Production of the antitumor drug epirubicin (4'-epidoxorubicin) and its precursor by a genetically engineered strain of Streptomyces peucetius.

A fermentation method that bypasses the low-yielding semisynthesis of epirubicin (4'-epidoxorubicin) and 4'-epidaunorubicin, important cancer chemotherapy drugs, has been developed for Streptomyces peucetius. This bacterium normally produces the anthracycline antibiotics, doxorubicin and daunorubicin; the 4'-epimeric anthracyclines are formed by introducing the heterologous Streptomyces avermitilis avrE or Saccharopolyspora eryBIV genes into an S. peucetius dnmV mutant blocked in the biosynthesis of daunosamine, the deoxysugar component of these antibiotics. Product yields were enhanced considerably by replacing the chromosomal copy of dnmV with avrE and by introducing further mutations that can increase daunorubicin and doxorubicin yields in the wild-type strain. This method demonstrates that valuable hybrid antibiotics can be made by combinatorial biosynthesis with bacterial deoxysugar biosynthesis genes.

New biosynthetic anthracyclines related to barminomycins incorporating barbiturates in their moiety.

Three new anthracyclines, FCE 21424 (2), FCE 24366 (3) and FCE 24367 (4), were isolated from culture broths of Streptomyces peucetius and its mutant strains after addition of sodium barbiturates during the fermentation. Structural assignment, achieved through spectroscopic and degradative studies, that the new anthracyclines had a common barminomycin-like structure incorporating different barbiturate moieties. The new anthracyclines were found to display outstanding cytotoxicity and remarkable potency "in vivo" against P388 ascitic leukemia.

13-Deoxycarminomycin, a new biosynthetic anthracycline.

A new antitumor antibiotic, 13-deoxycarminomycin, has been isolated from the anthracycline complex produced by Streptomyces peucetius var. carminatus (ATCC 31502), a biochemical mutant of Streptomyces peucetius var. caesius, the doxorubicin-producing microorganism. The new anthracycline, showing antibacterial and cytotoxic activity in vitro, was found active against P-388 murine leukemia.

Separation, characterization, and analysis of epirubicin (4'-epidoxorubicin) and its metabolites from human urine.

Three metabolites of the new antitumor anthracycline epirubicin (4'-epidoxorubicin, 4'-epiDX) detected by HPLC in urine of four patients treated with 75 mg/m2 of the drug, were isolated and identified. In an initial step, fluorescent metabolites present in urine, pooled during the first 24 hr after drug administration, were adsorbed on a column of a styrene divinylbenzene adsorbent. The subsequent gradient elution with aqueous methanol, followed by extraction with 1-butanol at different pH values, allowed separation of 4'-epiDX and its 13-dihydro derivative from the more polar metabolites. Each anthracycline was subsequently separated by reverse phase liquid chromatography, characterized by acid and enzymatic hydrolyses, and studied with chemical-physical analysis. In addition to the parent drug, its 13-dihydro derivative and 4'-O-beta-D-glucuronyl-4'-epiDX which were structurally characterized, a glucuronide conjugate of 13-dihydro-4'-epiDX was identified. In four patients analyzed, approximately 12% of the total administered dose was estimated, by reverse phase HPLC and fluorescence detection, to be excreted in the 0-24-hr pooled urine. 4'-EpiDX accounted for 57% of the total excreted fluorescent anthracyclines, its 4'-O-beta-D-glucuronide for 32%, 13-dihydro-4'-epiDX for 7%, and its glucuronide for 3%, average values.

New anthracycline glycosides: 4-O-demethyl-11-deoxydoxorubicin and analogues from Streptomyces peucetius var. aureus.

The new anthracyclines 4-O-demethyl-11-deoxydoxorubicin, 4-O-demethyl-11-deoxydaunorubicin along with its 13-dihydro and 13-deoxo analogues are the main components of the anthracycline complex produced by cultures of Streptomyces peucetius var. aureus. They were isolated by solvent partition, separated by column chromatography and characterized by chemical and physical methods. Among these new anthracyclines, displaying antibacterial and cytotoxic activity "in vitro", 4-O-demethyl-11-deoxydoxorubicin and the corresponding daunorubicin analogue were also active against experimental tumors.

New anthracycline glycosides from Micromonospora. II. isolation, characterization and biological properties.

Four glycosides, designated A, B, C and D, are the main components of the anthracycline complex produced by cultures of Micromonospora sp. nov. They were extracted by solvent partition, separated by column chromatography and characterized by chemical and physical methods as 11-deoxy analogues of daunorubicin. Among these new anthracyclines, displaying antibacterial and cytotoxic activity in vitro, 11-deoxydaunorubicin and 11-deoxydoxorubicin are also active against P388 leukemia in mice.