Pharmacokinetic studies in mice of two new thioxanthenones (183577 and 232759) that showed preferential solid tumor activity.

Two new thioxanthenones, 183577 and 232759, have rekindled interest in the development of representatives from this class of structures as useful anticancer agents. Although the mechanism of action is unknown, both compounds demonstrated a similar spectrum of solid tumor selectivity. 232759 was selected for clinical development because it showed no hepatotoxicity in preliminary studies, whereas 183577 showed hepatotoxicity but only at the maximum tolerated dose (MTD). The limiting toxicity for the clinical candidate was myelosuppression in preliminary studies. Plasma and tissue drug levels, as well as protein binding, were studied in mice using optimal administration times at the MTD for each drug (for 183577, this was a 4-h infusion at 1350 mg/m2 and for 232759, it was a 5-min injection at 240 mg/m2), as well as at one-half the MTD for the clinical candidate. The drugs were 96-100% bound by plasma proteins. The peak drug concentrations, half-life, and area under the concentration-time curve in plasma for 183577 were 3483 ng/ml, 465 min, and 2018 microgram/ml. min, respectively. The peak drug concentration, half-life, and area under the concentration-time curve in plasma for 232759 were 5257 ng/ml, 44 min, and 276 microgram/ml. min, respectively, at the MTD and 2810 ng/ml, 40 min, and 110 microgram/ml. min at one-half the MTD. In all instances of simultaneous measurements, drug concentrations were equal or higher in tissues than they were in plasma. Unlike the plasma and kidney concentrations of 183577, the liver concentrations did not show a declining trend over the 8-h observation period. Declines in plasma, liver, kidney, and tumor levels of 232759 were detected over the 8-h observation period. The sustained high 183577 concentration in liver is believed to be responsible for its prolonged half-life and hepatotoxicity. Evidence for metabolism of the parent drugs was based on the finding of additional peaks on the high-pressure liquid chromatography tracings. Future studies will focus on identification and antitumor studies of these presumed metabolites in hopes of a better understanding of the solid tumor activity profiles and toxic effects of these compounds.

Phase I pharmacokinetic study of the novel antitumor agent SR233377.

SR233377 is a novel thioxanthenone analogue that demonstrated solid tumor selectivity in vitro with activity confirmed in vivo against several murine tumors including those of colon, pancreas, and mammary origin. Its primary preclinical dose-limiting toxicities included myelosuppression and neurological toxicity. The neurological toxicity was acute and could be ameliorated in mice when the drug was administered as a 1-h infusion instead of rapid i.v. injection. As a result of its preclinical efficacy profile, SR233377 entered Phase I clinical investigation. The compound was administered i.v. over 2 h on day 1 repeated every 28 days. The starting dose was 33 mg/m2 (one-tenth the mouse LD10). Escalations continued to 445 mg/m2 (six escalations), where dose-limiting toxicity was observed. At this dose, acute ventricular arrhythmias, including one patient with torsades de pointes and transient cardiac arrest, occurred. Because this toxicity might have been related to the plasma peak, the protocol was amended to a 24-h infusion beginning at 225 mg/m2. With this dose, prolongation of the corrected QT interval (QTc) over the pretreatment levels resulted. Because prolonged QTc is a known forerunner to acute ventricular arrhythmias, clinical development of SR233377 was stopped. However, preclinical antitumor and toxicity studies with analogues are underway with hopes of identifying a new clinical candidate with similar antitumor effects that is devoid of cardiac toxic effects.

Identification of a novel mutation (867delA) in the glucose-6-phosphatase gene in two siblings with glycogen storage disease type Ia with different phenotypes.

We identified a novel mutation (867delA) in the glucose-6-phosphatase gene of two siblings with glycogen storage disease type Ia. Although both siblings share the same mutations, their phenotype regarding adult height and hepatomegaly differs. In glycogen storage disease type Ia, substantial heterogeneity in phenotype is observed. So far, no evidence for a clear genotype-phenotype correlation has been found. Hum Mutat 15:381, 2000.

Synthesis and bacterial DNA gyrase inhibitory properties of a spirocyclopropylquinolone derivative.

A novel conformationally restricted 1-cyclopropylquinolone (1) that incorporates structural features of both ofloxacin and ciprofloxacin has been prepared. Compound 1 was found to be a DNA gyrase inhibitor having potency similar to ofloxacin but less than ciprofloxacin. The cellular inhibitory and in vivo antibacterial potencies of 1 were found to be less than those of the two reference agents.

Mammalian topoisomerase II inhibitory activity of 1-cyclopropyl-6,8- difluoro-1,4-dihydro-7-(2,6-dimethyl-4-pyridinyl)-4-oxo-3-quinolinecarb oxylic acid and related derivatives.

1-Cyclopropyl-6,8-difluoro-1,4-dihydro-7-(2,6-dimethyl-4-pyridinyl)-4-ox o-3-quinolinecarboxylic acid (1), a previously reported potent inhibitor of bacterial DNA gyrase, was found to be interactive with mammalian topoisomerase II (topo II). In a DNA-cleavage assay using topo II isolated from HeLa cells, 1 exhibited an EC50 value of 7.6 microM (VP-16; EC50 = 0.81 microM). A series of analogues modified at the 1-, 2-, 3-, 5-, and 7-positions of 1 were subsequently made and assessed for topo II inhibition. Compound 1 was considerably more potent than derivatives where the 1-substituent was alkyl, aryl, or H, or when N-c-C3H5 was replaced with S. The descarboxyl (i.e., 3-H) analogue had potency comparable to that of 1; when both these compounds were substituted at the 2-position with methyl or phenyl, an interesting relationship between activity and the conformation of the carboxyl group emerged. Upon replacement of the 5-H of 1 with NH2 or F, sustained potency was seen. No enhancement of activity was evident upon replacing the 7-substituent of 1 with other pyridinyl groups, 4-methyl-1-piperazinyl, or pyrrolidinyl groups; however, the 7-(4-hydroxyphenyl) analogue (CP-115,953) was 6-fold more potent than 1. The topo II inhibitory properties of 1 translated to modest in vitro cytotoxicity and in vivo activity versus P388.

Synthesis and antitumor activity of 4-aminomethylthioxanthenone and 5-aminomethylbenzothiopyranoindazole derivatives.

Two new series of antitumor agents, 4-aminomethylthioxanthenones (6-50) and 5-aminomethylbenzothiopyranoindazoles (51-61), are described and compared. Nearly all members of both series display excellent in vivo activity versus murine pancreatic adenocarcinoma 03 (Panc03) although there is little to distinguish the two series from each other. In both series there is no discernible relationship between structure and in vivo efficacy. Selected analogues were evaluated in vitro; all were observed to have moderate to strong DNA binding via intercalation. However, varying degrees of in vitro P388 cytotoxicity and topoisomerase II inhibition were seen. In general, those molecules which exhibited strong topoisomerase II inhibition were significantly more cytotoxic than those which did not. In both series, those derivatives (48-50, 60, and 61) having a phenolic hydroxy substitution exhibited the most potent P388 cytotoxicity and topoisomerase II inhibition.

Evidence of enhanced in vivo activity using tirapazamine with paclitaxel and paraplatin regimens against the MV-522 human lung cancer xenograft.

PURPOSE: Tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide; SR 4233) is a bioreductive agent that exhibits relatively selective cytotoxicity towards cells under hypoxic conditions and can enhance the antitumor activity of many standard oncolytics. In the present study we examined the interaction between tirapazamine in vivo with paclitaxel and paraplatin in two- and three-way combination studies using the MV-522 human lung carcinoma xenograft model. METHODS: Agents were administered as a single i.p. bolus, with tirapazamine being given 3 h prior to paclitaxel, paraplatin, or their combination. Tumor growth inhibition (TGI), final tumor weights, partial and complete responses, and time to tumor doubling were determined after drug administration. RESULTS: Tirapazamine as a single agent was ineffective against this human lung tumor model. A substantial increase in TGI was seen in animals treated with the triple-agent regimen (tirapazamine-paclitaxel-paraplatin) compared to animals treated with double-agent regimens that did not include tirapazamine. The addition of tirapazamine to paclitaxel-paraplatin therapy resulted in a 50% complete response rate; there were no complete responses seen when only the paclitaxel-paraplatin combination was administered. Time to tumor doubling was also significantly improved with the addition of tirapazamine to the paclitaxel and paraplatin combinations. Tirapazamine did not increase the toxicity of paclitaxel, paraplatin, or their combinations as judged by its minimal impact on body weight and the fact that no toxic deaths were observed with tirapazamine-containing regimens. CONCLUSIONS: These results are important since recent studies have suggested that the combination of paclitaxel and paraplatin may be particularly active in patients with advanced stage non-small-cell lung cancer. Since tirapazamine can significantly improve efficacy, but does not appear to enhance the toxicity of paclitaxel and paraplatin, its evaluation in future clinical trials in combination with paclitaxel-paraplatin-based therapy appears warranted.

Neurotensin receptor-mediated inhibition of pancreatic cancer cell growth by the neurotensin antagonist SR 48692.

Second messenger calcium responses to the neuropeptide neurotensin and its non-peptide antagonist SR 48692 were studied in relation to the proliferation of pancreatic cancer cells. Neurotensin caused a transient increase in intracellular calcium in two pancreatic lines, MIA PaCa-2 and PANC-1, with EC50 values of 4.6 and 11.4 nM and peak calcium concentrations of 190% and 470% of basal levels, respectively. SR 48692 inhibited these calcium changes with an IC50 (at 25 nM neurotensin) of 4.9 and 4.1 nM in MIA PaCa-2 and PANC-1 cells, respectively. In MIA PaCa-2 cells, SR 48692 may act as an inverse agonist as it depressed basal calcium. SR 48692 inhibited growth of both MIA PaCa-2 and PANC-1 cells. Only in MIA PaCa-2 cells did neurotensin overcome this inhibition or stimulate proliferation. The results imply that, in MIA PaCa-2 cells, the neurotensin antagonist SR 48692 inhibits growth in a neurotensin receptor-mediated fashion.

Affinity of cefonicid, a long-acting cephalosporin, for the penicillin-binding proteins of Escherichia coli K-12.

The binding of cefonicid (SK&F 75073), a new parenteral cephalosporin, to the penicillin-binding proteins (PBPs) of Escherichia coli K-12 (strain KN-126) was determined by competitive binding studies versus benzyl[14C]penicillin. Cefonicid showed its greatest affinity for PBPs 1a greater than 3 greater than 1b, bound with low affinity to PBPs 4 greater than 2, and did not bind to PBPs 5 and 6. Provisional affinity constants (cefonicid concentration that gave 50% inhibition of [14C]penicillin binding) were determined: PBP 1a, less than 0.25 microgram/ml; PBP 3, 0.7 microgram/ml; PBP 1b, 10 micrograms/ml; PBP 4, 26 micrograms/ml; PBP 2, 90 micrograms/ml; PBPs 5 and 6 greater than 256 micrograms/ml. Direct binding studies with [14C]-cefonicid confirmed this pattern of binding. Subinhibitory concentrations of cefonicid (MIC, broth 0.2 microgram/ml, agar 0.4 microgram/ml) induced filamentation of E. coli KN-126. This implies that PBP 3 is the primary inhibitory site despite the higher affinity of PBP 1a for this cephalosporin.

Glycopeptide antibiotics: a mechanism-based screen employing a bacterial cell wall receptor mimetic.

The evolution of a highly targeted screening program for the discovery of antibiotics of the glycopeptide (vancomycin) class is described. A holistic approach was utilized which optimized not just screening techniques but also the selection of candidate producer cultures and their growth under conditions which enhanced production of target compounds. Two screen techniques were utilized; differential inhibition of a vancomycin-resistant strain and its susceptible parent, and a specific antagonism screen using the reversal of glycopeptide activity by a tripeptide analog of the glycopeptide receptor, diacetyl-L-lysyl-D-alanyl-D-alanine. The latter screen was 2- to 32-fold more sensitive to known glycopeptides than the former, and was absolutely specific, yielding no false positive responses. The use of the tripeptide antagonism assay, combined with optimized culture selection and growth conditions yielded novel glycopeptide antibiotics at a rate of 1 per 320 cultures screened. With a holistic approach to screening and properly optimized techniques, large numbers of cultures do not need to be examined in order to discover novel antibiotics.

A patient with common glycogen storage disease type Ib mutations without neutropenia or neutrophil dysfunction.

We describe a 16-year old boy with glycogen storage disease type Ib, homozygous for the common 1211-1212delCT mutation, who never experienced neutropenia, and did not suffer from frequent infections or inflammatory bowel disease. In addition, neutrophil function tests showed no abnormalities.

1H chemical shift imaging of the brain in guanidino methyltransferase deficiency, a creatine deficiency syndrome; guanidinoacetate accumulation in the gray matter.

MR spectroscopy results in a mild case of guanidinoacetate methyltransferase (GAMT) deficiency are presented. The approach differs from previous MRS studies in the acquisition of a chemical shift imaging spectral map showing gray and white matter with the corresponding spectra in one overview. MR spectroscopy revealed guanidinoacetate (GAA) in the absence of creatine. New is that GAA signals are more prominent in gray matter than in white. In the prevailing view, that enzyme deficiency is localized in liver and pancreas and that all GAA is transported into the brain from the blood and the cerebrospinal fluid, this would be compatible with a more limited uptake and/or better clearance of GAA from the white matter compared to the grey matter.

Normal very-long-chain fatty acids in peroxisomal D-bifunctional protein deficiency: a diagnostic pitfall.

We present a relatively mild case of peroxisomal D-bifunctional protein deficiency with inconsistent screening results in plasma for peroxisomal disorders.

Inhibition, but not uncoupling, of respiratory energy coupling of three bacterial species by nitrite.

The effect of nitrite on respiratory energy coupling of three bacteria was studied in light of a recent report that nitrite acted as an uncoupling agent with Paracoccus denitrificans grown under denitrifying conditions. Our determinations of proton translocation stoichiometry of Pseudomonas putida (aerobically grown), Pseudomonas aeruginosa, and P. denitrificans (grown both aerobically and under denitrifying conditions) showed nitrite inhibition of proton-to-oxidant stoichiometry, but not uncoupling. Nitrite both reduced the H+/O ratio and decreased the rate of proton resorption. Increased proton resorption rates, characteristic of authentic uncoupling agents, were not observed. The lack of enhanced proton permeability due to nitrite was verified via passive proton permeability assays. The H+/O ratio of P. aeruginosa increased when growth conditions were changed from aerobic to denitrifying. This suggested the induction of an additional coupling site in the electron transport chain of denitrifying P. aeruginosa.

Inhibition of platelet-derived growth factor-mediated signal transduction by transforming ras. Suppression of receptor autophosphorylation.

The function of ras protein and its relationship to growth-factor mediated signal transduction remain unclear. The demonstration that the expression of transforming ras (v-ras) can block the stimulation of growth-related gene expression and cell division mediated by the platelet-derived growth factor (PDGF) may provide a model for the functional interation of ras with growth factor receptors. In the current studies, we have demonstrated that this blockade by v-ras of PDGF-BB signal transduction occurs very early in signal transduction, at the level of PDGF receptor autophosphorylation. Although the expression of PDGF receptor as detected by Western blot with anti-PDGF receptor antibody was not diminished in v-ras-transformed murine Balb/c 3T3 fibroblasts, the autophosphorylation of PDGF receptor in response to ligand (recombinant PDGF-BB homodimer) stimulation was profoundly suppressed. This same phenomenon of v-ras-mediated PDGF receptor autophosphorylation inhibition was also demonstrated in normal rat kidney fibroblasts. Further, factor(s) present in v-ras-expressing fibroblasts found in the membrane fractions of these cells can dominantly inhibit the autophosphorylation of the PDGF receptor obtained from normal fibroblasts. These findings suggest a role for ras in one of the earliest steps of the signal transduction pathway.

Bacterial inhibitory effects of nitrite: inhibition of active transport, but not of group translocation, and of intracellular enzymes.

Nitrite inhibited active transport of proline in Escherichia coli but not group translocation of sugar via the phosphoenolpyruvate:phosphotransferase system. These results were consistent with previous results that nitrite inhibits active transport, oxygen uptake, and oxidative phosphorylation in aerobic bacteria. Nitrite also inhibited aldolase (EC 4.1.2.13) from E. coli, Pseudomonas aeruginosa, Streptococcus faecalis, and rabbit muscle. Thus, these various data showed that nitrite has more than one site of attack in the bacterial cell. These data also indicated that nitrite is inhibitory to a wide range of physiological types of bacteria.

Dissociation of platelet-derived growth factor (PDGF) receptor autophosphorylation from other PDGF-mediated second messenger events.

Activated p21ras alters the platelet-derived growth factor (PDGF) signal transduction pathway in fibroblasts by inhibiting autophosphorylation of the receptor as well as by inhibiting the induction of the growth-related genes c-myc, c-fos, and JE. To elucidate the cause and effect relationships between receptor autophosphorylation and other second messenger events in the PDGF signaling pathway we created revertants of v-ras transformed cells by two methods: 1) the use of cAMP analogues, and 2) the introduction of a gene, Krev-1, which has been reported previously to revert ras transformed cells to normal morphology. Analysis of the revertants shows that the PDGF-mediated tyrosine phosphorylation of the 180-kDa PDGF receptor remains inhibited; however, the PDGF-mediated activation of phospholipase C and the induction of the growth-related genes c-myc, c-fos, and JE have been restored. These data suggest the presence of parallel pathways for PDGF signal transduction which are not dependent on autophosphorylation of the PDGF receptor.

Effect of dihydrostreptomycin on active transport in isolated bacterial membrane vesicles.

Membrane vesicles prepared from bacterial cells grown in the absence of dihydrostreptomycin but subsequently incubated in the presence of dihydrostreptomycin transported proline normally, but vesicles prepared from cells grown in media to which dihydrostreptomycin was added 30 min before harvesting had a greatly impaired ability to accumulate proline. The latter cells extruded protons normally but were unable to maintain a proton gradient as effectively as normal cells. These data indicated that metabolism was required for dihydrostreptomycin to exert an effect on the bacterial cell membrane.

Characteristic growth pattern in male X-linked phosphorylase-b kinase deficiency (GSD IX).

Growth retardation is one of the clinical characteristics of glycogen storage disease (GSD) type IX. Initial growth retardation has been described in a few case reports, followed by a complete catch-up in growth. This study aimed to determine the growth pattern of patients with GSD IX. Growth charts of 51 male Dutch patients with GSD IX (age 0-33 years, median follow-up time 8.3 years (range 0-30.5 years)) were studied retrospectively and compared with Dutch standard growth charts. Patients had a normal height at birth, significant growth retardation between the ages of 2 and 10 years (mean z-score -1.96), delayed growth spurt in puberty and catch-up towards quite normal final height (mean z-score -0.55). We conclude that GSD IX patients have a specific growth pattern characterized by initial growth retardation, a late growth spurt and complete catch-up in final height. Intervention for growth retardation is therefore in general not warranted. It is speculated that mild hypoglycaemia related to the disorder may cause endocrine changes. Because the glucose need per kg bodyweight decreases with age, the enzyme defect becomes less important with ageing and the effect on growth diminishes.

The effect of phenazine methosulfate-ascorbate on bacterial active transport and adenosine triphosphate formation: inhibition of Pseudomonas aeruginosa and stimulation of Escherichia coli.

The artificial electron-donor system, phenazine methosulfate (PMS) ascorbate, inhibited active transport of glucose by Pseudomonas aeruginosa irrespective of whether the incubation systems were in air, flushed with oxygen, or gassed with nitrogen under anaerobic denitrifying conditions. Active transport of glucose by P. aeruginosa was also inhibited by reduced 5-N-methyl-phenazonium-3-sulfonate, a membrane-impermeable electron donor. PMS-ascorbate caused rapid depletion of intracellular adenosine triphosphate (ATP) when added to respiring cell suspensions of P. aeruginosa either in the presence or absence of glucose or succinate as oxidizable energy sources. In contrast, under identical conditions, Escherichia coli formed ATP with PMS-ascorbate as the sole oxidizable energy source and ATP formation continued when glucose or succinate was present in addition to PMS-ascorbate in the incubation system.