Local Effect of Intramuscular Injection of Meloxicam and Diclofenac: An Experimental, Comparative and Histological Study

Objectives: To compare the local effect of therapeutic doses of meloxicam and diclofenac on the muscle of rats at different periods by intramuscular injection. Materials and methods: Thirty six adult albino rats were divided equally into 6 groups. The 1 and 2 groups were injected intramuscularly with normal saline for 2 and 4 weeks respectively, the 3 rd and 4 th groups were injected intramuscularly with meloxicam for 2 and 4 weeks respectively, the last 2 groups were injected intramuscularly with diclofenac sodium also for 2 and 4 weeks respectively. The drugs were injected into the quadriceps femoris muscle of the right femur once each day till the end of the period stated. The specimens were histologically processed, stained with hematoxylin and eosin and examined with light microscope. Results: No changes were seen in quadriceps femoris muscles of groups 1, 2, 3 and 4, whereas group 5 showed congested blood vessels within normal muscle tissue. In Group 6 mononuclear inflammatory cells infiltration, vascular congestion, fibrosis, degenerative changes and muscle tissue necrosis were seen. Conclusion: Meloxicam is safer than diclofenac sodium on muscular tissue after a long-term daily intramuscular injection. Keyword: meloxicam, diclofenac, intramuscular injection, muscle tissue. ,ةيبيرجت ةسارد :كانيفولكدلاو ماكيسكوليملا يراقعل يلضعلا نقحلل يعضوملا ريثأتلا ةيجيسن و ةنراقم . نساق ماشه ذوحا * ، يذوح ضاير روع * ، بىيا نيحرلا ذبع فرشا * ، سيجرج رهاط ةيط ذوحه * عرف* ةجسنلااو حيرشتلا ةنجلااو ةيلك ، لا لصىولا ،لصىولا ةعهاج ، ةط قارعلا ،


INTRODUCTION
ystemic and topical nonsteroidal antiinflammatory drugs(NSAIDs) are the most commonly prescribed drugs in the treatment of inflammation and pain associated with chronic inflammatory conditions like rheumatoid arthritis and osteoarthritis 1-5 . They reduce inflammation by reducing prostaglandin synthesis through inhibition of cyclooxygenase enzyme(COX) 6 . COX-1 and COX-2 are isoforms of the enzyme, suppression of COX-2 is responsible for the therapeutic gain of NSAIDs, while their adverse effects result from inhibition of COX-1 7 .
The route of administration of NSAIDs is considered to be an important field for investigation, because these drugs can be given orally, intramuscularly, rectally and intra-articularly in addition, they can be applied locally on mucosa and skin [8][9][10] . Thus a lot of researches had been carried out with results showing that intramuscular administration had some advantages over oral route in that it has faster onset of action, avoids first pass metabolism, besides, it prevents direct contact of the drug with gastric mucosa 8,[11][12][13][14] . On the contrary, intramuscular route can cause tissue necrosis at the site of injection even after a short term use 15,16 . Since meloxicam is a preferentially selective COX-2 inhibitor, whereas diclofenac sodium is a nonselective COX inhibitor 17,18 . several researchers had studied the efficacy, tolerability, as well as the safety of meloxicam and compared it with diclofenac after a short period of intramuscular injection or after a long period of oral administration in both experimental animals and humans [19][20][21][22] . The aim of the present study is to compare the local effect of therapeutic doses of meloxicam and diclofenac on rats' muscle given for the same periods as an intramuscular injection.

Animals
Thirty six male adult albino rats were obtained from the animal house of Experimental Research Unit, College of Medicine, University of Mosul, Iraq. The animals were kept in the laboratory in special plastic cages under ideal conditions. The number of animals in each cage was three. Food and water were available ad libitum throughout the study.

Drugs
Meloxicam (Mobic ® , Boehringer Ingelheim, Germany) and diclofenac sodium (Refen ® , Hemofarm, Serbia) in the form of ampoules were purchased from a common commercial supply (Alradwan drug store). The drugs were administered in therapeutic doses similar to that given to human 23 . Which were calculated by diluting the drugs with normal saline.
(24) The drugs were injected intramuscularly into the quadriceps femoris muscle of the right femur of the rat every day till the end of the stated period according to the subdivided groups.

Groups And Experimental Design
After one week of acclimatization, the animals were divided into six groups, each consisted of six rats.

Control Groups
Groups 1 and 2: received a single daily intramuscular injection of normal saline (1ml/ Kg) for 2 and 4 weeks respectively. Treated Groups -Group 3: animals received a single daily intramuscular injection of 0.22mg/ kg of meloxicam for 2 weeks. -Group 4: is similar to that of group 3, but the drug is given for 4 weeks. -Group 5: rats were daily intramuscularly injected with a single dose of diclofenac sodium (2.2 mg/ kg) for 2 weeks. -Group 6: is similar to that of group 5, but the drug is given for 4 weeks.
Twenty four hours after the last injection, rats were killed by inhaling overdose of Ether. Dissection of the right hind limbs was done and the quadriceps femoris muscle was removed from each animal and prepared for histological processing.

Histological Study
The quadriceps femoris muscle tissue samples were fixed in 10% neutral buffered formalin for 24 hours, then they were dehydrated, cleared, embedded in paraffin and sectioned by the microtome into sections of 5µm thickness, thereafter the sections were mounted on glass slides and stained with hematoxylin and eosin. The histological slides were examined through Olympus light microscope, and several sections were chosen to obtain their photographs using a digital camera.

Control (Untreated) Groups:
In groups 1 and 2, the histological changes in the muscular biopsy were unremarkable ( Figure 1).

Treated Groups
In groups 3 and 4, microscopic examination was similar to that of control groups ( Figure 2). In animals of group 5, the histological finding was only congested skeletal muscle fibers( Figure 3). However, in group 6, the histological sections revealed mononuclear inflammatory cells infiltration, vascular congestion, fibrosis, degenerative changes and muscle tissue necrosis. Moreover, several areas showed hemorrhage which could be of traumatic origin (Figures 4 and 5, Table 1).

DISCUSSION
Long term administration of NSAIDs is preferable via the oral route, however intramuscular injection is beneficial for the short term relief of renal colic, acute pain associated with rheumatological disorders, dysmenorrhea, post operative pain and neoplastic conditions [25][26][27] . Furthermore, intramuscular formulations may be used for treating patients in whom oral as well as rectal administration is not possible 8 . This study demonstrates that a single daily intramuscular injection of therapeutic dose of meloxicam for 2 weeks and up to 4 weeks cause no damage to muscular tissue at site of injection. On the other hand, when diclofenac sodium is injected in a single daily therapeutic dose for 2 weeks will cause congestion of blood vessels in an otherwise normal muscle tissue, and increasing the duration of administration to 4 weeks will produce mononuclear inflammatory cells infiltration and vascular congestion associated with degenerative changes, necrosis of muscle tissue and fibrosis.
There are no observed data regarding the local histological effect of therapeutic doses of meloxicam and diclofenac on muscle tissue when used intramuscularly for period longer than one week.
Stei . observed that meloxicam when given intramuscularly in high doses for 4 days will cause minimal histological changes at site of injection. These findings in conjunction with the result of present study indicate that, meloxicam is safer than diclofenac when given intramuscularly. Moreover, many cases of muscle and skin necrosis (Nicolau syndrome) have been reported in patients who received a single intramuscular injection of diclofenac [29][30][31][32][33][34][35] . In contrast, no case of such syndrome has been recorded after meloxicam injection till now.
Sutton et al 36 . mentioned that muscle damage can be related to local drug concentration as well as its release from formulation and a drug with slow release has the ability to lessen muscle tissue damage. Meloxicam reaches its maximum plasma concentration 90 minutes after intramuscular injection 37 .
whereas diclofenac reaches its maximum concentration after 20 minutes 38. Thus the above findings can explain why meloxicam causes less muscle damage in comparison with diclofenac as it has a slower release rate. This explanation was supported by Lima and Oliveira 39 . Souza et al. (40) and Lopes et al.

41
. who investigated the protective effect of diclofenacloaded liposomes. They discovered that liposomes are able to reduce the rate of diclofenac release, acting as a drug reservoir, lowering diclofenac concentration in the local tissue and thus protecting the skeletal muscle from the toxic effect of the drug.
Other investigators who tried to explain the pathogenesis of muscle damage after diclofenac injection, reported that diclofenac which is a nonselective COX inhibitor, will inhibit both COX-1 and COX-2, reducing prostaglandins and resulting in vasoconstriction with subsequent ischemic necrosis 29,31,42 . Paulsen et al 43 . stated that when only COX-2 is inhibited, COX-1 produces sufficient level of prostaglandins, maintaining good blood flow to the muscles, and this may further explain why meloxicam produces no muscular damage in this study, since it inhibits COX-2 more than COX-1 at therapeutic dose. (17) However, this would be controversial to a study of Novak et al 44 . who affirmed that COX-2 activity is essential for skeletal muscle repair and growth. So the exact cause of the difference in the intramuscular tolerability between meloxicam and diclofenac sodium remains unclear and needs further research.

CONCLUSION
When meloxicam had been administered for 2 to 4 weeks, it produces no muscular damage, while diclofenac sodium causes damage to the muscle tissues. Therefore meloxicam is safer than diclofenac on skeletal muscle after a long period of daily intramuscular injection.