ALKYLATING AGENTS: Cyclophosphamide
If a patient does not show an initial response to corticosteroids, or if the disease is refractory to corticosteroids, an alkylating agent such as cyclophosphamide (Cytoxan) has been used for some diseases. Cyclophosphamide is one of the nitrogen mustards and is one of the most potent immunosuppressive drugs available. Cyclophosphamide undergoes extensive metabolism via cytochrome-P450 (CYP450) oxidation followed by spontaneous conversion. The immunosuppressive effects of the nitrogen mustards can be attributed to the metabolites phosphoramide mustard and acrolein. The cytotoxic effect on lymphocytes is caused by damage to DNA. Because cyclophosphamide is directly cytotoxic to lymphocytes, it can directly suppress B-cell activity and antibody formation. B-cells are affected more than T-cells because their rate of recovery from an alkylating agent is slower.
Cyclophosphamide is a potent immunosuppressive drug, but the adverse effects can be serious. In people, long-term therapy is discouraged because of the risk that cyclophosphamide will induce secondary malignancies. In dogs, the most serious toxic effect is bone marrow suppression, which can lead to secondary infections. Another adverse effect is sterile hemorrhagic cystitis, which is a severe toxic reaction involving the urinary bladder mucosa. The injury to the urinary bladder is caused by the toxic effects of metabolites on the bladder epithelium (especially acrolein) that are concentrated and excreted in the urine. Various strategies have been used to decrease the injury to the bladder epithelium. Corticosteroids are usually administered with cyclophosphamide to induce polyuria and decrease the concentration of the metabolite on the epithelium. In people, the drug mesna (Mesnex, mercaptoethanesulfonate) provides free active thiol groups to bind metabolites of cyclophosphamide in the urine. The bladder also has been irrigated with acetylcysteine, which binds thiol groups to reactive metabolites. Neither of these approaches has been used commonly in veterinary medicine. Cats are resistant to hemorrhagic cystitis from cyclophosphamide. Gastrointestinal effects, such as nausea, vomiting, and diarrhea, also are possible in any species treated.
When used clinically, the dose administered to dogs is 50 mg/m2, which is approximately 1.5 mg/kg PO for large dogs and 2.5 mg/kg PO for small dogs. It is available in 25 and 50 mg tablets. This dose has been administered on an every-other-day (EOD) basis, with corticosteroids administered on the alternate days.
As first-line therapy, or as an alternative to nitrogen mustard alkylating agents for treatment of immune-mediated disease, thiopurines have been administered. The most common drug used for this purpose is azathioprine (Imuran). It is metabolized in the liver to the active metabolite 6-mercaptopurine (6-MP). In vitro studies indicate that some metabolism may occur at target cells responsible for immune effects. Azathioprine interferes with de novo synthesis of purine nucleotides that are important for lymphocyte proliferation. 6-MP inhibits T-cell lymphocyte function and helper cell effects on antibody synthesis, with little direct effect on B-cells. In people, it has been suggested that azathioprine is more effective for Ig-G mediated disease whereas cyclophosphamide is more effective for Ig-M mediated disease. This theory has not been tested in animals.
Clinical Use. In veterinary medicine, azathioprine has been used for immune-mediated anemia, colitis, immune-mediated skin disease, and acquired myasthenia gravis. Azathioprine is available as 50 mg tablets. It is dosed at 2 mg/kg, orally, q24h. Long-term therapy is administered at a dose of 0.5 to 1.0 mg/kg EOD, with prednisolone administered on the alternate days. In people, the lag-period before successful treatment is recognized with azathioprine may be as long as two to eight months. In veterinary medicine, this lag-period is probably shorter and therapeutic benefits have been observed after only three to five weeks.
Adverse Effects. Bone marrow suppression is a concern in all animals. Leukopenia and thrombocytopenia can be serious. In addition, gastrointestinal toxicity and hepatotoxicity are possible. Gastrointestinal effects such as nausea and diarrhea may be only temporary and subside after several days of therapy. Sterile hemorrhagic cystitis, the complication cited for cyclophosphamide, has not been seen with azathioprine. There has also been association (but not well documented) between the administration of azathioprine plus prednisolone and the development of acute pancreatitis in dogs. It has been suggested that this effect is caused by azathioprine's effect on decreasing pancreatic secretion in animals.
Is azathioprine contraindicated in cats? (Beale, et al., 1992) Some veterinarians have administered azathioprine to cats at a total dose of about 6.25 mg per cat (1/8 tablet), PO EOD. Success has been reported with 1 mg/kg q48h for pemphigus (Caciolo, et al., 1984). However, doses of 2.2 mg/kg EOD produced profound neutropenia in cats (Beale et al., 1992). Some veterinarians have administered 1.1 mg/kg every day, or every other day, but other references have discouraged its use in cats because of the bone-marrow suppressing effects (Helton-Rhodes, 1995). Differences in metabolism may explain the susceptibility in cats (discussed below under metabolism). Because cats may be at a risk of bone marrow suppression from administration of azathioprine, currently recommended doses are as low as 0.3 mg/kg once daily or every other day. Careful monitoring of the CBC is recommended during treatment
Metabolism. After azathioprine is converted to 6-MP, it is further metabolized by three routes to other metabolites. One metabolic route is via xanthine oxidase to inactive metabolites. Allopurinol will decrease this route because it inhibits xanthine oxidase. Another metabolic route is via thiopurine methyltransferase (TPMT), which is responsible for conversion to non-toxic 6-MP nucleotides. In humans, there is genetic polymorphism that determines high or low levels of TPMT. People with low TPMT activity are more responsive to therapy, but have a high incidence of toxicity (myelosuppression); people with high levels of TPMT activity have low incidence of toxicity but lower efficacy (Lennard et al., 1989). Most of the human population has high TPMT activity, but about 11% have low levels and are more prone to toxicity. In people with low TPMT activity, doses must be lowered. It appears that in dogs, this proportion may be similar with 90% of dogs showing normal TPMT activity and 10% with low (White et al., 1998). Cats, as expected, have low TPMT activity (Foster et al., 1999). Animals that are treated with azathioprine should have their bone marrow function monitored during initial therapy to identify those that may have low TPMT activity so doses can be adjusted accordingly.
Cyclosporine is a fat-soluble, cyclic polypeptide fungal product with potent immunosuppressive activity. It has been an important drug used in humans, primarily to induce immunosuppression in organ transplant patients. This drug binds to a specific cellular receptor on calcineurin and inhibits the T-cell receptor-activated signal transduction pathway. Particularly important are its effects to suppress interleukin-2 (IL-2) and other cytokines, and block proliferation of activated T-lymphocytes. The action of cyclosporine is more specific for T-cells as compared to B-cells. One important advantage in comparison to other immunosuppressive drugs is that it does not cause significant myelosuppression or suppress nonspecific immunity.
Clinical Use. In veterinary medicine, cyclosporine has been used for various immune-mediated conditions (Vaden, 1995). It has suppressed immune-mediated reactions in transplant patients and in patients treated for dermatitis, perianal fistulas, keratoconjunctivitis sicca, faucitis, and immune-mediated anemia. In humans, it has been used successfully for treatment of atopic dermatitis (Camp et al., 1993), and although results of clinical trials are limited, there is evidence for a beneficial effect for atopic dermatitis in dogs at a dose of 5 mg/kg of Neoral (Olivry 2000; Fontaine 1999). The response for autoimmune skin disease has not been encouraging (Rosenkrantz et al., 1989).
Formulations and Pharmacokinetics. The pharmacokinetics of cyclosporine is complicated because of the differences between dosage forms, presence of many metabolites, influence of drug interactions, and variability in oral absorption. The formulation Sandimmune has been used for many years. Although effective, it exhibits variable rate and extent of oral absorption. The newer formulation is a microemulsion called Neoral, which has a much more consistent rate and extent of absorption that is less affected by influences such as feeding. With administration of Neoral, oral absorption is not improved if patients were already showing good absorption with Sandimmune, but poor absorbers of Sandimmune will have higher and more consistent absorption after switching to Neoral.
Cyclosporine is metabolized in the gut and liver to several metabolites. Twenty-five to 30 such metabolites have been identified. The pre-hepatic intestinal enzymes account for significant metabolism of cyclosporine (Wu et al., 1995), and systemic absorption is only 20–30% (Myre et al., 1991). The intestinal metabolism by cytochrome P-450 enzymes and the efflux caused by intestinal p-glycoprotein (p-GP), account for most of the loss in systemic availability after oral administration. One explanation for higher oral absorption from Neoral than Sandimmune formulation is that p-GP is inhibited by Neoral and less cyclosporine is transported back to the intestine (Lown et al., 1997). Drug enzyme inhibitors such as ketoconazole, diltiazem, or the flavonoids in grapefruit juice can inhibit the intestinal enzymes and produce a profound increase in the systemic availability of cyclosporine. For example, 5–10 mg/kg of ketoconazole once daily can decrease the dose of cyclosporine because clearance is reduced by 85% (Myre et al., 1991).
Administration and Monitoring. A common oral dose in people is 5–10 mg/kg/day to achieve targeted whole blood concentrations of 150-400 ng/ml. In people, dosages are adjusted to meet the needs of the individual patient based on clinical response and monitoring trough plasma concentrations. For animals, doses of 10–20 mg/kg/day were frequently cited in the literature, but more recent publications, in which newer formulations have been used, have cited lower doses. For organ transplantation in cats (Mathews and Gregory, 1997) a dose of 3 mg/kg q12h (Neoral formulation; dose was doubled for Sandimmune formulation) was used to achieve blood concentrations of 300–500 ng/ml. At NCSU, we routinely administer 25 mg/cat for suppression of immunity for transplantation, and modify as needed with monitoring. For organ transplant in dogs, 10 mg/kg q12h to achieve concentrations of 500–600 ng/mL has been used (Mathews et al., 2000). A report on treating perianal fistulas in dogs (Mathews and Sukhiani, 1997) found that an average dose of 6 mg/kg q12h was needed to achieve a targeted blood concentration of 400–600 ng/ml. However, this recommendation was later modified to 2.5 to 6 mg/kg/day (3 mg/kg q12h) to achieve an effective blood concentration of 100–300 ng/ml. For treatment of dogs with perianal fistulas, we have achieved adequate blood concentrations, without producing toxicity at a dose of 3 mg/kg q12h. For treating dermatitis in animals, doses of 3–6 mg/kg/day (dogs and cats) have been administered to achieve concentrations of 200–300 ng/mL. Concentrations above 600 ng/mL may increase the risk of toxicity.
Because of wide individual variation in cyclosporine pharmacokinetics, monitoring can be used to determine the optimum dose for each patient. One must be cognizant of the assay used when monitoring cyclosporine. Plasma values will be lower than whole blood assays because as much as 50–60%, and 10–20% of the dose is concentrated in erythrocytes and leukocytes, respectively. Nonspecific assays will report higher values than a more specific (monoclonal or HPLC) assay. Despite the hypothesized higher specificity of the monoclonal assay, important discrepancies between these assays and the more specific HPLC assay have been reported (Steimer 1999). For example, in humans, the difference between HPLC and the commonly used TDx monoclonal immunoassay was 57%. In cats, we found that the TDx assay overestimated the true cyclosporine concentration by a factor of approximately two fold. (That is, TDx assay reporting 500 ng/ml corresponded to an actual value of 250 ng/mL). In dogs, the TDx assay overestimates the true cyclosporine concentrations by a factor of 1.5 to 1.7. When using a specific radioimmunoassay or HPLC true concentrations are measured. But, when using a TDx fluorescence polarization assay (monoclonal whole blood) one must multiply the feline concentrations by 0.5 to get the true concentration, and multiply the canine concentration by 0.6 to get the true concentration.
Adverse Effects and Precautions. Cyclosporine can cause vomiting, diarrhea, anorexia, secondary infections, hair loss (hirsutism in people), and gingival hyperplasia (Vaden, 1995). Tremors or shaking have been observed in dogs administered high doses. Nephrotoxicity, once a problem with older forms, is rare with current formulations. Secondary malignancies are a possible complication to long-term therapy. One reference noted few adverse effects as long as blood concentrations were kept within accepted limits (Mathews and Sukhiani, 1997).
Several drugs may interact with cyclosporine. For example, co-administration of ketoconazole in people to treat secondary fungal infections will decrease metabolism of cyclosporine. Ketoconazole has been used deliberately to reduce the need for cyclosporine in some investigations. Cimetidine, erythromycin, grapefruit juice, and diltiazem also may inhibit cyclosporine metabolism and increase blood concentrations.
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