Hematological Complications of Phenylbutazone Therapy: Review of the Literature and Report of Two Cases

Two examples of hematological toxicity following phenylbutazone therapy are described, one of agranulocytosis and one of aplastic anemia. In the first case, prednisolone in a dosage of 20 mg. daily restored neutrophil percentage and the total leukocyte count to normal, but the patient with aplastic anemia, having shown no response to corticosteroid therapy, became dependent on repeated blood transfusion.The English literature on the hematological toxicity of phenylbutazone is reviewed. Ten fatal cases of agranulocytosis have been recorded, as have eight cases of aplastic anemia, of which five proved fatal. Other toxic effects noted have included leukopenia, depression of erythropoiesis, megaloblastic anemia, thrombocytopenia and leukemia.     

Agranulocytosis, aplastic anemia, and leukemia: relevance to arachidonic acid metabolism

Several agents including drugs, chemicals and viruses are known to induce agranulocytosis, aplastic anemia, and leukemia. The recent identification, characterization and cloning of several peptide regulatory factors, including granulocyte-macrophage-colony stimulating peptide regulatory factor (GM-CSF), erythropoietin, and interleukins and a study of their actions, suggest that agents producing agranulocytosis, aplastic anemia, and leukemia may interfere with the action of these factors. The agents that are capable of inducing these diseases and the various peptide regulatory factors have positive and/or negative actions on the prostaglandin system. Prostaglandins are known to be involved in the maturation and differentiation of the progenitor cells of the bone marrow and in erythropoietin-mediated erythropoiesis. Since prostaglandins influence immune response, modify genetic damage induced by drugs and chemicals, modulate gene action, and have feed-back control on the actions of peptic regulatory factors, it is likely that prostaglandins are involved in the pathogenesis of agranulocytosis, aplastic anemia, and leukemia. If so, this may lead to new therapeutic strategies in these hematological conditions.     

3D QSAR Markov model for drug-induced eosinophilia--theoretical prediction and preliminary experimental assay of the antimicrobial drug G1

The application of 3D-MEDNEs as a novel alternative technique to reduce the use of animal experimentation in toxicology in the early stages of medicinal chemistry research has been extended from agranulocytosis to chemically induced eosinophilia. Firstly, a heterogeneous series of organic compounds, which are classified either as eosinophilia inductors or noninductors, was collected. A linear discriminant analysis was subsequently used to obtain a QSTR that gave rise to a very good classification of 91.82% (110 chemicals within training series). Eosinophilia inductors (88.89%) composed the first group while the other one contained only harmless compounds (97.37%). The total predictability (88.1%) was tested by means of an external validation series (42 compounds). The model correctly classifies 88.89% of harmless compounds and 87.5% of toxic ones. Finally, comparison of predicted versus experimental results for G1 [2-bromo-5-(2-bromo-2-nitroethenyl)furan, which is a promising antibacterial-antifungal compound] illustrates the practical application of the method. A dose-dependent study of G1 (9.8-185.6 mg/Kg) at 48, 72 and 96 h after oral administration in rats is reported here for the first time. The study has shown that G1 does not affect the murine eosinophils count under these conditions--a situation in total agreement with the model prediction.     

Idiosyncratic NSAID drug induced oxidative stress

Many idiosyncratic non-steroidal anti-inflammatory drugs (NSAIDs) cause GI, liver and bone marrow toxicity in some patients which results in GI bleeding/ulceration/fulminant hepatic failure/hepatitis or agranulocytosis/aplastic anemia. The toxic mechanisms proposed have been reviewed. Evidence is presented showing that idiosyncratic NSAID drugs form prooxidant radicals when metabolised by peroxidases known to be present in these tissues. Thus GSH, NADH and/or ascorbate were cooxidised by catalytic amounts of NSAIDs and hydrogen peroxide in the presence of peroxidase. During GSH and NADH cooxidation, oxygen uptake and activation occurred. Furthermore the formation of NSAID oxidation products was prevented during the cooxidation indicating that the cooxidation involved redox cycling of the first formed NSAID radical product. The order of prooxidant catalytic effectiveness of fenamate and arylacetic acid NSAIDs was mefenamic acid>tolfenamic acid>flufenamic acid, meclofenamic acid or diclofenac. Diphenylamine, a common moiety to all of these NSAIDs was a more active prooxidant for NADH and ascorbate cooxidation than these NSAIDs which suggests that oxidation of the NSAID diphenylamine moiety to a cation and/or nitroxide radical was responsible for the NSAID prooxidant activity. The order of catalytic effectiveness found for sulfonamide derivatives was sulfaphenazole>sulfisoxazolez.Gt;dapsone>sulfanilic acid>procainamide>sulfamethoxazole>sulfadiazine>sulfadimethoxine whereas sulfanilamide, sulfapyridine or nimesulide had no prooxidant activity. Although indomethacin had little prooxidant activity, its major in vivo metabolite, N-deschlorobenzoyl indomethacin had significant prooxidant activity. Aminoantipyrine the major in vivo metabolite of aminopyrine or dipyrone was also more prooxidant than the parent drugs. It is hypothesized that the NSAID radicals and/or the resulting oxidative stress initiates the cytotoxic processes leading to idiosyncratic toxicity.     

Prognostic aspects of aplastic anemia in pregnancy. Experience on six cases and review of the literature

Our resent experience on six cases of aplastic anemia complicated with pregnancy is described. In addition, 43 similar cases were collected from the literature and reviewed to analyze some prognostic aspects of this relatively rare but potentially serious complication. Clinical and hematological data were treated to extract some clinically meaningful factors in relation to the success and failure of pregnancy. Among initial hematological parameters, no significant difference was found between successful and unsuccessful cases with an exception of hemoglobin concentration. The patients diagnosed as aplastic anemia prior to conception demonstrated an better outcome of pregnancy as well as survival rate of mother when compared with those diagnosed during pregnancy. Mortality has apparently improved after the late 1950's. Success rate of pregnancy before 1958 was 21%, while it was 67% and 71% in the era of 1959-1969 and after 1970, respectively. However, hemorrhage and infection remained to be two major causes of maternal death in both eras. Based on these observations, the currently recommendable attitude to this complication is discussed.     

Induction of apoptosis by remoxipride metabolites in HL60 and CD34+/CD19- human bone marrow progenitor cells: potential relevance to remoxipride-induced aplastic anemia.

The antipsychotic agent, remoxipride [(S)-(-)-3-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2,6-dimethoxybenz amide] has been associated with acquired aplastic anemia. We have examined the ability of remoxipride, three pyrrolidine ring metabolites and five aromatic ring metabolites of the parent compound to induce apoptosis in HL60 cells and human bone marrow progenitor (HBMP) cells. Cells were treated for 0-24 h with each compound (0-200 microM). Apoptosis was assessed by fluorescence microscopy in Hoechst 33342- and propidium iodide stained cell samples. Results were confirmed by determination of internucleosomal DNA fragmentation using gel electrophoresis for HL60 cell samples and terminal deoxynucleotidyl transferase assay in HBMP cells. The catechol and hydroquinone metabolites, NCQ436 and NCQ344, induced apoptosis in HL60 and HBMP cells in a time- and concentration dependent manner, while the phenols, NCR181, FLA873, and FLA797, and the derivatives formed by oxidation of the pyrrolidine ring, FLA838, NCM001, and NCL118, had no effect. No necrosis was observed in cells treated with NCQ436 but NCQ344 had a biphasic effect in both cell types, inducing apoptosis at lower concentrations and necrosis at higher concentrations. These data show that the catechol and hydroquinone metabolites of remoxipride have direct toxic effects in HL60 and HBMP cells, leading to apoptosis, while the phenol metabolites were inactive. Similarly, benzene-derived catechol and hydroquinone, but not phenol, induce apoptosis in HBMP cells [Moran et al., Mol. Pharmacol., 50 (1996) 610-615]. We propose that remoxipride and benzene may induce aplastic anemia via production of similar reactive metabolites and that the ability of NCQ436 and NCQ344 to induce apoptosis in HBMP cells may contribute to the mechanism underlying acquired aplastic anemia that has been associated with remoxipride.     

Risk of agranulocytosis and aplastic anaemia in relation to use of antithyroid drugs. International Agranulocytosis and Aplastic Anaemia Study.

The relation of the use of antithyroid drugs to the risk of developing agranulocytosis and aplastic anaemia was evaluated in a population based case-control study with patients from Israel and seven regions in Europe. Data were obtained from cases and hospital controls by interview. Use of antithyroid drugs in the week before the onset of illness was compared in 262 patients with agranulocytosis and 1771 controls. Forty five patients (17%) and five controls (0.3%) had used antithyroid drugs. The relative risk was estimated to be 102 (95% confidence interval 38 to 275) taking into account confounding by other factors, including the use of other drugs. The excess risk for use of antithyroid drugs in any one week was estimated to be 6.3 cases of agranulocytosis per million users. Use of antithyroid drugs in a five month period ending one month before admission to hospital was compared in 135 patients with aplastic anaemia and 2145 controls. Four patients (3%) and five controls (0.2%) had taken drugs; the estimate of relative risk was 9.2 (95% confidence interval 1.8 to 47) after control for confounding. The estimate of excess risk of agranulocytosis with the use of antithyroid drugs was lower than found previously. Although the excess risk for aplastic anaemia was not calculated, these data suggest that it is very low.     

ACOUSTIC IDENTIFICATION OF NINE DELPHINID SPECIES IN THE EASTERN TROPICAL PACIFIC OCEAN

Acoustic methods may improve the ability to identify cetacean species during shipboard surveys. Whistles were recorded from nine odontocete species in the eastern tropical Pacific to determine how reliably these vocalizations can be classified to species based on simple spectrographic measurements. Twelve variables were measured from each whistle ( n = 908). Parametric multivariate discriminant function analysis (DFA) correctly classified 41.1% of whistles to species. Non-parametric classification and regression tree (CART) analysis resulted in 51.4% correct classification. Striped dolphin whistles were most difficult to classify. Whistles of bottlenose dolphins, false killer whales, and pilot whales were most distinctive. Correct classification scores may be improved by adding prior probabilities that reflect species distribution to classification models, by measuring alternative whistle variables, using alternative classification techniques, and by localizing vocalizing dolphins when collecting data for classification models.     

Use of ordinal categorical variables in skeletal assessment of sex from the cranium

In anthropological studies, visual indicators of sex are traditionally scored on an ordinal categorical scale. Logistic and probit regression models are commonly used statistical tools for the analysis of ordinal categorical data. These models provide unbiased estimates of the posterior probabilities of sex conditional on observed indicators, but they do so only under certain conditions. We suggest a more general method for sexing using a multivariate cumulative probit model and examine both single indicator and multivariate indicator models on a sample of 138 crania from a Late Mississippian site in middle Tennessee. The crania were scored for five common sex indicators: superciliary arch form, chin form, size of mastoid process, shape of the supraorbital margin, and nuchal cresting. Independent assessment of sex for each individual is based on pubic indicators. The traditional logistic regressions are cumbersome because of limitations imposed by missing data. The logistic regression correctly classified 66/74 males and 46/64 females, with an overall correct classification of 81%. The cumulative probit model classified 64/74 males correctly and 51/64 females correctly for an overall correct classification rate of 83%. Finally, we apply parameters estimated from the logit and probit models to find posterior probabilities of sex assignment for 296 additional crania for which pubic indicators were absent or ambiguous. Am J Phys Anthropol 107:97–112, 1998. © 1998 Wiley-Liss, Inc.     

Multivariate statistics in analysis of data from the in vitro motility assay

A novel approach is described for classification of filaments as stationary or moving and for extraction of velocity data for smooth actin filament sliding in vitro. Moving and stationary filaments were effectively classified using four discriminating variables in a multivariate statistical analysis. The variables were (1) two different measures of the average filament distance from its starting point, (2) a measure of the variability in sliding direction, and (3) the coefficient of variation (CV) of the frame-to-frame sliding velocity (v(mean)). On the basis of this multivariate analysis we obtained correct classification of 98% of the stationary filaments and 94% of the moving filaments in a cross-validation data set. The same classification functions were useful throughout despite a 10-fold variation in the average sliding velocity in the cross-validation data. Further analysis of motile filaments suggested that the velocity of smooth sliding should, ideally, be obtained from the intercept on the velocity axis of a plot of v(mean) against CV. The velocity, so obtained, was between 10 and 30% (mean 20+/-3%; n=7; p<0.001) higher than if average sliding velocity was obtained for all moving filaments with CV<0.5.     

Febrifugine analogue compounds: synthesis and antimalarial evaluation

Febrifugine is an alkaloid isolated from Dichroa febrifuga Lour as the active component against Plasmodium falciparum, but exhibits toxic side effects. In this study novel febrifugine analogues were designed and efficiently synthesized. New compounds underwent efficacy and toxicity evaluation. Some compounds are much less toxic than the natural product febrifugine and existing antimalarial drugs and are expected to possess wide therapeutic windows. In Aotus monkeys infected with the chloroquine resistant FVO strain of P. falciparum, one interesting compound possesses a 50% curative dose of 2mg/kg/day and a 100% curative dose of 8 mg/kg/day. These compounds, as well as the underlying design rationale, may find usefulness in the discovery and development of new antimalarial drugs.     

原卟啉IX二钠盐的低能铁离子束辐照产物对实验性小鼠再生障碍性贫血的疗效初步研究

利用110keV Fe^ 离子注入原卟啉IX二钠盐薄膜,动物实验和细胞学观察等表明辐照混合产物对于治疗^60Coγ射线所致实验性小鼠再生障碍性贫血具有一定的疗效,且无明显的毒副作用,有望从中筛选治疗再生障碍性贫血的新药或其先导化合物。     

High-resolution and contextual analysis for the diagnosis of fine needle aspirates of breast.

A study was undertaken to confirm earlier work on a smaller number of patients that had suggested that medium-resolution contextual analysis complements high-resolution individual cell analysis for cytomorphometric classification of fine needle aspirate smears of breast. The objectives of this study were to improve and verify the method. Sixty-one biopsy-confirmed hematoxylin and eosin-stained aspirate smears of breast were restained using the Feulgen technique. Individual nuclei were digitized at a resolution of 0.25 micron. Features describing size, shape, density and texture were extracted from the images. Individual cell analysis correctly classified 84% of cases, contextual analysis correctly classified 70% of cases, and the combined use of both techniques resulted in 87% classification accuracy. However, if fibroadenoma cases are excluded, the combined correct classification rate is 93%. Geometric and densitometric features contributed most to correct classification in individual cell analysis, while the most important contextual feature was the number of clusters per scene. We conclude that the addition of quantitative measures of smear patterns, termed "contextual analysis," improves automated classification schemes.     

Predicting drug-induced agranulocytosis: characterizing neutrophil-generated metabolites of a model compound,DMP 406, and assessing the relevance of an in vitro apoptosis assay for identifying drugs that may cause agranulocytosis

DMP 406 is a clozapine analogue developed by Dupont-Pharma for the treatment of schizophrenia. Unfortunately it caused agranulocytosis in dogs during preclinical studies. Clozapine also causes agranulocytosis and this is believed to be due to a reactive nitrenium ion metabolite produced by neutrophils. We studied the oxidation of DMP 406 by activated neutrophils and found that the major reactive species that is produced is not a nitrenium ion but rather an imine. This metabolite is similar to the reactive metabolite that has been proposed to be responsible for mianserin-induced agranulocytosis. Therefore we also studied the oxidation of mianserin by activated neutrophils and found that, although the major species is an iminium ion, it also bears a lactam moiety in the piperazine ring resulting from further oxidation. We usually find that HOCl is a good model system for the production of reactive metabolites of drugs that are formed by activated neutrophils, but in the case of both DMP 406 and mianserin, the products produced were significantly different than those formed by activated neutrophils. In contrast, the combination of horseradish peroxidase and hydrogen peroxide (HRP/H(2)O(2)) formed a very similar pattern of products, and this system was used to produce sufficient quantities of metabolites to allow for identification. The reactive metabolites of both DMP 406 and mianserin reacted with a range of nucleophiles, but in many cases the reaction was reversible. The best nucleophile for trapping these reactive metabolites was cyanide. It has been demonstrated that the products of clozapine oxidation by HRP/H(2)O(2), presumably the nitrenium ion, induced apoptosis in neutrophils at therapeutic concentrations of clozapine. It has been suggested that this process is involved in the mechanism of clozapine-induced agranulocytosis. We tested DMP 406 and mianserin in this system to see if the ability of a reactive metabolite of a drug to cause apoptosis could predict the ability of that drug to cause agranulocytosis. We used clozapine as a positive control and we also tested olanzapine, a drug that forms a reactive metabolite similar to that of clozapine but is given at a lower dose and does not cause agranulocytosis. We found that DMP 406 did not increase apoptosis at concentrations below 50 microM, and although mianserin did increase apoptosis at 10 microM this is above the therapeutic concentration. Olanzapine caused an increase in apoptosis at the same concentration as clozapine (1 microM), but because its therapeutic concentration is lower, this concentration was above the pharmacological range. There was no increase in apoptosis with any drug in the absence of HRP/H(2)O(2). These results indicate that this assay is unable to reliably predict the ability of different types of drugs to cause agranulocytosis. This is not a surprising result given that different drugs may induce agranulocytosis by different mechanisms.     

Marrow transplantation for severe aplastic anemia associated with exposure to quinacrine

Severe aplastic anemia developed in a patient after administration of quinacrine for treatment of discoid lupus erythematosus. Marrow transplantation was performed from an HLA genotypically identical sister after conditioning with cyclophosphamide. Quinacrine which was accumulated in the patient tissues did not interfere with engraftment, suggesting that the drug has no direct cytotoxic effect on hematopoietic stem cells. This study extends our previous observation that severe aplastic anemia acquired after exposure to drugs or toxins can be cured by marrow transplantation.     

Acquired aplastic anemia: Is bystander insult to autologous hematopoiesis driven by immune surveillance against malignant cells?

We previously reported a serendipitous finding from a patient with refractory severe aplastic anemia who had gotten an unexpected hematological response to treatment with gut-cleansing preparations (GCPs). This patient experienced three recurrences over the ensuing one year of intermittent GCP treatments, with each recurrence occurring 7-8 wk from a GCP. After his third recurrence, he was prescribed successive treatment with rifampicin, berberine, and monthly administered GCP for 4 mo, and he developed an erythroid proliferative neoplasma and an overwhelming enteropathy, and eventually died of septic shock. Laboratory investigations had validated the resolution of myelosuppression and the appearance of malignant clonal hematopoiesis. From the treatment process and laboratory investigations, it is reasonably inferred that the engagement of gut inflammation is critically required in sustaining the overall pathophysiology of acquired aplastic anemia probably by creating a chronic inflammatory state. Incorporation of rifampicin, berberine, and monthly GCP into cyclosporine can enhance the immunosuppressive effect. In a subgroup of acquired aplastic anemia patients whose pathogenesis is associated with genotoxic exposure, the suppressed normal hematopoiesis may result from the bystander insult that is mediated by the soluble inflammatory cytokines generated in response to the immunogenic products of damaged hematopoietic cells in the context of chronic inflammatory state and may offer a protective antineoplastic mechanism against malignant proliferation.     

Use of the Analysis of the Volatile Faecal Metabolome in Screening for Colorectal Cancer

Diagnosis of colorectal cancer is an invasive and expensive colonoscopy, which is usually carried out after a positive screening test. Unfortunately, existing screening tests lack specificity and sensitivity, hence many unnecessary colonoscopies are performed. Here we report on a potential new screening test for colorectal cancer based on the analysis of volatile organic compounds (VOCs) in the headspace of faecal samples. Faecal samples were obtained from subjects who had a positive faecal occult blood sample (FOBT). Subjects subsequently had colonoscopies performed to classify them into low risk (non-cancer) and high risk (colorectal cancer) groups. Volatile organic compounds were analysed by selected ion flow tube mass spectrometry (SIFT-MS) and then data were analysed using both univariate and multivariate statistical methods. Ions most likely from hydrogen sulphide, dimethyl sulphide and dimethyl disulphide are statistically significantly higher in samples from high risk rather than low risk subjects. Results using multivariate methods show that the test gives a correct classification of 75% with 78% specificity and 72% sensitivity on FOBT positive samples, offering a potentially effective alternative to FOBT.     

The toxicity of adenosine analogues against Babesia bovis in vitro.

The toxicities of 20 analogues of deoxyadenosine or adenosine were tested in vitro against the intraerythrocytic parasite Babesia bovis. IC37 values (the concentration of compound required to reduce cell survival to 37%) were determined for each compound. Tubercidin (7-deaza-adenosine), 2-bromo-adenosine, 8-bromo-3-ribosyl adenine and 6-phenylamino-deoxyadenosine were shown to be the most toxic towards B. bovis. Comparison of the toxicity results for these compounds in B. bovis with those in human melanoma cell lines indicated a differential toxicity, in that many of the compounds were toxic towards B. bovis but were relatively non-toxic towards human melanoma cell lines and vice versa. These results suggest that the mechanism of toxicity of the deoxyadenosine and adenosine analogues, whose normal metabolism involves transport, metabolism and incorporation into nucleic acids, may vary significantly between B. bovis and mammalian cells, allowing such drugs to be considered for parasite chemotherapy.