Biological effects of acetomycin. I. Activity against tumor cells in vitro and in vivo.

The antibiotic acetomycin was active in vitro against HCT-8 human colon adenocarcinoma cells (IC50, 1.5 microgram/ml) and L1210 murine leukemia cells (IC50, 2.2 micrograms/ml). Acetomycin also had marked activity in the human tumor stem cell assay, with a 33% overall response rate (less than or equal to 30% survival) against 49 primary tumors. However, acetomycin was inactive in four in vivo tumor assay systems (L1210 and P388 leukemias, B16 melanoma and the MX-1 mammary xenograft system). This lack of in vivo activity may result from metabolic inactivation of acetomycin.

Anthrapyrazole anticancer agents. Synthesis and structure-activity relationships against murine leukemias.

Chromophore modification of the anthracenediones related to mitoxantrone in an attempt to provide agents with diminished or no cardiotoxicity has resulted in a novel class of DNA binders, the anthrapyrazoles. Their synthesis was carried out by a two-stage condensation sequence starting from requisite 1,4- or 1,5-dichloro-9,10-anthracenedione precursors. Reaction with a monoalkylhydrazine gave a chloroanthrapyrazole intermediate whose subsequent condensation with primary or secondary alkylamines provided the target "two-armed" anthrapyrazoles. A-ring 7,10-dihydroxy anthrapyrazoles were derived from amine condensation with intermediate 5-chloro-7,10-dihydroxyanthrapyrazoles or, alternatively, from intermediate 5-chloro-7,10-bis(benzyloxy)anthrapyrazoles followed by hydrogenolysis of the benzyl protecting groups to provide the target compounds. Potent in vitro activity was demonstrated against murine L1210 leukemia in vitro (IC50 = 10(-7)-10(-8) M) as well as against P388 leukemia in vivo over a wide range of structural variants. In general, activity against the P388 line was maximized by basic side chains at N-2 and C-5, two to three carbon spacers between proximal and distal nitrogens of the side chain, and A-ring hydroxylation. Besides having curative activity against the P388 line, the more active compounds were curative against murine B-16 melanoma in vivo. On the basis of their exceptional in vivo anticancer activity, A-ring dihydroxy compounds 71 and 74 reported in this study have been selected for development toward clinical trials.

Biochemical pharmacology and experimental chemotherapy studies with guanine-7-oxide, a novel purine antibiotic.

1. Guanine-7-oxide is a novel purine antibiotic produced by a Streptomyces species, ATCC 39364. 2. Guanine-7-oxide is cytotoxic to murine and human leukemia cells in vitro at sub-micromolar concentrations. Murine and human carcinoma cells are much less sensitive. 3. Guanine-7-oxide has significant in vivo antitumor activity, particularly against the intraperitoneal and subcutaneous L1210 leukemia systems. 4. Guanine-7-oxide, at highly cytotoxic concentrations, has little effect on biosynthesis of RNA and DNA. 5. There is preliminary evidence for an early effect of guanine-7-oxide on cellular protein synthesis. 6. Guanine, guanosine and hypoxanthine protect cells from the cytotoxicity of guanine-7-oxide. 7. Activation of guanine-7-oxide requires the presence of the enzyme hypoxanthine-guanine phosphoribosyltransferase in the target cells. 8. Cytotoxic concentrations of guanine-7-oxide do not cause depletion of cellular guanine nucleotides during a two hr incubation period. 9. Guanine-7-oxide is converted within mouse and human cells to a metabolite with chromatographic mobility corresponding to a ribonucleoside 5'-triphosphate.

In vitro activity of the novel antitumor antibiotic fostriecin (CI-920) in a human tumor cloning assay.

A human tumor cloning assay was utilized to evaluate the antineoplastic activity of the novel antitumor antibiotic fostriecin (CI-920). Initial screening with 10.0 mcg/ml continuous exposure against a variety of histologic tumor types resulted in 14/51 (27%) in vitro responses (defined as greater than 50% decrease in TCFUs). Further investigation of the compound was performed in 1-hr preincubation experiments. The in vitro response rate at a concentration of 1.0 mcg/ml (which was considered to correspond to a clinically achievable concentration) was 15/43 (35%). Response rates for specific tumor types included: 5/15 in ovarian cancer, 5/12 in breast, and 4/11 in human lung cancer. The impression of significant antitumor activity of the compound at this dose was further substantiated by comparing its in vitro activity with a variety of simultaneously tested standard anticancer agents. In addition, these data indicated the possibility of non-cross resistance of CI-920 to several established cytostatics. CI-920 is a compound with good in vitro activity which should be further developed for clinical trials.

Biological and biochemical activities of the novel antitumor antibiotic PD 114,759 and related derivatives.

A complex of novel and exceptionally potent antibiotics has been evaluated for antitumor activity in vitro and in vivo and characterized with regard to their ability to cause DNA strand scission. The major component, PD 114,759, was quite active against all in vitro tumor systems including the human tumors, MCF-7 breast, HCT-8 colon, and A549 lung and the murine tumors M16/c mammary, Lewis lung, Pan 02 pancreas and L1210 leukemia. ID50 values ranged from 2-57 pg/ml. In vivo this agent produced significant increases of host life spans in mice bearing L1210 leukemia, B16 melanoma and the M5076 sarcoma. Further, it inhibited growth of subcutaneous implants of the Ridgway osteogenic sarcoma by 80% and growth of the MX-1 human mammary xenograft by 90-95%. PD 114,759, however, had no activity against the colon adenocarcinoma 11a or mammary adenocarcinoma 16c. Chinese hamster ovary cells exposed for 24 hours to concentrations of PD 114,759 ranging from 18 to 37 pg/ml accumulated in the S and G2+M phases of the cell cycle with a corresponding decrease in G1. Higher concentrations of drug apparently stopped any progression through the cell cycle. PD 114,759 caused significant DNA single strand breaks in L1210 cells exposed for 1 hour to drug concentrations as low as 20 pg/ml and the frequency of these lesions increased in proportion to the drug concentration. A portion of these DNA breaks appeared to be associated with protein. In contrast, no double strand DNA breaks were detected at the highest drug concentration tested (100 pg/ml).

Anticancer activity of the structurally novel antibiotic Cl-920 and its analogues.

Cl-920 is a structurally novel phosphate ester antibiotic that contains an unsaturated lactone and a conjugated triene system. It has potent antileukemic activity in mice. At doses of 25 mg/kg given i.p. once daily for 5 days to mice bearing approximately 10(7) L1210 leukemia cells, Cl-920 is curative in about 10% of the mice. Life span increases in noncured mice are typically in excess of 150%. The unsaturated lactone and phosphate ester moieties are required for activity against L1210 leukemia. Ring hydroxylation or removal of the terminal hydroxyl group have only modest effects on activity. Schedule studies suggest that prolonged exposure to low levels of Cl-920 is considerably more toxic than is daily or intermittent administration. Daily administration produces optimal activity against L1210 leukemia. Administration i.p. and i.v. of Cl-920 produce roughly equal toxicity and equal activity against an i.p. implant of L1210 leukemia. Cl-920 is inactive when given p.o. or s.c. Cl-920 failed to show confirmed activity against the following tumors in mice: M5076 sarcoma, B16 melanoma, and Ridgway osteogenic sarcoma. The lack of solid tumor activity in mice may be caused by a transport deficiency similar to that found with methotrexate.

Adenosine deaminase inhibitors. Synthesis and biological evaluation of (+/-)-3,6,7,8-tetrahydro-3-[(2-hydroxyethoxy)methyl]imidazo[4,5-d] [1,3]diazepin-8-ol and some selected C-5 homologues of pentostatin.

The synthesis of several analogues of (8R)-3-(2-deoxy-beta-D-erythro- pentofuranosyl)-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol (pentostatin, 1a) is described. Ring closure of 2-amino-1-(5-amino-1H-imidazol-4-yl)ethanone dihydrochloride (3) with triethyl orthoacetate or triethyl orthopropionate gave the C-5 methyl and ethyl ketoaglycons, 6,7-dihydro-5-methylimidazo[4,5-d][1,3]diazepin-8(3H)-one (4b) and 5-ethyl-6,7-dihydroimidazo[4,5-d][1,3]diazepin-8(3H)-one (4c), respectively. Stannic chloride catalyzed condensation of the pertrimethylsilyl derivatives of 4b and 4c with a protected glycosyl halide afforded anomeric mixtures of ketonucleosides 3-(2-deoxy-3,5-di-O-p-toluoyl-beta- and -alpha-D-erythro-pentofuranosyl)-6,7-dihydro-5-methylimidazo[4,5-d] [1,3]diazepin-8(3H)-one (5b and 6b) and 3-(2-deoxy-3,5-di-O-p-toluoyl)-beta- and -alpha-D-erythro-pentofuranosyl)-5-ethyl-6,7-dihydroimidazo[4,5-d]- [1,3]diazepin-8(3H)-one (5c and 6c), respectively. Subsequent separation of the anomers, followed by deprotection and reduction of 5b, 6b, and 5c, afforded the respective 8R and 8S isomers. Stannic chloride catalyzed condensation of pertrimethylsilyl ketoaglycon 4a with 2-(chloromethoxy)-1-(p-toluoyloxy) ethane to give ketonucleoside 6,7-dihydro-3-[[2-(p-toluoyloxy)ethoxy] methyl]imidazo[4,5-d][1,3]diazepin-8(3H)-one (9a) was followed by deprotection to 6,7-dihydro-3[(2-hydroxyethoxy)methyl]imidazo[4,5-d][1,3] diazepin-8(3H)-one (9b) and then reduction to the racemic acyclic pentostatin analogue (+/-)-3,6,7,8-tetrahydro-3-[ (2-hydroxyethoxy)methyl]imidazo[4,5-d][1,3]diazepin-8-ol (2). Ki values for the in vitro adenosine deaminase (EC 3.5.4.4; type I; calf intestinal mucosa) inhibitory activities of 1b, 1c, and 2 were determined to be 1.6 X 10(-8), 1.5 X 10(-6), and 9.8 X 10(-8) M, respectively. When compounds 2 and 9b were tested in combination with vidarabine against herpes simplex virus, type 1, in an HEp-2 plaque reduction assay, only compound 2 was able to potentiate the antiviral activity of vidarabine.

Influenza vaccination in kidney transplant recipients: cellular and humoral immune responses.

Influenza infection in renal transplant recipients may cause either morbidity and mortality or acute allograft rejection; thus, routine annual influenza vaccination should be considered. We have studied the humoral and cellular immune responses to influenza virus antigens before and after trivalent vaccine administration in 13 patients and 16 control subjects. The patients, nine of whom were either on alternate-day or low-dose daily steroid therapy, showed highly significant serum hemagglutination-inhibition antibody responses to each influenza virus strain, There was no significant change in mean lymphocyte stimulation index to any influenza virus strain after vaccination in either group. There was no correlation in the patient group between hemagglutination-inhibition antibody titer or response, or lymphocyte stimulation index or response, and the degree of allograft function or dose or duration of immunosuppressive therapy. The vigorous antibody response and the evidence of cellular immunity support the efficacy of influenza vaccination in these patients.

Ether-treated, subunit Hsw1N1 influenza vaccines: response of immunologically primed subjects to two antigenic variants.

Two bivalent, ether-treated, subunit influenza vaccines were compared in adults greater than or equal to 45 years old. Both vaccines contained 200 chick cell-agglutinating (CCA) units of A/Victoria/3/75 antigen/dose. The Hsw1N1 components, also at a level of 200 CCA units/dose and designated A/Shope and A/X-53, were antigenically representative of the A/swine/1976/31 and A/New Jersey/8/76 viruses, respectively. A/Shope virus possessed about 100 times more neuraminidase activity than A/X-53 virus. The two vaccine groups had equivalent geometric mean titers (GMTs) of antibody to A/NJ virus, with about 95% of each group having titers of greater than or equal to 1:40 after vaccination. Group GMTs of antibody to A/Vic virus were also equivalent. Failure of the A/X-53 vaccinees to respond according to the dogma of original antigenic sin and a highly significant between-group difference in response to A/PR/8/34 antigen are interpreted as due to a difference in vaccine neuraminidase levels. It is suggested that, although A/Shope was as serologically effective against A/NJ virus as A/X-53 in this age group, under similar conditions a recombinant with the A/NJ hemagglutinin and the stable A/swine neuraminidase antigens might be more effective against A/NJ than either of the present vaccines.

Reactions and serologic responses after administration of inactivated monovalent influenza A/swine virus vaccines. I. Immunization of children and adults with influenza A/Shope virus vaccines.

Reactivity and immunogenicity of three inactivated, zonally purified, monovalent influenza A/swine virus vaccines were studied in children and adults. Each dose of vaccine contained either 400 chick cell-agglutinating (CCA) units/0.5 ml or 200 CCA units/0.25 ml. The vaccines contained either whole virus or ether-extracted, subunit virus with or without 1.5 mg of A1PO4/0.5 ml. Children younger than 10 years of age received a half dose. Substantial system reactions, including temperature increases of 2.2 F-4.9 F, were observed in all children who received whole-virus vaccines. In contrast, ether-extracted, subunit vaccines (with or without A1PO4) were minimally pyrogenic in 185 subjects. Two doses of subunit vaccine in subjects younger than 25 years of age were immunologically equivalent to a single dose in older subjects. We concluded that two doses of ether-extracted, subunit virus vaccine with Hsw1N1 antigen, administered at least four weeks apart, are serologically effective for immunization of seronegative subjects of any age and that this dosage regimen should be used in young children in whom whole-virus vaccines are unacceptably reactive.

Reactions and serologic responses after administration of inactivated monovalent influenza A/swine virus vaccines. II. Immunization of children with influenza A/New Jersey/X-53 virus vaccines.

Reactivity and immunogenicity of two inactivated, zonally purified, ether-extracted, influenza A/New Jersey/X-53 subunit virus vaccines were studied in 103 children three to 18 years of age. Children aged nine years of younger received doses of 100 or 200 chick cell-agglutinating (CCA) units, and those older than nine years received doses of 200 or 400 CCA units. Vaccines were given intramuscularly. Two doses were given at intervals of four weeks. The vaccines were minimally pyrogenic, causing only two instances of temperatures of greater than 100.0 F. Other systemic reactions were observed infrequently. Tenderness at the site of injection occurred relatively frequently but was of no medical consequence. The geometric mean titers of homologous antibody, which ranged from 1:52 to 1:75 after administration of two doses, were statistically equivalent in all treatment groups. Titers of antibody of greater than or equal to 1:40 to the influenza A/New Jersey/8/76 virus strain were achieved by 88% of the vaccinees. We concluded that two doses of ether-extracted, subunit influenza A/New Jersey/X-53 virus vaccine were well tolerated and, when given at least four weeks apart, were serologically effective for immunization of children aged three to 18 years.

Relevance of the immunoprecipitation assay to human immunogenicity of influenza vaccines.

Influenza vaccines representing each of the four U.S. manufacturers' output for the 1975-76 respiratory season were characterized clinically and assayed by immunoprecipitation. All vaccines contained 350 CCA units/dose each of the A/Port Chalmers/1/73 (H3N2) and A/Scotland/840/74 (H3N2) viruses plus 550 CCA units/dose of B/HK/5/72 virus. Two of the vaccines were whole virus while the other two were subunit products; one made by extraction with ethyl ether, the second by detergent treatment. The vaccines were compared for serologic efficacy in children naturally primed to the (H3N2) family of viruses and by immunoprecipitation techniques against monospecific goat antiserum to the viral hemagglutinin prepared at the Bureau of Biologics of the U.S. Food & Drug Administration. The subunit vaccines had significantly greater specific activity (human immunogenicity/unit mass of type-specific precipitable antigen) than the whole virus products. It is concluded that clinical immunogenicity is as much a function of antigen form (subunit vs whole virus) as it is of mass and that setting a level for precipitable antigen content alone is an insufficient criterion for potency standardization. Since antigen form, as well as mass, must be considered, immunoprecipitation may be useful for standardization of human immunogenicity only if candidate lots are compared by this technique to an homologous, reference vaccine of identical manufacture and form which is tested for potency in humans.