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

2. 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
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.

3. 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
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.

4. 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.

5. 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.

6. 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.

7. 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 Feb;316:10-16.

8. 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 Jul;58(1):

9. 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

10. 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

11. 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

12. 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

13. 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

14. References
Schizophrenia. National Institute of Mental Health [Internet]. 2016Feb; Available from:
Ojong M, Allen SN. Management and prevention of agranulocytosis in patients receiving clozapine. Mental Health Clinician. 2013;3(3):139–43.
Agranulocytosis, Acquired [Internet]. National Organization for Rare Disorders. [cited 2018 Nov 27]. Available from:
Williams, DP, Pirmohamed M, Naisbitt DJ, Uetrecht JP, Park BK. Induction of metabolism-dependent and -independent neutrophil apoptosis by clozapine. Mol pharmacol Jul;58(1):
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 Feb;316:10-16.
de Weide K, Loovers H, Pondman K, Bogers J, van der Straaten T, Langemeijer E, et al. Genetic risk factors for clozapine-induced neutropenia and agranulocytosis in a Dutch psychiatric population. Pharmacogenomics J May;17:471–478.
Uetrecht, JP. Metabolism of Clozapine by Neutrophils: Possible Implications for Clozapine-Induced Agranulocytosis. Drug Saf. 1992;7 Suppl 1:51-56.
Liu, ZC, Uetrecht, JP. Clozapine is Oxidized by Activated Human Neutrophils to a Reactive Nitrenium Ion that Irreversibly Binds to the Cells. J Pharmacol Exp Ther. 1995;275(3):
Gerson S, Lieberman J, Friedenberg W, Lee D, Marx J, Meltzer H. Polypharmacy in fatal clozapine-associated agranulocytosis. The Lancet Jul 27;338(8761):262–3.
Shuman MD, Trigoboff E, Demler TL, Opler LA. Exploring the potential effect of polypharmacy on the hematologic profiles of clozapine patients. J Psychiatr Pract Jan;20(1):50–8.
Wiciński M, Węclewicz MM. Clozapine-induced agranulocytosis/granulocytopenia: mechanisms and monitoring. Curr Opin Hematol Jan;25(1):22–8.
Wilson S, Hamilton R, Callender J, MacManus A, Howitt S, Okpo B. Clozapine antipsychotic polypharmacy: audit of use and patient monitoring. The Psychiatrist Oct;37(10):322–5.
Agranulocytosis Management and Treatment [Internet]. Cleveland Clinic [cited 2018 Nov 28]. Available from: agranulocytosis/management-and-treatment