Impact of p-glycoprotein inhibition and lipopolysaccharide administration on blood-brain barrier transport of colistin in mice.

The aim of this study was to investigate the factors limiting the blood-brain barrier (BBB) transport of colistin in healthy mice and to assess the impact of systemic inflammation on the transport of this antibiotic across the BBB. Colistin sulfate (40 mg/kg) was administered subcutaneously to Swiss outbred mice as single and multiple doses to determine any relationship between brain uptake and plasma concentrations of colistin. To assess the effect of P-glycoprotein (P-gp) on BBB transport, colistin sulfate (5 mg/kg) was concomitantly administered intravenously with PSC833 or GF120918 (10 mg/kg). Systemic inflammation was induced by three intraperitoneal injections of lipopolysaccharide (LPS; 3 mg/kg), and BBB transport of colistin was subsequently measured following subcutaneous administration and by an in situ brain perfusion. The brain uptake of colistin was low following single and multiple subcutaneous doses, with brain-to-plasma concentration ratios ranging between 0.021 and 0.037, and this was not significantly enhanced by coadministration of GF120918 or PSC833 (P > 0.05). LPS significantly increased the brain uptake of subcutaneously administered colistin with area under the brain concentration time curve (AUC(brain)) values of 11.7 ± 2.7 µg·h/g and 4.0 ± 0.3 µg·h/g for LPS- and saline-treated mice, respectively (mean ± standard deviation). Similarly, in situ perfusion of colistin led to higher antibiotic brain concentrations in LPS-treated animals than in saline-treated animals, with colistin brain-to-perfusate concentration ratios of 0.019 ± 0.001 and 0.014 ± 0.001, respectively. This study demonstrates that the BBB transport of colistin is negligible in healthy mice; however, brain concentrations of colistin can be significantly enhanced during systemic inflammation, as might be observed in infected patients.

A reexamination of PSC 833 (Valspodar) as a cytotoxic agent and in combination with anticancer agents.

BACKGROUND: The cyclosporins have been thought as being mainly immunosuppressive agents which interfere with the function of the MDR pump and thus play a role in resistance to drug anticancer effects. We reexamined their cytotoxicity in defined cell lines both as single agents and in combination with agents which may be of value in human malignant disease. METHODS: Cells were grown to confluence following inoculation at 5,000-8,000 cells/well in 96-well dishes, and growth patterns and death were determined by an MTT assay. Median effect analysis was used to look for synergy, additive effects, or antagonism between the cyclosporins and drugs with antitumor effects in humans. RESULTS: Cyclosporin A and PSC 833 were found to have cytotoxic activity at clinically achievable concentrations in breast, leukemia, and prostate cell lines. Synergistic or additive effects were demonstrated in all three prostate cell lines when PSC 833 was combined with estramustine, etoposide, ketoconazole, suramin, or vinorelbine in the prostate cancer cell lines. Cell line-selective additive effects or synergism were also identified with bicalutamide, carboplatin, cisplatinum, cis-retinoic acid, dexamethasone, 5-fluorouracil, liarozole, and trans-retinoic acid. CONLCLUSIONS: PSC 833 or cyclosporin alone or in combination with other agents may have an anticancer effect independently of their modulatory action on MDR. Several of the synergistic combinations which are not mediated by the MDR pump need to be tested in vivo for efficacy.

Influences of Ginkgo biloba on cyclosporin A induced lipid peroxidation in human liver microsomes in comparison to vitamin E, glutathione and N-acetylcysteine.

The in vitro effect of cyclosporin A (CsA) on lipid peroxidation in human liver microsomes was investigated, and efforts were made to prevent the resulting toxic effect of CsA. Microsomes were prepared from human liver resection material and incubated with CsA (0, 10, 30, 100, 300, 1000 micrograms/mL) for one hour (pH 7.4, 37 degrees, 95% O2, 5% CO2). Subsequently the resulting concentrations of malondialdehyde equivalents (MDA) were determined, a breakdown product of lipid peroxidation. Furthermore the duration of incubation was varied (0, 15, 30, 60, 90 min) using a CsA concentration of 300 micrograms/mL. CsA was shown to stimulate MDA-formation to up to 10-fold of the control value in both a time and concentration dependent manner. The dosage dependent experiment stated above was repeated, adding alpha-tocopherol (vitamin E, 1 mM), reduced glutathione (GSH, 1 mM), N-acetylcysteine (0.1, 0.3, 1, 3 mM), and Ginkgo biloba extract (Gbe, 15, 50, 150 micrograms/mL), respectively, to the medium of incubation. Vitamin E, a potent radical scavenger, proved to inhibit lipid peroxidation almost totally. Both GSH and N-acetylcysteine were also able to prevent lipid peroxidation, suggesting that the antioxidant effect of GSH might be caused by its thiol group and does not depend on the integrity of the whole molecule. Gbe inhibited CsA induced lipid peroxidation in a concentration dependent manner. This effect of Gbe was diminished yet not totally abolished when FeCl3 was added to the medium of incubation, whereas N-acetylcysteine even slightly enhanced CsA stimulated lipid peroxidation in the presence of iron. These results suggest that Gbe might be able to prevent radical mediated damage to human membranes caused by CsA.

Reversal of the toxic effects of cyclosporine on male reproduction and kidney function of rats by simultaneous administration of hCG + FSH.

We have shown earlier that the administration of cyclosporine impairs testicular function and causes a decrease in sperm counts, sperm motility and fertility. In order to determine whether or not the deleterious effects of CsA could be reversed by hormonal therapy, we injected sexually mature male Sprague Dawley rats with cremaphor + saline or CsA (40 mg./kg./d) alone or in combination with human chorionic gonadotropin (hCG; five micrograms./d/rat) and follicle stimulating hormone (FSH; five micrograms./d/rat). The injections were given subcutaneously for 14 days. As expected, CsA administration decreased the body and reproductive organ weights, testicular and epididymal sperm counts, sperm motility and fertilizing ability. Serum levels of LH were elevated and testosterone was decreased. The administration of FSH + hCG to the CsA treated rats restored the body and reproductive organ weights, sperm counts and motility. Seventy five percent of gonadotropin treated males were fertile as compared to 25% in the CsA treated group. In the hormone treated group, the blood levels of CsA were 50% of that of CsA treated group. In order to verify whether or not the decline in the blood levels of CsA was the cause for the amelioration of CsA-induced changes in the reproductive function, we compared the CsA + hormone treated group with another group treated with five mg./kg./d CsA which had blood levels of CsA comparable to the former group. In the five mg./kg./d group the reproductive functions were significantly lower than the CsA + hormone treated group suggesting, therefore, that the restoration of reproductive functions in the CsA + hormone treated group is a result of hormonal treatment. Administration of CsA (40 mg./kg./d) reduced the kidney weight and increased the levels of serum creatinine: these changes were also ameliorated by the administration of hCG + FSH.

Quantitative maintenance of spermatogenesis in cyclosporine-treated rats by exogenous administration of testosterone propionate.

The authors had previously shown that the subcutaneous administration of cyclosporine (CsA) resulted in an impairment of spermatogenesis. Testosterone levels declined and gonadotropin levels increased, suggesting that CsA primarily affects the synthesis and secretion of testosterone. In this study, the authors attempted to determine whether the exogenous administration of testosterone would maintain spermatogenesis in animals treated with a very high dose of CsA. Sexually mature, male Sprague-Dawley rats were treated subcutaneously with CsA (40 mg/kg per day) alone, or in combination with testosterone propionate (TP; 2 and 5 mg/d per rat), for 14 days. As expected, CsA reduced the body and reproductive organ weights and the levels of serum testosterone, while elevating the levels of follicles-stimulating hormone (FSH) and luteinizing hormone (LH). Quantitative analysis of spermatogenesis revealed a decline in all the different types of germ cells in tubules at stage VII of the cycle of the seminiferous epithelium. Administration of TP in 2 and 5 mg/d per rat doses restored the body and reproductive organ weights and the circulating levels of FSH. The serum levels of LH were below the assay's minimum level of detectability. Analysis of spermatogenesis revealed a dose-dependent increase in the germ cell counts after the administration of 2 and 5 mg of TP. The circulating levels of CsA were also significantly reduced after TP administration. These results revealed that CsA-induced alteration in spermatogenesis can be prevented by the exogenous administration of testosterone.

Effects of sex steroid hormones on sex-associated differences in the survival time of allogeneic skin grafts in rats. Evidence that testosterone enhances and estradiol reverses the immunosuppressive activity of cyclosporine.

Three-week-old DA (RT1a) male and female rats were pretreated with either orchiectomy or ovariectomy and administration of the opposite-sex steroid hormones, estradiol or testosterone. Skin from same-sex AO (RT1a) rats was then grafted when these pretreated rats reached 14 weeks of age, with a short course of immunosuppressive treatment of cyclosporine. The survival times of the grafts were reversed in that the male recipients pretreated to be like females acutely rejected the graft within 10 days as do normal adult female recipients. On the other hand, the female recipients pretreated to be like males accepted the graft as do normal adult male recipients. In addition, the synergistic effects of either testosterone or estradiol with CsA on the survival time of the graft were examined. Testosterone successfully prolonged graft survival on normal adult female and young male recipients, but negligible prolongation was observed on young females. In contrast, estradiol abrogated the immunosuppressive activity of CsA and accelerated graft rejection in both male and female recipients.

Prevention and complete reversal of cyclosporine A-induced renal vasoconstriction and nephrotoxicity in the rat by fenoldopam.

The primary mechanism of cyclosporine A-induced nephrotoxicity involves renal vasoconstriction. In the present study, we have tested the effects of fenoldopam, a dopamine DA1, receptor agonist with renal vasodilator properties, on the changes in renal function induced by acute and subacute administration of cyclosporine A. In inactin-anesthetized rats, acute administration of cyclosporine A (100 mg/kg i.p.) significantly decreased paraaminohippuric acid (PAH) and inulin clearances. Fenoldopam, at a dose (10 micrograms/kg.min) which alone significantly increased PAH and inulin clearances, completely prevented the cyclosporine A-induced reductions in renal function. Similarly, subacute administration of cyclosporine A (20 mg/kg.day for 3 days) resulted in significant reductions in base-line PAH and inulin clearances which were normalized by administration of fenoldopam. These data indicate that administration of fenoldopam can both prevent and completely reverse cyclosporine A-induced renal vasoconstriction and nephrotoxicity.

Cyclosporin A treatment enhances angiotensin converting enzyme activity in lung and serum of rats.

Nephrotoxicity and arterial hypertension are the most common side effects of treatment with cyclosporin A (CSA). Its effects on angiotensin converting enzyme (ACE) activity in the renal cortex, lung and serum of nephrotoxic rats have been investigated. Wistar rats were treated with CSA (20 mg kg-1 day-1 i.p.) or vehicle (olive oil containing 10% ethanol) for 14 days. On day 15, the rats were killed and ACE activity determined by radiometric assay using [3H]hippuryl-glycyl-glycine as substrate. CSA treatment resulted in a decrease in creatinine clearance, urine flow and body weight and a significant increase in serum and lung ACE activities (436 +/- 9 vs 391 +/- 7 nmol mL-1 min-1, P less than 0.001; 184 +/- 8 vs 142 +/- 10 nmol mg-1 min-1 P less than 0.01, respectively). In contrast, renal cortex ACE activity was reduced in the CSA-treated rats (0.35 +/- 0.02 vs 0.51 +/- 0.02 nmol mg-1 min-1, P less than 0.01). ACE activities in the renal cortex and serum were not affected by treatment with gentamicin (80 mg kg-1 day-1) for 11 days. In rats treated simultaneously with CSA and captopril (50 mg kg-1 day-1) ACE activity in the serum, lung and renal cortex was inhibited by 95, 93 and 92%, respectively. These changes in ACE activity were associated with a decreased systolic blood pressure in the rats receiving CSA and captopril. Therefore, ACE activity in the serum and lung of CSA-treated rats was increased, while its activity in the renal cortex was reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cyclosporin A on aldosterone production by dispersed rabbit adreno-capsular cells.

The effect of cyclosporin A on aldosterone production by dispersed adreno-capsular cells from rabbit was examined. Cyclosporin A significantly stimulated aldosterone production at concentrations of 10(-7) M and 10(-6) M. The maximum stimulation of aldosterone production by cyclosporin A (at 10(-6) M) was comparable to that by angiotensin II at 10(-8) M). This stimulating effect of cyclosporin A on aldosterone production was not accompanied by an increase in cyclic AMP production, and was not inhibited by a calcium-channel blocker, nicardipine. These results suggest that the aldosterone-stimulating action of cyclosporin A at these concentrations is not mediated by a known second messenger system such as channel-linked Ca2+ inflow or cyclic AMP.

Anti-CD3 antibody-induced expression of both p55 and p75 chains of the high affinity interleukin-2 receptor on human T lymphocytes is inhibited by cyclosporin A.

The inhibitory effect of cyclosporin (CsA) was investigated on human lymphocytes stimulated by anti-T-cell antibodies (anti-CD3 and -CD2) or mitogenic lectins. Whereas inhibition of cell proliferation (50%) occurred at 10 ng/ml CsA after cell activation via CD3 or CD2, higher CsA concentrations (300 ng/ml) were necessary to inhibit lectin-mediated cell activation (PHA, Con A). Exogenous recombinant interleukin-2 (rIL-2) partially reversed the inhibitory effect on antibody-stimulated cells only; however, at higher CsA concentrations (300 ng/ml) proliferation was again inhibited. Thus, CsA affected IL-2R expression and/or function at higher concentrations (300 ng/ml). CsA had no effect on receptor function as measured on IL-2-dependent cell growth of CTLL cells or preactivated lymphocytes. However, CsA inhibited both high and low affinity receptor expression as shown by [125I]IL-2 equilibrium binding studies on anti-CD3-stimulated cells. Cross-linking studies revealed that both p55 (TAC) and p75 chains of the IL-2R were not induced at low CsA concentrations (10 ng/ml). However, addition of rIL-2 reversed CsA inhibition of IL-2R expression. It is concluded that CsA, at least in anti-CD3-stimulated cells, inhibits IL-2R expression and cell proliferation with similar potency. Exogenous rIL-2 reverses CsA inhibition of IL-2R expression. This might be due to binding of rIL-2 to receptors which escape CsA inhibition, thereby up-regulating receptor expression which is drug resistant.

Acute cyclosporine renal dysfunction reversed by dopamine infusion in healthy subjects.

Up to now, no studies have been performed in normal humans to investigate the role of renal hemodynamic abnormalities in relation to acute-cyclosporin A (CsA) renal dysfunction and to verify whether the specific renal vasodilator, dopamine, can counteract these abnormalities. Eight normal subjects were examined both (A) after oral CsA (12 mg/kg body wt) and (B) after oral CsA + dopamine infusion (2 mg/kg body wt/min), under water diuresis. Both in protocols A and in B, four basal renal clearances were performed before CsA and every twenty minutes for four hours after CsA administration. In protocol A, after CsA, inulin (GFR) and PAH clearance (RPF) fell by up to 27% and to 41%, respectively, so that filtration fraction (FF) increased (P less than 0.01). A slight (not significant) hypertension occurred while renal resistances were markedly raised (P less than 0.001). Fractional urine and Na+ excretion as well as CH2O decreased, while UOsm increased (P less than 0.01). In protocol B, dopamine was infused from 120 to 180 minutes after CsA (that is, when the maximal adverse effects of CsA on renal hemodynamics had been observed in A). Dopamine infusion could reverse completely the effects of CsA on RPF, GFR, fractional urine output and CH2O; only UOsm remained higher than normal in conclusion with an increased fractional excretion of sodium (P less than 0.01). No changes were observed in plasma renin activity, aldosterone and in urinary epinephrine and norepinephrine excretion both in protocols.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-schistosomal activity of cyclosporin A: studies on murine spleen cells and the influence of a cyclosporin antagonist on resistance to infection.

Mice were treated with 5-day courses of cyclosporin A (CsA) around the time of infection with Schistosoma mansoni. Recovery of lung-stage worms 4-8 days post-infection (p.i.) was substantially reduced (80%) and no sexually mature adults were recovered from the hepatic portal system at 7 weeks p.i. Flow cytometric analysis of spleen cells from CsA-treated animals during the period of maximal parasite attrition revealed transient reductions in CD3+ and CD4+ cells and in the CD4+: CD8+ ratio compared with drug vehicle-treated, infected controls. No significant numerical changes in B cells, macrophages or eosinophils were detected relative to vehicle-treated infected mice. Transfer of spleen cells from CsA-treated donors 8 days after infection failed to confer increased resistance to S. mansoni infection on untreated recipients. Moreover, concomitant administration of CsA and an inducer of interleukin-2 production (ADA-202-718) did not interfere with the anti-schistosomal effect of CsA. Despite our incomplete understanding of the in vivo properties of CsA and reports of its paradoxical effects on immune responses, these new data indicate that the influence of CsA in schistosomiasis is unlikely to be mediated by modulation of host cell mediated immunity. This contrasts with certain other anti-parasitic effects of CsA which appear to be mediated by an action on T cells.

Anticancer potential of Viva-Natural, a dietary seaweed extract, on Lewis lung carcinoma in comparison with chemical immunomodulators and on cyclosporine-accelerated AKR leukemia.

Viva-Natural, extracted from a dietary seaweed, containing a macrophage-activating polysaccharide, has been confirmed to be active against intraperitoneally implanted Lewis lung carcinoma (LLC) and spontaneous AKR T cell leukemia. The antitumor potential against LLC has been evaluated in comparison with standard synthetic immunomodulators such as pyran copolymer (MVE-2), isoprinosine, levamisole, and tilorone while manipulating the immune systems by immunosuppressive agents, cyclophosphamide (CY) and 2-chloroadenosine. The anti-LLC activity of Viva-Natural has been found to be superior to that of isoprinosine but inferior to that of MVE-2. LLC-enhancing effect of CY could be partially reserved by the subsequent administration of Viva-Natural or MVE-2 but not by isoprinosine. 2-Chloroadenosine, a specific macrophage inhibitor, abrogated the anti-LLC activity of Viva-Natural and isoprinosine but not the activity of MVE-2. Levamisole and tilorone showed no anti-LLC activity. Ethanol-precipitable fraction of water-soluble part of Viva-Natural (crude polysaccharide) demonstrated curative activity similar to that of MVE-2. Viva-Natural reversed the potentiation effect of ciclosporin on the development of leukemia in AKR mice at preleukemic stage.

Rioprostil, a new prostaglandin E1, prevents cyclosporin A-induced damage to endocrine and exocrine pancreas.

The possibility that rioprostil can prevent cyclosporin A-induced damage to the pancreas is investigated. Cyclosporin A is given to rats once daily intragastrically in doses of 5, 10 and 20 mg/kg body weight. These doses cause dose dependent significant reduction of insulin release and enzyme secretion from the arterially perfused isolated pancreas. The subcutaneous injection of rioprostil, 15 micrograms/kg twice daily, could completely prevent the effects of 5 mg/kg cyclosporin A on insulin release and could completely prevent the effects of 5 mg/kg cyclosporin A on insulin release and could completely prevent the effects of 5 mg/kg cyclosporin A on insulin release and could also afford significant protection from the effects of 10 and 20 mg/kg cyclosporin A on both insulin release and enzyme secretion. The cytoprotective action of rioprostil on the pancreas may have therapeutic implications.