Remission induction therapy of untreated acute myeloid leukemia using a non-cytarabine-containing regimen of idarubicin, etoposide, and carboplatin.

BACKGROUND: The safety and efficacy of idarubicin, etoposide, and carboplatin as remission induction therapy for patients younger than 60 years with untreated acute myeloid leukemia was studied as an alternative to standard regimens based on cytarabine plus anthracycline. METHODS: Eligible patients received idarubicin (36-40 mg/m2), etoposide (500 mg/m2), and carboplatin (1000-1500 mg/m2) over 5 days. Those who achieved complete remission received a single course of cytarabine 1.5 gm/m2 every 12 hours for a total of 12 doses. D-xylose absorption was studied as a marker for cytotoxic therapy-induced gut mucosal damage. Cytogenetic and immunophenotyping studies were performed at the time of diagnosis and examined for prognostic importance. RESULTS: Remission was achieved in 29 (67%) of 43 patients with a single induction course. The median leukemia free and overall survival times were 15.4 months (95% CI 6.5-24.2) and 12.5 months (95% CI 5.9-19.1), respectively. Induction mortality was 14%. Karyotype (normal, simple, or complex vs. very complex) was the strongest predictor of remission (79% vs. 25%, P=0.01), leukemia free survival (odds ratio [OR] 19.3, 95% CI 2.7-138.9), and overall survival (OR 5.4, 95% CI 2.1-13.9). Dose-limiting gut mucosal toxicity was greatest during Weeks 2 and 3. Bloodstream infections occurred in 49% of patients at a median of 12 days. Grade 3-4 diarrhea, nausea, stomatitis, esophagitis/dysphagia, and vomiting developed in 33%, 26%, 23%, 9%, and 2% of cases, respectively, at a median of 17, 16, 11, 15.5, and 21 days, respectively. CONCLUSIONS: This regimen was active in adults younger than 60 years with untreated acute myeloid leukemia and normal, simple, or complex karyotypes. Remission duration was confounded by karyotype. Mucosal toxicity limited the tolerability of this regimen. These adverse effects might be overcome by increasing the intensity of postremission therapy and modifying the dosing schedule.

Therapy of untreated acute myeloid leukemia in the elderly: remission-induction using a non-cytarabine-containing regimen of mitoxantrone plus etoposide.

PURPOSE: The University of Manitoba Adult Acute Leukemia Study Group sought to examine the safety, efficacy, and impact on quality of life of a non-cytarabine-containing remission-induction regimen followed by intermediate-dose cytarabine (IDARA-C) postremission therapy for the management of untreated acute myeloid leukemia (AML) in patients age 60 to 80 years. PATIENTS AND METHODS: Eligible patients received mitoxantrone 10 mg/m2 and etoposide 100 mg/m2 on days 1 to 5. Complete remitters received a single course of cytarabine 0.5 mg/m2 every 12 hours on days 1 to 6. Cytogenetic and immunophenotyping studies were performed at diagnosis and were examined for prognostic importance. The Functional Living Index-Cancer (FLI-C) was used in the longitudinal assessment of quality of life. RESULTS: A total of 37 (55%) of 67 eligible patients achieved remission, 34 (92%) of whom did so with a single course. The induction mortality rate was 12%. The median disease-free and overall survival times were 8.4 and 9.2 months, respectively. CD34 stem-cell phenotype, poor performance status, and high cytogenetic complexity score were independent covariates of failure to achieve remission. Very complex karotype combined with CD34 stem-cell phenotype to predict induction death in 67% of cases (P = .0003). Cytotoxic therapy-related gut epithelial damage was maximal during weeks 2 and 3 of therapy. Complete remitters and partial responders exhibited significantly improved global FLI-C scores following completion of therapy. CONCLUSION: Mitoxantrone plus etoposide was an effective and well-tolerated first-line induction regimen for AML in the elderly that should be studied further in comparison to the standard cytarabine/anthracycline-based therapy.

Structural studies on bovine spleen heme oxygenase. Immunological and structural diversity among mammalian heme oxygenase enzymes.

Heme oxygenase is an Mr 32,000 microsomal enzyme which catalyzes the rate-limiting step in the oxidative catabolism of heme to yield equimolar quantities of biliverdin IX alpha, carbon monoxide, and iron. In the present investigation, evidence is presented suggesting that immunochemical and structural differences exist between bovine spleen heme oxygenase and heme oxygenase enzymes from other mammalian species. Using an antibody directed against bovine spleen heme oxygenase, enzyme-linked immunosorbent assays, Western blotting experiments, and cell-free translation immunoprecipitation studies showed that bovine spleen heme oxygenase is only weakly immunochemically related to heme oxygenase from rat spleen. This observation was supported by the fact that a rat spleen heme oxygenase cDNA probe did not hybridize significantly to bovine spleen heme oxygenase mRNA in Northern analyses nor to restriction fragments containing the bovine heme oxygenase gene in Southern analyses. Tryptic peptides were prepared from bovine spleen heme oxygenase and the amino acid sequences of nine peptides comprising 94 amino acid residues were determined, providing the first information on the primary structure of bovine spleen heme oxygenase. Comparison of the sequences of these tryptic peptides with regions of the deduced amino acid sequences of rat spleen and human macrophage heme oxygenase revealed sequence similarities ranging from 55 to 100%. Several peptides displaying the highest degree of sequence similarity were found to occur in regions of the heme oxygenase molecule postulated to contain the heme binding site, indicating that despite the immunochemical and apparent structural differences between bovine spleen heme oxygenase and the rat and human enzymes, functionally important amino acid residues have been conserved in the evolution of mammalian heme oxygenase genes.

Alterations in microsomal drug metabolism and heme oxygenase activity in isolated hepatic parenchymal and sinusoidal cells in Murphy-Sturm lymphosarcoma-bearing rats.

Previous studies have demonstrated that Murphy-Sturm lymphosarcoma-bearing rats have significantly decreased hepatic microsomal cytochrome P-450 and NADPH-cytochrome c reductase activity, a consequent decreased capacity for oxidative microsomal drug metabolism, and increased microsomal heme oxygenase activity. Splenic microsomes obtained from tumor-bearing rats did not display similar changes. To define the cellular locus of these changes, preparations of hepatic parenchymal and hepatic sinusoidal cells were obtained from control and Murphy-Sturm lymphosarcoma-bearing rats and examined for alterations in microsomal parameters of drug metabolism and heme oxygenase. In hepatic parenchymal cell populations, heme oxygenase activity was significantly increased in tumor-bearing rats (0.046 nmol/mg/min vs. 0.019 nmol/mg/min, control, p less than 0.01) while other microsomal parameters were all significantly decreased in activity (cytochrome P-450, 0.25 nmol/mg vs 0.70 nmol/mg, control, p less than 0.001; NADPH-cytochrome c reductase, 24.4 nmol/mg/min vs 42.6 nmol/mg/min, control, p less than 0.005; benzo(a) pyrene hydroxylase, 0.230 nmol/mg/min vs. 0.518 nmol/mg/min, control, p less than 0.001). These results are similar to those obtained with microsomes from whole liver. Conversely, while hepatic sinusoidal cell preparations also demonstrated increased heme oxygenase activity (0.134 nmol/mg/min vs 0.079 nmol/mg/min, control, p less than 0.01), all other hepatic sinusoidal cell microsomal parameters were not appreciably altered in tumor-bearing animals. The results indicate that the major effect of the tumor-bearing state on hepatic microsomal heme and drug metabolism is on the parenchymal cell, and not the sinusoidal cell population.

Methene bridge carbon atom elimination in oxidative heme degradation catalyzed by heme oxygenase and NADPH-cytochrome P-450 reductase.

Physiological heme degradation is mediated by the heme oxygenase system consisting of heme oxygenase and NADPH-cytochrome P-450 reductase. Biliverdin IX alpha is formed by elimination of one methene bridge carbon atom as CO. Purified NADPH-cytochrome P-450 reductase alone will also degrade heme but biliverdin is a minor product (15%). The enzymatic mechanisms of heme degradation in the presence and absence of heme oxygenase were compared by analyzing the recovery of 14CO from the degradation of [14C]heme. 14CO recovery from purified NADPH-cytochrome P-450 reductase-catalyzed degradation of [14C]methemalbumin was 15% of the predicted value for one molecule of CO liberated per mole of heme degraded. 14CO2 and [14C]formic acid were formed in amounts (18 and 98%, respectively), suggesting oxidative cleavage of more than one methene bridge per heme degraded, similar to heme degradation by hydrogen peroxide. The reaction was strongly inhibited by catalase, but superoxide dismutase had no effect. [14C]Heme degradation by the reconstituted heme oxygenase system yielded 33% 14CO. Near-stoichiometric recovery of 14CO was achieved after addition of catalase to eliminate side reactions. Near-quantitative recovery of 14CO was also achieved using spleen microsomal preparations. Heme degradation by purified NADPH-cytochrome P-450 reductase appeared to be mediated by hydrogen peroxide. The major products were not bile pigments, and only small amounts of CO were formed. The presence of heme oxygenase, and possibly an intact membrane structure, were essential for efficient heme degradation to bile pigments, possibly by protecting the heme from indiscriminate attack by active oxygen species.

Mechanism of action of heme oxygenase. A study of heme degradation to bile pigment by 18O labeling.

The formation of bile pigment from heme by a reconstituted heme oxygenase system containing purified bovine spleen heme oxygenase, NADPH-cytochrome P-450 reductase, and biliverdin reductase was studied under an atmosphere containing 18,18O2. The product, bilirubin, was isolated and subjected to mass spectrometry, which revealed incorporation of 18O consistent with a two-molecule mechanism, whereby the product bile pigment contains oxygen atoms derived from two different oxygen molecules.

Alterations in hepatic heme and cytochrome P-450 metabolism in Murphy-Sturm lymphosarcoma-bearing rats. Implications for drug metabolism.

Previous studies have shown that tumor-bearing rats have significantly decreased hepatic microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity with, consequently, significantly decreased capacity for microsomal oxidative drug metabolism. Subsequent investigations have revealed that the rates of hepatic cytochrome P-450 apo-protein synthesis and degradation are decreased significantly and hepatic microsomal heme oxygenase activity is increased significantly in rats bearing an extra-hepatic tumor. Further studies have been done to attempt to clarify the pathogenesis and significance of these observations. Hepatic delta-aminolevulinic acid (ALA) synthetase activity in male Wistar rats declined to a nadir of 162 +/- 34 (S.E.) pmoles ALA per mg protein per 30 min 6 days following i.m. transplantation of Murphy-Sturm lymphosarcoma (vs control = 218 +/- 36 pmoles per mg per 30 min). Turnover of 3H-labeled heme in microsomal CO-binding particles (i.e. cytochrome P-450 heme) was increased significantly 8 days following i.m. transplantation of Murphy-Sturm lymphosarcoma with a T 1/2 of 5.5 hr for the fast phase of hepatic cytochrome P-450 heme disappearance in tumor-bearing rats as compared with a T 1/2 of 7 hr in control rats. Hepatic cytochrome P-450 apo-protein concentration was slightly, but not significantly, increased in Murphy-Sturm lymphosarcoma-bearing rats as compared with control rats up to 10 days following tumor transplantation. These results suggest that, in Murphy-Sturm lymphosarcoma-bearing rats, decreased microsomal cytochrome P-450 concentration is the result of both decreased cytochrome P-450 apo-protein synthesis and increased cytochrome P-450 heme turnover. Apo-cytochrome P-450 concentration was not appreciably altered because increased cytochrome P-450 heme turnover and decreased cytochrome P-450 apo-protein degradation were balanced by decreased cytochrome P-450 apo-protein synthesis. Because of their effects on cytochrome P-450 concentration and action, these alterations in heme and hemoprotein metabolism may be of importance in regulating oxidative drug metabolism in the tumor-bearing state.

Effect of cholestasis produced by bile duct ligation on hepatic heme and hemoprotein metabolism in rats.

Cholestasis produced by bile duct ligation has been associated with decreased concentrations of hepatic microsomal cytochrome P450 and decreased hepatic microsomal oxidative drug metabolism. Bile duct ligation producing cholestasis results in a marked increase in hepatic microsomal heme oxygenase activity, with corresponding decreases in hepatic microsomal cytochrome P450 concentration, reduced form of nicotinamide adenine dinucleotide phosphate-cytochrome c reductase activity, and hepatic delta-aminolevulinic acid synthetase activity. As sham-operated rats also demonstrate a less prolonged decrease in cytochrome P450 concentration and reduced form of nicotinamide adenine dinucleotide phosphate-cytochrome c reductase activity, the metabolic effects of surgery and anesthesia must also be involved in these alterations in microsomal oxidative drug metabolism. The relative rate of hepatic cytochrome P450 synthesis and of degradation are both decreased after bile duct ligation. These data suggest that decreased hepatic microsomal cytochrome P450 concentrations in cholestasis are partly the result of decreased cytochrome P450 synthesis. Increased levels of heme oxygenase activity are not related to increased cytochrome P450 turnover, but may instead reflect enlargement and increased catabolism of a free heme pool resulting from decreased hemoprotein (cytochrome P450) synthesis.

The catalysis of heme degradation by purified NADPH-cytochrome C reductase in the absence of other microsomal proteins.

The report by Schacter et al. (J Biol Chem 247: 3601, 1972) that an antibody to NADPH-cytochrome c oxidoreductase inhibited NADPH-cytochrome c reductase and heme oxygenase activities in rat and pig liver and spleen microsomes demonstrated the role of this flavoprotein in microsomal heme oxygenation. Recent studies from other laboratories (Yoshida et al., J Biochem 75, 1187: 1974 and Bissell et al., Fed Proc 33: 1246, 1974) have strongly suggested that cytochrome P-450 is not involved in heme oxygenation. The availability of a homogeneous preparation of NADPH-cytochrome c reductase prompted us to test heme oxygenase activity in a system devoid of hemoprotein contamination. NADPH-cytochrome c reductase catalyzed biliverdin formation at a rate of 8.26 +/- 0.5 SEM nmole min-1mg-1 in the absence of biliverdin reductase. The rate of bilirubin formation in the presence of biliverdin reductase was less than 10% of the rate of biliverdin formation, suggesting that mixture of biliverdin isomers may be produced. Biliverdin production was potently (70--80%) inhibited by catalase, but was unaffected by superoxide dismutase. Epinephrine also inhibited heme oxygenation, presumably by utilizing O2. required for the formation of H2O2 by the reductase. By extrapolation, the NADPH oxidase activity due to NADPH-cytochrome c reductase can account for heme degradation occurring in microsomes. However, the specificity of ring scission at the IXalpha position must be due to another microsomal protein, perhaps the heme oxygenase of Yoshida et al., and not cytochrome P-450.

Human spleen heme oxygenase in normal, hemolytic and other pathological states.

Heme oxygenase (HO), the primary enzyme responsible for heme catabolism, was measured in spleens from 10 normal subjects, 14 patients with chronic hemolytic anemia (HA), 12 with idiopathic thrombocytopenic purpura (ITP), and 10 with various lymphoproliferative disorders (LD) to determine the splenic enzymatic capacity for heme catabolism and the response of splenic HO to hemolysis. Splenic NADPH-cytochrome c reductase activity and cytochrome P-450 levels were also measured. Splenic HO specific activity in normal spleens was 0.235 +/- 0.106 (SE) nmoles bilirubin/mg protein/min; in HA, 0.276 +/- 0.050; in ITP, 0.228 +/- 0.036; and in LD, 0.420 +/- 0.105. However, the total HO activity per spleen was significantly greater in HA (742.9 +/- 137.4 (SE) nmoles/min, p less than 0.001) and LD (681.9 +/- 180.3, p less than 0.005) than in normal spleens (137.1 +/- 55.0), but was not significantly increased in ITP (269.5 +/- 121.5). In normal spleens cytochrome P-450 was 0.052 +/- 0.006 (SE) nmoles/mg and NADPH-cytochrome c reductase activity was 8.3 +/- 2.2 (SE) nmoles/mg/min; neither of these specific activities was significantly altered in HA, ITP, or LD. Again, total activity was significantly increased in HA and LD associated with increased splenic size. Although total HO activity is greater in the larger spleens, HO activity does not increase per unit weight of tissue. In normal spleen the calculated capacity for bilirubin production by HO was 115 mg per day. This accounts for only 50% of normal daily production from erythroid sources and suggests that other sites are of major importance for hemoglobin degradation.