Demonstrating the reliability of in vivo metabolomics based chemical grouping: towards best practice.

While grouping/read-across is widely used to fill data gaps, chemical registration dossiers are often rejected due to weak category justifications based on structural similarity only. Metabolomics provides a route to robust chemical categories via evidence of shared molecular effects across source and target substances. To gain international acceptance, this approach must demonstrate high reliability, and best-practice guidance is required. The MetAbolomics ring Trial for CHemical groupING (MATCHING), comprising six industrial, government and academic ring-trial partners, evaluated inter-laboratory reproducibility and worked towards best-practice. An independent team selected eight substances (WY-14643, 4-chloro-3-nitroaniline, 17α-methyl-testosterone, trenbolone, aniline, dichlorprop-p, 2-chloroaniline, fenofibrate); ring-trial partners were blinded to their identities and modes-of-action. Plasma samples were derived from 28-day rat tests (two doses per substance), aliquoted, and distributed to partners. Each partner applied their preferred liquid chromatography-mass spectrometry (LC-MS) metabolomics workflows to acquire, process, quality assess, statistically analyze and report their grouping results to the European Chemicals Agency, to ensure the blinding conditions of the ring trial. Five of six partners, whose metabolomics datasets passed quality control, correctly identified the grouping of eight test substances into three categories, for both male and female rats. Strikingly, this was achieved even though a range of metabolomics approaches were used. Through assessing intrastudy quality-control samples, the sixth partner observed high technical variation and was unable to group the substances. By comparing workflows, we conclude that some heterogeneity in metabolomics methods is not detrimental to consistent grouping, and that assessing data quality prior to grouping is essential. We recommend development of international guidance for quality-control acceptance criteria. This study demonstrates the reliability of metabolomics for chemical grouping and works towards best-practice.

Preneoplastic lesions in the human breast.

A subgross sampling technique with histological confirmation was used to study the pathology of 119 whole human breasts, either cancer-associated (that is, containing cancer or contralateral to a cancer) or taken from random routine autopsies. Atypical lobules were observed much more frequently in the cancer-associated group than in the group of routine autopsy breasts. Atypical lobules showed varying degrees of anaplasia that formed a continuum between normal epithelium and carcinoma in situ, usually of the common ductal type. As apprarent markers for increased cancer risk, atypical lobules in the human breast may be homologous to hyperplastic alveolar nodules that are abundant in high mammary cancer strains of mice. This indirect evidence supports the hypothesis that atypical lesions are common preneoplastic lesions in the human mammary gland.

Pharmaceutical development, quality control, stability and compatibility of a parenteral lyophilized formulation of the investigational polymer-conjugated platinum antineoplastic agent AP5346.

AP5346 is a low molecular weight polymer-conjugated platinum antineoplastic agent. The lyophilized drug product has completed a phase I clinical trial. In order to guarantee a constant quality of AP5346 pharmaceutical products, quality control and analysis of the drug substance and final product were performed. The identity of AP5346 was confirmed using 1H NMR, 195Pt NMR and IR spectroscopy. Furthermore, the free platinum content, platinum release characteristics, molecular size and size distribution were established. With the selected analytical techniques, AP5346 could be distinguished very well from its polymeric analogues, such as AP5280 and AP5279. Stability experiments revealed that AP5346 final product is stable for 12 months at 5 degrees C, in the dark. For administration to patients, AP5346 final product is reconstituted with 5% w/v dextrose and diluted in infusion containers. To investigate the influence of container materials, the stability of AP5346 after reconstitution and dilution in infusion containers was determined. The infusion containers investigated were composed of glass, polyvinyl chloride (PVC, intraflex) and low density polyethylene (LD-PE, Ecoflac). AP5346 was shown to be stable after reconstitution and dilution with 5% w/v dextrose in these infusion containers for at least 96 h at 2-8 degrees C in the dark and at room temperature with ambient light conditions.

Effect of peroxidase inhibitors on an in vivo metabolite of the urinary bladder carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide in rats.

Peroxidase metabolism of 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) was evaluated in vitro and in vivo. In vitro metabolism of ANFT was characteristic of the hydroperoxidase activity of prostaglandin H synthase. The peroxidase inhibitors, 6-n-propyl-2-thiouracil and methimazole, significantly reduced ANFT binding to trichloroacetic acid precipitable material and glutathione conjugate formation. Isolated perfused kidneys rapidly converted the glutathione conjugate to its corresponding mercapturic acid (ANFT-MA). With both radiochemical and electrochemical techniques, ANFT-MA was identified in the urine of rats given N-[14C]-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide, the carcinogenic N-formyl analogue of ANFT. ANFT was the major urinary metabolite with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide not detected. A 30-min pretreatment with 6-n-propyl-2-thiouracil and methimazole significantly reduced urinary excretion of ANFT-MA in rats given N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (150 mg/kg) from 14.8 +/- 2.1 (SE) to 7.9 +/- 0.8 and 6.2 +/- 1.1 nmol/18 h, respectively. Peroxidase inhibitor pretreatment did not alter the excretion of ANFT or prostaglandin E2. These results provide further in vitro and in vivo support for the involvement of peroxidases, i.e., the hydroperoxidase activity of prostaglandin H synthase, in ANFT metabolism.

Pneumonitis complicating low-dose methotrexate therapy in rheumatoid arthritis.

Three of 95 patients with rheumatoid arthritis who were being treated with low-dose (5 to 15 mg/wk) methotrexate sodium developed the clinical, radiographic, and pathologic features of methotrexate-associated pulmonary injury. Marked hypoxemia emphasized the severity of illness in our patients; lowest oxygen pressure values for each patient were 35 mm Hg, 42 mm Hg, and 45 mm Hg. The management of our patients with a pulmonary toxic reaction to methotrexate included discontinuing the drug treatment, antibiotic therapy until an infectious cause was excluded, and high-dose methylprednisolone. Two patients recovered and one died. Contrary to an earlier report that suggested that pneumonitis occurred only with methotrexate sodium doses exceeding 15 mg/wk, our three cases demonstrate that a severe pulmonary toxic reaction may also complicate low-dose weekly methotrexate therapy of rheumatoid arthritis.

Improved targeting of platinum chemotherapeutics. the antitumour activity of the HPMA copolymer platinum agent AP5280 in murine tumour models.

AP5280 is a novel N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound platinum (Pt) therapeutic designed to increase the therapeutic index relative to conventional, small-molecule platinum agents. The platinum-polymer construct accumulates in solid tumours on the basis of increased capillary permeability. The bound platinum moiety is present as an N,O-Pt chelate at the distal end of a tetrapeptide linker, glycine-phenylalanine-leucine-glycine, and the weight-average molecular weight (Mw) of the construct is 22 kDa. The antitumour activity and toxicity of AP5280 were assessed in the syngeneic murine B16F10 and Lewis lung tumour models, and in the human ovarian carcinoma 2008 and head and neck squamous carcinoma UMSCC10b xenograft models. The maximum tolerated dose (MTD) of AP5280 was 6-fold greater than that of carboplatin (CBDCA) in vivo. AP5280 was active in all four tumour models, and it displayed a higher therapeutic index than CBDCA in each of these tumour models. The antitumour effect of AP5280 given at 16% of its MTD was equivalent to that produced by a MTD of CBDCA. Thus, consistent with the design goal for this drug, and despite being less potent than CBDCA, AP5280 produced less systemic toxicity relative to its antitumour activity and thus has a greater therapeutic index. On the basis of the improved therapeutic index evidenced in these models, AP5280 has been advanced into clinical trials.

Endobronchial telangiectasias and hemoptysis in scleroderma.

Hemoptysis is considered a rare event in scleroderma and to date only two previous cases could be identified. The occurrence of hemoptysis with bleeding and friable telangiectasias is reported in a patient with rapidly progressing systemic sclerosis. This represents the first report of this association, although bleeding telangiectasias have been reported in other systems. A brief review of the relevant literature is included.

Methotrexate-associated hepatotoxicity: retrospective analysis of 210 patients with rheumatoid arthritis.

PURPOSE: Beginning in the 1980s, methotrexate has been used successfully to treat rheumatoid arthritis. The magnitude and severity of short- and long-term methotrexate toxicity, however, have not been adequately investigated. Our study was performed to determine the prevalence of hepatotoxicity in patients with rheumatoid arthritis receiving long-term methotrexate therapy. PATIENTS AND METHODS: We conducted a retrospective, computer-assisted review of all Duke University Medical Center patients undergoing liver biopsy for methotrexate monitoring from January 1979 to January 1988. A total of 538 biopsies were performed in 399 patients, 259 of whom had inflammatory arthritis (210 with rheumatoid arthritis, 47 with psoriatic arthritis, and two with seronegative spondyloarthropathy). RESULTS: No evidence of cirrhosis was defined in the cohort with rheumatoid arthritis; however, six patients with rheumatoid arthritis had histologic changes of fibrotic liver disease (prevalence of 2.9 percent in the group with rheumatoid arthritis) while taking methotrexate. Five of the six patients were obese and three had glucose intolerance or overt diabetes mellitus, and one person admitted to alcohol usage. Only one patient with fibrotic liver disease had elevated liver function test results, and no person showed a declining serum albumin level at the time of biopsy. Sixty-one patients with rheumatoid arthritis underwent multiple samplings (44 with two, 13 with three, and four with four biopsies). Fourteen of these patients showed progressive hepatic disease, whereas four patients improved. CONCLUSION: Although the prevalence of methotrexate hepatotoxicity in this large cohort of patients with rheumatoid arthritis was low, a small but definite risk of hepatic fibrosis, not predictable by laboratory screening, still exists.

Pain in the rheumatic diseases. Practical aspects of diagnosis and treatment.

Patients with rheumatic disease experience pain that can be intense, persistent, and disabling. This pain is frequently multifactorial in origin and has both central and peripheral components. Because of the array of conditions that can cause musculoskeletal pain, patient management must begin with a complete clinical assessment that identifies possible etiologies and measures objective findings against subjective complaints. Especially in patients with known rheumatic disease, the possibility of concurrent pain of central origin must be considered and appropriate treatment given. By applying a comprehensive therapy plan of drugs, physical therapy, and patient education, significant benefits can often be achieved in this prevalent group of painful diseases.

"Fibrositis" syndrome.

There appears to be as yet undefined but significant and possibly multifactorial elements of personality, stress, or depression in the manifestations and possibly the pathogenesis of FS. If these factors, perhaps amplified by the neurophysiologic effects of disturbed sleep, produce a neurochemical disturbance in CNS function, and if this perturbation includes a reduction or impairment of function involving the pain-modulation pathways, then a simple and perhaps compelling explanation for the experience of pain in FS becomes apparent. Reduced midbrain/brainstem inhibition of ascending nociceptive impulses would clearly explain the finding of tender points in normal-appearing areas of the body, as well as the lack of segmental distribution of discomfort in FS. Local anesthetics, injected peripherally into tender points, would be expected, as is the case, to block pain and tenderness in the local area for the duration of action of the agent used. Analgesics with peripheral activity, such as aspirin and NSAIDs, are relatively ineffective in treating FS, and would be predictably so in a disorder involving reduced central pain inhibition as opposed to increased peripheral nociceptive input. It would not be surprising to find that centrally acting agents, particularly those producing enhancement of serotonergic neurons such as amitriptyline, would provide substantial or total pain relief as well as improvement in mood in a significant number of patients. Most importantly, this concept would highlight the real pain experienced by these patients and the obligation of involved physicians to appropriately diagnose and treat this common pain syndrome. Avoiding excessive conjecture, it is then permissible at the present time to conclude that: FS is a characteristic, clinically common pain syndrome in which aspects of the pain itself appear to be of physiologic origin. Although stress or inherent personality traits may play a role in FS, the relative uniformity in symptomatology virtually excludes conversion hysteria as a major factor in this disorder. The lack of evidence for a disturbance in muscle, fascia, and other soft tissues in FS, the lack of adequate response to NSAIDs, and the frequent response to TCAs suggest that specific dysfunction of the CNS may play a major role in the symptomatology of this entity. Impaired function of the pain-modulation system, located anatomically in the midbrain and brainstem, provides a plausible explanation for the pain and finding of tender points in FS, as well as a potentially rational basis for therapy.

Anthropomorphic 1H MRS head phantom.

An anthropomorphic 1H MRS head phantom has been developed which mimics the in vivo structure, metabolite concentrations, and relaxation times (for both water and metabolites) of human brain tissue. Different brain regions and two tumor types, fluid-containing ventricles, and air-filled sinus, and subcutaneous fat are all simulated. The main tissue-mimicking materials are gelatin/agar mixtures with metabolites and several other ingredients added. Their composition and method of production are thoroughly described. T1's and T2's of water in the phantom are very close to in vivo values, and metabolite T1's and T2's are considerably more realistic than those in aqueous solutions. Spectra and relaxation times for the pig brain were also acquired and compare well with those of the phantom. The realistic properties of this phantom should be useful for testing spectral quantitation and localization.

Lymphocytic lymphoma and systemic lupus erythematosus: their coexistence with antibody to the Sm antigen.

The serologic and clinical features of a 32-year-old women with coexistent systemic lupus erythematosus and lymphoma were studied. A cervical node biopsy specimen demonstrated nodular, poorly differentiated lymphocytic lymphoma; subsequent development of polyarthralgias prompted evaluation for connective-tissue disease, and a high titer of antinuclear antibody was found. Immunochemical studies proved that this antibody was directed to the Sm antigen, a serologic finding highly specific for systemic lupus erythematosus. This antibody has been maintained throughout the course of the patient's disease, characterized by persistent lymphadenopathy, pleuropericarditis, arthritis, hypocomplementemia, hyperglobulinemia, and a slight elevation of titers of anti-DNA antibody. The course of the lymphoproliferative disorder has been consistent with that of an indolent non-Hodgkin's lymphoma. In sera from other patients with lymphoproliferative disorders, anti-Sm antibody was not found. These findings emphasize the importance of establishing the antigenic specificity of antinuclear antibodies detected in the sera of patients with neoplasia.

Cyclosporin A therapy for Wegener's granulomatosis.

Five patients with active Wegener's granulomatosis were treated with the immunosuppressive agent Cyclosporin A, along with low dose prednisone. All five patients had previously taken cyclophosphamide, but further treatment with this agent was not desired, either due to patient choice, drug toxicity or malignancy. In initial doses of up to 5mg/kg/day, CyA showed efficacy but when lowered to 1-2mg/kg/day, mild disease flares occurred. CyA may provide an alternative to traditional therapy in selected patients with WG.

On neurobiological, neuro-fuzzy, machine learning, and statistical pattern recognition techniques.

In this paper, we propose two new neuro-fuzzy schemes, one for classification and one for clustering problems. The classification scheme is based on Simpson's fuzzy min-max method (1992, 1993) and relaxes some assumptions he makes. This enables our scheme to handle mutually nonexclusive classes. The neuro-fuzzy clustering scheme is a multiresolution algorithm that is modeled after the mechanics of human pattern recognition. We also present data from an exhaustive comparison of these techniques with neural, statistical, machine learning, and other traditional approaches to pattern recognition applications. The data sets used for comparisons include those from the machine learning repository at the University of California, Irvine. We find that our proposed schemes compare quite well with the existing techniques, and in addition offer the advantages of one-pass learning and online adaptation.

Endocrine control of inflammation: rheumatoid arthritis double-blind, crossover clinical trial.

A dysfunction in the endocrine control system for inflammation in rheumatoid arthritis serves as the theoretical basis for chronic inflammation in the study design described. Eighteen patients with rheumatoid arthritis, who acted as their own controls, were brought to a minimum symptom state through conventional means, trained, and allowed to control subsequent flares by a patient-initiated, flare-response prednisone regimen. The six-month trial was double-blind with a crossover at midpoint. While continuing stable non-steroidal anti-inflammatory and disease modifying antirheumatic drug therapies, the patients averaged additional 57% and 75% reductions from baseline in tender joint count and total pain score, respectively, on the prednisone therapy. The prednisone therapy was differentiated by improvement from that of a placebo by six of the nine parameters evaluated. The adverse events were no more frequent with prednisone than with placebo use. The efficacy of prednisone was increased threefold while reducing consumption by 40% when compared to the predecessor 5-mg prednisone/day clinical trial.

Patient predictors of caregiver burden, optimism, and pessimism in rheumatoid arthritis.

The authors of the present study investigated the relationship between rheumatoid arthritis (RA) patients' demographic, medical, and functional status and caregivers' burden, optimism, and pessimism. Subjects were 65 RA patients and their caregivers who were recruited from an outpatient rheumatology clinic. Each caregiver completed the Burden Interview to measure caregiver burden and the Life Orientation Test to measure optimism and pessimism. Each RA patient completed the Arthritis Impact Measurement Scale to measure pain and physical disability as well as a number of cognitive measures to assess two summary psychological cognitive factors labeled self-efficacy expectations and distorted cognitions. These cognitive factors were based on the following commonly used measures in RA research: the Cognitive Errors Questionnaire, the Arthritis Self-Efficacy Scale, the Coping Strategies Questionnaire, and the Pain Beliefs and Perceptions Inventory. Correlational analyses indicated that patients' functional and psychological measures (including poor self-efficacy expectations regarding symptoms) were related to caregiver burden, that patient self-efficacy expectations were related to caregiver optimism, and that patient physical disability was related to caregiver pessimism. Regression analyses revealed that, when competing with other demographic and disease severity variables, the relationships between patient self-efficacy expectations and caregiver burden and caregiver optimism, and patient physical function and caregiver pessimism remained significant. Taken together, these findings suggest that patient expectancies about control over arthritis-related symptoms (including pain) are strongly related to caregiver burden and caregiver optimism and that patient physical status is strongly related to caregiver pessimism.