Uptake Of Cyclosporin Research Articles

Role of micelle dynamics in enhancing cyclosporine uptake in hyaluronic acid-contact lenses for improved critical lens properties in dry eye management

Loading hydrophobic drugs, such as cyclosporine, into contact lenses for dry eye treatment is challenging due to low drug uptake during soaking and alterations in critical lens properties, including wettability, water content, and optical transmission. This study aimed to investigate the role of micelle dynamics in enhancing cyclosporine loading into hyaluronic acid (HA)-contact lenses while improving their critical properties. The findings indicate that Pluronic® F127 (342 nm) and Tween® 80 (296 nm) micelles could encapsulate cyclosporine within the lens core, with Pluronic® F127 demonstrating superior surface tension on Langmuir-Blodgett trough compared to Tween® 80. The uptake of cyclosporine-loaded micelles (13.43–20.92 µg) was higher compared to cyclosporine without micelles (CY-CL = 8.80 µg). Moreover, the cyclosporine-loaded micelles improved the lens’s contact angle (wettability), equilibrium water content, and optical transmission compared to cyclosporine without micelles. The in vitro test showed sustained release of cyclosporine from contact lenses over 120 h or 96 h using Pluronic®-micelles or Tween® 80-micelles, respectively. In animal study, the 0.05% w/v Pluronic® F127-CL and 0.0015% w/v Tween 80-CL lenses showed an initial release of 2.54 µg/ml and 3.59 µg/ml, respectively, with the release profile extending over 48 h. Wear of the micelle-laden lenses resulted in lower levels of IL-6 compared to cyclosporine eye drops. In conclusion, Pluronic® F127 micelles demonstrated greater stability, sustained release, and improved outcomes compared to Tween® 80 micelles. These findings suggest the potential of micelles-laden contact lenses as a promising alternative to eye drops for improved dry eye management.

Low-density Lipoprotein Receptor Activities, Lipids, Apolipoprotein, and Clinical Course of Patients with Steroid-resistant Nephrotic Syndrome Treated with Low-density Lipoprotein Apheresis: A Case Series.

We herein report three cases of steroid-resistant nephrotic syndrome successfully treated with low-density lipoprotein apheresis (LDL-A). All patients were treated with a combination of steroids, cyclosporine, and LDL-A. In all cases, the serum concentrations of LDL, total and high-density lipoprotein cholesterol, and triglycerides were significantly lowered following LDL-A administration. Furthermore, the estimated LDL receptor activity increased, while both serum LDL and total cholesterol levels decreased, suggesting that LDL-A increases LDL receptor activity by driving changes in serum cholesterol concentration. This case series suggests that LDL-A increases LDL receptor activity, which may improve the intracellular uptake of cyclosporine.

Decreased brain uptake of cyclosporin A and tacrolimus in the inflammatory bowel disease mice.

Decreased brain uptake of cyclosporin A and tacrolimus in the inflammatory bowel disease mice.

Effects of serum lipoproteins on cyclosporine A cellular uptake and renal toxicity in vitro

In-vitro studies were performed to shed light on previous findings that showed increased uptake of cyclosporine A in the kidneys and liver of hyperlipidemic rats, and increased signs of kidney toxicity. Hepatocytes were obtained from rats, cultured, and exposed to a diluted serum from hyperlipidemic rats. Some cells were also exposed to lipid-lowering drugs. After washing out the rat serum or lipid-lowering drugs, cells were exposed to cyclosporine A embedded in serum lipoproteins. Pretreatment with hyperlipidemic serum and lipid-lowering drugs was associated with an increased uptake of cyclosporine A. As expected, atorvastatin caused an increase in low density lipoprotein receptor and a decrease in MDR1A mRNA in the hepatocytes. A decrease in NRK-52E rat renal tubular cellular viability caused by cyclosporine A was noted when cells were preincubated with diluted hyperlipidemic serum. This was matched with evidence of hyperlipidemic-serum-associated increases in the NRK-52E cellular uptake of cyclosporine A and rhodamine-123. The findings of these experiments suggested that in hyperlipidemia the expression and (or) the functional activity of P-glycoprotein was diminished, leading to greater hepatic and renal uptake of cyclosporine A, and renal cellular toxicity.

Effect of HEPES Buffer on the Uptake and Transport of P-Glycoprotein Substrates and Large Neutral Amino Acids

HEPES has been widely employed as an organic buffer agent in cell culture medium as well as uptake and transport experiments in vitro. However, concentrations of HEPES used in such studies vary from one laboratory to another. In this study, we investigated the effect of HEPES on the uptake and bidirectional transport of P-gp substrates employing both Caco-2 and MDCK-MDR1 cells. ATP-dependent uptake of glutamic acid was also examined. ATP production was further quantified applying ATP Determination Kit. An addition of HEPES to the growth and incubation media significantly altered the uptake and transport of P-gp substrates in both Caco-2 and MDCK-MDR1 cells. Uptake of P-gp substrates substantially diminished as the HEPES concentration was raised to 25 mM. Bidirectional (A-B and B-A) transport studies revealed that permeability ratio of P(appB-A) to P(appA-B) in the presence of 25 mM HEPES was significantly higher than control. The uptake of phenylalanine is an ATP-independent process, whereas the accumulation of glutamic acid is ATP-dependent. While phenylalanine uptake remained unchanged, glutamic acid uptake was elevated with the addition of HEPES. Verapamil is an inhibitor of P-gp mediated uptake; elevation of cyclosporine uptake in the presence of 5 muM verapamil was compromised by the presence of 25 mM HEPES. The results of ATP assay indicated that HEPES stimulated the production of ATP. This study suggests that the addition of HEPES in the medium modulated the energy dependent efflux and uptake processes. The effect of HEPES on P-gp mediated drug efflux and transport may provide some mechanistic insight into possible reasons for inconsistencies in the results reported from various laboratories.

ATP-Binding Cassette Subfamily B Member 1 Polymorphisms Do Not Determine Cyclosporin Exposure, Acute Rejection or Nephrotoxicity After Heart Transplantation

We hypothesized that genetic variation of ATP-binding cassette subfamily B member 1 (ABCB1) that encodes P-glycoprotein (involved in the uptake of cyclosporin A [CsA]) contributes to trough drug concentrations and thereby to CsA's immunosuppressive and toxic effects. Three hundred thirty-seven adult heart transplant recipients were studied retrospectively. White recipients receiving CsA at month 3 and years 1 to 5 after transplantation (n=192, 168, 156, 130, 95, and 74, respectively) were then studied with respect to ABCB1 genotype or haplotype and CsA disposition. Genotyping was performed using a gel-based polymerase chain reaction method. Dose- and weight-adjusted CsA trough concentrations ([microg/L]/[mg/kg]), time to first endomyocardial biopsy-proven acute rejection episode (grade>or=3A), weaning from steroids at 1 year, and renal function at 1 year posttransplant were measured. An association between dose- and weight-adjusted CsA trough concentrations and ABCB1 haplotypes was found, with 12/1236, 21/2677, 26/3435 CC/GG/CC individuals having significantly higher concentrations than TT/TT/TT individuals at years 1 and 5 (68.9+/-26.9 vs. 54.9+/-19.5 and 70.6+/-35 vs. 50.0+/-12.2 [microg/L]/[mg/kg] P<0.05, respectively) There was no difference in the incidence of acute rejection, steroid weaning, or renal impairment between the genotype or haplotype groups. The association of ABCB1 12/1236, 21/2677, and 26/3435 CC/GG/CC haplotype with increased CsA dose- and weight-adjusted CsA trough concentrations in this group of adult white heart transplant recipients was not consistent over time and had no effect on the incidence of acute rejection or on the development of renal impairment.

Applications of LDL-apheresis in nephrology

LDL-apheresis (LA) was originally used for familial hyperlipidemia, and then in Japan extended to use for the treatment of patients with peripheral arterial disease (PAD) and nephrotic syndrome due to steroid-resistant focal glomerular sclerosis (FGS). The reason why this treatment is applicable for these disorders is due to the fact that LA exerts its favorable effects beyond the lipid-lowering effect. The main underlying mechanisms, for example, in the case of LA application in patients with PAD are: (1) improvement of hemorheology, (2) improvement of endothelial dysfunction, (3) elevations of serum levels of NO and bradykinin, (4) increase in serum levels of vascular endothelial growth factor, and (5) reduction of adhesion molecules on monocytes. Furthermore, we have reported that LA could have anti-inflammatory effects because LA reduces serum levels of P-selectin, which is known to play an important role in the development of atherosclerosis as well as a reduction of serum C-reactive protein levels as standard biomarker of atherosclerosis. Massive proteinuria is also an important challenge in nephrology. The possible mechanisms besides removal of toxic lipids are the reduction of the vasoconstrictive prostanoid and thromboxane A2 (TXA2) and an improvement in macrophage function evidenced by a significant amelioration of interleukin-8 production by lipopolysaccharide-stimulated peripheral blood mononuclear cells. It is intriguing to note that in terms of pharmacodynamics, LA improves steroid and cyclosporine uptake into lymphocytes. Although there are no randomized controlled trials, it is clear that LA has various effects beyond lowering lipids. Making the device more concise and changing it into a whole blood adsorption type, we need to collect more clinical cases and to study the underlying mechanisms further.

Site-dependent contributions of P-glycoprotein and CYP3A to cyclosporin A absorption, and effect of dexamethasone in small intestine of mice

We examined whether the oral bioavailability of cyclosporin A is controlled primarily by P-glycoprotein (P-gp) or CYP3A in the small intestine. In situ loop method was used to evaluate the uptake of cyclosporin A (40 nmol) at the upper and lower intestine of wild-type and mdr1a/1b knockout mice treated or not treated with dexamethasone (75 mg/kg/day, 7 days, i.p.). Expression of CYP3A mRNA in the control group was higher in the upper than the lower intestine, while that of the multidrug resistance-1a ( mdr1a) mRNA was in the opposite order. Dexamethasone administration potently induced CYP3A and mdr1a mRNAs in the lower and upper intestine, respectively. At 45 min after cyclosporin A administration into an upper intestinal loop of the control group of wild-type mice, the ratio of residual cyclosporin A to dose did not differ significantly from that of mdr1a/1b knockout mice, whereas in dexamethasone-treated wild-type mice, the residual ratio was increased significantly. The ratio of the cyclosporin A metabolite M17 to cyclosporin A in portal venous blood at an upper intestinal loop of mdr1a/1b knockout mice was much higher than that a lower intestinal loop. The M17/cyclosporin A ratio of portal venous blood at a lower intestinal loop in mdr1a/1b knockout mice was increased significantly by dexamethasone treatment. These results suggest that, under physiological conditions, the oral bioavailability of cyclosporin A is mainly controlled by CYP3A in the upper intestine, rather than liver, but when P-gp is induced by steroid, the intestinal absorption of cyclosporin A may be inhibited.

Nitric oxide mediates increased P-glycoprotein activity in interferon-{gamma}-stimulated human intestinal cells.

Patients with refractory inflammatory bowel disease (IBD) exhibit increased expression of intestinal P-glycoprotein (P-gp) as well as elevated luminal IFN-gamma and nitric oxide (NO) levels. Using the in vitro Caco-2 cell culture model, we investigated whether these pathological mediators associated with the etiology of IBD affect functional activity of intestinal efflux systems. IFN-gamma reduced cellular uptake of cyclosporin A (CysA) but not methotrexate (MTX) in a time- and concentration-dependent manner. Simultaneously, P-gp expression increased by approximately twofold. Coincubation with the inducible NO synthase inhibitor l-N(6)-(1-iminoethyl)lysine (l-NIL) dramatically reduced production of intracellular NO in response to IFN-gamma stimulus. The presence of l-NIL also abrogated the cytokine-mediated increase in P-gp expression and function suggesting that NO is required for IFN-gamma-mediated activation of this efflux system. Exposure of Caco-2 cells to the chemical NO donor S-nitroso-N-acetylpenicillamine (SNAP) produced a concentration-dependent decrease in intracellular CysA accumulation that was paralleled by an increase in P-gp expression. Both IFN-gamma and SNAP enhanced DNA binding of NF-kappaB, whereas inclusion of l-NIL dramatically decreased this cytokine-induced effect on NF-kappaB binding. These results suggest that NO mediates IFN-gamma-induced increase in expression and function of intestinal P-gp in the human Caco-2 cell culture model by altering DNA binding of NF-kappaB, which may enhance transcription of the ABCB1 gene encoding for this efflux system.

Neoral in de novo liver transplantation: Adequate immunosuppression without intravenous cyclosporine

Absorption of cyclosporine from the traditional oral formulation Sandimmune (Novartis Pharma, Basel, Switzerland) is particularly unpredictable in the early stages after liver transplantation. The absorption of cyclosporine is influenced by liver function, postoperative paralytic ileus, and graft dysfunction. Oral absorption of cyclosporine from Sandimmune is also bile dependent; cholestasis and external biliary drainage are associated with low cyclosporine absorption. Postoperative administration of intravenous Sandimmune is therefore often necessary to obtain adequate immunosuppression, despite the increased risk of renal and neurological toxicity. A microemulsion formulation of cyclosporine, Neoral (Novartis), has been developed to overcome the problems of poor and variable absorption of cyclosporine from Sandimmune. Uptake of cyclosporine from Neoral is rapid and less dependent on bile secretion so that higher peak concentrations are reached and absorption is less variable than with Sandimmune. A review of several open studies in which Neoral was administered to liver transplant patients immediately after transplantation is presented. The results suggest that the use of Neoral as a primary immunosuppressive therapy provides adequate cyclosporine trough levels, minimizing or obviating the need for intravenous cyclosporine administration. In addition, Neoral appears to reduce the risk of acute rejection episodes compared with immunosuppressive regimens involving intravenous cyclosporine. (Liver Transpl Surg 1997 Nov;3(6):571-7)

Neoral in de novo liver transplantation: adequate immunosuppression without intravenous cyclosporine.

Absorption of cyclosporine from the traditional oral formulation Sandimmune (Novartis Pharma, Basel, Switzerland) is particularly unpredictable in the early stages after liver transplantation. The absorption of cyclosporine is influenced by liver function, postoperative paralytic ileus, and graft dysfunction. Oral absorption of cyclosporine from Sandimmune is also bile dependent; cholestasis and external biliary drainage are associated with low cyclosporine absorption. Postoperative administration of intravenous Sandimmune is therefore often necessary to obtain adequate immunosuppression, despite the increased risk of renal and neurological toxicity. A microemulsion formulation of cyclosporine, Neoral (Novartis), has been developed to overcome the problems of poor and variable absorption of cyclosporine from Sandimmune. Uptake of cyclosporine from Neoral is rapid and less dependent on bile secretion so that higher peak concentrations are reached and absorption is less variable than with Sandimmune. A review of several open studies in which Neoral was administered to liver transplant patients immediately after transplantation is presented. The results suggest that the use of Neoral as a primary immunosuppressive therapy provides adequate cyclosporine trough levels, minimizing or obviating the need for intravenous cyclosporine administration. In addition, Neoral appears to reduce the risk of acute rejection episodes compared with immunosuppressive regimens involving intravenous cyclosporine.

The role of lipoproteins in the transport and uptake of cyclosporine and dihydro-tacrolimus into HepG2 and JURKAT cell lines

Objectives: We wish to examine the role of lipoproteins in the transport and cellular uptake of cyclosporine (CsA) and tacrolimus. Design and Methods: The distribution of tritiated CsA and tacrolimus among lipoproteins was determined in normo- and hypertriglyceridemic sera. The uptake of these two drugs into HepG2 and JURKAT cell lines was assessed in the presence of various concentrations of low density lipoproteins (LDL). Results: Our data showed that about 60% of these drugs were transported by high density lipoprotein in normolipidemic sera, while about 50–60% were carried by very low density lipoprotein in hypertriglyceridemic sera. Almost 80% of CsA and 70% of tacrolimus entered HepG2 and JURKAT cells within the first hour of incubation in lipoprotein free media. However, the uptake was decreased (CsA by 60% and tacrolimus by 40%) in the presence of LDL. Conclusions: Lipoproteins play a major role in the transport of CsA and tacrolimus, but not in their cellular uptake.

Transport of cyclosporin A in kidney epithelial cell line (LLC-PK1).

The characteristics of cyclosporin A transport have been studied in cultured kidney epithelial cell line LLC-PK1. The uptake of cyclosporin A by LLC-PK1 cells was time-dependent and reached a steady state at about 30 min. The initial uptake was saturable and was inhibited by cyclosporin A analogs, cyclosporin C and D and by verapamil, but not by metabolic inhibitors such as 2,4-dinitrophenol and rotenone. Cyclosporin A uptake as well as efflux was strongly dependent on temperature. The Arrhenius plot for cyclosporin A uptake was biphasic, whereas the Arrhenius plot for sulfanilamide uptake, which is transported by a simple diffusion, was linear. These results indicate that a specific mechanism is concerned in the transport of cyclosporin A in LLC-PK1, cells.

Cyclosporine uptake by cultured human proximal tubule cells

Characteristics of cyclosporine A (CsA) uptake in cultured normal human proximal tubule cells (PTC) have been studied. The uptake was very rapid, concentration dependent, and reached a steady state level within 10 min. Kinetic analysis yielded an apparent Km of 5.0 μM and Vmax of 66.7 pmoles/min μg DNA. Verapamil, a calcium channel blocker, at 0.1, 0.5, and 1.0 mM had no inhibitory effect on CsA uptake. Subcellular distribution of CsA following 1, 2, 5, and 10 min incubation of PTC suspension was determined by digitonin differential solubilization. The cytosol fraction contained 26% and the nuclear fraction contained 74% of CsA at all time periods studied. These studies demonstrate that in normal human renal PTC, uptake of CsA is rapid and saturable. Most of the CsA, once taken up by the human PTC, is concentrated in the nucleus.

Cyclophilin Content of Normal and Psoriatic Epidermis

The unique, immunosuppressive agent cyclosporine A has been shown to be of important therapeutic value in the treatment of psoriasis and other inflammatory dermatoses. To investigate the basis for its therapeutic efficacy, the tissue and cellular content of cyclophilin, a cytosolic receptor for cyclosporine A, has been determined in keratome biopsies from normal and psoriatic epidermis and in cultured, adult human keratinocytes. The mean cyclophilin content of normal (n = 10), involved (n = 10), and uninvolved (n = 10) psoriatic epidermal samples was not significantly different (0.126 +/- 0.016, 0.106 +/- 0.009, and 0.153 +/- 0.018 microgram cyclosporine A bound/mg cytosolic protein). Similarly, the cyclophilin content of keratinocytes cultured from normal and psoriatic epidermis (0.21 +/- 0.016 and 0.18 +/- 0.024 microgram/mg protein, respectively) did not differ significantly. Western blot analysis of normal and psoriatic epidermal extracts with monospecific rabbit anti-cyclophilin antisera revealed a single band of 17,000 daltons, which co-migrated with highly purified bovine cyclophilin. The intensity of this band was similar in normal and psoriatic samples, indicating that immunoreactive cyclophilin content was similar. Cyclophilin mRNA was readily detected in normal and involved psoriatic epidermis by RNA blot hybridization, and expression was not significantly different in normal and psoriatic epidermis. These findings indicate that cyclophilin is present in human keratinocytes and epidermis, where it may account for the accumulation of cyclosporine during therapy. However, the similar cyclophilin content of normal and diseased tissue suggests that differences in cyclosporine uptake probably do not account for its therapeutic efficacy in psoriasis.

IN VITRO CYCLOSPORINE TOXICITY

The epithelial cell line LLC-PK1, which expresses many proximal tubular characteristics, was used to investigate the relationship between calcium, the calcium channel blocker verapamil, and cyclosporine toxicity. The LLC-PK1 cells took up cyclosporine when this was added in a concentration of 2 micrograms/ml, and this uptake was maximal at 30 min (112 +/- 3 ng cyclosporine/mg cell protein). At 12 micrograms/ml it inhibited the sodium glucose cotransporter, as assessed by phlorizin-inhibitable 14C-alpha-methyl glucopyranoside (alpha-MG) uptake (control 37.2 +/- 6.3, 12 micrograms/ml 21.2 +/- 1.1 mumol/hr/mg protein). Cyclosporine at 2 micrograms/ml did not affect cell growth after 5 days (control 945 +/- 60 micrograms cell protein per 25 cm2 flask, 2 micrograms/ml cyclosporine/ml 1046 +/- 32 micrograms protein/flask), even in the presence of 7.6 mM ionized calcium (862 +/- 37 micrograms protein/flask). Cyclosporine at 12 micrograms/ml inhibited cell growth (286 +/- 27 micrograms protein/flask), and raising the ambient ionized calcium concentration to 7.6 mM reduced cell growth further (91 +/- 6 micrograms protein/flask). Cyclosporine at concentrations of 2 and 12 micrograms/ml produced increasing cell vacuolation, as seen in vivo. Short-term uptake of 2 micrograms/ml cyclosporine could be inhibited by 1.0 mM and 0.5 mM verapamil (49 +/- 9.5 and 71 +/- 6.4 ng cyclosporine/mg cell protein, respectively, at 30 min). However, in the presence of 2 micrograms/ml cyclosporine 0.1 mM verapamil was toxic to the cells grown over five days (44 +/- 5 micrograms protein/flask). At 0.01 mM verapamil was not toxic to cell growth (921 +/- 29 micrograms protein/flask), but raising the medium calcium to 7.6 mM reduced cell growth (652 +/- 96 micrograms/ml). Inhibition of cyclosporine uptake did not occur with 0.01 mm verapamil (control 145.6 +/- 12.3 vs. 0.01 mM verapamil 150.4 +/- 3.8 ng cyclosporine/mg cell protein). The LLC-PK1 cell line represents a good in vitro model for cyclosporine renal tubular toxicity, as the in vivo observation of glycosuria and proximal tubular cell vacuolation in cyclosporine nephrotoxicity can be reproduced. In vitro this is shown to be associated with inhibition of sodium-dependent glucose cotransport. Verapamil inhibited cyclosporine uptake, but only at concentrations that were toxic to the cells. Verapamil potentiated rather than reduced the increased cyclosporine toxicity produced by increasing the medium calcium concentration. The suggested protective effect of verapamil against cyclosporine nephrotoxicity is therefore unlikely to be due to inhibition of cyclosporine uptake or of calcium entry into proximal tubular cells.

FK506

WARTY, VIJAY; DIVEN, WARREN; CADOFF, EVAN; TPDO, SATORU; STARZL, THOMAS; SANGHVI, AJIT Author Information

Interactions of cyclosporine with renal proximal tubule cells and cellular membranes.

Cyclosporine-induced nephrotoxicity is a limiting factor in the clinical use of cyclosporine. Since the manner in which cyclosporine interacts with proximal tubule cells and their membranes may provide insight into the cellular pathophysiology of cyclosporine toxicity, experiments were undertaken to characterize the interactions of cyclosporine with proximal tubule cells, renal brush border membranes, and renal cortical mitochondria. Cyclosporine bound to isolated rat renal brush border membranes in a saturable manner with a Kd of 0.38 microM and an nmax of 0.33 nmoles/mg protein. Scatchard analysis suggested that the interaction of cyclosporine at low concentrations with brush border membranes was consistent with a partitioning process rather than binding to a specific membrane component. Cyclosporine inhibited rat renal cortical mitochondrial respiration in a dose-dependent manner, with 8 microM as a threshold dose. This inhibitory effect was greater for respiration supported by succinate than pyruvate-malate. TMPD-ascorbate-supported respiration was unaffected. Suspensions of rabbit renal proximal tubule segments were incubated in vitro with 0.5-500 microM 3H-cyclosporine to measure the kinetics of cyclosporine uptake. Uptake was rapid (80% after 10 min) and saturable at 100 microM, with 9 nmoles cyclosporine/mg protein accumulated. Incubation of suspensions of enriched in rabbit renal proximal tubule segments with 10 microM cyclosporine in vitro for 2 hr with or without 22.5 min of hypoxia, or for 16 hr without hypoxia, had no effect on a variety of quantitative metabolic parameters of cell injury, including basal and uncoupled tubule respiratory rates and tubule K+, Ca++ and adenine nucleotide levels. These results demonstrate that cyclosporine interacts with critical renal membrane components at low concentrations but this interaction does not result in proximal renal tubular cell injury acutely in vitro.

Hepatocellular uptake of cyclosporin A by simple diffusion

Cyclosporin A is known to be eliminated mainly via the biliar pathway after biotransformation. Whether liver cells take up the drug by simple diffusion across the lipid barrier or by carrier-mediated transport, as shown for some other peptides, was unknown up to the present. Experiments with [ 3H]cyclosporin A on isolated rat hepatocytes indicate that the uptake of cyclosporin A is neither saturable nor is driven by metabolic energy. Cholestasis caused by cyclosporin A treatment is therefore not the result of mutual competition for a carrier protein. Nevertheless, cyclosporin A interacts with the bile acid transport system by non-competitive inhibition of bile salt uptake.

The effect of verapamil on cellular uptake, organ distribution, and pharmacology of cyclosporine.

Verapamil has been shown to potentiate cyclosporine's effect in inhibiting lectin-stimulated proliferation of murine and human lymphocytes, and in prolonging graft survival in experimental heterotopic cardiac transplantation in rats. A series of experiments were designed to determine whether verapamil's effect occurred by increasing cyclosporine uptake or decreasing cyclosporine's clearance by lymphocytes utilizing human peripheral blood lymphocytes and radiolabeled cyclosporine. Verapamil had no effect. The distribution of radiolabeled cyclosporine was also studied in mice that had been given verapamil (10 mg/kg) 1 hr prior to cyclosporine injection. No significant changes in organ distribution occurred. Lectin-stimulated release of intracellular ionized calcium was studied using a flurometric technique (Quin-2 and Fura-2). Neither cyclosporine nor verapamil had any effect on either lectin-stimulated or phorbol ester-stimulated release of intracellular ionized calcium. Phorbol ester and subproliferative doses of lectin were used to determine the effect of cyclosporine and verapamil on protein kinase C-mediated lymphocyte activation. Cyclosporine inhibited phorbol ester stimulated proliferation and verapamil potentiated this inhibition. Verapamil does not change cell or organ uptake of cyclosporine, and it does not affect the initial increase in intracellular ionized calcium that occurs with lymphocyte activation. Verapamil potentiates cyclosporine in inhibiting protein kinase C-mediated events in lymphocyte activation.