Proinflammatory cytokine and human immunodeficiency virus RNA levels during early Mycobacterium avium complex bacteremia in advanced AIDS.

The relationship between Mycobacterium avium complex (MAC) bacteremia and proinflammatory cytokine and human immunodeficiency virus type 1 (HIV-1) RNA levels in AIDS was investigated. During a prospective study, blood samples were drawn monthly for mycobacterial cultures. Sera were available at baseline and onset of MAC bacteremia from 20 cases and at corresponding times from 19 controls. Mean interleukin-6 (IL-6) levels were 154% greater at the time of MAC bacteremia in cases than in controls. The IL-6 levels correlated with body temperature, serum tumor necrosis factor (TNF-alpha) levels, and alkaline phosphatase levels (P < or = .004 for each). Although TNF-alpha levels tended to rise more in MAC patients than in controls, the difference was not significant. However, among both cases and controls, serum TNF-alpha levels rose significantly from baseline to the time of last sample, irrespective of MAC infection (P = .015). Bacteremia was not associated with increased serum HIV-1 RNA levels. Thus, early MAC bacteremia is associated with increases in serum IL-6 levels, while TNF-alpha levels rise over time during advanced AIDS.

Flurbiprofen for the treatment of bone pain in patients with metastatic breast cancer.

Two investigators enrolled 26 women with metastatic breast carcinoma in a six-week, double-blind, placebo-controlled, crossover study of flurbiprofen (Ansaid, Upjohn) and placebo. The study was designed to determine the efficacy of flurbiprofen in reducing bone pain due to metastatic breast cancer. Pain score, overall performance, and concomitant use of narcotics were evaluated. The overall mean differences in pain scores between flurbiprofen and placebo showed better control of pain during treatment with flurbiprofen. None of these differences approached statistical significance. Evaluation of overall performance status reached statistical significance in one investigator's group. Three out of four patients reported decreased consumption of acetaminophen/aspirin plus codeine combinations while receiving flurbiprofen.

Synergistic combination of menogarol and melphalan and other two drug combinations.

Menogarol is a new anthracycline undergoing phase I clinical trial. We report here the lethality after 2 hr exposure to 2 drug combinations of menogarol and several antitumor agents. A new statistical procedure was used to identify synergistic combinations. Most of these combinations were additive, except for menogarol plus melphalan, which was synergistic. Adriamycin plus melphalan was also synergistic. The menogarol-melphalan combination wa studied in detail with regard to the effect of dose and drug-schedule, lethality for exponential and plateau phase cells and effect on cell cycle progression. Although the combination was synergistic for exponential cells it was additive for plateau phase cells. The combination exerted a synergistic effect in inhibiting progression of cells through the cell cycle. After 2 hr menogarol exposure cells were blocked in G2 for about 12 hr following which the block was reversed. This reversal was inhibited when menogarol was combined with melphalan. The uptake of menogarol or melphalan was not changed in the presence of the other drug.

Biochemical and cellular effects of didemnins A and B.

Didemnins are a new class of cyclic depsipeptides in which didemnin A is the major component, didemnin B the minor component, and a trace of didemnin C is present. Didemnin B was more potent than was didemnin A against B16 melanoma and P388 leukemia in vivo, and B was also approximately 20 times more cytotoxic than was didemnin A in vitro. Therefore, didemnin B was studied in greater detail for its biochemical and cellular effects. Didemnin B inhibited the in vitro growth of B16 greater than L1210 greater than V-79 cells = human foreskin fibroblast = 9L greater than Chinese hamster ovary cells. Didemnin B was more lethal to exponentially growing B16 cells (50% lethal dose for a 2-hr exposure, 17.5 ng/ml) than to plateau-phase cells (50% lethal dose for a 2-hr exposure, 100 ng/ml). After a 24-hr exposure, the 50% lethal dose for exponential- and plateau-phase B16 cells was 8.8 and 59.6 ng/ml, respectively. Chinese hamster ovary cells were not killed even at 25,000 ng/ml. Mitotic cells were the least sensitive to didemnin B, and cells became more sensitive as they progressed into G1 and S phase. However, since cells in all phases were killed, didemnin B cannot be considered a phase-specific agent. Didemnin B inhibited the synthesis of protein more than that of DNA, with much less inhibition of RNA synthesis. Cell progression studies showed that high doses (300 ng/ml for 2 hr or 100 ng/ml for 24 hr) of didemnin B "froze" the cells in their respective phases with complete inhibition of cell progression or growth. At low doses (10 ng/ml for 2 hr or 3 ng/ml for 24 hr), the cells were blocked at the G1-S border thereby increasing the percentage of G1 cells and decreasing the percentage of S-phase cells. Cells continued to progress from S phase to G2 + M and from G2 + M to G1. The cytotoxicity to different cell lines and inhibition of macromolecule synthesis by didemnin A is also reported.

Cell cycle effects of CC-1065.

CC-1065 is the most potent antitumor agent tested in our laboratory. It is lethal to B16 and CHO cells and to a variety of human tumors in the clonogenic assay at 1 ng/ml and is effective against L1210 leukemia and B16 melanoma in vivo at 1 to 50 micrograms/kg. CC-1065 inhibits DNA synthesis and binds to DNA in a nonintercalative manner in the minor groove. We report here the kinetics of inhibition of DNA synthesis and of cell progression and the phase-specific toxicity of the drug. To determine phase-specific toxicity, we started synchronous CHO cultures from mitotic cells harvested after Colcemid pretreatment. These cultures showed that mitotic cells were the most sensitive, and sensitivity decreased as the cells progressed through G1 to S and G2. Experiments with B16 and CHO mitotic cells harvested without Colcemid pretreatment also showed that mitotic cells were more sensitive than G1/S-phase cells. Cell progression studies showed that CC-1065 did not affect progression from mitosis to G1 or from G1 to S. Cells progressed slowly through S at low levels (1 ng/ml) of the drug but were blocked in S at 5 ng/ml. Cell progression from G2 to M was blocked by CC-1065. DNA synthesis in B16 cells was measured at different times after 2-hr exposure to CC-1065. The percentage of inhibition of DNA synthesis was minimum at 4 hr and maximum at 19 hr after drug exposure. Since B16 cell progression studies showed a marked change in percentage of S-phase cells during this time, the DNA synthesis rate was recalculated as cpm/S-phase cell. After this correction (i.e., expressing DNA synthesis as cpm/S-phase cell), the percentage of inhibition of DNA synthesis was minimum at 0 hr and gradually increased to maximum inhibition at 19 hr without the decrease seen previously at 4 hr.

CC-1065 (NSC 298223), a most potent antitumor agent: kinetics of inhibition of growth, DNA synthesis, and cell survival.

CC-1065 (NSC 298223) is the most cytotoxic agent tested against cells in culture in our laboratory. The 50% lethal doses for exponentially growing B16 melanoma and Chinese hamster ovary cells were 0.44 and 0.14 ng/ml, respectively, as compared to 35 and 500 ng/ml for Adriamycin. In the human tumor-cloning assay, 1-hr exposure to CC-1065 (0.1 ng/ml) caused greater than or equal to 50% lethality in a broad spectrum of tumors. The dose-survival curves for B16 and Chinese hamster ovary cells were characterized by an initial shoulder followed by an exponential decline with increasing dose. CC-1065 was more lethal to exponentially growing B16 cells (50% lethal dose = 0.44 ng/ml) than to plateau-phase cells (50% lethal dose = 1.2 ng/ml). CC-1065 inhibited DNA synthesis much more than did RNA or protein synthesis. After a 2-hr incubation with drug, inhibition of DNA synthesis was low immediately (0 hr) after drug exposure and reached maximum inhibition about 20 hr later. The doses for 50% inhibition of growth (0.18 ng/ml), survival (0.44 ng/ml), and DNA synthesis (0.15 ng/ml) were in the same range, whereas RNA synthesis was inhibited 50% at a much higher dose (5 ng/ml).

Effect of 7-con-O-methylnogarol on DNA synthesis, survival, and cell cycle progression of Chinese hamster ovary cells.

The effect of 7-con-O-methylnogarol (7-OMEN) on the survival of exponentially growing and plateau-phase Chinese hamster ovary cells was determined in a cloning assay. After 2 hr of exposure, the 50% lethal dose for exponential and plateau-phase cells was 0.3 and 1.5 microgram/ml, respectively. Drug doses for cell progression studies were based upon drug lethality; therefore, higher doses were used for plateau than for exponential populations. The effect of 7-OMEN on cell progression was studied by DNA flow cytometry under the following conditions: (a) during 24 hr of continuous exposure of exponentially growing cells; (b) during recovery of exponential cells after 2 or 7 hr of drug exposure; and (c) during recovery of plateau-phase cells after 2 hr of exposure. Exponential cells exposed continuously for 24 hr progressed normally from M to G1 phase and from G1 to S phase, progression through S phase was slowed, and cells were ultimately blocked in G2 + M. Inhibition of S-phase progression was dose dependent, 0.2 microgram/ml having only slight effect and 1.0 microgram/ml accumulating a large fraction in S phase. Inhibition of S-phase progression correlated with DNA synthesis inhibition. Similar inhibitory effects were observed after pulsed (2- or 7-hr) exposure of exponential cells. 7-OMEN also blocked plateau-phase cells in G2 + M after 2 hr of exposure, but higher doses (3.0 microgram/ml) were required. Simultaneous exposure of exponential cells to Colcemid (which blocks cells in metaphase) and 1.0 microgram 7-OMEN per ml completely inhibited the expected increase in mitotic index, indicating that the G2 + M block observed by DNA flow cytometry was a block in G2 or prophase.

Cell kill kinetics of several nogalamycin analogs and adriamycin for Chinese hamster ovary, L1210 leukemia, and B16 melanoma cells in culture.

Nogalamycin is an anthracycline antibiotic which was markedly cytotoxic in vitro and was active against several tumor systems in vivo. We compare here the lethality of several nogalamycin analogs against Chinese hamster ovary (CHO), mouse leukemia (L1210), and mouse melanoma (B16) cells in culture. 7-con-O-Methylnogarol (7-con-OMEN) was the most lethal of all the analogs tested. Thus, for CHO cells exposed for two hr to the drug, the 50% lethal doses of 7-con-OMEN, nogalamycin, and dis-nogamycin were 0.25, 2.7, and 5.8 micrograms/ml, respectively. In general, CHO cells were less sensitive than B16 or L1210 cells to most compounds. All compounds gave dose-survival curves which consisted of a shoulder region followed by a region of exponential decline in survival. The nogalamycin analogs nogalamycin, dis-nogamycin, 7-con-O-methylnogalarol, and 7-con-OMEN were selected for further study because of their greater lethality in vitro and antitumor activity in vivo. The lethality of these compounds was compared to that of Adriamycin. 7-con-OMEN was more toxic to CHO cells than was Adriamycin but was less toxic to B16 and L1210 cells. All of these compounds (except 7-con-O-methylnogalarol which was not tested) were more lethal to exponentially growing cells than to plateau-phase cells. The survival response after different periods of exposure to these drugs was compared. In order to make valid comparisons of the time-survival response to different drugs, the drug concentrations chosen were such that they were equitoxic after a two-hr exposure. Under these conditions, the order of lethality after long-term exposure (8 hr to 24 hr) was nogalamycin > dis-nogamycin > 7-con-OMEN, Adriamycin > 7-con-O-methylnogalarol. With all the drugs, the rate of cell death increased with increasing drug concentrations.

Antitumor activity and preliminary drug disposition studies on chartreusin (NSC 5159).

Chartreusin has exhibited significant therapeutic activity against three experimental mouse tumors (ascitic P388, L1210 leukemia, and B16 melanoma) when tumor cells were inoculated i.p. and drug was administered i.p. In further testing against P388 leukemia, no activity was observed when drug was administered p.o., s.c., or i.v. Chartreusin was very slowly absorbed from the small intestine, thus explaining the lack of activity when administered p.o. When given i.p., the drug precipitated in the peritoneal cavity and was slowly absorbed over several hr. The strong activity observed by this route was related to the prolonged and intimate contact of drug with tumor cells in the peritoneal cavity. Upon s.c. administration, extensive precipitation occurred. Subsequent dissolution and absorption from the injection site were very slow, and measured plasma and tissue levels were quite low. Biliary excretion of chartreusin, the predominant route of elimination. was very rapid, with 80 to 100% of the dose appearing as unchanged drug in the bile within 6 hr after i.v. administration. Rapid biliary excretion after i.v. administration was reflected in a rapid decline in plasma and tissue concentrations to levels (shown by in vitro cell kill experiments) less than those necessary to kill P388 cells. When the bile ducts of i.v.-dosed leukemic mice were ligated, therapeutic activity was observed, confirming that the physiological disposition of chartreusin exerts a major influence on its therapeutic utility.