Journal of Diabetes and Its Complications xxx (2015) xxx–xxx
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Effect of ketotifen in obese patients with type 2 diabetes mellitus, Sahar M. El-Haggar a, Wael F. Farrag b, Fedaa A. Kotkata a,⁎ a b
Clinical Pharmacy Department, Faculty of Pharmacy, Tanta University, Egypt Internal Medicine Department, Faculty of Medicine, Tanta University, Egypt
a r t i c l e
i n f o
Article history: Received 10 December 2014 Received in revised form 25 January 2015 Accepted 28 January 2015 Available online xxxx Keywords: T2DM Mast cells Ketotifen Obesity Inflammation
a b s t r a c t Aim: Mast cells are found to be an important contributor in obesity induced insulin resistance. We evaluate the effect of ketotifen in obese patient with type 2 diabetes (T2DM) treated with glimepiride. Method: In a randomized controlled study we recruited forty-eight obese patients with T2DM from Internal Medicine Department at Tanta University Hospital, Egypt. They were classified into three groups: group 1, those who received glimepiride (GL) 3 mg/d alone; group 2, those who received GL 3 mg/d + ketotifen 1 mg once daily; and group 3, those who received GL 3 mg/d + ketotifen 1 mg twice daily. Fasting blood samples were obtained before and 12 weeks after treatment for biochemical analysis of glycemic and inflammatory biomarkers. Data were statistically analyzed by paired Student's t-test and one way analysis of variance; p b 0.05 was considered statistically significant. Results: The obtained data suggested that the addition of ketotifen in twice daily dose has a beneficial effect on all measured parameters except adiponectin. However, glimepiride plus ketotifen once daily only affected the level of inflammatory biomarkers without any significant effect on other parameters. Conclusions: The co-administration of ketotifen twice daily plus glimepiride improves glycemic and inflammatory process in obese patients with T2DM. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Obesity is a major risk factor for insulin resistance and type 2 diabetes mellitus (T2DM). Although how obesity promotes insulin resistance is unclear, the inflammatory response is thought to be a potentially important mechanism, which could alter adipose tissue function, thus leading to systemic insulin resistance. The development of insulin resistance is associated with pro-inflammatory cytokines produced by infiltrating leukocytes and resident adipocytes within the adipose tissue in obese subjects (Sismanopoulos et al., 2013). A previous report has demonstrated that mast cells (MCs) are important inflammatory cells that participate in immune responses during allergic reactions. Recent studies, however, suggest that these cells also participate in other inflammatory diseases, such as cancers, inflammatory bowel disease, metabolic bone disease, renal injury, arthritis, atherosclerosis, abdominal aortic aneurysms, obesity, and diabetes (Wang & Shi, 2011). Higher numbers of mast cells were found in Wight adipose tissue from obese subjects compared with that of lean subjects; obese subjects also had significantly higher mast cell tryptase (MCT) concentrations in their serum than did lean
individuals. These observations suggest a possible association between mast cells and obesity (Liu et al., 2009). Ketotifen is a mast cell stabilizer used as anti-allergic drug. The most important finding of recent studies is that mast cell stabilization with cromolyn or ketotifen reduces body weight gain and improves glucose and insulin tolerance in mice without noticeable toxicity (Wang & Shi, 2011). Although the precise roles of mast cell interleukin-6 (IL-6) and interferon gamma (IFN-γ) in obesity and diabetes remain incompletely understood, the absence of these inflammatory cytokines in mast cells reduces body weight gain, glucose tolerance, and serum levels of insulin, and glucose (Wang & Shi, 2011). No clear data on the effect of mast cell stabilizers on human were reported, so the present study was designed to investigate the effect of ketotifen on glycemic parameters (fasting blood glucose (FBG) and hemoglobin A1c (HbA1c)) and some inflammatory biomarkers (IL-6 and leukotriene B4 (LTB4)) in obese patients with T2DM. 2. Patients and methods 2.1. Patients
Conflict of interest: There are no conflicts of interest. There is no source of financial support for the research (no fund from any organization). ⁎ Corresponding author at: 31111. Tel.: +20 1017872669; fax: +20 403335466. E-mail address: [email protected] (F.A. Kotkata).
From January 2013 to April 2014, we recruited forty-eight obese patients with T2DM from internal medicine department at Tanta University Hospital, Egypt. The inclusion criteria were patients who had
http://dx.doi.org/10.1016/j.jdiacomp.2015.01.013 1056-8727/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: El-Haggar, S.M., et al., Effect of ketotifen in obese patients with type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.01.013
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S.M. El-Haggar et al. / Journal of Diabetes and Its Complications xxx (2015) xxx–xxx
body mass index (BMI) ≥ 30 kg/m 2, had diabetes duration from 5 to 10 years, were treated with glimepiride alone and had ages ranging from 45 to 55 years. The exclusion criteria were patients who had any other inflammatory disease (bone, asthma, etc.), severe hepatic or renal disease, epilepsy and also pregnant or lactating females. 2.2. Study design A total number of 48 patients who fulfilled the selection criteria were enrolled in the study. The study has been approved by the national research ethics committee (Tanta University ethical committee) and has been performed in accordance with the ethical standards as laid down in the 1964 declaration of Helsinki and its later amendments or comparable ethical standards. An informed written consent was obtained from all patients included in the study. The study design was a parallel randomized controlled study to compare the effect of two different doses (once and twice daily) of ketotifen (Ketoti®, Pharco, Egypt) in addition to glimepiride (GL) (Amaryl®, Sanofi-Aventis, Berlin, Germany). The patients were divided into three groups (each group [n = 16]) as follows: group 1, those who received glimepiride (GL) 3 mg/d alone; group 2, those who received GL 3 mg/d plus ketotifen 1 mg once daily; and group 3, those who received GL 3 mg/d plus ketotifen 1 mg twice daily for 12 weeks. Patients were followed up at monthly intervals for assessment of compliance to the study medication and adverse events. All blood samples were obtained after a 10- to 12-hours fasting period. Blood samples were collected in tubes containing EDTA and centrifuged immediately. Plasma was separated, coded and stored at − 80 °C until analysis. 2.3. Demographic characters Patients' medical history was taken to ensure the absence of any interacting or interfering drugs. Demographic data were collected at baseline using questionnaire. Information collected included age, sex, diabetes duration, BMI, FBG and HbA1c (Table 1). 2.4. Anthropometric evaluations Weights were measured and recorded to the nearest 0.5 kg. Body heights were measured and recorded to the nearest centimeter. Body mass index was calculated which is defined as the weight in kilograms divided by the square of the height in meters; i.e., BMI = weight (kg)/height2 (m). Height and weight were measured using Detecto scale (Detecto Company, 203 East Daugherty Sheet, USA). 2.5. Biochemical assays 2.5.1. Blood glucose and hemoglobin A1c Fasting blood glucose (FBG) levels were assayed using glucose oxidase method (Kaplan, 1984) (Spinreact, Spain). Hemoglobin A1c% (HbA1c %) was assayed by ion exchange method (Bissé & Abraham, 1985) (Biosystems, Spain). 2.5.2. Assay of adiponectin, interleukin-6 (IL-6), leukotriene B4 (LTB4) and mast cell tryptase (MCT) Enzyme-linked immunosorbent assay (ELISA) using commercial kits was carried out according to the manufacturers' instructions for assay of plasma adiponectin (Assaypro, USA), MCT (Wuhan Elaab Science Company, China), IL-6 Platinum ELISA (eBioscience, San Diego) and LTB4 (R&D Systems, USA). 2.5.3. Lipid panel Plasma was used for determination of lipid profiles including total cholesterol (TC) which was measured by enzymatic colorimetric
Table 1 Demographic data of patients at baseline. Characteristics
Group 1
Group 2
Group 3
Number Sex (M/F) Age (years) Diabetes duration BMI (kg/m2) FBG (mg/dl) (mmol/l) HbA1c % (mmol/mol)
16 3/13 51.3 ± 4.5 7.9 ± 2.5 37.3 ± 5.4 190.3 ± 50.6 (10.6 ± 2.8) 7.9 ± 0.96 (63 ± 10.5)
16 3/13 50.1 ± 4.6 7.1 ± 2.7 37.5 ± 6.1 205.9 ± 38.1 (11.4 ± 2.1) 8 ± 0.76 (64 ± 8.3)
16 4/12 49.1 ± 4.9 8 ± 2.5 37.8 ± 5.3 183.2 ± 52 (10.2 ± 2.9) 7.6 ± 0.88 (60 ± 9.6)
Data presented as mean ± SD. Group 1: obese patients with type 2 diabetes treated with glimepiride 3 mg/d alone; Group 2: obese patients with type 2 diabetes treated with glimepiride 3 mg/d plus ketotifen 1 mg once daily; Group 3: obese patients with type 2 diabetes treated with glimepiride 3 mg/d plus ketotifen 1 mg twice daily; M: male; F: female; BMI: body mass index; FBG: fasting blood glucose; HbA1c: hemoglobin A1c.
method (Watson, 1960), triglycerides (TGs) which was measured by enzymatic-colorimetric method (Fossati & Prencipe, 1982) and high density lipoprotein (HDL-C) which was determined by precipitation method (Warnick & Wood, 1995) using commercial kits (BioMed, Germany). Low-density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald formula (Friedewald, Levy, & Fredrickson, 1972) where LDL-C = [TC − HDL-C − (TGs/5)] provided that TGs level is less than 400 mg/dl (Friedewald et al., 1972). 2.6. Statistical analysis Data were analyzed using SPSS statistical package version 22.0, IBM Corporation Software Group, USA. Paired Student's t-test was used to assess any significant difference between each group at baseline and after 12 weeks of treatment course. One way analysis of variance (ANOVA) test followed by Bonferroni or Tamhane tests was used to assess any significant difference among the three groups at baseline and after 12 weeks. Values were presented as mean ± standard deviation (SD). Pearson's correlation test was used to assess the correlation between measured parameters after the effective intervention. Fisher's exact test was used for statistical analysis of the reported side effects. All p values were two-tailed and p b 0.05 was considered significant for statistical analysis. 3. Results 3.1. Characteristics of patients At baseline, there were no significant differences between group 1 treated with glimepiride alone and intervention groups (group 2 and group 3) treated with glimepiride plus ketotifen once and twice daily respectively), in demographic or anthropometric parameters (Table 1). 3.2. Effect of ketotifen on glycemic and metabolic parameters A summary of the mean ± SD values of variables at baseline and after 12 weeks in all groups is presented in (Table 2). After 12 weeks of co-treatment with glimepiride plus ketotifen 1 mg twice daily (group 3), there were significant decreases in BMI, FBG, HbA1c, TC, TGs and LDL-C. While there was a significant increase in HDL, there was no significant change in plasma adiponectin level. For groups 1 and 2 treated with glimepiride alone or plus ketotifen once daily), there were no significant differences in these outcome variables measured over the intervention period. The comparison of the three groups before and 12 weeks after treatment revealed that, there were no significant differences in measured parameters between all groups at baseline. After 12 weeks,
Please cite this article as: El-Haggar, S.M., et al., Effect of ketotifen in obese patients with type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.01.013
S.M. El-Haggar et al. / Journal of Diabetes and Its Complications xxx (2015) xxx–xxx
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Table 2 Clinical variables at baseline and after 12 weeks in the three groups. Variables
Group 1 (n = 16) Baseline
BMI (kg/m2) FBG (mg/dl) (mmol/l) Adiponectin (μg/ml) TC (mg/dl) (mmol/l) TGs (mg/dl) (mmol/l) HDL-C (mg/dl) (mmol/l) LDL-C (mg/dl) (mmol/l) HbA1c % (mmol/mol)
37.3 190.3 10.6 6.8 140.2 3.6 123.2 1.4 32.4 0.84 83.1 2.2 7.9 (63
± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.4 50.6 2.8 1.1 20.7 0.54 20.1 0.23 6.6 0.17 17.2 0.45 0.96 10.5)
Group 2 (n = 16) 12 weeks
Baseline
37.6 207.7 11.5 6.9 147.8 8.7 127.8 1.5 31.9 0.83 87.8 2.3 8.7 (72
37.5 205.9 11.4 6.8 151.7 3.9 105.5 1.2 30.3 0.78 100.3 2.6 8 (64
± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.3 47.6 2.6 0.8 25 0.65 22.1 0.25 8.1 0.2 28.2 0.73 0.93 10.2)
± ± ± ± ± ± ± ± ± ± ± ± ± ±
6.1 38.1 2.1 1.2 30.1 0.78 30 0.34 2.7 0.07 27.4 0.71 0.76 8.3)
Group 3 (n = 16) 12 weeks
Baseline
37.6 199 11 6.8 146.4 3.8 97.3 1.1 31.2 0.81 95.8 2.5 8.2 (66
37.8 183.2 10.2 7.24 146.7 3.8 116.1 1.3 30.9 0.8 92.8 2.4 7.6 (60
± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.8 38 2.1 1.2 31.1 0.8 31.1c 0.35c 4.3 0.11 28.7 0.74 0.82 9)
± ± ± ± ± ± ± ± ± ± ± ± ± ±
12 weeks 5.3 52 2.9 1.2 29.9 0.77 42.6 0.49 4 0.1 32.8 0.85 0.88 9.6)
37.2 147.9 8.2 7.3 129.3 3.3 97 1.1 38.5 0.99 71.26 1.9 7.1 (54
± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.9⁎ 39.3a,b,⁎ 2.2a,b,⁎ 1.3 28.3⁎⁎ 0.73⁎⁎ 38.4a,⁎⁎ 0.44a,⁎⁎ 3.9a,⁎⁎ 0.1a,⁎⁎ 27.9⁎⁎ 0.72⁎⁎ 0.86a,b,⁎ 9.4)a,b⁎
Data presented as mean ± SD. Group 1: obese patients with type 2 diabetes treated with glimepiride 3 mg/d alone; Group 2: obese patients with type 2 diabetes treated with glimepiride 3 mg/d plus ketotifen 1 mg once daily; Group 3: obese patients with type 2diabetes treated with glimepiride 3 mg/d plus ketotifen 1 mg twice daily. BMI: body mass index, FBG: fasting blood glucose, HDL-C: high density lipoprotein, LDL-C: low density lipoprotein, TC: total cholesterol, TGs: triglycerides, HbA1c: hemoglobin A1c. a Comparing group 3 with group 1. b Comparing group 3 with group 2. c Comparing group 2 with group 1. ⁎ p b 0.05, compared with baseline values (paired sample t-test). ⁎⁎ p b 0.01, compared with baseline values (paired sample t-test).
comparing group 3 with group 1, there was a significant decrease in FBG (p b 0.01), HbA1c (p b 0.01) and TGs (p b 0.05), and an increase in HDL-C (p b 0.05). Comparing group 2 with group 1, there was a significant decrease in TGs (p b 0.05) only. Comparing group 3 with group 2 there was a significant decrease in FBG (p b 0.01) and HbA1c (p b 0.01), and an increase in HDL-C (p b 0.01). 3.3. Effect of ketotifen on inflammatory biomarkers A summary of the mean ± SD values of variables at baseline and after 12 weeks in all groups is presented in (Table 3). After 12 weeks of co-treatment with glimepiride plus ketotifen twice daily (group 3), there were significant decreases in MCT, IL-6 and LTB4. After 12 weeks of co-treatment with glimepiride plus ketotifen once daily (group 2) there were significant decreases in IL-6 and LTB4. In group 1 treated with glimepiride alone), there were no significant changes in these biomarkers measured over the intervention period. The comparison of the three groups before and 12 weeks after treatment revealed that, there were no significant differences in these biomarkers between all groups at baseline. After 12 weeks, comparing group 3 with group 1 there was a significant decrease in IL-6 (p b 0.01), LTB4 (p b 0.01) and MCT (p b 0.05). Comparing group 2 with group 1 there was a significant decrease in IL-6 (p b 0.01) and
LTB4 (p b 0.01). Comparing group 3 with group 2 there was no significant difference in these biomarkers.
3.4. Correlation between significantly changed variables A correlation study between parameters in group 3 treated with glimepiride plus ketotifen 1 mg twice daily) revealed that, posttreatment FBG showed a significant positive correlation with TGs; TGs showed a significant positive correlation with IL-6, IL-6 showed a significant negative correlation with adiponectin and HbA1c showed a significant positive correlation with BMI as shown in Fig. 1.
3.5. Analysis of side effects and interaction Statistical analysis of the obtained data revealed that sedation and drowsiness were statistically significant and common side effects of the used drug especially at twice daily dose (p = 0.001), and also drug may to some extent lead to irregularity in heart beats (p = 0.041). Other reported side effects were considered nonstatistically significant and not related to the used drug. Also none of participants experienced elevation in liver enzymes as a side effect or thrombocytopenia as an interaction with glimepiride.
Table 3 Inflammation biomarkers at baseline and after 12 weeks in the three groups. Biomarkers
Group 1 (n = 16)
Group 2 (n = 16)
Group 3 (n = 16)
Baseline
12 weeks
Baseline
12 weeks
Baseline
12 weeks
MCT (ng/ml) IL-6 (pg/ml) LTB4 (pg/ml)
3.2 ± 1.2 2.9 ± 0.9 13 ± 4.4
4.3 ± 1.5 3.2 ± 1.2 15.7 ± 5.1
3.7 ± 1.2 2.6 ± 0.8 16.4 ± 5.3
3.5 ± 1.1 2.1 ± 0.7b,⁎ 11.5 ± 4.3b,⁎
3.9 ± 1.1 2.8 ± 0.7 16.5 ± 6.7
3.1 ± 0.7a,⁎ 2.1 ± 0.8a,⁎⁎ 8.7 ± 2.5a,⁎⁎
Data presented as mean ± SD. Group 1: obese patients with type 2 diabetes treated with glimepiride 3 mg/d alone; Group 2: obese patients with type 2 diabetes treated with glimepiride 3 mg/d plus ketotifen 1 mg once daily; Group 3: obese patients with type 2diabetes treated with glimepiride 3 mg/d plus ketotifen 1 mg twice daily. IL-6: interleukin-6, LTB4: leukotriene B4, MCT: mast cell tryptase. ⁎ p b 0.05, compared with baseline values (paired sample t-test). ⁎⁎ p b 0.01, compared with baseline values (paired sample t-test). a Comparing group 3 with group 1. b Comparing group 2 with group 1.
Please cite this article as: El-Haggar, S.M., et al., Effect of ketotifen in obese patients with type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.01.013
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S.M. El-Haggar et al. / Journal of Diabetes and Its Complications xxx (2015) xxx–xxx
Fig. 1. Correlation between measured parameters after 12 weeks in group 3 treated with glimepiride plus ketotifen 1 mg twice daily. (a) Positive correlation between fasting blood glucose (FBG) and triglycerides (TGs). (b) Positive correlation between TGs and interleukin-6 (IL-6). (c) Negative correlation between adiponectin and IL-6. (d) Positive correlation between hemoglobin A1c (HbA1c) and body mass index (BMI).
Percentage of reported side effects in different study groups is shown in Fig. 2. 4. Discussion The present study evaluates whether the prescribed mast cell stabilizer (ketotifen) could have an impact on glycemia in obese patients with T2DM. Results of our study showed that twice daily dose showed a significant reduction in FBG and HbA1c % without a significant difference in BMI. Recent studies on animals are consistent with the effect observed in glycemic control but not the effect on BMI. Studies on mice that examined the effect of either MCs stabilization or deficiency on obesity and diabetes in mice emphasized that stabilization with cromolyn (another mast cell stabilizer) or ketotifen could
Fig. 2. Percentage of reported side effects in different study groups treated with glimepiride alone (group 1) or in combination with ketotifen 1 mg once (group 2) and twice daily (group 3).
significantly reduce body weight gain and improve glucose and insulin tolerance; also MC deficient mice gained significant less body weight than wild type and demonstrated greater glucose tolerance (Liu et al., 2009). Shi (unpublished data) showed that patients with T2DM showed improvement in serum glucose and HbA1c after few months of treatment with oral cromolyn (Wang & Shi, 2011). Results obtained from rats with diabetes treated with ketotifen also suppose these findings (Zi-miao, Lian-song, Mei-rong, Liang-liang, & Wan-li, 2013). On the other hand, studies on human that discussed weight change showed that weight gain is a common side effect in patients treated with ketotifen (Canny, Reisman, & Levison, 1997; Ockenga et al., 1996). Asl, Vaez, Imankhah, and Hamidi (2014) who demonstrated that ketotifen induced weight loss on mice also discussed the opposing effects observed in human using ketotifen which sometimes causes weight gain or no change in weight, and in animals themselves, different effects at different doses were observed in mice (Nemati, Habibi, & Sharifi, 2006). So the effect of ketotifen on weight is controversial and this may be due to different mechanisms at different doses (Nemati et al., 2006) or difference in metabolic pathway between animal and human (Asl et al., 2014), therefore further studies should be carried out to investigate the exact effect on BMI. Our study showed a positive correlation between BMI and HbA1c which is confirmed by the link between obesity and diabetes discussed by Klein et al. (2004) who demonstrated that, weight management by lifestyle modification can help in T2DM control, also by Sluik et al. (2011) who demonstrated the association between abdominal adiposity and T2DM and by Mechanick, Garber, Handelsman, and Garvey (2012) who demonstrated that obesity is a disease with genetic, environmental, and behavioral determinants that confer increased morbidity and mortality risk in patients with T2DM. Mast cells (MCs) are tissue-resident inflammatory regulators, steering the course of the local immune reaction toward different directions. There are two types of MC based on the expression profile of serine proteases—MCTs are mucosal MCs expressing only tryptase and MCTCs (mast cell tryptase/chymase) express both chymase and
Please cite this article as: El-Haggar, S.M., et al., Effect of ketotifen in obese patients with type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.01.013
S.M. El-Haggar et al. / Journal of Diabetes and Its Complications xxx (2015) xxx–xxx
tryptase (Chmelar, Chung, & Chavakis, 2013). They are normal residents within the pancreatic ducts in close proximity to the pancreatic islets and are associated with inflammatory conditions of the pancreas (Christy & Brown, 2007). Mast cells contribute directly to insulin resistance and type 2 diabetes (Liu et al., 2009), promoting diet-induced obesity and glucose intolerance by production of IL6 and IFN-γ (Liu et al., 2009). Our study showed that treatment with glimepiride plus ketotifen twice daily decreased the level of MCT which proves that ketotifen works through MC stabilization. This result is matched with the effect of ketotifen observed on MCT in rabbits (Monument et al., 2012) and on chymase (another mast cell protease) in rats (Sánchez-Patán, Aller, et al., 2008) and on ocular tryptase in human (Schoch, 2003). Elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and triglycerides (TGs) and low levels of high-density lipoprotein cholesterol (HDL-C) are strongly associated with increased risk for macrovascular events (e.g., myocardial infarction, ischemic stroke, and coronary mortality) among patients with T2DM (Toth et al., 2012). Our results showed that treatment with glimepiride plus ketotifen twice daily significantly decreased TGs and increased HDL-C. This observed decrease in TGs is consistent with result achieved in rats with T2DM treated with ketotifen (Zi-miao et al., 2013). Also, the observed decrease in TGs and increase in HDL-C are consistent with result achieved in mice with atherogenesis treated with cromolyn (Wang et al., 2013), but not consistent with the reductive effect on LDL-C as observed in these studies. Our correlation results indicated a positive correlation between TGs and FBG. This result is matched with Abdel-Gayoum (2004) who demonstrated that improvement in glycemia control in T2DM patients results in a lower TGs level. Adiponectin is highly expressed by adipocytes with potent anti-inflammatory properties (Kwon & Pessin, 2013). Adiponectin decreases insulin resistance and body weight by increasing lipid oxidation in muscles and other organs, such as the pancreas and liver (Jee et al., 2013). Our results showed that the adiponectin was no't changed after ketotifen intake. This result is in agreement with results achieved in mice (Wang & Shi, 2011). Other studies on human found that adiponectin did not significantly change after caloric restriction (Imbeault, Pomerleau, Harper, & Doucet, 2004), and not significantly correlate with insulin resistance (Silha et al., 2003). This may be due to the complicated configuration of adiponectin (high, moderate and low molecular weight) with different behaviors (Tilg & Moschen, 2008), which makes the available analytic methods such as ELISA and RIA, less specificity and we really do not know what we are exactly measuring (Sun et al., 2009). Interleukin-6 (IL-6) is an inflammatory mediator produced by macrophages in the adipose tissue (Vrachnis et al., 2012). It is one of mast cell cytokine mediators (Christy & Brown, 2007; Wang & Shi, 2011; Zhang & Shi, 2012); this cytokine is increased in obesity and has multiple effects on insulin sensitivity in muscles, liver, or beta cells of the pancreas, ultimately leading to insulin resistance (Vrachnis et al., 2012). Our results showed that treatment with glimepiride plus ketotifen twice daily reverses obesity-related increases in IL-6 in obese patients with T2DM. This effect is consistent with the result obtained from obese mice with T2DM (Liu et al., 2009), human conjunctival cells (Pauly, Blondin, Riancho, & Baudouin, 2004) and rats with T2DM (Zi-miao et al., 2013). Our correlation results indicated a positive correlation between IL-6 and TGs. This result is matched with results achieved in IL-6 deficient female mice (Wallenius et al., 2002), but did not match with the results achieved in IL-6 knockout mice in the study of Di Gregorio, Hensley, Lu, Ranganathan, and Kern (2004) which demonstrated that there were no differences in any of the parameters associated with lipid metabolism. Our study also indicated a negative correlation between IL-6 and adiponectin. This result is consistent with Nishida, Funahashi, and Shimomura (2007) who observed an inverse correla-
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tion between adiponectin and inflammatory markers such as IL-6, and with Im et al. (2006) who demonstrated that adiponectin attenuates the production and action of tumor necrosis factor α, which may influence IL-6. Also this result is in agreement with Di Gregorio et al. (2004) who demonstrated that IL-6 knockout mice showed adiponectin increase, and with results achieved in obese women (Esposito et al., 2003) and in subjects with diabetic foot where IL-6 was positively correlated with clinical and laboratory variables with which adiponectin was negatively correlated (Tuttolomondo et al., 2010). Leukotriene B4 (LTB4) is a proinflammatory lipid mediator generated from arachidonic acid through the sequential activities of 5-lipoxygenase, 5-lipoxygenase-activating protein, and leukotriene A4 hydrolase (Spite et al., 2011). It is released from MCs to increase migration of other cells (Christy & Brown, 2007). The potent biological actions of LTB4 are mediated primarily through a high-affinity interaction with a G protein-coupled receptor termed BLT-1 and it was reported that deficiency of BLT-1 protects against the development of insulin resistance in diet-induced obesity (DIO) by regulating ATM (adipose tissue macrophages) accumulation and inflammation in insulin-sensitive tissues (Spite et al., 2011). Our results showed that treatment with glimepiride plus ketotifen twice daily reduced the level of LTB4 in obese patients with T2DM. This result is in agreement with Eliakim, Karmeli, Okon, and Rachmilewitz (1992) who demonstrated that mucosal LTB4 generation was significantly reduced in ketotifen treated rats three weeks after mucosal damage induction. On the contrary, Sánchez-Patán, Anchuelo, et al. (2008) demonestrate that LTB4 increased in rats with portal hypertention treated with ketotifen. However, once daily dose has a significant reductive effect on inflammatory biomarkers only without exerting any significant effect on metabolic or glycemic parameters; this may be due to the low dose which was not sufficient to produce clinical effect, and this dose dependent effect of ketotifen is observed in previous studies on weight (Nemati et al., 2006), IL-6 (Pauly et al., 2004), histamine and tryptase release (Schoch, 2003). Our study results showed that sedation is a common side effect of ketotifen which is consistent with other studies which demonstrated that ketotifen increased the risk of sedation (Canny et al., 1997; Schwarzer, Bassler, Mitra, Ducharme, & Forster, 2004). The observed irregularity in heart beats was not reported before and may be related to the anticholinergic side effects of ketotifen. These observations implicate that ketotifen and other MC stabilizers could be a therapeutic drug target to treat obese subjects with T2DM and prevent complications. The limitation of our study includes the relatively small numbers of patients investigated. Also the common sedation limits the ability to increase dose and/or frequency. Furthermore, inability to measure other useful parameters is also a limitation.
5. Conclusion The present study demonstrated that MC stabilization with ketotifen could improve glycemic control in T2DM patients, and so ketotifen could be used as new line of treatment especially for uncontrolled patients or whom other modalities are unsuitable.
5.1. Recommendations Mast cell stabilization is a promising line for research. In this trend, higher doses of ketotifen should be studied to observe the possible effect at high dose and try to find a solution for the common sedation; other mast cell stabilizers should be also examined. Also other useful parameters should be measured like fasting insulin for HOMA-IR calculation which would be very useful for evaluating the effect on insulin sensitivity.
Please cite this article as: El-Haggar, S.M., et al., Effect of ketotifen in obese patients with type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.01.013
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Please cite this article as: El-Haggar, S.M., et al., Effect of ketotifen in obese patients with type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.01.013
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Effect of ketotifen in obese patients with type 2 diabetes mellitus, Sahar M. El-Haggar a, Wael F. Farrag b, Fedaa A. Kotkata a,⁎ a b
Clinical Pharmacy Department, Faculty of Pharmacy, Tanta University, Egypt Internal Medicine Department, Faculty of Medicine, Tanta University, Egypt
a r t i c l e
i n f o
Article history: Received 10 December 2014 Received in revised form 25 January 2015 Accepted 28 January 2015 Available online xxxx Keywords: T2DM Mast cells Ketotifen Obesity Inflammation
a b s t r a c t Aim: Mast cells are found to be an important contributor in obesity induced insulin resistance. We evaluate the effect of ketotifen in obese patient with type 2 diabetes (T2DM) treated with glimepiride. Method: In a randomized controlled study we recruited forty-eight obese patients with T2DM from Internal Medicine Department at Tanta University Hospital, Egypt. They were classified into three groups: group 1, those who received glimepiride (GL) 3 mg/d alone; group 2, those who received GL 3 mg/d + ketotifen 1 mg once daily; and group 3, those who received GL 3 mg/d + ketotifen 1 mg twice daily. Fasting blood samples were obtained before and 12 weeks after treatment for biochemical analysis of glycemic and inflammatory biomarkers. Data were statistically analyzed by paired Student's t-test and one way analysis of variance; p b 0.05 was considered statistically significant. Results: The obtained data suggested that the addition of ketotifen in twice daily dose has a beneficial effect on all measured parameters except adiponectin. However, glimepiride plus ketotifen once daily only affected the level of inflammatory biomarkers without any significant effect on other parameters. Conclusions: The co-administration of ketotifen twice daily plus glimepiride improves glycemic and inflammatory process in obese patients with T2DM. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Obesity is a major risk factor for insulin resistance and type 2 diabetes mellitus (T2DM). Although how obesity promotes insulin resistance is unclear, the inflammatory response is thought to be a potentially important mechanism, which could alter adipose tissue function, thus leading to systemic insulin resistance. The development of insulin resistance is associated with pro-inflammatory cytokines produced by infiltrating leukocytes and resident adipocytes within the adipose tissue in obese subjects (Sismanopoulos et al., 2013). A previous report has demonstrated that mast cells (MCs) are important inflammatory cells that participate in immune responses during allergic reactions. Recent studies, however, suggest that these cells also participate in other inflammatory diseases, such as cancers, inflammatory bowel disease, metabolic bone disease, renal injury, arthritis, atherosclerosis, abdominal aortic aneurysms, obesity, and diabetes (Wang & Shi, 2011). Higher numbers of mast cells were found in Wight adipose tissue from obese subjects compared with that of lean subjects; obese subjects also had significantly higher mast cell tryptase (MCT) concentrations in their serum than did lean
individuals. These observations suggest a possible association between mast cells and obesity (Liu et al., 2009). Ketotifen is a mast cell stabilizer used as anti-allergic drug. The most important finding of recent studies is that mast cell stabilization with cromolyn or ketotifen reduces body weight gain and improves glucose and insulin tolerance in mice without noticeable toxicity (Wang & Shi, 2011). Although the precise roles of mast cell interleukin-6 (IL-6) and interferon gamma (IFN-γ) in obesity and diabetes remain incompletely understood, the absence of these inflammatory cytokines in mast cells reduces body weight gain, glucose tolerance, and serum levels of insulin, and glucose (Wang & Shi, 2011). No clear data on the effect of mast cell stabilizers on human were reported, so the present study was designed to investigate the effect of ketotifen on glycemic parameters (fasting blood glucose (FBG) and hemoglobin A1c (HbA1c)) and some inflammatory biomarkers (IL-6 and leukotriene B4 (LTB4)) in obese patients with T2DM. 2. Patients and methods 2.1. Patients
Conflict of interest: There are no conflicts of interest. There is no source of financial support for the research (no fund from any organization). ⁎ Corresponding author at: 31111. Tel.: +20 1017872669; fax: +20 403335466. E-mail address: [email protected] (F.A. Kotkata).
From January 2013 to April 2014, we recruited forty-eight obese patients with T2DM from internal medicine department at Tanta University Hospital, Egypt. The inclusion criteria were patients who had
http://dx.doi.org/10.1016/j.jdiacomp.2015.01.013 1056-8727/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: El-Haggar, S.M., et al., Effect of ketotifen in obese patients with type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.01.013
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body mass index (BMI) ≥ 30 kg/m 2, had diabetes duration from 5 to 10 years, were treated with glimepiride alone and had ages ranging from 45 to 55 years. The exclusion criteria were patients who had any other inflammatory disease (bone, asthma, etc.), severe hepatic or renal disease, epilepsy and also pregnant or lactating females. 2.2. Study design A total number of 48 patients who fulfilled the selection criteria were enrolled in the study. The study has been approved by the national research ethics committee (Tanta University ethical committee) and has been performed in accordance with the ethical standards as laid down in the 1964 declaration of Helsinki and its later amendments or comparable ethical standards. An informed written consent was obtained from all patients included in the study. The study design was a parallel randomized controlled study to compare the effect of two different doses (once and twice daily) of ketotifen (Ketoti®, Pharco, Egypt) in addition to glimepiride (GL) (Amaryl®, Sanofi-Aventis, Berlin, Germany). The patients were divided into three groups (each group [n = 16]) as follows: group 1, those who received glimepiride (GL) 3 mg/d alone; group 2, those who received GL 3 mg/d plus ketotifen 1 mg once daily; and group 3, those who received GL 3 mg/d plus ketotifen 1 mg twice daily for 12 weeks. Patients were followed up at monthly intervals for assessment of compliance to the study medication and adverse events. All blood samples were obtained after a 10- to 12-hours fasting period. Blood samples were collected in tubes containing EDTA and centrifuged immediately. Plasma was separated, coded and stored at − 80 °C until analysis. 2.3. Demographic characters Patients' medical history was taken to ensure the absence of any interacting or interfering drugs. Demographic data were collected at baseline using questionnaire. Information collected included age, sex, diabetes duration, BMI, FBG and HbA1c (Table 1). 2.4. Anthropometric evaluations Weights were measured and recorded to the nearest 0.5 kg. Body heights were measured and recorded to the nearest centimeter. Body mass index was calculated which is defined as the weight in kilograms divided by the square of the height in meters; i.e., BMI = weight (kg)/height2 (m). Height and weight were measured using Detecto scale (Detecto Company, 203 East Daugherty Sheet, USA). 2.5. Biochemical assays 2.5.1. Blood glucose and hemoglobin A1c Fasting blood glucose (FBG) levels were assayed using glucose oxidase method (Kaplan, 1984) (Spinreact, Spain). Hemoglobin A1c% (HbA1c %) was assayed by ion exchange method (Bissé & Abraham, 1985) (Biosystems, Spain). 2.5.2. Assay of adiponectin, interleukin-6 (IL-6), leukotriene B4 (LTB4) and mast cell tryptase (MCT) Enzyme-linked immunosorbent assay (ELISA) using commercial kits was carried out according to the manufacturers' instructions for assay of plasma adiponectin (Assaypro, USA), MCT (Wuhan Elaab Science Company, China), IL-6 Platinum ELISA (eBioscience, San Diego) and LTB4 (R&D Systems, USA). 2.5.3. Lipid panel Plasma was used for determination of lipid profiles including total cholesterol (TC) which was measured by enzymatic colorimetric
Table 1 Demographic data of patients at baseline. Characteristics
Group 1
Group 2
Group 3
Number Sex (M/F) Age (years) Diabetes duration BMI (kg/m2) FBG (mg/dl) (mmol/l) HbA1c % (mmol/mol)
16 3/13 51.3 ± 4.5 7.9 ± 2.5 37.3 ± 5.4 190.3 ± 50.6 (10.6 ± 2.8) 7.9 ± 0.96 (63 ± 10.5)
16 3/13 50.1 ± 4.6 7.1 ± 2.7 37.5 ± 6.1 205.9 ± 38.1 (11.4 ± 2.1) 8 ± 0.76 (64 ± 8.3)
16 4/12 49.1 ± 4.9 8 ± 2.5 37.8 ± 5.3 183.2 ± 52 (10.2 ± 2.9) 7.6 ± 0.88 (60 ± 9.6)
Data presented as mean ± SD. Group 1: obese patients with type 2 diabetes treated with glimepiride 3 mg/d alone; Group 2: obese patients with type 2 diabetes treated with glimepiride 3 mg/d plus ketotifen 1 mg once daily; Group 3: obese patients with type 2 diabetes treated with glimepiride 3 mg/d plus ketotifen 1 mg twice daily; M: male; F: female; BMI: body mass index; FBG: fasting blood glucose; HbA1c: hemoglobin A1c.
method (Watson, 1960), triglycerides (TGs) which was measured by enzymatic-colorimetric method (Fossati & Prencipe, 1982) and high density lipoprotein (HDL-C) which was determined by precipitation method (Warnick & Wood, 1995) using commercial kits (BioMed, Germany). Low-density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald formula (Friedewald, Levy, & Fredrickson, 1972) where LDL-C = [TC − HDL-C − (TGs/5)] provided that TGs level is less than 400 mg/dl (Friedewald et al., 1972). 2.6. Statistical analysis Data were analyzed using SPSS statistical package version 22.0, IBM Corporation Software Group, USA. Paired Student's t-test was used to assess any significant difference between each group at baseline and after 12 weeks of treatment course. One way analysis of variance (ANOVA) test followed by Bonferroni or Tamhane tests was used to assess any significant difference among the three groups at baseline and after 12 weeks. Values were presented as mean ± standard deviation (SD). Pearson's correlation test was used to assess the correlation between measured parameters after the effective intervention. Fisher's exact test was used for statistical analysis of the reported side effects. All p values were two-tailed and p b 0.05 was considered significant for statistical analysis. 3. Results 3.1. Characteristics of patients At baseline, there were no significant differences between group 1 treated with glimepiride alone and intervention groups (group 2 and group 3) treated with glimepiride plus ketotifen once and twice daily respectively), in demographic or anthropometric parameters (Table 1). 3.2. Effect of ketotifen on glycemic and metabolic parameters A summary of the mean ± SD values of variables at baseline and after 12 weeks in all groups is presented in (Table 2). After 12 weeks of co-treatment with glimepiride plus ketotifen 1 mg twice daily (group 3), there were significant decreases in BMI, FBG, HbA1c, TC, TGs and LDL-C. While there was a significant increase in HDL, there was no significant change in plasma adiponectin level. For groups 1 and 2 treated with glimepiride alone or plus ketotifen once daily), there were no significant differences in these outcome variables measured over the intervention period. The comparison of the three groups before and 12 weeks after treatment revealed that, there were no significant differences in measured parameters between all groups at baseline. After 12 weeks,
Please cite this article as: El-Haggar, S.M., et al., Effect of ketotifen in obese patients with type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.01.013
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Table 2 Clinical variables at baseline and after 12 weeks in the three groups. Variables
Group 1 (n = 16) Baseline
BMI (kg/m2) FBG (mg/dl) (mmol/l) Adiponectin (μg/ml) TC (mg/dl) (mmol/l) TGs (mg/dl) (mmol/l) HDL-C (mg/dl) (mmol/l) LDL-C (mg/dl) (mmol/l) HbA1c % (mmol/mol)
37.3 190.3 10.6 6.8 140.2 3.6 123.2 1.4 32.4 0.84 83.1 2.2 7.9 (63
± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.4 50.6 2.8 1.1 20.7 0.54 20.1 0.23 6.6 0.17 17.2 0.45 0.96 10.5)
Group 2 (n = 16) 12 weeks
Baseline
37.6 207.7 11.5 6.9 147.8 8.7 127.8 1.5 31.9 0.83 87.8 2.3 8.7 (72
37.5 205.9 11.4 6.8 151.7 3.9 105.5 1.2 30.3 0.78 100.3 2.6 8 (64
± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.3 47.6 2.6 0.8 25 0.65 22.1 0.25 8.1 0.2 28.2 0.73 0.93 10.2)
± ± ± ± ± ± ± ± ± ± ± ± ± ±
6.1 38.1 2.1 1.2 30.1 0.78 30 0.34 2.7 0.07 27.4 0.71 0.76 8.3)
Group 3 (n = 16) 12 weeks
Baseline
37.6 199 11 6.8 146.4 3.8 97.3 1.1 31.2 0.81 95.8 2.5 8.2 (66
37.8 183.2 10.2 7.24 146.7 3.8 116.1 1.3 30.9 0.8 92.8 2.4 7.6 (60
± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.8 38 2.1 1.2 31.1 0.8 31.1c 0.35c 4.3 0.11 28.7 0.74 0.82 9)
± ± ± ± ± ± ± ± ± ± ± ± ± ±
12 weeks 5.3 52 2.9 1.2 29.9 0.77 42.6 0.49 4 0.1 32.8 0.85 0.88 9.6)
37.2 147.9 8.2 7.3 129.3 3.3 97 1.1 38.5 0.99 71.26 1.9 7.1 (54
± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.9⁎ 39.3a,b,⁎ 2.2a,b,⁎ 1.3 28.3⁎⁎ 0.73⁎⁎ 38.4a,⁎⁎ 0.44a,⁎⁎ 3.9a,⁎⁎ 0.1a,⁎⁎ 27.9⁎⁎ 0.72⁎⁎ 0.86a,b,⁎ 9.4)a,b⁎
Data presented as mean ± SD. Group 1: obese patients with type 2 diabetes treated with glimepiride 3 mg/d alone; Group 2: obese patients with type 2 diabetes treated with glimepiride 3 mg/d plus ketotifen 1 mg once daily; Group 3: obese patients with type 2diabetes treated with glimepiride 3 mg/d plus ketotifen 1 mg twice daily. BMI: body mass index, FBG: fasting blood glucose, HDL-C: high density lipoprotein, LDL-C: low density lipoprotein, TC: total cholesterol, TGs: triglycerides, HbA1c: hemoglobin A1c. a Comparing group 3 with group 1. b Comparing group 3 with group 2. c Comparing group 2 with group 1. ⁎ p b 0.05, compared with baseline values (paired sample t-test). ⁎⁎ p b 0.01, compared with baseline values (paired sample t-test).
comparing group 3 with group 1, there was a significant decrease in FBG (p b 0.01), HbA1c (p b 0.01) and TGs (p b 0.05), and an increase in HDL-C (p b 0.05). Comparing group 2 with group 1, there was a significant decrease in TGs (p b 0.05) only. Comparing group 3 with group 2 there was a significant decrease in FBG (p b 0.01) and HbA1c (p b 0.01), and an increase in HDL-C (p b 0.01). 3.3. Effect of ketotifen on inflammatory biomarkers A summary of the mean ± SD values of variables at baseline and after 12 weeks in all groups is presented in (Table 3). After 12 weeks of co-treatment with glimepiride plus ketotifen twice daily (group 3), there were significant decreases in MCT, IL-6 and LTB4. After 12 weeks of co-treatment with glimepiride plus ketotifen once daily (group 2) there were significant decreases in IL-6 and LTB4. In group 1 treated with glimepiride alone), there were no significant changes in these biomarkers measured over the intervention period. The comparison of the three groups before and 12 weeks after treatment revealed that, there were no significant differences in these biomarkers between all groups at baseline. After 12 weeks, comparing group 3 with group 1 there was a significant decrease in IL-6 (p b 0.01), LTB4 (p b 0.01) and MCT (p b 0.05). Comparing group 2 with group 1 there was a significant decrease in IL-6 (p b 0.01) and
LTB4 (p b 0.01). Comparing group 3 with group 2 there was no significant difference in these biomarkers.
3.4. Correlation between significantly changed variables A correlation study between parameters in group 3 treated with glimepiride plus ketotifen 1 mg twice daily) revealed that, posttreatment FBG showed a significant positive correlation with TGs; TGs showed a significant positive correlation with IL-6, IL-6 showed a significant negative correlation with adiponectin and HbA1c showed a significant positive correlation with BMI as shown in Fig. 1.
3.5. Analysis of side effects and interaction Statistical analysis of the obtained data revealed that sedation and drowsiness were statistically significant and common side effects of the used drug especially at twice daily dose (p = 0.001), and also drug may to some extent lead to irregularity in heart beats (p = 0.041). Other reported side effects were considered nonstatistically significant and not related to the used drug. Also none of participants experienced elevation in liver enzymes as a side effect or thrombocytopenia as an interaction with glimepiride.
Table 3 Inflammation biomarkers at baseline and after 12 weeks in the three groups. Biomarkers
Group 1 (n = 16)
Group 2 (n = 16)
Group 3 (n = 16)
Baseline
12 weeks
Baseline
12 weeks
Baseline
12 weeks
MCT (ng/ml) IL-6 (pg/ml) LTB4 (pg/ml)
3.2 ± 1.2 2.9 ± 0.9 13 ± 4.4
4.3 ± 1.5 3.2 ± 1.2 15.7 ± 5.1
3.7 ± 1.2 2.6 ± 0.8 16.4 ± 5.3
3.5 ± 1.1 2.1 ± 0.7b,⁎ 11.5 ± 4.3b,⁎
3.9 ± 1.1 2.8 ± 0.7 16.5 ± 6.7
3.1 ± 0.7a,⁎ 2.1 ± 0.8a,⁎⁎ 8.7 ± 2.5a,⁎⁎
Data presented as mean ± SD. Group 1: obese patients with type 2 diabetes treated with glimepiride 3 mg/d alone; Group 2: obese patients with type 2 diabetes treated with glimepiride 3 mg/d plus ketotifen 1 mg once daily; Group 3: obese patients with type 2diabetes treated with glimepiride 3 mg/d plus ketotifen 1 mg twice daily. IL-6: interleukin-6, LTB4: leukotriene B4, MCT: mast cell tryptase. ⁎ p b 0.05, compared with baseline values (paired sample t-test). ⁎⁎ p b 0.01, compared with baseline values (paired sample t-test). a Comparing group 3 with group 1. b Comparing group 2 with group 1.
Please cite this article as: El-Haggar, S.M., et al., Effect of ketotifen in obese patients with type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.01.013
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Fig. 1. Correlation between measured parameters after 12 weeks in group 3 treated with glimepiride plus ketotifen 1 mg twice daily. (a) Positive correlation between fasting blood glucose (FBG) and triglycerides (TGs). (b) Positive correlation between TGs and interleukin-6 (IL-6). (c) Negative correlation between adiponectin and IL-6. (d) Positive correlation between hemoglobin A1c (HbA1c) and body mass index (BMI).
Percentage of reported side effects in different study groups is shown in Fig. 2. 4. Discussion The present study evaluates whether the prescribed mast cell stabilizer (ketotifen) could have an impact on glycemia in obese patients with T2DM. Results of our study showed that twice daily dose showed a significant reduction in FBG and HbA1c % without a significant difference in BMI. Recent studies on animals are consistent with the effect observed in glycemic control but not the effect on BMI. Studies on mice that examined the effect of either MCs stabilization or deficiency on obesity and diabetes in mice emphasized that stabilization with cromolyn (another mast cell stabilizer) or ketotifen could
Fig. 2. Percentage of reported side effects in different study groups treated with glimepiride alone (group 1) or in combination with ketotifen 1 mg once (group 2) and twice daily (group 3).
significantly reduce body weight gain and improve glucose and insulin tolerance; also MC deficient mice gained significant less body weight than wild type and demonstrated greater glucose tolerance (Liu et al., 2009). Shi (unpublished data) showed that patients with T2DM showed improvement in serum glucose and HbA1c after few months of treatment with oral cromolyn (Wang & Shi, 2011). Results obtained from rats with diabetes treated with ketotifen also suppose these findings (Zi-miao, Lian-song, Mei-rong, Liang-liang, & Wan-li, 2013). On the other hand, studies on human that discussed weight change showed that weight gain is a common side effect in patients treated with ketotifen (Canny, Reisman, & Levison, 1997; Ockenga et al., 1996). Asl, Vaez, Imankhah, and Hamidi (2014) who demonstrated that ketotifen induced weight loss on mice also discussed the opposing effects observed in human using ketotifen which sometimes causes weight gain or no change in weight, and in animals themselves, different effects at different doses were observed in mice (Nemati, Habibi, & Sharifi, 2006). So the effect of ketotifen on weight is controversial and this may be due to different mechanisms at different doses (Nemati et al., 2006) or difference in metabolic pathway between animal and human (Asl et al., 2014), therefore further studies should be carried out to investigate the exact effect on BMI. Our study showed a positive correlation between BMI and HbA1c which is confirmed by the link between obesity and diabetes discussed by Klein et al. (2004) who demonstrated that, weight management by lifestyle modification can help in T2DM control, also by Sluik et al. (2011) who demonstrated the association between abdominal adiposity and T2DM and by Mechanick, Garber, Handelsman, and Garvey (2012) who demonstrated that obesity is a disease with genetic, environmental, and behavioral determinants that confer increased morbidity and mortality risk in patients with T2DM. Mast cells (MCs) are tissue-resident inflammatory regulators, steering the course of the local immune reaction toward different directions. There are two types of MC based on the expression profile of serine proteases—MCTs are mucosal MCs expressing only tryptase and MCTCs (mast cell tryptase/chymase) express both chymase and
Please cite this article as: El-Haggar, S.M., et al., Effect of ketotifen in obese patients with type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.01.013
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tryptase (Chmelar, Chung, & Chavakis, 2013). They are normal residents within the pancreatic ducts in close proximity to the pancreatic islets and are associated with inflammatory conditions of the pancreas (Christy & Brown, 2007). Mast cells contribute directly to insulin resistance and type 2 diabetes (Liu et al., 2009), promoting diet-induced obesity and glucose intolerance by production of IL6 and IFN-γ (Liu et al., 2009). Our study showed that treatment with glimepiride plus ketotifen twice daily decreased the level of MCT which proves that ketotifen works through MC stabilization. This result is matched with the effect of ketotifen observed on MCT in rabbits (Monument et al., 2012) and on chymase (another mast cell protease) in rats (Sánchez-Patán, Aller, et al., 2008) and on ocular tryptase in human (Schoch, 2003). Elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and triglycerides (TGs) and low levels of high-density lipoprotein cholesterol (HDL-C) are strongly associated with increased risk for macrovascular events (e.g., myocardial infarction, ischemic stroke, and coronary mortality) among patients with T2DM (Toth et al., 2012). Our results showed that treatment with glimepiride plus ketotifen twice daily significantly decreased TGs and increased HDL-C. This observed decrease in TGs is consistent with result achieved in rats with T2DM treated with ketotifen (Zi-miao et al., 2013). Also, the observed decrease in TGs and increase in HDL-C are consistent with result achieved in mice with atherogenesis treated with cromolyn (Wang et al., 2013), but not consistent with the reductive effect on LDL-C as observed in these studies. Our correlation results indicated a positive correlation between TGs and FBG. This result is matched with Abdel-Gayoum (2004) who demonstrated that improvement in glycemia control in T2DM patients results in a lower TGs level. Adiponectin is highly expressed by adipocytes with potent anti-inflammatory properties (Kwon & Pessin, 2013). Adiponectin decreases insulin resistance and body weight by increasing lipid oxidation in muscles and other organs, such as the pancreas and liver (Jee et al., 2013). Our results showed that the adiponectin was no't changed after ketotifen intake. This result is in agreement with results achieved in mice (Wang & Shi, 2011). Other studies on human found that adiponectin did not significantly change after caloric restriction (Imbeault, Pomerleau, Harper, & Doucet, 2004), and not significantly correlate with insulin resistance (Silha et al., 2003). This may be due to the complicated configuration of adiponectin (high, moderate and low molecular weight) with different behaviors (Tilg & Moschen, 2008), which makes the available analytic methods such as ELISA and RIA, less specificity and we really do not know what we are exactly measuring (Sun et al., 2009). Interleukin-6 (IL-6) is an inflammatory mediator produced by macrophages in the adipose tissue (Vrachnis et al., 2012). It is one of mast cell cytokine mediators (Christy & Brown, 2007; Wang & Shi, 2011; Zhang & Shi, 2012); this cytokine is increased in obesity and has multiple effects on insulin sensitivity in muscles, liver, or beta cells of the pancreas, ultimately leading to insulin resistance (Vrachnis et al., 2012). Our results showed that treatment with glimepiride plus ketotifen twice daily reverses obesity-related increases in IL-6 in obese patients with T2DM. This effect is consistent with the result obtained from obese mice with T2DM (Liu et al., 2009), human conjunctival cells (Pauly, Blondin, Riancho, & Baudouin, 2004) and rats with T2DM (Zi-miao et al., 2013). Our correlation results indicated a positive correlation between IL-6 and TGs. This result is matched with results achieved in IL-6 deficient female mice (Wallenius et al., 2002), but did not match with the results achieved in IL-6 knockout mice in the study of Di Gregorio, Hensley, Lu, Ranganathan, and Kern (2004) which demonstrated that there were no differences in any of the parameters associated with lipid metabolism. Our study also indicated a negative correlation between IL-6 and adiponectin. This result is consistent with Nishida, Funahashi, and Shimomura (2007) who observed an inverse correla-
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tion between adiponectin and inflammatory markers such as IL-6, and with Im et al. (2006) who demonstrated that adiponectin attenuates the production and action of tumor necrosis factor α, which may influence IL-6. Also this result is in agreement with Di Gregorio et al. (2004) who demonstrated that IL-6 knockout mice showed adiponectin increase, and with results achieved in obese women (Esposito et al., 2003) and in subjects with diabetic foot where IL-6 was positively correlated with clinical and laboratory variables with which adiponectin was negatively correlated (Tuttolomondo et al., 2010). Leukotriene B4 (LTB4) is a proinflammatory lipid mediator generated from arachidonic acid through the sequential activities of 5-lipoxygenase, 5-lipoxygenase-activating protein, and leukotriene A4 hydrolase (Spite et al., 2011). It is released from MCs to increase migration of other cells (Christy & Brown, 2007). The potent biological actions of LTB4 are mediated primarily through a high-affinity interaction with a G protein-coupled receptor termed BLT-1 and it was reported that deficiency of BLT-1 protects against the development of insulin resistance in diet-induced obesity (DIO) by regulating ATM (adipose tissue macrophages) accumulation and inflammation in insulin-sensitive tissues (Spite et al., 2011). Our results showed that treatment with glimepiride plus ketotifen twice daily reduced the level of LTB4 in obese patients with T2DM. This result is in agreement with Eliakim, Karmeli, Okon, and Rachmilewitz (1992) who demonstrated that mucosal LTB4 generation was significantly reduced in ketotifen treated rats three weeks after mucosal damage induction. On the contrary, Sánchez-Patán, Anchuelo, et al. (2008) demonestrate that LTB4 increased in rats with portal hypertention treated with ketotifen. However, once daily dose has a significant reductive effect on inflammatory biomarkers only without exerting any significant effect on metabolic or glycemic parameters; this may be due to the low dose which was not sufficient to produce clinical effect, and this dose dependent effect of ketotifen is observed in previous studies on weight (Nemati et al., 2006), IL-6 (Pauly et al., 2004), histamine and tryptase release (Schoch, 2003). Our study results showed that sedation is a common side effect of ketotifen which is consistent with other studies which demonstrated that ketotifen increased the risk of sedation (Canny et al., 1997; Schwarzer, Bassler, Mitra, Ducharme, & Forster, 2004). The observed irregularity in heart beats was not reported before and may be related to the anticholinergic side effects of ketotifen. These observations implicate that ketotifen and other MC stabilizers could be a therapeutic drug target to treat obese subjects with T2DM and prevent complications. The limitation of our study includes the relatively small numbers of patients investigated. Also the common sedation limits the ability to increase dose and/or frequency. Furthermore, inability to measure other useful parameters is also a limitation.
5. Conclusion The present study demonstrated that MC stabilization with ketotifen could improve glycemic control in T2DM patients, and so ketotifen could be used as new line of treatment especially for uncontrolled patients or whom other modalities are unsuitable.
5.1. Recommendations Mast cell stabilization is a promising line for research. In this trend, higher doses of ketotifen should be studied to observe the possible effect at high dose and try to find a solution for the common sedation; other mast cell stabilizers should be also examined. Also other useful parameters should be measured like fasting insulin for HOMA-IR calculation which would be very useful for evaluating the effect on insulin sensitivity.
Please cite this article as: El-Haggar, S.M., et al., Effect of ketotifen in obese patients with type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.01.013
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