Th2 cells. Contrariwise, all tested SRL doses (0.02, 0.04 or 0.08 mg/kg) did not affect cytokine mRNA expression. However, heart allografts from rat recipients treated with synergistic SRL/CsA doses displayed reduced levels of IFN-gamma (P < 0.01), IL-2 (P < 0.001) and IL-10 (P < 0.001) mRNA. Thus, because subtherapeutic doses of CsA reduce Th1/Th2 activity, thereby facilitating the inhibition of signal transduction by low does of SRL, the two agents act synergistically to inhibit allograft rejection.

Animals↗
PubMed1997

Sirolimus in transplantation.

Although sirolimus (SRL) alone was very effective in rodents and pigs, it produced toxic side effects in dogs. Low doses of SRL combined with cyclosporine/brequinar (CsA/BQR) combinations achieved potent immunosuppression in the CsA-resistant mouse model. Similarly, SRL/CsA/BQR therapy protected kidney allografts from rejection in dogs without producing toxic side effects. In the CsA-sensitive rat model SRL/CsA combinations produced a potent synergistic interaction. In addition, recent clinical trials document the beneficial effects of low SRL doses in human kidney transplant recipients. Sirolimus, when combined with standard immunosuppressive therapy, remarkably reduces the incidence of acute rejection and permits individual drug dose reduction.

Animals↗
PubMed1996

Effects of the pharmacokinetic interaction between orally administered sirolimus and cyclosporine on the synergistic prolongation of heart allograft survival in rats.

Oral administration, but not continuous intravenous infusion, of sirolimus (SRL) in combination with cyclosporine (CsA) produces a pharmacokinetic interaction, namely increases in the whole blood trough concentrations of SRL ([SRL(WB)]) and CsA ([CsA(WB)]). The effects of this pharmacokinetic interaction on the synergism between SRL and CsA was examined in Wistar Furth (RT1u) recipients of Buffalo (RT1b) heart allografts. A 14-day course of oral SRL produced dose-dependent prolongation of heart allografts: in untreated controls, 0.5 mg/kg SRL per day extended the mean survival time (MST) from 6.4+/-0.5 days to 12.3+/-3.8 days (P<0.05); SRL at 1.0 mg/kg per day prolonged the MST to 18.0+/-5.5 days (P<0.01); at 2.0 mg/kg SRL per day, MST was extended to 52.5+/-13.2 days (P<0.01); and 4.0 mg/kg SRL per day prolonged MST to 90.0+/-41.1 days (P<0.01). Comparison of the in vivo effects after oral versus continuous intravenous SRL administration suggested that the oral bioavailability of SRL is less than 10%. Combinations of oral SRL and CsA synergistically prolonged heart allograft survival, as documented by combination index values of 0.01-0.64 (combination index <1 indicates synergistic interaction). In rats treated with dual drug combinations, CsA increased the bioavailability of SRL by two- to elevenfold, and SRL increased the bioavailability of CsA by two- to threefold, thereby significantly decreasing the oral effective dose (ED) values for each drug. The ED50 for SRL alone is 2.4 mg/kg per day, which produces an average [SRL(WB)] of 13.2 ng/ml. The ED50 for CsA alone is 8.0 mg/kg per day, which produces an average [CsA(WB)] of 1642 ng/ml. However, when the two drugs are combined, the ED50 effect is achieved with only 0.34 mg/kg SRL per day ([SRL(WB)]=1.1 ng/ml) and 2.1 mg/kg CsA per day ([CsA(WB)] =326 ng/ml). Individually, 0.34 mg/kg SRL per day produces an ED9 with an average [SRL(WB)] of 0.6 ng/ml, and 2.1 mg/kg CsA per day produces an ED22 with an average [CsA(WB)] of 174 ng/ml. Thus, the pharmacokinetic interaction between oral SRL and CsA contributes to the in vivo synergism between the two drugs.

Administration, Oral↗
PubMed1996

Induction of transplantation tolerance by chimeric donor/recipient class I RT1.Aa molecules.

Donor-specific transplantation tolerance was induced by administration of chimeric antigens in which four donor immunogenic amino acids (a.a.) were substituted onto the host class I MHC protein. We constructed chimeric rat RT1.Aa cDNA molecules by substituting nucleotides in the alpha1 helical region that encode 10 Lewis (LEW; RT1.A1) a.a., namely Asp58, Arg62, Glu63, Gln65, Lys66, Gly69, Asn70, Asn73, Ser77, and Asn80 ([alpha(1h)1]-RT1.Aa). The chimeric [alpha(1h)1]-RT1.Aa cDNA sequence was verified before transfection into Buffalo (BUF; RT1b) hepatoma cells. Interestingly, the helical regions of LEW rats (alpha(1h)1) and Wistar Furth (WF; RT1u) rats (alpha(1h)u) share four a.a. (Arg62, Glu63, Gln65, and Gly69). Consequently, subcutaneous administration of [alpha(1)1]-RT1.Aa transfectants (20x10(6); day -7) immunized BUF rats to reject in rapid fashion either LEW heart allografts (mean survival time [MST] = 4.2+/-0.4 days vs. 5.6+/-0.5 days in controls; P<0.001) or WF heart allografts (MST=4.4+/-0.6 days vs. 6.0+/-0.0 days in controls; P<0.002). Subcutaneous immunization of ACI (RT1a) rats with [a(1)1]-RT1.Aa transfectants (bearing 10 LEW donor a.a.) accelerated the rejection of LEW hearts (MST=5.0+/-0.8 days vs. 8.2+/-0.4 days in controls; P<0.001). In contrast, the same [a(1)1]-RT1.Aa transfectants (bearing only four WF donor a.a.) injected subcutaneously into ACI rats modestly prolonged the survival of WF hearts to 14.0+/-10.3 days from 5.4+/-0.5 days in controls (P<0.001). Furthermore, ACI recipients were rendered tolerant to WF heart allografts by a single injection via the portal vein of soluble [a(1)1]-RT1.Aa (but not RT1.Aa, RT1.Au, or [a(2)1]-RT1.Aa) antigens in conjunction with brief oral gavage treatment with cyclosporine. Thus, selected donor immunogenic a.a. (Arg62, Glu63, Gln65, and Gly69) of class I MHC antigens become tolerogenic when flanked by host sequences.

Amino Acid Sequence↗
PubMed1996

Induction of specific allograft immunity by soluble class I MHC heavy chain protein produced in a baculovirus expression system.

Spodoptera frugiperda (Sf9) insect cells secreted a class I MHC RT1.Aa heavy chain protein when infected with baculovirus that bore a construct that contained a honeybee melittin secretion (ms) signal attached to RT1.Aa cDNA. The RT1.Aa heavy chain protein in the culture supernatant and cell lysate immunoprecipitated in the presence of 5 individual anti-RT1.Aa-specific mAb. As was revealed by densitometric analysis, the ms signal increased the production (7- to 17-fold) and secretion (20- to 47-fold) of RT1.Aa protein by Sf9 cells (compared with RT1Aa-Sf9 cells without the ms signal). Subcutaneous immunization with secreted RT1.Aa heavy chain protein of Wistar-Furth (WF; RT1u) rats (day -4) accelerated the rejection of ACI (RT1a), but not third-party Brown Norway (BN; RT1n), heart allografts from 5.9 +/- 0.5 days in controls to 4.0 +/- 0.0 days (P < 0.001); cell lysate from RT1.Aa-Sf9 or ms/RT1.Aa-Sf9 cells reduced ACI heart allograft survival to 3.8 +/- 0.4 days or 3.7 +/- 0.5 days, respectively (P < 0.001). Indirect presentation of RT1.Aa heavy chain proteins by syngeneic macrophages shortened the survival of RT1.Aa-disparate PVG.R8 (RT1.AaDuBuCu) heart allografts in PVG.1U (RT1u) hosts from 6.3 +/- 0.5 days in controls to 4.0 +/- 0.0 days (P < 0.01). Finally, RT1.Aa heavy chain proteins injected into the thymus or into the portal vein (day -14) in combination with anti-T cell receptor mAb (days -14 and -13) induced indefinite survival of ACI liver allografts in Lewis (RT1l) recipients ( > 250 days). Thus, indirect presentation of soluble class I MHC heavy chain proteins (produced in a baculovirus/Sf9 cell system) may either sensitize or induce tolerance in the same fashion as native class I MHC alloantigens expressed on donor tissues.

Animals↗
PubMed1996

Beneficial effect of graft perfusion with anti-T cell receptor monoclonal antibodies on survival of small bowel allografts in rat recipients treated with brequinar alone or in combination with cyclosporine and sirolimus.

In the present experiments, a multimodality regimen was developed that included an anti-T cell receptor R73 monoclonal antibody and the pharmacologic agents brequinar (BQR), cyclosporine (CsA), and sirolimus (rapamycin; RAPA) to prolong the survival of small bowel (SB) allografts. BQR was the most potent single drug: the 4.0 or 8.0 mg/kg/day BQR doses delivered every second day (q.o.d.) per gavage for 28 days prolonged the survival of Brown Norway (BN; RT1n) SB allografts in Lewis (LEW; RT1l) recipients from a mean survival time of 10.6 +/- 1.9 days in untreated controls to 29.2 +/- 5.8 days, respectively (both P < 0.001). When treatment was extended to 56 days, 8.0 mg/kg/q.o.d BQR produced a mean survival time of 83.8 +/0 33.8 days (P < 0.001), with 2/5 hosts surviving more than 100 days. In a host-versus-graft model, BQR (8.0 mg/kg/q.o.d) delivered for 28 days with CsA (2.0 mg/kg/day) and RAPA (0.04 mg/kg/day) delivered intravenously for 14 days prolonged the survival of BN SB grafts in LEW recipients to 54.4 +/- 21.0 days (P < 0.001). Extending triple-drug therapy to 42 days induced the prolongation of SB allograft survival to greater than 100 days in 5/7 recipients. Although pretransplant perfusion of the grafts with R73 mAb was ineffective alone, the combination of graft perfusion and a 28-day course of BQR (8.0 mg/kg/q.o.d) in the GVH model indefinitely prolonged LEW graft in F1 recipients. Alternatively, indefinite survival of SB allografts ( > 100 days; P < 0.001) was achieved by the combination of a 14-day course of a triple-drug regimen using each agent at subtherapeutic doses, namely BQR (2.0 mg/kg/q.o.d.), CsA (2.0 mg/kg/day), and RAPA (0.04 mg/kg/day). The state of transplantation tolerance is these hosts was documented by the acceptance of donor-type but not third-party heart allografts.

Animals↗