Acute and Subchronic Interaction between Metformin and Meloxicam in Mice

 

Falah Muosa Kadhim AL-Rekabi¹, Salma Jameel Asker¹, Mohammed Mosleh Shwaish^2*

 

ABSTRACT:

Acute and subchronic interaction and assessment of some biochemical parameters of metformine (antidiabetic agent) and meloxicam (nonsteroidal anti-inflammatory agent), were investigated in male Balb-C mice. Fifteen male mice were in-volved for studying acute toxicity and their interaction, further fifteen mice also were em-ployed for calculation LD₅₀ after subchronic exposure to both medicines alone and their co-mbination, up and down method had been carried out for evaluating the LD₅₀ after acute and subchronic exposure. Twenty  male mice divided equally into four groups  for studying orally  subchronic exposure and assigned as, group 1(G₁)  14 mg/Kg.BW metformine, group2(G₂) meloxicam 0.2 mg/Kg.BW group 3(G₃) combination of metformine)  14 mg/Kg-.BW metformine plus meloxicam 0.2 mg/Kg.BW group 3, while group 4 (C) dosed distilled waterand considered control group. The results revealed that LD₅₀ of metformine, melox-icam and their combination were 1511, 343.04 and 694.5 plus 110 mg/Kg. BW respectively, while the LD₅₀ of them after subchronic exposure were 1611, 343.03  and 897.5 plus 228.3 mg/Kg.BW respectively. Regarding to toxiclogical aspect the interaction sorts of both medicines were synergism and antagonism after acute and subchronic exposure respecti-vely. The results of subchronic exposure revealed/there were significant p<0.05 decrease in blood glucose, serum cholesterol in all treated groups, increase in triglyceride of group2, ALT and AST activity of group 2 and 3, decrease in serum uric acid of gro-up1 and 3, serum creatinine of group 2 and BUN of group3 incomparison to the cont-rol group, but the changes in the all these  biochemical parameters, being in the ran-ge of the normal values regarding to literatures, except BUN in mice of group2 which exceeded. The histopathological changes in both liver and kidney could be sumarized as, monnuclear infiltration (MNCs), multiple foci of necrotic area in live of group1. Degeneration, swollen of hepatic cells and MNCs  infiltration in group2.Multiple ne-crosis, severe perivascular cuffing of MNCs surrounded blood vessels in group3. The kidney lesions were represented by necrosis, inflammatory cells infiltration, nephritis, depletion of many glomerular tufts and hyaline cast filled many dilated renal tubules of group 1. Thickening of pelvis epithelium. pylitis ,atrophy of glomeruls and vacuolar degeneration in group2. Vacoular degeneration of renal epithelium, subcapsular and interlobular hemorrhage, enlargement of glomruler tuft and infiltration of MNCs in group3.

 

 

 

 

INTRODUCTION:

Metformin is an initially therapy for diabetes mellitus type ΙΙ⁽¹⁾, it is antih-yperglycemic agent which reduces blood glucose level by inhib-iting both liver out-put(inhibition gluconeogenesis) and reducing insulin resistance of liver and skeletal muscles through enhancing their glucose uptake⁽²⁾. The minimum effective dose for metformin is 500 mg/day, with an optimum dose of 2000 mg/day (1 g twice a day). The maximum dose should not exceed 2550 mg/day in divided doses^(3,4,5). Metform-in dose dependently reduced the production of both inflammatory mediators nitric o-xide (NO) and prostaglandin E2 (PGE2) and suppressed the mRNA and protein levels of inducible nitric oxide synthase (iNOS) and COX-2 in lipopolysaccharide activated macrophages⁽⁶⁾. Meloxicam is a non-steroidal anti-inflammatory drug widely used in treatment of various inflammatory conditions in both human and veterinary medicine, it has apparently high selectivity for inhibition COX-2 than COX-1⁽⁷⁾. It had been rep-rted that metformin can serve as potential drug to treat inflammation-related disord-ers. Metformin can serve as potential drug to treat inflammation-related disorders⁽⁸⁾. Administration of metformin in combination with meloxicam, in osteoarthritis pa-tients produced very well characterized analgesic and anti-inflammatory activities, and improves the therapeutic profile of meloxicam⁽⁹⁾ .

 

The present study is aimed to investigate the sort of metformin–meloxicam interaction and their consequence.

 

MATERIALS AND METHODS:

Medicines and chemicals:-

Mobic^® tablet contains 15 mg meloxicam obtained from BoehringerIngelheim (Germany) and Glucophage® film-coated tablet contains 500 mg metformine from Mereck Serono (France). Commercial Kits from BioSystems (Spain) used for assessment of random blood glucose, chlestrol, triglycerides, alanine aminotrnasferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), serum cre-atinineand serum uric acid.

 

Animals:

Balb-C male mice about three month age and weighed 27-32 gram, purchased from Pharmaceutical Quality Control, Ministry of Health, Iraq. They were housed under optimum condition of temperature 25±1ºC with photoperiod followed dark : light cycle of 12:12 hours along the period of experiment .Standard rodent pellet feed and drinking water provided ad libtum . They were left two weeks for acclimatization before starting the experiment.

 

Experimental design:

This experiment was carried out as approved by the Scientific Committee in physiology and pharmacology department / College of veterinary medicine ,university of Baghdad accordance to ethical standard of working on laboratory animals.

 

Dosages and dosing:

All medicines in question were calculated according to the body weight of animal as mg/Kg.BW .They were administered with dose volume 0.1 ml/10 gm .BW of mice by calculating and fitting all their concentrations for all experiments in our study.

 

Acute Toxicity assessment:

Median lethal dose (LD₅₀) of meloxicam , metformine and their combination had calculated after acute and subchronic orally administration by up and down method⁽¹⁰⁾.Depending on the outcome of each dose whether the animal dead or alive ,the medicines increased and decreased 20% for both medicines. The LD₅₀ for each medicines and their combination after acute and subchronic administration was calculated by the following equation.

 

LD₅₀ = XF+Kd

Xf=The last dose

K=Constant

D= difference between doses.

 

Assessment of interaction of both medicines:

The Isobolgraphy analysis used to determine the sort of interaction between meloxicam(drug A)and metformine (drug B) both after acute and subchronic dosing ,If we denote the intercepts by A for the LD₅₀ of Drug A and by B for the LD₅₀ of Drug B, then the isobole is expressed by the simple linear equation:

 

a                              b

--------------     -------------------

A                             B

here a is the dose of Drug A and b is the dose of Drug B when the 2 are present together, and the A and B are now the respective individual doses. The is obole expressed in equation above allows the assessment the interactions whether are synergism, antagonism or additive when actual combination doses are tested. If testing shows that the specified effect of a combination is achieved by a dose pair that plots asa point below the isobole, this means that the effect was attained with doses less than those on the line, a situation that denotes synergism. In contrast, an experiment may show that greater combination doses are needed to produce the specified effect and th-erefore this dose pair plots as a point above the isobole line denotes antagonism . Dose pairs that experimentally lie on the line (or not significantly off the line) are termed additive^(11).

 

Assessment the effects of subchronic dosing of both medicines:

Twenty male Balb-C mice divided equally into four groups administered all med-icine in question daily for two month and assigned as group one (G₁) metformine 14 mg/Kg.BW , group two (G₂) meloxicam 0.2 mg /Kg.BW ,group three (G₃) combina-tion of metformine plus meloxicam while the fourth group (C) dosed distilled water and considered control group.

 

Blood samples collected after one and two month of dosing through heart punc-ture technique, serum obtained by centrifugation with 3000 rpm for 10 minutes for clinical chemistry. Blood glucose measured specterophotometrically as coupled colored complex Quinoneimine⁽¹²⁾. Cholestrol measured specterophotometric method, briefly by converting cholesterol ester to cholestenone⁽¹³⁾. Triglycerides measure specrophotmetrically by converting them to glycerol ⁽¹⁴⁾. AST estimated specrophotometry through de-termination catalytic concentration of enzyme from the rate of decreased NADH⁽¹⁵⁾ Serum uric acid measure through coupled reaction which converts uric acid to all-ntoin in the presence uricase and colored complex that can be measured by spectrophotometer^(16,17). Blood urea nitrogen (BUN) measured specrophometrically through c-oupled reaction which mediated by urease and glutamate dehydrogenase to form glutamate and NAD which could be measured^(18,19). Serum creatinine measured by reaction with picric acid in alkaline medium and forming coloured complex which could be measured spectrophotometrically^(20,21).

 

RESULTS:

The results of Median lethal dose (LD₅₀) of metformine and meloxicam and their combination after acute single dose and suchronic repeated doses in male mice are summarized in table 1 and 2 respectively. Regarding to isobologaphy of two medicines after acute and subchronic administration , exhibited synergism with value 0.781 and antagonism with value 1.222 interaction respectively from toxicological aespect, Figure 1 and 2. Clinical observation of all mice subjected to both exposure (acute and subchronic) for 24 hours revealed restlessness or anxiety, raised tail, grooming included rapid respiration .tremor ,convulsions, laying down on one side and finally death along 24 hours of observation.

 

Table 1:The result of up and down method for calculation LD₅₀ of etformine , meloxicam and their combination after acute orally administeration

K of metformine =0.555 , K of meloxicam = 1.288 , K of combination =0.305

 

Table 2: The result of up and down method for calculation LD₅₀ of metformine , meloxicam and their combination after subcronic oral administered.

K of metformine= 0.555 , K of meloxicam =1.288 , K of combination= 0.71

 

 

Figure 1: Isobolographic of combination LD₅₀ of metformine plus meloxicam in mice after acute single dose.

 

Figure 2: Isobolographic of combination LD₅₀ of metformine plus meloxicam in mice after subchronic exposure .

The animals of G₁(metformine) and G₃ (metformine plus meloxicam), showed significant P< 0.05 decrease in their blood glucose after one and two months of treatment compared to both G₂(meloxicam) and control group, but they didn’t show significant P< 0.05 differences within these groups along the period of treatment (table 3). The animals of G₁ and G₂, showed significant P< 0.05 decrease of their serum cholesterol compared to both G₃ and control group along the period of experiment , when the serum cholesterol of animals of G₃ showed no significant P< 0.05 compared to control group. Although the serum cholesterol of G₁ and G₂ were significantly decreased but there weren’t significantly P< 0.05 changes within these two groups along the period of experiment (table3).The serum triglyceride (TG) of G₁, G₂and G₃ showed significant P< 0.05 increases after one month of treatment compared to TG of control group.But after two month of treatment only G₂ (meloxicam) showed significant P<0.05 increase in TG compared to control group. After two months of treatment, TG of both G₁and G3 showed no significant P< 0.05 compared to control group. All treated G₁,G₂ , G₃ and control group didn’t show any significant P< 0.05 changes proportional the period of treatment (table 3).

 

Table 3: Glucose m/dl ,Cholestrol mg/dl and triglyceride mg/dl in male mice exposed to metformine, meloxicam and their combination for one and two months.

Data = M±SE, Capital letters denote significant differences p<0.05 between groups , Small letters denote significant differences p<0.05 within groups

 

The serum activity of liver injury enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of both G₂(meloxicam) and G₃(metformine plus meloxicam) showed significant p<0.05 increase compared to both G₁(metformine) and control group.ALT and AST serum activity of G₂ and G₃ were significantly p<0.05 increases proportional to the period of treatment. The serum activity of ALT and AST of G₁ showed no significant p<0.05 changes compared to control group, also the showed no significant p<0.05 changes proportional to period of treatment (table 4 ).

 

Table 4:ALT and AST U/L in male mice exposed to metformine , meloxicam and their combination for one and two months.

 Data = M±SE , Capital letters denote significant differences p<0.05 between groups, Small letters denote significant differences p<0.05 within groups

 

There were significant p< 0.05 decrease in serum uric acid (UA) of G₁(metformine) and G₃ (metformine plus meloxicam ) along the period of experiment compared to UA of both G₂ (meloxicam) and control group. G₂ animals showed significant p< 0.05 increase in UA after two months of treatment only compared to control group. But UA of G₁,G₂ and G₃ showed no significant p< 0.05 changes proportion to dosage period (table 5). Only G₂ animals showed significant p< 0.05 decrease in serum creatinine ( Scr) after one and two months of treatment compared to G₁,G₃ and and control group. The treatment G₁, G₃ and control group showed no significant p< 0.05 changes in their Scr proportion to dosage period, but G₃ showed significant p< 0.05 decrease in Scr after two months compared to its Scr after one month of treatment (table 5). The animals of G₂ (meloxicam) showed significant p< 0.05 increase in blood urea nitrogen (BUN) compared to G₁(metformine), G₃(metformine plus meloxicam) and control group after one and two months of treatment, while G₁ and G₃ showed significant p< 0.05 decrease in BUN only after one month of treatment but not after two months compared to control group. The BUN of G₂ was significantly p< 0.05 increase when compared to its value after one month of treatment (table 5).

 

 

Table 5: Serum uric acid mg/dl ,serum creatinine mg/dl and BUN mg/dl in male mice exposed to metformine , meloxicam and their combinationfor one and two months.

Data = M±SE, Capital letters denote significant differences p<0.05 between group Small letters denote significant differences p<0.05 within groups

 

Histopathology:

Liver:

Group one (metformine) showed, multiple granulomatous lesions characterized by many mononuclear cells (MNCs) infiltrated within necrotic foci of hepatocytes after one month of treatment (figure 3). But after two month of treatment there were, multiple focal necrotic areas in liver parenchyma, congested sinusoidal capillaries seen dilated and contained MNCs (figure 4).

 

 

Figure 3. Liver section of mouse dosed orally 14 mg/kg BW metformine for one month showing focal hepatic necrosis with MNCs infiltration (←)H and (X40)

 

 

Figure 4. Liver section of mouse dosed orally 14 mg/kg BW metformine for two month, showing multiple focal necroticarea (→) and MNCs in filtration (→). H and E(X10)

 

While the liver histopathological pattern of group two(meloxicam), revealed hepatic cells degeneration appeared enlarge and swollen with mild infiltration of MNCs after one month of treatment only (figure 5).

 

 

Figure 5. Liver section of mouse dosed orally 0.2 mg/kg BW meloxicam for one month, showing enlargement and swollen of hepatic cells (→) and MNCs infiltration (→). (H and E .X40).

 

The hepatic tissue of group three ( metformine plus meloxicam) after one month of treatment showed, multiple necrotic granulomatous lesions and perivascular cuffing, necrosis and lysis of hepatocytes with small MN foci(figure 6). But after two months of treatment, there were severe perivascular cuffing of mononuclear cells surrounded intraedamatous blood vessels, also infiltrate the liver parenchyma and within sinusoids, also focal aggregations of mononuclear cells seen. Degenerative and necrotic hepatocytes occasionally noted (figure7).

 

 

FIGURE 6. Liver section of dosed orally 14mg/kg.BM metformine +0.2mg/kg.BM meloxicam for one month,showing multiple necrotic granulomatous lesion (→) and perivascular cuffing (→) (H&E) (X10)

 

Figure 7. Liver section of mouse dosed orally 14 mg/kg BW metformine

 

Figure 7. Liver section of mouse dosed orally 14 mg/kg bw metformine for two month, showing multiple focal               

Liver of control group histopathological appearance , is normal architecture of hepatic lobule from central vein radiated with hepatocytes.

 

Kidney:

The histological examination of renal tissue of group one (metformine) after one month of treatment showed, necrosis with inflammatory cells infiltration (Figure 8), while after two months,the renal tissue showed severe nephritis characterized by cortical necrosis, atrophy and depletion of many glomerular tufts with thickening of intertubular septa due to infiltration of inflammatory cells and proliferation of connective tissue, the hyaline casts (eosinophilic) filled many dilated renal tubules, (Figure 9). Other sections showed extensive necrosis in cortical area with heavy infiltration of inflammatory cells, atrophic glomeruli and necrosis of renal tubules with hyaline casts in dilated tubules (Figure 10).

 

 

Figure 8. kidney section of mouse dosed orally 14mg/kg. BW metformine for one month, showing necrosis with inflammatory cells infiltration (→) (H&E × 40)

 

 

Firure 9 kidney section of mouse dosed orally 14mg/kg BW metformine for two months, showing thickening cabsular and subcabsular tissue with heavy inflammatory cells (→) (H&E X10).

 

Firure 10 kidney section of mouse dosed orally 14mg/kg BW metformine for two months, showing hyaline cast within renal tubules (→) (H&E X40).

 

The Kidney sections of group two after one month of treatment (Meloxicam) showed ,great thickening of pelvic epithelium and heavy infiltration of mononuclear cells(pylitis) (figure11), mild periglomerularinfiltration seen (figure12).But after two months of treatment the lesion could be summarized as , severe atrophic of glomeruli with distention of bowman space and vacuolar degeneration of renal epithelium figure 13.

 

 

Firure 11 kidney section of mouse dosed orally 0.2mg/kg BW meloxicam for one months, showing thickening of pelvic epithelium (→) pylitis (→)(H&E X10).

 

 

Firure 12 kidney section of mouse dosed orally 0.2mg/kg BW meloxicam for one month, showing mild periglomerular infiltration (→)(H&E X20).

 

 

Firure 13 kidney section of mouse dosed orally 0.2mg/kg BW meloxicam for two month, showing severe atrophic of glomeruli with distention of bowman space (→)(H&E X40).

The histopathological findings in kidney of group three(metformine plus meloxicam) after one month of treatment including, severe vacuolar degeneration of renal epithelium (figure 14).

 

 

Firure 14 kidney section of mouse dosed orally 14mg/kg.BW metformine + 0.2mg/kg. BW meloxicam for one month, showing severe vacuolar degeneration of epithelium (→)(H&E X40).

 

While after two months representing by, subcapsular hemorrhageand intertubular hemorrhage in cortex ,enlargement of glomerular tuft due to congestion of their capillaries and infiltrated with lymphocytic cells. Infiltration of mononuclear cells in renal parenchyma cortex and medulla, focal periglomerular and glomerular tuft infiltration also peritubular infiltration, atrophic glomeruli occasionally seen and distended of bowman's space, severe degeneration of renal lining epithelium. Thickening of pelvic epithelium noted with heavyinfiltration of mononuclear cells (figure15). The kidney of control group showed nothing abnormal appearance

 

 

Firure 15 kidney section of mouse dosed orally 14mg/kg BW metformine + 0.2mg/kg BW meloxicam for two months, showing inter-tubular hemorrhage ((→) enlargement of glomerular tuft infltirated with MNC (→) (H&E X20)

 

DISCUSSION:

Enormous number of patients are receiving multitherapy for curing several diseases, such as hypertention, arthritis and diabetes. Some times metformineand meloxicam have been used to manage hyperglycemic patient with arthralgia and myalgia. An interaction may occurs between theme, sometimes has no clinical significant adverse effects .

 

Meloxicam LD₅₀ is343mg/Kg.BW in mice for 24 hours observation after single acute exposure and single dose after subchronic exposure for two months falls in category grade 4. So meloxicam classified as very toxic agent according to⁽²²⁾. It is matching finding of^(23,24) who found meloxicam fall in severely toxic category also. Metformine, also classified as severe toxic agent, since we found the LD₅₀ is 1511 and 1611 mg/Kg.BW after single acute and subchronic exposure orally in mice, following the same grade of metformine. The synergistic interaction between metformine and meloxicam after acute exposure may be due to physiochemical interaction between two medicine, when they were administered simultaneously and change their transportation into systemic circulation and target tissue⁽²⁵⁾.

 

The antagonist interaction between metformine and meloxicam after subchronic exposure, possibly due the antioxdant role of metformine through the improvement in the activities of antioxidant enzymes⁽²⁶⁾, and antioxidant and hepatoprotective effect of meloxicam which were assured in rat exposed to 5mg/kg.BW meloxicam twice weekly for eight weeks⁽²⁷⁾. The clinical signs which observed included both adrenergic (anxiety, tremor) and neuroglycopenia (seizures or convulsions), we supposed that because of hypoglycemic effect of metformine which were encountered by elevation of epinephrine and glucagon⁽²⁸⁾.

 

The serum biochemistry analysis is a complex of tools, were used in this study to evaluate the effects of metformine and meloxicam alone and incombination in some organs activities and tissues. Liver function assay through measuring the serum levels of AST, ALT, glucose, Cholestrol and triglyceride. Specially, mice which received meloxicam alone and incombination with metformine for one and two month showed signicant increases in serumTiglyceride (TG), ALT and AST activities. Despite the increases of AST, ALT, TG and cholesterol, arestill within the normal level in mice, since the range of AST between 54-298 IU/L⁽²⁹⁾, ALTis 26-120 IU/L, TG 71-164 mg/dL and Cholesterol 63-174 mg/dL⁽³⁰⁾. Blood glucose in treated groups which received meloxicam, although they didn’t show any significant changes when compared to blood glucose of control group, was within the normal limit of glucose in mice 106-278 mg/dL⁽³⁰⁾. But the decrease in blood glucose in mice, which received metformine alone and incombination with meloxicam may be attributed to the dual antihyperglycemic role of metformine ,through inhibition liver output of glucose⁽³¹⁾ and reducing liver and skeletal muscles resistance by increase their sensitivity to insulin⁽³²⁾. The thought is that the histopathological lesions which were approved in liver of all treated groups, not severe or sufficient enough to cause substantial changes in liver function, that improved when there were no significant changes in AS, ALT. Cholestrol. TG and glucose.

Blood urea nitrogen (BUN) is producing from breakdown of protein taken through food, while creatinine is waste comes from normal tear of muscle, their normal levels in mice are 19-34 mg/dL and 0.5-0.8 mg/dL respectively⁽³⁰⁾. Both of them are traditional tools for screening kidney function⁽³³⁾. All mice treated with metformine and meloxicam plus metformine, despite the were showed significant increase in serum level of both BUN and serum creatinine, but these changes fall into normal range. The only mice treated with meloxicam alone but not incombination with metformine showed prominent increase in BUN and serum creatinine, especially, after one and two month of treatment compared both to control, metformine, metformine plus meloxicam groups and also to reference value in literatures, with exception of BUN in mice exposed to meloxicam for two months which is exceeding the reference value in literature, the thought is due to the severe atrophy in glomeruli, which is assured in histopathological renal section (figure 13), could be as suggestive renal failure, but not fatal renal failure which was assured by⁽³⁴⁾ when they found diclofenac acid a nonsteroidal anti-inflammatory drug which is inhibiting both COX-1 and COX-2 had been reported fatal nephrotoxicty invultures incontrast to meloxicam which caused nonfatal nephrotoxicity, while⁽³⁵⁾ found that meloxicam single dose 0.2mg/Kg.BW intravenously to beagles has no significant effect on BUN and creatinine serum level.

 

Serum uric acid (SUA) is considered one of important tool to evaluate kidney function, the elevated level of SUA is reflecting the reduce in ability of kidney excretion⁽³⁶⁾. The level of serum uric acid is determined by a balance between purine dietary uptake and production and excretion of uric acid. It is thought that uric acid is mainly produced in the liver and almost excreted through the urinary tract. Because of the high reserve capacity of kidney, the changes in serum level of serum creatinine, SUA and BUN observed when the histopathological lesion involved more than two third of nephrons are damaged. So we couldn’t observed substantial changes in these parameters of all experimental groups because of the histopatholgical changes which observed in the kidney tissue were not do so, moreover there were no indications of chronic renal failure (reduction in kidney size, irregular subcapsular surface, hard to section with gritty consistency)⁽³⁷⁾.

 

In conclusion, interestingly, the simultaneous administration of meloxicam and metforminehas been showed no substantial deleterious effects, especially after subchronic exposure, the suggestion is, possibly due to hepatoprotective effect of metformine plus anti oxidant activity of meloxicam

 

ACKNOWLEDGEMENTS:

The authors are very grateful to Dr. Duraid A. Abbas of the Department of Physiology, Biochemistry and Pharmacology-College of Veterinary Medicine-University of Baghdad for his apparatus which was necessary to complete this research.

 

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Accepted on 06.03.2018           © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(6): 2336-2344.