Phase I study of TAS-115, a novel oral multi-kinase inhibitor, in patients with advanced solid tumors.

TAS-115 is a novel MET, VEGFR, FMS and PDGFR inhibitor, developed to improve the continuity of drug administration with a relatively short half-life. We assessed its tolerability, safety, pharmacokinetics, efficacy, and pharmacodynamics in patients with solid tumors. This open-label, dose-escalation phase I study of TAS-115 consisted of three parts: part 1 (TAS-115 was administered orally once daily [SID]); part 2 and an expansion part (SID in a 5 days on/2 days off [5-on/2-off] schedule for 21 days per cycle). In part 1 (200-800 mg SID administered to 21 patients), systemic exposure after single administration increased almost dose-proportionally. Three dose-limiting toxicities (DLTs) were observed in three patients: grade 3 rash (650 mg), thrombocytopenia with bleeding, and rash (800 mg). The maximum tolerated dose (MTD) was determined as 650 mg SID. In part 2, the 5-on/2-off schedule was evaluated at the MTD to improve treatment exposure. No DLTs were observed and no patients required treatment interruption in cycle 1. During part 2 and the expansion part (N = 61), grade ≥3 treatment-related adverse events were reported in 47 patients, with neutropenia (24.6%), hypophosphatemia (21.3%), anemia, and thrombocytopenia (14.8% each), and leukocytopenia (11.5%) occurring in ≥10% of patients. The best overall response was stable disease in 31 of 82 patients (37.8%). An apparent reduction in fluorodesoxyglucose-uptake and bone scan index was observed in some patients. TAS-115 was generally well tolerated, with manageable toxicities and recommended phase II dose was estimated as 650 mg SID, 5-on/2-off. Furthermore, promising antitumor activity was observed.

Salt-sparing diuretic action of a water-soluble urea analog inhibitor of urea transporters UT-A and UT-B in rats.

Inhibitors of kidney urea transporter (UT) proteins have potential use as salt-sparing diuretics ('urearetics') with a different mechanism of action than diuretics that target salt transporters. To study UT inhibition in rats, we screened about 10,000 drugs, natural products and urea analogs for inhibition of rat UT-A1. Drug and natural product screening found nicotine, sanguinarine and an indolcarbonylchromenone with IC50 of 10-20 µM. Urea analog screening found methylacetamide and dimethylthiourea (DMTU). DMTU fully and reversibly inhibited rat UT-A1 and UT-B by a noncompetitive mechanism with IC50 of 2-3 mM. Homology modeling and docking computations suggested DMTU binding sites on rat UT-A1. Following a single intraperitoneal injection of 500 mg/kg DMTU, peak plasma concentration was 9 mM with t1/2 of about 10 h, and a urine concentration of 20-40 mM. Rats chronically treated with DMTU had a sustained, reversible reduction in urine osmolality from 1800 to 600 mOsm, a 3-fold increase in urine output, and mild hypokalemia. DMTU did not impair urinary concentrating function in rats on a low protein diet. Compared to furosemide-treated rats, the DMTU-treated rats had greater diuresis and reduced urinary salt loss. In a model of syndrome of inappropriate antidiuretic hormone secretion, DMTU treatment prevented hyponatremia and water retention produced by water-loading in dDAVP-treated rats. Thus, our results establish a rat model of UT inhibition and demonstrate the diuretic efficacy of UT inhibition.

CETP genotype and concentrations of HDL and lipoprotein subclasses in African-American men.

Aim: To assess the association between the CETP Taq1B and I405V polymorphisms with levels of lipoprotein subclasses in African-American (AA) men with and without Type 2 diabetes (T2DM). Patients & methods: AA men, over 30 years of age, with (n = 54) or without T2DM (n = 50), and not receiving lipid-lowering agents, underwent advanced lipid analysis and genotyping. Results & conclusion: In the total patient population Taq1B B2-allele carriers had significantly higher levels of large HDL subclasses (HDL-2b [p = 0.017] and HDL-L [p = 0.019]), lower levels of small-HDL subclasses (HDL-3a [p = 0.004] and HDL-3b [p = 0.031]), and lower levels of LDL subclasses (LDL-IVa [p = 0.012] and LDL-IIIb [p = 0.009]). The only significant genotype-diabetes interaction occurred with the HDL-2a subclass (p = 0.015). No statistically significant associations were seen with I405V genotype. Our observations of lower levels of small-HDL and higher levels of large-HDL suggest that a potentially important HDL subclass-CETP relationship exists.

Permeability of rabbit polymorphonuclear leukocyte membranes to urea and other nonelectrolytes.

The diffusional permeability coefficients of rabbit polymorphonuclear leukocyte membranes to urea, methylurea and thiourea have been measured. It was found that the permeability coefficient of these membranes to urea is very low and that thiourea was more permeable than methylurea which was, in turn, more permeable than urea. These results suggest that there is no need to postulate a carrier-mediated mechanism for urea transport across biological membranes and that the concept of "aqueous pores" is not a general property of biological membranes but restricted only to certain cases.

Transferrin-bipyridine iron transfer mediated by haemoproteins.

Rabbit reticulocyte cytosol was able to mediate transferrin-bipyridine iron transfer in the presence of ATP. The cytoplasmic factor responsible for the mediation of iron transfer was identified as haemoglobin. Other cytoplasmic proteins and the membrane fraction were ineffective. Human alpha and beta subunits and human myoglobin were over three times more effective than human haemoglobin A. Carbon monoxide strongly inhibited the mediation of iron transfer. Oxidation of haemoglobin abolished it but methaemoglobin could be reactivated with NADH, even when azide was bound to the haem iron. Neither globin nor haem alone were able to mediate iron transfer, even when NADH was present. Together, the reconstituted methaemoglobin A could be reactivated wtih NADH. Although the physiological significance of this pehnomenon is not clear, the involvement of haemoproteins in intracellular iron metabolism seems likely.

Synthesis and evaluation of two positron-labeled nitric oxide synthase inhibitors, S-[11C]methylisothiourea and S-(2-[18F]fluoroethyl)isothiourea, as potential positron emission tomography tracers.

In an effort to develop a tracer for probing inducible nitric oxide synthase (iNOS) levels in vivo utilizing positron emission tomography, we have synthesized and evaluated two positron-emitting iNOS selective inhibitors: S-[11C]methylisothiourea (1b) and S-(2-[18F]fluoroethyl)-isothiourea (3b). Prior to fluorine-18 labeling, the nonradioactive fluoro derivative S-(2-fluoroethyl)isothiourea (3a) was prepared and determined to have a 9-fold higher selectivity for iNOS compared to endothelial NOS (eNOS). Radiochemical synthesis of both compounds, in high radiochemical purity and at high specific activity, was accomplished by the S-alkylation reaction of labeled precursors (11CH3I or 18FCH2CH2OTf) with thiourea. An in vitro model, J774 macrophage cell line, was used to assess the uptake of radiolabeled iNOS inhibitor in response to iNOS induction at the cellular level. Increased cell uptake of these two labeled compounds at stimulated iNOS levels, as well as blocking under controlled in vitro conditions, was observed. Lipophilicity (log P o/w), stability, and tissue biodistribution data of both compounds are reported. Serum stability studies indicate that 3b metabolized much more rapidly compared to the relatively stable 1b in vitro and in vivo. Based on in vitro cell uptake data, both tracers were further evaluated in lipopolysaccharide (LPS)-pretreated rats. LPS has been reported to induce iNOS protein expression in the liver, lung, heart, and kidney and other tissues. The uptake for LPS-pretreated rats (6 h post-treatment) was significantly increased in the liver, kidney, and heart for 3b at 10 min and in the liver and lung for 1b at 30 min. The results suggest that this first generation of radiolabeled inhibitors may be useful for assessing induction of iNOS in vivo with PET.

HMR1402, a potassium ATP channel blocker during hyperdynamic porcine endotoxemia: effects on hepato-splanchnic oxygen exchange and metabolism.

OBJECTIVE: To assess the effects of the potassium ATP (KATP) channel blocker HMR1402 (HMR) on systemic and hepato-splanchnic hemodynamics, oxygen exchange and metabolism during hyperdynamic porcine endotoxemia. DESIGN: Prospective, randomized, controlled study with repeated measures. SETTING. Animal laboratory. SUBJECTS: Eighteen pigs allocated to receive endotoxin alone (control group, CON, n=10) or endotoxin and HMR (6 mg/kg h(-1), n=8). INTERVENTIONS: Anesthetized, mechanically ventilated, and instrumented pigs receiving continuous i.v. endotoxin were resuscitated with hetastarch to maintain mean arterial pressure (MAP) >60 mmHg. Twelve hours after starting the endotoxin infusion, they received HMR or its vehicle for another 12 h. RESULTS: HMR transiently increased MAP by about 15 mmHg, but this effect was only present during the first 1 h of infusion. The HMR decreased cardiac output due to a fall in heart rate, and thereby reduced liver blood flow. While liver O(2) delivery and uptake remained unchanged, HMR induced hyperlactatemia [from 1.5 (1.1; 2.0), 1.4 (1.2; 1.8), and 1.2 (0.8; 2.0) to 3.1 (1.4; 3.2), 3.2 (1.6; 6.5), and 3.0 (1.0; 5.5) mmol/l in the arterial, portal and hepatic venous samples, respectively] and further increased arterial [from 8 (3; 13) to 23 (11; 57); p<0.05], portal [from 9 (4; 14) to 23 (14; 39); p<0.05] and hepatic vein [from 7 (0; 15) to 30 (8; 174), p<0.05] lactate/pyruvate ratios indicating impaired cytosolic redox state. CONCLUSION: The short-term beneficial hemodynamic effects of KATP channel blockers have to be weighted with the detrimental effect on mitochondrial respiration.

Identification of 1-methylthiourea as the metabolite of ethylenethiourea in rats by high-performance liquid chromatography.

The method is described for determination of a metabolite of ethylenethiourea (ETU) in rats. ETU and its metabolites were extracted from plasma into ethanol after collecting the blood from female rats treated orally with ETU. Then the extract was cleaned up on a silica gel column chromatography and examined by high-performance liquid chromatography (HPLC). About 0.014 ppm of 1-methylthiourea was detected as the metabolite of ETU in plasma collected 2 h after an oral administration of 200 mg/kg of ETU.

Liquid chromatographic determination of noxytiolin and 1-methyl-2-thiourea in serum: application to pharmacokinetic studies in rabbits and humans.

A high-performance liquid chromatographic method is described which determines noxytiolin and 1-methyl-2-thiourea concentrations in serum. Valid determination requires immediate ultracentrifugation of blood samples, rapid serum freezing, and injection into the chromatograph within 6 h. A number of pharmacokinetic parameters were calculated from serum concentration data in rabbits and humans. An unknown metabolite was detected in both species but its structure was not identified.

Differential handling of urea and its analogues suggests carrier-mediated urea excretion in freshwater rainbow trout.

The possible presence of urea transport mechanisms in the gill and kidney of the freshwater rainbow trout (Oncorhynchus mykiss) was investigated in vivo by comparing the branchial and renal handling of analogues acetamide and thiourea with the handling of urea. Trout were fitted with indwelling dorsal aortic catheters and urinary catheters and injected with an isosmotic dose of [(14)C]-labeled urea analogue (acetamide or thiourea) calculated to bring plasma analogue concentrations close to plasma urea concentrations. Urea and analogue concentrations were significantly greater in the urine than in the plasma. Branchial clearance rate of acetamide was only 48% of urea clearance, whereas the clearance of thiourea was only 22%, a pattern that was also observed in branchial uptake of these substances and was similar to our previous observations in toadfish and midshipmen. The renal secretion clearance rates of urea and acetamide were similar, and on average, both substances were secreted on a net basis, although reabsorption did occur in some cases. In contrast, thiourea was neither reabsorbed nor secreted by the kidney tubule. The secretion clearance rates of both acetamide and urea were well correlated with the secretion clearance rates of Na(+), Cl(-), and water, whereas there was no relationship between thiourea and these substances. The pattern of acetamide, thiourea, and urea handling by the gill of the trout is similar to that found in the gills of the midshipman and the gulf toadfish and strongly suggests the presence of a UT-type facilitated diffusion urea transport mechanism. The pattern of differential handling in the kidney is unlike that in the gill and also unlike that in the kidney of the midshipman and the gulf toadfish, suggesting a different mechanism. In addition, renal urea secretion occurs against a concentration gradient, suggesting the involvement of an active transport mechanism.

Lipopolythiourea transfecting agents: lysine thiourea derivatives.

Synthetic vectors represent an alternative to recombinant viruses for gene transfer. We have recently explored the transfection potential of a class of noncationic lipids bearing thiourea moieties as DNA-associating headgroups. The encouraging results obtained with lipopolythioureas derived from serinol prompted us to further investigate this family of vectors. In the present study, we considered the transfection properties of a series of derivatives based on a different thiourea polar headgroup bearing a lysine scaffold. The synthesis of these compounds could be readily achieved in three steps with good yields. We found that these lipopolythioureas (LPT) might be considered as alternative systems for gene transfection since their activity reached the same magnitude range as that of cationic vectors in the presence of serum. LPT with 14-carbon length chains appeared to be more efficient as a transfecting agent than the ones with shorter chains. Toxicity studies proved that the hydration film method led to particles well tolerated both by the cells in vitro and by the mice in vivo. The ability to induce gene expression in vivo was demonstrated by intratumoral injection. Finally, biodistribution studies showed that the quantity recovered in blood circulation, 2 h after systemic injection, was improved as compared to that in cationic lipids.

Comparative study of the thiourea carrier in erythrocytes.

A densimeter technique was used to measure the rate of exit of thiourea from erythrocytes of various species of mammals. The cells were first equilibrated with a 200 mM thiourea solution in 1% NaCl. An aliquot of these cells was added to 1% NaCl containing 4.6-23.1 mM thiourea. Facilitated diffusion was demonstrated in each case. Using exit times or initial rates, calculations of half-saturation constants (phi) in mM and maximum transport rates (K) in isotones per min were made by three different methods. The following values were obtained: human-phi=60, 42, 35; K=1.2, 2.9, 0.9; rabbit-phi=46, 33, 32; K=0.8, 2.1, 0.8; mouse-phi=46, 40, 30; K=3.4, 8.5, 3.2; rat-phi=65, 42, 23; K=6.1, 15.3, 3.7; ox-phi=107, 63, 88; K=0.6, 1.4, 0.4; sheep-phi=56, 38, 56; K=0.9, 2.2, 0.6; and pig-phi=110, 64, 49; K=1.6, 3.6, 1.1.

Urea transport deficiency in Jk(a-b-) erythrocytes.

The activity of the urea transporter was determined in human erythrocytes of the Kidd blood type Jk(a-b-) by measuring unidirectional urea and thiourea fluxes in tracer flux experiments and urea net fluxes in light-scattering experiments. When compared with control cells, Jk(a-b-) cells exhibited diminished urea and thiourea fluxes and lacked the kinetic characteristics of mediated transport, suggesting that in these cells urea and thiourea moved only by simple diffusion through the lipid bilayer. Control experiments showed that anion, water, and ethylene glycol permeabilities were the same in Jk(a-b-) and control cells. These experiments thus demonstrate that Jk(a-b-) cells lack mediated urea transport and strongly support the notion that urea transport function is separate from the transport for anions, water, and ethylene glycol, probably because different proteins are responsible for these transport functions.

Permeability of individual human erythrocytes to thiourea.

The osmotic swelling to haemolysis of individual red blood cells by isosmotic thiourea has been studied using microcine photography. 2. Crenation occurs immediately upon addition of isosmotic thiourea. The cell becomes a crenated sphere without volume decrease. 3. Subsequently, the cell volume increases linearly with time with maximum swelling occurring at about 102 sec which is 81% of the total haemolysis time. 4. At maximum swelling, the cell volume is 92% greater than the initial cell volume. This volume increase is about double that measured with other permeating substances. 5. The much larger maximum volume implies that thiourea increases the area of the cell membrane. This increase varies from 0 to 75% for individual cells, with a mean of 22%. 6. Membrane expansion varies inversely as the initial cell membrane area and cell volume (r=0-790). 7. Using the increased surface area, increased maximum volume and the swelling time, the mean permeability is calculated to be 5-52 X 10(-7) cm/sec (S.D. of mean=+/-1-19 X 10(-7) cm/sec). The distribution of permeabilities represents a normal distribution. 8. The pre-lytic potassium loss ranged from 0 to 36% with a mean value of 16-5%. This is consistent with values reported in the literature for slow haemolysis. As with other permeants the distribution is skewed towards lower values. 9. Membrane permeability of individual cells varies with the amount of membrane expansion observed. Coefficient of correlation between permeability and expansion index is 0-674. 10. There is no correlation between permeability and the reciprocal of the haemolysis time (r=-0-035). The correlation between permeability and the reciprocal of the swelling time is also poor (r=0-303), probably owing to the variability in membrane expansion by thiourea in individual cells. 11. As has been shown previously for faster permeants, the permeability coefficient cannot be calculated from the haemolysis time. Because thiourea alters the membrane area and the haemolytic volume, the coefficient cannot be calculated from the swelling time unless the changes in the membrane area are also taken into account.

Dimethylthiourea, a hydroxyl radical scavenger, impedes the inactivation of methionine synthase by nitrous oxide in mice.

Dimethylthiourea (DMTU), a potent scavenger of hydroxyl radicals, was studied to see if it attenuated the inactivation of methionine synthase produced by nitrous oxide in mice. Mice were given i.p. injections of DMTU 0.5-4.0 mg g-1 or saline and, 1 h after injection, were exposed to 66% nitrous oxide in oxygen for periods of 0.5-8 h. At given times after nitrous oxide exposure, higher methionine synthase activities were found in the livers, kidneys and brains of mice injected with DMTU than in the saline-injected animals. These higher methionine synthase activities in the DMTU-treated animals represented a delay in the enzyme inactivation produced by nitrous oxide, as the difference in activities between the DMTU-injected and saline-injected mice decreased with increasing duration of exposure to nitrous oxide. Greater differences in methionine synthase activities between the DMTU- and saline-injected animals were observed with increasing doses of DMTU. The rate of enzyme inactivation following exposure to nitrous oxide was greater in liver and least in brain, and the difference in activities between the two groups varied with the organ examined. DMTU exhibited its greatest effect in the kidney, where methionine synthase activities were nearly doubled in the DMTU 2.0 mg g-1-injected compared with the saline-injected mice after 1-h exposure to 66% nitrous oxide. Following a marked inactivation of methionine synthase by exposing mice to 66% nitrous oxide for 4 h, injection of DMTU 2.0 mg g-1 at the end of exposure to nitrous oxide did not enhance, but impaired, the recovery of enzyme activity. The findings are consistent with the hypothesis that nitrous oxide combines with the vitamin B12 molecule of methionine synthase to form a hydroxyl radical that reacts with an inactivates the enzyme, and that DMTU slows this inactivation by scavenging hydroxyl radicals.