INTRODUCTION:

Non-Steroidal Anti-Inflammatory drugs (NSAIDs) have been extensively used in the treatment of rheumatoid arthritis and other related conditions. However, they carry the risk of adverse systemic side effects and gastrointestinal irritation at the usual dose of oral administration¹. Topical and transdermal products are vital classes of drug delivery systems, and their use in therapy is becoming more common as they offer many increased patient acceptability (non-invasiveness), avoidance of gastrointestinal disturbances, and first pass metabolism of the drug².

Diclofenac is a nonsteroidal anti-inflammatory agent, widely used in musculoskeletal disorders, arthritis, for symptomatic relief of pain and inflammation. Diclofenac diethylamine (DDEA) is a novel NSAID of the aryl-acetic acid class. It is chemically 2- {2- [(2, 6-dichloro phenyl) amino] phenyl} acetic acid is a white crystalline powder, amphiphilic in nature, sparingly soluble in water and acetone, freely soluble in ethanol and methanol. Diclofenac diethylammonium inhibits cyclooxygenase (COX-1 and -2), which is responsible for production of prostaglandin and is used in the treatment of local pain, inflammation, soft tissue rheumatism, and degeneration of inflammatory lesions of the tendons, ligaments, and joints³.

Due to oral administration of DDEA, it has increased risk of serious to fatal cardiovascular events, heart attack or stroke, ulcers and perforation in the stomach and intestine⁴. It has analgesic and anti-inflammatory properties and also effective in relieving symptoms of muscle aches, back aches, dysmenorrhea, dental pain, menstrual cramps and sport injuries⁵.

A few types of research are reported determination of DDEA concentration in rabbit study using HPLC method. The present investigation by the author explains pharmacokinetic profile of diclofenac diethylamine in microsponges gel in rabbit compared with marketed gel, this type of dosage form is suitable for diclofenac diethylamine for its the unique property of penetration into skin barriers to reach joints muscles and synovial fluid due to the presence of dimethylammonium salts of diclofenac owing to its lipophilic nature as shown in figure 1, and topical application of gel is more suitable for geriatric patients with increased patients acceptability⁶.

Figure. 1: structure of Diclofenac Diethylamine

MATERIAL AND METHOD:

Diclofenac Diethylamine was a gift sample from Santa Cruz, Mumbai. India and Carbamazepine were obtained as a gift sample from Vasudha Pharma, Chem. Ltd., India. KH₂PO₄(Potassium dihydrogen phosphate), Orthophosphoric acid (OPA) and Acetonitrile was procured from Merck, Mumbai. Water for HPLC was procured from Sigma-Aldrich Chem Pvt. Ltd., Bangalore. Animal Ethical Committee has approved the study, and the studies were piloted in accordance with standard institutional guidelines.

Surgical blade, cotton adsorbent, gauze needle, syringe, Gloves, 1.5 mL Eppendorf tubes, Whatman filter paper, Buffers pH 2.5, 5.0, C18 column, Triethanolamine (TEA) from Qualikems Fine Chem Pvt. Ltd., Safe labs K3EDTA tubes, CML Biotech, Pvt. Ltd., Cyclomixer (Remi Model cm101. LC solution software 2.1 version. Voltarol (Marketed Gel). Centrifuge (Model R-8C, Remi Elektrotechnik, Vasai, India).

Preparation of Optimized Formulation of DDEA:

The DDEA microsponges were prepared by emulsion solvent diffusion technique using an internal phase consisted of Ethylcellulose as a polymer and Triethyl citrate (1% w/v) as a plasticizer dissolved in 10mL of dichloromethane alone (Internal Phase). This was followed by the addition of Drug: polymer ratio of 1:2. The mixture was then poured into an aqueous solution of Poly Vinyl alcohol which acts as the external phase with continuous stirring at 1000rpm for 180 min. Then the microsponges were formed due to the removal of dichloromethane from the system by evaporation⁷. Prepared microsponges were then filtered, washed with distilled water for thrice, and are left for drying under desiccator.

Preparation of Buffer:

10mM Potassium dihydrogen orthophosphate was prepared by taking 0.272mg of KH₂PO₄in 200mL of HPLC water, sonication is required for complete dissolution of KH₂PO₄, still, particles are removed by filtration through filter paper with a pore size of 0.45µm (Spectrum laboratories)⁸. Further buffer is adjusted to pH 2.5 with orthophosphoric acid and triethanolamine.

Preparation of Mobile Phase:

Mobile phase was prepared by mixing acetonitrile and freshly prepared 10mM KH₂PO₄ by using distilled water in the ratio of 70:30 v/v, the mobile phase was filtered through 0.45µm membrane filter and was degassed with the help of a bath sonicator (Ultrasonic).⁹

Preparation of Internal Standard:

Accurately weighed 10mg of carbamazepine as Internal standard (IS) was transferred into 100mL the volumetric flask containing 10mL of acetonitrile (HPLC grade) and sonicate for about 10 mins. The volume was made up to the mark with acetonitrile. The stock solution was further diluted with mobile phase to give the final concentration of 1000ng/mL of each¹⁰.

Detection of Peak:

From the various trails of Internal standard and drug concentration, the optimized concentrations of Internal standard (carbamazepine) 1000ng/mL and pure drug (DDEA) 100ng/mL was dissolved in a similar solvent and injected into HPLC to elute the peak, for identification of individual peaks of ACN, Carbamazepine (Internal standard), and DDEA (pure drug).

Chromatographic Conditions:

The chromatographic system consists of a Shimadzu LC (Liquid chromatography)-20AD Toyoko Japan, solvent delivery pump equipped with a 20µL loop and rheodyne sample injector, Hypersil RP-C18 column (250mm x 4.6 mm) Purosphur Star, encapped (5µm particle size) Darmstadt, Germany. Thermo scientific-analytical column was used. The detector equipped with SPD (Shimadzu Prominence Detector) 20-A, Toyoko Japan, dual-wavelength UV-Visible detector, and the eluate was measured at 276nm, the sensitivity was set at 0.0001 AUFS, the isocratic flow rate was kept at 1 mL/min, the data were recorded using LC solution software version 2.1 (Liquid chromatography).

A 20µL Hamilton injection syringe was used for sample injection. The flow rate of mobile was increased gradually with maintained constant pressure Kilogram-force (Kgf) and finally flow rate was stabilized constant at 1mL/min, the column temperature was maintained at 25±0.5°C, and the detection of the drug was carried out at 276nm. HPLC grade Acetonitrile, water and analytical grade potassium dihydrogen phosphate of Merck Limited, Mumbai. India.

Construction of Calibration Curve:

The column was equilibrating with the mobile phase for at least 30 min to the injection of serial concentrations of the drug. The linearity of the peak area response was determined by taking measurement at nine concentrations point including zero concentration, each concentration is measured in triplicate (n=3)

Calibration curve was constructed with rabbit plasma, these involves various concentrations of DDEA (0, 50, 100, 200, 300, 400, 500, 600, 750ng/mL as given in table 1) and a fixed concentration of carbamazepine (internal standard) were prepared by taking suitable samples of the working standard solution in different 2 mL Eppendorf tubes and dilution was made up to the mark with the mobile phase. 15µL of the dilution sample was taken and injected into the column at an isocratic flow rate of 1 mL/min, each dilution was injected three times into the column. The drug eluates are monitored at 276 nm and the corresponding chromatograms were obtained. From these chromatograms mean peak area was calculated and a plot of concentration over the peak area was constructed. A linear relationship in the range was found to be 50-750 ng/mL. the correlation coefficient R² = 0.9976 and y= 201.74x+1381.9 from the above equation concentration of drug was estimated and tabulated. The slope of the plot was determined by the method of least square regression analysis and was used to calculate the DDEA concentration in the unknown samples as shown in figure 2.

Table. 1: Calibration Curve of Diclofenac Diethylamine at 276nm wavelength

S. No	Concentration of DDEA (ng/mL)	Mean Peak Area (n=3)
1	0	0
2	50	10495.3
3	100	20990.6
4	200	41981.2
5	300	62450.4
6	400	83962.3
7	500	104321.9
8	600	125943.2
9	750	147350.0

Figure. 2: Calibration curve of DDEA at 276nm wavelength

Preliminary Study:

Two days before the study a 2×2cm area on the abdominal of each rabbit was trimmed using a scissors one day before the study, a depilatory cream was applied for 15 min to the trimmed on the back and to both ears and then thoroughly washed off, to ensure complete removal of the hair in the respective areas¹¹.

Skin Irritation Test:

Skin irritation test was evaluated by the Draize method. Six healthy rabbits were used in this test. The rabbits were trimmed at abdominal skin both left and right side of the rabbit¹². An aqueous solution of sodium lauryl sulfate (SLS) of 10% w/v was used as an irritant for the skin of the rabbit and its area was calculated according to the following law Area ₌ ᴨr². The desired amount of drug containing microsponges gel was applied on the right side of the rabbit, standard irritant (10% w/v SLS) was applied at the left side of the rabbit, then the microspongial gel was removed after a period of 24 hrs, 48hrs, 72hrs. Scores were graded after removal of microsponges gel from the skin and observed for the development of erythema or any sign of edema for each rabbit compared with standard irritant¹³.

Sample Collection:

Albino male rabbits of bearing weight 1.75 to 1.85kg were used in the study. During in vivo pharmaceutical analysis rabbits were given for free access to food and normal water, until night, prior to dosing fasted for 10 hrs, before the experiment weight of each rabbit was measured¹⁴. An in vivo pharmacokinetic study was conducted in accordance with the ethical guidelines for investigations in laboratory animals and approved by the Institutional Animal Ethics Committee (IAEC), at Vaagdevi Institution of Pharmaceutical Sciences Reg No. 1663/PO/Re/S/2012/CPCSEA.

The experiments were designed as a crossover trial for this the same rabbit was used twice in the experiments including marketed gel (Reference) and finalized microsponge gel (Test). Initially, rabbits were divided into the two groups, each group contains three rabbits. Group-1 (n=3) were subjected to the application of a Marketed gel of diclofenac diethylamine as a reference drug for both the groups at the abdominal side of the body. Group-2 (n=3) were subjected to the application of microsponge gel containing diclofenac diethylamine (optimized formulation) for both the groups, before the gel was applied, rabbits were trimmed and cleaned with normal saline. The skin of the rabbit was examined in case of any damage¹⁵. Each rabbit in the group was received a marketed gel and medicated microsponge gel containing DDEA 58 mg. The sample collected points were 1, 2, 3, 4, 6, 8, 10, 12, 24, 48 hrs. About 0.5mL of blood was collected from the marginal ear vein and placed in the K3EDTA tubes¹⁶. Plasma samples were separated by centrifugation at 3000rpm for 10 min at room temperature and stored at -20°C until analysis.

Preparation of Sample for Injection into HPLC:

Blank plasma sample (rabbit which is not treated) preparation method was validated from taking 10µL of internal standard (1000ng/mL of carbamazepine) and 100µL of acetonitrile was added into 10µL plasma sample in the centrifuge tube. the mixture was vortexed for 2 mins and extracted with 0.5mL of supernatant was taken after centrifugation of the Eppendorf tube at 10,000rpm for 10 min¹⁷. into another empty tube and 15 µL of the sample were injected into the HPLC for the analysis as a blank¹⁸.

10µL of plasma was taken from the previously stored plasma sample (rabbit which is treated) which is subjected to drug analysis of that 10µL internal standard carbamazepine (1000ng/mL) was added followed by the addition of 100µL of ACN. The entire mixture was mixed with Cyclomixer (Remi, cm 101) and separation was done by centrifugation for 10 min at 1000rpm, about 15µL of supernatant was collected and injected into HPLC¹⁹.

Table. 2: Comparative in vivo plasma drug concentration

	Marketed gel	Formulated gel
Time (hrs)	Plasma Concentration (ng/mL)
0	0	0
1	174.4367	80.0513
2	235.5383	151.4824
3	299.2667	196.0589
4	291.61	262.1312
6	246.7033	229.4923
8	203.6017	185.7894
10	181.9767	144.9577
12	151.265	111.4987
24	6.635333	9.324939

Figure. 3: Drug penetration profile of MG and FMG

Table. 3: Intra-day and Inter-day precision for quantification of DDEA

Concentration (ng/mL)	Intraday			Mean	S.D.	% RSD
250.00	52296.50	52450.30	+-56421.32	53722.70667	2338.332537	4.35
350.00	73212.00	74545.23	69587.33	72448.18667	2565.687278	3.54
550.00	115052.33	121458.30	100032.65	112181.0933	10997.61881	9.80
Concentration (ng/mL)	Interday			Mean	S.D.	% RSD
250.00	54569.66	56206.75	51097.05	53957.82	2609.218209	4.83
350.00	75487.99	77752.63	70684.21	74641.61	3609.419573	4.83
550.00	121548.32	125194.77	113813.43	120185.5067	5811.76997	4.83

Table. 5: Pharmacokinetic Profile of Marketed gel (Reference)

Parameters	Units	R1	R2	R3	R4	R5	R6	Mean ±SD
C_max	ng/mL	252.46	320.08	357.99	388.27	296.98	292.63	318.06±48.83
T_max	hrs	4	3	3	3	4	3	3.33±0.51
T_1/2	hrs	2.82	2.59	2.67	3.64	2.53	2.25	2.75±0.47
AUC _(0-24)	ng-hr/mL	3101.38	3645.35	3417.07	4116.74	3329.56	3449.23	3509.88±345.92
AUC _(0-∞)	ng-hr/mL	3126.25	3663.23	3435.35	4204.33	3346.34	3458.84	3539.05±369.52
Vd	mL	65180.95	51131.21	56222.71	62567.12	54579.24	47003.85	56114.18±6841.67
Clearance	mL/hr	15993.6	13649.15	14554.55	11892.5	14941.68	14455.69	14247.86±1383.64
AUMC	hr.hr ng/mL	25195.04	26518.27	25115.49	35556.38	26656.46	26825.52	27644.53±3947.37
MRT	hrs	8.05	7.23	7.31	8.45	7.96	7.75	7.79±0.464
K_E	hrs^-1	0.245	0.266	0.258	0.190	0.273	0.307	0.256±0.038
C_last	ng/mL	6.103	4.774	4.732	16.65	4.595	2.958	6.63±5.00

K_E = Elimination Rate Constant, C = Concentration,

Table. 6: Pharmacokinetic profile of Formulated Microspongial gel (Test)

Parameters	Units	R1	R2	R3	R4	R5	R6	Mean ±SD
C_max	ng/mL	253.99	257.50	282.18	248.14	255.61	275.33	262.13± 13.43
T_max	hrs	4	4	4	4	4	4	4± 0
T_1/2	hrs	3.463	2.721	2.213	4.102	3.898	4.169	3.42±0.8
AUC _(0-24)	ng-hr/mL	2719.66	2874.26	2816.71	2648.86	2825.73	2781.03	2777.70±81.40
AUC _(0-∞)	ng-hr/mL	2764.66	2893.50	2823.42	2725.64	2898.80	2865.06	2828.51±7097
Vd	mL	90361.05	67857.33	56555.00	108570.03	97022.92	104972.9	83353.86±20381
Clearance	mL/hr	18085.36	17280.06	17708.98	18344.25	17248.47	17451.61	17686.46±448
AUMC	hr.hr ng/mL	22497.83	23060.42	21622.94	23265.19	25032.46	24801.17	23380±1321.45
MRT	hrs	8.13	7.96	7.65	8.53	8.63	8.65	8.258±0.410
K_E	hrs^-1	0.20	0.25	0.31	0.16	0.17	0.16	0.208±0.06
C_last	ng/mL	9.00	4.90	2.10	12.97	12.99	13.97	9.321±4.90

Figure. 4: Marketed gel and Formulated Microspongial Gel

Figure. 5: Similarity factor between concentrations of MG and FMG

RESULTS AND DISCUSSION:

Since microsponges are prepared by quasi-emulsion solvent diffusion technique the drug has slightly soluble in the solvent and remaining drug is entrapped into the microsponges due to its porous nature of the carrier the drug is slowly released from the matrix to outer environment gel, from gel to a deeper layer of the skin through sweat pores which act as the main entrance of the drug into the skin, as a carrier called microsponges are larger in their structure which cannot penetrate into the pores and are trapped in the hooks of the skin thereby release the drug slowly into the pore, as the drug is absorbed into the pores, passes into the deeper layers of the skin through the pore column. At the end of the column several bloods vessels are surrounded which helps in absorption of the drug into systemic circulation²¹.

After 72 hrs of treated DDEA based gel, animals show no skin irritation such as redness of skin or inflammation at the site of application, both the areas, when applied with vehicle or selected formulation were found to be free of any sign of irritation thus it is conducted that is the following are safe for topical application of gel products, hence it is needed to confirm safety after repeated application also.

Pharmacokinetics parameters calculated by WinNonlin using a Non-compartmental model (NCM) from the pharmacokinetic studies appear that there is a change in C_max AUC _(0-24), AUMC after microsponge gel treatment, there is a considerable decrease in C_max with prolonged release of DDEA after formulating into microsponges, this work was carried out in rabbit and that same results may also be excepted in humans.

Due to emulsion diffusion method of preparation of microsponges the excepted prolonged release was occurred compared to marketed gel which is required during the several joint pains and continuous release of inflammatory mediators, the absorption of the drug into systemic circulation is very close to marketed formulation due to the presence of diethyl ammonium salts of diclofenac act as a permeation enhancer, designed for topical application moreover the presence of triethanolamine help in faster penetration of the drug into the systemic circulation.

The area under the curve was measured by trapezoid rule in which the drug has an action for longer period of time. The prolonged-release of drug is due to entrapment of microsponges in nooks of the skin surface, the novel delivery system of microsponges act as reservoir system by maintaining sink condition, slowly drug is release from microsponges into the gel, from there to a deeper layer of skin, the study was conducted for 24 hrs. in this study we measure pharmacokinetic parameters of plasma concentration of diclofenac diethylamine.

In this study we measured DDEA concentration in plasma for both marketed and formulated microsponges gel at a maximum time points up to 24 hrs, the C_max after topical application of marketed gel and formulated microsponges were reported as 318.06±48.83 ng/mL and 261.13±13.43ng/mL, a decrease in C_max is due to slow release of medicament from the microsponges thereby preventing sudden increases in the concentration of drug in the plasma which leads to allergic reactions. Slower absorption may be the reason which is clear from the shift in T_max of the formulated gel was found to 4.0±0 hrs, has slight increase in T_max when compared with marketed gel 3.33±0.51 as given in table 5 and 6. The sensitivity of developed method for quality control analysis of DDEA proved by LOD and LOQ values were found to be 38.25ng/mL, 115.91ng/mL as given in table 4. Percentage RSD was used to determine precision for quantification of DDEA for intra-day and inter-day as shown in the table 3. the repeatability of newly method is proved by very less values are obtained as % RSD confirming the method is satisfactorily precise under the same conditions.

The T_1/2 value of the plasma drug concentration for marketed gel and formulated microsponges gel were 2.75±0.47 hrs and 3.42±0.8 hrs the increased in volume of distribution found to be 83353.86 ± 20381.68mL this is due to active absorption of the drug into deeper layers of the skin and into the systemic circulation. The mean residence time for marketed formulation and formulated was found to be 7.79±0.464 hrs and 8.258±0.410 hrs the long stay of the drug in plasma is due to its high protein binding nature of the drug, microsponges as a carrier system act as a depot for DDEA, the C_last the concentration found in the formulated microsponges gel was more when compared to marketed gel, still, there is a drug to delivery into the systemic circulation. So it must be important to design a study after repeated application of microsponge gel.

Since diclofenac diethylamine is having a high tendency to penetrate into deeper layers of the skin, the pharmacokinetics property has shown a difference in their parameters, due to a formulation difference since we required less amount of the drug that enter into systemic circulation for a prolonged period of time since there is the larger difference was observed with in vitro and in vivo studies in term of action of the drug for 24 hour’s period of time. Finally, the similarity factor was applied between marketed formulation and formulated microsponge gel was found to be dissimilarity due to the values obtained as f2 ₌ 12.6889 and f1 ₌ 23.70427 respectively as shown in figure 5. A significant changed was observed between marketed gel and formulated microspongial gel, where drug absorption was rapid in plasma drug concentration of marked gel compared to formulated gel as shown in table 2 and figure 3 and 4. The basic points which shows prolonged action of the drug is due to its protein binding and found to be 99.7 % from the literature survey. the duration of action of the drug from marketed formulation and formulated microsponge gel is observed for 24 hrs in rabbit model that same can be estimated in the human model.

The purpose of this study is to identify the drug in blood for a prolonged period of time due to its high level of protein binding. The purpose of design of formulation into novel microsponges are to prevent its sudden increase in plasma drug the concentration which leads to severe adverse effects likes gastrointestinal problems, allergic reactions including kidney damage, this can be prevented by a novel delivery system which releases the drug slowly into the systemic circulation for a prolonged time.

CONCLUSION:

On the present the study, with respect to the in vivo studies conducted. Gel administration of Diclofenac diethylamine produces better prolonged release when compared to topically applied marketed gel. This could be due to the slow and continuous supply of Diclofenac diethylamine at a desirable rate to the systemic circulation by microsponge gel without sudden increase in concentration.

Further the slow and sustained release of the drug from the transdermal system might reduce the release of inflammatory mediators for a prolonged time. The present study shows that topical application of DDEA gel exhibits better control of inflammations more effectively reversing the complication associated with inflammation release than normal gel application.

CONFLICT OF INTEREST:

There is no conflict of interest in publishing this article.

ACKNOWLEDGMENT:

The author would like to thank Professor. Yamsani Madhusudan Rao Director of Vaagdevi group of colleges for this continuous support in building this article and Mr. Phaniendra for his help in conducting the experiment. The Author also shows special thanks to Dr Yamsani Shravan Kumar H.O.D. of Pharmaceutics, Vaagdevi College of Pharmacy for his valuable suggestions. The Author also shows special thanks to Dr. Rajan Surulivel S Manipal College of Pharmacy, for analyzing the sample with WinNonlin software V 8.1.

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Received on 03.09.2020 Modified on 27.11.2020

Research J. Pharm.and Tech 2021; 14(12):6385-6391.

DOI: 10.52711/0974-360X.2021.01104

Limit Of Detection (LOD)	38.25 ng/mL
Limit Of Quantification (LOQ)	115.91 ng/mL