A Pharmacological Analysis of Sodium-Glucose Cotransporter-2 Inhibitors for the Treatment of Diabetes and the Complications Associated

Jahnavi Dave¹, Pravin Tirgar¹, Bhoomi Patel²

¹Department of Pharmacology, School of Pharmacy, RK University, Rajkot - 360020, Gujarat, India.

²Department of Quality Assurance, School of Pharmacy, Rai University, Saroda,

Dholka Road, Ahmedabad - 382260, Gujarat, India.

*Corresponding Author E-mail: jdave785@rku.ac.in, pravin.tigar@rku.ac.in, bhoomipatel2512@gmail.com

ABSTRACT:

Background: Diabetes is a metabolic disorder, if not managed properly can lead to cardiovascular, cerebrovascular, nephropathy, neuropathy, retinopathy, cataract, and foot injury issues. New medicines are remain in demand despite the advent of biguanides, sulfonylureas, and thiazolidinediones. New oral anti-diabetic medications like sodium-glucose co-transporter inhibitors can ameliorates diabetes also the risk of diabetic cardiovascular complications. The goal of this study was to compare effect of SGLT2 inhibitors like Canagliflozin, Dapagliflozin, Empagliflozin, and Remogliflozin in an in-vivo model of diabetes and its major complications. Method: The Streptozotocin model was used to induce the diabetes and complications in rats. Various parameters were analysed for diabetes (blood glucose, HbA1C level) and its related complications like nephropathy (creatinine, CK-MB levels), neuropathy (Tail flick test) for cardiovascular complications lipids levels like LDL, VLDL, Cholesterol levels and monitoring blood pressure throughout the experiment, Retinopathy (Transparency of lens) At the end, histopathology of different organs were also studied. Results: All selected SGLT2 inhibitors, Canagliflozin, Dapagliflozin, Empagliflozin and Remogliflozin shown excellent in-vivo antioxidant potential and having protective effects against diabetes and its complications like Cardiovascular, nephropathy and Neuropathy. In our study among other SGLT2 inhibitors Remogliflozin showed significant effect managing blood glucose levels, HbA1C, creatinine, CK-MB, lipid levels, increased latency time in tail flick test and decrease in the progression of lens abnormalities and maturation of cataract.

KEYWORDS: Diabetes mellitus, Diabetic Complications, SGLT-2 inhibitors, Nephropathy, Neuropathy, Retinopathy.

INTRODUCTION:

Diabetes mellitus (DM) is a collection of metabolic illnesses. It is due to insulin resistance and deficiency¹. It can be due to some genetic factors and exacerbated by environmental variables such as lack of activity, excess weight, and mental anguish². Diabetes, if not properly managed, can cause variety of diseases, including retinopathy, nephropathy, and neuropathy, as well as macrovascular disorders like stroke, ischemic heart and peripheral vascular diseases^3-5. Diabetes was estimated to cause 1.6 million deaths in 2016.

Diabetes is a growing problem in India, with an average 8.7% diabetic population between the ages of 20 and 70^3,6. Diabetes mellitus can give indications like thirst, blurring of vision, and weight loss⁷. Primary prevention of type II diabetes is lifestyle changes pointed at weight control and increased physical activity⁸. Secondary prevention of diabetes can be done by controlling blood glucose levels⁹ by using medications like Metformin, which is the first drug that is used to treat type 2 diabetes¹⁰.

An SGLT-2 inhibitor or glucagon-like peptide 1 receptor agonist along with proven CVD treatment is advised as an aspect of the glucose-reducing treatment plan in patients with diabetes mellitus who have formed atherosclerotic CVD or heart failure and also severe renal failure^12-15. In people with T2DM, SGLT2 have become a class of glucose-lowering medications that also dramatically reduce CVD risk in T2DM^11,16. The SGLT-2, which promotes reabsorption of glucose in the kidney, is blocked by SGLT2 inhibitors¹⁷. which is a novel family of oral diabetes medications that decreases glucose levels in the blood by raising urine glucose excretion independently of insulin secretion or activity¹⁸. As a result, there is a reduction in renal glucose reabsorption, an improvement in renal glucose excretion, and a reduction in blood glucose levels¹⁹. The focus of the current investigation is on SGLT-2 inhibitors' preclinical applications in the treatment of diabetes and its related disorders^20,21.

MATERIALS AND METHODS:

Drugs and Chemicals: All of the essential chemicals were obtained from the Sigma-Aldrich Chemicals Co. in St. Louis, Missouri. These chemicals were of analytical grade and were used in all of the tests.

Animals and Diet: In the study, healthy Sprague-Dawley rats of either sex were used and housed under controlled temperature, humidity, and 12hr. of light and dark sets for throughout the experiment.

Experimental design: Streptozotocin Induced model: The institutional animal ethics committee will approve the experiment's protocol under its direction. I.P. injection of freshly prepared streptozotocin (60mg/kg) in 0.1mol/L citrate buffer (pH 4.5), after fasting for 12h was given to rats to induced diabetes^22,23. To reverse the drug-produced hypoglycaemia, the animals were given 5 % sucrose solution throughout the night. After three days of injection, rats with fasting blood glucose levels more than 200 mg/dL were deemed diabetic. The study began one week following STZ administration. Ten groups of eight Sprague Dawley rats (150 and 200g), were formed at random in accordance with:

Group I: Normal control	Group II: Diabetic control
Group III: Diabetic control treated with Canagliflozin (30 mg/kg)	Group IV: Diabetic control treated with Dapagliflozin (1 mg/kg)
Group V: Diabetic control treated with Empagliflozin (5mg/kg)	Group VI: Diabetic control treated with Remogliflozin (30mg/kg)
Group VII: Normal control treated with Canagliflozin (30 mg/kg)	Group VIII: Normal control treated with Dapagliflozin (1 mg/kg)
Group IX: Normal control treated with Empagliflozin (5mg/kg)	Group X: Normal control treated with Remogliflozin (30mg/kg)

At the end of the study, animals were euthanized using ether anaesthesia. The kidneys,pancreas brain and heart were excised and washed in a saline buffer (0.9% NaCl). The blood was collected for biochemical studies.

In vivo antioxidant activity of homogenate tissues:

After the completion of study, tissues were taken from the control and experimental groups. After homogenising the tissues in 0.1M Tris-HCl buffer having a pH of 7.4 and centrifuging them at 3000rounds per minute for 10minutes, the supernatant was filtered for further research after the tissues had been washed with cold water²².

Superoxide dismutase estimation:

Using 300micro-litre of 17mM sodium pyrophosphate buffer and 100 micro-litre of phenazine methosulphate, the amount of SOD activity was assessed. To acquire the enzyme in the top layer, the tissue was first centrifuged at 1500 rounds per minute for 10min, and then at 10,000rpm for 15 min. The supernatant was gathered, and 150micro-litre of it was transferred to an aliquot that included 0.5ml of 186μm phenazine methosulphate and 600μL of 0.052mM sodium pyrophosphate buffer (pH 7.0). Finally, 100μL of 780μM of NADH was added to initiate the enzymatic reaction. After 1 minute, adding 500micro-litre of glacial acetic acid ended the reaction. The colour intensity was measured using absorbance at 560nm^23,24.

Estimation of reduced glutathione (GSH):

The incorporation of 0.5ml of 4 percent sulfosalicylic acid precipitated the tissue homogenate (500 microlitre). The specimen were centrifuged for 20 minutes at 1200 rounds per minute after being kept for 1hour at 4°C. 33 microlitre of the supernatant was gathered, and aliquots of 900 microlitre of 0.1M potassium phosphate buffer and 66 microlitre of 100mM dithiobis (2-nitrobenzoic acid) (DTNB) were then added. DTNB and GSH combine to form a complex that is yellow in hue. At 412 nm, absorbance was measured^23,25.

Examination of tissues by histopathology:

Freshly prepared lung specimens were cut into puny sections and preserved in 10% formalin for 3–4hours. The preserved samples were washed using an escalating alcohol concentration (50, 70, 90 and 100percent). A 3–4 micrometer piece of the tissue was cut out and stained in haematoxylin and eosin for slide processing. Finally, slides were captured on camera using a light microscope (DIALUX 20 EB) set to 40X.

Blood Collection and Biochemical Assays:

Collected Blood samples were centrifuged at 3,000g for 10minutes at 4⁰C and used for the further analytical procedures. Biochemical markers of serum The hexokinase/glucose-6-phosphate dehydrogenase method was employed to assess the glucose level while fasting ²⁶. The levels of serum total cholesterol (TC), serum triglycerides (TG), LDL, HDL, VLDL and collagen level were measured using enzymatic-colorimetric techniques^27,28. Serum concentrations of CK-MB, lactate dehydrogenase and creatinine were measured using colorimetric techniques^29,30. Using commercial diagnostics kits (ERBA Diagnostics) and Spectrophotometer 1200 UNICO Instruments, Inc., Dayton, NJ, all serum parameters were evaluated.

Glycosylated Haemoglobin:

The concentration of glycosylated haemoglobin (HbA_1c) was determined using the Glycohemoglobin Reagent Set from Pointe Scientific Inc. (USA) in accordance with the manufacturer's instructions.

Non-invasive Blood pressure:

The blood pressure was determined using CODA monitor (Kent Scientific) and by placing the VPR sensor cuff through the tail to the base of tail without force. After all the animals have been placed in the holder and their tails were cuffed, they were allowed at least 5 min to acclimate the surroundings. Recorded the blood pressure of the animal. The blood pressure of animals should between 120 systolic and 80 diastolic mmHg³¹.

Slit lamp examination and cataract grading:

Eyes were examined every week using a slit lamp biomicroscope (Kowa Portable; Kowa, Ltd., Tokyo, Japan) on dilated pupils. Beginning and development of lens opacity was divided into five categories³⁷. Briefly, lens opacity was graded as clear - clear lenses with no vacuoles; stage 1 - vacuoles cover approximately one-half of the surface of the anterior pole; stage 2 - some vacuoles have disappeared and the cortex haziness; stage 3 - a hazy cortex remains and dense nuclear opacity is present; stage 4 - a mature cataract.

Tail Flick test:

The water bath was maintained at 52°C used to immerse the distal 2/3 of a tail of rat. The latency until withdrawal (rapid flick) was measured prior to drug treatment (baseline) and compared among treatment groups. Typically a 10 s cut off time was utilized in order to avoid damage to the tail. Temperatures should not increase over 55°C as this temperature will cause tissue damage if tails remain in the water over 10 s³².

Statistic evaluation: The computerised GraphPad Prism programme 5.0 was used to gather and analyse the experimental data from the experiments. Using Statistix 8, a one-way ANOVA was carried out to determine the impact of various drugs given to animals in in-vivo research. Tukey's multiple comparison test was used for comparison between treatments, with a probability level of less than equal to 0.01.

RESULTS:

Antioxidant Parameters:

There was significant decrease in GSH levels by using Empagliflozin in diabetic control rats with **p<0.01 in heart, kidney, brain and in eye with *p<0.05. In SOD parameters we have found that there is a increase in SOD levels with different anti-diabetic drug treated rats when equated to the diabetic control group. There was significant increase in SOD levels by using Remogliflozin in diabetic control rats with **p<0.01 in kidney, and in heart, brain and in eye with *p<0.05. In the diabetic control rat group, there was a constant upsurge in GSH levels during the whole study and decrease in the levels of SOD [Figure 1].

Nephropathy parameters: Results showed a decrease in creatinine levels, CK-MB (Creatine Kinase-Myoglobin Binding) level, Lactate Dehydrogenase (LDH) level in different anti-diabetic drug treated rats when equated with diabetic control group. There was significant decrease in creatinine levels by using Remogliflozin with *p<0.05, CK-MB levels with **p<0.01 and Lactate Dehydrogenase (LDH) with *p<0.05 in diabetic control rats. In the diabetic control rat group, there was a constant upsurge in creatinine levels, CK-MB and Lactate Dehydrogenase levels during the whole study [Figure 5].

Neuropathy Parameters: Results showed a substantial increase in the tail flick time period in different anti-diabetic drug treated rats when compared with diabetic control group. There was significant increase in tail flick time period by using Remogliflozin and Empagliflozin with *p<0.05 in diabetic control rats. In the diabetic control rat group, there was a constant decrease in tail flick time period during the whole study [Figure 6 (b)]. The onset of cataract was observed in diabetic control group and in control group all the lenses are appeared to be normal and free of opacities during the experimental period, there was significant decrease progression of lens abnormalities and maturation of cataract after the administration of various anti-diabetic drugs used in the study [Figure 6 (a)].

Figure 6: Neuropathy and Retinopathy Parameters

Histopathological Parameters: Hematoxylin-eosin staining of the LV sections of the diabetic groups revealed the features of diabetes cardiomyopathy [Figure 7]. In the LV myocardium of diabetic rat models, myofiber damage and rupture as well as myocardial deterioration could be seen. Normal glomerulus was found in the kidney histopathology of normal control rats when compared to diabetic control where glomerulus of a diabetic STZ rat with irregular capillaries and modest mesangial proliferation was found [Figure 8]. Comparing the diabetes control group to the normal control rats, histological analysis of the brain tissues in the diabetic control group showed substantial symptoms of cognitive deficits, with substantial damage and damage to hippocampal neurons, along with gliocyte proliferation [Figure 9]. After providing the aforementioned anti-diabetic medications, the degree of substantial damage, damage to hippocampal neurons, and gliocyte proliferation were considerably reduced.

DISCUSSION:

The body's oxidant radicals cause stress that has an impact on glucose metabolism. Complications result from an unwanted condition of hyperglycemia. Streptozotocin causes diabetes by creating free radicals and damaging the insulin-secreting islets cells of the pancreas, causing hyperglycemia and the complications, such as nephropathy, neuropathy, retinopathy, and different cardiac illnesses. The objective of the present research was to observe the protective effect of SGLT2 inhibitors Canagliflozin, Dapagliflozin, Empagliflozin, and Remogliflozin's in diabetes and complications. Both GSH and SOD concentrations are significant indicators of antioxidant activity, and they are related to glucose levels in diabetes mellitus³³. Comparing our data to previously published studies, there was a significant drop in GSH and an increase in SOD activity in the treatment groups, which relates to reduction of oxidative stress indirectly²⁶. This evidence shows that the antidiabetic activity of all the drugs used in the study may be due to their potent antioxidant potential that may help in the reduction of metabolic oxidative stress in the body.

In their MOA, SGLT2 inhibitors are highly distinctive since they enhance glycemic control by utilising glucosuria, which is often only seen as a side effect of hyperglycemia³⁴. It has been established that canagliflozin and other SGLT2 inhibitors are harmless, well-tolerated, and effective for the management of diabetes and its associated cardiovascular problems³⁵. Furthermore, Results showed that SGLT2 inhibitors offer benefits other than glycemic management; they maintain body weight and blood pressure, and it may be due to improve insulin sensitivity and beta-cell activity, along with minimizing vascular problems, which allow for a variety of combination therapies with other hypoglycemic medications. Due to these benefits, SGLT2 inhibitors are anticipated to be integrated into diabetes treatment methods and widely utilised as monotherapy and in amalgamation therapies. SGLT2 inhibitors appear to be promising for the treatment of diabetes and related conditions in animal and human research³⁶ and further research is needed to examine the advantages and dangers in bigger populations.

CONCLUSION:

With lifestyle changes and medicines, a considerable majority of people with diabetes mellitus fail to meet glycemic targets. Earlier several therapies have been shown to reduce blood glucose levels and lower the risk of diabetic problems. SGLT2 inhibitors are a prospective add-on to existing anti-diabetic medications for the reason that they target a pathophysiologic constituent of T2DM and decrease the reabsorption of glucose in the kidney. Alone or in combination with other diabetes medications, SGLT2 inhibitors enhance glycaemic control in an insulin-independent way. The results of a long-term trial (EMPA-REG OUTCOME^®) that looked at the cardiovascular properties of empagliflozin in over 7000 persons having T2DM and an elevated risk of CVD problems found that empagliflozin also reduced the risk of cardiovascular events. Also from present investigation, we can concluded that among other SGLT2 inhibitors, Remogliflozin significantly reduces the levels of blood glucose, HbA1C level, creatinine, CK-MB levels, Lipid levels and blood pressure and increased the latency time in tail flick test and in addition to that delayed progression and maturation of cataract was noticed in this study. In the histopathological study we have found the substantial decrease in all the disorders present in tissue samples of Brain, Heart and Kidney in comparison to the other said drugs of choice taken for the study. Research is also being conducted to assess the potency and safety of SGLT2 inhibitors in diabetic patients. The current data on SGLT2 inhibitors available supports its practice as a therapy choice in people with diabetes and related disorders.

REFERENCES:

1. Bastaki, S., Diabetes mellitus and its treatment. Dubai Diabetes and Endocrinology Journal. 2005; 13: 111-134.

2. Medalie, J.H., et al., Major factors in the development of diabetes mellitus in 10,000 men. Archives of Internal Medicine. 1975; 135(6): 811-817.

3. Organization, W.H., Global report on diabetes: World Health organization. Report No.: 9789241565257. WHO, 2016.

4. Chawla, A., R. Chawla, and S. Jaggi, Microvasular and macrovascular complications in diabetes mellitus: distinct or continuum? Indian Journal of Endocrinology and Metabolism. 2016. 20(4): 546.

5. Zhang, X.-X., J. Kong, and K. Yun, Prevalence of diabetic nephropathy among patients with type 2 diabetes mellitus in China: a meta-analysis of observational studies. Journal of Diabetes Research. 2020.

6. Singla, S., Diabetes mellitus: Etiology, prevalence and effects on quality of life of diabetic patients. Published online, 2022.

7. Stalin, P., S. Jayaramachandran, and G. Sathya, Smoking and Diabetes: A Case Control Study in a Rural Area of Kancheepuram District of Tamil Nadu. Research Journal of Pharmacy and Technology, 2018.

8. Palacios, O.M., M. Kramer, and K.C. Maki, Diet and prevention of type 2 diabetes mellitus: beyond weight loss and exercise. Expert Review of Endocrinology and Metabolism,. 2019; 14(1): 1-12.

9. Fitriyani, N.L., et al., A Comprehensive Analysis of Chinese, Japanese, Korean, US-PIMA Indian, and Trinidadian Screening Scores for Diabetes Risk Assessment and Prediction. Mathematics. 2022; 10(21): 4027.

10. Foretz, M., B. Guigas, and B. Viollet, Understanding the glucoregulatory mechanisms of metformin in type 2 diabetes mellitus. Nature Reviews Endocrinology. 2019. 15(10): 569-589.

11. Association, A.D., 1. Improving care and promoting health in populations: Standards of Medical Care in Diabetes—2020. Diabetes Care. 2020. 43(Supplement_1): S7-S13.

12. Langat, M.K., Prevention of diabetes mellitus complications among adults. Research J. Pharmacology and Pharmacodynamics. 2011.

13. Patel BD, Chaudhary AB, Parmar PJ, Patel VN. Development and validation of reversed phase high performance liquid chromatography method for simultaneous estimation of Nebivolol HCl and Cilnidipine in combined tablet dosage form. Pharm. and Bio. Eval. 2016; 3(2): 208-214. DOI:04.01.2016/2016/575238.

14. Garcia-Ropero, A., J.J. Badimon, and C.G. Santos-Gallego, The pharmacokinetics and pharmacodynamics of SGLT2 inhibitors for type 2 diabetes mellitus: the latest developments. Expert Opinion on Drug Metabolism and Toxicology. 2018. 14(12): 1287-1302.

15. Brown, E., et al., Weight loss variability with SGLT2 inhibitors and GLP-1 receptor agonists in type 2 diabetes mellitus and obesity: Mechanistic possibilities. Obesity Reviews. 2019; 20(6): 816-828.

16. Olson, J., et al., Soluble leucocyte adhesion molecules in diabetic retinopathy stimulate retinal capillary endothelial cell migration. Diabetologia. 1997; 40(10): 1166-1171.

17. Patel BD, Vekaria HJ. Central Composite Design Expert-Supported RP-HPLC Optimization and Quantitative Evaluation of Efonidipine Hydrochloride Ethanolateand Chlorthalidone in Tablet. Journal of Chromatographic Science. 2023; 1-8. DOI: https://doi.org/10.1093/chromsci/bmad077.

18. Shivani Chib, Neha Kumari, Sukhbinder. Role of Vasopressin and Desmopressin in Diabetes Insipidus. Int. J. Tech. 2020; 10(1): 7-12.

19. Vandna Dewangan, Himanshu Pandey. Pathophysiology and Management of Diabetes: A Review. Res. J. Pharmacology and Pharmacodynamics. 2016; 8(3): 219-222.

20. Neuen, B.L., et al., SGLT2 inhibitors for the prevention of kidney failure in patients with type 2 diabetes: a systematic review and meta-analysis. Research Journal of Pharmacy and Technology. 2019; 7(11): 845-854.

21. Mohan, V., et al., Remogliflozin etabonate in the treatment of type 2 diabetes: design, development, and place in therapy. Drug Design, Development and Therapy. 2020; 14: 2487.

22. Rathi S. Patel D. Shah S. Physicochemical Characterization and In-Vitro Dissolution Behavior of Artemether and Lumefantrine: Hydroxypropyl-Β-Cyclodextrin Inclusion Complex. Research Journal of Pharmacy and Technology. 2020; 13(3): 1137- 1141. DOI: 10.5958/0974-360X.2020.00209.7.

23. Tahir, I., et al., Evaluation of phytochemicals, antioxidant activity and amelioration of pulmonary fibrosis with Phyllanthus emblica leaves. BMC Complementary and Alternative Medicine, 2016; 16(1): 1-12.

24. Chauhan, S., et al., Antihyperglycemic and Antioxidant Potential of Plant Extract of Litchi chinensis and Glycine max. International Journal of Nutrition, Pharmacology, Neurological Diseases. 2021; 11(3): 225.

25. Jasim, R.H. and H.H. Abdualameer, A Biochemical Study for Evaluation of Some Cellular Oxidation parameters in Sera Samples of Gastrointestinal Tumors. Journal of Kufa for Chemical Science . 2020; 2(6).

26. Deepika. V, Harsha. L, Aishwarya Ravishankar. Treatment of Diabetes Insipidus. Research J. Pharm. and Tech. 8(6): June, 2015; 767-771.

27. Merry Raphael, Vijayanarayana K, GirishThunga, Karthik Rao N, Sreedharan N. Utilization Pattern of Anti-Diabetic Drugs in Type 2 Diabetes Mellitus in Tertiary Care Hospital. Research Journal of Pharmacy and Technology. 2017; 10(7): 2063-2068.

28. Fossati, P. and L. Prencipe, Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clinical Chemistry. 1982. 28(10): 2077-2080.

29. Vaja MD, Patel RR, Patel BD, Chaudhary AB. Development and Validation of RP-HPLC Method for Estimation of Lurasidone and its impurities Lurasidone 1 and Lurasidone 8. Research Journal of Pharmacy and Technology. 2022; 15 (11): 4999-5004. DOI: 10.52711/0974-360X.2022.00840.

30. Landt, Y., et al., Semi-automated direct colorimetric measurement of creatine kinase isoenzyme MB activity after extraction from serum by use of a CK-MB-specific monoclonal antibody. Clinical Chemistry, 1988; 34(3): 575-581.

31. Wang, Y., S.E. Thatcher, and L.A. Cassis, Measuring blood pressure using a noninvasive tail cuff method in mice, in The Renin-Angiotensin-Aldosterone System. 2017, Springer. 69-73.

32. Hanlon, K.E. and T.W. Vanderah, Constitutive activity at the cannabinoid CB1 receptor and behavioral responses, in Methods in Enzymology. 2010, Elsevier. 3-30.

33. Zhang, P., et al., Oxidative stress and diabetes: antioxidative strategies. Frontiers of Medicine. 2020; 14(5): 583-600.

34. Cowie, M.R. and M. Fisher, SGLT2 inhibitors: mechanisms of cardiovascular benefit beyond glycaemic control. Nature Reviews Cardiology. 2020. 17(12): 761-772.

35. Patel BD, Mishra A, Chaudhary A. RP-HPLC Method Development and Validation for Simultaneous Estimation of Pyrantel Pamoate, Praziquantel, Febantel in Tablet. Research Journal of Pharmacy and Technology. 2022; 15 (8): 3535-3539. DOI: 10.52711/0974-360X.2022.00593

36. Bhargava Vyasa. Cardiovascular Death: Leading cause of death in Diabetes. Research J. Pharmacology and Pharmacodynamics. 2013; 5(4). 249-256.

Received on 23.10.2023 Modified on 06.01.2024

Research J. Pharm. and Tech 2024; 17(8):3625-3632.

DOI: 10.52711/0974-360X.2024.00566