INTRODUCTION:

The first approved human immuno deficiency virus type 1 integrase inhibitor is Raltegravir. Raltegravir (RAL) is an antiretroviral medication^1-3 to treat HIV/AIDS⁴ and used as a part of post exposure prophylaxis^5-7. It is taken orally by mouth. Common side effects include feeling tired, trouble sleeping^8-9, nausea¹⁰, high blood glucose, and headaches. Severe side effects may include allergies ^{11- 12}including Stevens Johnson syndrome^13-14, muscle breakdown and liver problems. Raltegravir is an HIV integrase strand transfer inhibitor ¹⁵ which blocks the functioning of HIV integrase which is required for viral replication. Raltegtavir has a half life of 9 hours and molecular formula C₂₀H₂₁FN₆O_5.The Chemical structure of Raltegravir is given in Figure 1. The present study was planned with an objective to conduct in vivo pharmacokinetic studies for raltegravir extended release tablets in rabbit. Experimental Raltegravir extended release tablet formulation were tried to compare with pure raltegravir API. Very few analytical methods for the estimation of Raltegravir were reported in literature^16-20. Hence, for the estimation of Raltegravir in rabbit plasma it was proposed to develop a new HPLC method.

Figure 1: Structural representation of Raltegravir

MATERIALS AND METHODS:

Raltegravir (99.9% pure) was obtained from M/s. Aurobindo Pharma Ltd., Hyderabad, India as gift sample. HPLC marked acetonitrile, ortho phosphoric acid and tri ethyl amine were procured from Merck, Mumbai, India. HPLC marked water was obtained from milli Q (Milli Q system, USA) water purification.

PHARMACOKINETIC STUDY:

Selection of animals

In vivo pharmacokinetic studies, 6 healthy white New Zealand rabbits (2.0-2.5 kg) were used. All animal experiments approved and performed in GITAM Institute of Pharmacy, Visakhapatnam accord with principles of Institutional Animal Ethics Committee (CPCSEA No: 1287/ac/09/CPCSEA) vide approval IAEC/GIP-1287/LS-F/Approved/1/2019.

Analytical method and Instrumentation:

In this method, chromatographic condition of HPLC (Waters Alliance e2695) were X-bridge phenyl hexyl column (5μm, 150mm×4.6mm). acetonitrile Tri ethyl amine buffer (30:70 v/v) is used as a mobile phase at 1.0 ml/min flow rate under ambient temperature of 30°C was maintained and detection was done at 312 nm wavelength.

Preparation of Stock Solutions:

Raltegravir 200 μg/ml of stock solution was made with acetonitrile by dissolving 5 mg in 25 ml. An intermediate stock of 2 μg/ml was prepared with mobile phase from stock. From this solution, 25, 50, 125, 250, 375, 500, 625, 750 & 1000 ng/ml working standard solutions were made which were used for standard calibration curve. Ritonavir standard solution at a concentration of 500 ng/ml was prepared for internal standard (IS).

Sample Preparation:

White New Zealand rabbits 2.0-2.5 kg weights were used for the blood collection. Blood was collected in Eppendorf containing 10% EDTA solution from rabbit marginal ear vein. The blood centrifuged for 30 min in cooling centrifuge (Remi, India) at a speed of 5000 rpm and yellow supernatant plasma was collected subsequently after centrifugation. The clear supernatant plasma was used for analysis. For sample preparation 200 µl of plasma was taken and 500 µl of internal standard and 500 µl of standard stock solutions were added and mixed. To this mixture 500 µl of acetonitrile and 300 µl diluent was added to precipitate all the proteins and blend within the vortex cyclo mixture. These samples further subjected for Centrifuge at 5000 rpm for 30 min. Collect the supernatant solution and filter through 0.2 µ nylon syringe filter and the clear solution was placed in HPLC vial for injection into the chromatogram.

Specificity:

Six rabbit blank plasma samples were collected randomly and they were subjected for protein precipitation. They were chromatographed to identify the endogenous components of plasma which could interfere with either standard drug or internal standard.

System suitability:

The system suitability was assured by determining peak retention time, peak area, tailing and plate count of Raltegravir.

Linearity:

Calibration samples of different concentration (5%, 10%, 25%, 50%, 75%, 100%, 125%, 150% and 200%) of Raltegravir were prepared by appropriate amount and dilution of standard drug into control plasma. The samples were further subjected for protein precipitation and chromatographed.

Accuracy:

Analytical method accuracy is a trial result mean of the analyte nominal value that can be measured by minimum of 3 concentrations with 5 determinations of each concentration. The mean nominal value (relative error) deviation presents the measure of accuracy.

Precision:

Analytical method precision is the individual analyte nominal value that can be calculated by minimum of 3 concentrations with 5 determinations of each concentration. It is subdivided into Within-day and Between-day precision which determines precision w.r.t time, equipment, different analysts and reagents etc.

Limit of detection and qualification:

LOD and LOQ are lowest analyte detectable and lowest analyte quantifiable concentration. Which are calculated by the formula 3.3σ/s and 10σ/s respectively. Where σ- slope intercept standard deviation (SD) & S- calibration curve slope.

PHARMACOKINETIC STUDY:

Animals are separated into two groups in such a way that each group contains 3 rabbits. Before experimentation all animals are starved overnight and had water ad-libitum. Topical anesthetic procedure was used. Pharmacokinetic evaluation was performed for Raltegravir experimental formulations and pure drug. The samples were administered to each rabbit under fasting conditions. Group 1 animals was treated with experimental formulation. Group 2 animals was treated with control (Pure raltegravir). After a single oral administration of raltegravir, blood samples were collected from rabbit marginal ear vein with volume of 0.5 ml to 1.0 ml at 0.5, 1, 2, 3, 6, 9, 12, 16, 20 and 24 hrs. The blood was collected in Eppendorf containing 10% EDTA solution. Blood was centrifuged at 5000 rpm for 30 mins at 2-8°ϲ temperature. The clear supernatant plasma were collected & stored at -30°ϲ till its analysis. The plasma samples were treated for liquid- liquid phase extraction and analyzed for drug content with developed analytical method. The pharmacokinetic parameters for raltegravir oral administration were determined from plasma concentration data. Pharmacokinetic parameters like AUC, C_max, T_max the time at which C_max occurred, Kel, t_½, K_a and MRT were calculated using the data. Data was measured by the trapezoidal rule method from time zero to infinity of concentration-time curve. C_max and T_max were obtained from the graph. All values are expressed in mean ± SD.

RESULTS AND DISCUSSION:

Specificity

No interfering peaks were found in blank plasma sample at the retention time of both Raltegravir and ISTD. The Chromatogram blank rabbit plasma was shown in Figure 2 and Chromatogram blank plasma spiked with analyte and IS shown in Figure 3.

Figure 2: Chromatogram blank rabbit plasma

Figure 3: Chromatogram blank plasma spiked with analyte and IS

Standard calibration curve and linearity:

From the calibration curve, it had been clear that the height area ratios were proportional to the concentration. The concentration range of Raltegravir is 25-1000 ng/ml. Calibration curve was plotted between peak area of Raltegravir against the plasma concentration of the drug. The calibration curve was linear and was found to be 0.999. The regression coefficient value is y=0.0021x+0.0053. The Linearity of Raltegravir was shown in Table 1 and Figure 4.

Figure 4: Linearity plot for Raltegravir

Precision:

In precision the Quality control sample (MQC), concentration taken was 500 (ng/ml). Precision was determined by %RSD. Intra and inter batch % precision for Raltegravir was found to be 100.11 and 99.92 and % RSD varies from 0.64 and 1.32. The %CV and precision results were found to be within the suitable limits.

Accuracy:

In Accuracy the Quality control sample (LLQC, LQC, HQC), concentration taken was 25, 250, 1000 (ng/ml). The intra-day RSD was less than 5.87% and the accuracy ranged from 97.26% to 101.35%. Inter day RSD was less than 8.26% and the accuracy ranged from 98.87% to 100.58%. The %CV and accuracy results were found to be within the suitable limits. The extraction recovery of raltegravir from the spiked rabbit plasma samples were within 95.68 to 97.87% with % RSD at each concentration level less than 4.68. Thus, the proposed liquid - liquid phase extraction technique was found to be accurate with high recovery values.

Limit of detection and qualification:

LOD and LOQ was determined from the signal to noise ratio. It is defined as the lowest peak area concentration with signal to noise ratio 3:1 for detection and 10:1 for qualification. The LOD value was found to be 2.5 ng/ml and LOQ values is 25 ng/ml

IN VIVO PHARMACOKINETIC EVALUATION:

The plasma concentration time profiles of raltegravir in rabbit for Experimental formulation and pure drug, are shown in Table 2 and Figure 5. The graph indicated bell shaped curve in both the cases of experimental formulation and pure drug. Raletegravir could be traced to be present in the blood for 20 h after oral administration y, which indicates the effectiveness of drug release from the formulation.

The pharmacokinetic parameters C_{max ,}Tmax, T_1/2,K_el,K_a, AUC_{0-t ,}AUC_0-∞,AUMC _0-24,AUMC_t-∞,MRT_0-24,MRT_0-∞were calculated and the data is shown in Table 3. The C_max for experimental formulation and pure drug were found to be 205.09 µg/ml and 248.36 µg/ml respectively. The T_max for experimental formulation and pure drug were found to be 6h and 2h respectively. The t_½values were 6.268h and 5.471h respectively for experimental formulation and pure drug. The K_elfor experimental formulation and pure drug were 0.11044 and 0.12666 h^-1. The Ka values of experimental formulation and pure drug were found to be 0.2977 and 2.41 h^-1 respectively.The AUC_0-t for experimental formulation and pure drug were found to be 2830.65 and 1965.5 respectively. The values of AUMC_0-_¥ and AUMC_0-24for experimental formulation and pure drug were found to be 31637.18 µg h² ml^-1,12466.14 µg h² ml^-1and 18854.07, 7991.63. The MRT_0-24and MRT_0-_¥for experimental formulation and pure drug were found to be 6.66h ,4.065h and 9.67h, 5.86h respectively shown in table 3. formulation and pure drug were 0.11044 and 0.12666 h^-1. The Ka values of experimental formulation and pure drug were found to be 0.2977 and 2.41 h^-1 respectively.The AUC_0-t for experimental formulation and pure drug were found to be 2830.65 and 1965.5 respectively. The values of AUMC_0-_¥ and AUMC_0-24for experimental formulation and pure drug were found to be 31637.18 µg h² ml^-1,12466.14 µg h² ml^-1and 18854.07, 7991.63. The MRT_0-24and MRT_0-_¥for experimental formulation and pure drug were found to be 6.66h, 4.065h and 9.67h, 5.86h respectively shown in table 3.

Figure 5: In vivo plasma profiles of Raltegravir in rabbit

Table 2: In vivo Plasma Profiles of Raltegravir in Rabbits

Time (hrs)	R1 (Experimental 200mg)	R2 (Control 200mg)
0	0.00	0.00
0.5	8.15±12.4	17.11±10.6
1	40.25±8.2	172.21±9.5
2	118.53±14.9	248.36±8.9
3	132.14±13.4	182.16±14.8
6	205.09±9.1	119.19±11.7
9	190.82±6.4	71.42±9.2
12	143.02±12.9	40.97±13.4
16	95.03±13.4	27.05±12.5
20	48.65±10.6	20.32±11.9
24	0.00	0.00

Table 3: Pharmacokinetic parameters of Raltegravir

S. No.	Pharmacokinetic Parameter	R1 (Experimental)	R2 (Control)
1	C_max(ng/ml)	205.09	248.36
2	Tmax (h)	6	2
3	T_1/2(h)	6.268	5.471
4	K_el (h^-1)	0.110544	0.12666
5	K_a (h^-1)	0.2977	2.41
6	AUC_0-t (ng h/ml)	2830.65	1965.51
7	AUC_0-∞(ng h/ml)	3270.75	2125.93
8	AUMC _0-24 (ng h/ml)	18854.07	7991.63
9	AUMC_t-∞(ng h/ml)	31637.18	12466.14
10	MRT_0-24	6.66	4.065
11	MRT_0-∞	9.67	5.86

CONCLUSION:

The higher sensitive HPLC method was validated and determined by using Raltegravir in rabbit plasma. Here the method is fast, rugged, reproducible bio-analytical method. Simple and efficient method was developed and utilized in pharmacokinetic studies to see the investigated analyte in body fluids. The in vivo data showed the experimental formulation has highest peak plasma concentration of 205.09±9.1µg/ml and maximum time of 6 h to reach maximum plasma peak concentration when compared with Pure drug. The half life was also more for experimental formulation, when compared with Pure drug after oral administration. The results also showed that AUC and area under the first moment of concentration (AUMC) was more for experimental formulation when compared with Pure drug. The mean residence time (MRT) was also more for experimental formulation and this indicates that raltegravir stayed for a long time period in the body and having extended release pattern.

CONFLICTS OF INTEREST:

Authors declare that there was no conflict of interest. This research did not receive any specific grant from any funding agencies.

ACKNOWLEDGEMENT:

The authors are grateful to GITAM Institute of Pharmacy, GITAM (Deemed to be University), Visakhapatnam for providing necessary facilities and support.

ABBREVIATIONS:

RP-HPLC: Reverse Phase High performance liquid chromatography; NM: Nano Meter; USFDA: United States Food and Drug Administration; USA: United States of America; AUC: Area under the ROC curve; ISTD: Internal Standard ; %RSD: Relative Standard Deviation ; %CV :Cumulative variation.

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Received on 07.09.2020 Modified on 30.01.2021

Research J. Pharm.and Tech 2022; 15(4):1800-1804.

DOI: 10.52711/0974-360X.2022.00302

Linearity	Raltegravir concentration (ng/ml)	Raltegravir peak response	IS response	Ratio response
1	25	3942	78541	0.050
2	50	8223	78263	0.105
3	125	20678	78254	0.264
4	250	41062	78655	0.522
5	375	61816	78353	0.789
6	500	80541	78656	1.024
7	625	101654	78492	1.295
8	750	125487	78351	1.602
9	1000	165302	78369	2.109
Slope		0.0021
Intercept		0.0053
CC		0.99949