Clozapine-Induced Agranulocytosis Eddy Huang, Christine Shi, Steven Fong, Sarina Sharma PHM142 Presentation November 28, 2018 Instructor: Dr. J. Henderson

Clozapine and Schizophrenia Schizophrenia is a mental disorder that affects the person at a cognitive, behavioural, and emotional level Typical and atypical antipsychotics used to treat Schizophrenia Clozapine is an atypical antipsychotic with high efficacy used to treat positive and negative symptoms of Schizophrenia Positive symptoms: Losing touch with reality Negative symptoms: Reduced emotions Adverse effects of clozapine may outweigh its benefits Agranulocytosis https://www.healthline.com/health/schizophrenia-types Before we begin with Clozapine, we need to understand Schizophrenia. Schizophrenia is a mental disorder that usually begins at the ages of 16 to 30. It is characterized as a disorder that affects how a person thinks, behaves, and feels. Typical and atypical antipsychotics are used to treat Schizophrenia. There are numerous drugs that can alleviate the symptoms of Schizophrenia but Clozapine is our focus today. Clozapine is used in cases where high efficacy is needed. It is also effective because there is a lower relapse with the drug, and there are lower rates of discontinuation. It also treats both positive and negative symptoms of Schizophrenia. Positive symptoms of Schizophrenia disconnect the patient's mind from reality, and patients often experience hallucinations while having a delusional mind. Negative symptoms of Schizophrenia lead to a decrease in emotion, and these patients experience reduced feelings of pleasure. Because Clozapine alleviates both positive and negative symptoms while maintaining high efficacy, it is very effective for the treatment of Schizophrenia. However, there are adverse effects that may outweigh its benefits.

Clozapine and Agranulocytosis Rare but fatal agranulocytosis can be induced by clozapine Agranulocytosis is the depletion of leukocytes Severe cases of a clozapine-induced agranulocytosis results in an ANC < 500 cells/uL and WBC < 2000 cells/uL Symptoms of Agranulocytosis: Ulcer development in mucous membranes that line the mouth and GI tract Susceptible to bacterial infections Sepsis occurs to bacterial contamination https://www.news-medical.net/health/Sepsis-Prevention.aspx A rare but fatal adverse effect of Clozapine, is Agranulocytosis. In general terms, Agranulocytosis is the depletion of leukocytes in a patient. In cases of severe agranulocytosis, the Absolute Neutrophil Count is less than 500 cells per microlitre, and the leukocyte count is less than 2000 cells per microlitre. The resulting decrease in immune cells lead to a suppression of the immune system. Common symptoms of Agranulocytosis include ulcer development in membranes that line the mouth and GI tract, and patients will also become susceptible to bacterial infections due to immunosuppression. As a result, something as deadly as sepsis can occur in the body.

Drug Metabolism by Neutrophils Neutrophils use respiratory burst to fight pathogens such as bacteria NADPH oxidase converts oxygen to superoxide, a ROS Superoxide spontaneously converts to hydrogen peroxide (H2O2), another ROS Granules release myeloperoxidase (MPO) MPO is oxidized by H2O2 into its active form (Compound I), a strong oxidant Compound I metabolizes drugs Converts chloride and H2O2 to hypochlorous acid (HOCl), a strong oxidizing antibacterial agent Leukocytes, particularly neutrophils, may be affected by their metabolism of clozapine. Neutrophils employ the use of respiratory burst to fight pathogens such as bacteria. Respiratory burst occurs when reactive oxygen species are rapidly released from the activated neutrophil to either the outside of the cell, or into a phagolysosome during phagocytosis. These reactive oxygen species attack pathogens, but can also harm surrounding host tissues by indiscriminately damaging DNA, RNA, lipids, and proteins. During neutrophil activation, membrane-associated enzymes known as NADPH oxidases convert oxygen to superoxide, a reactive oxygen species, which can then spontaneously convert to hydrogen peroxide, another reactive oxygen species and also an oxidizing agent. Activated neutrophils may also release myeloperoxidase from their azurophilic granules. Myeloperoxidase is then oxidized by hydrogen peroxide to its active enzymatic form, known as Compound I. Compound I is a strong oxidant that is known to be able to metabolize drugs. Compound I converts chloride ions and hydrogen peroxide into hypochlorous acid, a strong oxidizing antibacterial agent.

Clozapine is oxidized to nitrenium ion by hypochlorous acid Mechanism Clozapine is oxidized to nitrenium ion by hypochlorous acid Dehydrogenation leads to formation of nitrenium ion Very reactive, extremely short half-life, covalently binds to glutathione (GSH), as well as sulfhydryl groups in cell membrane proteins May affect neutrophil progenitors cells, which are also capable of metabolizing clozapine to nitrenium Hypochlorous acid is known to react rapidly with clozapine. It oxidizes clozapine to the nitrenium ion in a dehydrogenation reaction. The nitrenium ion is very reactive and has an extremely short half-life, usually spontaneously reacting with water to form phenols when there are no other nucleophiles present. However, it will bind covalently to strong nucleophiles such as glutathione, as well as sulfhydryl groups in cell membrane proteins. Another note of interest is that leukocyte progenitors in the bone marrow are also capable of metabolizing clozapine to nitrenium.

Road to clozapine-induced agranulocytosis Nitrenium implicated in two routes that lead to agranulocytosis Binds to neutrophil surface proteins and acts as a hapten Immunomediated cell apoptosis Clozapine-induced agranulocytosis Nitrenium ion Binds to and depletes GSH, cell is more susceptible to oxidative stress Cellular apoptosis Nitrenium has been implicated in two routes that lead to agranulocytosis. When a neutrophil undergoes respiratory burst, the ion is released outside of the cell. Here, it can bind to neutrophil membrane proteins. The ion can then act as a hapten, activating the adaptive immune response, priming and causing neutrophils to be targeted by T cells and induced to undergo apoptosis. Similarly, in the neutrophil itself, the nitrenium ion can bind to and deplete GSH, making the neutrophil more susceptible to oxidative stress. wherein with enough stress, the neutrophil can undergo apoptosis and eventually agranulocytosis.

Clozapine exposure induces lymphocyte proliferation There is evidence that the immune system plays a role in agranulocytosis HC: healthy controls SZ: schizophrenic patient CIAC: clozapine induced agranulocytosis While the mechanism behind agranulocytosis is not known, there is evidence for both routes. A recent study by Regen et al. gave evidence toward the immune system hypothesis. Lymphocytes taken from schizophrenic patients with a history of agranulocytosis proliferated when incubated with clozapine. Lymphocytes taken from healthy controls and schizophrenic patients without agranulocytosis did not. This was reminiscent of a secondary immune response that occurs when the immune system is re-exposed to a previously encountered insult. The assay measures the proliferation of antigen specific T-cells after being incubated for 96 hours with a varying concentrations of clozapine (no clozapine to 80 micromolar). All the assays were also stimulated with a non-specific mitogen as a control (to ensure that the cells could proliferate). MTS OD refers to the assay where live cells reduce MTS tetrazolium to a colored dye. Lymphocytes from patients that have a history of clozapine-induced agranulocytosis greatly proliferate when exposed to clozapine, implicating an immune response component to agranulocytosis. Figure from Regen, F, Herzog I, Hahn E, Ruehl C, Le Bret N, Dettling M, et al. Clozapine-induced agranulocytosis: evidence for an immune-mediated mechanism from a patient-specific in-vitro approach. Toxicol appl pharmacol. 2017 Feb;316:10-16.

Clozapine metabolites correlated with apoptosis Activating system consists of horseradish peroxidase and hydrogen peroxide Enables clozapine to be oxidized to nitrenium ion (amongst other things) With regard to the oxidative route, an early in vitro study done here at UofT showed that neutrophils incubated with clozapine underwent apoptosis when also incubated with reagents that oxidized clozapine to its metabolites, including nitrenium. Importantly, they found that apoptosis occurred for clozapine concentrations in the therapeutic range up to 3 micromolar. The annexin-V assay was used for detecting apoptotic vs necrotic cells by binding to phosphotidylserine residues exposed on the cell surface of apoptotic cells. Necrotic cells did not display annexin-V binding. Figure from: Williams, DP, Pirmohamed M, Naisbitt DJ, Uetrecht JP, Park BK. Induction of metabolism-dependent and -independent neutrophil apoptosis by clozapine. Mol pharmacol. 2000 Jul;58(1):207-216.

Treatment and Prevention Treatment includes weekly, biweekly or monthly granulocyte counts for those on clozapine (based on severity of known disease) Treatment of resulting infection, prevention and precautionary measures against infection If agranulocytosis becomes too severe, therapy may have to be discontinued either temporarily (2 weeks to recover) or permanently May need to switch to less effective alternatives like Olanzapine Transfusion of Granulocytes currently not a viable solution as cells short lived

The Future: Ongoing Research Key areas of research of interest to Pharmacists: 1) Drug Drug Interactions Goals: Safe use of clozapine with other medications 2) Risk factors of Agranulocytosis Goals: Knowing who is more or less susceptible, who should and should not take medications with this as a possible side effect

Antipsychotic Drug Interactions Many patients taking clozapine are on other drugs Drug-drug interaction may play a role in risk of agranulocytosis A 2014 study found an increased risk of agranulocytosis in patients taking clozapine with autonomous nervous agents and various GI drugs, including PPIs Exact mechanisms of DI not known Pharmacists could use this to engage in better MTM practice with pts on clozapine https://www.ncbi.nlm.nih.gov/pubmed/24419309 https://www.thelancet.com/journals/lancet/article/PII0140-6736(91)90410-Q/fulltext https://www.cambridge.org/core/services/aop-cambridge-core/content/view/84D019A344785253BA48550EE94CBAD2/S175832090000408Xa.pdf/clozapine_antipsychotic_polypharmacy_audit_of_use_and_patient_monitoring.pdf

Research on Risk Factors People of Asian descent, elderly, and women at higher risk of Clozapine induced agranulocytosis Autoimmune link too, may be of autoimmune origin May also be genetic risks too, as CYP3A4 and FM-O3 polymorphisms associated with risk While genetic factors are not modifiable, one day gene testing and demographic analysis may be able to stratify risk + help clinicians know if a pt is a good candidate for clozapine https://pmj.bmj.com/content/postgradmedj/81/959/546.full.pdf https://www.ncbi.nlm.nih.gov/pubmed/28984748

Summary Clozapine is an atypical antipsychotic that treats positive and negative symptoms of Schizophrenia Agranulocytosis leads to susceptibility to bacterial infections Clozapine is oxidized to nitrenium ion through dehydrogenation of the piperazine ring by neutrophil NADPH oxidase/myeloperoxidase-generated hypochlorous acid The nitrenium ion has been implicated in two mechanisms that may lead to agranulocytosis The nitrenium ion can act as a hapten and cause the immune system to target and induce apoptosis in neutrophils The nitrenium ion depletes the neutrophil’s GSH levels, making it more susceptible to oxidative stress and eventually leading to apoptosis Current treatment for agranulocytosis involves strict monitoring of neutrophil levels and treating complications as they arise. May need to switch to an alternative antipsychotic Drug-drug interactions between clozapine and other regimens maybe implicated in agranulocytosis, mechanisms unknown Risk factors for agranulocytosis include: female, Asian descent, elderly age Genetic risk factors are also implicated, but no predictive algorithm

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