Interferon-alpha 2a in the treatment of acquired immunodeficiency syndrome-related Kaposi's sarcoma.

In a series of studies, recombinant interferon-alpha 2a (rIFN alpha 2a, Roferon-A) was administered alone (273 men) or combined with vinblastine (91 men) to patients with acquired immunodeficiency syndrome (AIDS)-related Kaposi's sarcoma (KS). Patients were treated with daily doses of rIFN alpha 2a ranging from 3 to 54 million international units (I.U.) administered intramuscularly. A dose of 36 million I.U. daily for approximately 10 weeks followed by a three times weekly maintenance schedule with the same dose resulted in the best overall therapeutic benefit. An escalating-dose regimen of 3, 9, and 18 million I.U. daily, each for 3 days, followed by 36 million I.U. daily, produced equivalent therapeutic benefit with amelioration of acute toxicity in some patients. Response was more likely in patients without a history of opportunistic infection or B symptoms (fever, night sweats, or weight loss). Response rate increased with increasing baseline CD4 lymphocyte count and was 45.5% in patients with a CD4 count of greater than 400/mm3. Responding patients with a CD4 count of greater than 200/mm3 had a distinct survival advantage over patients who had similar CD4 counts but whose tumors did not regress with therapy. The addition of vinblastine increased toxicity and did not improve the response rate or prolong survival. Side effects included fatigue, fever, chills, myalgias, headaches, anorexia, nausea, diarrhea, and dizziness. Mild abnormalities in hematologic and liver function tests occurred in some patients. Most adverse effects diminished or resolved with continued therapy. We conclude that rIFN alpha 2a offers important therapeutic benefit in a select group of patients with AIDS-related KS.

Drug residue formation from ronidazole, a 5-nitroimidazole. I. Characterization of in vitro protein alkylation.

The metabolic activation of [14C]ronidazole by rat liver enzymes to metabolite(s) bound to macromolecules was investigated. The alkylation of protein by [14C]ronidazole metabolite(s) was catalyzed most efficiently by rat liver microsomes, in the absence of oxygen utilizing NADPH as a source of reducing equivalents. Based on a comparison of total ronidazole metabolized versus the amount bound to microsomal protein, approximately one molecule alkylates microsomal protein for every 20 molecules of ronidazole metabolized. Protein alkylation was strongly inhibited by sulfhydryl-containing compounds such as cysteine and glutathione whereas methionine had no effect. Based on HPLC analysis of ronidazole, cysteine was found not to inhibit microsomal metabolism of ronidazole ruling out a decrease in the rate of production of the reactive metabolite(s) as the mechanism of cysteine inhibition.

Drug residue formation from ronidazole, a 5-nitroimidazole. II. Involvement of microsomal NADPH-cytochrome P-450 reductase in protein alkylation in vitro.

Purified liver microsomal NADPH-cytochrome P-450 reductase is able to catalyze the activation of [14C]ronidazole to metabolite(s) which bind covalently to protein. Like the reaction catalyzed by microsomes, protein alkylation catalyzed by the reductase is (1) sensitive to oxygen, (2) requires reducing equivalents, (3) is inhibited by sulfhydryl-containing compounds and (4) is stimulated several fold by either flavin mononucleotide (FMN) or methytlviologen. A cytochrome P-450 dependent pathway of ronidazole activation can be demonstrated as judged by the inhibition of the reaction by carbon monoxide, metyrapone and 2,4-dichloro-6-phenylphenoxyethylamine but the involvement of specific microsomal cytochrome P-450 isozymes has not been definitively established. Milk xanthine oxidase is also capable of catalyzing ronidazole activation. Polyacrylamide sodium dodecyl sulfate (SDS)-gel electrophoresis reveals that the reactive intermediate(s) of ronidazole does not alkylate proteins selectively.

Drug residue formation from ronidazole, a 5-nitroimidazole. III. Studies on the mechanism of protein alkylation in vitro.

Ronidazole (1-methyl-5-nitroimidazole-2-methanol carbamate) is reductively metabolized by liver microsomal and purified NADPH-cytochrome P-450 reductase preparations to reactive metabolites that covalently bind to tissue proteins. Kinetic experiments and studies employing immobilized cysteine or blocked cysteine thiols have shown that the principal targets of protein alkylation ara cysteine thiols. Furthermore, ronidazole specifically radiolabelled with 14C in the 4,5-ring, N-methyl or 2-methylene positions give rise to equivalent apparent covalent binding suggesting that the imidazole nucleus is retained in the bound residue. In contrast, the carbonyl-14C-labeled ronidazole gives approx. 6--15-fold less apparent covalent binding indicating that the carbamoyl group is lost during the reaction leading to the covalently bound metabolite. The conversion of ronidazole to reactive metabolite(s) is quantitative and reflects the amazing efficiency by which this compound is activated by microsomal enzymes. However, only about 5% of this metabolite can be accounted for as protein-bound products under the conditions employed in these studies. Consequently, approx. 95% of the reactive ronidazole metabolite(s) can react with other constituents in the reaction media such as other thiols or water. Based on these results, a mechanism is proposed for the metabolic activation of ronidazole.

Activating enzymes: multiple monooxygenase forms with different substrate preferences as related to toxic effects.

The presence of multiple molecular forms of cytochrome P-450 in experimental animals and perhaps is of considerable importance in dealing with the metabolism of drugs, mutagens, carcinogens and other toxic compounds. Differences in substrate specificity, positional specificity and stereoselectivity of various forms of cytochrome P-450 play an important role in regulating the balance between activation and inactivation pathways of a given chemical. The relative proportions of various forms of cytochrome P-450 in a given tissue or individual may be an important factor in determining the cytotoxic and carcinogenic action of many toxic compounds.

Studies on the association of cytochrome P-450 and NADPH-cytochrome c reductase during catalysis in a reconstituted hydroxylating system.

The interaction between cytochrome P-450 and NADPH-cytochrome c reductase during catalysis has been investigated with a reconstituted monooxygenase system composed of the two purified enzyme components and synthetic phospholipid. Steady state kinetic data are consistent with a scheme in which the formation of a binary complex between the two proteins precedes catalysis. The formation of this binary complex is described by a simple mass action equation. In agreement with this equation, the observed Vmax for benzphetamine N-demethylation was found to be directly proportional to the calculated concentration of the cytochrome P-450 . reductase complex. Furthermore, with appropriate reductase/cytochrome P-450 mole ratios, the Vmax could be shown to be linearly dependent on either the reductase or the cytochrome P-450 concentration alone. In contrast, the Km parameter is independent of the complex concentration, indicating that no change in the rate-limiting step has occurred. Thus a distinction should be made between a rate-limiting enzyme component and the rate-limiting step in this multienzyme system.

Separation, purification, and properties of multiple forms of cytochrome P-450 from the liver microsomes of phenobarbital-treated mice.

Four distinct cytochrome P-450 fractions (A1, A2, C1, and C2) have been separated and purified from the liver microsomes of phenobarbital-treated hybrid mice (B6D2F1/J). Fractions A2 and C2 were highly purified with specific contents of 16.5 and 17.5 nmol of cytochrome P-450/mg of protein, respectively, based on their amino acid compositions. The major hemeprotein bands of A2 and C2 have different minimum molecular weights (50,000 and 56,000, respectively) on polyacrylamide gels in the presence of sodium dodecyl sulfate. All four fractions with respect to their spectral and catalytic properties, thereby demonstrating that mouse liver microsomes from phenobarbital-treated hybrid mice contain at least four forms of cytochrome P-450.

Multiple forms of rat liver cytochrome P-450. Immunochemical evidence with antibody against cytochrome P-448.

Purified hepatic cytochrome P-448 from 3-methylcholanthrene-treated rats was used to produce antibody in rabbits. The cytochrome P-448 antibody (IgG fraction) isolated from immune rabbit serum is quite specific and precipitates purified rat liver cytochrome P-448 at low antibody to protein ratios when assayed by the Ouchterlony double diffusion technique. Purified hepatic cytochrome P-450 from phenobarbital-treated rats cross-reacts poorly with the cytochrome P-448 antibody as do purified rabbit hepatic cytochrome P-448 and P-450. No cross-reaction is observed with purified cytochrome P-450 from beef adrenal mitochondria or from Pseudomonas putida in Ouchterlony double diffusion experiments. The cytochrome P-448 antibody produces a single distinct precipitin band with purified rat cytochrome P-448. In contrast, purified liver cytochrome P-450 from phenobarbital-treated rats gives three precipitin bands, all of which contain hemeprotein as judged by benzidine staining. At least two of the three precipitin bands are immunochemically different from the precipitin band formed with cytochrome P-448. When added to the reconstituted system, the cytochrome P-448 antibody inhibits purified rat cytochrome P-448- and P-450-supported N-demethylation of benzphetamine, O-deethylation of ethoxycoumarin, hydroxylation of benzo[a]pyrene, and the hydroxylation of testosterone at the 6beta, 7alpha, and 16alpha positions. Antibody inhibits cytochrome P-448-supported metabolism more than cytochrome P-450-supported metabolism except for benzo[a]pyrene hydroxylation at low antibody to hemeprotein ratios. In addition, the pattern and extent of inhibition of the cytochrome P-450 system depends on the substrate used, suggesting that multiple forms of the hemeprotein are present in the purified preparation from phenobarbital-treated rats. The observed patterns of immunoprecipitation and inhibition of catalytic activity indicate that (a) cytochrome P-448 from 3-methylcholanthrene-treated rats is immunochemically different from cytochrome P-450 from phenobarbital-treated rats, and (b) there appear to be at least three hemeprotein forms in the purified cytochrome P-450 preparation from phenobarbital-treated rats.