A physiologically based pharmacokinetic model for capreomycin.

The emergence of multidrug-resistant tuberculosis (MDR-TB) has led to a renewed interest in the use of second-line antibiotic agents. Unfortunately, there are currently dearths of information, data, and computational models that can be used to help design rational regimens for administration of these drugs. To help fill this knowledge gap, an exploratory physiologically based pharmacokinetic (PBPK) model, supported by targeted experimental data, was developed to predict the absorption, distribution, metabolism, and excretion (ADME) of the second-line agent capreomycin, a cyclic peptide antibiotic often grouped with the aminoglycoside antibiotics. To account for interindividual variability, Bayesian inference and Monte Carlo methods were used for model calibration, validation, and testing. Along with the predictive PBPK model, the first for an antituberculosis agent, this study provides estimates of various key pharmacokinetic parameter distributions and supports a hypothesized mechanism for capreomycin transport into the kidney.

Characterization of "peak E," a novel amino acid associated with eosinophilia-myalgia syndrome.

Epidemiologic studies strongly associate eosinophilia-myalgia syndrome (EMS) with ingestion of tryptophan containing a contaminant ("peak E"). Prior reports have suggested that peak E is the di-tryptophan N alpha-animal of acetaldehyde. Spectral and chemical studies now demonstrate that peak E is 1,1'-ethylidenebis[tryptophan]. This novel amino acid may be the etiological agent responsible for EMS, or it may be a marker of a still unidentified causal agent.

Granule-associated flavin adenine dinucleotide (FAD) is responsible for eosinophil autofluorescence.

Unstained human eosinophils exhibit marked autofluorescence in comparison to other leukocytes due to a granule-associated fluorescent substance. Fluorescence spectroscopy of granule extracts reveals excitation maxima at approximately 380 and approximately 450 nm with a single emission at approximately 520, characteristic of flavins. The fluorescent material from eosinophil granule extracts was characterized by fluorescence, high-performance liquid chromatographic, and enzymatic analyses. First, acidification to pH 2.6 resulted in increased fluorescence, indicative of flavin adenine dinucleotide (FAD). Second, because flavin mononucleotide (FMN) and riboflavin cannot be distinguished by acidification, high-performance liquid chromatography was performed and revealed a predominance of FAD and smaller amounts (< 15%) of both FMN and riboflavin. Third, the presence of FAD was clearly demonstrated by reconstitution of the activity of D-amino acid oxidase, a FAD-dependent enzyme, when granule extracts were added to the apoenzyme. Thus, we have identified FAD as the predominant fluorophore in eosinophil granules. The small amounts of FMN and riboflavin detected may result from the hydrolysis of FAD under the acidic conditions of granule extraction. Because fluorescent material is deposited onto target cells by eosinophils, it is possible that granule-associated flavoproteins may act as a source of hydrogen peroxide and/or superoxide, which, in conjunction with eosinophil peroxidase, could yield potent cytotoxic agents.

Eosinophilia-myalgia syndrome and tryptophan production: a cautionary tale.

An epidemic of a new disease, termed eosinophilia-myalgia syndrome, occurred in the USA in 1989. This syndrome was linked to the consumption of L-tryptophan manufactured by a single company utilizing a fermentation process. All the findings indicate that the illness was probably triggered by an impurity formed when the manufacturing conditions were modified. This outbreak highlights the need for close monitoring of the chemical purity of biotechnology-derived products, and for rigorous testing of such products following any significant changes to the manufacturing process.

1,1'-Ethylidenebis[tryptophan] induces pathologic alterations in muscle similar to those observed in the eosinophilia-myalgia syndrome.

1,1'-Ethylidenebis[tryptophan] (EBT), a derivative of L-tryptophan (LT), is a trace contaminant in batches of LT implicated by epidemiologic evidence in the pathogenesis of the eosinophilia-myalgia syndrome (EMS). We treated female Lewis rats with EBT or unimplicated LT (4 mg per 100 grams daily) by intraperitoneal injection. No rash or weakness occurred in either group. All three EBT rats had a few necrotic muscle fibers. In two rats, perimysium and fascia were abnormally thickened and infiltrated with lymphocytes, macrophages, and sparse eosinophils; two rats had sparse perineurial inflammatory cells. Rats treated with unimplicated LT showed no abnormality. These findings replicate an important feature of human EMS and support the epidemiologic evidence linking EBT to the pathogenesis of the human disease.

3-(Phenylamino)alanine, a novel aniline-derived amino acid associated with the eosinophilia-myalgia syndrome: a link to the toxic oil syndrome?

The eosinophilia-myalgia syndrome (EMS) is an inflammatory disease that occurred in epidemic proportions in the United States during 1989. Cases of EMS were also reported in Europe and elsewhere. Clinically, EMS resembles the Spanish toxic oil syndrome. EMS has been associated with ingestion of manufactured L-tryptophan and, more specifically, with lots of tryptophan that contained the trace contaminant 1,1'-ethylidenebis(tryptophan) (EBT). Another trace contaminant ("peak UV-5") has been reported, but the strength of its association with EMS has not been demonstrated. Herein we report independently that peak UV-5 is 3-(phenylamino)alanine (PAA). Patients with EMS ingested significantly greater amounts of both PAA and EBT than did control tryptophan users. PAA is chemically similar to 3-phenylamino-1,2-propanediol, an aniline derivative isolated from samples of oil that were consumed by persons in whom the toxic oil syndrome developed. The discovery of an aniline-derived contaminant in tryptophan raises the possibility that EMS and toxic oil syndrome may have a common etiologic trigger.

EBT, a tryptophan contaminant associated with eosinophilia myalgia syndrome, is incorporated into proteins during translation as an amino acid analog.

The tryptophan dimer 1,1'-ethylidenebis[L-tryptophan] was identified as a contaminant of tryptophan preparations associated with Eosinophilia-Myalgia Syndrome. In this paper, we describe experiments examining the hypothesis that 1,1'-ethylidenebis[L-tryptophan] acts as an amino acid analog replacing L-tryptophan during the synthesis of proteins. We propose further that proteins containing 1,1'-ethylidenebis[L-tryptophan] are rejected in an autoimmune process identified clinically as Eosinophilia-Myalgia Syndrome. Rabbit reticulocyte lysates containing an estimated 1 microM L-tryptophan were used to assay the ability of 1,1'-ethylidenebis[L-tryptophan] to compete with 3H-L-tryptophan for incorporation into proteins translated from BMV RNA. 1,1'-Ethylidenebis[L-tryptophan] in concentrations of 40, 80 and 110 microM reduced lysate 3H-L-tryptophan incorporation to 81%, 76% and 75% of control incorporation obtained in the absence of 1,1'-ethylidenebis[L-tryptophan]. In the presence of 20 microM L-tryptophan, 110 microM 1,1'-ethylidenebis[L-tryptophan] reduced 3H-L-tryptophan incorporation to 56% of control incorporation. In contrast, ethyl-L-tryptophan did not significantly reduce 3H-L-tryptophan incorporation. In the presence of 110 microM 1,1'-ethylidenebis[L-tryptophan] and 20 microM L-tryptophan, 3H-L-leucine incorporation was not significantly reduced compared to incorporation in the absence of 1,1'-ethylidenebis[L-tryptophan], demonstrating that proteins were translated to full length during elongation. These findings suggest that 1,1'-ethylidenebis[L-tryptophan], but not ethyl-L-tryptophan, reduced 3H-L-tryptophan incorporation into proteins by substituting for L-tryptophan rather than by causing premature termination or significant slowing of nascent protein chains.

On-line HPLC-tandem mass spectrometry analysis of contaminants of L-tryptophan associated with the onset of the eosinophilia-myalgia syndrome.

The structural characterization of a number of contaminants of L-tryptophan (Trp) associated with eosinophilia myalgia syndrome has been performed for the first time by the powerful structural elucidation technique of tandem mass spectrometry coupled with on-line HPLC (LC-ESI-MS/MS). The identity of the contaminants: peaks UV-5, 3-(phenylamino)alanine, (PAA); E 1,1'-ethylidenebis(tryptophan); 200, 2-(3-indolylmethyl)-L-tryptophan; (all identified as case related) and peaks 1, 3-carboxy-1,2,3,4-tetrahydro-beta-carboline; 2, 3-carboxy-1-methyl-1,2,3,4-tetrahydro-beta-carboline; 100, 2-(2,3 dihydroxy-1-[3-indolyl]propyl)-L-tryptophan; and 300 and 400, diastereomers of 3-carboxy-1-[3-indolyl-methyl]-1,2,3,4-tetrahydro-beta-carboline, have been confirmed by this technique. By comparison of tandem MS (MS/MS) data from these compounds with the MS/MS data of several other impurities, we have structurally characterized peaks CC, KK and OO, as well as two previously unreported components labeled as peak P18 and peak P31. Peak P18 was unresolved from the large Trp peak and has been characterized as indole-3-ethylamine. Peak P31 was previously unresolved from peak 200, a case related compound and therefore its structure is of extreme importance. This compound has been tentatively identified as 2-(3-indolyl)-L-tryptophan.

Studies with 1,1'-ethylidenebis(tryptophan), a contaminant associated with L-tryptophan implicated in the eosinophilia-myalgia syndrome.

L-Tryptophan binds to a rat liver nuclear envelope protein, and this binding is saturable, stereospecific, and of high affinity. Utilizing an in vitro assay of [3H]tryptophan binding to rat hepatic nuclear envelopes, we have previously determined that the L-tryptophan obtained from Showa Denko and which was implicated in cases of the eosinophilia-myalgia syndrome (EMS) inhibited [3H]tryptophan binding differently than did control L-tryptophan (not implicated in EMS). Therefore, in this study we investigated whether the addition of 1,1'-ethylidenebis(tryptophan) (EBT), a contaminant or impurity in L-tryptophan implicated in EMS, would have an effect. Our results indicate that EBT alone has little inhibitory binding effect compared with that of control L-tryptophan and that when EBT was added to control L-tryptophan the inhibitory binding effort was similar to that of control L-tryptophan alone. On the other hand, in vitro addition of EBT plus L-tryptophan to nuclei of cultured murine macrophages (WLG5) results in less inhibition of [3H]-tryptophan binding than does addition of L-tryptophan alone. Similar in vitro additions to nuclei of rat brain reveal little effect on binding, as was also the case for hepatic nuclear envelopes. Adding EBT to an in vitro hepatic protein synthesis system and measuring [3H]tryptophan incorporation into acid-precipitable proteins reveal that it competes similarly to that found with equimolar concentrations of unlabeled L-tryptophan. It does not affect [14C]leucine incorporation into proteins. [14C]EBT becomes incorporated in vitro into proteins (acid-precipitable), and this incorporation is diminished in the presence of equimolar concentrations of unlabeled EBT or L-tryptophan. This suggests that EBT or possibly a breakdown product becomes incorporated into proteins. Speculation as to how EBT may affect tissues in experimental animals is presented.

1,1'-Ethylidenebis[L-tryptophan], an impurity in L-tryptophan associated with eosinophilia-myalgia syndrome, stimulates type I collagen gene expression in human fibroblasts in vitro.

Eosinophilia-myalgia syndrome (EMS), a recently described inflammatory disorder characterized by myalgia, peripheral eosinophilia, and multisystem inflammation is associated with L-tryptophan consumption. Fibrosis of various tissues due to excessive accumulation of type I collagen is a prominent late manifestation of the syndrome. 1,1'-Ethylidenebis[L-tryptophan] (EBT), an impurity distinct from L-tryptophan found in case-associated lots, has been implicated in function in vitro. Incubation of confluent fibroblasts with EBT, but not its hydrolysis product 1-methyl-tetrahydro-beta-carboline-3-carboxylic acid, caused a dose-dependent increase in collagen synthesis and in type I collagen mRNA levels independent of its effect on proliferation. In contrast, expression mRNA for fibronectin was not affected. These findings indicate that EBT stimulates type I collagen production by human fibroblast, and suggest that EBT may be involved in the development of fibrosis in EMS.

Eosinophils preferentially use bromide to generate halogenating agents.

Human eosinophils preferentially utilize bromide to generate a brominating agent, even at physiological halide concentrations, where chloride (140 mM) is over 1000-fold greater than bromide (20-100 microM). Under the same conditions, neutrophils use chloride to generate a chlorinating agent. The total amount of active halogen trapped by 1,3,5-trimethoxybenzene from eosinophils increases by over 2-fold as the added bromide concentration increases from 0 to 100 microM, with approximately 40 nmol of halogen trapped per million cells at the highest bromide level. At least 25-35% of the oxygen consumed by stimulated eosinophils is directed toward the generation of halogenating species. Since the relative halogenating behavior of eosinophil peroxidase and neutrophil myeloperoxidase in this bromide range is essentially identical to that of the cells, the specificity of eosinophils toward bromide is intrinsic to eosinophil peroxidase and not to any special cellular properties. These results suggest that human eosinophils use bromide in vivo and that a deficiency of bromide may influence their ability to produce halogenating agents.

Biotransformation of 3-(phenylamino)-1,2-propanediol to 3-(phenylamino)alanine: a chemical link between toxic oil syndrome and eosinophilia-myalgia syndrome.

During late 1989, the eosinophilia-myalgia syndrome (EMS) developed as an epidemic in the United States, with numerous additional cases reported in several other countries worldwide. Eight years earlier, a closely-related disease, the toxic oil syndrome (TOS), occurred in Spain as a massive food-borne epidemic. Although EMS was linked to the ingestion of tainted L-tryptophan, and TOS to aniline-denatured rapeseed oil, the etiologic agent(s) responsible for both diseases remains undetermined. Contaminants in these foodstuffs are believed to have triggered the diseases. Aniline contaminants, including 3-(phenylamino)-1,2-propanediol (PAP), have been reported in oil used by patients who developed TOS. A related aniline derivative, 3-(phenylamino)-L-alanine (PAA), was recently isolated from L-tryptophan associated with the onset of EMS. Here, we demonstrate the biotransformation of PAP into PAA by both rat hepatocytes and human liver tissue. The structural characterization of PAA was unequivocally determined using on-line HPLC coupled with atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS). This finding is the first reported chemical link between TOS and EMS and suggests that these two related diseases share a common etiology, namely, PAA.

Lidocaine in bronchoalveolar lavage fluid (BALF) is an inhibitor of eosinophil-active cytokines.

Eosinophils and eosinophil granule proteins may play an important role in the pathogenesis of asthma. BALF from 40 patients with symptomatic asthma were analysed for cytokine activity by the eosinophil survival assay. BALF from 15 patients showed increased survival activity. Survival activities in BALF from four of these patients were almost completely blocked by anti-IL-5 MoAb, and the remaining activities were blocked by anti-granulocyte-macrophage colony-stimulating factor (GM-CSF), anti-IL-3 antibody, or both. Surprisingly, BALF samples from the other 25 patients decreased eosinophil viabilities below the levels of medium control. The inhibitory factor in these BALF was of low molecular weight, was heat-stable, was largely overcome by excess exogenously added cytokines, and was positively correlated with the concentrations of lidocaine in the BALF. Lidocaine itself inhibited eosinophil survival at concentrations less than those present in the BALF. These findings indicate that lidocaine is an inhibitor of cytokines in the eosinophil survival assay, and they suggest the need for caution in analyses of BALF containing lidocaine or other local anaesthetics.

Eosinophil-active cytokine from mononuclear cells cultured with L-tryptophan products: an unexpected consequence of endotoxin contamination.

BACKGROUND: The eosinophilia-myalgia syndrome, caused by a contaminant or contaminants in epidemiologically implicated L-tryptophan products, is characterized by eosinophilia and eosinophil degranulation. We hypothesized that immune cells are stimulated by implicated L-tryptophan and produce eosinophil-active cytokines. OBJECTIVES: This study was designed to identify substances in L-tryptophan causing the eosinophilia-myalgia syndrome. METHODS: Peripheral blood mononuclear cells were cultured with L-tryptophan products, and supernatants were tested for their ability to enhance eosinophil degranulation and survival in vitro and for their cytokine content. Subsequently, 46 different L-tryptophan lots were analyzed for their in vitro biologic activities. RESULTS: After peripheral blood mononuclear cells were cultured with implicated L-tryptophan, their supernatants enhanced eosinophil degranulation and survival. These activities were blocked by anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody; immunoreactive GM-CSF was measurable in the supernatants. Monocytes, but not T lymphocytes, were the responding cells. However, no correlation was observed between the in vitro biologic activity and lots of epidemiologically implicated L-tryptophan products. This biologic activity in the L-tryptophan products was characterized as endotoxin. CONCLUSION: Although L-tryptophan products stimulate peripheral blood mononuclear cells to produce GM-CSF, this response is caused by endotoxin contamination of the L-tryptophan products and not by a specific L-tryptophan contaminant. Endotoxin contamination must be considered as a possible cause of eosinophil-active cytokine production by peripheral blood mononuclear cells.

An investigation of the cause of the eosinophilia-myalgia syndrome associated with tryptophan use.

BACKGROUND: The eosinophilia-myalgia syndrome is a newly recognized illness that has been associated with the consumption of tryptophan products. It is not known whether the cause is related to the tryptophan itself or to chemical constituents introduced by the manufacturing process. METHODS: To describe the epidemiology of the eosinophilia-myalgia syndrome further and elucidate a possible association with the manufacturing process, we conducted surveillance for the syndrome in Minnesota, a community survey of tryptophan use in Minneapolis-St. Paul, and a case-control study to assess potential risk factors, including the use of tryptophan from different manufacturers. We performed high-performance liquid chromatography on tryptophan samples to identify other chemical constituents. RESULTS: The prevalence of tryptophan use increased from 1980 to 1989 and was highest among women. Among the subjects for whom the source of the tryptophan was known, 29 of 30 case patients (97 percent) and 21 of 35 controls (60 percent) had consumed tryptophan manufactured by a single company (odds ratio, 19.3; 95 percent confidence interval, 2.5 to 844.9; P less than 0.001). This company used a fermentation process involving Bacillus amyloliquefaciens to manufacture tryptophan. Analysis of the manufacturing conditions according to the retail lot demonstrated an association between lots used by case patients and the use of reduced quantities of powdered carbon in a purification step (odds ratio, 9.0; 95 percent confidence interval, 1.1 to 84.6; P = 0.014), as well as the use of a new strain of B. amyloliquefaciens (Strain V) (odds ratio, 6.0; 95 percent confidence interval, 0.8 to 51.8; P = 0.04). There was a significant correlation (r = 0.78, P less than 0.001) between the reduced amount of powdered carbon used during manufacturing and the use of the new bacterial strain. High-performance liquid chromatography of this company's tryptophan demonstrated one absorbance peak (peak E) that was present in 9 of the 12 retail lots (75 percent) used by patients and 3 of 11 lots (27 percent) used by controls (odds ratio, 8.0; 95 percent confidence interval, 0.9 to 76.6; P = 0.022). CONCLUSIONS: The outbreak of the eosinophilia-myalgia syndrome in 1989 resulted from the ingestion of a chemical constituent that was associated with specific tryptophan-manufacturing conditions at one company. The chemical constituent represented by peak E may contribute to the pathogenesis of the eosinophilia-myalgia syndrome, or it may be a surrogate for another chemical that induces the syndrome.