Blonanserin N-Oxide Lowers Glucose Levels in Animal Models

1School of Chemistry, University of Hyderabad, Prof. C. R. Rao Road, Gachibowli, Central University, Hyderabad, India 2Technology Business Incubator, University of Hyderabad, Prof. C. R. Rao Road, Gachibowli, Hyderabad, India 3Virchow Biotech Pvt. Ltd., Animal House Facility, S. V. Co-op Industrial Estate, IDA Jeedimetla, Hyderabad, India 4Life Science Incubator, IKP Knowledge Park, Genome Valley, Turkapally, Shameerpet, Hyderabad, India

Type-2 Diabetes mellitus (DM) is a metabolic disorder caused by insulin resistance (dysfunction of pancreatic beta cells) and obesity. It is now recognized as a major health problem worldwide and affects adults of working age in developing countries. WHO estimates on the global prevalence of diabetes are expected to increase from 171 million in 2000 to 366 million in 2030. As a matter of fact 21.7% of these (~80 million) will be from India [1]. A first-line treatment for diabetes is lifestyle changes such as low calorie diet, weight control, and regular physical activity. If blood sugar (glucose) levels still remain high, then the patient is administered anti-diabetic drugs (for Type-2 diabetes) or insulin (Type-1 diabetes mellitus). A major challenge in T2DM therapy is how to make the pancreas more insulin sensitive. The other goal is to increase endogenous insulin levels in T2DM patients. One way to address the problem is to target pancreatic β-cells for increasing endogenous insulin levels via small molecule drugs (bigunides, sulfonylureas, meglitinides, thiazolidinediones, alpha-glucosidase inhibitors, dipeptidyl-peptidase IV inhibitors, incretin-based therapies, etc.) [2][3][4]. Metformin (bigunide), is the classic example of a T2DM drug which is an insulin sensitizer. However metformin requires circulating insulin levels for drug action but does not activate insulin secretion. The sulfonylureas, or ATP channel blockers, gliclazide, glimepiride, glipizide, tolbutamide, etc. promote the release of insulin from the β-cells by blocking the ATP-sensitive K + -channels, thereby resulting in depolarization with Ca +2 -influx which activates insulin secretion. However, a serious side-effect of the sulfonyl urea drugs is hypoglycaemia, which happens due to excess insulin release. This means that the patient has to have additional sugar or carbohydrate to normalize insulin levels. The dipeptidyl peptidase IV inhibitors (also known as incretin enhancers or DPP-IV inhibitors, [5] are the latest generation T2DM drug molecules, e.g. sitagliptin, linagliptin, saxagliptin, and vildagliptin [6]. The glucagon like peptide (GLP-1 and 2) are natural incretin hormones which are released from the gut in response to food intake, and they are collectively responsible for glucose-dependent insulin release [7]. The physiological activity of the incretin hormones is limited by DPP-IV peptidase enzyme which cleaves the terminal end of active GLP to inactive Introduction peptide. This is reversed by the gliptin drugs, which are tight binding inhibitors of the DPP-IV enzyme. An advantage of the gliptins, compared to say the glitazones, is that since GLP levels are modulated by food intake, insulin release occurs post prandial and hence hypoglycemic condition is avoided. However, complete studies on the potential side effects of incretin-based gliptins are still ongoing [8].
The N-oxide of anti-psychotic drug clozapine (CLNO, clozapine N-oxide) is a natural metabolite and it was studied in transgenic mice for the activation of β-cell signaling pathway in vivo [13]. When transgenic (Tg) mice were injected with glucose alone, the blood glucose and insulin levels remained similar to the wild type set. However, co-injection of glucose (3 g/kg, i.p.) with CLNO (1 mg/kg i.p.) resulted in an immediate increase in insulin release (2 min) and subsequent time points (up to 15 min), at levels almost 12 times higher than the first 10 min, compared to the control group. There was a two-fold increase in islet density and 30% increase in β-cell mass size after a 2 weeks treatment [14]. Thus the pharmacological effects of CLNO are fundamentally different compared to previous drug classes, in that it not only activates insulin secretion instantaneously but also increases the size and mass of the pancreatic islet β-cells [15]. In the same class of anti-psychotic drugs is blonanserin (BLN), an agent with dopamine D2 and serotonin 5-HT2A receptor antagonist properties, [15] similar to SNRI drug clozapine (CLN) (serotonin norepinephrine reuptake inhibitors); both drugs are used in the treatment of schizophrenia and central nervous system (CNS) disorders [16]. Based on the structural similarity of these two molecules (Figure 1), we undertook the synthesis of the natural metobolite BLNO and to screen its anti-diabetic activity.
There is increasing evidence in the past decade that patients taking anti-psychotic drugs for schizophrenia run an increased risk of diabetes mellitus [9]. That anti-psychotic drugs cause weight gain, and together with reduced physical activity in such patients, are additional factors for the high incidences of T2DM in psychotic patients [10,11]. A recent study showed that phosphorylation of M3-muscarinic receptor plays an important mechanistic role in facilitating insulin release from pancreatic islets by coupling of the receptor to protein kinase D1 [12]. These muscarinic acetylcholine receptors are a subfamily of GPCRs recognizing the neurotransmitter acetylcholine and signaling through G proteins of the Gq/11 class. These receptors are also targets for the treatment of diabetes and have been implicated in the treatment of cognitive disorders such as Alzheimer's disease and schizophrenia.
Blonanserin (200 mg, 0.54 mmol) was dissolved in 15 mL of methanol and stirred at room temperature until it dissolved completely. To this reaction mixture 1.44 mL of 30% H 2 O 2 was added and refluxed at 70 °C for 2 h. After completion of the reaction (by TLC), methanol was removed by rotary evaporator resulting in a viscous liquid. The reaction mixture was diluted with water and extracted with CHCl 3 . Drying of the organic layer with Na 2 SO 4 evaporation of volatiles and drying on a vacuum pump for 2 h afforded a

Synthesis of Blonanserin N-oxide (BLNO)
Blonanserin was purchased from Mesochem Technology Co. Ltd., China, and used without further purification. Clozapine was purchased from Shanghai Xunxin Chemical Co Ltd, China. All other chemicals and reagents were purchased from domestic suppliers and are of analytical grade purity. Metformin hydrochloride (MHCL) and Sitagliptin phosphate monohydrate (SPMH) were purchased from drugs suppliers. Streptozotocin and solvents and reagents were purchased from Sigma-Aldrich and Merck (Hyderabad, India).
white solid which was purified by recrystallization from n-pentane. Yield 150 mg (70%); m.p. 153-155 °C (decomposition) [17]. 30 mg of this BLNO was dissolved in 5 mL DMSO: H 2 O (1:1 v/v) and left for slow evaporation at room temperature. Good quality single crystals were harvested after about one week for single crystal X-ray diffraction analysis.

X-ray crystallography
X-ray reflections were collected on Oxford CCD X-ray diffractometer (Yarnton, Oxford, UK) equipped with Mo-Kα radiation (λ = 0.71073 Å) and Cu-Kα X-radiation (λ = 1.5406 Å) source. Data reduction was performed using CrysAlisPro 171.33.55 software. Crystal structures were solved and refined using Olex 2-1.0 with anisotropic displacement parameters for non-H atoms. Hydrogen atoms were experimentally located through the Fourier difference electron density maps in all crystal structures. All O-H and C-H atoms were geometrically fixed using HFIX command in SHELX-TL program of Bruker-AXS. X-Seed was used to prepare packing diagrams.

Synthesis of Clozapine N-oxide (CLNO)
The preparation and characterization of BLNO and CLNO is described. Streptozotocin and solvents and reagents were purchased from Sigma-Aldrich and Merck (Hyderabad, India).

Materials and Methods
Male and female Wistar rats of body weight 200-250 g were procured from Sainath Agency, Hyderabad. Animals were acclimatized for 10 days to our prior experiment, maintained at temperature of 21 to ±2 º C. The animals were standardized by 12 hours light and 12 hours dark schedule. Six animals were housed per cage. All the animals were quarantined for 4 days and had free access to sterile formulated feed pellets and filtered potable clean water. The study was conducted in compliance with standard animal use practices at Virchow Biotech Private Limited, Department of preclinical toxicology, Hyderabad, India (Registration No. 546/02/A/ CPSCEA, India). Animal Ethics Committee Approval dated. 4 April 2014.

Procurement of animals and maintenance
After noting the insulinogenic activity of novel antidiabetic compounds CLNO and BLNO, we tested these N-oxides in Wistar rats.

Effect of BLNO and CLNO in STZ induced rats
The rats were subjected to fasting overnight, and then diabetes condition was induced with intraperitoneal injection (i.p.) of Streptozotocin (STZ, 40 mg/kg dose). The STZ was freshly dissolved in citrate buffer (0.01M, pH 4.5) [19]. The injection volume was 1.0 mL/kg [13] After 3 days of standard STZ induction, blood glucose levels were measured and the animals with a glucose concentration of more than 300 mg/dL were classified as diabetic [19] and taken for the experiment. Administration of the BLNO, CLNO was initiated on 4 th day after STZ injection and this was considered to be the 1 st day of treatment, which was continued for 28 days for 1X dosage and 18 days for 5X dosage.

Experimental induction of diabetes in Wistar rats
Volume 4 | Issue 1  Oral glucose tolerance test (OGTT) for non-diabetic female rats were performed according to the standard method [20]. In short, Group 1 to 5 animals (each group consisted of 6M + 6F) were selected for OGTT test after starving for 16 hours (water only). Baseline glucose levels were measured by glucometer to the glucose fed (3 g/kg body weight) rats. The standard and test compounds were administered in oral form for MHCL and SPMH and intraperitoneal for BLNO and CLNO.
Group 1: Positive control rats (3M + 3F) were administered STZ but no drug (diabetic rats) Group 2: Male diabetic rats received BLNO at 1 mg/kg dose (intraperitoneal, i.p.) Group 3: Male diabetic rats received CLNO at 1 mg/kg dose (i.p.) Group 4: Female diabetic rats received BLNO at 1 mg/kg dose (i.p.) Group 5: Female diabetic rats received CLNO at 1mg/kg dose (i.p.) Blonanserin-N-oxide single crystals suitable for X-ray diffraction were obtained from DMSO: H2O (1:1 v/v) mixture. The crystal structure was solved and refined in space group Pbca with one molecule of BLN-O and two water molecules in the asymmetric unit. Blonanserin is a selective antagonist of serotonin 5-HT2A and dopamine D2 receptors [15]. It has a binding affinity for the dopamine D2 receptor that is superior to that for clozapine. Both clozapine and blonanserin acutely impair insulin sensitivity to the pancreas [15,21] But the natural metabolite of clozapine (N-oxide of clozapine) studied in the treatment of Tg mice with CLNO (1mg/kg i.p. route) in streptozotocin induced model led to the activation of β-cell signaling pathway leading to in reduced glucose levels.. In this study, bolanserin N-oxide was treated at 1 mg/kg dosage (~ 1 mg/kg of BLNO) and 5 mg/kg dosage in both STZ induced model and Oral Glucose tolerance test (OGTT).

In vivo drug efficacy
Fasting blood glucose level and body weight were measured at 1st, 3 rd , 6 th , 9 th , 12 th , 15 th , 18 th , 21 st , 24 th , and 27 th day. The pancreas of rats was impaired by STZ treatment (40 mg/kg) and then study was started after 3 days. Blood samples were obtained by pricking the tail vein. The N-oxides of antipyschotic drugs BLNO and CLNO showed positive results at 1 mg/kg dosage. Plasma glucose values decreased significantly (P < 0.0001) from about 380 mg/dL to 90 mg/dL at the end of 28 days, while for the control group (labeled CTRL, no drug) the value increased to 550-600 mg/dL (Figure 3; values are listed in Table S1). The dramatic increase in glucose levels in the control group was due to their impaired pancreas by STZ. The average body weight of rats reduced by about 10 g during this study period, from 205 g to 195 g ( Figure S1, Supporting Information).

X dose study
The N-oxide drugs BLNO and CLNO were compared with known anti-diabetic drugs Metformin HCl and Sitagliptin phosphate monohydrate as positive controls. The hypoglycemic effect of BLNO in glucose-induced hyperglycaemic Wistar rats provides novel lead molecule for the control of diabetes.
Volume 4 | Issue 1 Figure 4: Glucose levels in 18 day study on STZ treated rats with drug administered daily at 5 mg/kg dose (i.p.). The control group CTRL was not treated with STZ White male and female Wistar rats of body weight of 200-250 g were taken. The animals were kept fasting overnight providing only water, after which the BLNO and CLNO drugs were administrated (i.p.) to four groups each consisting of 6 rats. If mortality was observed in 4 out of 6 animals per group, then the dose administered was assigned as a toxic dose. Body weight before and after administration were noted ( Figure S2) and behavior pattern were observed and also signs of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma were seen. Treatment with 5 mg/kg of BLNO and CLNO showed significant decrease in plasma glucose values, much faster than the 1X dose study. Plasma glucose levels dropped significantly (P < 0.0001) from 390 mg/dL to 90 mg/dL at the end of 9 th day ( Figure 4 and Table S2). However, no signs of toxicity or side effects were observed. BLNO and CLNO were found to be safe at dose of 5 mg/kg body weight in Wistar rats.
5X dose study OGTT Figure 5: Comparison of test drugs CLNO and BLNO (at 5 mg/kg, i.p.) with the standard drugs Metformin HCl and Sitagliptin phosphate monohydrate (100 mg/kg, oral) Blood sample drawn from the tail vein was measured by using glucometer at 0, 15, 30, 45, 60, 90 and 120 min ( Figure 5 and Table  S3). Induction of hyperglycemia by intragastric ingestion of glucose resulted in a two-fold increase in plasma glucose levels (con-