Involvement of Extracellular Vesicles in the Proinflammatory Response to Clozapine: Implications for Clozapine-Induced Agranulocytosis.

Most idiosyncratic drug reactions (IDRs) appear to be immune-mediated, but mechanistic events preceding severe reaction onset remain poorly defined. Damage-associated molecular patterns (DAMPs) may contribute to both innate and adaptive immune phases of IDRs, and changes in extracellular vesicle (EV) cargo have been detected post-exposure to several IDR-associated drugs. To explore the hypothesis that EVs are also a source of DAMPs in the induction of the immune response preceding drug-induced agranulocytosis, the proteome and immunogenicity of clozapine- (agranulocytosis-associated drug) and olanzapine- (non-agranulocytosis-associated drug) exposed EVs were compared in two preclinical models: THP-1 macrophages and Sprague-Dawley rats. Compared with olanzapine, clozapine induced a greater increase in the concentration of EVs enriched from both cell culture media and rat serum. Moreover, treatment of drug-naïve THP-1 cells with clozapine-exposed EVs induced an inflammasome-dependent response, supporting a potential role for EVs in immune activation. Proteomic and bioinformatic analyses demonstrated an increased number of differentially expressed proteins with clozapine that were enriched in pathways related to inflammation, myeloid cell chemotaxis, wounding, transforming growth factor-ß signaling, and negative regulation of stimuli response. These data indicate that, although clozapine and olanzapine exposure both alter the protein cargo of EVs, clozapine-exposed EVs carry mediators that exhibit significantly greater immunogenicity. Ultimately, this supports the working hypothesis that drugs associated with a risk of IDRs induce cell stress, release of proinflammatory mediators, and early immune activation that precedes severe reaction onset. Further studies characterizing EVs may elucidate biomarkers that predict IDR risk during development of drug candidates. SIGNIFICANCE STATEMENT: This work demonstrates that clozapine, an idiosyncratic drug-induced agranulocytosis (IDIAG)-associated drug, but not olanzapine, a safer structural analogue, induces an acute proinflammatory response and increases extracellular vesicle (EV) release in two preclinical models. Moreover, clozapine-exposed EVs are more immunogenic, as measured by their ability to activate inflammasomes, and contain more differentially expressed proteins, highlighting a novel role for EVs during the early immune response to clozapine and enhancing our mechanistic understanding of IDIAG and other idiosyncratic reactions.

A Reactive Metabolite of Clozapine Induces Hematopoietic Toxicity in HL-60 Cells Undergoing Granulocytic Differentiation through Its Effect on Glutathione Metabolism.

Clozapine is an atypical antipsychotic with several advantages over conventional antipsychotics, in addition to its well-known efficacy in treatment-resistant schizophrenia. However, the high risk of agranulocytosis associated with clozapine therapy limits its clinical application. Clozapine bioactivation to an unstable protein-reactive metabolite, identified as a nitrenium intermediate, has been implicated in cytotoxicity toward neutrophils. Clozapine affects myeloid precursor cells rather than neutrophils; however, the impact of its reactive metabolite on myeloid precursor cells undergoing granulocytic differentiation remains unclear. Herein, we used hydrogen peroxide (H2O2) to generate the reactive metabolite and compared reactive metabolite-induced cytotoxicity between HL-60 cells undergoing granulocytic differentiation and differentiated HL-60 cells. In addition, we examined the role of oxidative stress in this type of cytotoxicity. The reactive metabolite of clozapine induced rapid cytotoxicity in HL-60 cells undergoing granulocytic differentiation, but not in differentiated HL-60 cells, with the metabolite exhibiting more potent cytotoxicity than clozapine. No cytotoxicity was observed following incubation with olanzapine, a structural analog of clozapine, even after exposure of the drug to H2O2. The reactive metabolite of clozapine decreased the levels of reduced glutathione, while addition of reduced glutathione attenuated the reactive metabolite-induced cytotoxicity. These findings indicate that glutathione metabolism plays a role in the hematopoietic toxicity induced by the reactive metabolite of clozapine. Oxidative stress may potentially increase susceptibility to the hematopoietic toxicity induced by the reactive metabolite of clozapine.

Genome-Wide Association Study of Metamizole-Induced Agranulocytosis in European Populations.

Agranulocytosis is a rare yet severe idiosyncratic adverse drug reaction to metamizole, an analgesic widely used in countries such as Switzerland and Germany. Notably, an underlying mechanism has not yet been fully elucidated and no predictive factors are known to identify at-risk patients. With the aim to identify genetic susceptibility variants to metamizole-induced agranulocytosis (MIA) and neutropenia (MIN), we conducted a retrospective multi-center collaboration including cases and controls from three European populations. Association analyses were performed using genome-wide genotyping data from a Swiss cohort (45 cases, 191 controls) followed by replication in two independent European cohorts (41 cases, 273 controls) and a joint discovery meta-analysis. No genome-wide significant associations (p < 1 × 10-7) were observed in the Swiss cohort or in the joint meta-analysis, and no candidate genes suggesting an immune-mediated mechanism were identified. In the joint meta-analysis of MIA cases across all cohorts, two candidate loci on chromosome 9 were identified, rs55898176 (OR = 4.01, 95%CI: 2.41-6.68, p = 1.01 × 10-7) and rs4427239 (OR = 5.47, 95%CI: 2.81-10.65, p = 5.75 × 10-7), of which the latter is located in the SVEP1 gene previously implicated in hematopoiesis. This first genome-wide association study for MIA identified suggestive associations with biological plausibility that may be used as a stepping-stone for post-GWAS analyses to gain further insight into the mechanism underlying MIA.

Clozapine-induced agranulocytosis/granulocytopenia: mechanisms and monitoring.

PURPOSE OF REVIEW: Clozapine-induced agranulocytosis/granulocytopenia (CIAG) is an uncommon condition, but potentially fatal in consequences. The pathogenesis, despite multiple experiments, is not fully elucidated. The current theory suggests reactive oxygen species - nitrenium ion as the most important factor of CIAG. In this review, mechanism and monitoring of CIAG will be discussed. RECENT FINDINGS: The mechanism of CIAG seems to have an autoimmune background, rather than toxic. Clozapine has a high potential to undergo biochemical activation to nitrenium ion. The role of the primary metabolite of clozapine - N-desmethylclozapine - is in decline. Nitrenium ion is mainly synthesized by CYP3A4, CYP2D6, and myeloperoxidase system in leukocytes. An important component of CIAG pathogenesis is genetic aberration in human leukocyte antigen genes, and also genes associated with apoptosis and ubiquitination. Clozapine monitoring regimes differ between countries. US-derived clozapine Risk Evaluation and Mitigation Strategy is the most tolerant in the aspect of blood parameter thresholds. Therefore, it provides the opportunities for physician to continue the treatment and also to rechallenge the drug after the episode of CIAG. SUMMARY: Each patient with the episode of CIAG should be assessed individually, with special attention to risk factors and drug-drug interactions. Upon that, the decision about clozapine rechallenge or withdrawal should be made.

Mechanisms of action that contribute to efficacy of omalizumab in chronic spontaneous urticaria.

The monoclonal anti-immunoglobulin E (IgE) antibody, omalizumab, was the first drug approved for use in patients with chronic idiopathic/spontaneous urticaria (CIU/CSU) who remain symptomatic despite H1 -antihistamine treatment. Omalizumab binds to free IgE, which lowers free IgE levels and causes FcεRI receptors on basophils and mast cells to be downregulated. It has been shown to improve symptoms of CIU/CSU, but its mechanism of action is not currently understood. Potential mechanisms in CIU/CSU include reducing mast cell releasability, reversing basopenia and improving basophil IgE receptor function, reducing activity of IgG autoantibodies against FcεRI and IgE, reducing activity of IgE autoantibodies against an antigen or autoantigen that has yet to be definitively identified, reducing the activity of intrinsically 'abnormal' IgE, and decreasing in vitro coagulation abnormalities associated with disease activity. However, none of these theories alone or in combination fully account for the pattern of symptom improvement seen with omalizumab therapy, and therefore, no one mechanism is likely to be the definitive mechanism of action. Additional research is needed to further clarify the involvement of omalizumab in relieving symptoms associated with the complex, multifactorial pathogenesis of CIU/CSU.

Genetic determinants of cocaine-associated agranulocytosis.

BACKGROUND: Drug-induced agranulocytosis is a recognized adverse drug event associated with serious infectious complications. Levamisole is an antihelminthic and immunomodulator withdrawn from North American markets in 2005 after reports of agranulocytosis. Previous studies of levamisole, suggest that the human leukocyte antigen (HLA)-B27 confers a genetic predisposition to this adverse drug event. Since 2009, emergency room visits due to agranulocytosis in individuals consuming levamisole-adulterated crack-cocaine have increased. METHODS: We performed a case-control study using a genotyping assay and novel gene chip to test the association between cocaine-associated agranulocytosis (CAA) and HLA-B27 and to identify pharmacokinetic and pharmacodynamic gene variants associated with the phenotype. RESULTS: Fifty-one CAA cases were identified through a provincial physician reporting system between 2008 and 2011. We examined eight of these cases and 26 matched controls. Genotyping revealed a significant association between HLA-B27 and CAA (odds ratio [OR] 9.2, 95% confidence interval [CI], 1.54-54.6). We also observed a similar association with the HLA-B27 tag single nucleotide polymorphism rs4349859 (OR 9.2, 95% CI 1.5-54.6) and rs13202464 (OR 6.7, 95% CI 1.1-41). Further associations were identified with variants in the ARBCC12 (OR 10.0, 95% CI 2.7-36.8) and CYP11A1 (OR 7.4, 95% CI 2.1-26.6) genes, while a novel protective association was observed with variants in the ARDB1 gene (OR 0.06, 95% CI 0.007-0.46). CONCLUSIONS: We confirmed the association of HLA-B27 with CAA and identified additional susceptibility variants. Health care providers should inform people who are identified as having CAA that it is genetically determined and can recur with continued cocaine use. The severity of infections and subsequent hospitalization, and the risk of recurrence may prompt health-promoting behaviour changes of the affected individuals. These genetic associations warrant the attention of public health and knowledge translation efforts to highlight the implications for susceptibility to this severe adverse drug event.

Effect of clozapine and olanzapine on neutrophil kinetics: implications for drug-induced agranulocytosis.

Clozapine is effective in the treatment of schizophrenia; however, its use is limited by a relatively high incidence of idiosyncratic agranulocytosis. The mechanism of clozapine-induced idiosyncratic agranulocytosis is unknown. Although most patients treated with clozapine do not develop agranulocytosis, most do have an immune response with an increase in inflammatory cytokines such as IL-6 and a release of immature neutrophils with neutrophilia rather than agranulocytosis. We have previously shown that treatment of rabbits with clozapine also causes an early release of neutrophils. Clozapine is oxidized to a reactive nitrenium ion that covalently binds to neutrophils, and this reactive metabolite may be responsible for the observed effects. Olanzapine and clozapine have very similar structures, and olanzapine is also oxidized to a reactive nitrenium ion; however, if it ever causes agranulocytosis, the incidence is much lower than that of clozapine. One possible basis for the difference in incidence of agranulocytosis between clozapine and olanzapine is that the therapeutic dose of olanzapine is much lower than that of clozapine. In this study, we compared the effects of clozapine and olanzapine in Sprague-Dawley rats at an equimolar dose and found that only clozapine had a significant effect on neutrophil kinetics. This suggests that the immune response and effects on neutrophil kinetics induced by clozapine are related to its ability to cause agranulocytosis.

Effect of aminoglutethimide on neutrophils in rats: implications for idiosyncratic drug-induced blood dyscrasias.

Aminoglutethimide (AMG) is an aromatic amine aromatase inhibitor associated with a high incidence of idiosyncratic blood dyscrasias, especially agranulocytosis. Animal models of idiosyncratic drug reactions (IDRs) represent essential tools to study these reactions; however, there is currently no valid model of idiosyncratic drug-induced agranulocytosis. Although AMG does not cause agranulocytosis in most animals or humans, drugs associated with serious IDRs generally cause a higher incidence of mild reactions that resolve despite continued treatment. Therefore, the effects of AMG on neutrophils and bone marrow in rats were studied to understand the mechanisms of more serious IDRs. An increase in peripheral blood neutrophils occurred as early as 24 h after AMG treatment with minimal changes to the total leukocyte count. Further investigation using 5-bromo-2'-deoxyuridine (BrdU) found an increased release of neutrophils from the bone marrow. Histologically, this corresponded to an increase in myeloid cells in the bone marrow, which was confirmed by differential staining with CD45 and CD71. AMG treatment stimulated an increase in colony forming unit granulocyte-macrophage and colony forming unit granulocyte ex vivo. There was also a marked increase in the number of activated neutrophils in the circulation expressing the extravasation marker CD62L. These findings indicate that AMG affects neutrophil production, release, and function. Similar effects on neutrophil kinetics in clozapine-treated rats have previously been found, and transient neutrophilia has been observed in patients taking other drugs associated with idiosyncratic agranulocytosis; therefore, the changes observed with AMG may be biomarkers to predict the risk that a drug will cause agranulocytosis.

Potential role of a quetiapine metabolite in quetiapine-induced neutropenia and agranulocytosis.

Clozapine was the first of the atypical antipsychotics to be developed, but its use has been restricted because of toxicity issues, particularly the risk of potentially life-threatening drug-induced neutropenia and agranulocytosis, which occurs in about 1% of patients. Bioactivation of clozapine by peroxidases forms a reactive nitrenium ion, which covalently adducts to protein and leads to neutrophil toxicity. The current generation of clozapine-inspired atypical antipsychotics has reduced toxicity through improved potency/decreased dose or through structural modification to prevent peroxidase-catalyzed nitrenium ion formation. Through the substitution of sulfur for the bridging nitrogen found in clozapine, quetiapine does not directly form a nitrenium ion when incubated with myeloperoxidase/H(2)O(2). We present evidence that cytochrome P450 2D6 catalyzes the formation of 7-hydroxyquetiapine, which can be oxidized by human myeloperoxidase to form a reactive quinone-imine and a reactive radical, which may account for the continued, although reduced, neutrophil toxicity. In the presence of myeloperoxidase/H(2)O(2) and glutathione, covalent 7-hydroxyquetiapine-glutathione adducts were formed. Bioactivation of quetiapine was verified in vivo in rat where three 7-hydroxyquetiapine-mercaptate adducts and a 7-hydroxyquetiapine-glutathione adduct were detected from bile after oral dosing. The decreased incidence of agranulocytosis with quetiapine over clozapine is postulated to be due to the lower exposure of the toxic precursor, 7-hydroxyquetiapine versus clozapine, respectively.

Immune-mediated agranulocytosis caused by the cocaine adulterant levamisole: a case for reactive metabolite(s) involvement.

The United States Public Health Service Administration is alerting medical professionals that a substantial percentage of cocaine imported into the United States is adulterated with levamisole, a veterinary pharmaceutical that can cause blood cell disorders such as severe neutropenia and agranulocytosis. Levamisole was previously approved in combination with fluorouracil for the treatment of colon cancer; however, the drug was withdrawn from the U.S. market in 2000 because of the frequent occurrence of agranulocytosis. The detection of autoantibodies such as antithrombin (lupus anticoagulant) and an increased risk of agranulocytosis in patients carrying the human leukocyte antigen B27 genotype suggest that toxicity is immune-mediated. In this perspective, we provide an historical account of the levamisole/cocaine story as it first surfaced in 2008, including a succinct review of levamisole pharmacology, pharmacokinetics, and preclinical/clinical evidence for levamisole-induced agranulocytosis. Based on the available information on levamisole metabolism in humans, we propose that reactive metabolite formation is the rate-limiting step in the etiology of agranulocytosis associated with levamisole, in a manner similar to other drugs (e.g., propylthiouracil, methimazole, captopril, etc.) associated with blood dyscrasias. Finally, considering the toxicity associated with levamisole, we propose that the 2,3,5,6-tetrahydroimidazo[2,1-b]thiazole scaffold found in levamisole be categorized as a new structural alert, which is to be avoided in drug design.

Suppression of the stem cell antigen-1 response and granulocyte lineage expansion by alcohol during septicemia.

OBJECTIVE: Granulocytopenia frequently occurs in alcohol abusers with severe bacterial infection, which strongly correlates with poor clinical outcome. Knowledge of the molecular mechanisms underlying the granulopoietic response to bacterial infection remains limited. This study investigated the involvement of stem cell antigen-1 expression by granulocyte lineage-committed progenitors in the granulopoietic response to septicemia and how alcohol affected this response. DESIGN: : Laboratory investigation. SETTING: University laboratory. SUBJECTS: Male Balb/c mice. INTERVENTIONS: Thirty mins after intraperitoneal injection of alcohol (20% ethanol in saline at 5 g of ethanol/kg) or saline, mice received an intravenous Escherichia coli challenge. MEASUREMENTS AND MAIN RESULTS: E. coli septicemia activated stem cell antigen-1 expression by marrow immature granulocyte differentiation antigen-1 precursors which correlated with an increase in proliferation, granulocyte macrophage colony-forming unit production, and expansion of this granulopoietic precursor cell pool. Acute alcohol treatment suppressed stem cell antigen-1 activation and inhibited the infection-induced increases in proliferation, granulocyte macrophage colony-forming unit production, and expansion the of immature granulocyte differentiation antigen-1 precursor cell population. Consequently, recovery of the marrow mature granulocyte differentiation antigen-1 cell population after E. coli challenge was impaired. Stem cell antigen-1 was induced in sorted granulocyte differentiation antigen-1, stem cell antigen-1' cells by lipopolysaccharide-stimulated C-Jun kinase activation that was also inhibited by alcohol. Furthermore, stem cell antigen-1 knockout mice failed to expand the marrow immature granulocyte differentiation antigen-1 cell pool and demonstrated fewer newly produced granulocytes in the circulation after the E. coli challenge. CONCLUSIONS: Alcohol suppresses the stem cell antigen-1 response in granulocyte lineage-committed precursors and restricts granulocyte production during septicemia, which may serve as a novel mechanism underlying impaired host defense in alcohol abusers.

Investigating the mechanisms of aromatic amine-induced protein free radical formation by quantitative structure-activity relationships: implications for drug-induced agranulocytosis.

Aromatic amine drugs have been associated with agranulocytosis (neutrophil depletion) for which the mechanism is unknown. We have previously shown that the metabolism of two aromatic amine drugs by human myeloperoxidase (MPO) results in phenyl radical metabolite formation and also in protein free radical formation on MPO. Because the concentration of drug required to produce a maximum signal for MPO protein free radical (MPO*) detection was different for each drug, this prompted us to consider that other aromatic amines may also show varying degrees of ability to induce MPO* formation. Immunoassay experiments using the immuno-spin-trapping technique were performed, which evaluated the potency of different aromatic amines containing the aniline substructure to generate the MPO*. Each reaction contained equal amounts of H(2)O(2), 5,5-dimethyl-1-pyrroline-N-oxide, MPO, and variable concentrations of aniline derivatives. Several physicochemical parameters for aniline derivatives were used to derive quantitative structure-activity relationship equations, which showed that the Hammett constant (sigma) best correlated with the MPO* formation for all aniline derivatives. More statistically robust equations were derived if the anilines were separated into mono- and disubstituted groups. However, some aniline derivatives did not induce MPO* formation. Using electron spin resonance spectroscopy, we evaluated the ability of all aniline derivatives tested to produce phenyl radical metabolites, as previously shown by spin trapping for the aromatic amine drugs. Interestingly, we found that only those aniline derivatives that produced a phenyl radical also formed MPO*. We propose that the phenyl radical is the reactive free radical metabolite responsible for generating the MPO*.

Aminoglutethimide-induced protein free radical formation on myeloperoxidase: a potential mechanism of agranulocytosis.

Aminoglutethimide (AG) is a first-generation aromatase inhibitor used for estrogen-dependent breast cancer. Unfortunately, its use has also been associated with agranulocytosis. We have investigated the metabolism of AG by myeloperoxidase (MPO) and the formation of an MPO protein free radical. We hypothesized that AG oxidation by MPO/H2O2 would produce an AG cation radical that, in the absence of a biochemical reductant, would lead to the oxidation of MPO. We utilized a novel anti-DMPO antibody to detect DMPO (5,5-dimethyl-1-pyrroline N-oxide) covalently bound to protein, which forms only by the reaction of DMPO with a protein free radical. We found that AG metabolism by MPO/H2O2 induced the formation of DMPO-MPO, which was inhibited by MPO inhibitors and ascorbate. Glutethimide, a congener of AG that lacks the aromatic amine, did not cause DMPO-MPO formation, indicating the necessity of oxidation of the aniline moiety in AG. When analyzed by electron spin resonance spectroscopy, we detected a phenyl radical adduct, derived from AG, which may be involved in the free radical formation on MPO. Furthermore, we also found protein-DMPO adducts in MPO-containing, intact human promyelocytic leukemia cells (HL-60). MPO was affinity-purified from HL-60 cells treated with AG/H2O2 and was found to contain DMPO. These findings were also supported by the detection of protein free radicals with electron spin resonance in the cellular cytosolic lysate. The formation of an MPO protein free radical is believed to be mediated by one of two free radical drug metabolites of AG, one of which was characterized by spin trapping with 2-methyl-2-nitrosopropane. These results are the first demonstration of MPO free-radical detection by the anti-DMPO antibody that results from drug oxidation. We propose that drug-dependent free radical formation on MPO may play a role in the origin of agranulocytosis.

[Dapsone-induced agranulocytosis. The role of xenobiotic-metabolizing enzymes demonstrated by a case report]. / Dapson-induzierte Agranulozytose. Die Rolle fremdstoffmetabolisierender Enzyme am Beispiel einer Kasuistik.

A 34-year-old female patient with a three year history of generalized granuloma annulare was treated systemically with dapsone (DADPS). Six weeks after the onset of treatment, the patient developed an extensive tonsillitis of the base of the tongue with fever and malaise. Routine laboratory work showed a leukocytopenia with agranulocytosis. Further investigation revealed a marked decrease of the enzyme activity of N-acetyltransferase 2, which plays an important role in dapsone metabolism. Treatment included the cessation of dapsone, antibiotic coverage, and G-CSF leading to the rapid improvement of symptoms and normalization of leukocyte counts. Dapsone-induced angina agranulocytotica is a rare event and is interpreted as an idiosyncratic reaction. Depending on genetic polymorphisms of various enzymes, dapsone can be metabolized to immunologically or toxicologically relevant intermediates. Because of the risk of severe hematologic reactions, dapsone should only be employed for solid indications and with appropriate monitoring.

Expression of CD117 and CD11b in bone marrow can differentiate acute promyelocytic leukemia from recovering benign myeloid proliferation.

The morphologic characteristics of bone marrow aspirates from patients recovering from acute agranulocytosis may be closely similar to the pattern observed in cases of acute promyelocytic leukemia (APL). The clinical manifestation also can be ambiguous in a substantial number of cases. The immunophenotypic features of bone marrow from 5 patients recovering from acute agranulocytosis, showing an increase in the percentage of promyelocytes (26%-66%), were compared with the immunophenotype of 31 consecutive patients with APL whose diagnosis was confirmed by PML-RAR alpha gene rearrangement. All markers were similarly expressed, except for CD117 and CD11b. CD117 was positive in 24 (77%) of the APL cases and in none of the acute agranulocytosis cases. On the other hand, CD11b was positive in 5 (100%) of the acute agranulocytosis cases and in only 2 (6%) of the APL cases. Thus, the CD117-CD11b+ phenotype was detected in all patients recovering from agranulocytosis and in only 1 (3%) of 31 APL cases. Therefore, we suggest that the combination of both markers is helpful in the differentiation of APL from recovering benign myeloid proliferation.

Influence of immunosuppression on the pharmacokinetics and pharmacodynamics of azithromycin in infected mouse tissues.

Azithromycin has been shown to preferentially distribute to infection loci. Due to the potential contribution of phagocytes as transporters of drug to these sites, there has been some concern that immunosuppression of the cellular arm of the host defence system would greatly reduce the delivery of azithromycin to sites of infection and hence impair efficacy. Therefore, we evaluated the pharmacokinetics and pharmacodynamics of azithromycin in a Staphylococcus aureus intramuscular infection model in normal and immunosuppressed mice, employing therapeutic and prophylactic regimens. Immunosuppression was induced by daily doses of cyclophosphamide that culminated in leucopenia with an underlying granulocytopenic condition, with circulating peripheral granulocytes numbering from < or = 0.1-0.3 x 10(9)/L. Azithromycin tissue levels were not reduced in infection loci in granulocytopenic mice but moderate increases in Cmax and AUC values were observed, relative to similar tissues from normal mice. The tissue half-life of azithromycin in infected tissues in a therapeutic mode (75 h) was three-fold longer than in a prophylactic mode (25 h); this correlated with the degree of inflammation (therapy was withheld until inflammation was evident; i.e., prophylaxis reduced inflammation). Histological examination of infected tissues from normal and leucopenic mice was indistinguishable despite a 70%-85% reduction in circulating granulocytes. Compared with untreated infected controls, bactericidal activity was noted following prophylaxis with azithromycin and bacteraemia was suppressed in mice receiving azithromycin therapeutically. In summary, these data indicate that azithromycin delivery and efficacy in a moderately immunosuppressed animal model are unimpaired.

Reactive metabolites and agranulocytosis.

Central to most hypotheses of the mechanism of idiosyncratic drug-induced blood dyscrasias is the involvement of reactive metabolites. In view of the reactive nature of the majority of such metabolites, it is likely that they are formed by, or in close proximity to the blood cells affected. The major oxidative system of neutrophils generates hypochlorous acid. We have demonstrated that the drugs associated with the highest incidence of agranulocytosis are oxidized to reactive metabolites by hypochlorous acid and/or activated neutrophils. There are many mechanisms by which such reactive metabolites could induce agranulocytosis. In the case of aminopyrine-induced agranulocytosis, most cases appear to involve drug-dependent anti-neutrophil antibodies, and these are likely to be induced by cell membrane antigens modified by the reactive metabolite of aminopyrine. The target of agranulocytosis associated with many other drugs is usually neutrophil precursors and may involve cytotoxicity or a cell-mediated immune reaction induced by a reactive metabolite. In the case of aplastic anaemia, there is evidence in some cases for involvement of cytotoxic T cells, which could either be induced by metabolites generated by neutrophils, or more likely, by reactive metabolites generated by stem cells.

Production of tumor necrosis factor-alpha in granulocytopenic mice with pulmonary candidiasis and its modification with granulocyte colony-stimulating factor.

In the present study, a lethal model of pulmonary candidiasis was established using granulocytopenic mice with cyclophosphamide. These mice started to die 1 day after infection and had all died within the next 48 hr. The counts of live C. albicans in the lung gradually increased with time, while the organisms were quickly eliminated in the normal mice. From the histology and measurements on bronchoalveolar lavage fluid (BALF), polymorphonuclear cells (PMN) response was almost zero up to 24 hr; and then a weak but significant response was observed at 48 hr, while a marked accumulation of PMN was detected from as early as 6 hr in normal mice. In contrast, macrophages had accumulated in BALF by 48 hr in granulocytopenic mice, but not in normal mice. Both in serum and BALF, a considerable level of tumor necrosis factor-alpha (TNF-alpha) was detected from 6 hr, peaking at 24 to 48 hr, while in normal mice the quantity was under the detection limit in serum and very low in BALF. The effects of administering granulocyte colony-stimulating factor (G-CSF) on these parameters were next examined. G-CSF significantly prolonged the survival time of granulocytopenic mice, promoted the clearance of organisms through increasing the counts of PMN in the lung, and strongly inhibited the production of TNF-alpha both in BALF and serum. These results suggest that this cytokine does not protect them, but plays some role in their death due to candidial infection in granulocytopenic mice.