Phase I study of the orally administered butyrate prodrug, tributyrin, in patients with solid tumors.

Butyrates have been studied as cancer differentiation agents in vitro and as a treatment for hemoglobinopathies. Tributyrin, a triglyceride with butyrate molecules esterified at the 1, 2, and 3 positions, induces differentiation and/or growth inhibition of a number of cell lines in vitro. When given p.o. to rodents, tributyrin produces substantial plasma butyrate concentrations. We treated 13 patients with escalating doses of tributyrin from 50 to 400 mg/kg/day. Doses were administered p.o. after an overnight fast, once daily for 3 weeks, followed by a 1-week rest. Intrapatient dose escalation occurred after two courses without toxicity greater than grade 2. The time course of butyrate in plasma was assessed on days 1 and 15 and after any dose escalation. Grade 3 toxicities consisted of nausea, vomiting, and myalgia. Grades 1 and 2 toxicities included diarrhea, headache, abdominal cramping, nausea, anemia, constipation, azotemia, lightheadedness, fatigue, rash, alopecia, odor, dysphoria, and clumsiness. There was no consistent increase in hemoglobin F with tributyrin treatment. Peak plasma butyrate concentrations occurred between 0.25 and 3 h after dose, increased with dose, and ranged from 0 to 0.45 mM. Peak concentrations did not increase in three patients who had dose escalation. Butyrate pharmacokinetics were not different on days 1 and 15. Because peak plasma concentrations near those effective in vitro (0.5-1 mM) were achieved, but butyrate disappeared from plasma by 5 h after dose, we are now pursuing dose escalation with dosing three times daily, beginning at a dose of 450 mg/kg/day.

Intravenous pamidronate disodium treatment of bone metastases in patients with breast cancer. A dose-seeking study.

BACKGROUND: Treatment of the symptoms of bone metastases currently involves the use of narcotic medication, radiation therapy, or hormonal therapy. Pamidronate disodium, a bisphosphonate, may prove helpful in the palliative treatment of bone metastases in patients with breast cancer as demonstrated in this multicenter, dose-ranging trial. METHODS: Ambulatory female patients age 18 years or older with breast cancer metastatic to bone and a life expectancy of at least 3 months were eligible for the study. Bone metastases were confirmed by bone scan or bone survey within 6 months of enrollment. Sixty-one patients were treated as outpatients and were randomized to receive one of four intravenous pamidronate regimens for 12 weeks: 30 mg administered every 2 weeks, 60 mg every 4 weeks, 60 mg every 2 weeks, or 90 mg every 4 weeks. The primary efficacy parameter for this study was pain score. The change from baseline in pain score was determined for each patient at each study visit and at endpoint, defined as the last postbaseline evaluation for each patient before or at week 12. Secondary efficacy variables included narcotic scores, urinary calcium/creatinine and hydroxyproline/creatinine ratios, serum osteocalcin and bone alkaline phosphatase concentrations, and bone lesion (radiologic) response. RESULTS: At 3 months, the regimens of 60 mg every 4 weeks, 60 mg every 2 weeks, and 90 mg every 4 weeks resulted in significant reduction in bone pain beginning by week 6 of treatment. The regimen of 30 mg every 2 weeks was not effective. Narcotic use, as reflected by narcotic scores, did not parallel the pain scores, because there was little evidence of any effect for any of the treatment groups. Reduction in bone pain was accompanied by decreases in urinary calcium/creatinine and hydroxyproline/creatinine ratios, and bone alkaline phosphatase concentrations. Side effects of pamidronate were mild and transient. Radiographic changes consistent with healing of lytic lesions were observed in 15 patients (25%). CONCLUSION: Intravenous pamidronate is a well tolerated treatment that produced significant relief of bone pain in the majority of patients with metastatic breast cancer at the three highest doses tested.

Cisplatin and platinum analogues: recent advances.

Cisplatin is a critically important antineoplastic agent employed in treating patients who have a broad spectrum of neoplasms. Resistance to this agent poses a serious clinical dilemma. The objective of this review is to summarize the principal mechanisms that contribute to cytotoxicity by this agent and underlie resistance. Knowledge of these processes provides a rational basis for developing therapeutic strategies to circumvent resistance.

Preirradiation chemotherapy of supratentorial malignant primary brain tumors with intracarotid cis-platinum (CDDP) and i.v. BCNU. A phase II trial.

Thirty patients with histologically verified malignant supratentorial gliomas were treated with a preirradiation chemotherapy protocol consisting of two courses of intracarotid (i.c.) CDDP, 90 mg/m2, followed by i.v. BCNU, 200 mg/m2. Side effects from therapy were mild and self-limiting; no irreversible retinal or neurologic toxicity could be attributed to i.c. chemotherapy. Of the 27 patients who completed the chemotherapy portion of the protocol, tumor size on postchemotherapy computed tomography (CT) was decreased by greater than 50% in 13% as compared to the postoperative CT scan; in only 4% was the CT scan unequivocally increased in size. Twenty-five (83%) patients completed the entire protocol. Median time to tumor progression and survival in patients who completed the protocol was 53 (range of 13-130+) and 61 (range of 29-130+) weeks, respectively. Twenty-four percent of patients still have not demonstrated tumor progression at intervals greater than 1 year after diagnosis as judged by clinical and radiographic criteria. Tumor recurrences were always contiguous to the original tumor bed. We conclude that preirradiation chemotherapy may be administered safely and with low morbidity. Further study to determine an optimal timing between chemotherapy and radiation therapy is warranted.

Mechanisms underlying resistance to streptozotocin in Mer+ and Mer- human tumor lines.

Streptozotocin (STZ) is a monofunctional nitrosourea employed in the treatment of patients with islet cell tumors. To analyze the role of DNA repair mechanisms in causing resistance to STZ, we evaluated the cytotoxicity by this agent in three human tumor lines that differ with respect to their abilities to repair N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) damaged virus (the Mer phenotype). HT-29, A2182, and BE human tumor lines are high, intermediate and low, respectively, with regard to features that define the Mer phenotype. Our results demonstrated that the order of resistance to STZ is HT-29 greater than A2182 greater than BE. The degree of inhibition of DNA synthesis by STZ was in the following order: BE greater than A2182 greater than HT-29. O6-Alkyltransferase activity was increased markedly in HT-29 cells compared to A2182 cells which, in turn, had significantly increased levels compared to the BE line. Other potential factors such as 3-methyladenine DNA glycosylase activity, the induction by STZ of single-stranded DNA breaks, and the kinetics of repair of these breaks do not clearly underlie differences in cytotoxicity among the three tumor lines. However, increased topoisomerase II activity, as well as enhanced sensitivity to agents that interact with topoisomerase II, was present in A2182 cells compared to BE cells. These findings demonstrate that while O6-alkyltransferase contributes to resistance to STZ in some Mer+ tumor lines, other mechanisms may also contribute to resistance to this agent.

Schedule dependence of buthionine sulfoximine in reversing resistance to cisplatin.

We have found that the potentiation of antiproliferative effects by buthionine sulfoximine (BSO) of cell growth inhibition induced by cisplatin are highly schedule dependent in resistant BE colon carcinoma cells. Maintenance of low GSH levels during the 12-h interval after cisplatin (cis-DDP) treatment is critical. A schedule of BSO exposure that results in low GSH levels for 12 h after cisplatin exposure is associated with a marked increase in DNA interstrand cross-link formation as analyzed by alkaline elution. These findings are consistent with a central role of GSH in interfering with the conversion of cis-DDP DNA monoadducts to DNA interstrand cross-links and may prove relevant to the design of clinical trials of BSO with cisplatin.

Characterization of acquired resistance to cis-diamminedichloroplatinum (II) in BE human colon carcinoma cells.

To study mechanisms underlying resistance to cis-diamminedichloroplatinum (II) (cis-DDP) we have induced resistance to this agent in BE human colon carcinoma cells. A 5-fold increase in the IC50 of resistant compared to sensitive cells was noted as analyzed by the inhibition of cellular growth. Up to a 4-fold reduction in interstrand cross-link formation by cis-DDP in resistant compared to sensitive cells was present as measured by alkaline elution. No significant differences were detectable either in the extent of DNA platination as analyzed by atomic absorption spectroscopy or in the induction of cis-DDP DNA adducts as evaluated by an enzyme-linked immunosorbent assay employing antiserum that detects intrastrand cross-links formed by cis-DDP. Further, no differences in the kinetics of excision of DNA interstrand cross-links, cis-DDP DNA adducts, or total platinum in DNA were present. Levels of glutathione, however, were increased about threefold in resistant compared to sensitive cells. Loss of resistance was associated with increased interstrand cross-link formation and declines in glutathione levels. Our results are consistent with a critical role of glutathione in preventing platinum monoadduct rearrangements resulting in lower levels of interstrand cross-links and resistance to cis-DDP in resistant BE cells.

DNA repair mechanisms affecting cytotoxicity by streptozotocin in E. coli.

Mechanisms underlying cytotoxicity by the monofunctional nitrosourea streptozotocin (STZ) were evaluated in DNA repair-deficient E. coli mutants. Strains not proficient in recombinational repair which lack either RecA protein or RecBC gene products were highly sensitive to STZ. In contrast, cells that constitutively synthesize RecA protein and cannot initiate SOS repair mechanisms because of uncleavable LexA repressor (recAo98 lexA3) were resistant to this drug compared to a lexA3 strain. Further, E. coli cells lacking both 3-methyladenine DNA glycosylases I (tag) and II (alkA) also were highly sensitive to STZ. DNA synthesis was most inhibited by STZ in recA and alkA tag E. coli mutants, but was suppressed less markedly in wild-type and recBC cells. DNA degradation was most extensive in recA E. coli after STZ treatment, while comparable in recBC, alkA tag, and wild-type cells. Although increased single-stranded DNA breaks were present after STZ treatment in recA and recBC mutants compared to the wild type, no significant increase in DNA single-stranded breaks was noted in alkA tag E. coli. Further, DNA breaks in recBC cells were repaired, while those present in recA cells were not. These findings establish the critical importance of both recombinational repair and 3-methyladenine DNA glycosylase in ameliorating cytotoxic effects and DNA damage caused by STZ in E. coli.

Sequence specificity of streptozotocin-induced mutations.

The isolation and characterization of streptozotocin (STZ)-induced mutations in the phage P22 mnt repressor gene is described. Cells carrying the plasmid-borne mnt gene were exposed to STZ to give 10-20 percent survival and at least an eleven-fold increase in mutation frequency. DNA sequence analysis showed that 50 of 51 STZ-induced mutations were GC to AT transitions, and one was an AT to GC transition. We have also compared the STZ mutational spectrum to that for N-methyl-N'-nitro-N-nitroso-guanidine (MNNG). There are sites in the mnt gene which are mutated only by STZ; only by MNNG, or by both agents. Sites at which only STZ induced GC to AT transition mutations occur were in sequences that are pyrimidine rich 5' to the mutated site and purine rich 3' to the mutated site. Induction of mutations by both STZ and MNNG should be considered to maximize the number of mutable sites.

Gene expression caused by alkylating agents and cis-diamminedichloroplatinum(II) in Escherichia coli.

Previous work has demonstrated heterogeneous effects of methylating agents on induction of DNA damage inducible genes in Escherichia coli. These studies employed E. coli mutants that have fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents. These mutants were selected from random insertions of the Mu-dl (Apr lac) phage by screening for induction of beta-galactosidase activity in the presence of methylmethanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine. The current report extends these findings by analyzing gene expression caused by mechlorethamine, chloroethylnitrosoureas and cis-diamminedichloroplatinum(II) (cis-DDP). The results demonstrate heterogeneous effects by these agents on gene expression. While 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea induces alkA, other nitrosoureas, mechlorethamine, and cis-DDP do not cause expression of this gene. Further, while all nitrosoureas caused expression of aidC, mechlorethamine and cis-DDP did not. Lastly, cis-DDP caused marked expression of a sulA fusion mutant while not inducing any of the other E. coli fusion mutants.

Gene expression in E. coli after treatment with streptozotocin.

Gene induction by the methylating agents streptozotocin (STZ), N-methyl-N-nitrosourea (MNU), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was evaluated in E. coli fusion mutants. These mutants have fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents and were previously selected from random insertions of the Mu-dl (Apr lac) phage by screening for induction of beta-galactosidase activity in the presence of methyl methanesulfonate or MNNG. The results demonstrate that STZ differs from MNNG and MNU in failing to induce aidC expression. Further, expression of aidC after exposure to MNU and MNNG occurs only in nonaerated cultures; aeration blocks the induction. Induction of aidD, alkA, aidB, and sfiA expression occurs with all 3 agents although at markedly lower concentrations of MNNG and STZ compared to MNU. alkA and to a lesser extent aidD mutants of E. coli strains were more sensitive to these agents, while no differences were evident between wild-type and aidB or aidC fusion mutants.

Interactions of cis-diamminedichloroplatinum(II) with 1-beta-D-arabinofuranosylcytosine in LoVo colon carcinoma cells.

Prior reports demonstrated more than additive cytotoxic effects of cis-diamminedichloroplatinum(II) (CDDP) and 1-beta-D-arabinofuranosylcytosine (ara-C) in LoVo colon carcinoma cells. We have extended these findings by analyzing mechanisms that may underlie the effect of ara-C on CDDP-induced cytotoxicity. In contrast to a previous study, ara-C neither enhances DNA interstrand cross-link formation by CDDP nor affects the excision of platinum from DNA. Features peculiar to ara-C, such as its misincorporation into DNA, probably contribute since more than additive cytotoxic effects do not occur by combinations of CDDP with inhibitors of DNA synthesis that are not incorporated into DNA. Also, while ara-C does not significantly enhance the degree of inhibition of DNA synthesis caused by CDDP, the recovery of DNA synthesis after drug removal is significantly slowed when cells are exposed to both drugs. These findings contrast with those obtained with CDDP and aphidicolin (the latter agent resembles ara-C in competing with dCTP for binding to DNA polymerase alpha but, unlike ara-C, is not incorporated into DNA). Lastly, ara-C is incorporated into LoVo cell DNA undergoing replicative synthesis as well as into DNA undergoing repair synthesis after CDDP-induced induced DNA damage.

Potentiation of ara-C induced cytotoxicity by hydroxyurea in LoVo colon carcinoma cells.

The present study was undertaken to determine whether cytotoxicity by 1-beta-D-arabinofuranosylcytosine (ara-C) in LoVo colon carcinoma cells, which are resistant to high concentrations of ara-C, would be enhanced by concurrent exposure to hydroxyurea (HU). Since mechanisms underlying the effects of HU on ara-C induced cytotoxicity are unclear, we also evaluated the effect of HU on the incorporation of ara-C into DNA, as well as potential consequences of misincorporation. Our results demonstrate that HU synergistically enhances cytotoxicity by ara-C in these cells. This effect was not present when HU was combined with aphidicolin, an agent that resembles ara-C in competing with dCTP for binding to polymerase alpha but that is not incorporated into DNA. Further, cells exposed to HU and ara-C incorporated up to 5-fold as much ara-C into DNA as cells solely treated with ara-C. While the extent of inhibition of DNA synthesis was comparable with cells exposed to HU and aphidicolin as those treated with HU and ara-C, recovery of DNA synthesis was delayed more significantly by the latter combination. These findings suggest that HU synergistically potentiates ara-C induced cytotoxicity by enhancing incorporation of ara-C in LoVo cell DNA.

Mutagenesis and repair of DNA damage caused by nitrogen mustard, N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), streptozotocin, and mitomycin C in E. coli.

Cytotoxicity and mutagenesis by streptozotocin, BCNU, nitrogen mustard, and mitomycin C were evaluated in E. coli mutants deficient in SOS repair, SOS-mediated mutagenesis, the adaptive response, and mutants that engage in aberrant mismatch repair. The results demonstrate that premutagenic lesions are caused by nitrogen mustard, BCNU and streptozotocin that are not repaired by ada or recognized by umuDC. Further, recA mutants were hypomutable after exposure to nitrogen mustard, BCNU, and streptozotocin compared to wild type. With the exception of the monofunctional nitrosourea, streptozotocin, both recA and uvrA gene products contribute to the repair of DNA damage caused by the alkylating agents tested. In the case of streptozotocin, although recA mutants were more sensitive than wild type, uvrA mutants were not. Moreover, while ada and alkA E. coli mutants showed increased sensitivity to streptozotocin, they were not more sensitive to the other alkylating agents evaluated.

Studies on mutagenesis and repair induced by platinum analogs.

Mutagenesis and cytotoxicity were studied in Escherichia coli by iproplatin and carboplatin, two analogs of cisplatin (CDDP) currently undergoing clinical trial. As with CDDP, mutagenesis by these agents was mediated by the umuDC gene product. In contrast to CDDP, however, mismatch repair did not substantially contribute to survival of cells after exposure to these agents since dam-3 E. coli were not more sensitive than wild type E. coli. UvrA- E. coli, however were more sensitive to these analogs demonstrating that as with CDDP, uvr endonuclease-mediated excision contributes to the repair of DNA damage induced by platinum compounds.

Mismatch repair of cis-diamminedichloroplatinum(II)-induced DNA damage.

Because cytotoxicity by an alkylating agent such as N-methyl-N'-nitrosoguanidine is markedly increased in adenine methylase-deficient dam-3 Escherichia coli, it was of interest to assess whether mismatch repair was similarly important in the repair of DNA damage induced by cis-diamminedichloroplatinum(II) (CDDP). The results demonstrate that after exposure to 5-40 microM CDDP, dam-3 E. coli are 2-15-fold more sensitive to the cytotoxic effects of this agent. Further, dam-3 mutL451 E. coli deficient in mismatch repair was as resistant as wild type. trans-Diamminedichloroplatinum(II) treatment did not cause marked increments in cytotoxicity in dam-3 E. coli compared to wild type. The rate of excision of platinum was significantly reduced in dam-3 E. coli compared to wild type, demonstrating that differences in the repair of CDDP-induced DNA damage underlie enhanced cytotoxicity by this agent. Lastly, mutagenesis by CDDP was abrogated in umuDC- E. coli, showing that this gene product mediates mutagenesis by this agent.