The Molecular Effects of Acrolein

Cyclophosphamide is an extensively used anticancer and immunosuppressive agent. It is a prodrug undergoing a complicated process of metabolic activation and inactivation. Technical difficulties in the accurate determination of the cyclophosphamide metabolites have long hampered the assessment of the clinical pharmacology of this drug. As these techniques are becoming increasingly available, adequate description of the pharmacokinetics of cyclophosphamide and its metabolites has become possible. There is incomplete understanding on the role of cyclophosphamide metabolites in the efficacy and toxicity of cyclophosphamide therapy. However, relationships between toxicity (cardiotoxicity, veno-occlusive disease) and exposure to cyclophosphamide and its metabolites have been established. Variations in the balance between metabolic activation and inactivation of cyclophosphamide owing to autoinduction, dose escalation, drug-drug interactions and individual differences have been reported, suggesting possibilities for optimisation of cyclophosphamide therapy. Knowledge of the pharmacokinetics of cyclophosphamide, and possibly monitoring the pharmacokinetics of cyclophosphamide in individuals, may be useful for improving its therapeutic index.

Clinical Pharmacokinetics of Cyclophosphamide

Cyclosphosphamide is widely used for cancer chemotherapy and for immunosuppression. The parent compound is inactive in vitro and exerts its biological activities through metabolites generated by hepatic microsomal enzymes. The drug may be administered either parenterally or orally. Systemic availability after oral administration is greater than 75% at the low doses which have been studied. Cyclophosphamide is minimally protein bound but some of its metabolites are more than 60% protein bound.

Summary The human pharmacology of cyclophosphamide was investigated in 26 patients who received cyclophosphamide 14 C in doses of 6 to 80 mg/kg i.v. Levels of the intact drug in plasma and urine and excretion of 14 C label in breath and stools were determined by liquid scintillation counting. Plasma and urine alkylating activity was measured by reaction with 4-(4-nitrobenzyl)pyridine. Protein binding of cyclophosphamide and plasma alkylating metabolites were determined by plasma ultrafiltration. Injected cyclophosphamide distributed rapidly into 64% of body weight, and plasma cyclophosphamide half-life in patients without prior drug exposure was 6.5 hr. Not more than 20% of injected cyclophosphamide was excreted intact in urine at any dose level. Plasma alkylating metabolites were 56% bound to plasma proteins. After a 40-mg/kg dose, peak unbound alkylating activity averaged 13.3 mµmoles/ml, and in most patients at this dose alkylating activity in the plasma was measurable for 24 hr. Sixty-eight % of injected 14 C label was excreted in urine. Breath and fecal excretion were negligible. In a regimen of five consecutive daily cyclophosphamide administrations, cyclophosphamide half-life was shorter and peak alkylating levels were constantly higher on the 5th day than on the 1st day. Prior patient treatment with allopurinol resulted in significantly longer cyclophosphamide half-life, but concomitant prednisolone treatment had no effect. The effect of hepatic metastases on cyclophosphamide metabolism was unclear. Moderate renal failure in one patient resulted in prolonged retention of alkylating materials in plasma and severe toxicity. Although patients with and without prior exposure to microsomal enzyme-inducing drugs demonstrated marked variation in plasma cyclophosphamide half-life and peak alkylating levels, the total concentration × time product remained relatively constant for a given cyclophosphamide dose, suggesting that alterations in the rate of cyclophosphamide metabolism by drugs or liver metastases in the absence of renal failure will not change toxicity or therapeutic effect.

https://cancerres.aacrjournals.org/content/canres/33/2/226.full.pdf

Clinical pharmacology of cyclophosphamide.

Metabolism of oxazaphosphorines

Experiments were designed to investigate the metabolism of cyclophosphamide in vitro and in vivo following the administration of known stimulators and depressors of rat hepatic microsomal mixed-function oxidase activity, the antitumor efficacy of cyclophosphamide as a function of its metabolism, and the toxicity of cyclophosphamide as a function of its metabolism. Regarding the metabolism of cyclophosphamide by hepatic microsomal preparations, the following observations were made. ( a ) Pretreatment with Phenobarbital of male and female rats and of female mice increased the rate of metabolism 7-, 23-, and 7-fold, respectively. ( b ) pretreatment of male rats with 3-methylcholanthrene depressed metabolism to 33% of that of controls, but similar pretreatment of female mice did not alter the rate of metabolism. ( c ) The Km for cyclophosphamide metabolism by microsomes obtained from male rat liver changed from 1.39 mm to 0.57 and 0.58 mm after phenobarbital and 3-methylcholanthrene pretreatment, respectively. ( d ) Pretreatment of male rats with thioacetamide, morphine, or cobalt chloride depressed metabolism to 12, 48, and 8% of control, respectively. ( e ) Male rats bearing the Walker 256 carcinosarcoma i.m. showed a depressed ability to metabolize cyclophosphamide. In vivo cyclophosphamide metabolism in rats paralleled in vitro metabolism. Thus, at early time points, blood levels of alkylating activity were ( a ) increased following pretreatment with phenobarbital, ( b ) decreased following pretreatment with 3-methylcholanthrene or cobalt chloride, ( c ) decreased when the animal bore the Walker 256 carcinosarcoma i.m., and ( d ) lower in female compared with male rats. Walker 256 carcinosarcoma cells grown i.m. in the hindlegs of male and female rats were used to evaluate therapeutic efficacy. The dose of cyclophosphamide that inhibits tumor growth in male rats by 50% was 0.7 mg/kg. Similar median effective doses were obtained in female rats and in male rats pretreated with phenobarbital or cobalt chloride. 3-Methylcholanthrene pretreatment increased the median effective dose of cyclophophamide to 3.5 mg/kg. For estimation of the toxicity of cyclophosphamide, blood leukocyte counts were made at various intervals following injection of cyclophosphamide. Little difference in the decline of the number of leukocytes or in their subsequent return to normal levels was observed between control male rats; female rats; and phenobarbital-, 3-methylcholanthrene-, or cobalt chloride-pretreated male rats. Pretreatment with phenobarbital did accelerate leukocyte depression and also increased the magnitude of depression. In methylcholanthrene-pretreated male rats and in female rats, the magnitude of depression was somewhat less and recovery rates were somewhat altered. Administered by itself, phenobarbital, 3-methylcholanthrene, or cobalt chloride had no effect on tumor growth or blood leukocyte levels. The data demonstrate the futility of trying to improve the therapeutic efficacy of cyclophosphamide by pretreatment with drugs that alter its rate of activation. In addition, the data provide a rational basis for the ineffectiveness of such an effort.

https://cancerres.aacrjournals.org/content/canres/32/3/535.full.pdf

N. E. Sladek

Papers

Therapeutic Efficacy of Cyclophosphamide as a Function of Its Metabolism