Unexpectedly high affinity of a novel histamine H(3) receptor antagonist, GSK239512, in vivo in human brain, determined using PET.

BACKGROUND AND PURPOSE: This study aimed to investigate the relationship between the plasma concentration (PK) of the novel histamine H3 receptor antagonist, GSK239512, and the brain occupancy of H(3) receptors (RO) in healthy human volunteers. EXPERIMENTAL APPROACH: PET scans were obtained after i.v. administration of the H(3) -specific radioligand [(11) C]GSK189254. Each subject was scanned before and after single oral doses of GSK239512, at 4 and 24 h after dose. PET data were analysed by compartmental analysis, and regional RO estimates were obtained by graphical analysis of changes in the total volumes of distribution of the radioligand, followed by a correction for occupancy by the high affinity radioligand. The PK/RO relationship was analysed by a population-modelling approach, using the average PK of GSK239512 during each scan. KEY RESULTS: Following administration of GSK239512, there was a reduction in the brain uptake of [(11) C]GSK189254 in all regions, including cerebellum. RO at 4 h was higher than at 24 h, and the PK/RO model estimated a PK associated with 50% of RO of 0.0068 ng·mL(-1) . This corresponds to a free concentration of 4.50 × 10(-12 ) M (pK = 11.3). CONCLUSIONS AND IMPLICATIONS: The affinity of GSK239512 for brain H3 receptors in humans in vivo is much higher than that expected from studies in vitro, and higher than that observed in PET studies in pigs. The study illustrates the utility of carrying out PET studies in humans early in drug development, providing accurate quantification of GSK239512 RO in vivo as a function of time and dose.

Pharmacological differentiation of opioid receptor antagonists by molecular and functional imaging of target occupancy and food reward-related brain activation in humans.

Opioid neurotransmission has a key role in mediating reward-related behaviours. Opioid receptor (OR) antagonists, such as naltrexone (NTX), can attenuate the behaviour-reinforcing effects of primary (food) and secondary rewards. GSK1521498 is a novel OR ligand, which behaves as an inverse agonist at the µ-OR sub-type. In a sample of healthy volunteers, we used [(11)C]-carfentanil positron emission tomography to measure the OR occupancy and functional magnetic resonance imaging (fMRI) to measure activation of brain reward centres by palatable food stimuli before and after single oral doses of GSK1521498 (range, 0.4-100 mg) or NTX (range, 2-50 mg). GSK1521498 had high affinity for human brain ORs (GSK1521498 effective concentration 50 = 7.10 ng ml(-1)) and there was a direct relationship between receptor occupancy (RO) and plasma concentrations of GSK1521498. However, for both NTX and its principal active metabolite in humans, 6-ß-NTX, this relationship was indirect. GSK1521498, but not NTX, significantly attenuated the fMRI activation of the amygdala by a palatable food stimulus. We thus have shown how the pharmacological properties of OR antagonists can be characterised directly in humans by a novel integration of molecular and functional neuroimaging techniques. GSK1521498 was differentiated from NTX in terms of its pharmacokinetics, target affinity, plasma concentration-RO relationships and pharmacodynamic effects on food reward processing in the brain. Pharmacological differentiation of these molecules suggests that they may have different therapeutic profiles for treatment of overeating and other disorders of compulsive consumption.

Application of a generic physiologically based pharmacokinetic model to the estimation of xenobiotic levels in human plasma.

Estimation of xenobiotic kinetics in humans frequently relies upon extrapolation from experimental data generated in animals. In an accompanying paper, we have presented a unique, generic, physiologically based pharmacokinetic model and described its application to the prediction of rat plasma pharmacokinetics from in vitro data alone. Here we demonstrate the application of the same model, parameterized for human physiology, to the estimation of plasma pharmacokinetics in humans and report a comparative evaluation against some recently published predictive methods that involve scaling from in vivo animal data. The model was parameterized through an optimization process, using a training set of in vivo data taken from the literature, and validated using a separate test set of published in vivo data. On average, the vertical divergence of the predicted plasma concentrations from the observed data, on a semilog concentration-time plot, was 0.47 log unit. For the training set, more than 80% of the predicted values of a standardized measure of the area under the concentration-time curve were within 3-fold of the observed values; over 70% of the test set predictions were within the same margin. Furthermore, in terms of predicting human clearance for the test set, the model was found to match or exceed the performance of three published interspecies scaling methods, all of which showed a distinct bias toward overprediction. We conclude that the generic physiologically based pharmacokinetic model, as a means of integrating readily determined in vitro and/or in silico data, is potentially a powerful, cost-effective tool for predicting human xenobiotic kinetics in drug discovery and risk assessment.

Application of a generic physiologically based pharmacokinetic model to the estimation of xenobiotic levels in rat plasma.

The routine assessment of xenobiotic in vivo kinetic behavior is currently dependent upon data obtained through animal experimentation, although in vitro surrogates for determining key absorption, distribution, metabolism, and elimination properties are available. Here we present a unique, generic, physiologically based pharmacokinetic (PBPK) model and demonstrate its application to the estimation of rat plasma pharmacokinetics, following intravenous dosing, from in vitro data alone. The model was parameterized through an optimization process, using a training set of in vivo data taken from the literature and validated using a separate test set of in vivo discovery compound data. On average, the vertical divergence of the predicted plasma concentrations from the observed data, on a semilog concentration-time plot, was approximately 0.5 log unit. Around 70% of all the predicted values of a standardized measure of area under the concentration-time curve (AUC) were within 3-fold of the observed values, as were over 90% of the training set t1/2 predictions and 60% of those for the test set; however, there was a tendency to overpredict t1/2 for the test set compounds. The capability of the model to rank compounds according to a given criterion was also assessed: of the 25% of the test set compounds ranked by the model as having the largest values for AUC, 61% were correctly identified. These validation results lead us to conclude that the generic PBPK model is potentially a powerful and cost-effective tool for predicting the mammalian pharmacokinetics of a wide range of organic compounds, from readily available in vitro inputs only.

Combined antisense and pharmacological approaches implicate hTASK as an airway O(2) sensing K(+) channel.

Neuroepithelial bodies act as airway oxygen sensors. The lung carcinoma line H146 is an established model for neuroepithelial body cells. Although O(2) sensing in both cells is via NADPH oxidase H(2)O(2)/free radical production and acute hypoxia promotes K(+) channel closure and cell depolarization, the identity of the K(+) channel is still controversial. However, recent data point toward the involvement of a member of the tandem P domain family of K(+) channels. Reverse transcription-polymerase chain reaction screening indicates that all known channels other than hTWIK1 and hTRAAK are expressed in H146 cells. Our detailed pharmacological characterization of the O(2)-sensitive K(+) current described herein is compatible with the involvement of hTASK1 or hTASK3 (pH dependence, tetraethylammonium and dithiothreitol insensitivity, blockade by arachidonic acid, and halothane activation). Furthermore, we have used antisense oligodeoxynucleotides directed against hTASK1 and hTASK3 to suppress almost completely the hTASK1 protein and show that these cells no longer respond to acute hypoxia; this behavior was not mirrored in liposome-only or missense-treated cells. Finally, we have used Zn(2+) treatment as a maneuver able to discriminate between these two homologues of hTASK and show that the most likely candidate channel for O(2) sensing in these cells is hTASK3.

Stigma and depression: a double whammy.

The burden of depression is increased by the stigma of mental illness and the widely held idea that psychoactive medication is useless and addictive. These preconceptions may delay and obscure diagnosis and reduce treatment adherence. A sensitive clinician should be able to recognise the difficulties in the individual patient and overcome them.

The effect of dietary caffeine manipulation on blood caffeine, sleep and disturbed behaviour.

The effect of withdrawal of caffeine from the diet of a group of highly disturbed severely retarded adult patients was studied. Two weeks withdrawal produced no improvement in sleep pattern or behaviour, but re-introduction of normal diet was accompanied by a highly significant increase (P < 0.001) in ward disturbance ratings.

Fluorescence detected magnetic resonance (FDMR) of green sulfur photosynthetic bacteria Chlorobium sp.

Fluorescence Detected Magnetic Resonance (FDMR) spectra have been measured for whole cells and isolated chlorosomal fractions for the green photosyntheic bacteria Chlorobium phaeobacteroides (containing bacteriochlorophyll e, and isorenieratene as major carotenoid) and Chlorobium limicola (containing bacteriochlorophyll c, and chlorobactene as major carotenoid). The observed transition at 237 MHz (identical in both bacteria) and > 1100 MHz can be assigned, by analogy with published data on other carotenoids, to the 2E and D + E transitions, respectively, of Chlorobium carotenoids. Their zero field splitting (ZFS) parameters are estimated to be: |D|=0.0332 cm(-1) and |E|=0.0039 cm(-1) (chlorobactene), and |D|=0.0355 cm(-1) and |E|=0.0039 cm(-1) (isorenieratene). In the intermediate frequency range 300-1000 MHz the observed transitions can be assigned to chlorosomal bacteriochlorophylls c and e, and to bacteriochlorophyll a located in the chlorosome envelope and water-soluble protein. The bacteriochlorophyll e triplet state measured in 750 nm fluorescence (aggregated chlorosomal BChl e) is characterised by the ZFS parameters: |D|=0.0251 cm(-1) and |E|=0.0050 cm(-1).

Fluorescence detected magnetic resonance of the primary donor and inner core antenna chlorophyll in Photosystem I reaction centre protein: Sign inversion and energy transfer.

The Photosystem I reaction centre protein CP1, isolated from barley using polyacrylamide gel electrophoresis showed an EPR (Electron Paramgnetic Resonance) spectrum with the polarisation pattern AEEAAE, typical of the primary donor triplet state (3)P700, created via radical pair formation and recombination. (3)P700 could also be detected by Fluorescence Detected Magnetic Resonance (FDMR) at λf > 700 nm even in the presence of a large number of chlorophyll antennae. Its zero field splitting parameters, D=282.5×10(-4) cm(-1) and E=38.5×10(-4) cm(-1), were independent of the detection wavelength, and agreed with ADMR (Absorption Detected Magnetic Resonance) and EPR values. The signs of the (3)P700 D+E and D-E transitions were positive (increase in fluorescence intensity on applying a resonance microwave field). In contrast, in the emission band 685 < λf < 700 nm FDMR spectra with negative D+E and D-E transitions were detected, and the D value was wavelength-dependent. These FDMR results support an excitation energy transfer model for CP1, derived from time-resolved fluorescence studies, in which two chlorophyll antenna forms are distinguished, with fluorescence at 685 < λf < 700 nm (inner core antennae, F690), and λf > 700 nm (low energy antenna sites, F720), in addition to the P700. The FDMR spectrum in F690 emission can be interpreted as that of (3)P700, observed via reverse singlet excitation energy transfer and added to the FDMR spectrum of the antenna triplet states generated via intramolecular intersystem crossing. This would indicate that reversible energy transfer between F690 and P700 occurs even at 4.2 K.

Inhibition of endogenous lactate turnover with lactate infusion in humans.

The extent to which lactate infusion may inhibit endogenous lactate production, though previously considered, has never been critically assessed. To examine this proposition, single injection tracer methodology (U-14C Lactate) has been used for the estimation of lactate kinetics in 12 human subjects under basal conditions and with the infusion of sodium lactate. The basal rate of lactate turnover was measured on a day before the study with lactate infusion, and averaged 63.7 + 5.5 mg/kg/h. Six of these individuals received a stable lactate infusion at an approximate rate of 160 mg/kg/h, while the remaining six individuals were infused at the approximate rate of 100 mg/kg/h. It has been found that stable lactate infused at rates approximating 160 mg/kg/h consistently produced a complete inhibition of endogenous lactate production. Infusion of lactate at 100 mg/kg/h caused a lesser and more variable inhibition of endogenous lactate production (12% to 64%). In conclusion, lactate infusion significantly inhibits endogenous lactate production.

The use of drugs for physical conditions in adults with mental retardation.

A survey of the drugs given for physical complaints in two mental handicap hospitals is described. Thirty-six per cent of 537 adults in hospital A and 43% of 944 adults in hospital B received medications and, of those who did, over half in each hospital received only one drug. The hospital populations differed significantly but both showed a significant increase in total drug usage with increasing age in both sexes, higher among females in every group. This increase was greatest with C.V.S. drug usage, but it did not reach significance for the three most frequently prescribed groups, which were gastrointestinal drugs (to 13% of the total patients), vitamins and nutritional supplements (11%), dermatological (10%) and cardiovascular (10%) drugs. Mental level was significantly indirectly related to usage of gastrointestinal drugs, drugs for anaemia, and vitamins and nutritional supplements, and directly to usage of cardiovascular drugs.

500 MHz 1H NMR of chlorophylls in the major light-harvesting chlorophyll-protein complex of photosystem II.

500 MHz 1H NMR spectra were obtained of solutions containing oligomeric and monomeric forms of Chl a/b-P2, the major light-harvesting chlorophyll a/b-protein complex of photosystem II, isolated from thylakoid membranes of barley (Hordeum vulgare). Oligomers showed only a broad unresolved spectrum, but for monomers several downfield-shifted chlorophyll proton resonances were observed, assigned to the alpha and beta methine protons and the formyl proton of Chl-b. Identifying the observed shifts as ring-current shifts, these NMR data can be matched with previously obtained optical data confirming the trimeric arrangement of Chl-b in Chl a/b-P2 protein, with a distance between the chromophore centers of approximately 12 A.

Role of the kidneys in the metabolism of circulating mevalonate in humans.

Previous studies in several animal species have demonstrated that the kidneys are the primary site of mevalonate metabolism by the oxidative or shunt pathway. To determine the role of the human kidney in mevalonate oxidation, we studied mevalonate shunt activity in patients undergoing hemodialysis for varying degrees of renal failure. Surprisingly, at least half of the uremic patients and even anephric patients had normal ability to oxidize mevalonate by the shunt pathway. In addition, we found a strong negative correlation (R = -0.94) between mevalonate shunt activity and serum phosphorus levels in uremic patients. The resulting inhibition of mevalonate oxidation by high serum phosphorus levels was reversed by lowering the serum phosphorus in one patient. Finally, a positive correlation was found between mevalonate oxidation and serum PTH levels. The results of this study suggest that, in humans, extrarenal tissues can be major contributors to mevalonate oxidation. It is therefore probable that in humans, in contrast to other animals, the kidney is not the primary site of mevalonate metabolism by this oxidative pathway. Finally, the strong negative correlation between serum phosphorus levels and the ability of uremic patients to oxidize mevalonate suggests a regulatory role for the phosphate ion in the mevalonate shunt pathway.

Erythrocyte insulin receptors remain intact through three days of storage.

The status of the erythrocyte insulin receptor was investigated prior to and during storage at 4C in acid-citrate dextrose (ACD) solution. The receptors on cells that were obtained from both resting and exercised fasting subjects and stored for up to three days in ACD were unchanged as evaluated by both maximal specific hormone binding and the concentration of insulin required to one half maximally inhibit specific binding. These findings indicate, therefore, that the binding of insulin to its receptor on erythrocytes can be assayed in samples of stored blood and that this assay reflects the status of the receptor at the time of sampling.

Acute effects of dichloroacetate in the depancreatized dog: glucose synthesis and turnover.

Blood glucose turnover (entry and removal rates) and the rate of recycling of radiolabelled glucose carbon into newly synthesized blood glucose have been evaluated before and acutely after the administration of dichloroacetate to depancreatized dogs. Blood glucose concentration began to decline immediately after dichloroacetate administration and fell to new steady state levels within 1.5-3 h. Analysis of blood glucose kinetics during the decline demonstrated a 52% (average) reduction in the rate of hepatic glucose supply. Glucose supply remained reduced over the duration of these studies (3-4.5 h). Glucose turnover in the steady state following dichloroacetate administration averaged 62% of pretreatment values. Cori cycle activity was depressed by 63% after dichloroacetate administration. The results of these studies are consistent with the hypothesis that a major mechanism underlying the hypoglycaemic action of this drug is the inhibition of glucose synthesis.

Myocardial lactate metabolism: evidence of lactate release during net chemical extraction in man.

Myocardial blood flow has been recognized to be heterogeneous in patients with coronary artery disease. Traditional arterial-coronary sinus sampling methods cannot demonstrate comparable heterogeneity of myocardial metabolism. In this study we used a tracer technique to investigate possible heterogeneity of myocardial lactate metabolism. Twenty-one patients with symptoms of ischemic heart disease were studied. We injected 14C-1-lactate intravenously as a constant infusion after a priming dose. Coronary sinus and arterial samples were obtained for chemical and radioisotopic analyses. At rest, myocardial lactate extraction by chemical analysis was 24.6 +/- 8.5% (mean +/- SD). By radioisotopic analysis, the lactate extraction was 41.0 +/- 10.2% (p less than 0.001). Thus, certain areas of the myocardium were releasing lactate despite global net extraction of lactate. In the 12 patients with significant left main or both left anterior descending (LAD) and left circumflex (LCX) lesions, the calculated amount of lactate released at rest was 0.136 +/- 0.045 mumol/ml of blood (mean +/- SD). In contrast, the amount released in the six patients with a significant lesion in only the LAD or LCX was 0.076 +/- 0.019 mumol/ml, and in the three patients without left coronary arterial lesions it was 0.039 +/- 0.004 mumol/ml. Using a tracer method, myocardial lactate metabolism was demonstrated to be heterogeneous at rest in patients with ischemic heart disease. A significant amount of lactate can be released by the myocardium at a time when chemical arterial-coronary sinus analysis indicates global myocardial extraction. The amount of lactate released appears to be related to the severity of the coronary artery disease.

Sex difference in human mevalonate metabolism.

Two pathways of mevalonate metabolism have been demonstrated: the major (sterol) pathway leads to cholesterol synthesis, whereas the second shunts mevalonate away from sterol production and ultimately results in its oxidation to CO2. Previous studies have demonstrated that the female rat metabolizes circulating mevalonate by the shunt pathway at twice the rate of the male, whereas the male rat converts significantly more circulating mevalonate to cholesterol than the female. The present study extends these observations to humans. Six men and five premenopausal women with normal renal function were injected with R,S-[5-14C]mevalonate, and 14CO2 expired in the breath of the subjects was monitored continuously with an ionization chamber. On an average, the female subjects expired 16.5% and the males 9.8% of the injected R-[5-14C]mevalonate (P less than 0.001). No differences were observed in the plasma and erythrocyte [14C]cholesterol levels. These data demonstrate, in human beings, a sex difference in mevalonate metabolism. The overall impact of the greater mevalonate shunt activity on cholesterol balance in women is unknown.

The mechanism of the acute hypoglycemic action of phenformin (DBI).

The mechanisms by which biguanide (phenformin) acutely brings about a reduction in blood glucose in diabetic subjects has been studied with the aid of C-6 14C glucose. Six diabetic subjects were studied, each at three separate dose levels of phenformin. Two of these same subjects were studied with placebo. Consistent and increasingly pronounced effects of drug versus placebo were noted as the level of biguanide was increased. Biguanide consistently lowered hepatic glucose output while not significantly affecting the removal of glucose from the circulation. It was noted that glucogenesis from lactate was not significantly curtailed. However, a lack of stimulation in Cori Cycle activity in the presence of significant elevations of circulating lactate were taken as an indication of inhibition of glucogenesis from this substrate. On balance, it is concluded that the acute hypoglycemic action of this biguanide is mediated primarily through a restriction in the supply of glucose from the liver to the circulation. The data support the contention that these drugs inhibit hepatic glucogenesis even though Cori Cycle activity may be increased and also suggest that a portion of the decrease in hepatic glucose supply may be the result of impaired glycogenolysis.