Glucan phosphate potentiates endotoxin-induced interferon-gamma expression in immunocompetent mice, but attenuates induction of endotoxin tolerance.

Glucan phosphate has been shown to enhance antimicrobial immunity in a variety of experimental models. However, the mechanisms by which glucans enhance resistance to infection remain largely unknown. Interferon-gamma (IFN-gamma) is a key regulator of both innate and acquired immunity. Suppression of IFN-gamma production is a prominent feature of the altered immune response that follows major trauma or sepsis. The present studies were designed to determine the effect of glucan phosphate on IFN-gamma expression in normal mice and endotoxin [lipopolysaccharide (LPS)]-tolerant mice. The model of LPS tolerance was used because it results in patterns of cytokine expression similar to those commonly observed following severe trauma or sepsis. Glucan treatment potentiated LPS-induced IFN-gamma expression in control mice. The induction of LPS tolerance resulted in marked suppression of LPS-induced IFN-gamma production. However, co-administration of glucan with LPS, during the tolerance induction phase, attenuated the LPS-tolerant response. Interleukin-12 (IL-12) and IL-18 are important mediators of LPS-induced IFN-gamma production. LPS-induced IL-12 p40 mRNA expression was increased in the spleens of glucan-treated mice compared with controls. Induction of LPS tolerance caused marked suppression of IL-12 production, a response that was attenuated by glucan treatment. IL-18 was constitutively expressed in both control and LPS-tolerant mice, and LPS-induced serum levels of IL-18 were increased in mice treated with glucan. T cells isolated from glucan-treated mice exhibited increased IFN-gamma expression in response to IL-12 and IL-18, as well as increased expression of the IL-12 and IL-18 receptors. The ability of glucan to potentiate IFN-gamma expression in control mice provides a potential mechanism by which glucan enhances antimicrobial immunity. The ability of glucan to attenuate suppressed IFN-gamma expression in LPS-tolerant mice denotes its potential benefit for the treatment of trauma and sepsis-induced immunosuppression.

Phase I and pharmacokinetic study of LU79553, a DNA intercalating bisnaphthalimide, in patients with solid malignancies.

PURPOSE: To determine the maximum-tolerated dose and characterize the pharmacokinetic behavior of LU79553, a novel bisnaphthalimide antineoplastic agent, when administered as a daily intravenous infusion for 5 days every 3 weeks. PATIENTS AND METHODS: Patients with advanced solid malignancies received escalating doses of LU79553. Plasma sampling and urine collections were performed on both days 1 and 5 of the first course. RESULTS: Thirty patients received 105 courses of LU79553 at doses ranging from 2 to 24 mg/m(2)/d. Proximal myopathy, erectile dysfunction, and myelosuppression precluded the administration of multiple courses at doses above 18 mg/m(2)/d. These toxicities were intolerable in two of six patients after receiving three courses at the 24-mg/m(2)/d dose level. At the 18-mg/m(2)/d dose, one of six patients developed febrile neutropenia and grade 2 proximal myopathy after three courses of LU79553. The results of electrophysiologic, histopathologic, and ultrastructural studies supported a drug-induced primary myopathic process. A patient with a platinum- and taxane-resistant papillary serous carcinoma of the peritoneum experienced a partial response lasting 22 months. Pharmacokinetics were dose-independent, optimally described by a three-compartment model, and there was modest drug accumulation over the 5 days of treatment. CONCLUSION: Although no dose-limiting events were noted in the first two courses of LU79553, cumulative muscular toxicity precluded repetitive treatment with LU79553 at doses above 18 mg/m(2)/d, which is the recommended dose for subsequent disease-directed evaluations. The preliminary antitumor activity noted is encouraging, but the qualitative and cumulative nature of the principal toxicities, as well as the relatively small number of patients treated repetitively, mandate that rigorous and long-term toxicologic monitoring be performed in subsequent evaluations of this unique agent.

A phase I clinical and pharmacokinetic study of the dolastatin analogue cemadotin administered as a 5-day continuous intravenous infusion.

PURPOSE: The dolastatins are a class of naturally occurring cytotoxic peptides which function by inhibiting microtubule assembly and tubulin polymerization. Cemadotin is a synthetic analogue of dolastatin 15 with potent antiproliferative and preclinical antitumor activity. This report describes a phase I study to evaluate the administration of cemadotin to adult cancer patients by a 5-day continuous intravenous (CIV) infusion. METHODS: All patients had histologically confirmed refractory solid tumors. The dose was escalated from an initial level of 2.5 mg/m2 (0.5 mg/m2 daily) according to a modified Fibonacci algorithm. A minimum of three patients was evaluated at each dose level until the maximum tolerated dose (MTD) was established. Treatment was repeated every 21 days until patients were removed from the study due to toxicity or disease progression. Drug-related toxicities were evaluated and graded by the U.S. National Cancer Institute's Common Toxicity Criteria. A radioimmunoassay (RIA) that detected both the parent drug and its metabolites with an intact N-terminal region of the molecule was used for pharmacokinetic studies. RESULTS: Twenty heavily pretreated patients received a total of 40 courses of cemadotin over five dose levels ranging from 2.5 to 17.5 mg/m2. Reversible dose-related neutropenia was the principal dose-limiting toxicity and 12.5 mg/m2 was established as the MTD. Nonhematologic toxicities attributed to the drug were moderate, and there was no evidence of the cardiovascular toxicity noted in the prior phase I studies of cemadotin given IV as a 5-min injection or 24-h infusion. There were no objective antitumor responses. Time courses of the cemadotin RIA equivalent concentration in whole blood were defined in 14 patients during the first cycle of therapy. The RIA-detectable species exhibited apparent first-order pharmacokinetics across the entire range of doses. The mean +/- SD of the observed steady-state blood concentration at the 12.5 mg/m2 MTD was 282 +/- 7 nM (n = 3). Blood levels decayed monoexponentially following the end of the infusion, with a mean half-life of 13.2 +/- 4.3 h (n = 14) in all patients. Mean values (n = 14) of the total blood clearance and apparent volume of distribution at steady state were 0.52 +/- 0.09 lh/m2 and 9.9 +/- 3.3 l/m2, respectively. CONCLUSIONS: The cardiotoxic effects of cemadotin were completely avoided by administering it as a 120-h CIV infusion. Thus. cardiovascular toxicity appears to be associated with the magnitude of the peak blood levels of the parent drug or its metabolites, whereas myelotoxicity is related to the duration of time that blood levels exceed a threshold concentration. Nevertheless, the data acquired during the extensive clinical experience with cemadotin requires careful examination to assess whether advancing this compound into disease-oriented efficacy studies is merited.

Hypercalcemia and T-cell lymphoma with acquired immunodeficiency syndrome: occurrence without human T-cell leukemia virus-I.

We describe the case of a patient with acquired immunodeficiency syndrome (AIDS) who had a CD4 cell count of 60/microL, bilateral hilar adenopathy, and hypercalcemia. Transbronchial biopsy showed T-cell anaplastic large cell lymphoma. Serology was negative for human T-cell leukemia virus-I (HTLV-I). This appears to be the first case of T-cell anaplastic large cell lymphoma occurring in an AIDS patient with hypercalcemia who was seronegative for HTLV-I.

Phase I and pharmacokinetic study of the water-soluble dolastatin 15 analog LU103793 in patients with advanced solid malignancies.

PURPOSE: To determine the maximum-tolerated dose (MTD), dose-limiting toxicities (DLTs), and pharmacokinetic profile of the dolastatin 15 analog LU103793 when administered daily for 5 days every 3 weeks. PATIENTS AND METHODS: Fifty-six courses of LU103793 at doses of 0.5 to 3.0 mg/m2 were administered to 26 patients with advanced solid malignancies. Pharmacokinetic studies were performed on days 1 and 5 of course one. Pharmacokinetic variables were related to the principal toxicities. RESULTS: Neutropenia, peripheral edema, and liver function test abnormalities were dose-limiting at doses greater than 2.5 mg/m2 per day. Four of six patients developed DLT at 3.0 mg/m2 per day, whereas two of 12 patients treated at 2.5 mg/m2 per day developed DLT. Pharmacokinetic parameters were independent of dose and similar on days 1 and 5. Volume of distribution at steady-state (Vss) was 7.6 +/- 2.0 L/m2, clearance 0.49 +/- 0.18 L/h/m2, and elimination half-life (t1/2) 12.3 +/- 3.8 hours. Peak concentrations (Cmax) on day 1 related to mean percentage decrement in neutrophils (sigmoid maximum effect (Emax) model). Patients who experienced dose-limiting neutropenia had significantly higher Cmax values than patients who did not, whereas nonhematologic DLTs were more related to dose. CONCLUSION: The recommended dose for phase II evaluations of LU103793 daily for 5 days every 3 weeks is 2.5 mg/m2 per day. The lack of prohibitive cardiovascular effects and the generally acceptable toxicity profile support the rationale for performing disease-directed evaluations of LU103793 on the schedule evaluated in this study.

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.

The role of forensic anthropology in the recovery and analysis of Branch Davidian compound victims: assessing the accuracy of age estimations.

Age-at-death estimations of 44 individuals (27 adults, 17 children) from the Branch Davidian sample were compared with their actual ages. Estimations were evaluated for bias and accuracy for the actual age at death. Although the overall average estimates correlated well with the actual ages (r = 0.946), several individuals displayed high residual requiring further analysis and review. These individuals displayed age-related features that did not correspond with the expected morphology for individuals of their ages. Several age estimation techniques scored these individuals with all bias in the same direction. These examples should serve as cautionary reminders that biology does not always correlate with expected outcomes, particularly in such multifaceted traits such as age.

The role of forensic anthropology in the recovery and analysis of Branch Davidian Compound victims: recovery procedures and characteristics of the victims.

Anthropological contributions to the investigation of the events at the Branch Davidian Compound near Waco, Texas in early 1993, were of two major types: the recovery of human remains from the site and the analysis of most of those individuals at the Medical Examiner's Office in Fort Worth, Texas. This paper describes the role of forensic anthropology in the recovery and analysis of Branch Davidian Compound victims and the recovery procedures and characteristics of the victims.

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.

Multiple fluorescent ligands for dopamine receptors. I. Pharmacological characterization and receptor selectivity.

We report the synthesis and pharmacological characterization of novel fluorescently labeled ligands with high affinity and specificity for D1 and D2 dopamine receptors. D1-selective antagonist probes have been synthesized using (R,S)-5-(4'-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-[1H]-3- benzazepin-7-ol, the 4'-amino derivative of the high affinity D1-selective antagonist, SCH-23390, while D2-selective antagonist probes were synthesized using the high affinity, D2-selective agonist, N-(p-aminophenethyl)spiperone (NAPS). In addition, we have synthesized fluorescent probes using an amino-derivative of the high affinity, D2-selective agonist, 2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin (PPHT or N-0434). These ligands were coupled to the fluorescent moieties, fluorescein, rhodamine, coumarin, Texas red, Cascade blue, or Bodipy. This resulted in a wide variety of dopaminergic ligands which fluoresce at different wavelengths: Cascade blue and coumarin are blue fluorophores, fluorescein and Bodipy, are yellow-green, and Texas red and rhodamine are red. The interaction of these fluorescent ligands with dopamine and serotonin receptors was evaluated by examining their ability to compete for radioligand binding to D1 and D2 dopamine receptors and 5-HT1A, 5-HT1C and 5-HT2 serotonin receptors. We report here that these novel fluorescent ligands exhibit high affinity and, in general, selectivity for either D1 or D2 dopamine receptors. In addition, we demonstrate that the fluorescent derivatives of PPHT retain the full agonist efficacy exhibited by the parent compound.(ABSTRACT TRUNCATED AT 250 WORDS)

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.