Steady-state bioequivalence study of clozapine psychotic patients in Brazil

The present work aimed to compare the relative bioavailability of Zolapin with 100 mg tablet Leponex as a reference formulation, through a simple, robust and low-cost bioequivalence assays method. The study design was multiple-dose, randomized, crossover with patients in steady-state, and was performed with 24 schizophrenic male patients. Subjects received 100 mg twice a day of either Leponex or Zolapin for 10 days. At day 10 days of each study phase, blood samples were collected at different times during 12 h after drug administration and the clozapine concentration was determined by high performance liquid chromatography (HPLC). The individual peak plasma concentrations (Cmax) and the area under the concentration-time curve (AUC0-12h) ratios were calculated. The evaluated pharmacokinetic parameters were quite similar for both formulations. The 90% confidence interval for mean ratio of lnCmax (0.9677 to 0.9937) and lnAUC0-12h (0.9811 to 1.0029) were within accepted international guidelines. The results demonstrate that this methodological approach was able to identify Zolapin as bioequivalent to Leponex when orally administered, both in terms of the rate and extent of absorption, and therefore, suitable as a potential low-cost alternative to branded antipsychotic drugs.


INTRODUCTION
The American Psychiatric Association Guidelines for the Treatment of Schizophrenia establish that, after the failure of two or three treatments with atypical antipsychotics, the patient should be considered as bearer of refractory schizophrenia (RS).This means that the patient is a good candidate for treatment with *Corresponding author.E-mail: lquerinoac@gmail.com.Tel: +55-21-26299255 Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License bearer of refractory schizophrenia (RS).This means that the patient is a good candidate for treatment with clozapine, the treatment of choice in refractory schizophrenia (third-line agent) in the last two decades (Warnez and Alessi-Severini, 2014).Clozapine is a tricyclic dibenzodiazepine derivative (Figure 1), that was introduced in clinical studies in the United States in the 70s, as the first atypical antipsychotic (Wohlfarth et al., 2011) and has been serving as the best alternative medication for patients with treatment-resistant schizophrenia who failed to respond to other antipsychotics (Suzuki et al., 2011;Kane and Correll, 2010).It is also effective for the treatment of aggressive behavior of schizophrenic and demented patients and in the management of psychosis and aggression in Parkinson's disease (Thomas and Friedman, 2010).
Even though clozapine has been very effective to schizophrenic patients refractory or intolerant to classical antipsychotic therapy, and also induced a good recovery during therapy in some patients with chronic psychosis, a relative risk of serious blood disorders such as agranulocytosis has restricted its use (Asenjo et al., 2010;Meltzer, 2012).Other haematological effects include leucopenia, neutropenia and eosinophilia (Herceg et al., 2010).However, these side effects can be detected, prevented, minimized and treated, and the number of fatalities associated with these effects is actually much reduced (Warnez and Alessi-Severini, 2014;Meltzer, 2012;Herceg et al., 2010;Drew, 2013).The use of clozapine at doses higher than 600 mg daily should follow published recommendations, in order to minimize the risk of seizures; these include anticonvulsant regimens based on blood levels (Cohen et al., 2012).
The success of clozapine treatment is also strongly dependent on the drug´s bioavailability.In this context, 90 to 95% of clozapine is rapidly absorbed following oral administration, and is subject to first-pass metabolism, resulting in an absolute bioavailability of 50 to 60%.The time of onset of maximum plasma clozapine concentration after a single oral dose (75 or 100 mg) is approximately 1.5 h (average, at 2 to 3 h) and its halflife of elimination reaches 4 to 12 h (average of 8 h), in general.Clozapine's plasma concentration may vary widely, depending on several factors, especially at higher doses, above 500 mg/day (Tassaneeyakul et al., 2005).Individual factors may also change the response to this drug, such as cigarette smoking, that induces the metabolism of clozapine (Lucas and Martin, 2013).Clozapine is extensively metabolized by CYO1A2 in the liver and the major metabolites detected in urine and in the plasma are norClozapine (N-desmethylClozapine) and (Clozapine N-oxide) (Wohlfarth et al., 2011).The desmethyl metabolite has only limited pharmacological activity while the hydroxylated and N-oxide derivatives are inactive.
Usually, studies involving the determination of bioequivalence of clozapine employ Leponex (Novartis Pharma AG) as a reference drug.Leponex is an important and widely employed treatment option for many cases of schizophrenia, even though it remains a relatively expensive drug.Therefore, the availability of similar formulations on the market offers an option for patients, especially on developing and low-income countries.In the healthcare scenario of developing countries such as Brazil, in order to deal with high cost of medicaments for the general low-income population, generic drug policies have been implemented since the 90´s, with the aim of encouraging commercial competitiveness, improve the quality of medicines given its interchangeability with the brand drug and increase the population's access to treatment, reducing significantly the cost of drug therapy (Araújo et al., 2010).This legislation recommends that the generic drugs are at least 35% cheaper than the reference brand name drug (Brazil, 1999).Clozapine, despite the availability of its formula in the public domain, has a high cost for Brazilian health programs.Even though the Brazilian government encourages public-private partnerships for the production of generic drugs, there are few options of neuroleptics similar to clozapine, and any novel medicament proposed as a low-cost alternative to branded antipsychotic drugs would have initially pass in criterious assessments of quality and pharmacological performance through parameters such as bioavailability.
Many methods for evaluation of bioequivalence of clozapine formulations in plasma are available in the literature (Wohlfarth et al., 2011;da Fonseca et al., 2013;do Carmo Borges et al., 2012).Amongst the similar generic drugs commercially available at the time this study was started, Zolapin was chosen as being a potential low cost antipsychotic substance under the scrutiny of the Brazilian Health Authorities.In the present work, we aimed to present and validate an alternative methodology, with simple, reproducible steps for the performance of effective and low cost evaluation of the relative bioavailability of Zolapin (Meizler Biopharma), a formulation of clozapine, employing Leponex, a brand name of clozapine in Brazil, as a reference drug.

Drugs and chemicals
Clozapine (Figure 1) was purchased from USP reference standards (cat nr.114107).Dothiepin was purchased from British Chemical Reference Substances (cat nr.134).Both clozapine and Dothiepin were kindly provided by Meizler, São Paulo, Brazil.Other chemicals used in the study were of analytical grade and purchased from Tedia Brazil.Clozapine formulations employed in this study were 100 mg Leponex® tablets (Novartis Pharma AG, Stein, Switzerland, batch no.ZO 007), and 100 mg Zolapin® tablets (Synthon BV -Nijmegen -Holand -batch no.04H 13 GAC).For disambiguation, it is important to observe that another drug, manufactured by Konark Life Sciences (India), employs the similar name Zolapin MD, but it is a generic formulation containing Olanzapine, and was not used in the present study.

Solutions
Stock solutions of the analytes were prepared by dissolving suitable amounts of each pure substance in methanol (673.300µ/ml clozapine) and water (0.0236 µ/ml Dothiepin, internal standard).Standard solutions were prepared by diluting stock solutions with the mobile phase.

Subjects
The study protocol was approved by the Ethical Committee of the Bonsucesso General Hospital at Rio de Janeiro, Brazil, under the no.06/04.Thirty-six male patients regularly admitted and institutionalized in the private EGO Psychiatric Clinic, Itaboraí, Rio de Janeiro, Brazil, were enrolled in the study, 34 of whom completed the clinical steps and 24 were included in the statistical analysis.The test subjects or the person legally responsible for decisions on the subject's behalf were informed, both verbally and in writing, about the experimental procedures and the purposes of the study.A written informed consent was obtained from each participant in the study, following the operational procedures of the Clinical Research Center, nowadays renamed as Clinical Research Unit, at the Fluminense Federal University -UFF -Niterói, Brazil.The personal data were: all male patients (n = 24) aging between 27 to 52 years (mean ± SD, 40.6 ± 7.6); body weight range from of 71.7 to 95.0 kg (mean ± SD, 82.43 ± 12.4), and their body mass indices were in the range of 18 to 34.Exclusion criteria consisted of medical history of allergy to clozapine (or to other anti-psychotic drugs), liver disease, kidney disease, gastrointestinal disorders, cardiovascular diseases, blood disorders, hepatitis, drug abuse, alcoholism, coexisting psychiatric disorders in Parkinson's disease, other psychotic disorders, affective disorders, dyskinesias and related disorders, AIDS or HIV sero-positive (Agência Nacional de Vigilância Sanitária (ANVISA), 2002; Food and Drug Administration (US-FDA), 2014).The blind code was disclosed after all measurements, calculation and plots made as in order to characterize the double-blind study.

Study design
The study was conducted using a multiple-dose, randomized, twoway crossover study design, in three steps: Step 1: Entry treatment.All the patients received increasing doses of the reference formulation -Leponex (Novartis Pharma AG, Stein, Switzerland) for seven days according to the following schedule: day-1, 12.5 mg after breakfast (8:00 am) and 12.5 mg after dinner (8:00 pm); day-2 and day-3, 25 mg the same time as the first day; day-4, 50 mg after breakfast and 75 mg after dinner; day-5 and day-6, 75 mg twice daily and the day-7, 100 mg twice daily.Drug administration was performed by the nurses of the EGO Psychiatric Clinic.
Step 2: Randomized Exposure.Patients were randomly divided into 2 groups: (i) one group received 100 mg twice daily of the Leponex (Novartis Pharma AG, Stein, Switzerland) and (ii) the other received 100 mg twice daily of the Test formulation, Zolapin (Synthon BV-Nijmegen-Holand), for 10 days, in order to reach the steady-state.At the end of this period, on day-11, the patients were transported to the Clinical Research Center, under medical supervision, and blood samples were taken for analysis by venipuncture in heparinized Vacutainer® blood collectors' tubes.This first sample was collected after 12 h fasting, just before the single morning dose administration of either the test or the reference drug.The subjects were subsequently given one tablet of 100 mg clozapine orally with 120 ml water and blood samples were collected at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10 and 12 h after drug administration (via IV catheter).Blood samples were centrifuged at 1200 × g for 10 min and the supernatant plasma was separated, transferred into polypropylene test tubes and frozen at -20°C until the assay.
Step 3: Crossover.In this phase, a crossover exposure was performed as previously described (Mercolini et al., 2007;Rosland et al., 2007;Golden and Honigfeld, 2008).Patients from each group received a similar treatment, but changing the clozapine formulation from the previous step, that is, patients who received Leponex during Step 2 now received Zolapin during Step 3, and vice-versa.There was no period of "washout" between the two treatments (Steps 2 and 3).During the study, patients received close assistance of the medical team.Every meal was standardized by an accredited nutritionist according to the protocol study and the main course was around 2.500 calories.The patients were allowed to drink water freely, except one hour before and one hour after drug administration.After the study, at the discretion of physician, the patients continued to receive 100 mg twice daily of the clozapine reference formulation (Leponex) or had their dose adjusted for other clinically more effective drugs.

Calibration curves
Aliquots of standard solutions at six different concentrations, as follows: 3.905, 1.953; 0.976; 0.488; 0.0976 and 0.0195 µg/ml, were added to blank plasma.Three calibration curves were obtained with two aliquots of each concentration in each curve.Blank plasma was obtained from the blood of healthy volunteers and subjected to the same procedures reported above for patients' plasma.

Determination of Clozapine in plasma
Plasma concentration of clozapine was assayed by an isocratic HPLC method with UV absorbance detection -HPLC PerkinElmer, Series 200 (PerkinElmer, USA).Initially the plasma was heated at 37°C in water-bath, being placed in polypropylene tubes by shaking for 10 min in vortex, followed by centrifugation (1 min at 1200 g).Subsequently, 0.5 ml of each plasma sample were transferred into Eppendorf tubes and 100 μl of the internal standard (Dothiepin 23.6 mg/ml) were added and mixed briefly.After that, 1 ml 50 mM phosphate buffer solution adjusted to pH 10.0 with NaOH (4 M) was added and vortexed briefly.
Plasma samples were pre-treated by means of solid phase extraction (SPE) with Sep-Pak® Vac 100 mg tC18 cartridges (Waters, USA).The cartridges were equilibrated with 1 ml of methanol and 0.3 ml of phosphate buffer under gentle vacuum.The prepared plasma samples were loaded into the cartridges (2 × 0.7 ml).Washing was carried out with 0.5 ml of phosphate buffer and a 1:1 mixture of methanol and water (2 × 0.75 ml), under gentle vacuum.The analytes were eluted with 0.3 ml of a mixture of mobile phase and acetonitrile (6:4) also on gentle vacuum and 20 μl were injected into the HPLC system.The mobile phase was composed of 7:3 10 mM o-phosphoric acid and acetonitrile, 4 ml/L triethylamine and 2 mg/L N,N,N',N'tetramethylethylenediamine, the pH being adjusted to 3.55 using 85% H3PO4.
The analytical column was a Lichrospher RP 18e (5 μm) stainless steel (125 × 3 mm) (Macherey-Nagel, Germany).Flow rate of the mobile phase was set at 1.0 ml/min and absorbance was monitored at 245 nm.Standard curves were constructed in the clozapine concentration range of 19 to 4000 ng/ml.The standard curve samples were treated in the same manner as the plasma samples collected from the volunteers.

Data analysis
Pharmacokinetic parameters of bioequivalence were set from drug concentration-time curves set by the study protocol.The outstanding measures were: AUC(0-12h), Maximum stabilized plasma concentration (Cmax), Minimum stabilized plasma concentration (Cmin) and time of maximum plasma concentration (Tmax).The area under the clozapine concentration-time curves from 0 to 12 h (AUC0-12h) were calculated using the trapezoidal method and the elimination rate constant (Ke) was estimated using straight regression adjusted in 4 to 6 last log-transformed concentration values.
The relationship between pharmacokinetic variables such as Cmax, Cmin, Tmax and (AUC0-12h) were used to compare the reference and test, determined using analysis of variance (ANOVA) after logtransformed data.The points were estimated at a 90% confidence interval.According to the standard criteria of the Brazilian National Health Surveillance Agency (ANVISA) and the United State Food and Drug Administration (US-FDA), bioequivalence of the two formulations was established when formulation or treatment effect of AUC0-12h and Cmax should not be different at alpha level of 0.05 and the 90% confidence interval of the mean ratio of AUC0-12h and Cmax between the tested product and the reference product should stand within the range of 0.80 to 1.25 for log-transformed data.(ANVISA, 2002; US-FDA, 2014).

RESULTS
Under described chromatographic conditions, the retention times for clozapine and internal standard (Dothiepin) were 1.75 at 2.29 and 4.17 at 5.69 min, respectively (Figure 2).Selectivity was evaluated by injecting samples drawn from six different healthy volunteers and none of them produced peaks which could interfere with the analysis.The calibration curves were linear over the range of 20 to 4000 ng/ml.No other peak appeared at the retention times of the compounds of interest (Figure 2B).Within-day coefficients of variation (determined in 5 replicated samples) were 8.45, 6.80 and 7.24% at clozapine concentrations of 40, 1800 and 3200 ng/ml, respectively.

Patients and steady-state determination
All subjects well tolerated both clozapine formulations.No serious adverse effects were reported during the study.Physical examination, laboratory tests and electrocardiograms revealed that none of the subjects suffered from any serious side effect from the clozapine products used in the study and all were tolerant to both formulations.It was allowed concomitant therapies that had been used during the stabilization of serum concentrations of clozapine (step 1).The steady-state drug concentration was determined in each subject at day-5 and day-6 (Step 1) and day-12 and day-13 (Step 2) prior to the morning dose of the drug.The mean of clozapine concentration-time profiles after administration of the test and reference formulations in 24 subjects are showed in Figure 3.
All of the pharmacokinetic parameters calculated for the Test formulation were close to those of the Reference formulation and there were no statistically significant differences between the two products (Table 1).The mean ratios for lnC max and lnAUC 0-12h between the Zolapin and Leponex formulations were 0.9807 ± 0.0391 and 0.9920 ± 0.0327, respectively.
Analysis of variance (ANOVA) of the lnAUC 0-12h and lnC max obtained from the Test and Reference formulations revealed that the sequence, period or formulation was not significantly different at p<0.05.The 90% confidence interval for lnAUC 0-12h ranged from 0.9811 to 1.0029 and for lnC max ranged from 0.9677 to 0.9937.It should also be noted that none of these values obtained from 24 subjects were outside the range of 0.8 to 1.25.In addition, ANOVA of lnT max revealed that these two Clozapine products were not significantly different from each other (Table 2).

DISCUSSION
Recent reviews and meta-analysis on clinical practice guidelines and prescribing trends on schizophrenia   The HPLC method used in this study is simple and straightforward, also providing appropriate sensitivity, specificity for the pharmacokinetic study of clozapine.Previous bioequivalence studies of clozapine (Pokorny at al., 1994) made with healthy patients caused serious adverse effects such as hypotension, bradycardia, and syncope asystole.For this reason, the US-FDA recommends that any bioequivalence study of clozapine should be conducted only in psychiatric patients.Therefore, the present study was carried out following the recent US-FDA Guidance and ANVISA -Brazil Guidance (ANVISA, 2002; US-FDA, 2014), using a multiple-dose, steady-state bioequivalence study in schizophrenic patients.The validation method is a simple, fast and robust HPLC assay for the determination of clozapine and can be applicable to pharmacokinetic studies and therapeutic drug monitoring, which may be useful in order to determine the optimal dosing for each individual (Cohen et al., 2012).
In the present results, comparing the mean transformed C max and AUC of the 24 volunteers in Table 2, lnC max (0.9677 to 0.9937) and lnAUC 0-12h (0.9811 to 1.0029), we verify that they are within the 90% CI, satisfying the condition of bioequivalence between products within the parameters recommended by ANVISA.
Employing the proposed method, a wide interpatient variability in pharmacokinetic parameters was also observed for both clozapine formulations.The large variability in plasma concentrations of clozapine is both due to individual differences in bioavailability and to the fact that clozapine is metabolized by the highly variable enzyme CYP1A2.This enzyme varies among individuals due to induction, inhibition and probably genetic factors (Cohen et al., 2012).The sources of these variabilities also could be explained by age, smoking and simultaneous use of others drugs, which are known for their influence on plasma Clozapine concentrations (Lucas and Martin, 2013).Bioequivalence assays enable the manufacture and distribution of generic drugs for the Brazilian health system at a price much lower than the brand name drug.As clozapine´s reference formulation (Leponex) is an expensive drug and therefore finds limited use in the Brazilian public health system, it is very important to assess possible new formulations of generic clozapine for reducing the cost of treatments (Lewis et al., 2006).
In this context, the present work has shown that candidate generic drugs can have validated analytic methods, following the preconized standards of international studies, providing safe and interchangeable alternatives for patients.The validation of simple methods which can be performed at university and public laboratories must also be an incentive to more initiatives on the production and testing of novel low-cost generic drugs for important clinical conditions.Since the inclusion and exclusion criteria for studies with clozapine are already well stablished, this incentive becomes more straightforward to the generic drug policies in countries like in Brazil, where healthcare services related to the use of this neuroleptic cannot reach the majority of the affected population due to high-cost of medicaments (Araújo et al., 2010;Rumel et al., 2006).Therefore, we believe that simpler and low-cost bioequivalence assays may facilitate the registration of novel products, with their safety incurred by laboratory analyses in governmental laboratories, for widespread distribution to low-income patients.
It is also important to note that not only patients with refractory schizophrenia, but also with Parkinson disease-related psychosis could benefit for generic dlozapine formulations.

Conclusion
The bioavailability of Zolapin is equivalent to that of the reference drug Leponex, when orally administered, both in terms of the rate and extent of absorption, as measured on a simplified and low-cost methodology.

Figure 2 .
Figure 2. Determination of retention times of Clozapine.A. Chromatogram of a blank plasma sample.B. Plasma sample containing clozapine and Internal Standard.CLZ= Clozapine IS= Internal Standard (Dothiepin).

Table 1 .
Mean pharmacokinetic parameters of Clozapine obtained from 24 subjects after administration of either test or reference formulation.

Table 2 .
Comparative mean ratio of ln-transformed Cmax and ln-transformed AUC0-12h of Clozapine in 24 subjects after administration of Zolapin and Leponex.