Acclimation to decompression sickness in rats.

Protection against decompression sickness (DCS) by acclimation to hyperbaric decompression has been hypothesized but never proven. We exposed rats to acclimation dives followed by a stressful "test" dive to determine whether acclimation occurred. Experiments were divided into two phases. Phase 1 rats were exposed to daily acclimation dives of hyperbaric air for 30 min followed by rapid decompression on one of the following regimens: 70 ft of seawater (fsw) for 9 days (L70), 70 fsw for 4 days (S70), 40 fsw for 9 days (L40), 40 fsw for 4 days (S40), or unpressurized sham exposure for 9 days (Control). On the day following the last exposure, all were subjected to a "test" dive (175 fsw, 60 min, rapid decompression). Both L70 and S70 rats had significantly lower incidences of DCS than Control rats (36% and 41% vs. 62%, respectively). DCS incidences for the other regimens were lower than in Control rats but without statistical significance. Phase 2 used the most protective regimen from phase 1 (L70); rats were exposed to L70 or a similar regimen with a less stressful staged decompression. Another group was exposed to a single acclimation dive (70 fsw/30 min) on the day before the test dive. We observed a nonsignificant trend for the rapidly decompressed L70 dives to be more protective than staged decompression dives (44% vs. 51% DCS incidence). The single acclimation dive regimen did not provide protection. We conclude that protection against DCS can be attained with acclimating exposures that do not themselves cause DCS. The deeper acclimation dive regimens (70 fsw) provided the most protection.

Physicochemical considerations in the preparation of amorphous ritonavir-poly(ethylene glycol) 8000 solid dispersions.

A systematic study of the properties of ritonavir and the influence of polyethylene glycol 8000 (PEG) on ritonavir revealed that amorphous ritonavir dispersions in PEG would have an improved dissolution profile and could exhibit long-term stability. Ritonavir, a human immunodeficiency virus (HIV) protease inhibitor, is highly lipophilic [distribution coefficient (log D)= 4.3, 25 degrees C, pH 6.8], poorly water soluble (400 microg/mL in 0.1 N HCl, 1 microg/mL at pH 6.8, 37 degrees C), and exhibits an exceedingly slow dissolution rate (0.03 mg/cm(2)-min in 0.1 N HCl at 37 degrees C). These properties indicated that a solid dispersion containing ritonavir might be useful for overcoming problems associated with slow dissolution. In addition, ritonavir is a good glass former [glass-transition temperature (T(g))/melting point (T(m)) > 0.7]. Amorphous ritonavir has an apparent solubility of 4 mg/mL in 0.1 N HCl at 37 degrees C and shows reasonable stability at 25 degrees C. Amorphous ritonavir, therefore, has properties desirable for preparing a solid dispersion containing this phase. Since PEG, a commonly used polymer, improved the aqueous solubility of crystalline ritonavir, it was expected to have a positive influence on the dissolution rate of ritonavir. Moreover, PEG was found to have negligible plasticizing effect on amorphous ritonavir, which was beneficial for the stability of the dispersion. Finally, solid dispersions of amorphous ritonavir in PEG were prepared, and these dispersions had improved in vitro dissolution rate and were physically stable for > 1.5 years at 25 degrees C when protected from moisture. The performance of this solid dispersion has been attributed to the physicochemical properties of amorphous ritonavir.

Decompression comparison of helium and hydrogen in rats.

The hypothesis that there are differences in decompression risk between He and H2 was examined in 1,607 unanesthetized male albino rats subjected to dives on 2% O2-balance He or 2% O2-balance H2 (depths < or = 50 ATA, bottom times < or = 60 min). The animals were decompressed to 10.8 ATA with profiles varying from rapid to slow, with up to four decompression stops of up to 60 min each. Maximum likelihood analysis was used to estimate the relative decompression risk on a per unit pressure basis (termed "potency") and the rate of gas uptake and elimination, both factors affecting the decompression sickness risk, from a specific dive profile. H2 potency for causing decompression sickness was found to be up to 35% greater than that for He. Uptake rates were unresolvable between the two gases with the time constant (TC) estimated at approximately 2-3 min, leading to saturation in both cases in < 15 min. Washout of both gases was significantly slower than uptake, with He washout (TC approximately 1.5-3 h) substantially slower than H2 washout (TC approximately 0.5 h). It is unknown whether the decompression advantage of the faster washout of H2 or the disadvantage of its increased potency, observed in the rat, would be important for human diving.

Chemical safety of U.S. Navy Fleet soda lime.

Contamination was suspected of U.S. Navy Fleet soda lime (High Performance Sodasorb) when an ammonia-like odor was reported during its use in August 1992. This material contained indicator dye and was used for carbon dioxide absorption during diving. This incident had a major impact on the U.S Navy diving program when the Navy temporarily banned use of Sodasorb and authorized Sofnolime as an interim replacement. The Naval Medical Research Institute was assigned to investigate. Testing involved sampling from the headspace (gas space) inside closed buckets and from an apparatus simulating conditions during operational diving. Volatile organic compounds were analyzed by gas chromatography and mass spectrometry; ammonia and amines were measured by infrared spectroscopy. Significant amounts of ammonia (up to 30 ppm), ethyl and diethyl amines (up to several ppm), and various aliphatic hydrocarbons (up to 60 ppm) were detected during testing of both Sodasorb and Sofnolime. Contaminants were slowly removed by gas flow and did not return. The source(s) of the ammonia and amines are unknown, although they may result from the breakdown of the indicator dye. Hydrocarbon contamination seems to result from the materials of which the bucket is constructed. Unfortunately, evaluation of potential hazards associated with this contamination is difficult, due in part to the large number of variables of operational use and the absence of appropriate exposure limits. Based on these findings, the U.S. Navy has begun to phase in, for all diving, non-indicating soda lime that will be required to meet defined contaminant limits.

Physicochemical properties of A-75998, an antagonist of luteinizing hormone releasing hormone.

The physicochemical properties of A-75998, a synthetic antagonist of luteinizing hormone releasing hormone with potential for treatment of hormone-sensitive cancers and endometriosis, are described. An accelerated solution stability study indicated that the compound is relatively stable and showed a U-shaped pH-rate profile, with maximum stability between pH 4.5 and 6.5. The acid dissociation behavior of A-75998 was examined by UV-visible spectrophotometry at 25 degrees C in a series of buffers ranging from pH 1 to 13. The data were fit to a model in which the dissociations of all four ionizable groups contributed to changes in the absorbance. The estimated macroscopic acid dissociation constants were p beta 1 = 3.230 +/- 0.022, p beta 2 = 4.885 +/- 0.030, p beta 3 = 9.871 +/- 0.022, and p beta 4 = 11.026 +/- 0.157. The corresponding microscopic dissociation constants were pk1 = 3.24 (nicotinyl), pk2 = 4.88 (pyridyl), pk5 = 9.91 (tyrosyl), and pk6 = 10.99 (isopropyllysyl). The apparent n-octanol/water partition coefficients were measured from pH 2 to 13, and the profile was consistent with the expected acid-dissociation behavior. While appearing fairly water-soluble at pH < 5, dynamic light scattering of A-75998 in pH 4.5 buffer indicated the formation of aggregates of nonuniform size distribution. A-75998 exhibited reverse or thermal gelation; sodium chloride exacerbates this gel formation and self-association. Surface activity was pH-dependent, but no evidence was found for micelle formation. Based on the results, development of a parenteral formulation of A-75998 appears feasible, provided that aggregation can be minimized.

Thermal stability of low molecular weight urokinase during heat treatment. II. Effect of polymeric additives.

Turbidimetric or light scattering assays can be used to determine the extent of aggregation in protein formulations. Using low molecular weight urokinase (LMW-UK) as a model protein, the effect of polymeric additives on heat-induced aggregation was evaluated. Previous work has shown that under 60 degrees C heat treatment, LMW-UK initially denatures and the unfolded protein associates to form soluble aggregates. Eventually, these aggregates associate to form a precipitate. The effects of polymers on the initial aggregation phase was examined. Hydroxyethyl (heta) starch, polyethylene glycol 4000, and gelatin were found to be effective, concentration-dependent inhibitors of aggregation, whereas polyvinylpyrrolidone (PVP) and polyethylene glycol 300 were ineffective. Overall, the effect of polymeric additives on the stability of thermally-stressed LMW-UK can be accounted for by preferential exclusion of the solute from the surface of the protein.

Thermal stability of low molecular weight urokinase during heat treatment. I. Effects of protein concentration, pH and ionic strength.

Exposure of low molecular weight urokinase (LMW-UK) to prolonged heating (60 degrees C, 10 hours) is used to inactivate possible viral contaminants. This process leads to a significant loss of active enzyme. Amidolytic activity was monitored following heat treatment in order to establish the conditions for maintaining the optimal stability of LMW-UK. The effects of pH, ionic strength, protein concentration, and various ionic additives were examined. While LMW-UK is stable across a wide pH range (pH 2-11), heating LMW-UK in aqueous solution leads to complete loss of activity except between pH 4 and 7.5. The mechanism of inactivation was delineated using activity assays as well as turbimetric and spectroscopic methods. Thermal inactivation occurs via aggregation of unfolded LMW-UK, followed by subsequent precipitation. Threshold effects upon the thermally-induced aggregation of LMW-UK were observed.

Effects of tannic acid on antigenicity and membrane contrast in ultrastructural immunocytochemistry.

We have modified our previous method for immunogold staining of unosmicated, plastic-embedded tissue by addition of tannic acid as a post-fixative to increase membrane contrast. Overall cell ultrastructure and organelle membranes, in particular, appeared well preserved after this treatment. We evaluated quantitatively the effect of tannic acid on the antigenicity of several membrane proteins in rat liver and intestine. For all antigens tested, significant antigenicity was retained on both intracellular and plasma membranes. However, the level of antigenicity decreased with increased concentrations of tannic acid. This effect was most apparent on the apical and basolateral membranes of hepatocytes and on the apical membrane of enterocytes, surfaces that had been in direct contact with the tannic acid fixative. The results indicate that when low concentrations of tannic acid are employed, this method yields greatly enhanced membrane contrast while preserving sufficient antigenicity to facilitate the ultrastructural localization of many membrane and other antigens.

The major subunit of the rat asialoglycoprotein receptor can function alone as a receptor.

Mammalian hepatic asialoglycoprotein receptors (ASGP-R) are composed of two unique, but closely related polypeptides, which in the rat are designated rat hepatic lectins 1 and 2/3 (RHL 1, RHL 2/3). Despite numerous studies, the composition of a functional ASGP-R has remained unclear. We examined this question in rat hepatoma tissue culture (HTC) cells (which lack endogenous ASGP-R) that were co-transfected with cDNAs for both RHL 1 and RHL 2/3. The original population was cloned, but derivatives were unstable. We therefore used fluorescence-activated cell sorting to separate a subpopulation of cells (positive) that specifically endocytosed fluoresceinated asialoorosomucoid (ASOR) from one that did not (negative). We then used indirect immunofluorescence with polypeptide-specific ASGP-R antibodies, immunoanalysis, and binding and uptake studies with two Gal ligands (ASOR and NAc-galactosylated poly-L-lysine (Gal-Lys] to further define the ASGP-R status in these two populations. As reported by others, we found that expression of both RHL 1 and RHL 2/3 in the positive cells resulted in binding, uptake and degradation of ASOR, the most commonly used ASGP-R ligand. The negative cells expressed only RHL 1 and neither bound nor processed ASOR. However, the presence of RHL 1 was sufficient for specific high affinity binding and processing of the synthetic ligand, Gal-Lys, by negative cells. These results show that RHL 1 can function as an ASGP-R, given a highly galactosylated ligand, and that RHL 2/3 must play an important role in the organization of native ASGP-R in the membrane.

Variation in light transmission properties of amber oral syringes.

Four brands of amber oral syringes were tested for compliance with compendial standards for light-resistant packaging. United States Pharmacopoeia (USP) specifications state that light-resistant packaging must not transmit more than 10% of incident light with wavelengths of 290-450 nm. The amount of light transmitted by samples of the barrels of 200 amber plastic oral syringes from each of four manufacturers was determined by spectrophotometry. Samples of both the printed and unprinted sides of the barrel were tested (after removal of all ink). The thickness of each syringe-barrel sample was measured, and the apparent density was determined. All of the Becton-Dickinson syringes tested met the USP standard for light transmittance, and none of the syringes from Baxa or Solopak met the compendial standards. Of the Burron syringes tested, 70 syringes met the standard, 83 did not meet the standard, and 47 could not be classified. The samples of the printed side of the syringe barrel had light transmission properties similar to those of the unprinted side with the exception of the syringes from Baxa, which had substantially higher apparent densities at all wavelengths for the printed side. Only one manufacturer's oral syringes consistently met the USP standard for light-resistant packaging. Further testing is needed to determine the clinical importance of these findings.

Chemical and statistical considerations in the determination of partition coefficients of weakly ionizable drugs and poisons.

Equations have been developed that relate the concentration (or a parameter directly proportional to concentration, such as optical absorbance) of a weakly ionizable solute in a water-immiscible phase, in equilibrium with an aqueous phase, to the pH of the aqueous phase, the partition coefficient of the unionized solute and the phase volume ratio. These relationships have been used in the design of experimental methods for determining partition coefficients, which require measurement of solute concentration in only one phase. Data obtained in this way permit ready recognition of deviations from assumptions made in the development of the model; these assumptions include insolubility of the ionized solute in the water-immiscible phase and lack of interaction between buffer components and solute. Conditions for optimal liquid-liquid extraction of weakly ionizable solutes are more easily recognized. With these techniques, the negative logarithm of the acid dissociation constant (pK'a) and the logarithm of the octanol-water partition coefficient (log P) have been measured for warfarin (pK'a = 5.15 +/- 0.04; log P = 2.82 +/- 0.06), strychnine (pK'a = 8.29 +/- 0.02; log P = 2.23 +/- 0.04), phenol (pK'a = 9.88 +/- 0.02; log P = 1.75 +/- 0.05), procaine (pK'a = 8.11 +/- 0.04; log P = 1.10 +/- 0.08), and ephedrine (pK'a = 9.92 +/- 0.01; log P = 1.65 +/- 0.04) at 21 degrees C.

Adsorption of insulin to the surface of peritoneal dialysis solution containers.

Continuous ambulatory peritoneal dialysis (CAPD) is becoming widely used in diabetics with end-stage renal disease. One of the proposed advantages of this technique is the ability to administer insulin intraperitoneally. The administration of insulin by this route provides acceptable plasma glucose control with no need for subcutaneous injections. Because adsorption of insulin to glass and polyvinyl chloride (PVC) surfaces is known to occur with intravenous fluid systems, this study was conducted to measure insulin adsorption to dialysis fluid systems. The effects of time, temperature, heparin concentration, dextrose concentration, and insulin concentration in 2-L glass and PVC dialysis solution containers were studied. While all factors significantly affected adsorption to the PVC containers, the effects of heparin and dextrose were considered to be clinically unimportant. The percentage of insulin adsorbed to the PVC surface increased with increasing time and temperature and decreased with increasing insulin concentrations. Under the conditions studied, 7.1% to 9.6% of insulin added to PVC containers was lost within the first minute. Since 15 to 30 minutes is required by most CAPD patients to prepare and instill the dialysate, 10% to 20% of added insulin may be adsorbed to the PVC bad. The adsorption of insulin to the glass surfaces was rapid with 39.6% to 42.8% being lost within the first minute. All factors studied significantly affected adsorption to the glass, but the effects of time, temperature, dextrose, and heparin were considered to be of minor clinical importance.(ABSTRACT TRUNCATED AT 250 WORDS)

Resolution of valproic acid from deuterated analogues and their quantitation in plasma using capillary gas chromatography.

Quantitation of valproic acid and a deuterated analogue in the same plasma sample by capillary gas chromatography without mass spectrometry was illustrated. Specificity was accomplished solely with a 60 m X 0.25 mm fused silica WCOT column coated with OV-351. A hexadeutero and two tetradeutero analogues of valproic acid had resolutions of at least 1.2 from valproic acid. Plasma samples were extracted with carbon tetrachloride following the addition of 2-ethylhexanoic acid as the internal standard. The method is sensitive to at least 0.5 microgram/ml and provides the capability of conducting absolute bioavailability and pulsed dosing studies with deuterated drug analogues without a mass spectrometer. The technique was applied to the analysis of plasma samples from dogs simultaneously administered valproic acid and a deuterated analogue.

Effect of thioacetamide-induced hepatic necrosis on the regioselective metabolism of S-warfarin by rat liver mixed-function oxidase enzymes.

The biotransformation of S-warfarin was examined using liver microsomes prepared from rats 6-96 hr after treatment with a necrotizing dose (5.6 mmoles/kg) of thioacetamide. Four catalytically distinct classes of enzyme activity were observed which declined in activity with different half-lives after thioacetamide intoxication. S-Warfarin 7-hydroxylase activity was destroyed with a half-life of 16.6 +/- 3.1 hr. 6-Hydroxylase activity was destroyed with a half-life of 25.3 +/- 3.0 hr. 4'-Hydroxylase activity was destroyed with a half-life of 34.6 +/- 4.8 hr, which paralleled the loss of total hepatic cytochrome P-450 with a half-life of 33.4 +/- 3.6 hr. Production of an unidentified metabolite was not affected by thioacetamide intoxication during the first 48 hr. The ratio of rates of product formation were used as an alternative method to test the homogeneity of distinct enzyme catalytic activities. The ratio of measured responses (e.g. chromatographic peak heights) was used directly to determine the product ratios, provided that the rate of formation of each product was directly proportional to the experimentally measured response for each product. The use of product response ratios to discriminate between catalytic activities was inherently more precise because calibration errors were eliminated. Differences in the rates of destruction of warfarin hydroxylases provided further evidence of the multiplicity of hepatic mixed-function oxidases and suggested topographical differences in their location within the liver lobule.

Sublingual lorazepam: a better premedication?

The efficacy of a sublingual preparation of lorazepam was compared with an i.m. injection of lorazepam as premedication for 150 patients undergoing minor gynaecological surgery. Anxiety, arousability and recall of auditory, visual and tactile stimuli were used as measurements of the degree and rate of onset of the effects. Anxiety scores decreased in both groups after medication. Patients who received the drug sublingually showed less recall, an earlier onset of sedation, more drowsiness and a longer recovery time than those who received the drug i.m. It is concluded that lorazepam given by the sublingual route is superior to that given i.m. because of more rapid absorption resulting in earlier drowsiness and more amnesia. An additional advantage is the absence of the discomfort of an injection.

Effects of dose and dosing regimen on tissue distribution and elimination kinetics of [14C] dithiobiuret in rats.

[14C] Dithiobiuret (DTB)-derived radioactivity is eliminated by adult male rats with an approximate plasma half-life of 8-10 hr. About 65-75% of an i.p. dose appears in the urine within 24 hr after treatment and about 2-4% appears in the feces during the same time period. Less than 1% is recovered as [14C] carbon dioxide in expired air. The elimination kinetics for plasma and the % dose of DTB eliminated as the parent compound and metabolites in urine and feces, respectively, are the same for 1 mg/kg and 25 mg/kg acute treatments and for 1 mg/kg/day chronic treatment. Dose and dosing regimen dependent changes in tissue distribution are evident for most tissues with the thyroid gland, lung, stomach and fat being the most affected. Accumulation of DTB or its metabolites in the thyroid gland is greater than in any other tissue and is saturable at 25 mg/kg. The lung and stomach have a lower concentration of [14C] DTB equivalents than the thyroid, but they show the greatest relative increase in concentrations, about 5 fold, when [14C] DTB is administered chronically. Conversely, fat does not accumulate DTB-derived radioactivity during chronic dosing. These findings suggest that DTB metabolism is altered by changes in dose and dosing regimen.