Simulated workplace protection factor study of powered air-purifying and supplied air respirators.

A study protocol was developed to obtain simulated workplace protection factor (SWPF) data for eleven models of powered air-purifying respirators (PAPRs) and supplied-air respirators (SAR) with hoods and helmets. Respirators were tested in a chamber that allowed the simulation of 12 exercises, including 2 exercises of interest to the pharmaceutical industry. Each respirator was tested by 12 volunteers, and a total of 144 sets of test results were obtained for each device. The testing protocol allowed SWPFs up to 250,000 to be measured (limit of quantification). Median SWPFs for all respirators, except one SAR, were at or above this reporting limit. Lower fifth percentiles were above 100,000, except for one SAR previously noted. An assigned protection factor (APF) was estimated for each respirator by dividing the lower fifth percentile by a safety factor of 25. APFs ranged from 6000-10,000 for PAPRs (including one loose-fitting PAPR) and 3400-10,000 for SARs, with one exception. This SAR had a lower fifth percentile of less than 20 and an estimated APF of 1. Results indicated that most respirators tested could provide a high degree of protection for workers, although one National Institute for Occupational Safety and Health-approved SAR provided minimal, if any, protection. Direct testing in a simulated workplace seems the only method that will assure employers of choosing an adequate SAR. This may be true for other classes of respirators. Furthermore, the historical approach of establishing APFs for classes of respirators, rather than individual models, may not provide adequate protection to the wearer. This is also a serious problem for regulatory agencies seeking to promulgate respirator standard provisions such as APFs for classes of respirators.

Potent, orally active GPIIb/IIIa antagonists containing a nipecotic acid subunit. Structure-activity studies leading to the discovery of RWJ-53308.

Although intravenously administered antiplatelet fibrinogen receptor (GPIIb/IIIa) antagonists have become established in the acute-care clinical setting for the prevention of thrombosis, orally administered drugs for chronic use are still under development. Herein, we present details from our exploration of structure-activity surrounding the prototype fibrinogen receptor antagonist RWJ-50042 (racemate of 1), which was derived from a unique approach involving the gamma-chain of fibrinogen (Hoekstra et al. J. Med. Chem. 1995, 38, 1582). Our analogue studies culminated in the discovery of RWJ-53308 (2), a potent, orally active GPIIb/IIIa antagonist. To progress from RWJ-50042 to a suitable candidate for clinical development, we conducted a series of optimization cycles that employed solid-phase parallel synthesis for the rapid, efficient preparation of nearly 250 analogues, which were assayed for fibrinogen receptor affinity and inhibition of platelet aggregation induced by four different activators. This strategy produced several promising analogues for advanced study, including 3-(3,4-methylenedioxybenzene)-beta-amino acid analogue 3 (significant improved in vivo potency) and 3-(3-pyridyl)-beta-amino acid 2 (significantly improved potency, oral absorption, and duration of action). In dogs, 2 displayed significant ex vivo antiplatelet activity on oral administration at 1.0 mg/kg, 16% systemic oral bioavailability, minimal metabolic transformation, and an excellent safety profile. Additionally, 2 was found to be efficacious in three in vivo thrombosis models: canine arteriovenous (AV) shunt (0.01-0.1 mg/kg, iv), guinea pig photoactivation-induced injury (0.3-3 mg/kg, iv), and guinea pig ferric chloride-induced injury (0.3-1 mg/kg, iv). On the basis of its noteworthy preclinical data, RWJ-53308 (2) was selected for clinical evaluation.

Macrocyclic hexapeptide analogues of the thrombin receptor (PAR-1) activation motif SFLLRN.

The thrombin receptor (PAR-1) is activated by alpha-thrombin to stimulate various cell types, including platelets, through the tethered-ligand sequence SFLLRN. Macrocyclic peptide analogues of SFLLRN were synthesized and evaluated in vitro. In general, the compounds were much less potent in inducing platelet aggregation relative to SFLLRN-NH2 and did not act as antagonists of alpha-thrombin. Derivative 3c was the most potent macrocycle in activating PAR-1, with an EC50 of 24 microM.

Functional diversity and clonal frequencies of reactivity in the available antibody repertoire.

The present experiments address functional antibody diversity and clonal distribution in murine available repertoires. IgM-containing supernatants were prepared by unbiased, polyclonal stimulation of resting splenic B cells from C57BL/6 mice, to ensure similar numbers of responding clones/culture and equivalent growth and maturation of all clones. The repertoires of clones and clonal mixtures were quantitatively assayed by limiting dilution analysis (LDA) on immunoblots of sodium dodecylsulfate polyacrylamide gel electrophoresis of homologous liver extracts, allowing to determine specific clonal frequencies towards the many hundred blotted antigens. The clonal frequency of reactivity of B cells with the extract was shown to be a bi-modal distribution of specific frequencies between 1/220 and 1/100,000. Cross-correlation analysis of reactivity to different bands in individual supernatants revealed low levels of cross-reactivity, suggesting that the blotted extract provides a very diverse set of antigens. Investigation of the affinity/concentration thresholds for detection of antigen-antibody interactions of our assay supports the notion that global repertoire analyses on immunoblots were highly discriminative and non-degenerate. Furthermore, reactivity patterns obtained with complex antibody mixtures correlated with the frequency of clonal reactivities as determined by LDA. The results demonstrate a large functional diversity of resting B lymphocytes, indicating a minimal repertoire size that is orders of magnitude higher than previous theoretical proposals suggested, and extensively heterogeneous in the size of clonal specificities.

Microsatellite instability and K-ras mutations associated with pancreatic adenocarcinoma and pancreatitis.

ras oncogene mutations and microsatellite instability (MIN) have been described in pancreatic cancer studies from paraffin blocks and fresh frozen tissue. We sought to determine whether they could be detected in endoscopic retrograde cholangiopancreatography-derived pancreatic juice. ras mutations were detected in the pancreatic juice of 40% (2 of 5) of patients with pancreatic cancer and 2 of 5 patients with pancreatitis. MIN was detected at a single locus in the pancreatic juice of 40% of pancreatic cancer patients and at > or = 2 loci of 100% of pancreatitis patients. The finding of MIN in pancreatitis specimens was verified in studies performed on paraffin blocks. MIN was not detected in normal pancreas controls. All of the cancer patients who had ras mutations in their pancreatic juice also had evidence of MIN at one or more loci (P < or = 0.05), suggesting that MIN is associated with the development of a ras mutation. More importantly, the finding of MIN in pancreatitis specimens suggests that MIN can occur in nonneoplastic conditions of the pancreas and may represent the saturation of an intact mismatch repair system.

Setting health-based residue limits for contaminants in pharmaceuticals and medical devices.

A procedure for determining health-based residue limits for impurities in drug substances and medical devices is described. The procedure is based upon the concept of setting residue limits that correspond to the intended usage of the drug or device, i.e., short-term use, prolonged use, and/or lifetime use. Data pertaining to chemical and physical properties, occurrence and use, biodisposition, pharmacology, toxicology, and effects in people are used. After evaluation of these data, acceptable daily intake (ADI) values are derived using a safety margin approach for short-term and prolonged exposure limits. The safety margin approach combines the use of safety factors and professional judgment. ADI values for lifetime exposure are calculated using the safety margin approach for noncarcinogens and for some carcinogens, and they are calculated using risk assessment procedures that provide ADI values corresponding to no more than a 1 in 10,000 excess lifetime cancer risk based upon maximum likelihood risk levels for other carcinogens. A weight-of-evidence test determines the use of each approach. Finally, ADI values from relevant routes and endpoints are compared and a residue limit or residue limits are estimated. The standard is expressed in terms of maximum dose per exposure period and/or dose per day and is applicable to medical products intended for short-term use, for prolonged use, and/or for lifetime use as a major clinical indications dictate.

Detection of distinct isoform patterns of the beta-amyloid precursor protein in human platelets and lymphocytes.

Cerebral deposition of the amyloid beta-protein (A beta P), approximately 40 residue fragment of the integral membrane protein, beta-amyloid precursor protein (beta APP), has been implicated as the probable cause of some cases of familial Alzheimer's disease (AD). The parallels between A beta P deposition in AD and the deposition of certain plasma proteins in systemic amyloid diseases has heightened interest in the analysis of beta APP in circulating cells and plasma. Here, we describe distinct isoform patterns of beta APP in peripheral platelets and lymphocytes. PCR-mediated amplification of mRNA from purified platelets demonstrated the expression of all three major beta APP transcripts (beta APP770,751,695). The full-length, approximately 140 kDa form of beta APP751,770 was detected in membranes of resting and activated platelets but very little immature, approximately 122 kDa beta APP751,770 was found, suggesting a different processing of beta APP in platelets than that described in a variety of cultured cells and tissues. Platelets stimulated with thrombin, calcium ionophore, or collagen released the soluble, carboxyl-truncated form of beta APP (protease nexin-II), but no evidence for the shedding of full-length beta APP associated with platelet microparticles was found, in contrast to previous reports. As a positive control marker for microparticles, the fibrinogen receptor subunit, GPIIIa, was readily detected in platelet releasates. Resting and activated platelets contained similar amounts of the approximately 10 kDa carboxyl terminal beta APP fragment that is retained in platelet membranes following the constitutive cleavage of protease nexin-II. Nonstimulated peripheral B and T lymphocytes contained small amounts of membrane-associated mature and immature beta APP751,770. The potentially amyloidogenic full-length beta APP molecules present in circulating platelets and lymphocytes but not in microparticles could serve as a source of the microvascular A beta P deposited during aging and particularly in AD.

Internal wall losses of pharmaceutical dusts during closed-face, 37-mm polystyrene cassette sampling.

A current practice for the determination of personal exposures to dusts involves the aspiration of known quantities of air through membrane filters held in 37-mm plastic cassettes. Samples are collected with the cassettes in the closed-face configuration. A major negative bias error has been identified with this sampling procedure for low-level pharmaceutical dusts. For the pharmaceuticals studied, on average, 62% of the active dust collected in each sample was found on the inside surface of the cassette top. Only 22% of the total active ingredient of the dust was found on the filters. The remaining 16% was found on the inside of the cassette bottoms; electrostatic attraction appears to be the reason that pharmaceutical dusts adhere to the inside surface of the cassette. Adherence to the inside surfaces of the polystyrene cassette occurs without regard to the type of material used to seal the two-piece cassette together. The use of shrink wrap versus plastic tape versus using no sealing material had no effect on where or how much of the active ingredient was found on the inside cassette surfaces. Because very little active ingredient was identified in backup cassettes, it is hypothesized that the active ingredient found on the inside of the bottom portion of the cassettes (past the filter and support pad) got there by falling off the filter during filter removal from the cassette prior to analysis. To eliminate both of these errors, an internal cassette extraction procedure was developed that (1) negates the error caused by static charging and (2) eliminates the need for opening the cassettes prior to analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Development and validation of a protocol for field validation of passive dosimeters for ethylene oxide excursion limit monitoring.

An exposure and analysis protocol is described for the field validation of passive dosimeters for ethylene oxide (EtO) excursion limit monitoring. The protocol calls for the use of a field exposure chamber with concurrent sampling using Tedlar air-sampling bags. The bags are analyzed immediately after sampling by gas chromatography with flame ionization detection (GC-FID). The chamber design allows all monitors to be exposed for the exact same time in the field. The sampling and analysis procedure not only determines the actual concentration of EtO present during the monitor's exposure but estimates if concentrations of EtO vary from point to point in the monitor array during the exposure. In chamber operation, the accuracy of the standard generator used to calibrate the GC-FID was independently verified in the field by the standard additions method. The sampling bias of the sampling train was determined to be -3.5% in the 2.4 ppm to 14.3 ppm concentration range. To estimate the stability of collected EtO samples in Tedlar bags, the rate of EtO loss in the bags was determined to be 0.011 ppm/hr at 2.57 ppm and 0.066 ppm/hr at 8.07 ppm. Sampling bias of the passive methods by additional EtO exposure of the monitors in the closed chamber after sampling and during purging was determined to be +1.5%. The Tedlar bag sampling method with subsequent GC-FID determination demonstrated a coefficient of variation of 1.8% at 2.43 ppm.

Field validation of three passive dosimeters for excursion limit monitoring of ethylene oxide.

The Occupational Safety and Health Administration (OSHA) set a 5-ppm excursion limit (EL) for ethylene oxide (EtO) in April 1988. Both active and passive sampling methods have been proposed for monitoring workers against this new standard. Passive dosimetry has considerable advantages over active sampling for monitoring short-term exposures to EtO, including reduced sampling and analysis complexity, increased chemical stability, and reduced cost. The major disadvantage of these passive methods is their questionable ability to meet the OSHA requirement for the test result to fall within +/- 35% of the "true" result with 95% confidence at the EL over a 15-min sampling period. A field validation study was performed to estimate the accuracy of three EtO EL passive dosimeters: 3M 3550/3551, Crystal Diagnostics AirScan, and Assay Technology EO CHEM CHIP. Area samples were taken at four unique concentration areas within a hospital products sterilization facility. A specially designed field exposure chamber was used to expose 12 dosimeters of each type concurrently at each sampling location while concurrently collecting six Tedlar bag samples from locations surrounding the dosimeter array. The Tedlar bag samples were analyzed on-site by gas chromatography with flame ionization detection (GC-FID). To enhance the strength of this validation study, manufacturers of the dosimeters were requested to take part in the investigation. Their input was used during the design of the exposure chamber and study protocol and in the interpretation of the results. Two of the three dosimeter types were analyzed by the investigators.(ABSTRACT TRUNCATED AT 250 WORDS)

Malnutrition in patients with AIDS.

Malnutrition is a frequent problem in persons infected with the human immunodeficiency virus. The origin of malnutrition in patients with AIDS may be multifactorial. The primary mechanisms include disorders of food intake, alterations in intermediary metabolism, and nutrient malabsorption. Attention to the problems of malnutrition in patients with AIDS is of paramount importance because the timing of death in these patients may be more closely related to degree of body cell mass depletion than to any specific underlying infection. Nutritional support can improve nutritional status in selected patients, and repletion of body cell mass may be associated with functional improvement. Early assessment, attention to nutritional requirements, and prompt intervention can minimize wasting and replete body cell mass. This article examines the evidence for malnutrition in patients with AIDS, reviews the studies of nutritional support, and presents an approach to the management of malnutrition in AIDS.

Laboratory and field validation of a JXC charcoal sampling and analytical method for monitoring short-term exposures to ethylene oxide.

A sampling and analytical method for the measurement of ethylene oxide (EtO) short-term exposure limits (STEL) was validated under both laboratory and field conditions. These studies were designed to examine the method against both the Occupational Safety and Health Administration (OSHA) EtO permissible exposure limit (PEL) method requirements and the National Institute for Occupational Safety and Health (NIOSH) industrial hygiene method validation criteria. The method's pooled accuracy was shown to be within both OSHA requirements and NIOSH guidelines. The EtO was collected on a JXC charcoal tube at a sample flow rate of 100 mL/min for 15 min. The samples were shipped on dry ice and were stored in a freezer until analyzed. The EtO was desorbed by carbon disulfide and the eluent was analyzed by gas chromatography with flame ionization detection (GC-FID). The desorption efficiency of EtO from JXC charcoal tubes was determined to be 84% over the 15-min time-weighted average concentrations: 2.5, 5.0 and 10 ppm EtO. The method limit of detection was determined to be 1.0 ppm. The coefficient of variation of the combined sampling and analytical method was 5.7%. A -7% method bias was calculated. Field validation of the method included data from a portable GC-FID for the determination of method bias. Results of the field validation study over the concentration range of 2.4 ppm to 19.9 ppm generated a field precision of 8.1% with an absolute bias of 3.9%. The method accuracy was determined to be +/- 20%.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of epsilon-hydroxycaproate and epsilon-aminocaproate from N-nitrosohexamethyleneimine: evidence that microsomal alpha-hydroxylation of cyclic nitrosamines may not always involve the insertion of molecular oxygen into the substrate.

The formation of the products of microsomal metabolism of the cyclic nitrosamine, nitrosohexamethyleneimine (NO-HEX) were studied. Information on the origins of the oxygen atoms in four major metabolites of NO-HEX was obtained by metabolizing this compound in an 18O2 atmosphere using microsomes and cytosol, beta- and gamma-Hydroxy-NO-HEX are formed as a result of the insertion of a hydroxyl group derived from molecular oxygen into NO-HEX. All of the oxygen atoms in epsilon-aminocaproate (EAC) were derived from water. Approximately half of the molecules of epsilon- hydroxycaproate ( EHC ) contain an 18O atom; thus, half of the alpha-hydroxy-NO-HEX formed incorporates a hydroxyl group derived from molecular oxygen with the remainder of the hydroxyls being from water. To account for the above data and the related metabolic origins of EAC and EHC ( Hecker and McClusky , Cancer Res., 42 (1982) 59; Hecker et al., Teratogen. Carcinogen. Mutagen (1982) in press), we have proposed a mechanism for the formation of these compounds from cyclic nitrosamines catalyzed by microsomal and cytosolic enzymes.

Mutagenicity of potassium alkanediazotates and their use as model compounds for activated nitrosamines.

The mutagenicity of a series of potassium alkanediazotates in the Ames assay was studied. These compounds were isolated as solids and are soluble in dimethyl sulfoxide. Upon addition to water, they form diazohydroxides (which are postulated intermediates in the decomposition of alpha-hydroxylated nitrosamines). The diazohydroxides decompose to electrophilic intermediates which may react with macromolecules or water. In the Ames assay, potassium diazotates produced his+ revertants in Salmonella typhimurium strains TA 100 and TA 1535 but not in strains TA 98, TA 1537, or TA 1538. Methane, methane-d3, ethane, propane, and phenylmethanediazotates were mutagenic in strain TA 100, and all diazotates with the exception of phenylmethanediazotate, produced revertants in TA 1535. The order of mutagenic potency of these compounds was: methane approximately equal to methane-d3 greater than ethane, greater than phenylmethane (TA 100) greater than propane greater than phenylmethane (TA 1535) = 0. All diazotates were direct-acting mutagens and produced revertants even when no liver 9000 X g supernatant (S9) fractions were present. S9 fractions inhibited the mutagenicity of potassium diazotates, and equivalent concentrations of S9 fractions (3 mg protein per plate) from either rat or hamster liver, whether induced or not, were equally effective. Bovine serum albumin was not as effective as S9 fractions in inhibiting diazotate mutagenesis, but heat-inactivated (70 degrees for 20 min) S9 fractions were as inhibitory of methanediazotate mutagenicity as native S9 fractions were at low protein concentrations. The half-lives of mutagenicity of methane- and ethanediazotates in aqueous solutions were identical (less than or equal to 15 sec); after less than 2 min in solution, these diazotates were rendered completely inactive. The implications of these studies for mechanisms of nitrosamine action and the use of potassium alkanediazotates as model compounds for activated nitrosamines are discussed.

Metabolism and mutagenicity of N-nitrosohexamethyleneimine and its hydroxylated derivatives.

There is a direct relationship between the metabolism and mutagenicity of N-nitrosohexamethyleneimine (NO-HEX) in the presence of uninduced and AC- and PB-induced S8 and S9 fractions from rats and hamsters. Although alpha-hydroxylation is the most important process in the formation of mutagens, NO-HEX may be hydroxylated on the beta- and gamma-carbon atoms as well. beta- and gamma-hydroxyNO-HEX do not appear to play a significant role in the total mutagenicity of NO-HEX. Using rat liver subcellular fractions, beta- and gamma-hydroxyNO-HEX are only marginally mutagenic compared with NO-HEX. With hamster S9 fractions, beta-hydroxyNO-HEX is equally as mutagenic as NO-HEX itself, but gamma-hydroxyNO-HEX is a much less potent mutagen. However, beta-hydroxyNO-HEX is produced in small amounts and therefore does not contribute greatly to the total mutagenicity of NO-HEX.

The nucleotide sequence of blue-green algae phenylalanine-tRNA and the evolutionary origin of chloroplasts.

Phenylalanine tRNA from the blue-green alga, Agmenellum quadruplicatum, has been purified to homogeneity. The nucleotide sequence of this tRNA was determined to be: (see tests) Comparisons of the sequence and the modified nucleosides of this tRNA with those of other tRNAPhes thus far sequenced, indicate that this blue green algal tRNAPhe is typically prokaryotic and closely resembles the chloroplast tRNAPhes of higher plants and Euglena. The significance of this observation to the evolutionary origin of chloroplasts is discussed.

The metabolism of nitrosopyrrolidine by hepatocytes from Fischer rats.

Nitrosopyrrolidine (NO-PYR), an hepatocellular carcinogen, is rapidly metabolized to CO2 by hepatocytes freshly isolated from the livers of male Fischer rats. Using CO2 evolution as a measure of NO-PYR metabolism, we observed two kinetic constants; a high affinity component (Km = 0.11 mM), and a lower affinity component (K m = 3.2 mM). The high affinity component has similar kinetic constants to those observed for in vitro reactions with microsomes plus cytosol (Km = 0.36 mM). Therefore, it is probable that the microsomal reaction is the limiting factor in the metabolism of NO-PYR in hepatocytes. NO-PYR may be metabolized to CO2 through normal anaplerotic sequences. Some metabolites of NO-PYR which have been tentatively identified are gamma-hydroxybutyrate, succinic semialdehyde, 3,4-dihydroxybutyric acid lactone, lactate, acetate, pyruvate, glyoxylate, gamma-aminobutyrate and alanine. 2-Hydroxytetrahydrofuran (2-hydroxy-THF). a product of alpha-hydroxylation was detected at low levels in only one of four reactions. 3-Hydroxy-NO-PYR is present but represents only a small percentage of the total metabolism and is probably of little significance in the overall catabolism of NO-PYR in hepatocytes.

Comparison of the in vitro metabolism of N-nitrosohexamethyleneimine by rat liver and lung microsomal fractions.

The in vitro metabolism of N-nitrosohexamethyleneimine by lung and liver microsomes and cytosol from uninduced male Fischer rats is described. Metabolites produced by both organs appeared to be identical. The liver subcellular fractions had a lower Km (0.6 mM) than did lung fractions (3 mM) and metabolized 2.5 to 5 times as much nitrosamine per mg protein. Our results, together with those from our earlier studies, indicate that, as the size of the carbon ring increases from nitrosopyrolidine to nitrosohexamethyleneimine, lung microsomes had an increased affinity for the cyclic nitrosamines; they was only a small effect with liver enzymes. Ths suggests that microsomal enzymes that metabolize cyclic nitrosamines in rat livers and lungs are not the same. The first stable alpha-hydroxylation product, 6-hydroxyhexanal, was not detected in reactions involving microsomes alone. Apparently, this compound is rapidly converted to 1,6-hexanediol by liver or lung microsomes. The presence of cytosol was needed for the full conversion of these metabolites to xi-hydroxycaproate and maximal alpha-hydroxylation activity. xi-Aminocaproate was always found in direct proportion to the hydroxyacid, suggesting that both acids arise from the same alpha-hydroxylation event by different breakdown mechanisms. beta- and gamma-hydroxynitrosohexamethyleneimine were not metabolized significantly by rat liver enzymes and thus, in this species, may be "detoxification products" of N-nitrosohexamethyleneimine.

The nucleotide sequence of Euglena cytoplasmic phenylalanine transfer RNA. Evidence for possible classifications of Euglena among the animal rather than the plant kingdom.

The nucleotide sequence of cytoplasmic phenylalanine tRNA from Euglena gracilis has been elucidated using procedures described previously for the corresponding chloroplastic tRNA [Cell, 9, 717 (1976)]. The sequence is: pG-C-C-G-A-C-U-U-A-m(2)G-C-U-Cm-A-G-D-D-G-G-G-A-G-A-G-C-m(2)2G-psi-psi-A-G-A-Cm -U-Gm-A-A-Y-A-psi-C-U-A-A-A-G-m(7)G-U-C-*C-C-U-G-G-T-psi-C-G-m(1)A-U-C-C-C-G-G- G-A-G-psi-C-G-G-C-A-C-C-A. Like other tRNA Phes thus far sequenced, this tRNA has a chain length of 76 nucleotides. The sequence of E. gracilis cytoplasmic tRNA Phe is quite different (27 nucleotides out of 76 different) from that of the corresponding chloroplastic tRNA but is surprisingly similar (72 out of 76 nucleotides identical) to that of tRNA Phe from mammalian cytoplasm. This extent of sequence homology even exceeds that found between E. gracilis and wheat germ cytoplasmic tRNA Phe. These findings raise interesting questions on the evolution of tRNAs and the taxonomy of Euglena.