A.W. Thomson

Toxicity of rapamycin : a comparative and combination study with cyclosporine at immunotherapeutic dosage in the rat

Sprague-Dawley rats were treated for 14 days with rapamycin (RAP; 1.5 mg/kg/day i.p.), cyclosporine (15 mg/kg/day by gavage), both drugs in combination, or appropriate drug vehicles. Hematological parameters and biochemical indices of renal and hepatic function were determined throughout the experimental period, at the end of which the rats were killed and tissues examined histologically. There was a significant reduction in weight gain in RAP- but not CsA-treated animals, while rats given both drugs showed a reduction in body weight over the 14-day experimental period. There were no significant alterations in absolute or differential white blood cell counts or in T or B cell numbers, except in the drug combination group, in which an absolute lymphopenia was detected on day 14. Small but significant increases in urinary flow rate (UFR) were found with either drug alone, and there was a marked (4-fold) increase in UFR in response to drug combination. Both RAP and CsA caused a small elevation in serum creatinine concentrations, but only with CsA was there a significant elevation in urinary enzyme activity and reduction in 51Cr. EDTA clearance. The drug combination exacerbated renal impairment, the extent of which was greater than the additive effect of either drug alone. Hyperbilirubinemia of similar magnitude was observed in rats receiving either CsA alone or in combination with RAP. In contrast to its effect on renal function, however, the CsA+RAP combination was without additional effect on liver function compared with the minor changes seen with either drug alone. Plasma and urinary glucose levels were elevated in all drug treatment groups and especially in animals given both drugs. RAP administration did not significantly affect whole-blood CsA concentrations, although the possibility of a pharmacokinetic interaction cannot be totally excluded. Histological studies revealed striking thymic medullary atrophy in all drug-treated animals. In addition, all rats given RAP showed focal myocardial necrosis of overall mild-moderate severity. Kidneys of RAP-treated rats appeared normal, whereas mild, focal, acute tubular necrosis was evident in all CsA-treated animals. Pancreases of all drug-treated animals were normal.

Cyclosporine: immunology, toxicity and pharmacology in experimental animals.

Cyclosporine (CsA) is the first of a new order of pharmacological immune suppressants and represents a significant advance in the clinical control of graft rejection. In laboratory animals, its capacity to prolong allograft survival has been well documented, including reports of indefinite donor-specific immunological tolerance after shortterm CsA treatment. There is also evidence that CsA can inhibit the onset or progress of a variety of experimental autoimmune diseases. Underlying these properties of the drug is its capacity to selectively interfere with T helper cell activation and lymphokine production, although some direct effects on B cells have also been reported. In addition, sparing of suppressor cells may in part explain the mode of action of CsA which, at the molecular level, is not understood.CsA-induced nephrotoxicity in the rat has been extensively studied and is characterized by reversible proximal tubular cell damage. This problem may be aggravated by concomitant administration of other potentially nephrotoxic drugs, such as gentamicin, or by therapeutic agents which interfere with the metabolism of CsA.CsA is metabolized in the liver and excreted in the bile. Although the pathway of hepatic metabolism of CsA has not been precisely elucidated, animal studies suggest that agents capable of inducing metabolism of the drug CsA could be used to alleviate its nephrotoxic properties.

FK 506: an immunosuppressant for the 1990s?

The novel macrolide immunosuppressant FK 506 is a powerful and selective anti-T-cell agent which has a similar mode of action to that of cyclosporin. Clinical studies of FK 506 in liver allograft recipients indicate a lower risk/benefit ratio than with cyclosporin, and wider evaluation of FK 506 in transplant recipients is now under way in multicentre, prospective, controlled trials in both Europe and North America.

A toxicological study in rats receiving immunotherapeutic doses of cyclosporin A.

SUMMARY Cyclosporin A (Cy A, 25 or 50 mg.kg-1/48 hr) administered during the course of the response, markedly suppressed graft-versus-host (GVH) reactivity in the rat, as assessed by the 7-day popliteal lymph node weight assay. Serum biochemical studies revealed small, but statistically significant increases in serum urea levels at both doses of Cy A and, at 50 mg.kg-1/48 hr, reduction in total serum protein, alkaline phosphatase, serum transaminase, and iron. In an additional experiment, Cy A (50 mg.kg-1/48 hr) was administered to rats over 4 weeks. Serum biochemical and hematological investigations were conducted at weekly intervals and at 28 days tissue (liver, kidney, spleen, lymph nodes, and small intestine) was taken for histological and ultrastructural examination. Glomerular function, monitored by creatinine and urea clearance was unaffected by Cy A treatment, but serum urea and creatinine levels were elevated. Hepatic function was not affected and hematological, histological, and ultrastructural observations, apart from evidence of hepatic fatty change, did not differ from those in vehicle-treated controls.

Adhesion molecule expression in psoriatic skin lesions and the influence of cyclosporin A.

Normal skin of healthy individuals and both lesional and uninvolved skin from patients with psoriasis before and after receiving cyclosporin A (CsA; 25 or 5 mg/kg per day) was examined by immunocytochemistry for differences in expression of adhesion‐relevant epitopes. Normal, lesional and uninvolved skin all showed staining of basal keratinocytes for CD29 (the common β chain of the βl‐integrin family). No other adhesion molecule investigated was detected on structural components of normal skin. In uninvolved skin, weak expression of CD54 (intercellular adhesion molecule I, ICAM‐1) was noted on vascular endothelium. Uninvolved keratinocytes were found to stain with anti‐CD58 (leucocyte function‐associated antigen3, LFA‐3) and there was weak expression of CD1 Ib (α chain of complement C3bi receptor) and CD11c (αchain of p150, 95 molecule) but not CD1 la (leucocyte function‐associated antigen1, LFA‐I, α chain) on those cells. In lesional skin, in addition to expression of CD58, there was also enhanced expression of CD11c. Weak expression of CD54 on keratinocytes was also observed. Lesional blood vessels were found to stain strongly with anti‐CD54, CD29 and CD58. CDlla was expressed only on infiltrating mononuclear cells. CsA treatment produced marked clinical improvement, accompanied by the loss of CD54 expression on keratinocytes. However, despite the loss of T cells from lesional skin with CsA treatment, CD54 persisted on blood vessels. CsA was found to have no effect on keratinocyte expression of CD29. CD58 or CD11bande. The persistence of CD54 on vascular endothelium and of adhesion molecule expression on keratinocytes, despite resolution of the skin lesions, may explain the universal and rapid recurrence of psoriasis on cessation of CsA administration.

Immunosuppressive activity, lymphocyte subset analysis, and acute toxicity of FK-506 in the rat. A comparative and combination study with cyclosporine.

The immunosuppressive and toxic properties of the recently discovered macrolide antibiotic FK506 were examined in comparison and in conjunction with cyclosporine administration in the rat. Male Sprague-Dawley rats were immunized systemically with sheep erythrocytes and received, from the same time, either FK506 (1 mg/kg/day) intramuscularly or CsA (25 mg/kg/day) by gavage, or both drugs in combination. Seven days after immunization, the splenic plaque-forming cell response and circulating antibody titers were reduced greater than 90% in animals receiving either FK506 or CsA and in the drug combination group. These immunosuppressive effects of FK506 and CsA were accompanied by significant increases in the incidences of splenic OX-8+ cells and by corresponding reductions in the W3/25+:OX-8+ ratio. No further changes in T cell populations were observed in animals given both drugs. A progressive monocytosis was found in response to CsA, but not in FK506-treated rats. Increases in plasma urea were observed in FK-506 and drug-combination or CsA-treated rats on day 7, whereas creatinine levels were raised only in the FK-506 groups. Elevated bilirubin levels and alterations in liver enzyme activities were observed in CsA-treated rats by day 4, whereas FK-506 alone produced no similar effects. CsA-treated rats also exhibited elevated blood and urinary glucose levels from day 4. No biochemical evidence of additive drug toxicity was detected. The only histological abnormalities observed were thymic medullary atrophy in all drug-treated animals, together with very minor reductions in bone marrow cellularity in a proportion of those rats given FK-506. These findings show that, at the dosage selected, the powerful immunosuppressive activities of FK-506 were associated with little evidence of acute toxicity and with no indications of additive toxicity with CsA.

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, ≤0.5 ng/ml; CsA ≤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; CD7I) 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, ≤0.1 ng/ml; CsA ≤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.

ENHANCED PERCUTANEOUS ABSORPTION OF A NOVEL TOPICAL CYCLOSPORIN A FORMULATION AND ASSESSMENT OF ITS IMMUNOSUPPRESSIVE ACTIVITY

No clinically successful topical cyclosporin A (CyA) formulation has been produced, mainly due to the apparent lack of drug penetration. This study has produced the first in vitro kinetic data on CyA penetration across human cadavar stratum corneum and has shown that addition of the penetration enhancers (PE) Azone and propylene glycol to the CyA vehicle significantly enhanced drug permeation across the skin barrier. Using flow‐through permeability cells with 5% w/v CyA (Sandimmun®) alone (CyA) or with PE (CyA+PE) in olive oil in the donor chamber, the penetration rate (mean ± SD μg/cm2/h) into receptor fluid was 53 ± 43 (n=13) for CyA and 660±175 (n=7) for CyA+PE.

The effect of rapamycin on renal function in the rat : a comparative study with cyclosporine

Male adult Sprague-Dawley rats were treated for 14 days with either rapamycin (RAP, 1.5 mg/kg/d i.p.) in carboxymethylcellulose (RAP/CMC) or polyethyleneglycol (RAP/PEG), cyclosporine (CsA, 15 mg/kg/d by gavage) or with the appropriate drug vehicles. Biochemical indices of renal function and integrity were determined throughout the experimental period, at the end of which the rats were killed and kidneys examined histologically. All animals gained weight at a similar rate to untreated animals except those treated with RAP; RAP/PEG animals were lighter on day 14 compared with day 0 values, whilst RAP/CMC animals were lighter only in comparison with CMC-only controls on day 14. Significant increases in urinary flow rate (UFR) were found in each drug treatment group. RAP/CMC, RAP/PEG and CsA caused mild renal functional impairment, but only with CsA was there a significant reduction in 51Cr-EDTA clearance. Significant enzymuria, resulting from drug but not vehicle administration, was observed only in the CsA-treatment group. Increased plasma and urinary glucose levels, elevated in all drug-treatment groups, were related to increased UFR. Kidneys of RAP-treated rats appeared normal, whereas mild, focal, acute tubular necrosis was evident in all CsA-tested animals. Pancreases of all drug-treated animals were histologically normal.

The immunosuppressive macrolides FK-506 and rapamycin

Cyclosporin (CsA) and FK-506 are structurally distinct fungal metabolites, which exert powerful inhibitory effects on CD4+ T (helper) cell activation and on the secretion of interleukin-2 (IL-2) and other cytokines, including various cell growth factors and interferon-gamma. Both drugs also inhibit IL-2 receptor expression on T cells. Consequently, when administered from the time of transplant surgery, both CsA and FK-506 inhibit the generation and proliferation of cytotoxic T cells which would otherwise mediate allograft rejection; T-cell dependent antibody responses are also inhibited by both drugs. CsA and FK-506 however, differ markedly in immunosuppressive potency. FK-506 is at least 100 times as potent as CsA in inhibiting human mixed lymphocyte reactions in vitro, whilst the ID50 of FK-506 for inhibition of allograft survival in animals is approximately one tenth that of CsA. FK-506 and CsA, both of which are highly lipophilic molecules, bind to distinct cytosolic proteins, each of which is a peptidyl-prolyl isomerase and the activities of which may play critical roles in signal transduction within activated T cells. The precise molecular mechanism by which these drugs selectively inhibit cytokine gene expression at a pretranscriptional level is not understood but a transcription activator has been implicated as the target. Compared with CsA, the inhibitory action of FK-506 appears more difficult to reverse, e.g., in response to pre-formed IL-2. Both drugs are however, ineffective in inhibiting directly the cytotoxic activity of cytotoxic T cells.(ABSTRACT TRUNCATED AT 250 WORDS)