Methods for treating a mental disorder with clozapine comprising genomic testing steps

Abstract

The present disclosure relates to methods for treating patients with the drug clozapine and particularly relates to methods for providing an effective treatment dose of the drug in order to provide an effective dose in a patient's bloodstream.

Description

[0001] METHODS FOR TREATING A MENTAL DISORDER WITH CLOZAPINE COMPRISING GENOMIC TESTING STEPS

[0002] Introduction

[0003] This disclosure relates to methods for treating patients with the drug clozapine and particularly relates to methods for providing an effective dose of the drug in a patient’s bloodstream.

[0004] Background

[0005] Clozapine is an atypical antipsychotic. It is a weak dopamine antagonist at the dopaminergic receptors DI, D2, D3 and D5, and shows potent antagonistic effects on the D4 receptor. It also has potent anti-a- adrenergic, anticholinergic, antihistaminic, arousal-lowering and anti-serotonergic properties. It is primarily used to treat patients with schizophrenia and schizoaffective disorder who have had an inadequate response to other antipsychotics or who have been unable to tolerate other drugs. It is also used for the treatment of psychosis in Parkinson's disease and has been used for the treatment of a number of other psychiatric disorders off label.

[0006] Clozapine is a uniquely effective antipsychotic, but its use is complicated by factors such as potentially serious haematological side effects and the absence of a reliable correlation between dose and plasma concentration. Since the plasma concentration of clozapine determines both treatment response and the severity of adverse effects, therapeutic drug monitoring (TDM) is widely used to optimise clozapine dose for a given individual. Data from TDM services suggest that biological sex, body mass index and smoking status account for a substantial proportion of the variability in plasma concentration of clozapine between individuals. Using these data, algorithms have been developed, see for example Rostami-Hodjegan et al. (2004), which predict the plasma concentration that should be obtained for a given dose based on biological sex and smoking status, but the low reproducibility of results obtained makes these approaches suboptimal.

[0007] Metabolism of clozapine in the liver by cytochrome P450 enzymes (P450s; CYPs) plays a significant role in the pharmacokinetics of the drug, which is almost completely metabolised prior to excretion by enzymes CYP1A2 and CYP3A4, and to some extent by CYP3A5, CYP2C19 and CYP2D6. Of the major metabolites, only the desmethyl metabolite is active. Its pharmacological actions are reminiscent of those of clozapine, but are considerably weaker and of shorter duration.

[0008] The ability to achieve a predictable plasma concentration of the drug following administration is important for ensuring that the patient responds in a clinically significant way to the drug and that their likelihood of suffering potentially dangerous adverse effects is minimised. It would therefore be desirable to provide methods of more reliably determining the appropriate amounts of clozapine with which to treat patients. Summary of Invention

[0009] In a first aspect, the present disclosure provides clozapine for use in a method for treating a mental disorder in a patient, wherein the method comprises administering to the patient an effective dose of clozapine and wherein the effective dose of clozapine is determined by a method comprising the steps of:

[0010] (i) obtaining a biological sample from the patient, wherein the sample comprises genomic DNA of the patient;

[0011] (ii) determining the presence in the genomic DNA of:

[0012] CYP1A2 alleles selected from * 1A, * 1C and * IF;

[0013] CYP3A4 alleles selected from * 1, * 1B, *3, * 10, and *22;

[0014] CYP3A5 alleles selected from * 1, *3, *6 and *7; and NFIB rs28379954 alleles selected from C or T;

[0015] (iii) determining whether the patient smokes tobacco; and

[0016] (v) determining the effective dose of clozapine based on steps (ii) and (iii).

[0017] In a second aspect the disclosure provides a method for determining an effective dose of clozapine for treating a patient having a mental disorder, the method comprising the steps of:

[0018] (i) obtaining a biological sample from the patient, wherein the sample comprises genomic DNA of the patient;

[0019] (ii) determining the presence in the genomic DNA of:

[0020] CYP1A2 alleles selected from * 1A, * 1C and * IF;

[0021] CYP3A4 alleles selected from * 1, * 1B, *3, * 10, and *22;

[0022] CYP3A5 alleles selected from * 1, *3, *6 and *7; and NFIB (rs28379954) alleles selected from C or T;

[0023] (iii) determining whether the patient smokes tobacco; and

[0024] (v) determining the effective dose of clozapine based on steps (ii) and (iii).

[0025] In a third aspect the disclosure provides a method for treating a patient with clozapine, wherein the patient is suffering from a mental disorder, the method comprising the steps of:

[0026] (i) obtaining a biological sample from the patient, wherein the sample comprises genomic DNA of the patient;

[0027] (ii) determining the presence in the genomic DNA of:

[0028] CYP1A2 alleles selected from * 1A, * 1C and * IF;

[0029] CYP3A4 alleles selected from * 1, * 1B, *3, * 10, and *22;

[0030] CYP3A5 alleles selected from * 1, *3, *6 and *7; and an NFIB (rs28379954) allele selected from C or T;

[0031] (iii) determining whether the patient smokes tobacco;

[0032] (v) determining an effective dose of clozapine based on steps (ii) and (iii); and (vi) treating the patient with the effective dose of clozapine.

[0033] In some embodiments, determining an effective dose of clozapine at step (v) comprises:

[0034] - assigning a score to each CYP1A2 allele present in the genomic DNA, wherein any CYP1A2 * IF allele is assigned a higher score if the patient is a smoker;

[0035] - assigning a score to each CYP3A4 allele present in the genomic DNA;

[0036] - assigning a score to each CYP3A5 allele present in the genomic DNA;

[0037] - summing the score for each CYP1A2 allele, each CYP3A4 allele and each CYP3A5 allele to obtain an overall activity score;

[0038] - increasing the overall activity score if the NFIB C allele is present in the genomic DNA;

[0039] - multiplying the overall activity score by a basal dose to obtain the effective dose of clozapine.

[0040] In some embodiments, the score assigned to each CYP1A2* IF allele is a first score if the patient is a smoker, and a second score if the patient is a non-smoker; and the score assigned to each CYP1A2* 1A, CYP3A4* 1, CYP3A4* IB, CYP3A4*3, CYP3A4* 10 or CYP3A5* 1 allele is the second score; and the score assigned to each CYP1A2* 1C or CYP3A4*22 allele is a third score; and the score assigned to each CYP3A5*3, CYP3A5*6 or CYP3A5*7 allele is a fourth score.

[0041] In some embodiments, the first score, the second score, the third score and the fourth score each have a different numerical value, preferably wherein: the first score is greater than the second score; and the second score is greater than the third score; and the third score is greater than the fourth score.

[0042] In some embodiments, the increase in the overall activity score if the NFIB C allele is present in the genomic DNA is an equivalent numerical value to that of the third score.

[0043] In some embodiments, the method further comprises the following step: (iv) determining whether the patient is being treated with omeprazole; and wherein step (v) further comprises determining the effective dose of clozapine based on step (iv). Suitably, determining the effective dose of clozapine based on step (iv) further comprises: reducing the overall activity score where the patient is being treated with omeprazole; optionally wherein the overall activity score is reduced by an equivalent numerical value to that of the third score.

[0044] In some embodiments, the method further comprises determining the presence or absence in the genomic DNA of:

[0045] HLA-B alleles selected from B*38:01:01, HLA-B 158T and B*59:01:01:01;

[0046] HLA-DQB1 alleles selected from 6672G>C, 126Q and *05:02; and / or the SLCO1B3 allele g.21083862C>T (rsl49104283-T); and wherein the presence or absence of the HLA-B, HLA-DQB1 and / or SLCO1B3 alleles is used to determine the risk of agranulocytosis in the patient, optionally wherein the method further comprises modifying the clozapine treatment or the monitoring of the patient based on the risk of agranulocytosis.

[0047] In some embodiments, the method further comprises determining the presence or absence in the genomic DNA of: the ACKR1 (DARC) allele c.-67T>C (rs2814778-C); and wherein the presence or absence of the CC (Duffy-null) genotype is used to determine the risk of benign ethnic neutropenia (BEN) in the patient, and wherein the method further comprises modifying the clozapine treatment or the monitoring of the patient based on the risk of BEN.

[0048] In some embodiments, the mental disorder comprises one or more of: schizophrenia, paranoid schizophrenia, schizoaffective disorder, a personality disorder, EUPD, mixed and other personality disorder, persistent delusional disorder, refractive schizophrenia, a psychotic disorder, a delusional disorder, schizophreniform disorder, or psychosis in Parkinson’s disease.

[0049] In some embodiments, the basal dose is 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg, preferably 75 mg.

[0050] In some embodiments, the effective dose of clozapine is that required to give a plasma concentration of clozapine of 0.35 ng / ml 12 hours post-administration.

[0051] In some embodiments, the method comprises the steps of:

[0052] (i) obtaining a biological sample from the patient, wherein the sample comprises genomic DNA of the patient;

[0053] (ii) determining the presence in the genomic DNA of:

[0054] CYP1A2 alleles selected from * 1A, * 1C and * IF;

[0055] CYP3A4 alleles selected from * 1, * 1B, *3, * 10, and *22;

[0056] CYP3A5 alleles selected from * 1, *3, *6 and *7; and NFIB rs28379954 alleles selected from C or T;

[0057] (iii) determining whether the patient smokes tobacco;

[0058] (iv) determining whether the patient is being treated with omeprazole; and

[0059] (v) determining the effective dose of clozapine based on steps (ii), (iii) and (iv); wherein determining the effective dose of clozapine at step (v) comprises:

[0060] - assigning a score to the CYP1A2 alleles present in the genomic DNA, wherein: each CYP1A2 allele * 1A is assigned a score of 1.0; each CYP1A2 allele * IF is assigned a score of 1.0 if the patient does not smoke tobacco; each CYP1A2 allele * IF is assigned a score of 2.0 if the patient smokes tobacco; and each CYP1A2 allele * 1C is assigned a score of 0.5;

[0061] - assigning a score to the CYP3A4 alleles present in the genomic DNA, wherein: each CYP3A4 allele * 1, * IB, *3 or * 10 is assigned an activity score of 1.0; and each CYP3A4 allele *22 is assigned an activity score of 0.5;

[0062] - assigning a score to the CYP3A5 alleles present in the genomic DNA, wherein: each CYP3A5 allele * 1 is assigned an activity score of 1.0; and each CYP3A5 allele *3, *6 or *7 is assigned an activity score of 0;

[0063] - summing the score for each CYP1A2 allele, each CYP3A4 allele and each CYP3A5 allele to obtain an overall activity score;

[0064] - increasing the overall activity score by 0.5 if the NFIB C allele is present in the genomic DNA;

[0065] - reducing the overall activity score by 0.5 if the patient is being treated with omeprazole;

[0066] - multiplying the overall activity score by the basal dose, optionally wherein the basal dose is 75 mg, to obtain the effective dose of clozapine.

[0067] Detailed Description of the Invention

[0068] Clozapine treatment

[0069] Clozapine is the most effective antipsychotic. In the UK and in many other parts of the world, clozapine is licenced to treat schizophrenia in patients aged 18 years or over, recurrent suicidal behaviour associated with schizophrenia or schizoaffective disorder and for the treatment of psychotic disorders occurring during the course of Parkinson's disease. Clozapine remains the only medication with proven efficacy in and licenced to treat schizophrenia that does not respond to other antipsychotics (treatment refractory schizophrenia; treatment resistant schizophrenia; TRS). Clozapine is therefore usually used when there has been failure of two previous antipsychotic drugs. Clozapine is also indicated in patients with schizophrenia who show severe, untreatable adverse neurological reactions to other antipsychotics, including second-generation antipsychotics.

[0070] Off-label uses of clozapine include the treatment of schizophrenia in patients younger than 18 years, bipolar disorder, depressive disorders, borderline personality disorder, substance misuse disorders, suicidality, aggression, tardive dyskinesia, and tardive dystonia. Despite the benefits of clozapine treatment, it remains underutilized in part due to concerns regarding adverse effects. The underuse of clozapine has been recognised for more than 25 years and is detailed in numerous publications, see for example Howes et al., 2012.

[0071] Adverse effects of clozapine treatment

[0072] Rare but potentially serious haematological side effects are associated with clozapine use. There is a recognized risk of blood dyscrasias especially in the first 3 months of clozapine treatment, including neutropenia, which may progress to agranulocytosis. This means that patients taking clozapine must be closely monitored by haematological monitoring. Clozapine use is limited to patients with initially normal white blood cell counts (white blood cell count > 3,500 / mm3(3.5xlO9 / L), and absolute neutrophil count > 2,000 / mm3(2.0xl09 / L). In some non-Caucasian patients, benign ethnic neutropenia (BEN) occurs wherein the patient’s neutrophil count is naturally persistently low (below 1,500 / mm3) without an accompanying increase in the incidence of infections. Clozapine may still be dispensed to such individuals, often in consultation with a haematologist.

[0073] In the U.K., a full blood count must be performed before clozapine treatment is initiated, and then weekly for the first 18 weeks of treatment, fortnightly for the next 34 weeks, and monthly thereafter. Continued dispensing of clozapine is conditional upon satisfactory haematological results.

[0074] The risk of metabolic syndrome, common to almost all new antipsychotics, can be attenuated with strategies on diet and physical activity in patients. Clozapine use is also associated with an increased risk of myocarditis. Its anticholinergic effect profile is associated with the development of constipation, in some cases severe (ileus) especially if associated with other anticholinergic treatments. Clozapine also has a dramatic hypotensive effect when first initiated. Further information on side effects may be obtained from a number of sources including https: / / bnf.nice.org.Uk / drugs / clozapine / #side-effects.

[0075] Due to the adverse effects associated with clozapine treatment, it is advisable to titrate the dose upwards over the first 2-4 weeks of treatment, whilst monitoring the patient’s mental and physical health (e.g. via temperature, pulse, blood pressure, blood lipids, fasting blood glucose and liver function). This slow titration must be balanced against the need to improve the patient’s mental health symptoms by reaching an effective dose as quickly as possible.

[0076] The effective dose

[0077] Dosing with clozapine is non-trivial; dose escalation / titration is typically employed, often with concomitant blood sampling to determine actual plasma levels (also known as therapeutic drug monitoring (TDM)). The British National Formulary (BNF) recommendation for clozapine dose escalation in adults betweenl8 and 59 years of age is the following: “12.5 mg 1 2 times a day for day 1, then 25- -50 mg for day 2, then increased, if tolerated, in steps of 25- -50 mg daily, dose to he increased gradually over 14 21 days, increased to up to 300 mg daily in divided doses, larger dose to he taken at night, up to 200 mg daily may he taken as a single dose at bedtime; increased in steps of 50-100 mg 1- 2 times a week if required, it is preferable to increase once a week; usual dose 200-450 mg daily, max. 900 mg per day, if restarting after interval of more than 48 hours, 12.5 mg once or twice on first day (but may be feasible to increase more quickly than on initiation) — extreme caution if previous respiratory or cardiac arrest with initial dosing. ” (https: / / bnf.nice.org.uk / drugs / clozapine).

[0078] The actual dose that is effective to treat the patient varies widely due to a variety of factors such as biological sex, weight, whether or not the patient smokes and so on. As disclosed herein, the genotype of the patient is also an important determinant of the effective dose of clozapine.

[0079] As defined herein, the effective dose is that dose which provides a plasma concentration of clozapine of the desired level at a predetermined time point after administration. The actual desired level is determined by clinical need. The effective dose is the minimum dose predicted to result in significant therapeutic benefit for the patient, thereby balancing symptom improvement with the potential for development of side / adverse effects. For example, in some embodiments the desired plasma level is a selected level between 0.25 to 0.5 ng / ml. Typically, the desired level is approximately equal to, or is 0.35ng / ml. In some embodiments the predetermined time point is 12hrs after administration, although any suitable time point may be chosen, for example based on clinical need and / or practical considerations

[0080] Whilst clozapine may be used in an injectable form, the present disclosure preferably provides methods for determining the effective dose when the drug is administered orally for example as a tablet, an orally disintegrating tablet or an oral suspension.

[0081] Pharmacogenetics

[0082] A number of Cytochrome P450 enzymes are understood to have a role in clozapine pharmacokinetics.

[0083] CYP1A2 (Cytochrome P450 1A2): the enzyme largely responsible for clozapine metabolism. Caution when prescribing clozapine is recommended in patients with functional impairment of the metabolic pathway. Certain alleles of the enzyme are highly inducible by the action of tobacco, drugs (e.g. omeprazole); several genotypes have been identified with different inducing capacities. Alleles include: CYP1A2* 1A (wild-type, normal activity), CYP1A2* 1C (low activity), CYP1A2* 1K (low activity), CYP1A2* IF (highly inducible, high activity). CYP3A4 (Cytochrome P450 3A4): Clozapine main metabolism enzyme (together with CYP1A2). Caution when prescribing clozapine is recommended in patients with functional impairment of the CYP3A4 metabolic pathway. Alleles include: CYP3A4* 1A (wild-type), CYP3A4* 1B, CYP3A4*3, CYP3A4* 10, *22CYP3A4*20 (contains a premature stop codon resulting in complete loss of function), and CYP3A4*22 (low activity). Over 90% of individuals are carriers of two alleles with full function and are considered normal metabolizers.

[0084] CYP3A5 (Cytochrome P450 3A5): Minor clozapine metabolism enzyme. Poor CYP3A5 metabolizers do not express functional CYP3A5 and do not metabolize the drug through this pathway. Over 85% of the general population are considered poor metabolizers of CYP3 A5. CYP3 A5 poor metabolizers have therapeutic plasma concentrations of clozapine in the absence of other clinical or genetic factors. Alleles include CYP3A5* 1 (normal metabolism), CYP3A5*3 (non-functional allele, poor metabolism), CYP3A5*6 (non-functional allele, poor metabolism), and CYP3A5*7 (non-functional allele, poor metabolism).

[0085] CYP2D6 (Cytochrome P450 2D6): the DPWG (Royal Dutch Association for the Advancement of Pharmacy - Pharmacogenetics Working Group, 2018) clinical practice guideline considers CYP2D6 genotype to have no clinical relevance to clozapine pharmacokinetics. However, the FDA pharmacogenetic biomarker table indicates that a clozapine dose adjustment may be necessary in patients carrying variants associated with alterations in function. Can be considered a minor metabolism route for clozapine. A large number of different alleles are known, the genotype accounts for normal, reduced, and non-existent CYP2D6 function in subjects.

[0086] CYP2C19 (Cytochrome P450 2C19): FDA and AEMPS drug labels attribute a secondary role in clozapine metabolism. Alleles include: CYP2C19* 1 (wild-type), CYP2C19*2 (non-functional allele, poor metabolism), CYP2C19*3 (non-functional allele, poor metabolism), and CYP2C19* 17 (increased activity).

[0087] In addition, the transcription factor NFIB (Nuclear factor 1 B-type; NF1B) genotype of an individual may also have a bearing on their clozapine metabolism, possibly by affecting the transcription of genes involved in clozapine metabolism. The NFIB rs28379954 C allele has been associated with reduced clozapine plasma concentration (Smith et al., 2020). Non-carriers of the NFIB rs28379954 C allele (e.g. carriers of the T allele) do not have an increased risk of sub-therapeutic clozapine serum concentrations in the absence of other clinical or genetic factors.

[0088] The Sample

[0089] Any biological sample from the patient which comprises genomic DNA may be usable. Conveniently, samples include buccal swab samples, saliva samples and blood samples. Determining the presence of alleles in the genomic DNA

[0090] The presence within the genomic DNA of the alleles of interest (i.e. those which are found to affect clozapine metabolism or plasma concentration) can be conveniently determined in a number of ways well known in the art. The most convenient approach is the use of DNA sequencing. This is due to its accuracy and its applicability to determining of the presence of multiple portions of DNA in a parallel fashion. Preferred approaches include (but are not limited to) the so-called “next generation” sequencing technologies. A variety of so-called next generation sequencing approaches are available including, without limitation, the Illumina HiSeq™ or MiSeq™ platforms, the Roche 454 platform and the Life Technologies ion proton system.

[0091] Typically DNA fragments are generated and used to provide sequencing libraries by clonal amplification. In some embodiments fragments may be enriched for genomic regions of interest by employing capture protocols, typically using immobilized capture oligos, before library preparation. In some approaches libraries of captured fragments are arrayed. The DNA is then typically sequenced by synthesis, such that the DNA sequence is determined by the addition of nucleotides to the complementary strand rather than through chain-termination chemistry. The arrayed, amplified DNA templates are sequenced simultaneously in a massively parallel fashion without the requirement for a physical separation step. These steps are followed in most NGS platforms, but each utilizes a different strategy.

[0092] Allelic targets

[0093] In the present approach, determination of the appropriate dosing for the patient includes determining the presence of specific alleles of at least one, at least two or all three of the following cytochrome P450 enzyme genes: CYP1A2, CYP3A4 and CYP3A5; and optionally the Nuclear Factor IB (NFIB) gene. In one embodiment, the at least one cytochrome P450 is CYP1A2.

[0094] Further embodiments may comprise determining the presence of specific alleles of other genes known or suspected to be involved in clozapine metabolism or to affect its plasma concentration directly or indirectly, such as CYP2D6 or CYP2C19. An alternative approach would be to determine the presence of alleles of genes known to be in strong linkage disequilibrium (LD) with such alleles.

[0095] Genomic regions which may include any one or more of the coding exons, intronic boundaries, deep intronic regions and untranslated regions (UTR) of the genes are sequenced and the presence or absence of any one or more of the following alleles is identified:

[0096] CYP1A2 alleles are selected from * 1A, * IC and * IF; CYP3A4 alleles are selected from * 1, * 1B, *3, * 10, and *22; CYP3A5 alleles are selected from * 1, *3, *6 and *7. Typically however, the presence (or absence) of each allele of each of these P450s is determined. Within the NFIB gene, the rs28379954 polymorphism is identified. Particularly the presence of the T>C polymorphism. Further determinations

[0097] In some embodiments, additional information may be used to modulate the dose, including: (i) the smoking status of the subject / patient, i.e. whether or not the subject is a smoker; (ii) the biological sex of the subject, and (iii) whether or not the subject is also being treated with omeprazole.

[0098] A determination of the effective dose of clozapine can be made or adjusted by a method that includes determining the presence or absence of CYP1A2 alleles selected from * 1A, * 1C and * 1F; CYP3A4 alleles selected from * 1, * 1B, *3, * 10, and *22; CYP3A5 alleles selected from * 1, *3, *6 and *7; and NFIB (rs28379954) alleles selected from C or T;

[0099] This determination maybe adjusted based on the smoking status of the patient and may also be adjusted based on their biological sex. For example, a dose may be higher in patients who smoke, particularly those who possess the * IF allele of CYP1A2. Smokers who are homozygous for the CYP1A2* IF allele have increased clozapine metabolism and may require significantly higher doses to reach therapeutic plasma concentrations. This is due to tobacco smoke producing polyaromatic hydrocarbons which induce CYP1A2. The dose may also be higher in male patients as opposed to female patients.

[0100] CYP1A2 inducibility method

[0101] In one embodiment, the determination of the effective dose may be made by a method comprising the following steps

[0102] (a) determining the biological sex of the subject,

[0103] (b) determining the CYP1A2 inducibility status of the subject according to the presence of CYP1A2* 1A and * IF alleles;

[0104] (c) determining the CYP1A2, CYP3A4 and CYP3A5 metabolizer status of the subject according to the presence of particular alleles

[0105] (d) determining a provisional dose with which to treat the subject according to biological sex and smoking status; and

[0106] (e) applying an adjustment to the provisional dose based on:

[0107] CYP1A2 inducibility status of the subject;

[0108] CYP1A2, CYP3A4 and CYP3A5 metabolizer status of the subject and the presence or absence of the NFIB rs28379954 C>T polymorphism.

[0109] CYP1A2 inducibility

[0110] The CYP1A inducibility status of the subject is selected from highly inducible, moderately inducible and minorly inducible based on the presence of the * 1A and / or the * 1F alleles according to Table 1 below: Table 1

[0111] The CYP1A2 metabolizer status is determined to be normal if the subject is homozygous for the CYP1A2* 1A allele, and either intermediate or poor respectively depending on whether the subject / patient is a carrier of one or two reduced activity alleles, for example CYP1A2* 1C.

[0112] The CYP3A4 metabolizer status is determined to be normal if the subject is homozygous for the CYP3A4* 1A allele, and intermediate or poor respectively depending on whether they are a carrier of one or two reduced activity alleles, for example CYP3A4*22.

[0113] The CYP3A5 metabolizer status is determined to be normal if the subject is homozygous for the CYP3A5* 1A allele, and intermediate or poor respectively depending on whether they are a carrier of one or two reduced activity alleles, for example CYP3A5*3.

[0114] Provisional dose

[0115] In one embodiment, the provisional dose is 275mg for a female non-smoker and 325 mg for a male non-smoker.

[0116] Adjustments

[0117] A first adjustment may be made to the provisional dose according to the CYP1A2 genotype of the patient in combination with the smoking status of the patient; and / or

[0118] A second adjustment may be made to the provisional dose according to CYP3A4 genotype; and / or A third adjustment may be made to the provisional dose according to the CYP3A5 genotype; and / or A fourth adjustment may be made to the provisional dose according to the NFIB rs28379954 genotype.

[0119] CYP1A2

[0120] In one embodiment the first adjustment to the provisional dose according to the CYP1A2 genotype is calculated as follows: if the patient has CYP1A2 intermediate or poor metaboliser genotype then a negative adjustment is made to the provisional dose in non-smoking males; and a negative adjustment is made in both smoking and non-smoking females. If the patient does not carry a CYP1A2 intermediate or poor metaboliser genotype then a positive adjustment may be made to the provisional dose in male smokers who are CYP1A2 * IF / * IF,

[0121] * IF / * 1A or * 1A / * 1A genotypes; and a positive adjustment may be made to the provisional dose in female smokers who are CYP1A2 * IF / * IF or * IF / * 1A genotypes.

[0122] In one embodiment, if the patient has a CYP1A2 intermediate or poor metaboliser genotype then, in non-smoking males the provisional dose is reduced by 25mg and is unchanged in smokers; and in females the provisional dose is reduced by 25mg in both smokers and non-smokers.

[0123] If the patient does not carry a CYP1A2 intermediate or poor metaboliser genotype then: for a female subject, 200 mg clozapine is added to the provisional dose in CYP1A2 highly inducible subjects, 100 mg in moderately inducible subjects, and zero mg in minorly inducible subjects.

[0124] For a male subject, 250 mg is added in CYP1A2 highly inducible subjects, 150 mg in moderately inducible subjects, and 50 mg in minorly inducible subjects.

[0125] Table 2 below summarises one approach of the CYP1A inducibility model in which the provisional dose for female subjects is 275mg and the provisional dose for male subjects in 325mg.

[0126] Table 2

[0127] These doses may be adjusted according to CYP3A4, CYP3A5 genotypes and NFIB rs28379954 variant status as follows:

[0128] • Patients classified as intermediate or poor CYP3A4 metabolisers reduce the dose by 25 mg,

[0129] • Patients classified as intermediate or normal CYP3A5 metabolisers increase the dose by 25 mg,

[0130] • Patients classified as NFIB rs28379954 T>C carriers increase the dose by 50 mg

[0131] Gene Variant Activity Score method

[0132] In a further embodiment, the determination of the effective dose may be made by a method comprising the following steps

[0133] (a) determining a basal dose; and

[0134] (b) applying an adjustment to the basal dose to obtain the effective dose of clozapine; wherein the adjustment is derived from the presence of CYP1A2, CYP3A4 and CYP3A5 alleles, the smoking status of the subject, and the presence of the NFIB rs28379954 T>C polymorphism in the genomic DNA of the subject.

[0135] The basal dose

[0136] In the (gene variant) activity score method disclosed herein the basal dose is the dose that is adjusted by the adjustment to provide the desired plasma level of clozapine at a predetermined time-point after administration. The desired plasma level of clozapine is described herein.

[0137] Adjusting the basal dose according to the CYP genotype

[0138] In some embodiments, applying an adjustment to the basal dose may include applying a scoring system in which an individual activity score is assigned to each of the CYP alleles relevant to clozapine metabolism determined to be present within the genomic DNA, particularly a scoring system in which an individual activity score is assigned to each of the CYP1A2, CYP3A4 and CYP3A5 alleles determined to be present within the genomic DNA; deriving an overall activity score from the individual activity scores; and adjusting the basal dose according to the overall activity score.

[0139] An overall activity score may be derived by (i) summing the individual activity scores of each of the CYP1A2, CYP3A4 and CYP3A5 alleles; (ii) increasing the activity score assigned to the CYP1A2* IF allele if the patient is a smoker; and (iii) increasing the overall activity score if the NFIB T>C allele is present in the genomic DNA.

[0140] In one embodiment the overall activity score can be derived by summing the (activity) score for each CYP1A2, CYP3A4 and CYP3A5 allele present to obtain the overall activity score. In some embodiments CYP3A5 *3, *6 and *7 may be ignored for the purposes of scoring. In some embodiments they may be given a null score. In some embodiments the CYP1A2 * 1F allele may be assigned a higher score if the patient is a smoker.

[0141] CYP1A2* IF may be considered a Rank 1 allele; CYP1A2* 1A, CYP3A4* 1, CYP3A4* IB, CYP3A4*3, CYP3A4* 10 and CYP3A5* 1 may be considered as Rank 2 alleles; CYP1A* 1C and CY3A4*22 may be considered Rank 3 alleles; and CYP3A5*3, CYP3A5*6 and CYP3A5*7 may be considered Rank 4 alleles.

[0142] In an embodiment where the patient / subject does not smoke, equal weighting (or approximately equal weighting) in the scoring system is given to each Rank 1 and Rank 2 allele, whilst a lower score is attached to Rank 3 and Rank 4 alleles. In one embodiment the score attached to Rank 3 alleles is lower than that attached to Rank 1 and Rank 2 alleles, for example it may be half that or approximately half that applied to Rank 1 and Rank 2 alleles. In some embodiments the Rank 4 alleles are assigned a lower score than the Rank 3 alleles and particularly may be assigned a null score (zero). In particular, Rank 1 and Rank 2 alleles may be assigned a score of 1, Rank 3 alleles may be assigned a score of 0.5, and Rank 4 alleles may be assigned a null score (0).

[0143] Table 3 below gives one possible scoring system for use when the patient does not smoke.

[0144] Table 3

[0145] The activity score of a genotype is the sum of the (activity) score assigned to each allele of one gene (e.g., CYP3A5* l / *3 and CYP3A5*3 / *3 genotypes have activity scores of 1.0 and 0 respectively).

[0146] Smoking

[0147] In an embodiment where the patient / subject smokes, a Rank 1 allele (for example CYP1A2* 1F) may be assigned a score which is higher than the Rank 2 alleles, for example the score may be twice or approximately twice the score of the Rank 2 alleles. This is due to the higher inducibility of the CYP1A2* IF allele in smokers. The score attached to Rank 3 alleles may be lower than that attached to Rank 2 alleles, for example it may be half that or approximately half that applied to Rank 2 alleles. In some embodiments the Rank 4 alleles are assigned a lower score than the Rank 3 alleles and particularly may be assigned a null score (zero). In particular, Rank 1 alleles may be assigned a score of 2, Rank 2 alleles may be assigned a score of 1, Rank 3 alleles may be assigned as score of 0.5, and Rank 4 alleles may be assigned a null score (0).

[0148] Table 4 below gives one possible scoring system for use when the patient / subject smokes.

[0149] Table 4 In the exemplary scoring system presented in Table 4 for a patient who smokes, an individual whose genotype is CYP1A2 * 1A / * 1F, CYP3A4 * l / *3, CYP3A5 * l / *3, NFIB would have an overall activity score of 6.0

[0150] Adjusting the basal dose according to the NFIB rs28379954 genotype

[0151] In some embodiments, applying an adjustment to the basal dose may include adjusting the overall activity score dependent on which NFIB rs28379954 alleles are determined to be present within the genomic DNA. NFIB rs28379954 alleles include T and C. Suitably, if one or more C allele is determined to be present within the genomic DNA, then the overall activity score may be increased. The extent to which the overall activity score is increased if one or more NFIB rs28379954 C allele is determined to be present within the genomic DNA may be similar to that of a Rank 3 allele of a CYP gene, particularly the activity score may be increased by 0.5. The NFIB rs28379954 adjustment may be applied to the overall activity score regardless of whether the subject / patient smokes.

[0152] Determining the effective dose of clozapine

[0153] Once the overall activity score has been obtained, the effective clozapine dose in (mg) to obtain the desired plasma level is determined by multiplying the basal dose by the overall activity score. The basal dose may be, for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg, preferably 75 mg. The basal dose may be between 65 and 85 mg, preferably between 70 and 80 mg, most preferably 75 mg. In one embodiment the basal dose is approximately equal to or is 75 mg.

[0154] Therefore, for the example individual given above who smokes and whose genotype is CYP1A2

[0155] * 1A / * IF, CYP3A4 * l / *3, CYP3A5 * l / *3, using the gene variant activity score method with a basal dose of 75 mg would lead to a prediction of an effective clozapine dose of 450 mg.

[0156] Omeprazole Correction

[0157] In a further embodiment, the activity score method may be augmented by incorporating an adjustment in the case that the patient is also treated with omeprazole. In some embodiments, in the case that a patient is also treated with omeprazole the overall activity score is reduced. In one embodiment where the patient is also treated with omeprazole the overall activity score is reduced by a score equal to that of a third rank allele. For example, the overall activity score is reduced by 0.5 where a patient is also treated with omeprazole. Enzyme Inhibitors

[0158] In some embodiments, the presence of particular CYP alleles in the genomic DNA can be used in the selection of enzyme inhibitors with which the patient may be additionally treated. For example, if the presence of CYP3A5 alleles * 1 and *3 is determined in the genomic DNA, the patient may be further treated with cimetidine (see Okon-Rocha et al., 2022).

[0159] Figures

[0160] Figures 1 and 2. show a Passing Bablok plot and a Bland Altman plot, respectively, for the doses derived using the Rostami-Hodjegan method i.e. the prior art dose prediction method. Mean difference was - 49.9mg / day ((SD 155.9), r = 0.36) from the actual dose required to give a plasma concentration of 0.35ng / ml.

[0161] Figures 3 and 4 show a Passing Bablok plot and a Bland Altman plot, respectively, for the doses derived using the gene variant activity score dose prediction method. The mean difference was -43.5mg / day ((SD 140.1), r=0.55) from the actual dose required to give a plasma concentration of 0.35ng / ml.

[0162] Figures 5 and 6 show a Passing Bablok plot and a Bland Altman plot, respectively, for doses derived using the gene variant activity score with omeprazole correction dose prediction method. The mean difference was - 31.0mg / day ((SD 140.8), r=0.54) from the actual dose required to give a plasma concentration of 0.35ng / ml.

[0163] Figures 7 and 8 show a Passing Bablok plot and a Bland Altman plot, respectively, for doses derived using the gene variant CYP1A2 inducibility predicted dose. The mean difference was -44.9mg / day ((SD 160.8), r = 0.32) from the actual dose required to give a plasma concentration of 0.35ng / ml.

[0164] Figure 9 shows anonymised patient data listing primary and secondary diagnosis, age, biological sex and smoking status as well as list of present medications.

[0165] Figure 10 shows clozapine dosage data for each of 18 patients.

[0166] Figure 11 shows an activity score for each allele identified in screening, using the present approach.

[0167] Figure 12 shows doses based on sex, smoking status and CYP1A2 inducibility.

[0168] Experimental Examples

[0169] To facilitate a better understanding of the embodiments of the present invention, the following examples of preferred or representative embodiments are given. In no way should the following examples be read to limit, or to define, the scope of the invention. Data were obtained from patients at St Andrews Hospital, Northampton, UK. All participants were prescribed clozapine as part of normal clinical practice. Blood sampling for plasma level determination was also undertaken according to clinical requirements. The method of clozapine assay was LCMS- MS. Patients receiving clozapine for any indication who had been stable on the same clozapine dose for at least two months were included. All participants were hospital in-patients whose clozapine intake was supervised. Compliance was not in doubt.

[0170] No patient’s treatment was altered in any way for the purposes of this investigation. Verbal patient consent was given by each of the participants. All data were collected by clinicians normally involved in the care of participants and transferred to a central computer where patients’ details were made anonymous. This study was approved by the institution’s Research Peer Review Committee and ethics approval was provided by the Reading Independent Ethics Committee.

[0171] Participant clozapine dose and concentration determination

[0172] For each patient, the daily dose and the date and time of the last blood sample taken for analysis of clozapine concentration were recorded. Each dose was adjusted according to the measured plasma concentration and the time of sampling to give a dose that would provide a 12-hour concentration of 0.35ng / ml.

[0173] Dose adjusted for plasma concentration = (0.35 / patient clozapine concentration) x patient’s actual dose (=A).

[0174] Dose adjusted for time of sample = A x 0.9 for 10-11 hours post dose; A x 1 for 11.1-12.9 hours;

[0175] A x 1.1 for 13-14 hours.

[0176] Thus for a patient on 300 mg clozapine a day, showing a plasma clozapine concentration of 0.7 ng / ml in a sample taken 14 hours post dose, the adjusted dose = (0.35 / 0.7)xl. l = 0.55x300 = 165 mg / day. This adjusted dose is the dose needed in that individual to give a plasma clozapine concentration of 0.35 ng / ml in a 12-hour sample.

[0177] Genetic testing

[0178] Coding exons and intronic boundaries, deep intronic regions and untranslated regions (UTR) of CYP1A2, CYP3A4, CYP3A5 genes and NFIB rs28379954 were captured using a custom probe library (SureSelect Target Enrichment Kit for Illumina paired-end multiplexed sequencing method, Agilent Technologies, Santa Clara, California) and sequenced using the HiSeq 1500 platform (Illumina, San Diego, California), following Illumina protocols. The read depth (the number of times that a base was sequenced by independent reads) of every nucleotide belonging to a defined genomic region of interest was >30* (mean 296*). Bioinformatic analysis

[0179] Sequence data analysis was performed using a proprietary bioinformatics pipeline that includes sample demultiplexing as well as all the steps necessary to obtain a report of annotated variants together with their coverage and corresponding quality parameters. CYP1A2, CYP3A4, CYP3A5 alleles were assigned according to the Pharmacogene Variation Consortium (Gaedigk et al., 2021).

[0180] Rostami-Hodjegan method (prior art approach)

[0181] A clozapine dose was provided by estimating the dose needed to give a 12-hour post-dose plasma clozapine concentration of 0.35ng / ml for each participant using the published charts for male and female smokers and non-smokers according to Rostami-Hodjegan et al., 2004.

[0182] Genetic variant-based clozapine dose

[0183] We estimated the clozapine dose needed to give a 12-hour post-dose plasma clozapine concentration of 0.35 ng / ml for each participant using three approaches based on genetic variants for cytochromes CYP1A2, CYP3A4, CYP3A5 and for NFIB rs28379954 variants.

[0184] The three approaches were as follows:

[0185] (i) Activity Score method (without correction for concomitant treatments)

[0186] Clozapine dose (mg)=75 x(AScYPiA2+AScYP3A)

[0187] (ii) Activity Score method with omeprazole correction

[0188] Clozapine dose (mg) =75 X(ASCYPIA2+ ASCYP3A-O) where ASCYP3A-O is the activity score adjusted for omeprazole (see above)

[0189] (Hi) CYP1A2 inducibility model

[0190] Scoring system

[0191] For approaches (i) and (ii) activity scores for CYP3A4, CYP3A5 and CYP1A2 were calculated as follows:

[0192] For CYP1A2 an activity score of 1.0 was assigned to the * 1A (wild-type) and the *1F (higher inducibility) alleles. In smoking patients, an activity score of 2.0 was assigned for the * IF allele instead.

[0193] In carriers of the NFIB rs28379954 T>C allele, the CYP1A2 activity score was increased by 0.5 regardless of the patient's smoking status. An aggregate CYP3A activity score was calculated as the sum of CYP3A4 and CYP3A5 activity. In patients receiving concomitant treatment with omeprazole the CYP3A activity score was reduced by 0.5 points.

[0194] Table 5 shows CYP1A2, CYP3A4 and CYP3A5 identified alleles and associated activity scores. Table 5

[0195] The activity score of a genotype is the sum of the score assigned to each allele (e.g., CYP3A5*l / *3 and CYP3A5*3 / *3 genotypes have activity scores of 1.0 and 0 respectively).aOnly observed alleles are listed.bActivity score 2.0 in smokers.

[0196] (Hi) CYP1A2 inducibility model

[0197] This model assigns a dose according to CYP1A2 inducibility, patient biological sex and smoking status.

[0198] Table 6 shows CYP1A2 genotypes grouped by inducibility level.

[0199] Table 7 shows doses determined using the CYP1A2 inducibility model method.

[0200] Table 7 These doses are adjusted according to CYP3A4, CYP3A5 genotypes and NFIB rs28379954 variant carrier status as follows:

[0201] • Patients classified as intermediate or poor CYP3A4 metabolisers reduce the dose by 25 mg,

[0202] • Patients classified as intermediate or normal CYP3A5 metabolisers increase the dose by 25 mg,

[0203] • Patients classified as NFIB rs28379954 carriers increase the dose by 50 mg

[0204] Data analysis

[0205] Dose estimates obtained with the different approaches were compared with the dose adjusted for plasma concentration and time of sample using the Passing-Bablok regression and Bland-Altman plot methods. Calculations were performed with R software version 4. 1.3 (R Foundation for Statistical Computing, Vienna, Austria).

[0206] Results

[0207] In total, 18 patients met the inclusion criteria for the study. Clinical details are given in Figure 9 and dose, dose adjustments and predicted dose (by the four disclosed methods) are given in Figure 10.

[0208] For the prior art algorithm-predicted dose (Rostami-Hodjegan et al., 2004) the mean difference was - 49.9mg / day ((SD 155.9), r = 0.36) from the actual dose required to give a plasma concentration of 0.35ng / ml. See Figures 1 and 2.

[0209] For the gene variant activity score predicted dose (method (i)) the mean difference was -43.5mg / day ((SD 140.1), r=0.55) from the actual dose required to give a plasma concentration of 0.35ng / ml. See Figures 3 and 4.

[0210] For the gene variant activity score with omeprazole correction predicted dose (method (ii)) the mean difference was -31.0 mg / day ((SD 140.8), r=0.54) from the actual dose required to give a plasma concentration of 0.35ng / ml, See Figures 5 and 6.

[0211] For the gene variant CYP1A2 inducibility predicted dose (method (iii)) the mean difference was - 44.9mg / day ((SD 160.8), r = 0.32) from the actual dose required to give a plasma concentration of 0.35ng / ml. See Figures 7 and 8.

[0212] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0213] All documents mentioned in this specification are incorporated herein by reference in their entirety. “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0214] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular embodiment and apply equally to all embodiments which are described. The embodiments disclosed herein suitably may be practiced in the absence of any element that is not specifically disclosed herein and / or any optional element disclosed herein.

[0215] It will further be appreciated by those skilled in the art that although the invention is described by way of example with reference to several embodiments, it is not limited to the disclosed embodiments and that alternative embodiments could be constructed without departing from the scope of the invention as defined in the appended claims.

[0216] Also, the terms in the claims have their plain, ordinary meaning unless otherwise explicitly and clearly defined by the patentee. Moreover, the indefinite articles “a” or “an,” as used in the claims, are defined herein to mean one or more than one of the element that it introduces.

[0217] Unless otherwise indicated, all numbers expressing quantities of, for example, patient variables, dose, blood plasma concentration, time, and so forth, used in the present specification and associated claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the embodiments of the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Uikewise, the term "about" in relation to a numerical value x is optional and means, for example, x±10%.

[0218] One or more illustrative embodiments are presented herein. Not all features of the implementation are described or shown in this application for the sake of clarity. It is understood that in the development of an implementation incorporating the embodiments disclosed, numerous implementation-specific decisions must be made to achieve the developer's goals, such as compliance with system-related, business-related, government-related and other constraints, which vary by implementation and from time to time. Whilst a developer's efforts might be time-consuming, such efforts would be, nevertheless, a routine undertaking forthose of ordinary skill the art and having benefit of this disclosure.

[0219] Whilst methods are described herein in terms of “comprising” various components or steps, the methods can also “consist essentially of’ or “consist of’ the various components and steps. Methods disclosed herein may be performed in a step-wise manner. It will be understood by the person skilled in the art that a number of steps of a method may be performed in any practical order. Alternatively, two or more steps may be conducted contemporaneously. Unless specifically stated, a process or method comprising steps may be performed in any suitable order. Thus steps can be performed in any appropriate order, including contemporaneously.

[0220] References

[0221] Gaedigk A, Casey ST, Whirl-Carrillo M, et al. Pharmacogene Variation Consortium: A Global Resource and Repository for Pharmacogene Variation. Clin Pharmacol Ther. Sep 2021 ; 110(3) :542- 545.

[0222] Howes OD, Vergunst F, Gee S, McGuire P, Kapur S, Taylor D. Adherence to treatment guidelines in clinical practice: study of antipsychotic treatment prior to clozapine initiation. Br J Psychiatry. 2012 Dec;201(6):481-5.

[0223] Okon-Rocha E, Ramudo-Cela L, Taylor D. Genetic Analysis of Clozapine Metabolism in a Patient With Subtherapeutic Clozapine Plasma Concentrations-The Importance of CYP3 A5 : A Case Report. J Clin Psychopharmacol. 2022 Nov-Dec 01;42(6): 604-606.

[0224] Rostami-Hodjegan A, Amin AM, Spencer EP, et al. Influence of dose, cigarette smoking, age, sex, and metabolic activity on plasma clozapine concentrations: a predictive model and nomograms to aid clozapine dose adjustment and to assess compliance in individual patients. J Clin Psychopharmacol. Feb 2004;24(l):70-78.

[0225] Smith RL, O'Connell K, Athanasiu L, Djurovic S, Kringen MK, Andreassen OA, Molden E. Identification of a novel polymorphism associated with reduced clozapine concentration in schizophrenia patients-a genome-wide association study adjusting for smoking habits. Transl Psychiatry. 2020 Jun 19; 10(1): 198. doi: 10.1038 / s41398-020-00888-l . Erratum in: Transl Psychiatry. 2020 Nov 2;I0(I):366.

Claims

Clozapine for use in a method for treating a mental disorder in a patient, wherein the method comprises administering to the patient an effective dose of clozapine and wherein the effective dose of clozapine is determined by a method comprising the steps of:(i) obtaining a biological sample from the patient, wherein the sample comprises genomic DNA of the patient;(ii) determining the presence in the genomic DNA of:CYP1A2 alleles * 1A, * 1C and * IF;CYP3A4 alleles * 1, * IB, *3, * 10, and *22;CYP3A5 alleles * 1, *3, *6 and *7; andNFIB rs28379954 alleles selected from C or T;(iii) determining whether the patient smokes tobacco; and(v) determining the effective dose of clozapine based on steps (ii) and (iii) wherein determining an effective dose of clozapine at step (v) comprises: - assigning a score to each CYP1A2 * 1A, * 1C and * 1F allele present in the genomic DNA, wherein any CYP1A2 * IF allele is assigned a higher score if the patient is a smoker; assigning a score to each CYP3A4 * 1, * 1B, *3, * 10, and *22 allele present in the genomic DNA; assigning a score to each CYP3A5 * 1, *3, *6 and *7 allele present in the genomic DNA; summing the score for each CYP1A2 allele, each CYP3A4 allele and each CYP3A5 allele to obtain an overall activity score; increasing the overall activity score if the NFIB C allele is present in the genomic DNA; and multiplying the overall activity score by a basal dose to obtain the effective dose of clozapine.

2. A method for determining an effective dose of clozapine for treating a patient having a mental disorder, the method comprising the steps of:(i) obtaining a biological sample from the patient, wherein the sample comprises genomic DNA of the patient;(ii) determining the presence in the genomic DNA of:CYP1A2 alleles * 1A, * 1C and * IF;CYP3A4 alleles * 1, * IB, *3, * 10, and *22;CYP3A5 alleles * 1, *3, *6 and *7; andNFIB rs28379954 alleles selected from C or T;(iii) determining whether the patient smokes tobacco; and(v) determining the effective dose of clozapine based on steps (ii) and (iii);wherein determining an effective dose of clozapine at step (v) comprises: - assigning a score to each CYP1A2 * 1A, * 1C and * 1F allele present in the genomic DNA, wherein any CYP1A2 * IF allele is assigned a higher score if the patient is a smoker; assigning a score to each CYP3A4 * 1, * 1B, *3, * 10, and *22 allele present in the genomic DNA; assigning a score to each CYP3A5 * 1, *3, *6 and *7 allele present in the genomic DNA; summing the score for each CYP1A2 allele, each CYP3A4 allele and each CYP3A5 allele to obtain an overall activity score; increasing the overall activity score if the NFIB C allele is present in the genomic DNA; and multiplying the overall activity score by a basal dose to obtain the effective dose of clozapine.

3. A method for treating a patient with clozapine, wherein the patient is suffering from a mental disorder, the method comprising the steps of:(i) obtaining a biological sample from the patient, wherein the sample comprises genomic DNA of the patient;(ii) determining the presence in the genomic DNA of:CYP1A2 alleles * 1A, * 1C and * IF;CYP3A4 alleles * 1, * IB, *3, * 10, and *22;CYP3A5 alleles * 1, *3, *6 and *7; andNFIB rs28379954 alleles selected from C or T;(iii) determining whether the patient smokes tobacco;(v) determining an effective dose of clozapine based on steps (ii) and (iii); and(vi) treating the patient with the effective dose of clozapine; wherein determining an effective dose of clozapine at step (v) comprises: - assigning a score to each CYP1A2 * 1A, * 1C and * 1F allele present in the genomic DNA, wherein any CYP1A2 * IF allele is assigned a higher score if the patient is a smoker; assigning a score to each CYP3A4 * 1, * 1B, *3, * 10, and *22 allele present in the genomic DNA; assigning a score to each CYP3A5 * 1, *3, *6 and *7 allele present in the genomic DNA; summing the score for each CYP1A2 allele, each CYP3A4 allele and each CYP3A5 allele to obtain an overall activity score; increasing the overall activity score if the NFIB C allele is present in the genomic DNA; and multiplying the overall activity score by a basal dose to obtain the effective dose of clozapine.

4. Clozapine for use according to claim 1, or a method for determining an effective dose of clozapine according to claim 2, or a method for treating a patient with clozapine according to claim 3, wherein: the score assigned to each CYP1A2* IF allele is a first score if the patient is a smoker, and a second score if the patient is a non-smoker; and the score assigned to each CYP1A2* 1A, CYP3A4* 1, CYP3A4* IB, CYP3A4*3, CYP3A4* 10 or CYP3A5* 1 allele is the second score; and the score assigned to each CYP1A2* 1C or CYP3A4*22 allele is a third score; and the score assigned to each CYP3A5*3, CYP3A5*6 or CYP3A5*7 allele is a fourth score.

5. Clozapine for use according to claim 5, or a method for determining an effective dose of clozapine according to claim 5, or a method for treating a patient with clozapine according to claim 5, wherein the first score, the second score, the third score and the fourth score each have a different numerical value, preferably wherein: the first score is greater than the second score; and the second score is greater than the third score; and the third score is greater than the fourth score.

6. Clozapine for use according to claim 1, or a method for determining an effective dose of clozapine according to claim 2, or a method for treating a patient with clozapine according to claim 3 wherein the method further comprises the following step:(iv) determining whether the patient is being treated with omeprazole; and wherein step (v) further comprises determining the effective dose of clozapine based on step (iv).

7. Clozapine for use according to claim 7 or a method for determining an effective dose of clozapine according to claim 7, or a method for treating a patient with clozapine according to claim 7, wherein determining the effective dose of clozapine based on step (iv) further comprises:- reducing the overall activity score where the patient is being treated with omeprazole; optionally wherein the overall activity score is reduced by an equivalent numerical value to that of the third score.

8. Clozapine for use according to claim 1 or claims 4 to 7, or the method for determining an effective dose of clozapine according to claim 2 or claims 4 to 7 , or the method for treating a patient with clozapine according to claim 3 to 7 , wherein the mental disorder comprises one or more of: schizophrenia, paranoid schizophrenia, schizoaffective disorder, a personality disorder, EUPD, mixedand other personality disorder, persistent delusional disorder, refractive schizophrenia, a psychotic disorder, a delusional disorder, schizophreniform disorder, or psychosis in Parkinson’s disease.

9. Clozapine for use according to claim 1 or claims 4 to 8, or the method for determining an effective dose of clozapine according to claim 2 or claims 4 to 8 , or the method for treating a patient with clozapine according to claim 3 to 8, wherein the basal dose is 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg, preferably 75 mg.

10. Clozapine for use according to claim 1, or claims 4 to 9, or the method for determining an effective dose of clozapine according to claim 2 or claims 4 to 9 , or the method for treating a patient with clozapine according to claim 3 to 9, wherein the effective dose of clozapine is that required to give a plasma concentration of clozapine of 0.35ng / ml 12 hours post-administration.

11. Clozapine for use in a method for treating a mental disorder in a patient, or a method for determining an effective dose of clozapine for treating a patient, or a method for treating a patient with clozapine wherein the patient is suffering from a mental disorder, the method comprising the steps of:(i) obtaining a biological sample from the patient, wherein the sample comprises genomic DNA of the patient;(ii) determining the presence in the genomic DNA of:CYP1A2 alleles selected from * 1A, * 1C and * IF;CYP3A4 alleles selected from * 1, * 1B, *3, * 10, and *22;CYP3A5 alleles selected from * 1, *3, *6 and *7; andNFIB rs28379954 alleles selected from C or T;(iii) determining whether the patient smokes tobacco;(iv) determining whether the patient is being treated with omeprazole; and(v) determining the effective dose of clozapine based on steps (ii), (iii) and (iv); wherein determining the effective dose of clozapine at step (v) comprises:- assigning a score to the CYP1A2 alleles present in the genomic DNA, wherein: each CYP1A2 allele * 1A is assigned a score of 1.0; each CYP1A2 allele * 1F is assigned a score of 1.0 if the patient does not smoke tobacco; each CYP1A2 allele * IF is assigned a score of 2.0 if the patient smokes tobacco; and each CYP1A2 allele * 1C is assigned a score of 0.5;- assigning a score to the CYP3A4 alleles present in the genomic DNA, wherein: each CYP3A4 allele * 1, * IB, *3 or * 10 is assigned an activity score of 1.0; and each CYP3A4 allele *22 is assigned an activity score of 0.5;- assigning a score to the CYP3A5 alleles present in the genomic DNA, wherein:each CYP3A5 allele * 1 is assigned an activity score of 1.0; and each CYP3A5 allele *3, *6 or *7 is assigned an activity score of 0;- summing the score for each CYP1A2 allele, each CYP3A4 allele and each CYP3A5 allele to obtain an overall activity score;- increasing the overall activity score by 0.5 if the NFIB C allele is present in the genomic DNA;- reducing the overall activity score by 0.5 if the patient is being treated with omeprazole;- multiplying the overall activity score by the basal dose, optionally wherein the basal dose is 75 mg, to obtain the effective dose of clozapine.

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