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Friction at the molecular level is examined with a novel scanning probe microscope that observes the onset of energy dissipation and adhesive forces simultaneously. Friction is monitored by measuring the damped vibrational amplitude of an oscillating probe tip, analogous to shear-force feedback commonly used in near-field scanning optical microscopes. A mechanically stable interfacial force sensor is used to measure normal forces at the tip independently and decoupled from the lateral forces, allowing one to observe friction from the early stages of the purely adhesive tensile regime to the latter stage of repulsive compression. Measurements on model lubricant silane and alkanethiol self-assembled monolayers indicate that friction dramatically increases with the strength of adhesive interactions between the tip and the monolayers. These adhesive interactions occur over an appreciable (7 ± 1 Å) displacement range, suggesting tensile reorientation of the lubricant chains and subsequent energy losses due to collective chain motion as well as dissipative hydrogen-bond breaking.

The ECVAM Task Force on Integrated Testing Strategies was established in December 1996, with the remit of assessing the current status of integrated toxicity testing, and of making proposals regarding the design and implementation of integrated testing strategies. The first step in an integrated testing strategy is usually to determine the chemical functionality of a substance, on the basis of its structure and physicochemical properties. The biokinetic and dynamic behaviours of the chemical in various in vitrosystems are then assessed. The various elements are then integrated, in either a parallel or a stepwise fashion, to make predictions of the local or systemic toxicity of the chemical of interest. In this report, a generic scheme for local/systemic toxicity, and a specific scheme for target organ toxicity, are proposed. The scope and limitations of the approaches are discussed. The task force hopes that its proposals will stimulate a discussion on the feasibility of this type of approach and it welcomes any feedback. It is planned that the discussion points will be elaborated in a second task force report.

To evaluate the theory that within precision-cut liver slices intercellular transport occurs in parallel with cellular metabolism and to illustrate the constraints this places on clearance predictions, the kinetics of ethoxycoumarin O-deethylation have been determined under varying conditions of hepatic cytochrome P-450 activity. Liver slices, isolated hepatocytes, and microsomes were obtained from rats treated with the inducers phenobarbital (PB) and beta-naphthoflavone (betaNF) and the inhibitor aminobenzotriazole (ABT). In hepatocytes and microsomes, a two-site kinetic model with a high-affinity, low-capacity site and an unsaturated low-affinity, high-capacity site described the hydroxycoumarin formation data. There were marked increases in Vmax (2- to 5-fold and 50- to 70-fold for PB and betaNF, respectively) in both systems and in CLint (3- and 9-fold for PB and betaNF, respectively) in hepatocytes and substantial decreases in both parameters (3-8 and 12-23% of control, respectively) in ABT hepatocytes and microsomes. A qualitatively similar response was evident in slices obtained from livers of rats treated with phenobarbital and ABT, but although slices from betaNF livers produced high metabolic rates (comparable to slices obtained from livers of rats treated with phenobarbital), these showed a linear increase with substrate concentration without indication of a high-affinity site. The intrinsic clearance parameters were scaled to full liver capacity using hepatocellularities and microsomal recovery indices to allow direct comparison of these responses. The slice system consistently underestimated the effects of the modifiers. When compared with hepatocytes, estimates of 30, 15, and 1% for ABT, PB, and betaNF, respectively, were observed and the degree of underestimation was dependent on the magnitude of intrinsic clearance and was consistent with the above theory.

The kinetics of metabolism of diazepam, phenytoin, and caffeine have been determined in rat liver slices of 260 microns thickness. The formation of 4'-hydroxy metabolites of diazepam and phenytoin are described by one- and two-site Michaelis-Menten equations, respectively. The other oxidative pathways for diazepam are less readily saturable than the 4'-hydroxylation. This kinetic behavior is consistent with that previously reported for other in vitro systems. In contrast, the metabolism of caffeine, assessed by total metabolism, showed differences from that observed with other systems, and this is believed to result from the incubation conditions used. By determining the hepatocellularity of the standard slice used, CLint (Vmax/KM) data were expressed per million cells and compared with the same parameters derived from incubations with freshly isolated hepatocyte suspensions. Data on diazepam, phenytoin, and caffeine were combined with previously published data on tolbutamide, ethoxycoumarin, and ondansetron to give a total of nine CLint values representing different pathways. CLint values in slices are consistently less than those in hepatocytes (ratio differing from 0.4 to 0.05). The CLint ratio decreased in a regular fashion as the hepatocyte CLint increased from 1.4 microliters/min (caffeine) to 105 microliters/min (ondansetron). It was also observed that the KM value for a particular pathway in slices always exceeded the corresponding value in isolated hepatocytes (ratios differing from 1.09 to 7.59). The KM ratio was positively related to hepatocyte CLint. These observations are consistent with the delayed accessibility of substrate to all the cells within a slice. It is proposed that the parallel processes of drug transport and metabolism within the slice do not allow a distribution equilibrium to be achieved between all the cells within a slice and the incubation media.

The ability of two azole antifungal agents, ketoconazole and fluconazole, to inhibit hepatic cytochrome P450 activity in vivo in the rat has been determined. To make a valid comparison, differences in pharmacokinetic properties between the azoles were accounted for by using an infusion approach to maintain steady-state plasma concentrations over a range of 1-48 mg/liter. Both compounds showed a maximum inhibitory effect, assessed by a reduction in antipyrine clearance, of approximately 75%. The relationship between steady-state plasma concentration and the degree of inhibition of antipyrine clearance was nonlinear for both azoles. However, the inhibitory effect resulted at lower concentrations for ketoconazole than for fluconazole. Analysis of these data provided Ki values of 3 and 10 microM, for ketoconazole and fluconazole, respectively, based on plasma concentration of azole. This difference in activity is 2 orders of magnitude greater when Ki values are expressed in terms of unbound concentration in the blood, which may be more representative of hepatic tissue concentrations. Ki values based on unbound drug concentration are 0.07 and 8.7 microM for ketoconazole and fluconazole, respectively. These data confirm the conclusions based on in vitro findings that ketoconazole is a more inhibitory of mammalian cytochrome P450 isoenzymes than fluconazole.

The glucuronidation of morphine, naloxone, and buprenorphine by the liver and intestine has been assessed both in vitro and in vivo in the rat. Using microsomes, Vmax/Km ratios were estimated as measures of intrinsic enzyme activity, and a wide range of ratios were obtained (500-fold). The ratio for the intestine was consistently less than for the liver, and the rank order of activities within each tissue was morphine less than naloxone less than buprenorphine. Using various routes of administration, plasma concentration-time profiles for each compound were determined and used to estimate hepatic and intestinal extraction ratios. These extraction ratios were dose-independent and more substantial for the liver than the intestine. For each tissue, buprenorphine and naloxone showed similar extraction, whereas morphine was less. Plasma binding and blood/plasma concentration ratios were determined, and perfusion models were used to calculate intrinsic clearance. This in vivo parameter of enzyme activity showed a wide range (200-fold), comparable to the spread of microsomal Vmax/Km ratios. Although the in vivo parameters consistently gave measures 10- to 30-fold higher than the in vitro parameters, the rank order was identical for the two sets. These data suggest that a comparative approach to correlate in vitro and in vivo data has advantages over the use of absolute drug characteristics.

Cimetidine inhibition of antipyrine elimination has been studied in the rat over a range of steady state cimetidine concentrations. Cimetidine is a potent inhibitor of antipyrine metabolism with a concentration of about 1.25 mg/liter causing a 50% decrease in the total plasma clearance of antipyrine. The degree of inhibition of antipyrine clearance caused by cimetidine is dependent upon its plasma concentration, but the relationship is not linear. The formation clearances of all antipyrine metabolites measured--3-hydroxymethylantipyrine, 4-hydroxyantipyrine, and norantipyrine--are inhibited by cimetidine at all concentrations used. The susceptibility of the different metabolites to cimetidine inhibition does vary. The renal clearance of antipyrine was also decreased by cimetidine by an unknown mechanism. Analysis of the individual formation clearances suggests that the inhibition of oxidative metabolism due to cimetidine is caused by its binding to two classes of enzyme sites--a high affinity, low capacity and a low affinity, high capacity site. These two different sites would appear to be responsible for the production of different metabolites of antipyrine. The true nature of the selectivity of cimetidine inhibition of antipyrine metabolism is apparent from the formation clearances but not from the urinary metabolite patterns.

Ethoxycoumarin (EC) and tolbutamide (TOL) were selected as examples of high- and low-clearance drugs, respectively, to investigate suitable methodologies for obtaining kinetic data on metabolism by precision-cut rat liver slices. A number of characteristics of the slice incubation were compound-dependent. TOL showed linear rates of metabolism over a longer time period than EC and was insensitive to the method of incubation (rotating vials and gyrating culture plates). Also, the need for complete tissue disruption (e.g. via sonication) before analysis was essential for EC but unimportant for TOL. This may suggest that conjugates do not freely diffuse out of the liver slice unlike oxidative metabolites. Both drugs showed rates of metabolism that were dependent on slice thickness (150-530 microns). A high turnover (intrinsic clearance 7.9 microliters/min) and low KM (1.3 microM) for EC, and a low turnover (intrinsic clearance 0.8 microliter/min) and high KM (707 microM) for TOL were determined in slices. These differences in kinetic behavior are comparable with those seen in hepatic microsomes, freshly isolated hepatocytes, and in vivo.

The kinetics of caffeine metabolism has been investigated in freshly isolated hepatocytes, hepatic microsomes, and in vivo in male Sprague-Dawley rats. A simple Michaelis-Menten model provides an adequate description of each of the three sets of data. There is reasonable agreement between the KM values for the three systems (56-200 microM). Vmax values for hepatocytes and microsomes show good agreement when expressed in the same units using scaling factors for hepatic cellularity and microsomal protein yield [315 and 420 nmol/min/standard rat weight (SRW), respectively]. Both values slightly exceed the in vivo-determined Vmax (190 nmol/min/SRW). Taking the Vmax/KM ratio (intrinsic clearance) as the basis for scaling, the in vitro data from both the hepatocyte (2.6 ml/min/SRW) and microsomal (2.7 ml/min/SRW) studies provide a good prediction of the in vivo total body clearance (3.4 ml/min/SRW).

The usefulness of determining aminopyrine demethylation kinetics via monitoring of metabolite exhalation rats had been assessed. In rats receiving [N-dimethyl-14C]aminopyrine, a biexponential decline in the 14CO2 exhalation rate is apparent when monitoring is continued over a 5 to 6-hr period. Both the fast phase (representing the first demethylation) and the slower phase (representing the second demethylation) are markedly influenced by phenobarbital and promethazine pretreatment. These changes are consistent with enhanced mixed-function oxidase activity. Examination of the urinary excretion products of aminopyrine obtained in these studies support this claim. The sensitivity of the above index of mixed-function oxidase activity is increased considerably by the use of crossover experimental designs. Considerable interanimal variation is observed in the metabolite production in both exhaled air and urine from rats administered aminopyrine.

Plasma concentration-time profiles for amidopyrine and its primary desmethyl metabolite have been determined in control rats and in rats pretreated with phenobarbitone. The kinetics of both compounds indicate enhanced clearance following pretreatment. Metabolite excretion in urine, bile and breath support the plasma data. It would appear that phenobarbitone pretreatment results in quantitative but not qualitative changes in the metabolism of amidopyrine in rat.

The absorption of [14C] carbenoxolone from everted rat ileum in vitroand from rat stomach and ileum in situhas been examined. The rate of its mucosal to serosal transfer in vitroincreases as pH increases from 5 to 8 whereas the amount bound to ileum tissue decreases with increased pH; absorption closely parallels the drug's solubility. The uptake of carbenoxolone in situis bi-exponential and the rate constants for the two processes, have been calculated. Absorption in situ, and biliary excretion, of the drug increases with increasing pH from 5·0 to 7·4. Tissue binding to the ileum in situis not dependent on pH except below pH 5·0 when extensive tissue accumulation of carbenoxolone occurs because of its low solubility. Tissue binding to the stomach increases markedly with decrease of pH from 7·4 to 6·5 and at pH 6·5 is 80 times greater than binding to the intestine. The rate of absorption from the stomach, at pH 6·5–7·4, was much less than that from the intestine in situ. When allowance is made for the binding of carbenoxolone to the stomach, contrary to the pH-partition hypothesis, correlation is apparent between its absorption and the amount present in the ionized form.

The inhibition of prostaglandin E2(PGE2) synthesis by S-(+)-ibuprofen and piroxicam have been assessed following intravenous and regional (intrapouch) drug delivery using the rat air-pouch model of inflammation. Anti-inflammatory response was defined as the decrease in the area under the exudate PGE2concentration-time curve between 3 and 10 h, following regional administration of the irritant carrageenan.Dose-response studies indicated that bolus regional administration of S-(+)-ibuprofen increased potency 30-fold compared with systemic administration and could be further improved 10-fold by regional infusion, whereas regional administration of piroxicam showed no therapeutic advantage. Examination of the concentration-response using AUC revealed that for a given response, average pouch concentrations for S-(+)-ibuprofen during the PGE2inflammatory response (3 to 10 h) was similar, irrespective of route or mode of administration. In contrast, an advantage following systemic rather than regional administration was revealed for piroxicam, based on plasma concentration-response data, indicating a major systemic anti-inflammatory component for piroxicam but not for S-(+)-ibuprofen.These observations stress the need to take account of both pharmacodynamics and pharmacokinetics when considering the potential advantages of regional administration.

Promethazine blood concentration-time curves have been determined in 7 rabbits following intravenous, oral and hepatic portal vein administration of promethazine. This phenothiazine has a large volume of distribution and a high metabolic clearance resulting in low blood concentrations particularly when the oral route is used. Analysis of the areas under the blood concentration-time curves indicates that hepatic first-pass metabolism is the major determinant of promethazine’s low oral availability. Absorption from the gastrointestinal tract is essentially complete in most rabbits and the contribution of metabolism by the intestinal mucosa is minimal. The present findings are compared with the literature on other phenothiazines.

During a multiple dosing regimen, the area under the promethazine blood concentration-time profile progressively decreased indicating auto-induction of metabolism. The increase in promethazine clearance (mean 35%) was not reflected in changes in either elimination half-life or minimum blood concentrations during the dosing interval. This was attributed to a deep compartment in the disposition of promethazine in the rabbit. The changes in promethazine clearance were accompanied by proportionally larger changes in the clearance of the monodesmethylpromethazine metabolite. The effect of promethazine pretreatment on the clearance of antipyrine was also studied and was found to be significantly increased by a mean of 17% following pretreatment with promethazine. However, the changes in the clearance of antipyrine did not highly correlate with those of promethazine and monodesmethylpromethazine. This may indicate that promethazine induces metabolic systems in the rabbit for which antipyrine is not a good substrate.

The effect of cold stress and ether stress on mixed function oxidase activity in vivo has been studied using the aminopyrine-−14CO2exhalation rate (CER) method. Cold stress following both acute and chronic exposure resulted in enhanced rate of demethylation of both aminopyrine (CER α half-life) and its monomethyl metabolite (CER β half-life). However the time course of the response to cold stress differs for the two demethylations. The characteristics of the cold-induced enhancement of aminopyrine-CER would appear to differ in several aspects from that reported for phenobarbitone pretreatment. In contrast, ether exposure did not produce any consistent effect on drug metabolizing status of the rat.

A procedure for the determination of promethazine in plasma and urine has been developed which involves solvent extraction and gas chromatography using an alkaline flame ionisation detector. This procedure is sensitive and reproducible and can be applied to samples from humans receiving therapeutic doses of promethazine.

The construction of a small library of mouse repetitive DNA has been previously reported (Pietras et al., Nucleic Acids Res. 11:6965-6983, 1983). Here we report that the 35 plasmids in this library corresponding to highly repeated (greater than 30,000 copies per genome) dispersed DNA sequences can be grouped into no more than 5 distinct families. These families together comprise 8 to 10% of the mouse genome. They include the previously described small elements B1, B2, and R and the large MIF-1 element. Twelve of the 35 clones contain evolutionarily conserved (EC) sequences. One EC clone in our library mostly consists of alternating dCdT residues; another consists of tandem repeats of the sequence CCTCT. The majority of B1s and B2s in the genome appear to be homogeneous, whereas R sequences, ECs, and MIF-1s are heterogeneous. Two earlier reports showed highly repeated mammalian DNA sequences in the herpesvirus genome (Peden et al., Cell 31:71-80, 1982; Puga et al., Cell 31:81-87, 1982). We show that sequences homologous to our EC clones are present in the herpesvirus genome, although these polypyrimidine stretches are not detected in poxvirus, adenovirus, and simian virus 40 genomes. We detect transcripts containing homology to all of these sequences in a nuclear transcription assay. Also, we show that small, polyadenylated RNA molecules homologous to B2 sequences are expressed in undifferentiated embryonal carcinoma cells but not in their differentiated derivatives. The significance of these findings is discussed.

The depth dependence of microstructural damage and swelling in pure nickel during nickel-ion bombardment at 0.51 Tm is measured and is compared with the predictions of the E-DEP-1 code. Cavities are developed to depths 30 to 40° greater than the maximum depths calculated for a significant level of displacement events. It is argued that diffusional spreading of point defects contributes to this extension. A helium distribution superimposed on the heavy ion damage profile greatly increases nucleation of cavities and reduces their growth. The swelling profile is modified in a complex manner by the local concentration of helium and by the method of implanting it. In general, helium preimplanted at room temperature strongly over-stimulates cavity formation causing enhanced swelling at low heavy ion dose and depressed swelling at high dose. Helium implanted simultaneously with heavy ion damage gives more gradual promotion of cavities and continuously increasing swelling with dose. A band of deep dislocations is found at twice the predicted maximum depth for damage, and is attributed to relief of the nonuniform swelling by plastic deformation.

Adenylate cyclase [ATP pyrophosphate lyase (cyclizing), EC 4.6.1.1] was shown to be present in cultured human articular chondrocytes. Optimal conditions of incubation time, protein and substrate concentrations and pH were determined in whole cell lysates. Maximal activity occurred at pH 8.5 with no decrease in activity up to pH 10.0. Adenylate cyclase activity of particulate membrane preparations was enhanced by the addition of crude cytosol preparations. The prostaglandins E1, E2, F1 alpha, F2 alpha, D2, B1, B2, A1 and A2, as well as adrenaline and isoprenaline, stimulated adenylate cyclase derived from either adult or foetal chondrocytes. No significant stimulation was observed in the presence of human calcitonin or glucagon. Bovine parathyroid hormone always significantly stimulated the adenylate cyclase derived from foetal chondrocytes, but not from adult chondrocytes. Preincubation of the chondrocytes in culture with indomethacin and with or without supernatant medium from cultured mononuclear cells increased the responsiveness of the adenylate cyclase to prostaglandin E1.

We have used the rat air pouch model of inflammation and S(+)ibuprofen as an experimental model system to enable the quantitative assessment of the pharmacokinetic determinants of site specific drug delivery. S(+)ibuprofen (50 & 1mg/kg) was administered directly into six day old air pouches immediately following the injection of the irritant carrageenan. Serial exudate and plasma samples were collected and analysed for ibuprofen by HPLC. The procedure was repeated following administration of S(+)ibuprofen (20 & 5mg/kg) intravenously. The parameters describing events in the air pouch and plasma indicated linear kinetics over the doses employed. The dose normalised AUCs were then used to formulate a quantitative measure of benefit for S(+)ibuprofen delivered directly to the air pouch. A Drug Targeting Index (DTI) was calculated from the ratio of AUC in the air pouch and plasma following direct inrrapouch administration divided by the same ratio following intravenous administration and gave a value of 130. This pharmacokinetic measure of benefit represents the maximum advantage afforded by the site specific delivery of S(+)ibuprofen as the whole of the administered dose is delivered directly to the site of action.

An N-acetyl-D-[14C]glucosamine radiolabel incorporation assay has been used to monitor chitin biosynthesis in whole cells of Candida albicans both in vitro and in vivo in two different mouse infection models, one using the peritoneal cavity as a chamber in which to add and retrieve cells and the other using infected kidneys. Specific labeling of chitin in alkali-insoluble material was confirmed by chitinase digestion, analysis of acid hydrolysates, and the use of nikkomycin Z as a probe. Nikkomycin Z was shown to strongly inhibit chitin biosynthesis in C. albicans grown in vitro and in vivo in both models. This demonstrates that nikkomycin Z-susceptible chitin synthase activity is present in C. albicans when the fungus is in its pathogenic state in vivo. The limited use of nikkomycin as a therapeutic agent is discussed.

Using laser sources at wavelengths of 1.06 and 10.6 μm, transmitted beam profiles, integrated backscatter, and range-resolved backscatter were measured in laboratory-generated water droplet clouds. Clouds with carefully controlled properties were produced in a specially designed cloud chamber. Inhomogeneities were introduced by partitioning the cloud chamber into three adjacent sections separated by air screens. The measurements show the influence of multiple-scattering effects in both the forward and backward measurement geometries, and these are investigated as functions of optical depth, cloud inhomogeneity, and receiver field of view. These data are unique in many ways, and they provide a great deal of insight to the scattering processes which directly affect lidar-type measurements. As well, these measurements provide a well-documented and detailed database for model validation. Very good agreement is demonstrated with the solutions derived from the multiscattering propagation model described in a companion paper [ Appl. Opt.27, 2478 ( 1988), same issue].

In order to examine the validity of models proposed previously for the gastrointestinal absorption of foreign compounds, the absorption behavior of a series of N-methylated carbamates has been studied in the rat. An optimal partition coefficient for the intestinal absorption of these compounds was demonstrated but this value was approximately one order higher than that reported earlier for a related series of unsubstituted carbamates. It is suggested that this difference is due to the modification in hydrogen bonding arising from N-methyl substitution. In contrast, an optimal partition coefficient for gastric absorption could not be demonstrated within the range of the apparent partition coefficient studied. The absorption behavior of the N-methylated carbamates tends to confirm the different trends previously observed between drug absorption from the intestine and that from the stomach.

The effect of route of administration on competitive drug biotransformation interactions was explored with respect to the interaction between salicylamide and ascorbic acid. Previous studies in man have shown that this interaction involves competition for available sulfate and consequent inhibition of drug sulfate formation. Adult rats received salicylamide (100 mg/kg i.v. or 500 mg/kg orally) alone or with ascorbic acid (500 mg/kg i.v. or orally). Plasma concentrations and urinary excretion rates of salicylamide, salicylamide glucuronide and salicylamide sulfate were determined as a function of time. With i.v. salicylamide, i.v. ascorbic acid caused a modest decrease of salicylamide sulfate formation and thereby of salicylamide body clearance. Orally administered vitamin had a somewhat more pronounced effect. The vitamin had no effect on the apparent volume of distribution of salicylamide and on the renal clearances of its conjugates. With orally administered salicylamide, oral ascorbic acid caused a very pronounced decrease of salicylamide sulfate formation and salicylamide body clearance, whereas intravenous vitamin had little or no effect. These observations are consistent with the assumption that the magnitude of competitive drug biotransformation interactions, being a function of concentration, is most pronounced when the interactants are co-administered orally, due to their high concentrations in the intestinal wall and liver during the first pass.

The National Institute of Standards and Technology (NIST) is establishing an infrared detector calibration facility to improve radiometric standards at infrared wavelengths. The absolute response of the cryogenic bolometer that serves as the transfer standard for this facility is being linked to the NIST high- accuracy cryogenic radiometer (HACR) at a few laser wavelengths. At the 10.6-µm CO_2 laser line, this link is being established through a pyroelectric detector that has been calibrated against the HACR. We describe the apparatus, methods, and uncertainties for the calibration of this pyroelectric detector.

Using the National Institute of Standards and Technology high-accuracy cryogenic radiometer (HACR), we have realized a scale of absolute spectral response between 406 and 920 nm. The HACR, an electrical-substitution radiometer operating at cryogenic temperatures, achieves a combined relative standard uncertainty of 0.021%. Silicon photodiode light-trapping detectors were calibrated against the HACR with a typical relative standard uncertainty of 0.03% at nine laser wavelengths between 406 and 920 nm. Modeling of the quantum efficiency of these detectors yields their responsivity throughout this range with comparable accuracy.

Laboratory measurements are reported for optical extinction at a wavelength of 1.06 µm in water droplet clouds. The extinction coefficient, σ_T, is determined using the two-way attenuation of a target reflected signal and comparing it to the extinction coefficient σ determined by a single-pass transmission measurement. As well as solid targets, layers of the clouds have been used as a reflector by employing a selective chopping method to provide range-resolved backscattering information and replicate in the laboratory a lidar configu-ration. It is found that multiple scattering can lead to substantial differences between σ_T and σ and that these differences depend upon the properties of the scattering medium and the target as well as on the field of view of the backscatter receiver used for the reflectance measurements. By keeping the field of view very small, the two methods of measuring the extinction coefficient give the same values.

Although there are numerous approaches to the treatment of spasticity, many patients are still unable to find a satisfactory method of managing their spasms with acceptable side effects. In the course of our fertility studies using rectal probe electrostimulation (RPES) in SCI men to produce ejaculation, we observed that a majority of the men experienced significant relief of their spasticity for many hours. This report describes a prospective, single-blinded study of this phenomenon in six SCI men and three SCI women who underwent RPES a total of 71 times. The mean age of the subjects was 28.2 years (21-41), the mean time from injury was 6.0 years (0.5-15); there were three paraplegic and six quadriplegic persons: four were Frankel class A and five were class B. Although all subjects had moderate to severe spasticity, only four took antispasm medications; one had undergone surgery for implantation of an epidural stimulator. The effectiveness of RPES on spasticity was evaluated by each subject for frequency of spasms and interference of daily activities and by independent, blinded assessors for tone, frequency of spasms and DTRs; four patients underwent quantitative videotape analysis of the pendulum test and two underwent somatosensory evoked potentials (SSEPs) to evaluate electrical activity in the central nervous system. Treatment variables included varying probe sizes and number of stimulations. All subjects experienced good to excellent decrease in tone, frequency of spasms and interference with ADL from 3 to 24 hours depending on treatment variables used. Mean duration of relief was 8.2 hours. Only one man ejaculated consistently with stimulation and there was no difference in spasticity relief between men and women and between the anejaculatory and ejaculatory males. There was no relation between subject age, age of injury, level or completeness of injury and relief of spasticity. All subjects felt RPES was more effective than medications, stretching or other modalities in relieving spasms, including the one subject with the epidural stimulator. No untoward effects were reported.

A high-accuracy cryogenic radiometer has been developed at the National Institute of Standards and Technology to serve as a primary standard for optical power measurements. This instrument is an electrical-substitution radiometer that can be operated at cryogenic temperatures to achieve a relative standard uncertainty of 0.021% at an optical power level of 0.8 mW. The construction and operation of the high-accuracy cryogenic radiometer and the uncertainties in optical power measurements are detailed.

Two carbohydrate‐phosphoric acid solutions, one a widely used beverage (Solution C) and the other a pharmaceutical product used as an antiemetic (Solution E), administered together with riboflavin‐5′‐phosphate or salicylamide to healthy human adults, significantly increased the bioavailability of riboflavin and appreciably altered the metabolic fate of salicylamide (increased conversion to the sulfate and decreased formation of the glucuronide). A beverage containing phosphoric acid but no carbohydrates (Solution T) also increased the bioavailability of riboflavin but not as much as Solution C. These effects are attributed to a decrease of the gastric emptying rate caused by carbohydrates and phosphoric acid, consistent with the empirical use of Solution C syrup and Solution E as antinauseants and antiemetics. The results demonstrate also that the choice of beverage to be taken with medication can affect the bioavailability and/or metabolic fate of medicinals with saturable absorption and/or biotransformation characteristics.

The kinetics of oxidation of phenytoin and tolbutamide were determined in freshly isolated hepatocytes and hepatic microsomes from male Sprague-Dawley rats. Similar enzyme kinetic models are applicable to the data from both in vitro systems; a two-site model for phenytoin with a high affinity (Km = 1-5 microM, based on unbound drug concentration), low capacity site and a low affinity, high capacity site, and a one-site model for tolbutamide. Steady-state infusion studies were performed to characterize the Michaelis-Menten parameters for phenytoin disposition in vivo, these data could also be described by a two-site metabolism model (Km 1.3 microM, intrinsic clearance 62 ml/min for unbound drug for the high affinity site). Comparison of in vivo and in vitro parameters (after scaling the latter parameters for either hepatocyte yield or microsomal recovery) showed excellent prediction of in vivo clearance of unbound drug from hepatocyte data (55 ml/min) but underprediction from microsomal data (17 ml/min). In contrast to phenytoin, the in vivo clearance of tolbutamide (1.5 ml/min for unbound drug) was equally well predicted by both hepatocyte (2.4 ml/min) and microsomal (3.1 ml/min) studies. The difference between the utility of in vitro systems to predict the in vivo clearance of these two drugs, which show similar pharmacrokinetic properties (low clearance restricted to unbound drug concentration in blood), may be a consequence of the particular terminal metabolite formed in each in vitro system.(ABSTRACT TRUNCATED AT 250 WORDS)

The pharmacokinetics of ethoxycoumarin have been characterized using steady-state plasma concentrations achieved after administration of this compound, at a series of infusion rates, into the hepatic portal vein of rats. The clearance of ethoxycoumarin could be described by a one-site Michaelis-Menten kinetic model with Vmax and unbound KM values of 495 nmol/min/standard rat weight (SRW) and 3.6 microM, respectively, and an intrinsic clearance (CLint, Vmax/KM ratio) of 137 ml/min/SRW (where SRW is 250 g). Urinary excretion experiments, using both ethoxycoumarin and hydroxycoumarin, demonstrated that 7-hydroxycoumarin, the metabolite frequently measured in in vitro studies, accounted for 26% of the metabolism of ethoxycoumarin. In vitro studies with hepatic microsomes and isolated hepatocytes were undertaken to characterize the kinetics of both hydroxycoumarin formation and ethoxycoumarin depletion and to compare the utility of these methods for predicting in vivo clearance. In both in vitro systems, hydroxycoumarin formation displayed biphasic kinetics, with a high-affinity/low-capacity component (with Vmax, KM, and CL1 terms) and a low-affinity/high-capacity component (with a CL2 term) that was not saturated over the substrate concentration range studied (0.5-100 microM). The use of scaling factors to relate in vitro and in vivo data showed that, although microsomal and hepatocyte Vmax values were comparable (26 and 17 nmol/min/SRW, respectively), both were substantially lower than the in vivo value. However, scaling of the in vitro CLint values, by taking into account the fraction of ethoxycoumarin metabolized to hydroxycoumarin, yielded in vivo predictions of 127 and 122 ml/min/SRW (representing 93 and 89% of the observed CLint value) for microsomes and hepatocytes, respectively. The depletion of ethoxycoumarin (1-1.5 microM) with time in both microsomes and hepatocytes displayed a monoexponential decline and predicted in vivo CLint values of 53 and 117 ml/min/SRW (representing 39 and 85% of the observed value), respectively. Therefore, both in vitro systems can accurately predict ethoxycoumarin CLint values using hydroxycoumarin formation rates, providing the importance of this pathway in total clearance is taken into account. Moreover, these results demonstrate that, even when the complete metabolic fate of the compound under investigation is unknown, isolated hepatocytes can be successfully used to predict in vivo CLint values by measurement of substrate depletion with time.

Microsomal protein recovery and hepatocellularity have been determined and investigated as scaling factors for interrelating clearance by hepatic microsomes, freshly isolated hepatocytes and whole liver from untreated (UT) rats and rats treated with either the cytochrome P450 inducer phenobarbital (PB) or dexamethasone (DEX). Hepatocellularity in UT rats (1.1 x 10(8) hepatocytes/g liver) was not significantly different after either PB or DEX induction (1.1 and 1.3 x 10(8) hepatocytes/g liver, respectively). However the microsomal protein recovery index, which provides a scaling factor that is inversely related to the efficiency of the microsomal preparation procedure, was 47 mg/g liver in both PB and DEX microsomes and differs from UT rats (60 mg/g liver). These contrasting findings are consistent with the interlaboratory trends in the literature, indicating that, although hepatocellularity estimates are in good accord, microsomal recovery can vary 2-fold; this has implications for scaling. The oxidation of diazepam to its three primary metabolites was measured in PB and DEX microsomes and hepatocytes and the scaling factors were applied to these data and previously reported UT data. Marked changes in kinetics occur on induction resulting in a shift in the major pathway. In particular, 3-hydroxylation is induced over 20-fold by DEX. Diazepam CL(int) was determined in vivo after administration of a bolus dose into the hepatic portal vein of UT, PB, and DEX rats; values of 127, 191, and 323 ml/min/SRW (where SRW is a standard rat weight of 250 g), respectively, were obtained. Using these scaling factors, the hepatocyte predictions of CL(int) were excellent (99, 144, and 297 ml/min/SRW for UT, PB, and DEX, respectively), whereas only the DEX prediction (248 ml/min/SRW) was accurate for the microsomal system, with a substantial underprediction for UT and PB (46 and 68 ml/min/SRW, respectively). Evidence is presented for product inhibition, resulting from accumulation of primary metabolites within the microsomal preparation, as the mechanism responsible for this underprediction. These results illustrate that the scaling factor approach is applicable to induced livers in which both cytochrome P450 complement and zonal distribution are altered. These data, together with our previous studies, demonstrate that CL(int) in cells (2.4-297 ml/min/SRW), microsomes (2.7-248 ml/min/SRW), and in vivo (1.5-323 ml/min/SRW) are related in a linear fashion and hence inherently both in vitro systems are of equal value in predicting in vivo CL(int).

The time course for distribution of five compounds (caffeine, tolbutamide, phenytoin, ondansetron, and diazepam) was studied in precision-cut rat liver slices. Transport of these compounds differed greatly, with caffeine being distributed rapidly, but not accumulating above the media concentration. Although tolbutamide similarly was not accumulated within the tissue, its uptake rate was slower. The rate of phenytoin, ondansetron, and diazepam distribution (with appropriate corrections for metabolism) was slower still; yet, these drugs were accumulated within the cells of the slice to a concentration approximately 15-fold that of the media. Examination of the physicochemical properties of these compounds demonstrated that the extent of accumulation positively correlated with lipophilicity, whereas the rate of uptake was not statistically correlated with log D. The extent of accumulation within the slice was assessed by an apparent volume of distribution parameter (ranging from 26 to 195 microliters/slice). Using cell:media partition coefficients determined in hepatocytes and the intra- and extracellular spaces within the slice (as measured with the markers tritiated water and sucrose), it was possible to predict apparent volumes of distribution for each drug in the liver slice. Comparison of observed and predicted apparent volumes of distribution gave ratios of 0.34-1. Intrinsic clearance values for these five drugs are available for slices and cells (slice: cell intrinsic clearance ratios 0.05-0.43; Worboys et al., Drug Metab. Dispos. 24, 676-681, 1996). Drugs that demonstrate low intrinsic clearance ratios also have low apparent volume ratios, thus indicating that reduced drug uptake and clearance in slices, relative to hepatocytes, are interdependent. Both phenomena may be rationalized by the existence of a drug concentration gradient within the slice. At very high drug concentrations, Vmax operates, and the consequence of the gradient is minimal. Therefore, it is possible to speculate upon the fraction of hepatocytes within the slice contributing to clearance by considering Vmax values. For six pathways of metabolism, Vmax in slices averages 35% of the corresponding parameter in isolated hepatocytes. This is most likely due to limited oxygen and compromised metabolic function of the core cells. These distribution phenomena severely complicate the possibility of using a scaling factor based on the theoretical number of slices obtainable from a liver to predict in vivo intrinsic clearance.