Introduction

Ketorolac is a prevalent agent within the 1^st cohort of non-steroidal anti-inflammatory agents (NSAIDs), providing therapeutic indications as analgesia, antipyretic, and anti-inflammatory properties ( 1 , 2 , 3 ). Ketorolac exerts its action by the reversible non-selective suppression of the cyclooxygenase (COX) enzymes, i.e., COX1 and COX2 isoforms, therefore obstructing the conversion of arachidonic acid to prostaglandins, which alleviates pain, fever, and inflammation ( 4 ). Ketorolac serves as a peripheral analgesic for the treatment of moderate to severe pain, exhibiting side effects ( 5 ) . It is recognised as an effective, licensed parenteral medication that is both cost-efficient and effective. In contrast to the central opioid analgesics such as tramadol and morphine, which induce severe adverse effects like respiratory depression and have potential for abuse ( 5 ), the main adverse effects of NSAIDs are on the gastrointestinal mucosa, including increased mucosal permeability, resulting in inflammation, ulceration, bleeding, and gut perforation ( 6 , 7 ). Ketorolac, is indicated for the treatment of acute moderate-to-severe pain ( 8 , 9 ). It is available in many dosage forms, including oral, nasal spray, intravenous, intramuscular, and ophthalmic solution ( 10 , 11 , 12 , 13 ). Ketorolac, extensively used in postoperative pain management ( 14 , 15 ), especially when co-administered with opioids, results in a significant decrease in opiate consumption. This combination approach not only diminishes the need for opioids but also decreases the occurrence of adverse effects, such as emesis and decreased gastrointestinal motility ( 15 , 16 ). In human medicine, the administration of ketorolac should not exceed 5 days due to the risk of cardiac thrombotic events, renal failure ( 17 ), peptic ulcers ( 18 ), and internal bleeding risk ( 7 ). This research seeks to ascertain the median lethal dosage (LD₅₀) and median effective dose (ED₅₀), as well as to calculate the therapeutic index, depending on the analgesic effectiveness of ketorolac in rats.

Materials and Methods

Laboratory animals

This research used mature male Albino rats weighing between 200 and 250 grams,obtained from the animal house of the College of Veterinary Medicine, Duhok University. Animals were kept under conventional circumstances at a temperature of 18-24ºC, with a light/dark cycle of 10/14 hours respectively, and they were provided withfood and water ad libitum. Animals were permitted to adapt to the new environment for 30 minutes before the beginning of the experimental. The Ethics committee of the Veterinary Medicine College, University of Duhok approved this research and the use of the experimental rats model.

Experiments

1- Assessment of the median lethal dose (LD50) of ketorolac (I.M) by the up-down method:

This experiment had been done according to the dixon method ( 19 ). Seven male rats were used in this experiment.The dose of ketorolac (Darnista, Ukraine 30 mg/ml,vial) was 500 mg/kg administered IM injection, based on a pilot study. The response of rats to ketorolac was evaluated as either stay alive or dead (O toward alive and X toward dead) for each animal 24 hours post-injection. The onset of acute toxicity signs and mortality was documented. The variation in Ketorolac dosage was maintained at a constant value of 125 mg/kg. By calibrating this dosage in both the upward and downward order among multiple rats, we were able to determine the LD50 of Ketorolac using the following equation:

LD50=Xf+Kd ,

where the symbols are analogous to the following:

LD50: Median lethal dosage

Xf: Final administered dose

K: A constant obtained from the Dixon table, d: the constant dosage range (both up and down) is derived.

2- Determining the median analgesic effective dose (ED50) after i.m injection of ketorolac by using electrical stimulator.

In this experiment, five rats were used. The principle of this test is based on the electrical pain response using an electrostimulator device (Scientific and Research Ltd., United Kingdom). This device is able to stimulate the sensation of rats by electro-stimulation process ( 20 ) The presence of a sensation reflect point to pain perception in rats ( 21 ). Ketorolac was administered intramuscularly to the rats at 20 mg/kg, depending on 1_st experiment. The dose was then adjusted using the up-and-down method by increments or decrements of 5 mg/kg, depending on the presence or absence of analgesia. After 30 minutes, the pain response was re-examined using the electrical stimulator. An increase in response time indicates the onset of the analgesic effect, and the rat is marked with the symbol (X); if there is no increase, the rat is marked with the symbol (O). After completing the first and second experiments, the safety indices of ketorolac were assessed using the following equation to evaluate and extrapolate its safety profile depending on the calculated ED50 and LD50 values:

Therapeutic Index (TI) = LD₅₀ / ED₅₀

A greater TI value indicates a greater margin of safety for the medicine

3- The analgesic activity of ketorolac against the visceral pain model-writhing test

Twenty-eight rats were divided into 4 groups. The first group administered 5 ml/kg of normal saline by intramuscular injection. The second, third, and fourth groups received ketorolac 20,40, and 80 mg/kg (I.M), respectively. One hour post-dosing, all groups received an intraperitoneal injection of acetic acid (1%) at a volume of 0.1 ml. The onset and frequency of writhing reactions throughout a 30-minute interval were recorded ( 22 )

Statistical analysis :IBM SPSS Statistics (Version 27) was used to analyse the data. Data expressed as mean ± standard error (mean ± SE) was shown for each outcome. One-way analysis of variance (ANOVA) was employed to determine significant variance among the groups, followed by Duncan's multiple comparisons at a probability level of (P≤0.05) ( 23 )

Results

1-Determinating (LD50) of ketorolac I.M injection by the up-and-down method

The median lethal dose was 693 mg/kg,i.m , B.W, where the rats displayed signs of acute toxicity within 10 to 15 minutes after administration. The symptoms were lethargy, increased breathing, and Straub's tail, leading to nervous tremors and death. (Table 1).

2-Determining the median analgesic effective dose (ED50) after i.m injection of ketorolac by using an electrical stimulator.

The principle of this test depends on the electrical pain response, which was generated by using an electrical stimulator.The acute median analgesic dosage was 14.39 mg/kg body weight, as shown in Table 2. A therapeutic index of 48indicated that the median effective dosage must be doubled 48 times to achieve a 50% mortality rate in rats.

3- The analgesic activity of ketorolac against the visceral pain model-writhing method

Ketorolac was injected (20,40, and 80 mg/kg) into each rat/group I.M, then after 1 hr Acetic acid 1% (0.1ml) I.P was injected, onset and number of writhing were recorded for 30 minutes. The onset of action at 80mg/kg (5.1 ± 0.49 min) and 40 mg\kg (4.8 ± 0.45 min) significantly increased compared to the control group (2.6±0.18 min), but the dose 20 mg/kg (3.1 ± 0.48 min) did not induce a significant differences compared with the control group (Table 3) . All groups showed a significantly difference in the number of writhes compared to the control group that received normal saline (103 ± 4.63). Number of writhing faded significantly at 40 mg/kg of ketorolac (59.8 ± 1.82) compared to the 20 mg/kg (81.6 ± 1.94), but the dose 80 mg/kg (39.4 ± 1.77) induce a significantly analgescic compared with 20 ,and 40 mg\kg (981.6 ± 1.94 and 59.8 ± 1.82) respectively. The analgesic efficacy percentages of 20,40 and 80 mg\kg were 21, 42, and 62% respectively. (Table 3).

Discussion

Ketorolac is a medication that is applicable across several administration routes, exhibiting significant analgesic efficacy as seen by the current research. The analgesic effect of ketorolac has been thoroughly investigated for the treatment of moderate to severe pain (). Analgesia was equivalent to or superior to aspirin or acetaminophen ( 24 )). In contrast to other NSAIDs, ketorolac is a powerful analgesic with higher water solubility and does not cause tissue irritation. It's delivered by different routes such as intravenous, intramuscular, oral, topical, and rectal ( 5 ). Ketorolac is a nonsteroidal anti-inflammatory agent (NSAID) recognised for its analgesic properties ( 25 ). Determining the median lethal dose is crucial in drug dosing, since it provides insight into the dosages applicable for experimentation and therapy ( 26 ). In this study, the median lethal dose of ketorolac was calculated using the up-and-down Dixon technique, which is recognised as ethically approved because it uses a minimal number of animals compared to other methods ( 27 ), the lethal dose of ketorolac in rats was 637 mg/kg. Our findings closely align with those of ( 5 , 28 ), they used dixone method indicated an ED₅₀ was 9.1 mg/kg,i.m in chicks and 5.62 mg/kg, in rats when administered by intramuscular injection of ketorolac. In the current study, the analgesic activity of ketorolac was assessed using the acetic-acid writhing method, which measures abdominal muscular contractions resulting from irritation of the serous membrane after an intraperitoneal injection of acetic acid solution ( 29 ). The pain-relieving effect of ketorolac in the visceral pain model, measured by the writhing test, is indicated by a notable decrease in the number of writhing movements in subjects treated with ketorolac compared to the control group, which is similar to our findings. Its action leads to temporary and non-selective blocking of cyclooxygenase (COX) enzymes (COX-1 and COX-2). Its activity results in reversible non-selective suppression of cyclooxygenase (COX) enzymes (COX-1 and COX-2) ( 8 , 31 , 32 ).

Conclusion

The study concludes that ketorolac exhibits a wide therapeutic window in the rat model, indicated by the substantial range between its therapeutic and lethal doses. Furthermore, ketorolac demonstrated peripheral and visceral analgesic activity, confirming its primary mechanism of action via inhibition of prostaglandin synthesis in peripheral tissues and internal organs rather than central nervous system mediation. This suggests the potential for safe use of ketorolac as an analgesic under veterinary supervision.

Acknowledgments

The authors wish to express their appreciation to the head of the Department of Physiology, Biochemistry, and Pharmacology for providing the necessary resources for completing this study.

Conflicts of interest

The authors declare that there is no conflict of interest.

Ethical Clearance

This work is approved by The Research Ethical Committee.

References

1. Abdulah, R.L. and Mousa, Y.J. (2024). Oxidative stress alters the therapeutic effects of ketorolac in the chicks model. Military Medical Science Letters, 93(3), 247-254.https://doi.org/10.31482/mmsl.2023.029.DOI
2. Alia, , Al- Bader, A. (2008). Effect of Aspirin as antigungal drug vagainst some opportunistic fungi.,  Basrah Journal of Veterinary Research, 7(2): 101-107. doi: 10.33762/bvetr.2008.55508.DOI
3. Jawad k A. (2007). Effect of Lidocaine , Diclofenac and their mixture on some blood parameters in experimental mice ,  Basrah Journal of Veterinary Research, 6(1): 29-37.doi: 10.33762/bvetr.2007.58552.DOI
4. Viegas, A., Manso, J., Corvo, M. C., Marques, M. M. B., and Cabrita, E. J. (2011). Binding of ibuprofen, ketorolac, and diclofenac to COX-1 and COX-2 studied by saturation transfer difference NMR. Journal of Medicinal Chemistry, 54(24), 8555–8562.https://doi.org/10.1021/jm201090k&#8203.DOI
5. Mousa, Y.J.(2019). Analgesic, antipyretic and anti-inflammatory efficacy of ketorolac in the chicks. Indian Journal of Animal Sciences. 89(10):1086–1090. https://doi 10.56093/ijans.v89i10.95003.DOI
6. Aly, S., Mahmoud, M.F., Hassan, S.H.M. and Fahmy, A. (2015). Evaluation of the analgesic activity and safety of ketorolac in whole body fractionated gamma irradiated animals. Future Journal of Pharmaceutical Sciences, 1(1): 8-15.https://doi.org/10.1016/j.fjps.2015.05.002.DOI
7. Hussein, A., Kamel A., Majeed, S.K. (2015). Histopathological and biochemical study to effect of Codeine-paracetamolen Sprague dawlwy rats,  Basrah Journal of Veterinary Research, 14(1):167-175. doi: 10.33762/bvetr.2015.100145.DOI
8. Hilal-Dandan, R. and Brunton, L.L., (2014). Goodman& Gilman's Manual of Pharmacology and Therapeutics (2nd ed.). McGraw-Hill Education. USA.
9. Guan J, Feng N, Yang K, Abudouaini H and Liu P(.2024). The efficacy and safety of ketorolac for postoperative pain management in lumbar spine surgery: a meta-analysis of randomized controlled trials. Syst.Rev.13(1):275-288 doi: 10.1186/s13643-024-02685-z..DOI
10. Vacha, M.E., Huang, W. and Mando-Vandrick, J.(2015). The role of subcutaneous ketorolac for pain management. Hospital Pharmacy, 50(2): 108-112. doi: 10.1310/hpj5002-108.DOI
11. Wongrakpanich, A., Khunkitchai, N., Achayawat, Y. and Suksiriworapong, J. (2023). Ketorolac-loaded PLGA-/PLA-based microparticles stabilized by hyaluronic acid: Effects of formulation composition and emulsification technique on particle characteristics and drug release behaviors. Polymers, 15(2):266-281. https://doi.org/10.3390/polym15020266.DOI
12. Martin, J. R., Yu, M., and Erstad, B. L. (2023). Adverse effects of nonsteroidal anti-inflammatory drugs in critically ill patients: A scoping review.  American journal of health-system pharmacy : AJHP : official journal of the American Society of Health-System Pharmacists, 80(6), 348–358. https://doi.org/10.1093/ajhp/zxac377.DOI
13. Mahmoodi, A.N., Patel, P. and Kim, P.Y.(2024). Ketorolac.National center for biotechnology information. StatPearls Publisher.USA.
14. Fillingham, Y.A., Hannon, C.P., Roberts, K.C., Hamilton, W.G. and Della Valle, C.J. (2020). Nonsteroidal anti-inflammatory drugs in total joint arthroplasty: The clinical practice guidelines of the American Association of Hip and Knee Surgeons, American Society of Regional Anesthesia and Pain Medicine Journal of Arthroplasty, 35(10):2704-2708. doi: 10.1016/j.arth.2020.04.022.DOI
15. Crotty, K., Freedman, K.I. and Kampman, K.M.(2020). Executive summary of the focused update of the ASAM national practice guideline for the treatment of opioid use disorder. Journal of Addiction Medicine, 14(2):99-112. doi: 10.1097/ADM.0000000000000619.DOI
16. Scherf-Clavel, M., Treiber, S., Deckert, J., Unterecker, S. and Hommers, L.(2020). Drug-drug interactions between lithium and cardiovascular as well as anti-inflammatory drugs. Pharmacopsychiatry, 53(5) :229-234. doi:10.1055/a-1157-9431.DOI
17. Lefebvre, C., Hindié, J., Zappitelli, M., Platt, R.W. and Filion, K.B.(2020). Non-steroidal anti-inflammatory drugs in chronic kidney disease: A systematic review of prescription practices and use in primary care. Clinical Kidney Journal, 13(1):63-71. doi: 10.1093/ckj/sfz054.DOI
18. Barrett, S., Habibullah, M., Tenembaum, D., Rubin, M.& Kim, S. (2014) 'Short-term use of intravenous ketorolac leading to giant duodenal ulcers', American Journal of Gastroenterology, 109(S1): S345. doi.org/10.14309/01.ajg.0000452641.11602.3a.DOI
19. Dixon, W.J.,(1980). The Up-and-Down Method for Small Samples. Journal of the American Statistical Association, 75(370):1-10.
20. Mousa Y J. and Mahmood B. M (2022).Effect of meloxicam coadministration on the anaesthetic potency of thiopental sodium in a chick model. Veterinarska stancia 53 (2): 155-163. https://doi.org/10.46419/vs.53.2.5.DOI
21. Mousa Y.J. and ,  Al-Zubaidy M.H. (2019) Anesthetic efficacy of ketamine, ketamine-tramadol and ketamine-ketorolac in chicks. Iran J Vet Res;20(1):33–38.doi: 10.22099/ijvr.2019.31725.4626.DOI
22. Naktal M.S.(2023) Evaluation of the pharmacological effects of Flurbiprofen in mice M.Sc. Thesis, college of Veterinary Medicine / UOmosul.Iraq.
23. Katz MH (2011). A practical guide for clinicians and public health researchers. New York: Cambridge University Press; USA. doi.org/10.1017/CBO9780511974175.DOI
24. Rahman, M. M., Begum, N., Rouf, M. A., Masood, S., and Dhaka, B. (2021). Synergistic antinociceptive, Anti-inflammatory Interaction between Vitamin B12 and Ketorolac in Long Evans Rats. Europian journal of clinical medicine. 2 (3), 105-110. doi.org/10.24018/ejclinicmed.2021.2.3.94.DOI
25. Ong, C.K., Lirk, P., Tan, C.H. and Seymour, R.A., )2007(. An evidence-based update on nonsteroidal anti-inflammatory drugs. Clinical Medicine Research, 5(1), 19-34.doi.org/10.3121/cmr.2007.698.DOI
26. Pillai, S. K., Anwar, F., Gaba, M., Chari, S., and Raj, S. (2021). John William Trevan's concept of Median Lethal Dose (LD50/LC50)–more misused than used. Journal of Pre-Clinical and Clinical Research, 15(3), 137–141. doi.org/10.26444/jpccr/139588.DOI
27. Rispin, A., Farrar, D., Margosches, E., Gupta, K., Stitzel, K., Carr, G., Greene, M., Meyer, W., and McCall, D. (2002). Alternative methods for the median lethal dose (LD50) test: The up-and-down procedure for acute oral toxicity. ILAR Journal, 43(4), 233–243. https://doi.org/10.1093/ilar.43.4.233.DOI
28. Medina-Santillán, R., Reyes-García, G., Rocha-González, H.I. and Granados-Soto, V. (2004). B vitamins increase the analgesic effect of ketorolac in the formalin test in rat. Proceedings of the Western Pharmacology Society, 47: 95–99. doi: 10.1371/journal.pone.0005853.DOI
29. Gawade, S. (2012) 'Acetic acid induced painful endogenous infliction in writhing test on mice', Journal of Pharmacology and Pharmacotherapeutics, 3(4) : 348. https://doi.org/10.4103/0976-500x.103699.DOI
30. Krivokolysko, D. S., Dotsenko, V. V., Bibik, E. Yu., Samokish, A. et al (2022). New Hybrid Molecules Based on Sulfur-Containing Nicotinonitriles: Synthesis, Analgesic Activity in Acetic Acid-Induced Writhing Test, and Molecular Docking Studies. Russian Journal of Bioorganic Chemistry, 48(3), 628–635.https://doi.org/10.1134/S1068162022030104.DOI
31. Smyth E M and Fitz Gerald G A. (2009). The eicosanoids:prostaglandins, thromboxanes, leukotrienes, and related compounds, Basic and Clinical Pharmacology.McGrew-Hill Co. Inc., New York, USA. https://doi.org/10.1055/s-0030-1267855.DOI
32. Botting R M. (2006). Inhibitors of cyclooxygenases: mechanisms,selectivity and uses. Journal of Physiology and Pharmacology, 57:113–124.https://doi.org/10.1134/0006297920060010.DOI