Pharmacokinetic Profiles of Metamizole Metabolites after Intramuscular and Intravenous Administration in Healthy Arabian Horses.

Metamizole sodium (MT) is an analgesic and antipyretic drug molecule used in humans, horses, cattle, swine, and dogs. Metamizole rapidly hydrolyzes and turns into methylamino antipyrine (MAA), an active primary metabolite of MT. The present study aims to determine the pharmacokinetic (PK) profiles of MT metabolites after intravenous (IV) and intramuscular (IM) administration into sex of Arabian horses (Equus ferus caballus) using a cross-over study design. The plasma samples were extracted by solid-phase extraction (SPE) method, and plasma concentrations of MT metabolites were analyzed by high-performance liquid chromatography (HPLC). After administrations of MT, plasma concentrations of methylamino antipyrine (MAA), amino antipyrone (AA), and acetylamino antipyrone (AAA) were determined within range of 15 min-12 h. Plasma concentrations of AA and AAA were lower than the plasma concentrations of major metabolite MAA at each sampling point. The PK parameters were statistically evaluated for MT's metabolites between male and female horses and also between IM and IV administrations of PK parameters such as Cmax , tmax , t1/2λz , AUC0-t , AUC0-∞ , λz, Cl and Vss (p < .05). The AUCIM /AUCIV ratio in female and male horses for MAA was 1.19 and 1.13, respectively. The AUCIM /AUCIV ratio for AA was lower than those found for MAA. AUCIM /AUCIV ratio was statistically significantly different between male and female horses for AA (p < .05). According to these results, some PK parameters such as Cmax, AUC, and MRT, MAA and AA concentrations have shown statistically significant differences by MT administrations.

4-Methylamino antipyrine determination in human plasma by high-performance liquid chromatography tandem mass spectrometry.

In the present study, a simple and rapid method for metamizole metabolite 4-methylamino antipyrine (MAA) determination in human plasma was developed, validated and successfully applied to a clinical trial. Chromatographic separation was achieved in HILIC mode on a YMC-Pack SIL column (100 × 2.0 mm; S-5 µm, 30 nm), with a mobile phase consisting of acetonitrile, water and formic acid. Protein precipitation of a small plasma volume using acetonitrile was selected for sample preparation. The multiple reaction monitoring transitions in the positive ionization mode were m/z 218.2 → 56.2 for MAA and m/z 221.2 → 56.2 for MAA-d3 (IS, internal standard). Concentration levels of MAA calibration standards were in the range of 0.100-20 µg/ml. Metamizole conversion into MAA in both water and organic media was investigated, and the level of the conversion in commercially available injection solutions was estimated.

Evaluating Lean Liver Volume as a Potential Scaler for In Vitro-In Vivo Extrapolation of Drug Clearance in Obesity Using the Model Drug Antipyrine.

BACKGROUND: In vitro-in vivo extrapolation (IVIVE) of hepatic drug clearance (CL) involves the scaling of hepatic intrinsic clearance (CLint,uH) by functional liver size, which is approximated by total liver volume (LV) as per the convention. However, in most overweight and obese patients, LV includes abnormal liver fat, which is not thought to contribute to drug elimination, thus overestimating drug CL. Therefore, lean liver volume (LLV) might be a more appropriate scaler of CLint,uH. OBJECTIVE: The objective of this work was to assess the application of LLV in CL extrapolation in overweight and obese patients (BMI >25 kg/m2) using a model drug antipyrine. METHODS: Recently, a model to predict LLV from patient sex, weight, and height was developed and evaluated. In order to assess the LLV model's use in IVIVE, a correlation-based analysis was conducted using antipyrine as an example drug. RESULTS: In the overweight group (BMI >25 kg/m2), LLV could describe 36% of the variation in antipyrine CL (R2 = 0.36), which was >2-fold higher than that was explained by LV (R2 = 0.17). In the normal-weight group (BMI ≤25 kg/m2), the coefficients of determination were 58% (R2 = 0.58) and 43% (R2= 0.43) for LLV and LV, respectively. CONCLUSION: The analysis indicates that LLV is potentially a more appropriate descriptor of functional liver size than LV, particularly in overweight individuals. Therefore, LLV has a potential application in IVIVE of CL in obesity.

Organic Anion-Transporting Polypeptide Genes Are Not Induced by the Pregnane X Receptor Activator Rifampin: Studies in Hepatocytes In Vitro and in Monkeys In Vivo.

Induction potentials of the pregnane X receptor (PXR) activator rifampin (RIF) on transporter genes [e.g., organic anion-transporting polypeptides (OATPs)] are still in its infancy or remain controversial in the field. The present investigations characterized changes in transporter gene expression by RIF in sandwich-cultured hepatocytes from multiple donors of human and cynomolgus monkey using real-time quantitative reverse transcription polymerase chain reaction method. Three-day treatment of RIF significantly induced CYP3A4 (∼60-fold induction), but not CYP1A2 and CYP2D6 genes. SLC51B was the most highly induced uptake transporter gene (>10-fold) in both human and monkey hepatocytes. A greater induction of CYP2C9 was observed in monkey hepatocytes than that in humans. ATP-binding cassette (ABC)B1 and ABCC2 were induced slightly above 2-fold in human and monkey hepatocytes and appeared to be dose-dependent. The induction of OATP and other transporter genes was generally less than 2-fold and considered not clinically relevant. SLCO2B1 was not detectable in monkey hepatocytes. To investigate in vivo OATP induction, RIF (18 mg/kg per day) was orally dosed to cynomolgus monkeys for 7 days. Pitavastatin and antipyrine were intravenously dosed before and after RIF treatment as exogenous probes of OATP and CYP activities, respectively. Plasma coproporphyrin-I (CP-I) and coproporphyrin-III (CP-III) were measured as OATP endogenous biomarkers. Although a significant increase of antipyrine clearance (CL) was observed after RIF treatment, the plasma exposures of pitavastatin, CP-I, and CP-III remained unchanged, suggesting that OATP function was not significantly altered. The results suggested that OATP transporters were not significantly induced by PXR ligand RIF. The data are consistent with current regulatory guidances that the in vitro characterization of transporter induction during drug development is not required. SIGNIFICANCE STATEMENT: Organic anion-transporting polypeptide (OATP) genes were not induced by rifampin in sandwich-cultured human and monkey hepatocytes OATP functions measured by OATP probe pitavastatin and endogenous marker coproporphyrins were not altered in monkeys in vivo by 7-day rifampin treatment. The data suggested that OATP transporters are unlikely induced by the pregnane X receptor ligand rifampin, which are consistent with current regulatory guidances that the in vitro characterization of OATP1B induction during drug development is not required.

Placental transfer and tissue accumulation of dolutegravir in the ex vivo human cotyledon perfusion model.

OBJECTIVE: To determine the transplacental pharmacokinetics of the HIV integrase inhibitor dolutegravir. STUDY DESIGN: Maternal-to-fetal transfer across the term human placenta was investigated with the ex-vivo dually perfused cotyledon model, in 5 closed-circuit, recirculating experiments. Dolutegravir was added to a maternal perfusate containing antipyrine, a marker to validate the cotyledon's viability, and 2 g/liter of human albumin. RESULTS: After 3h of recirculating perfusion, the mean (± SD) DTG concentrations in the maternal and in the fetal compartments were respectively 2450 ± 286 ng/mL and 715 ± 369 ng/mL, with a fetal-to-maternal ratio of 34% ± 18% and a clearance index (in comparison with antipyrine transfer) of 79% ± 23%. The mean cotyledon accumulation index was 153% ± 25%. CONCLUSION: Fetal transplacental exposure to dolutegravir was considerable as well as accumulation in placental tissue. Whether this may lead to risks for the exposed fetus requires more investigation.

Effects of Temperature and Humidity on the Skin Permeation of Hydrophilic and Hydrophobic Drugs.

The humidity was a well-known method to hydrate the skin; however, the published data were varied, and systemic experiments in the previous papers were few. Therefore, the in vitro permeation of excised porcine ear skin by drugs with different polarities [aminopyrine (AMP), antipyrine (ANP), methylparaben (MP), and ibuprofen (IP)] was analyzed under a constant skin surface temperature with different temperatures and humidities to reveal the effects of temperature and humidity on the skin permeation enhancement effects. Applied formulations were prepared by mixing the drug and a hydrophilic vehicle containing glycerin. The disposition-distance profiles of water and the humectant glycerin in the stratum corneum were also investigated using confocal Raman microscopy. High absolute humidity (AH) significantly contributed to the high skin penetration of the hydrophilic penetrants AMP, ANP, and MP but not the hydrophobic penetrant IP. An increase in the partition parameter and a decrease in the diffusivity parameter occurred with an increase in AH, independent of drug polarity. Moreover, we found that dew condensation induced by high AH on temperature-controlled skin surface may effectively increase water content and may provide higher glycerin distribution in the skin barrier, the stratum corneum. Increasing the amount of water and hydrophilic vehicles such as glycerin in the stratum corneum may enhance the permeation of hydrophilic penetrants AMP, ANP, and MP. These data suggested a dew condensation on the skin surface induced by high AH at a constant skin surface temperature would be important to enhance hydrophilic penetrants.

Changes in the Expression of Aquaporin-3 in the Gastrointestinal Tract Affect Drug Absorption.

Aquaporin-3 (AQP3) plays an important role in water transport in the gastrointestinal (GI) tract. In this study, we conducted a Caco-2 cell permeability assay to examine how changes in the expression and function of AQP3 affect the rate at which a drug is absorbed via passive transport in the GI tract. When the function of AQP3 was inhibited by mercuric chloride or phloretin, there was no change in warfarin permeability. In contrast, when the expression of AQP3 protein was decreased by prostaglandin E2 (PGE2) treatment, warfarin permeability increased to approximately twice the control level, and membrane fluidity increased by 15%. In addition, warfarin permeability increased to an extent comparable to that after PGE2 treatment when cell membrane fluidity was increased by 10% via boric acid/EDTA treatment. These findings suggest the possibility that the increased drug absorption under decreased AQP3 expression was attributable to increased membrane fluidity. The results of this study demonstrate that the rate of water transport has little effect on drug absorption. However, our findings also indicate that although AQP3 and other similar transmembrane proteins do not themselves transport drugs, changes in their expression levels can cause changes in cell membrane fluidity, thus affecting drug absorption rates.

Edaravone: a new hope for deadly amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's disease, is a fatal motor neuron degenerative disorder leading to paralysis and eventual death. At present, we do not have any specific cure for this deadly disorder. Current drug therapy can only reduce morbidity in ALS patients. In 1995, riluzole was the first drug approved by the U.S. Food and Drug Administration (FDA) for ALS. After a long gap of 22 years, Mitsubishi Tanabe Pharma America got U.S. FDA approval for edaravone (Radicava) in May 2017 for the management of ALS. Edaravone, a novel neuroprotective agent, is indicated to slow down progression of ALS. In 2015, Mitsubishi Tanabe Pharma launched edaravone (Radicut) for the treatment of stroke and ALS in Japan. The U.S. FDA approved edaravone following clinical evidence from three clinical trials conducted in 368 ALS patients in Japan. Edaravone is awaiting approval by the European Medicines Agency (EMA) in Europe. Edaravone (60 mg) is administered by very slow intravenous infusion (60 minutes) in 28-day cycles. It has been shown to slow down the loss of physical function in ALS patients by 33% as compared to placebo. Edaravone is a strong antioxidant that prevents oxidative stress from inducing motor neuron death in ALS patients. Being a potent free radical scavenger, it has been shown to inhibit nitration of tyrosine residues in the cerebrospinal fluid and improve motor functions in mouse models of ALS. The product has been patented and the FDA has not approved any generic version of edaravone. This article discusses the preclinical pharmacology, pharmacokinetics, safety profile, clinical studies and drug interactions of edaravone (Radicava) in ALS.

Gastric retention pellets of edaravone with enhanced oral bioavailability: Absorption mechanism, development, and in vitro/in vivo evaluation.

Absorption mechanism of edaravone (EDR) was studied to inform the preparation of gastric retention pellets with the aim to enhance its oral bioavailability. Three different models, namely, Caco-2 cells model, in situ single-pass intestinal perfusion model, and everted gut sac model in rats, were employed to characterize the gastrointestinal absorption kinetics of EDR. And it was found that passive transfer plays a vital role for the transport of EDR, and acidic condition is preferable for EDR absorption. Further, it is likely that EDR acts as a substrate for P-glycoprotein and multidrug-resistance protein. And hence, an orally available gastric retention pellets were developed accordingly. Pharmacokinetic experiments performed with rats and beagles showed that the absolute bioavailability of EDR solution and enteric-coated pellets following oral administration were 33.85% ±â€¯2.45% and 7.64% ±â€¯1.03%, indicating that stomach absorption is better than intestinal adsorption for EDR. However, the gastric retention pellets resulted in 68.96% absolute bioavailability and about 200% relative bioavailability in comparison to EDR solution, which was 9 times that of enteric-coated pellets. The present work demonstrates that gastric retention pellets has excellent potential as oral administration route for EDR.

Pharmacokinetic profile of edaravone: a comparison between Japanese and Caucasian populations.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) affects persons of all races, and there continues to be a need for effective therapies to treat the disease. OBJECTIVE: To compare the pharmacokinetics (PK) of edaravone between Japanese and Caucasian populations. METHODS: Data from five PK studies among Japanese and Caucasian healthy volunteers were pooled and evaluated. In population PK (PPK) modelling, compartment models and other models with linear elimination were evaluated for appropriateness. Covariate effects by race, sex, weight, and age were investigated to explain variability in PK parameters. Simulations of the final PPK model were performed using a virtual population based on ALS clinical trials. RESULTS: The analysis included 86 subjects. A three-compartment model with Michaelis-Menten plus linear elimination was selected as the best fit model. Race was statistically detected as a covariate for the second peripheral volume of distribution (V2), indicating a 26% increase for Caucasian subjects compared to Japanese subjects. However, based on simulation of PPK model for a virtual ALS population, the small difference of V2 was associated with a difference of Ctau around 1 ng/mL after infusion, which was minimal compared to Cmax of approximately 1000 ng/ml. CONCLUSION: The PPK analyses demonstrated no clinically relevant difference in the PK profiles of edaravone by race, sex, weight, or age.

Prediction of sustained fetal toxicity induced by ketoprofen based on PK/PD analysis using human placental perfusion and rat toxicity data.

AIM: We encountered a case of fetal toxicity due to ductus arteriosus (DA) constriction in a 36-week pregnant woman who had applied multiple ketoprofen patches. The aim of the present study was to present the case and develop a model to predict quantitatively the fetal toxicity risk of transdermal administration of ketoprofen. METHODS: Human placenta perfusion studies were conducted to estimate transplacental pharmacokinetic (PK) parameters. Using a developed model and these parameters, human fetal plasma concentration profiles of ketoprofen administered to mothers were simulated. Using pregnant rats, DA constriction and fetal plasma drug concentration after ketoprofen administration were measured, fitted to an Emax model, and extrapolated to humans. RESULTS: Transplacental transfer value at the steady state of ketoprofen was 4.82%, which was approximately half that of antipyrine (passive marker). The model and PK parameters predicted almost equivalent mother and fetus drug concentrations at steady state after transdermal ketoprofen administration in humans. Maximum DA constriction and maximum plasma concentration of ketoprofen after administration to rat dams were observed at different times: 4 h and 1 h, respectively. The model accurately described the delay in DA constriction with respect to the fetal ketoprofen concentration profile. The model with effect compartment and the obtained parameters predicted that use of multiple ketoprofen patches could potentially cause severe DA constriction in the human fetus, and that fetal toxicity might persist after ketoprofen discontinuation by the mother, as observed in our case. CONCLUSION: The present approach successfully described the sustained fetal toxicity after discontinuing the transdermal administration of ketoprofen.

Integration and validation of the ex vivo human placenta perfusion model.

INTRODUCTION: The ex vivo human placenta perfusion model is an effective and non-invasive method to study transplacental passage of drugs and environmental compounds in humans. Due to many challenges and its high complexity it remains difficult to incorporate it routinely into laboratories. METHODS: This article describes a step-by-step protocol for the implementation and validation of a closed-closed ex vivo perfusion model. Antipyrine, a small molecule that passes the placental barrier by passive diffusion, was used as a measurement of overlap between foetal and maternal circulation. The pressure and the flow rate in the foetal circulation, glucose consumption and pH were implemented to ensure the integrity, viability and functionality of the method. RESULTS: In total 89 placenta were collected of which 34 placentas were successfully perfused with antipyrine and fulfilled all quality control measurements. A foetal/maternal antipyrine concentration ratio of 0.75 was reached within 89±21 min, while 210 min were required to achieve equilibrium. The foetal pressure remained under 70 mmHg during the entire experiment. The end foetal flow was 98% of the foetal starting flow. The average glucose consumption was 0.30±0.15 µmol/min/g. Every 30 min the maternal pH declined to 7.29±0.06 and was adjusted to 7.4. The foetal pH stayed stable at 7.30±0.05. DISCUSSION: Based on the assessment of multiple quality control measurements, the described method of a closed human ex-vivo placenta perfusion model was validated. The success rate (38%) was more than twice the success rate reported in literature (15%).

Osmolality of Orally Administered Solutions Influences Luminal Water Volume and Drug Absorption in Intestine.

Intestinal water absorption is reportedly influenced by luminal osmolality. In this study, we examined whether differences in the osmolality of the vehicle used for oral administration of drugs influence luminal water volume and drug absorption in the gastrointestinal (GI) tract, by means of in situ rat intestinal closed loop studies using solutions of fluorescein isothiocyanate dextran 4000 (a non-absorbable compound), atenolol (a low-permeability drug), and antipyrine (a high-permeability drug) in various solvents. Determination of the remaining fraction of water revealed the following rank order for water absorption in rat jejunum: purified water > saline > phosphate buffer = isosmotic mannitol solution. The luminal concentration of fluorescein isothiocyanate-dextran 4000 after administration in purified water was significantly increased to 2.5 times the initial dosing concentration. Thus, osmolality-dependent changes in GI water absorption can cause significant changes of drug concentration in the GI fluid, potentially resulting in altered drug absorption characteristics. Indeed, the fraction absorbed of atenolol in jejunum was significantly greater when the drug was administered in purified water than in isosmotic solution. In contrast, no significant change in fraction absorbed of antipyrine was observed. Our results indicate that osmolality-dependent changes in GI water volume may influence drug absorption, especially of low-permeability drugs.

Edaravone-Encapsulated Agonistic Micelles Rescue Ischemic Brain Tissue by Tuning Blood-Brain Barrier Permeability.

Thrombolysis has been a standard treatment for ischemic stroke. However, only 2-7% patients benefit from it because the thrombolytic agent has to be injected within 4.5 h after the onset of symptoms to avoid the increasing risk of intracerebral hemorrhage. As the only clinically approved neuroprotective drug, edaravone (EDV) rescues ischemic brain tissues by eradicating over-produced reactive oxygen species (ROS) without the limitation of therapeutic time-window. However, EDV's short circulation half-life and inadequate cerebral uptake attenuate its therapeutic efficacy. Here we developed an EDV-encapsulated agonistic micelle (EDV-AM) to specifically deliver EDV into brain ischemia by actively tuning blood-brain barrier (BBB) permeability. The EDV-AM actively up-regulated endothelial monolayer permeability in vitro. HPLC studies showed that EDV-AM delivered more EDV into brain ischemia than free EDV after intravenous injection. Magnetic resonance imaging also demonstrated that EDV-AM more rapidly salvaged ischemic tissue than free EDV. Diffusion tensor imaging indicated the highest efficiency of EDV-AM in accelerating axonal remodeling in the ipsilesional white matter and improving functional behaviors of ischemic stroke models. The agonistic micelle holds promise to improve the therapeutic efficiency of ischemic stroke patients who miss the thrombolytic treatment.

A Novel LC-MS-MS Method With an Effective Antioxidant for the Determination of Edaravone, a Free-Radical Scavenger in Dog Plasma and its Application to a Pharmacokinetic Study.

The objective of this study was to investigate the stability of edaravone in dog plasma by using an added antioxidant stabilizer, with an ultimate goal of developing and validating a sensitive, reliable and robust LC-MS-MS method for determination of edaravone in plasma samples. Edaravone was unstable in plasma, but it presented a good stability performance in the plasma with sodium metabisulfite (SMB), an effective antioxidant. The blood sample was collected in the heparinized eppendorf tube containing SMB and plasma sample was deproteinized using acetonitrile containing 20 ng/mL of phenacetin (Internal standard). The chromatographic separation was performed on a Zorbax Extend-C18 analytical column (2.1 mm × 150 mm I.D., particle size 3.5 µm, Agilent Technologies, USA). The mobile phase consisted of 0.1% formic acid in water (v/v) and methanol, and gradient elution was used. The analyte detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization by multiple reaction ion monitoring mode of the transitions at m/z [M + H]+ 175.1 → 77.1 for edaravone, and m/z [M + H]+ 180.2 → 110.0 for phenacetin. The linearity of this method was within the concentration range of 10-1000 ng/mL for edaravone in dog plasma. The lower limit of quantification was 10 ng/mL. The relative standard deviations of intra- and inter-precision were <10%. This method was successfully employed in the pharmacokinetics evaluation of edaravone in beagle dogs after intravenous administration.

In vivo use of the CYP inhibitor 1-aminobenzotriazole to increase long-term exposure in mice.

1-Aminobenzotriazole (ABT) is a well-known in vivo nonspecific inhibitor of cytochrome P450 (CYP) enzymes. An effective dosing regimen of ABT for a multiple-administration study is needed to conduct pharmacological studies for proof-of-concept, although it has been established for single-administration study, to characterize the pharmacokinetics of drug candidates. This study demonstrated a suitable dosing vehicle of ABT for continuous administration and increased exposure to antipyrine, which is a nonspecific probe of CYP, using ABT for a long period in mice. The dosing vehicle of ABT was 0.5% (w/v) hydroxypropyl methylcellulose and 0.5% (v/v) Tween 80 in N,N-dimethylacetamide/20% hydroxypropyl-ß-cyclodextrin aqueous solution (2:8, v/v) based on the duration of apparent solubility. After implantation of an ALZET osmotic pump with ABT, the plasma concentrations of ABT were maintained at more than 4.1 µg/ml over 336 h. Compared with the vehicle group, the CLtot of antipyrine with ABT decreased to approximately one-fourth, and the BA of antipyrine with ABT increased up to 3-fold. In addition, the enhancement of exposure of antipyrine by ABT was maintained over the 336 h. The body weight, food consumption and hematological parameters of mice did not change with ABT administration for 16 days. These findings demonstrated that pretreatment of ABT can increase long-term exposure using continuous administration with the ALZET osmotic pump in mice with no overt toxicity. It is concluded that the in vivo use of 1-aminobenzotriazole can be applied to pharmacological studies for proof-of-concept, thus contributing to the selection of drug candidates at an early drug discovery stage. Copyright © 2016 John Wiley & Sons, Ltd.

Application of Osmotic Pumps for Sustained Release of 1-Aminobenzotriazole and Inhibition of Cytochrome P450 Enzymes in Mice: Model Comparison with the Hepatic P450 Reductase Null Mouse.

The effectiveness of controlled release 1-aminobenzotriazole (ABT) administration to inhibit cytochrome P450 (P450) enzymes has been evaluated in mice. To maximize the duration of P450 inhibition in vivo, ABT was administered via an osmotic pump. The degree of P450 inhibition was compared with that achieved with a single bolus dose of ABT. Two-hour prior subcutaneous treatment of mice with ABT (50 mg/kg) inhibited antipyrine clearance by 88%. A less pronounced inhibitory effect (29% reduction in clearance) was observed when ABT was administered 24-hours before antipyrine administration, indicating partial restoration of P450 activity during this longer pretreatment time. The duration of ABT in mice was very short (mean residence time = 1.7 hours) after subcutaneous bolus administration. When the inhibitor was delivered by an osmotic pump, maximum blood concentrations of the inhibitor were observed 24 hours after device implantation and were maintained at steady state for 6 days. Inhibition of P450 activity, as measured by antipyrine clearance, was confirmed at 24 hours and 120 hours after pump implantation, highlighting the utility of this method as a longer-term model for P450 inhibition in mice. The magnitude of P450 inhibition in ABT-treated mice was compared with that in hepatic P450 reductase null mice and both models were comparable. In vivo ABT administration by an osmotic pump offers an effective approach for longer-term P450 inhibition in mice and avoids the necessity for multiple dosing of the inhibitor.

Evidence of functional brain reorganization on the basis of blood flow changes in the CAG140 knock-in mouse model of Huntington's disease.

Neuroimaging, especially functional brain mapping, may provide insights into the distributed involvement of multiple brain regions and loops in disorders classically associated with pathology of a localized region. One example is Huntington's disease (HD), typically classified as a basal ganglia disorder. Here, we report genotypic differences in cerebral perfusion mapping in an HD mouse model characterized by a gene knock-in (KI) of a human exon 1 CAG140 expansion repeat (CAG140 KI mice). Animals were examined at 6 months and compared with wild-type littermates. Regional cerebral blood flow (rCBF) was mapped in the awake, nonrestrained, male mouse at rest using [C]-iodoantipyrine autoradiography and analyzed in three-dimensionally reconstructed brains by statistical parametric mapping. Our results showed significant changes in rCBF between CAG140 KI and WT mice, such that CAG140 KI animals showed hypoperfusion of the basal ganglia motor circuit and hyperperfusion of cerebellar-thalamic and somatosensory regions. Significant hypoperfusion was also noted in CAG140 KI mice in the prelimbic and cingulate cortex (medial prefrontal area) and the hippocampus - areas associated with cognitive processing and mood. Changes in rCBF were apparent in the absence of motor deficits (rotarod test) or atrophy in the striatum (caudate-putamen) or hemispheric volume. Our results suggest a functional reorganization of whole-brain networks at a presymptomatic stage in the life of the CAG140 KI mouse. Functional brain mapping in animals may, in the future, serve as a translational biomarker for identifying sites of early synaptic change in the HD brain and for directing targeted preclinical molecular studies and clinical therapies.

Effects of rapamycin on cerebral oxygen supply and consumption during reperfusion after cerebral ischemia.

Activation of the mammalian target of rapamycin (mTOR) leads to cell growth and survival. We tested the hypothesis that inhibition of mTOR would increase infarct size and decrease microregional O2 supply/consumption balance after cerebral ischemia-reperfusion. This was tested in isoflurane-anesthetized rats with middle cerebral artery blockade for 1h and reperfusion for 2h with and without rapamycin (20mg/kg once daily for two days prior to ischemia). Regional cerebral blood flow was determined using a C(14)-iodoantipyrine autoradiographic technique. Regional small-vessel arterial and venous oxygen saturations were determined microspectrophotometrically. The control ischemic-reperfused cortex had a similar blood flow and O2 consumption to the contralateral cortex. However, microregional O2 supply/consumption balance was significantly reduced in the ischemic-reperfused cortex. Rapamycin significantly increased cerebral O2 consumption and further reduced O2 supply/consumption balance in the reperfused area. This was associated with an increased cortical infarct size (13.5±0.8% control vs. 21.5±0.9% rapamycin). We also found that ischemia-reperfusion increased AKT and S6K1 phosphorylation, while rapamycin decreased this phosphorylation in both the control and ischemic-reperfused cortex. This suggests that mTOR is important for not only cell survival, but also for the control of oxygen balance after cerebral ischemia-reperfusion.

POST-ANESTHETIC REHABILITATION PERIODS AND ANESTHESIA DOSAGE FOR LAPAROSCOPIC CHOLECYSTECTOMY: RELATIONSHIP TO THE TOTAL OXIDATIVE CAPACITY OF LIVER.

Analysis of the results of pharmacological phenotyping using antipyrine test prior to providing anesthesia for laparoscopic cholecystectomy showed that trimeperidine (promedol) dosing with allowance for the total oxidative capacity of liver and the patient mass allows the periods of post-anesthetic rehabilitation to be controlled. Clear algorithm of trimeperidine dosing based on established indices of the total oxidative capacity of liver and is yet nor developed because of restricted sampling set. The obtained results show expediency of using and studying antipyrine test as a simple, cheap, and informative method of individual anesthesia dosing for increasing the adequacy of general anesthesia.