International Journal of Pharmaceutical Sciences and Nanotechnology Volume 3 Issue 1 April June 2010 Research Paper Design and Characterization of a Parenteral Formulation of Meloxicam P.V. Swamy*, Neelima Tyagi, S.B. Shirsand, and S.A. Raju H.K.E. Society s College of Pharmacy, Gulbarga, India. ABSTRACT: Parenteral formulation of meloxicam was developed using ph 9.2 glycine-sodium hydroxide buffer containing 10% v/v of dimethylacetamide as the solvent. Various batches of meloxicam injection formulation were prepared in order to assess the influence of light, atmospheric oxygen and antioxidants (sodium bisulfite; sodium sulfite and thiourea) on the stability of the drug and the formulations were also subjected to accelerated stability testing (at 40 o, 50 o and 60 o C) in order to predict the shelf-life of the product. Since light and atmospheric oxygen reduced the stability of the formulation, air in the ampoules was replaced with an inert gas (carbon dioxide) and the product was filled in amber coloured containers (ampoules). Sodium bisulfite (0.15% w/v) as an antioxidant gave a shelf-life of approximately 200 days under ambient temperature conditions. KEYWORDS: Meloxicam, parenteral formulation; dimethylacetamide; glycine-sodium hydroxide buffer; shelf-life; sodium bisulfite; sodium sulfite; thiourea Introduction In spite of a few limitations such as pain on injection and inconvenient administration requiring a trained person, parenteral formulations are widely used especially when an immediate physiologic response is needed in lifethreatening emergency conditions and for administering those drugs that are destroyed by digestive secretions. These are the drug delivery systems of choice for noncooperative, nauseous or unconscious patients. Most of the anti-inflammatory drugs come under class II (drugs with low solubility and high permeability) according to the biopharmaceutical classification system (Amidon GL et al, 1995). Meloxicam (MX) is a nonsteroidal anti-inflammatory drug (NSAID) with a highly selective cyclo-oxygenase-2 (COX-2) inhibitory action. It is used in the treatment of rheumatoid arthritis, osteoarthritis, dental pain, in the management of acute postoperative pain, post-orthopedic surgical pain and primary dysmenorrhea (Sweetman SC, 2002). Its side effects include diarrhea, nausea, vomiting, gastrointestinal bleeding, ulceration, perforation (Narjes H et al, 1996) and moreover the onset of action with oral dosage form is relatively slow (Insel PA, 1996). Therefore, in order to overcome these disadvantages and to facilitate its administration to uncooperative, nauseous patients and to provide fast relief from pain, the present study was undertaken with an intention to develop a stable and effective parenteral formulation containing the drug meloxicam. Literature review shows that meloxicam was well tolerated by animal tissues without any signs of * For correspondence: P.V. Swamy, E-mail: vspadavala@rediffmail.com necrosis upon intramuscular and subcutaneous administration (Stei P et al, 1996). Materials and Methods All the chemicals were used as received from manufacturer or distributor without any further purification or processing. Meloxicam was obtained as gift sample from Sun Pharma, Mumbai. Glycine, dimethylacetamide (DMA), propylene glycol, polyethylene glycols 300 and 400 were obtained from SD Fine Chem Ltd, Mumbai;. sodium sulfite and sodium bisulfite from Ranbaxy, Mumbai; sodium chloride from CDH, New Delhi; methanol from Qualigens, Mumbai and thiourea from Nice Chemicals; Cochin. All other chemicals were of analytical reagent grade. Solubility Studies. Meloxicam is practically insoluble in water. To make a clear and thermodynamically stable solution, solubility studies with different solvents/ combinations of solvents (propylene glycol, polyethylene glycols 300 and 400, glycerol, dimethylacetamide, ethanol and water) were performed. Excess meloxicam (50 mg) was added to stoppered conical flasks containing 10 ml solvent. The flasks were kept on a rotary gyratory shaker (Dolphin, Mumbai) at 150 rpm for 12 h and kept aside overnight at room temperature to attain equilibrium conditions. Suitable aliquots were withdrawn from filtered solutions (Whatman filter paper with a pore size of 1.5 microns) and analyzed for drug content spectrophotometrically at 362 nm against solvent blank. The experiments were run in triplicate. A summary of the data obtained from solubility studies is presented in Table 1. 844
P. V. Swamy et al. : Design and Characterization of a Parenteral Formulation of Meloxicam 845 Table 1 Solubility studies. Sl.No Solvent * Saturation Solubility (mg/ml) 1 Distilled Water 0.041±0.01 2 PG:Et:DW (4:1:5) 1.05±0.11 3 DMA:DW (1:1) 3.46±0.14 4 DMF:DW (1:1) 3.51±0.08 5 PEG 400:DMA:DW (2:2:1) 3.52±0.06 6 PEG 400:DMA (4:1) 8.49±0.12 7 PEG 400:DMA (7:3) 11.52±0.14 8 PEG 400:DMA:DW (4:5:1) 11.50±0.04 9 PEG 400:DMA (3:2) 13.45±0.08 10 PEG 400:DMF:DMA (5:1:4) 17.51±0.17 11 PEG 400:DMF:DMA (2:1:2) 20.01±0.24 12 GSB (0.05M) ph 10 1.04±0.08 13 GSB (0.05M) ph 10.2 1.54±0.09 14 GSB (0.05M) ph 10.6 8.49±0.17 15 GSB (0.05M) ph 10.6:DMA (9:1) 22.48±0.54 16 GSB (1.125%w/v glycine) ph 9.2:DMA (9:1) Final ph after dissolving drug = 8.4 7.53±0.26 * All the ratios are expressed as v/v. PG-propylene glycol, Et-ethanol, DW-distilled water, DMA-dimethylacetamide, DMFdimethylformamide, PEG 400-polyethylene glycol 400, GSB-glycine sodium hydroxide buffer. Formulation of Injection. Glycine (1.125 g) and sodium chloride (0.58 g) were dissolved in about 80 ml of freshly boiled and cooled distilled water in a 250 ml beaker and the ph of the solution was adjusted to 9.2 with 1N sodium hydroxide solution. Then 10 ml of dimethylacetamide (DMA) was added and the volume made up to 100 ml with freshly boiled and cooled distilled water. Meloxicam (0.75 g) was dissolved in the above solution (250 ml volumetric flask) by heating on a waterbath for 5 minutes with intermittent shaking. After cooling to room temperature, the ph of the solution was checked, which was found to be 8.4. The solution (2.15 ml) was filled in 2 ml ampoules, sealed and immediately sterilized by autoclaving (30 minutes at 15 lbs per square inch pressure) Drug Content Estimation An accurately measured volume (0.5 ml) of injection formulation was appropriately diluted with methanol and the absorbance was measured at 363.2 nm (Rawat S et al, 2004) using methanol as blank on a UVspectrophotometer (Shimadzu-1700). Average of three determinations was taken as the drug content of the formulation. The drug obeys Beer s law in the concentration range of 4-20 µg /ml. Characterization of the Injection Various batches of meloxicam injection formulation were prepared (Table 2) in order to assess the influence of light, atmospheric oxygen and antioxidants on the stability of the drug and the formulations were also subjected to accelerated stability testing in order to predict the shelf-life of the product. Table 2 Coding of various batches of meloxicam injection. Sl. No. Code Name Air-replacement Antioxidant 1. Batch-I -- -- 2. Batch-II Yes -- 3. Batch-III Yes 0.15% w/v sodium bisulfite 4. Batch-IV Yes 0.01% w/v Thiourea 5. Batch-V Yes 0.15% w/v Sodium sulfite
846 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 3 Issue 1 April - June 2010 The effect of oxygen, light and temperature on the formulation was studied by storing the ampoules under specified conditions over a period of 5 weeks. To assess the effect of oxygen, the injection (2.15 ml) was filled in 2 ml and 10 ml ampoules. The air in 10 ml capacity ampoules was not displaced before sealing (condition A ), whereas the air present in the 2 ml capacity ampoules was replaced by flushing with carbon-dioxide and sealed (Condition B ). Samples from both sets of ampoules were withdrawn periodically at 5 day intervals and the drug content was estimated. Two sets of ampoules were used to study the effect of light on the formulation (Batch-I, Table- 2). The first set of ampoules were wrapped in aluminum foil and kept in a dark place. The second set of ampoules was stored in such a manner that they were exposed to daylight. The drug content of the samples was estimated periodically at intervals of 5 d. To determine the effect of temperature on the formulation, sufficient number of ampoules filled with the formulation (Batches I to V) were stored at different temperatures, i.e., 4 o (refrigerator), 40 o, 50 o and 60 o C (in a thermostatically controlled hot-air oven). Samples were withdrawn at intervals of 5 d, and the content of drug remaining was estimated spectrophotometrically. Similarly, the effect of antioxidants was assessed by including three antioxidants at the specified concentration, viz., sodium bisulfite (0.15% w/v), thiourea (0.01% w/v) and sodium sulfite (0.15% w/v). Drug-excipient Interaction Studies (FTIR) Infra red spectroscopy is one of the most powerful analytical techniques when it comes to the determination of presence of various functional groups involved in making up the molecule. It provides good accountable spectral data regarding any change in the functional group characteristics of a drug molecule occurring while in the processing of a formulation. IR spectra of MX and its promising parenteral formulation (Batch III) were obtained by potassium bromide pellet method using Perkin-Elmer FTIR series (model 1615) spectrometer in order to rule out drug-excipient interactions occurring during the formulation process. Results and Discussion In the present study, an attempt has been made to formulate a stable, effective parenteral formulation containing MX with a view to provide fast relief from acute pain and to provide a convenient means of administration to uncooperative, nauseous or unconscious patients. Since, meloxicam is practically insoluble in water, various techniques such as co-solvency, hydrotrophy (sodium benzoate and ascorbic acid), and salt formation (hydrochloride salt) of drug have been attempted. Sufficient solubility of drug (7.5 mg/ml) could not be achieved by these methods. A solution of meloxicam in ph 9.2 glycine-sodium hydroxide buffer (Seedher N et al, 2003) with 10% v/v DMA (Sol Motola et al, 1984) was prepared. The final ph of the solution was found to be 8.4, which is well within the physiologically acceptable limits of 4 to 9. The prepared parenteral formulation of meloxicam was found to withstand autoclaving. Therefore, autoclaving was selected as a method of choice for sterilization. Various batches of meloxicam injection formulations were prepared in order to assess the influence of different parameters such as light, atmospheric oxygen and antioxidants on the stability of the drug, and five of the formulations as shown in Table 1 were subjected to accelerated stability testing by storing the samples at 4º (refrigerator), 40º, 50º and 60º C over a period of 5 weeks and the data obtained is presented in Table 3 and figures 1 to 3. Table 3 Kinetic data for various batches of meloxicam formulations. Temperature k (day 1 ) Batch-I Batch-II Batch-III Batch-IV Batch-V 4º C 0.005435 0.005082 0.001121 0.001842 0.002994 40º C 0.004933 0.002513 0.001041 0.001166 0.001834 50º C 0.005129 0.003132 0.001544 0.001496 0.002080 60º C 0.005147 0.003961 0.001932 0.001934 0.002125
P. V. Swamy et al. : Design and Characterization of a Parenteral Formulation of Meloxicam 847 25 Percent Meloxicam Lost 20 15 10 5 0 0 5 10 15 20 25 30 35 40 Time (Days) Condition-A (10 ml Ampoules) Condition-B (2 ml Ampoules) Fig. 1 Effect of atmospheric oxygen on parenteral formulation of meloxicam. Percent Meloxicam Lost 18 16 14 12 10 8 6 4 2 0 0 5 10 15 20 25 30 35 40 Time (Days) Exposed to day light Wrapped in aluminium foil Fig. 2 Effect of light on parenteral formulation of meloxicam.
848 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 3 Issue 1 April - June 2010 12 10 Percent Meloxicam Lost 8 6 4 2 0 0 5 10 15 20 25 30 35 40 Time (Days) 4ºC 40ºC 50ºC 60ºC Fig. 3 Effect of various temperatures on promising meloxicam parenteral formulation (Batch-III formulation using sodium bisulfite as antioxidant) Effect of Atmospheric Oxygen on Meloxicam Formulation: From figure 1, it is evident that the amount of meloxicam degraded to a greater extent in those ampoules which were stored under condition A (i.e., 2 ml volume of formulation stored in 10 ml capacity ampoules without displacing air) than those stored under condition B (i.e., 2 ml volume of formulation stored in 2 ml capacity ampoules and displacing air with carbon dioxide before sealing). This indicates that the greater amount of oxygen present in the ampoules stored at condition A has an appreciable effect on the stability of the formulation. Effect of Light on Meloxicam Formulation: Figure 2 shows that light also has an effect on the stability of the meloxicam injection formulation. This is evident from the comparison of the amount of drug loss in both cases. It was found that the periodical reduction in the drug content was more in those ampoules which were exposed to day light than those which were stored in a dark place, protected from light, i.e., wrapped in aluminum foil. Effect of Temperature on Meloxicam Formulation: From the data obtained from the study of the effect of temperature on the formulations prepared, as shown in Table 3, it is evident that higher temperatures have deleterious effect on the formulation. The first-order degradation rate constants (k values) at 4º C for Batch I and Batch II formulations (with no antioxidants) are much higher than those at 40º, 50º and 60º C, which can partly be attributed to the separation of drug from solution at this temperature. In case of Batch III formulation, the k value is similar to that at 40º C and this formulation shows no separation of drug at any of the above test temperatures. Batch IV and Batch V formulations also displayed a slight separation of the drug at 4º C with the consequent lower assays and higher values for their k values. The percent drug lost (in 35 days in Batch III formulation) was low in the ampoules stored at 4º and 40º C (7.2 and 6.4%, respectively), whereas the percent drug lost was high in the ampoules stored at higher temperatures of 50º and 60º C (8.4 and 10.3%, respectively) Effect of antioxidants on Meloxicam Formulation: The effect of three antioxidants, viz., sodium bisulfite, thiourea and sodium sulfite along with air replacement on the stability of meloxicam parenteral formulation was studied at 4º, 40º, 50º and 60º C for 5 weeks. The results (kvalues) were summarized in Table 3. From the results obtained, it can be seen that sodium bisulfite enhanced the stability of meloxicam formulation. It increases the shelf-life from 70 days (Batch-II, with no antioxidants, but only air replacement) to approximately 200 days (Batch-III) under ambient temperature conditions. The stability profile of the promising formulation (Batch-III) is depicted in figure 3 and the stability data (k 25 and shelf-life values) of all the injection formulations (Batch-I to Batch-V) are shown in Table 4.
P. V. Swamy et al. : Design and Characterization of a Parenteral Formulation of Meloxicam 849 Batch Table 4 Data from Arrhenius plots Correlation Coefficient (r) k 25 Shelf-life (days) I 0.9141 0.0044 23.8 II 0.9935 0.0015 70.0 III 0.9990 0.00053 198.1 IV 0.9938 0.00070 150.0 V 0.9396 0.0016 65.0 Confirmation of undisturbed structure of meloxicam: The pure drug meloxicam shows a broad peak at 3435.16 cm 1 due to OH group and peaks at 1620.67 and 1530.28 cm 1 due to CONH functional group. The peaks at 1456.99 and 1346.60 cm 1 are due to asymmetric and symmetric methyl groups respectively, and the peak at 1153.13 cm 1 is due to SO 2 N group. Batch-III formulation of meloxicam injection (containing sodium bisulfite as an antioxidant) also shows broad peak at 3435.05 cm 1 due to OH group and peaks at 1614.76 cm 1 and 1511.66 cm 1 due to CONH functional group; the peaks at 1422.42 and 1329.74 cm 1 are due to asymmetric and symmetric methyl groups respectively, and the peak at 1195.91 cm 1 is due to SO 2 N group. This data confirms the undistributed structure of the drug in the formulation (Batch-III). The FTIR spectra of meloxicam, glycine, DMA and injection formulation (Batch-III) are shown in figure 4. Fig. 4 FTIR Spectra of (A) Meloxicam; (B) Glycine; (C) Dimethyl acetamide; (D) Injection Formulation (Batch-III)
850 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 3 Issue 1 April - June 2010 Conclusion Glycine-sodium hydroxide buffer (ph 9.2) containing 10% v/v DMA gives adequate solubility for the drug MX, making it possible to develop a parenteral formulation in an aqueous medium. Replacement of air above the solution in the containers (ampoules) with an inert gas enhances the stability of the product and gives a shelf-life of about 200 days, when sodium bisulfite is used as an antioxidant. Further studies are needed in enhancing the stability of this promising parenteral formulation of meloxicam, so that a final product with a shelf-life of at least one and a half-year is obtained. Acknowledgements The authors are thankful to M/s.Sun Pharma Pvt. Limited, Mumbai, for providing the gift sample of meloxicam. References Amidon GL, Lennernäs H, Shah VP, Crison JR. A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm. Res. 12: 413-20 (1995). Insel PA. Analgesics-Antipyretic and Anti-inflammatory agents. In; The Pharmacological Basis of Therapeutics, Gilman A.G., Goodman L.S., Hardman J.G (eds)., 9 th Edition, Volume II, McGraw Hill, New York, 1996, p.641. Narjes H, Turck D, Busch U, Heinzel G, Nehmiz G. Pharmacokinetics and tolerability of meloxicam after intramuscular administration. Br. J. Clin. Pharmacol. 41(2): 135-9 (1996). Rawat S, and Jain S.K., A calorimetric method for determination of meloxicam in pharmaceutical dosage forms. Indian Drugs 41(2): 106 (2004). Seedher N and Bhatia. Solubility enhancement of COX-2 inhibitors using various solvent systems. AAPSPharmSciTech. 4(3): Article 33 (2004). Sol Motola and Shreeram Agharkar. Preformulation research of parenteral medication. In: Pharmaceutical Dosage forms: Parenteral medication, volume 1, Kenneth E. Avis, Leon Lachman and Herbert A Liebermann (eds.). Marcel Dekker Inc., New York, 1984, pp.105-107. Stei P, Kruss B, Weigleb J and Trach V. Local tissue tolerability of meloxicam. Rheumatology 35: 45-50 (1996). Sweetman SC (ed). Martindale: The Complete Drug Reference, 33 rd edition, Pharmaceutical Press, London, 2002; pp.51-52.