Quantitative evaluation of cerebral vascular permeability using multi-slice dynamic CT.

We developed a method to measure regional permeability of cerebral capillary vessels, extracellular fluid space volume, and vascular bed volume using multi-slice dynamic CT with iodinated contrast medium. We implemented the method as a novel software "BBB study", and evaluated it in four cases of brain tumor. The examination time was about 20 minutes, and the resulting functional maps had quality sufficient to distinguish area of irregular permeability enhancement.

New possibilities for stereotaxis. Information-guided stereotaxis.

Information-guided stereotaxis, assisted by visualization of medical information, will become the next generation of neurosurgical systems. We performed 76 open MRI surgeries at Tokyo Women's Medical University between March 3, 2000 and April 12, 2001. Of them, comparisons of pre- and post-operative MR images for malignant gliomas in 21 cases revealed an average resection rate of 90.3% (the maximum 100%, the minimum 55%). In this article we describe real time updated navigation, augmented reality navigation, three-dimensional navigation, chemical navigation, information-guided navigation system (High definition visual Computer Aided Surgery System: HivisCAS), and open MRI-guided surgery that we are developing.

Isozymic nature of spore coat-associated alanine racemase of Bacillus subtilis.

Spore coat-associated alanine racemase of Bacillus subtilis, which converts L-alanine to D-alanine, that is, the germinant to the competitive inhibitor, to regulate spore germination for survival of the organism under unfavorable growth conditions, was examined. The dormant spores, L-alanine-initiated germination of which is inhibited by diphenylamine, were used to characterize the enzyme in the native form because of its unextractablility from dormant spores. The presence of isozymes, Enz-I and Enz-II with Km for L-alanine of about 20mM and 50mM and optimum activity at around 40 degrees C and 65 degrees C, respectively, was proposed. The enzymes were selectively used depending on the L-alanine concentration and the temperature. The pH profiles of the activity (optimun at pH 9.0) and the stability (stable between pH 6-11 at 60 degrees C) were similar, but Enz-II was more heat-stable than Enz-I and the denaturation curve demonstrated a two-domain structure for Enz-II. Sensitivity to D-penicillamine, hydroxylamine and HgCl2 was similar between Enz-I and Enz-II, while that to D-cycloserine, L- and D-aminoethylphosphonic acid, monoiodoacetate and N-ethylmaleimide was different; HgCl2 was the most effective inhibitor among these compounds.

Effects of pH, temperature, and alcohols on the remarkable activation of thermolysin by salts.

The activity of thermolysin in the hydrolysis of N-[3-(2-furyl)acryloyl] (FA)-dipeptide amides and N-carbobenzoxyl-L-aspartyl-L-phenylalanine methyl ester is remarkably enhanced by high concentrations (1-5 M) of neutral salts. The activation is due to an increase in the molecular activity, k(cat), while the Michaelis constant, K(m), is not affected by the addition of NaCl. In the present study, the effect of NaCl on the thermolysin-catalyzed hydrolysis of FA-glycyl-L-leucine amide (FAGLA) has been examined by changing the pH and temperature, and by adding alcohols to the reaction mixture. The enzyme activity, expressed by k(cat)/K(m), is pH-dependent, being controlled by two functional residues with pK(a) values of 5.4 and 7.8 in the absence of NaCl. The acidic pK(a) is shifted from 5.4 to 6.7 by the addition of 4 M NaCl, while the basic one is not changed. The degree of activation at a given concentration of NaCl is pH dependent in a bell-shaped manner with the optimum pH around 7. Although the activity increases in both the presence and absence of NaCl with increasing temperature from 5 to 35 degrees C, the degree of activation decreases. Alcohols inhibit thermolysin, and the degree of activation decreases with increasing alcohol concentration. The degree of activation tends to increase with increasing dielectric constant of the medium, although it varies considerably depending on the species of alcohol. Electrostatic interactions on the surface and at the active site of thermolysin are suggested to play a significant role in the remarkable activation by salts.

Cerebral blood flow: measurement with xenon-enhanced dynamic helical CT.

PURPOSE: To improve CT number reproducibility in helical CT, a newly developed controlled-orbit helical scan mechanism was applied. The authors developed a method for volumetric measurement of cerebral blood flow (CBF) with dynamic helical computed tomography (CT) after inhalation of xenon gas. MATERIALS AND METHODS: In three subjects (two healthy men aged 22 and 38 years and a 7-year-old girl with moyamoya disease), six helical scans were obtained during 6-minute xenon inhalation, and CT scans with 41 sections in 2-mm increments were reconstructed from each scan. Conventional CBF calculation was then applied to the CT images. RESULTS: CBF maps of 40 sections for each subject of sufficient diagnostic quality were obtained with a 39-cGy radiation dose. The spatial resolution was 2.5 line pairs per centimeter. CONCLUSION: This volumetric CBF mapping method is applicable in clinical practice because of the short examination time and acceptable radiation dose.

A method for quantitative measurement of cerebral vascular permeability using X-ray CT and iodinated contrast medium.

Cerebral vascular permeability is an important consideration in treatment for intracranial tumours. We have developed a new method to measure the cerebral vascular permeability quantitatively using a conventional X-ray CT scanner and iodinated contrast medium. We have already applied our method in 50 cases of intracranial tumour and 5 cases, which establish the methodology, are demonstrated. Dynamic CT scanning of a section including the tumour and the superior sagittal sinus was performed over 40 min after bolus injection of contrast medium, and 25 images were acquired. Our theoretical model of contrast enhancement was applied to analyse time-density curves, and the following parameters were obtained: Ki (inward flux constant), Kb (backward flux constant), Vp (vascular plasma volume), and lambda (extracellular fluid space volume). Furthermore, functional maps were generated from parameters for each pixel. Changes in intra-arterial iodine concentration, required in our model, were measured from CT numbers in the superior sagittal sinus. We have investigated several aspects of our method. Histological findings in surgical specimens of intracranial tumours agreed well with the parameters obtained by our method. Vp was verified quantitatively by single photon emission computed tomography. Our method was shown to be reproducible. These results show that the parameters are useful for assessing tumours and in planning chemotherapy. Our method, which employs no special equipment, is readily available at any institution.

Disposition and metabolism of [14C]sparfloxacin in the rat.

Disposition and metabolism of [carbonyl-14C]sparfloxacin SPFX, 5-amino-1-cyclopropyl-7-(cis-3,5-dimethyl-1-piperazinyl)-6,8-difluoro- 1,4-dihydro-4-oxoquinoline-3-carboxylic acid, AT-4140; CAS 110871-86-8), a novel antimicrobial quinolone, were studied in rats mainly after oral administration at 10 mg/kg. SPFX was absorbed from the whole area of small intestine as shown by the loop method. The extent of absorption was around 70% when estimated by AUC, urinary excretion and biliary excretion. Plasma level of radioactivity reached Cmax of 1.32 micrograms eq/ml within 1 h after oral administration and decreased with a half-life of about 4 h. Higher levels of radioactivity than that in plasma were seen in kidney, liver, submaxillary gland, lung, trachea and many other tissues and lower levels, in eye ball, brain and some others. Most tissue levels decreased with time essentially in parallel with plasma level. In pregnant rats, levels of fetal radioactivity amounted to about 60% of maternal plasma level. In lactating rats, milk was found to contain radioactivity several times as high as plasma level, which decreased with a similar half-life. SPFX was bound to plasma protein, mainly to albumin, at about 40%. Unchanged SPFX and its glucuronide were found in the plasma, milk, bile and urine. Within 48 h, about half of the dosed radioactivity was excreted in the bile, and part of which was re-absorbed. Within 96 h, about 20 and 80% of dose were found in the urine and feces, respectively.

Disposition and metabolism of [14C]-amezinium metilsulfate in rats.

Disposition and metabolism of [14C]-amezinium metilsulfate (4-amino-6-methoxy-1-phenylpyridazinium methylsulfate, Risumic) were systematically studied in rats after intravenous (5 mg/kg) or oral (20, 100 mg/kg) administration. After oral administration at 20 mg/kg, blood level reached the maximum (Cmax) of 0.65 microgram eq/ml at 3 h (tmax) and decreased with t1/2 of 8.1 h. Levels in plasma and most tissues elevated to the Cmax at 3 h. The liver level was the highest (61 times as high as plasma level) of all examined tissues. Most tissue levels decreased thereafter essentially in parallel with plasma levels. The findings by whole-body autoradiography essentially agreed with those by radiometry. In lactating rats, milk levels were virtually similar to plasma levels. [14C]-Amezinium metilsulfate radioactivity in fetus and fetal blood was around 0.3 microgram eq/g, being about 1/10 level of maternal plasma level. About 24, 72 and 42% were excreted in urine, feces and bile, respectively. Re-absorption of biliary metabolites accounted for about 31%, being about 13% of orally given [14C]-amezinium metilsulfate. Plasma and aorta contained unchanged amezinium and glucuronide of hydroxyl amezinium MIII. In the brain, the major metabolite was O-demethyl amezinium MV and unchanged drug was not detected. Urinary metabolites were largely MIII glucuronide and the unchanged drug. Biliary metabolite was found composed mostly from MIII glucuronide. In feces, MIII and the unchanged amezinium were found. MIII and its glucuronide were novel metabolites which were identified by thin-layer chromatography and mass spectrometry.

Mode of antitumor action of recombinant human tumor necrosis factor on the sarcoma Meth A transplanted in the mouse.

The mode of antitumor action of rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation with respect to time course, dose-response relationships and selectivity of the effects. The maximal cytotoxic effect on tumor cells revealed by inhibition of DNA synthesis and maximal lesional effect on tumor vasculature revealed by change in blood pool-size in the tissue were detected at 30 min and 1 h after administration of rHu-TNF, respectively. The dose-response relationship between cytotoxic and tumoricidal effects of rHu-TNF was irrespective of administration route. ED50s of these antitumor effects after i.v. administration of rHu-TNF were about 50 times as high as ED50s after i.t. administration. ED50 of i.t. given rHu-TNF for vascular effect was about 20 times as high as that for cytotoxicity while ED50 of i.v. rHu-TNF for vascular effect was only 2-3 times as high as that for cytotoxicity. The whole body autoradiographies with [125I]HSA given i.v. to see the blood influx into tumor tissue and [14C]thymidine given i.v. to see DNA synthesis in the whole body after administration of rHu-TNF revealed that the distribution of radioactivity was markedly changed in the tumor alone without any detectable change in other whole body tissues. In conclusion, the in vivo antitumor effect of rHu-TNF given i.t. or i.v. appears to be exerted through the direct action on Meth A sarcoma rather than indirectly on tumor vasculature. Under present conditions, the effect of rHu-TNF in the whole body tissues seems rather selective on cells and vasculature of the tumor.

Disposition of 125I-labeled recombinant human tumor necrosis factor in the tumor-bearing mouse.

Disposition of [125I]rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation. After i.v. administration, [125I]rHu-TNF measured by radioactivity and immunoreactivity biphasically decreased in plasma. Tumor level of [125I]rHu-TNF was the maximum at 1 h, then decreased and finally remained essentially constant. After i.t. administration, plasma level reached the maximum at 1 h. Tumor level decreased quickly and then became essentially constant. [125I]rHu-TNF was suggested to be degraded to small fragments in the tumor. Significant distribution of [125I]rHu-TNF was found in the kidney, lung, liver and tumor. Most tissue levels decreased with time in parallel with plasma levels. [125I]rHu-TNF radioactivity was found in proximal convoluted tubules of kidney and in those areas of tumor consisting of degenerating cells with pyknotic nuclei. Urine contained most of administered radioactivity, which being neither immunoreactive nor protein-bound.

Hydrolysis of haloperidol decanoate in vitro by cultured cells.

[14C]Haloperidol decanoate was hydrolysed by partially purified carboxylesterase but not in plasma, blood, lymph and lymphatic liquid. These fluids inhibited the enzyme-mediated hydrolysis of the ester. Within the same incubation period as above, the ester was found hydrolysed to various extents in cell cultures of isolated rat liver cells, of human and rat lymphocytes and of established cell lines (BGM cells, WI-38 cells and L6 cells). Thus, the hydrolysis of the ester was demonstrated in vitro with use of viable cell cultures instead of enzyme preparation. From the time course study on the metabolism of haloperidol decanoate in cell cultures, it was concluded that haloperidol decanoate was first concentrated in the cells and hydrolysed to haloperidol. Based on these results, the metabolic sequences in vivo leading to the formation of active principle haloperidol after intramuscular administration of its decanoate were discussed.

Absorption of intramuscularly administered [14C]haloperidol decanoate in rats.

When [14C]haloperidol decanoate, an ester of haloperidol and decanoic acid, was given intramuscularly to rats, levels of total radioactivity and haloperidol decanoate in medial iliac and hypogastric sacral lymph nodes nearest to injection sites were the highest in examined lymph nodes and plasma. These lymph node levels became maximum 16 days after administration and declined gradually with half-life (around 14 days) similar to those of plasma total radioactivity, haloperidol decanoate and haloperidol. However, when the labelled ester was given intravenously, plasma total radioactivity disappeared far more rapidly. Much more radioactivity was found in hind limbs whose femoral muscles had been injected than in other body parts, even at late stages after administration. Haloperidol alone was found in the brain after [14C]haloperidol decanoate was given either intramuscularly or intravenously. It was concluded that haloperidol decanoate injected in rat femoral muscle was rate-limitedly distributed in lymph circulation and that the absorbed ester did not penetrate the brain through the blood-brain barrier but formed haloperidol did.

Enzymatic hydrolysis of haloperidol decanoate and its inhibition by proteins.

When [14C]haloperidol decanoate, a long-acting neuroleptic and an ester of haloperidol and decanoic acid, was incubated in human whole blood and plasma and in rat plasma and homogenates of rat brain, lung, liver, kidney, pancreas and muscle, no hydrolysis of the ester was seen. Although the decanoate was hydrolyzed by partially purified carboxylesterase, addition of rat plasma or liver homogenate to the enzymic reaction mixture resulted in marked inhibition of hydrolysis, whereas addition of the defatted residues of plasma or liver produced only partial inhibition. The enzymic hydrolysis was inhibited also by beta-lipoprotein and albumin, depending on their concentrations. The assumption that interaction between haloperidol decanoate and protein resulted in inhibition of the hydrolytic reaction mediated by the enzyme was validated by kinetic models and experimental data. The kinetics were apparently competitive. Based on the kinetic analysis, the interaction between the decanoate and albumin or beta-lipoprotein was investigated by measuring their equilibrium constants and extent of protein binding. Haloperidol decanoate appeared to interact with several proteins; this was exemplified by other measures of protein binding, an increasing effect of proteins on the solubility, and the partition ratio of the ester. The interaction between haloperidol decanoate and proteins caused marked stabilization of this ester against enzymatic hydrolysis and, thereby, influenced its metabolism.

Disposition and metabolism of [14C]-haloperidol in rats.

Absorption, distribution, excretion and metabolism of [14C]-haloperidol in rats were studied after administration of 5 mg/kg. After oral and intramuscular administration, plasma levels of [14C]-haloperidol radioactivity reached the maximum at 1 h and decreased biphasically. The biphasic elimination was also observed after intravenous administration. The area under plasma level-time curve (AUC) from 0 to 24 h after intramuscular and oral administration was 110 and 87% of AUC after intravenous route, respectively. After intramuscular administration, levels of radioactivity in all tissues examined were higher than plasma and blood levels at 1 h. The lung, Harderian gland, pancreas, kidney, liver, spleen and adrenal had higher levels than other tissues. Most of tissue levels decreased virtually with similar half lives to plasma level and lowered to less than 1 microgram eq/g at 48 h, when negligible recoveries were found in most tissues. Findings concerning distribution obtained by whole-body autoradiography essentially agreed with those by above radiometry. Levels in the placenta and fetus in the pregnant rat were similar to each other and obviously higher than maternal blood level at 1 h after intramuscular administration but no radioactivity was seen in fetus at 48 h. In the lactating rat, milk levels were several times as high as plasma levels and decreased virtually in parallel with plasma levels after intramuscular administration of [14C]-haloperidol. Within 96 h after intramuscular administration, about 99% of administered radioactivity was excreted in urine (about 46%) and feces (about 53%), respectively. About 54% of radioactivity was excreted in the rat bile within 48 h after intramuscular administration. Administration of the bile obtained and thus containing [14C]-haloperidol radioactivity into the duodenum revealed that partial enterohepatic circulation occurred. After intramuscular administration, plasma contained p-fluorobenzoylpropionic acid and p-fluorophenylaceturic acid with comparable concentrations to unchanged haloperidol. Haloperidol alone could be detected in the brain. The liver, kidney, lung and submaxillary gland were also analyzed for their metabolites. Their metabolite compositions differed from each other. Unchanged haloperidol concentration was much higher in all tissues examined than that in plasma. The major urinary and biliary metabolite was p-fluorophenylaceturic acid and conjugates (glucuronide and sulfate) of haloperidol, respectively.

Excretion and metabolism of intramuscularly administered [14C]-haloperidol decanoate in rats.

Urinary, fecal and biliary excretion, together with enterohepatic circulation, of radioactivity were studied after intravenous (50 mg eq/kg) and intramuscular (5 and 50 mg eq/kg) administration of [14C]-haloperidol decanoate in rats. The composition of urinary and biliary metabolites was also examined. The rate of excretion after intravenous administration lowered rapidly with the half-life of about 1.5 days and about 95% of dose was excreted in excreta within 10 days. Shortly after intramuscular administration, the rate of excretion lowered rapidly but then more gradually later (half-lives after administration of 5 and 50 mg eq/kg were 16.4 and 11.2 days, respectively). About 90% of dose was excreted within 42 days after intramuscular administration. About 1.6% of dose/day was excreted in the bile during 15-17 days after intramuscular administration, of which about 30% was reabsorbed within 24 h (enterohepatic circulation). The major urinary metabolite was p-fluorophenylaceturic acid and the biliary metabolite, glucuronide and sulfate of haloperidol. No unchanged decanoate was detected in the excreta.

Tissue levels, tissue angiotensin converting enzyme inhibition and antihypertensive effect of the novel antihypertensive agent alacepril in renal hypertensive rats.

Tissue levels, tissue angiotensin I converting enzyme (ACE) inhibition and hypotension were examined 20 min, 1, 5 and 14 h after oral administration of 1-[(S)-3-acetylthio-2-methylpropanoyl]-L-prolyl-L-phenylalanine (alacepril, DU-1219) (37.5 mg (92 mumol)/kg) or 1-[(S)-3-mercapto-2-methylpropanoyl]-L-proline (captopril) (20.0 mg (92 mumol)/kg) in renal hypertensive rats, using 14C-labeled compounds. Alacepril exerted a more gradual and more sustained antihypertensive effect than captopril. The maximal hypotension was observed 1 and 5 h after administration of captopril and alacepril, respectively. After administration of [14C]captopril, serum level reached the maximum at 20 min and then decreased rapidly. After administration of [14C]alacepril, serum level reached the maximum at 1 h and decreased more slowly than after [14C]captopril. Time course patterns of tissue levels were essentially in parallel with those of serum levels. Captopril exerted the maximal reduction of ACE activity in tissues 20 min after oral administration and thereafter, the reduction was diminished with time rapidly. [14C]Alacepril showed gradual reduction (the maximum at 1 h) and recovery of ACE activity relative to captopril. After oral administration of [14C]alacepril, tissue unbound fractions contained captopril and its derived metabolites while serum unbound fraction contained the intermediate metabolite desacetyl-alacepril (DU-1227) as well. Correlations between ACE inhibition and tissue levels and between changes in tissue ACE inhibition and in blood pressure with time after oral administration of the two agents were discussed. Furthermore, the direct comparison of alacepril and captopril was attempted by the difference in blood pressures and in ACE inhibitions induced after oral administration of the agents.

Metabolism of protein conjugate of desacetyl-alacepril and its effect on angiotensin converting enzyme in renal hypertensive rats.

The fate of protein conjugate of desacetyl-alacepril (DU-1227) and its effect on angiotensin I converting enzyme (ACE) activity in renal hypertensive rats were studied. [14C]DU-1227-protein conjugate was prepared by ultrafiltration method and administered intravenously in rats. Elimination of radioactivity of [14C]DU-1227-protein from plasma after injection seemed much slower than that reported of [14C]alacepril (1-[(S)-3-acetylthio-2-methylpropanoyl]-L-prolyl-L-phenylalanine, DU-1219) given orally. In the plasma unbound fraction, captopril and captopril-cysteine were detected. Most tissue levels were higher than plasma levels. Significant reduction of tissue ACE activity was seen after administration of the conjugate. Radioactivity was mostly excreted in feces. Captopril, captopril disulfide and captopril-cysteine were found as urinary metabolites. These findings indicate that protein-bound DU-1227 readily dissociated and released DU-1227 was converted to captopril in vivo and can therefore participate in prolonged hypotensive effect exerted by alacepril.

[Disposition and metabolism of 14C-dehydrocorydaline in mice and rats].

Dehydrocorydaline, an alkaloid from Corydalis bulbosa possessing anti-ulceric activity, was labeled by methyl-14C at position 13, and its disposition and metabolism were studied in mice and rats after oral (50 mg/kg) or intravenous (6.8 mg/kg) administration. Blood levels were as low as 0.5 (mice) and 0.06 (rats) microgram eq./ml at maximum, conceivably due to its poor absorption from digestive tracts. Radiometric and autoradiographic studies after oral administration of 14C-dehydrocorydaline revealed that appreciable radioactivity was distributed in rather restricted organs, namely, digestive tracts, liver and kidney. Comparison of distribution with that after intravenous administration suggested that the compound is subject to the significant first pass effect by the liver. The distribution pattern of radioactivity in the liver was heterogeneous regardless to administration routes. Radiometry and autoradiography also revealed that the disposition did not differ significantly from each other in normal rats and those with experimentally induced gastric ulcer. Excretion of radioactivity occurred largely in feces. Excretion via urine and bile was quite minor. Metabolite analysis suggested that dehydrocorydaline was metabolized by O-demethylation and subsequent conjugation with glucuronic acid. However, metabolites were minor relative to the unchanged compound.

Autoradiographic and biochemical studies of drug distribution in the liver. I. [14C]Dehydrocorydaline.

Whole body autoradiography revealed that the distribution pattern of [14C]dehydrocorydaline in the mouse and rat liver was heterogeneous (or reticular) regardless of time after intravenous administration of the labeled agent. Microautoradiography by dry-mounting method revealed that the macroscopic heterogeneous pattern was due to the periportal localization of the radioactive compound in the hepatic lobule. By comparison with [14C]salicylid acid, [14C]diphenylhydantoin and [14C]p-chlorophenoxyacetic acid whose distribution pattern are homogeneous in the liver, the present studies indicated that the existence and persistence of heterogeneous distribution of [14C]dehydrocorydaline in the liver had the following causes: 1. Shortly after intravenous administration, the amount of [14C]dehydrocorydaline circulated to the liver was greatly restricted by its significant distribution in non-hepatic tissues. This was shown by the whole body autoradiography, radiometry of tissues and quantitative comparison of volumes of distribution in non-hepatic tissues. Therefore, 2. perilobular hepatocytes alone could take up [14C]dehydrocorydaline and consequently, centrilobular cells were unavailable to it: heterogeneous distribution pattern is formed. This was shown by microautoradiography as described above, and by the rapid and significant uptake of [14C]dehydrocorydaline by isolated hepatocytes in vitro and by the liver to which the labeled agents were continuously administered in situ. It was also substantiated by the more homogeneous distribution pattern in the liver of the rat to which greater amount of [14C]dehydrocorydaline was gradually given into the portal vein and of the mouse with allyl formate-induced perilobular damage. 3. Redistribution of [14C]dehydrocorydaline scarcely occurred in the whole body and therefore radioactive substance was not significantly supplied to the liver: the distribution pattern remained unchanged. This was shown by the whole body autoradiography and radiometry of tissues.