Concentrations Of CsA Research Articles

EFFECTS OF RAPAMYCIN ON GROWTH FACTOR-STIMULATED VASCULAR SMOOTH MUSCLE CELL DNA SYNTHESIS INHIBITION OF BASIC FIBROBLAST GROWTH FACTOR AND PLATELET-DERIVED GROWTH FACTOR ACTION AND ANTAGONISM OF RAPAMYCIN BY FK506

Rapamycin (RPM) is a potent and effective immunosuppressant which we have shown previously to inhibit intimal thickening in rat allograft and balloon-injured arteries. In this report, we have examined the effects of RPM on growth factor-induced vascular smooth muscle cell (VSMC) DNA synthesis. RPM potently inhibited platelet-derived growth factor (PDGF) (IC50 = 5 x 10(-9) M) and basic fibroblast growth factor (bFGF) (IC50 = 8 x 10(-10) M)-induced VSMC DNA synthesis. In contrast, only the highest concentrations of FK506 and CsA significantly altered PDGF- or bFGF-induced VSMC DNA synthesis. Addition of RPM (10(-9) M) at as late as 46 hr after growth factor addition still effectively suppressed bFGF- or PDGF-induced DNA synthesis by 76% and 54%, respectively. The extent of the antagonism of RPM's inhibition of bFGF-induced VSMC DNA synthesis by FK506 was inversely proportional to RPM concentration and directly proportional to FK506 concentration.

EFFECTS OF RAPAMYCIN ON GROWTH FACTOR-STIMULATED VASCULAR SMOOTH MUSCLE CELL DNA SYNTHESIS INHIBITION OF BASIC FIBROBLAST GROWTH FACTOR AND PLATELET-DERIVED GROWTH FACTOR ACTION AND ANTAGONISM OF RAPAMYCIN BY FK506

Rapamycin (RPM) is a potent and effective immuno-suppressant which we have shown previously to inhibit intimai thickening in rat allograft and balloon-injured arteries. In this report, we have examined the effects of RPM on growth factor-induced vascular smooth muscle cell (VSMC) DNA synthesis. RPM potently inhibited platelet-derived growth factor (PDGF) (IC50=5×10-9 M) and basic fibroblast growth factor (bFGF) (IC50=8×10-10 M)-induced VSMC DNA synthesis. In contrast, only the highest concentrations of FK506 and CsA significantly altered PDGF- or bFGF-induced VSMC DNA synthesis. Addition of RPM (10-9 M) at as late as 46 hr after growth factor addition still effectively suppressed bFGF- or PDGF-induced DNA synthesis by 76% and 54%, respectively. The extent of the antagonism of RPM's inhibition of bFGF-induced VSMC DNA synthesis by FK506 was inversely proportional to RPM concentration and directly proportional to FK506 concentration.

Mitochondrial Ca 2+ overload in primary cultures of rat renal cortical epithelial cells by cytotoxic concentrations of cyclosporine: a digitized fluorescence imaging study

Cyclosporine (CsA) has been reported to disrupt Ca 2+ efflux from mitochondria, which suggests that CsA interference with Ca 2+ homeostasis may be related to its nephrotoxicity. Therefore, the purpose of this study was (1) to determine intracellular free Ca 2+ concentration ([Ca 2+]i) and mitochondrial free Ca 2+ concentration ([Ca 2+]m) after primary cultures of rat renal cortical epithelial cells were exposed to cytotoxic concentrations of CsA; and (2) to explore the role of disruption of intracellular and mitochondrial Ca 2+ homeostasis in CsA-induced cytotoxicity. [Ca 2+]i in single kidney cells was examined by digitized fluorescence imaging (DFI) of the Ca 2+ fluorescent probe, fura-2, and [Ca 2+]m in single cells was observed by DFI of fura-2 entrapped in mitochondria after selective permeabilization of plasma membrane and other non-mitochondrial organelles by digitonin. Mitochondrial membrane potential (ΔΨ) in single kidney cells was examined by rhodamine 123 (Rh-123) with DFI. Intracellular ATP in kidney cells was determined by a HPLC method. CsA resulted in an elevation in [Ca 2+]i and [Ca 2+]m, dissipation of ΔΨ and depletion of ATP in a dose- and time-dependent manner. The elevation of [Ca 2+]i and [Ca 2+]m and depletion of ATP preceded CsA-induced cytotoxicity in kidney cells as measured by lactate dehydrogenase (LDH) leakage. We conclude that CsA-induced alterations in mitochondrial Ca 2+ homeostasis and a subsequent loss of energy supply may play a key role in CsA-induced cytotoxicity in primary cultures of rat renal cortical epithelial cells.

CYCLOSPORINE INCREASES THE OXIDIZABILITY OF LOW-DENSITY LIPOPROTEINS IN RENAL TRANSPLANT RECIPIENTS

Blood specimens from twenty-six renal transplant recipients treated with cyclosporine (CsA) were collected at weekly intervals, two months after transplantation. Specimens were grouped according to their CsA concentrations. Group I consisted of ten specimens with CsA concentration of >400 ng/ml; group II consisted of ten specimens with CsA concentrations ranging from 120-300 ng/ml; and group III consisted of six specimens with CsA concentrations of < 100 ng/ml. In addition, specimens from five renal transplant patients who, instead of CsA, received the immunosuppressant FK506 (group IV), and from six healty individuals were included. Plasma low-density lipoproteins (LDL) were isolated and their susceptibility to oxidation was studied by continuously monitoring the formation of conjugated dienes during copper ion-mediated oxidation. Patients with higher blood concentrations of CsA (groups I and II) had significantly higher oxidizability of LDL, as indicated by the shorter time required to start the oxidation (lag phase). The oxidizability of samples with low concentration of CsA (group III) was not significantly different from that of FK506-treated patients or healthy individuals. There was a negative correlation (r = -0702, P < 0.01) between oxidizability (lag phase) and CsA concentration in LDL. No correlation between blood CsA and plasma cholesterol or triglyceride concentration was evident during a three-month period postoperatively. Similarly, no correlation between the degree of oxidizability and plasma cholesterol or triglycerides was found at the time of the experiment. These findings suggest a prooxidant effect of CsA to plasma LDL, and may indicate that CsA is an important risk factor in the accelerated atherosclerosis of renal transplant recipients.

Cyclophilin-B is an abundant protein whose conformation is similar to cyclophilin-A

Cyclophilin-B (bCyP-20) was isolated in a relatively high quantity from calf brain and spleen tissues consecutively applying weak cation exchange, chromatofocusing and strong cation exchange chromatographies. Edman degradation yielded the N-terminal sequence NH 2-DEKKKGPKVTVK-VYFDLRIGDEDIGRVVIGLFGKTVPKTVDNFVAL. Bovine cyclophilin-B possesses the peptidylproline cis-trans isomerase activity which is inhibited by nM concentrations of CsA. bCyP-20 has a strong tendency to bind to cation exchangers including DNA and heparin. It could be released from DNA affinity column at concentrations of NaCl higher than 200 mM. Circular dichroism spectroscopy revealed that bovine cyclophilin-A (bCyP-18) and bCyP-20 in aqueous solution have similar conformations.

Phase I/II trial of cyclosporine as a chemotherapy-resistance modifier in acute leukemia.

To determine the toxicities and maximum-tolerated dose of cyclosporine (CsA) administered with daunorubicin as a modulator of multidrug resistance (MDR) in acute leukemia, and to evaluate response to treatment and its relationship to mdr1 gene expression. Patients with poor-risk acute myeloid leukemia (AML) received sequential treatment with cytarabine (3 g/m2/d intravenously [i.v.]) days 1 to 5, and daunorubicin (45 mg/m2/d) plus CsA as a 72-hour continuous infusion (CI) days 6 through 8 in a phase I/II trial. A loading dose of CsA administered over 1 to 2 hours preceded the CI. CsA dose escalations ranged from 1.4 to 6 mg/kg (load) and 1.5 to 20 mg/kg/d (CI). Whole-blood concentrations of CsA were monitored by immunoassay; plasma concentration of daunorubicin and daunorubicinol were determined by high-pressure liquid chromatography (HPLC). Specimens were analyzed for P-glycoprotein expression, and results confirmed by a quantitative RNA polymerase chain reaction (PCR) assay for the mdr1 gene transcript. Forty-two patients are assessable for toxicity and response. P-glycoprotein was detected in 70% of cases. Dose-dependent CsA toxicities included nausea and vomiting (22%), hypomagnesemia (61%), burning dysesthesias (21%), and prolongation of myelosuppression. Transient hyperbilirubinemia developed in 62% of treatment courses and was CsA-dose-dependent. Reversible azotemia occurred in three patients receiving concurrent treatment with potentially nephrotoxic antibiotics. Steady-state blood concentrations of CsA > or = 1,500 ng/mL were achieved in all patients receiving CI doses > or = 16 mg/kg/d. Mean plasma daunorubicin, but not daunorubicinol, levels were significantly elevated in patients who developed hyperbilirubinemia (P = .017). Twenty-six (62%) patients achieved a complete remission (CR) or restored chronic phase and three patients achieved a partial remission (PR) for an overall response rate of 69% (95% confidence interval, 54% to 84%). The response rate was higher in patients who developed hyperbilirubinemia (P = .001), whereas MDR phenotype did not influence response to treatment. Among five patients with MDR-positive leukemia, cellular mdr1 mRNA decreased (n = 1) or was absent from relapsed specimens (n = 4), while mdr1 RNA remained undetectable at relapse in two patients who were MDR-negative before treatment. High doses of CsA, which achieve blood concentrations capable of reversing P-glycoprotein-mediated anthracycline resistance in vitro, can be incorporated into induction regimens with acceptable nonhematologic toxicity. Transient hyperbilirubinemia occurs commonly with CsA administration and may alter daunorubicin pharmacokinetics. Recommended doses of CsA for phase II and III trials are a load of 6 mg/kg and CI of 16 mg/kg/d.

Molecular monitoring of the immunosuppressive effects of cyclosporine in renal transplant patients by using a quantitative polymerase chain reaction

Abstract We have developed a rapid (24 hours) quantitative PCR assay to measure the direct effect of cyclosporine on IL-2 mRNA production by activated PBMC cultures from renal transplant patients. The PBMCs were purified from normal laboratory volunteers (group A, n = 26), CsA-treated renal transplant patients with good renal function, tested between 3 and 8 weeks (group B, n = 14) or between 2 and 8 years (group C, n = 15) after surgery, and stimulated with PHA in media supplemented with either patient serum or pooled commercially obtained AB serum. The mRNA was then isolated and, using semiquantitative PCR or quantitative PCR with a competitive inhibitor, the relative levels or exact levels of IL-2 mRNA (in attomoles) could be measured. A 3-day confirmatory lymphoproliferation assay of [ 3H]thymidine incorporation was also performed on the samples. Kinetic analysis of the data from group A showed that the peak level of IL-2 transcription into mRNA occurred at 6 hours after mitogen stimulation. Increasing in vitro concentrations of CsA in this group resulted in lower IL-2 mRNA levels and a shift in the peak time to 12–24 hours. In the transplant recipients, there was no correlation between individual CsA blood levels and proliferation responses. However, some correlation was found between CsA blood levels and IL-2 mRNA levels. Although there was no difference in mean proliferation responses or in mean whole blood CsA levels between groups B and C, mean IL-2 mRNA levels were 38% and 68% of the group A levels, respectively, indicating that standard CsA blood level alone may not reflect the immunosuppressive effect of the drug. Therefore, it is proposed that this, a rapid, sensitive, and quantitative PCR analysis of IL-2 mRNA should be useful in longitudinal monitoring of patients who may not be appropriately suppressed at “therapeutic” CsA blood levels.

The effects of cyclosporine on HILDA/LIF gene expression in human T cells.

We examined the effect of cyclosporine on HILDA/LIF gene expression in alloreactive human T lymphocyte clones (ATLCs) 2B11 and 2F7 obtained from cells infiltrating a rejected human kidney graft. Both ATLCs were stimulated either by the specific antigen or by PMA + calcium ionophore in the presence of various concentrations of CsA (10-500 ng/ml). Inhibition of HILDA/LIF gene expression was analyzed at the protein level using a proliferative assay on the HILDA/LIF-dependent Da-1a cell line and by RNA blotting using a specific probe. Without CsA, the kinetics of mRNA accumulation for both ATLCs peaked at 5 and 10 hr, respectively, after mitogenic and antigenic stimulations. HILDA/LIF activity peaked at 24 and 72 hr, respectively, after mitogenic and antigenic stimulation in supernatants from both ATLCs and decreased thereafter. Subsequent experiments with CsA were thus performed at these time points. Our results show that HILDA/LIF mRNA accumulation and protein secretion in 2B11 and 2F7 clones were strongly inhibited in a dose-dependent manner by CsA, in both stimulation conditions. Maximal inhibition of HILDA/LIF transcripts and protein secretion (60-90%) was observed within the range of 75-500 ng/ml CsA.

The acute effect of FK506 and cyclosporine on endothelial cell function and renal vascular resistance.

We have previously documented that cyclosporine exerts a direct cytotoxic effect on endothelial cells and causes an increase in renal vascular resistance (RVR) in the rat. In the present study we investigated whether FK506, a novel immunosuppressive agent thought to be less nephrotoxic than CsA, impairs endothelial cell function in vitro and affects RVR in vivo. In vitro eicosanoid release and endothelin release were measured in bovine aortic endothelial cells in culture exposed for 1, 6, and 24 hr to increasing concentrations of FK506 (1 nM to 10 microM) or CsA (0.5, 10 microM). No significant changes in TxB2 and 6-keto-PGF1 alpha (the stable breakdown products of TxA2 and PGI2, respectively) and endothelin release were found after 1 and 6 hr of incubation with all the concentrations of FK506 and CsA considered. FK506 did not affect endothelin release even after 24 hr of incubation. In contrast, cell exposure to CsA was associated with a dose-dependent increase in TxB2, 6-keto-PGF1 alpha, and endothelin release that reached statistical significance after incubation with 10 microM CsA. FK506 did not induce cell detachment or lysis at any concentration and time considered, while 10 microM CsA induced a significant reduction in cell count accompanied by cell lysis. In vivo studies showed that a single i.v. injection of FK506 to rats within a broad range of doses (28 ng/kg to 2.8 micrograms/kg) did not modify RVR. This was true even for a dose as high as 20 mg/kg, while 20 mg/kg CsA caused a dramatic increase in RVR. We conclude that FK506, unlike CsA, does not induce endothelial cell injury in vitro. Whether this explains the differences in renovascular resistance observed in vivo after acute injection of FK506 and CsA is an attractive possibility that needs to be further explored.

Is the Monoclonal Fluorescence Polarization Immunoassay for Cyclosporine Specific? Comparison with Specific Radioimmunoassay

Trough concentrations of cyclosporine (CsA) in whole blood were measured by specific monoclonal 3H-radioimmunoassay (S-RIA) and compared with those obtained by monoclonal fluorescence polarization immunoassay (S-FPIA) in 89 transplant recipients. Differences in the S-RIA:S-FPIA relationship between kidney transplant (KT, n = 59) and heart transplant (HT, n = 24) recipients were investigated. Mean concentrations of CsA were significantly higher by S-FPIA than by S-RIA [191 +/- 127 vs. 166 +/- 124 ng/ml (mean +/- SD), p less than 0.001], with a S-FPIA:S-RIA ratio of 1.25 +/- 0.33. Higher ratios were observed either in patients with low CsA concentrations or in those with a high proportion of metabolites. HT recipients had higher S-FPIA:S-RIA ratios than did KT recipients (1.37 vs. 1.22, t = 1.87, p = 0.065). Within-assay coefficients of variation were lower for S-FPIA than for S-RIA (2.4% vs. 12.3%, p less than 0.001). Monoclonal FPIA is a precise and fast method, more suitable than RIA for therapeutic CsA monitoring in clinical practice. However, our results indicate a 25% higher mean CsA concentration by S-FPIA than by S-RIA (37% in HT recipients), which should be borne in mind until therapeutic and toxic ranges are established or a more specific FPIA method is developed.

Therapeutic monitoring of cyclosporin ? an update

The success of organ transplantation is closely related to clinical use of the immunosuppressive drug cyclosporin (CsA). The dosage of CsA is complicated by the large intra- and interindividual variability in its pharmacokinetics, as well as by the narrow concentration range between insufficient immunosuppression and toxicity. Potential sources of error in the sampling procedure and the advantages and disadvantages of the available analytical methods are discussed. Traditionally, 12 or 24 hour trough concentrations of CsA are monitored. Recently, peak concentrations or estimation of AUCs by a limited sampling strategy have been tried to improve the relatively weak concentration-effect and concentration-toxicity relationships found with trough CsA concentration monitoring. Studies of the CsA concentration-effect relationships for various treatment indications are reviewed.

Cyclosporine-induced synthesis of endothelin by cultured human endothelial cells.

Endothelin (ET), a peptide synthesized by endothelial cells (EC), causes a decreased renal blood flow and glomerular filtration rate and an increased mean arterial pressure when infused in animals. In tissue culture, ET causes smooth muscle cell (SMC) proliferation and contraction by influx of extracellular calcium, which is inhibited by calcium channel antagonists. Infusion of cyclosporine (CSA) hemodynamically parallels ET action, and knowing that CSA effects EC, we hypothesize that the vasoconstrictive effects of CSA are a result of ET synthesis by EC. Varying concentrations of CSA were incubated with EC resulting in ET present in the supernatants in a dose-dependent manner peaking at 75% above basal activity. Coincubation of either cremophor alone or cycloheximide with CSA resulted in minimal ET present in the EC supernatants (P less than 0.01 each). Incubation of conditioned media from CSA-treated EC with SMC caused proliferation at 114% above basal activity, which did not occur in the presence of CSA alone (P less than 0.01). This activity is specifically inhibited in the presence of an anti-ET antibody or nonspecifically in the presence of calcium channel antagonists (P less than 0.01 each). Therefore, CSA stimulates EC synthesis of ET which in turn causes SMC proliferation. This action is inhibited by the coincubation of a specific antibody to ET or a calcium channel antagonist. These findings may help in the understanding of CSA-induced hypertension and vasculopathy.

THE ANTIPROLIFERATIVE EFFECT OF CYCLOSPORINE ON HEMATOPOIETIC AND LYMPHOBLASTOID CELL LINES-COMMON MECHANISTIC ELEMENTS WITH INTERFERON-ALPHA

Cyclosporine, but not its nonimmunosuppressive analog cyclosporine H (CsH), caused in a variety of hematopoietic cell types a growth arrest in the G0/G1 phase of the cell cycle. This arrest was associated with a significant reduction in the c-myc mRNA levels, which could be observed already 1 hr following CsA treatment. Similarity between the antiproliferative effects of CsA and IFN-alpha was observed. Thus, the IFN-alpha sensitive human B-lymphoblastoid cell line Daudi was also sensitive to CsA while an IFN-alpha resistant variant of Daudi cells was found to be resistant to CsA as well. Inhibition of protein synthesis with cycloheximide during IFN-alpha or CsA treatment blocked their ability to reduce the expression of c-myc. Depletion of protein kinase C (PKC) activity from cells by pretreatment of Daudi cells with phorbol.12-myristate 13-acetate (PMA) abolished the G0/G1 arrest induced by both CsA and IFN-alpha. Combinations of low concentrations of CsA and IFN-alpha had synergistic effects on cell-cycle distribution and on c-myc mRNA level, suggesting that CsA and IFN-alpha differ in some features of their antiproliferative action. This conclusion was supported by the observation that a CsA-resistant variant of Daudi cells was found to retain its sensitivity to IFN-alpha. In addition, reduction of ornithine decarboxylase mRNA expression was obtained with IFN-alpha but not with CsA. Taken together, our results suggest that CsA and IFN-alpha share some common element(s) in the pathways of their antiproliferative activity. The possible mechanisms of their antigrowth effects and the clinical significance of our findings are discussed.

Quality assessment of cyclosporine monitoring by 32 Canadian laboratories

The Canadian Quality Assurance Program was initiated in June 1989, and is a voluntary program which currently encompasses all 32 laboratories involved in the measurement of cyclosporine (CsA) across Canada. Two whole blood samples from control or clinical patients (kidney, liver and heart) containing unknown concentrations of CsA are circulated to each participating laboratory monthly, and analyzed by all techniques employed within that laboratory. Four analytical methods are currently employed: HPLC (n = 4). Sandimmun SP (n = 3), CycloTrac SP (n = 27) and TDx (n = 3). Four laboratories reported survey results in more than one methodology. Results from all participating centers are analyzed monthly. The mean, SD, standard deviation index and range are reported to each laboratory with information coded to preserve confidentiality. Accuracy, precision, recovery, analytical specificity, linearity and blank studies have been performed. This report covers the period from June 1989 to April 1990.

The Immunosuppressive Activity of the Aldehydic Transformation of Cyclosporine on Alloreactive T‐Cells

Cyclosporine (CsA) is a potent immunosuppressive compound, and its metabolites have previously been shown to have pharmacologic activity. The aldehydic metabolites have been isolated and are a metabolic intermediate after the conversion of CsA to its most active hydroxylated metabolite. The in vitro sensitivity of alloreactive T-lymphocytes, which are generated from a mixed lymphocyte reaction and propagated from organ transplant biopsy specimens to the aldehydic metabolites of CsA, was tested. In secondary proliferative assays in the presence of varying concentrations of CsA and the aldehydes, the concentration required to inhibit proliferation by 50% was 50 to 150 ng/mL for CsA and 3150 to 3500 ng/mL for the aldehydes. Pretreatment of alloreactive cells with CsA or the aldehydes did not alter cell viability, as tested with dye exclusion, or cell reactivity on reculturing. These studies concluded that the structural modification formed by metabolism of CsA to the aldehydic structure eliminates its antiproliferative activity on T-lymphocytes.

Pharmacological modification of multi‐drug resistance (mdr) in vitro detected by a novel fluorometric microculture cytotoxicity assay. Reversal of resistance and selective cytotoxic actions of cyclosporin a and verapamil on mdr leukemia t‐cells

A novel fluorometric microculture cytotoxicity assay (FMCA), based on measurements of fluorescein diacetate (FDA) hydrolysis and DNA staining by Hoechst 33342, was used for drug sensitivity testing and detection of resistance reversal in acute lymphoblastic leukemia (ALL) cell lines. The 72-hr assay was found to be sensitive, reproducible and linearly related to the number of viable cells within a broad range of cell concentrations. At clinically achievable drug concentrations, the calcium channel blocker Verapamil (ver) and the immunosuppressant Cyclosporin A (csA) were found to partly reverse acquired Vincristine (vcr) resistance in multi-drug resistant (MDR) T-ALL L100 cells with little or no effect on the drug-sensitive parental L0 cell line. By combining the fluorometric indices, we found that low concentrations of csA were growth-inhibitory, whereas higher concentrations (greater than 10 micrograms/ml) were progressively cytotoxic for drug-sensitive L0 cells. In MDR L100 cells, on the other hand, csA produced significant cell kill even at low drug concentrations. Ver had no effects on sensitive L0 cells but showed considerable cytotoxic action towards MDR L100 cells. There was no apparent relationship between drug reversal of vcr resistance and the cytotoxic actions of the drug per se since the calcium channel blocker diltiazem (dil) significantly potentiated the actions of vcr on MDR L100 cells without being more toxic to these cells (compared to vcr-sensitive L0 cells).

LOW-DOSE IMMUNOSUPPRESSION BY CYCLOSPORINE OPERATING VIA ANTIGEN-PRESENTING DENDRITIC CELLS

The inhibitory effect of cyclosporine on the stimulation of lymphocytes was tested in 20 microliters hanging drop cultures. Cells were stimulated by concanavalin A, or by allogeneic dendritic cells in a mixed leukocyte culture (MLC). Significant inhibition of lymphocyte proliferation was obtained with low concentrations of CsA (10(-9)-10(-7) micrograms/ml) in addition to the well-documented, dose-dependent inhibition at concentrations greater than or equal to 10(-1) micrograms/ml. Intermediate doses (10(-6)-10(-2) micrograms/ml) caused little inhibition. To establish whether the CsA was acting on the antigen-presenting cells or on the responding T cells, each population was pulsed with CsA before adding to the MLC. Higher doses of CsA were required to cause inhibition using this short-term pretreatment rather than continuous presence of CsA; however, when DC were pulsed with CsA, biphasic effects of the drug were seen. The time of DC pretreatment with 1 microgram/ml CsA was varied, and inhibition was seen after 1-3 hr and 16-24 hr pretreatment but not at intermediate time points. Similarly, a biphasic dose-response developed when DC-pretreatment time was fixed (2 hr) and drug concentration was varied. By contrast, on pulsing responder lymphocytes with CsA there was a biphasic effect in some experiments, but more often a single dose-dependent inhibition occurred, particularly when DC were removed from the responder cells. Major dose-dependent inhibitory effects of CsA may therefore act on both antigen-presenting DC and on lymphocytes, but this work reveals an additional low-dose inhibition that can operate via DC.

Differential sensitivity of human T helper cell pathways by in vitro exposure to cyclosporin A.

Cyclosporin A (CsA) is a widely used agent for the prevention of tissue allograft rejection in human transplantation. As a result of the recent demonstration that the allospecific Th cell response of human PBL can be generated by three distinct pathways of Th cell and APC interactions, we investigated the sensitivity of these three Th-APC pathways, as well as the Th response to recall Ag, to different concentrations of CsA. PBL from healthy volunteer donors were set up as primary in vitro cultures either without antigenic stimulation, or with influenza A virus, tetanus toxoid, or HLA alloantigenic (ALLO) stimulation. Ag-stimulated IL-2 production and proliferation were measured to assess Th cell function. To study the effect of CsA on Th function, different concentrations of CsA (0.001 to 0.1 micrograms/ml final) were added to the cultures at the time of stimulation. Th responses to influenza A virus and tetanus toxoid were more sensitive to CsA than the Th response to ALLO. By selective depletion of either responder or stimulator APC and/or of CD4+ or CD8+ cells, we 1) verified that the human ALLO Th response can be mediated by three distinct Th-APC pathways; 2) demonstrated that the ALLO response mediated by CD4+ Th and self-APC (the same helper pathway used by recall Ag) is as sensitive to CsA as the responses to recall Ag; and 3) showed that there is a hierarchy of sensitivity of these three allospecific pathways. The results are discussed with respect to the potential significance of the differential sensitivity of these allospecific Th-APC pathways to CsA for prevention of tissue allograft rejection.

The Influence of FK‐506 and Low‐Concentration Ciclosporin on Human Lymphocyte Activation Antigen Expression and Blastogenesis: a Flow Cytometric Analysis

The influence of FK-506 and ciclosporin (CsA), either alone or in combination, on PHA- and alloantigen-induced human blood lymphocyte transformation was investigated. The concentrations of FK-506 and CsA required to cause 50% inhibition of mitogen-induced thymidine incorporation were 1.0 and 200 ng/ml respectively. Evidence of synergistic inhibition of DNA synthesis was obtained when doses of each drug below these concentrations were combined (FK-506, less than or equal to 0.5 ng/ml; CsA less than or equal to 50 ng/ml). In contrast, FK-506 and CsA failed to inhibit PHA-induced increases in cell size and granularity determined by flow cytometric analysis at 1 and 3 days of culture. Moreover, no significant changes in the expression of the interleukin 2 receptor (IL-2R; CD25), transferrin receptor (TR; CD71) or HLA-DR were observed in terms of percentage positive lymphocytes or mean cell surface membrane fluorescence intensity. In primary mixed lymphocyte cultures, 50% inhibition of thymidine incorporation was obtained with FK-506 and CsA concentrations of approximately 0.25 and 25 ng/ml respectively. Evidence of synergy was obtained when lower doses of each drug were combined (FK-506, less than or equal to 0.1 ng/ml; CsA less than or equal to 2 ng/ml). On their own, these concentrations of CsA were ineffective. In contrast to corresponding PHA-stimulated cells, FK-506-treated alloactivated lymphocytes exhibited reductions in IL-2R, TR and HLA-DR antigen expression, which in the cases of IL-2R and TR were further reduced by combination of FK-506 with low-concentration CsA. These data provide further evidence of the potential of combined low-dosage FK-506 and CsA for the control of human T-cell activation and proliferation in response to allostimulation.

Pharmacokinetic and pharmacodynamic evaluation of liposomal cyclosporine

The pharmacokinetics and renal toxicity of two liposomal intravenous preparations of cyclosporine (CSA) (A, dimyristoylphosphatidylcholine (DMPC): stearylamine molar ratio 7:1; and B, DMPC: dimyristoylphosphatidylglycerol molar ratio 4:1) were assessed in the murine model and compared to the commercially available intravenous formulation of CSA (IV) and drug-free controls. No significant differences in steady-state area beneath the whole blood concentration-time curves were found between formulations and IV following 10 mg/kg per day CSA for 10 days. However, the apparent volume of distribution was significantly greater in A compared to B or IV formulations (13.82 ± 2.9 vs. 7.23 ± 3.98 and 7.67 ± 3.01 l/kg, p < 0.05), most likely due to the significantly prolonged biologic half-life of this formulation. Although the glomerular filtration rate was not significantly different between A and saline controls (1056 ± 536 and 1130 ± 550 μl/min per g KW, respectively), dosing with B or IV resulted in significant impairment (522 ± 429 and 572 ± 353 μl/min per g kidney weight (KW), respectively; p < 0.05). Triglyceride levels remained unchanged in all groups. However, cholesterol concentrations were significantly increased compared to baseline in rats administered formulation A (98 ± 31 vs. 70 ± 19 mg/dl; p < 0.05). In summary, dosing of liposomal formulation A resulted in equivalent concentrations of CSA as the IV formulation without the dose-limiting nephrotoxicity. These data offer a nontoxic delivery system for intravenous CSA.