INTRODUCTION:

Liposomal formulations are widely utilised to enhance the effectiveness, absorption, and side effects of medications. Such particulate drug formulations enable the modulation of a range of pharmacological parameters, including drug circulation time and selective accumulation, which are dependent on the physicochemical characteristics of the formulation and the physiopathology of the target tissue. Since liposomal drug formulations behave differently from free pharmaceuticals, their pharmacokinetic evaluation is distinct from that of free medications.

Many medications exhibit inadequate in vivo target tissue accumulation. A number of doses of the free medication are frequently needed for an effective systemic therapy because of the free drug's quick elimination (short half-life) and wide distribution volumes¹. Encapsulation in long-circulating liposomes has been demonstrated to be beneficial for enhancing the in vivo performance of many medications. These liposomes are known because of their carefully chosen size (about 100 nm) and linked lecithin coating.

The use of such a formulation leads to a number of benefits, including increased circulation time, decreased free drug concentration in blood (which significantly reduces side effects), selected liposome size for selective accumulation in target tissues, and localised drug release from liposomes for long-lasting high concentration in target tissue².

A variety of assumptions deriving from liposome features are required in order to develop an appropriate pharmacokinetic model that will appropriately depict drug distribution as a function of time and the subsequent analysis of drug concentration in the target tissue³. In this study, we provide a necessary approach to the paclitaxel liposomal formulation pharmacokinetics. Finally, a recommended interpretation of the resulting pharmacokinetic parameters is made in correlation with the information gathered from research using a rat model.

Evaluation parameters like % yield, swelling index, % entrapment efficiency and in-vitro drug release studies were carried out and optimized batch were identified. Further, scanning electron microscopy (SEM) study of the optimized batch were also done⁴. Optimized batch was then subjected to the present study to evaluate the pharmacokinetic parameters and to compare the comparative bioavailability of paclitaxel liposome with paclitaxel raw drugs in two groups having 6 rats.

Animal Model And Treatment Protocols:

Materials:

Paclitaxel was provided by Fresenius Kabi Oncology Ltd, India as a gift sample.

AIM:

A method has been developed for validation assay of Paclitaxel in Rats plasma using Fluconazole as internal standard (IS).

OBJECTIVES:

To describe the development of proposed method of analysis of Paclitaxel from Rats plasma.

CHEMICALS:

Table 1: Chemicals used for the study

Methanol	HPLC Grade
Potassium Dihydrogen Phosphate	HPLC Grade
Phosphoric acid	AR Grade
Perchloric acid	AR Grade

CHROMATOGRAPHIC PARAMETERS:

Table 2: Chromatographic parameters

Component Name	HPLC
Component ID	Binary gradient
Manufacturer	Shimadzu Corp.
Model	SPD-M20A 230V
Serial Number	L20154705630
Column	Thermo 250-4,6 5µm C18
Mobile Phase	Phosphate buffer (pH 3) : Methanol (30:70)
Flow rate	1.0 mL/min.
Temparature	Ambient

Shimadzu HPLC Properties:

Table 3:HPLC Specifications

Shimadzu HPLC system Injection Volume	20.00µL
Pumps
Pump A Model	LC-20AD
Pump A Model	LC-20AD
Pumping Mode:	Binary Flow
Total Flow	1 mL/min.
Pump B Pct:	70.0 %
B Curve:	0
Pressure Range (Pump A/B)	0.0 – 6000 psi

Animals and Diet:

Male albino Rats (200-250g) body weight were used for the study. Animals were purchased from Saha Enterprise, 386/2, Nilachalbirati, Kolkata-700051, West Bengal and housed individually in animal cages (Tarsons) in a room. Animals were acclimated for one week in a typical laboratory conditions like temperature 25±1⁰C and humidity 50% - 60%⁵. Animals were exposed to a 12:12 h light/dark cycle with unrestricted access of both demineralized drinking water and commercially available rat chow diet throughout the duration of the study⁴. Throughout the experiment, all animal-related procedures were carried out in accordance with the recommendations of the "Institutional Animal Ethical Committee” for the care and use of laboratory animals. (CPCSEA Regn. No. 1938/PO/Rc/S/17/CPCSEA).

DOSE:

With the test preparation, the dose was one paclitaxel and with the reference preparation, the dose was one Paclitaxel raw drugs. The dose was 50mg/kg in rats.

BLOOD COLLECTION:

A total of 7 blood samples of each rats were collected at 0 h. (before drug administration) then 1, 2, 3, 4, 5 and 6 h⁶. (After drug administration) in the test tubes with K₂EDTA at each time respectively after drug ingestion. On the study days rats were permitted normal activities.

Collected blood samples were centrifuged immediately and plasma was separated and stored frozen at –20⁰C with appropriate labelling of volunteer code no with study date and collection time. Abnormal signs/ symptoms were monitored, during the study period and for one week after the study period and if noticed, their details were entered in the case report sheets and tabulated at the end of the study.

STUDY DESIGN:

The study was carried out in 12 healthy Male albino Rats (200-250g) and the animals were fasted overnight (12hrs) prior to dosing with free access to water. The protocol of the study was approved by the Institutional Animal Ethics Committee.Healthy albino Rats were obtained from registered breeder and were acclimatize for 10 days⁷. Each animal were fur marked for identification and allocated in separate cages. A zero hour fasting blood samples were withdrawn early in the morning. All the animals were administered orally both microsphere formulation and raw drugs at a dose of 50mg/kg using oral feeding syringe by oral route. 0.5 mL of blood was withdrawn by retro- orbital puncture at the time interval of 1, 2, 3, 4, 5 and 6 h. The washout period was kept minimum 7 days. The samples were collected into 0.5mL heparinized centrifuge tubes. The tubes were immediately centrifuged for 10min at 5000 rpm to collect the plasma which is been stored at -20°C until further analysis⁸. Blood samples were processed using acetonitrile protein precipitation method. 0.1mL blood sample cyclo-mixed for 10mins by adding 0.5mL of acetonitrile to extract the drug and subjected to centrifugation at 6000rpm for 5 min and the supernatant collected by using micropipette. Filtered the solution through 0.45µ filter and injected 20 microliter into HPLC system⁹.

PLASMA EXTRACTION PROCEDURE:

150µl subjects plasma, 50µL of IS were transferred into 1.5mL polypropylene micro-centrifuge tubes. Samples were deproteinized by addition of 300µl of ACN, vortexed for 5 minutes and then centrifuged at 6000rpm for 5minutes. Some 20µL of the clean upper layer was injected directly into the chromatographic system.

STATISTICAL ANALYSIS:

All data is presented as mean±SEM. Graph Pad Prism 5.0 was employed to perform statistical analysis of the data. One-factor analysis of variance (one-way ANOVA) followed by Tukey's multiple comparison test was used to test the statistical significance of differences between groups.

RESULTS AND DISCUSSIONS:

Table 4: Plasma Concentration (µg/ml) of Paclitaxel Raw Drug

Rat No.	Time (in hours)
Rat No.	0	1.0	2.0	3.0	4.0	5.0	6.0
1	0	0.60	1.95	8.90	4.37	3.92	2.76
2	0	0.80	2.25	6.25	10.29	6.35	3.21
3	0	0.43	2.48	9.35	6.52	4.10	1.84
4	0	0.59	4.87	8.77	4.24	2.95	1.65
5	0	0.54	3.92	7.20	9.34	5.94	2.73
6	0	0.39	4.59	9.21	6.45	3.04	1.79
Mean	0	0.56	3.34	8.28	6.87	4.38	2.33
S. D.	0	0.15	1.27	1.26	2.50	1.45	0.65
C.V.%	0	26.11	38.07	15.20	36.41	32.97	27.90

Table 5: Plasma Concentration (µg/mL) of Liposome Paclitaxel

Rat No.	Time (in hours)
Rat No.	0.0	1.0	2.0	3.0	4.0	5.0	6.0
1	0	0.80	10.90	19.80	19.95	4.23	3.12
2	0	1.05	8.75	21.58	16.95	6.61	2.98
3	0	0.95	7.95	15.52	23.70	9.03	2.71
4	0	1.60	10.36	18.81	10.95	6.02	1.96
5	0	0.83	5.29	20.50	15.66	8.38	3.23
6	0	0.90	12.30	21.35	8.52	4.77	3.52
Mean	0	1.02	9.26	19.59	15.96	6.51	2.92
S. D.	0	0.30	2.49	2.24	5.61	1.91	0.54
C.V.%	0	29.07	26.84	11.44	35.16	29.42	18.54

Table 6: Pharmacokinetic Parameters of Paclitaxel

Rat No.	Cmax		Tmax		AUC 0-t
Rat No.	Paclitaxel Raw Drug	Liposome Paclitaxel	Paclitaxel Raw Drug	Liposome Paclitaxel	Paclitaxel Raw Drug	Liposome Paclitaxel
1	8.90	19.95	3.0	4.0	21.12	57.24
2	10.29	21.58	4.0	3.0	27.55	56.43
3	9.35	23.70	3.0	4.0	23.80	58.51
4	8.77	18.81	3.0	3.0	22.25	48.72
5	9.34	20.50	4.0	3.0	28.30	52.28
6	9.21	21.35	3.0	3.0	24.58	49.60
N	6	6	6	6	6	6
Mean	9.31	20.98	3.33	3.33	24.60	53.80
SD	0.54	1.67	0.52	0.52	2.85	4.16
CV%	5.75	7.95	15.5	15.49	11.60	7.74

Continue Table no. 6

*Rat No.*	AUC _0-α		t_1/2		Kel
*Rat No.*	Paclitaxel Raw Drug	Liposome Paclitaxel	Paclitaxel Raw Drug	Liposome Paclitaxel	Paclitaxel Raw Drug	Liposome Paclitaxel
1	28.74	60.60	1.91	0.75	0.362	0.928
2	33.06	60.76	1.19	1.01	0.582	0.688
3	27.25	61.00	1.30	0.64	0.534	1.084
4	25.32	51.37	1.29	0.94	0.537	0.738
5	32.74	57.51	1.13	1.12	0.615	0.617
6	27.73	55.48	1.22	1.16	0.567	0.599
N	6	6	6	6	6	6
Mean	29.14	57.79	1.34	0.94	0.533	0.776
SD	3.12	3.84	0.29	0.21	0.089	0.192
CV%	10.71	6.64	21.51	22.06	16.68	24.72

Fig. 1: Chromatogram for paclitaxel

Table 7: Pharmacokinetic Parameters of Paclitaxel in 6 Rats

Pharmacokinetic Parameters	Paclitaxel Raw Drug		Liposome Paclitaxel
C_max (µg/mL.)	Mean	9.31	Mean	20.98
C_max (µg/mL.)	± S.D.	0.54	± S.D.	1.67
t_max (h)	Mean	3.33	Mean	3.33
t_max (h)	± S.D.	0.52	± S.D.	0.52
AUC 0-t (µg. h./mL)	Mean	24.60	Mean	53.80
AUC 0-t (µg. h./mL)	± S.D.	2.85	± S.D.	4.16
AUC 0-¥ (µg. h. /mL)	Mean	29.14	Mean	57.79
AUC 0-¥ (µg. h. /mL)	± S.D.	3.12	± S.D.	3.84
k_el (h.^-1)	Mean	0.533	Mean	0.776
k_el (h.^-1)	± S.D.	0.089	± S.D.	0.192
t_1/2 (h)	Mean	1.34	Mean	0.94
t_1/2 (h)	± S.D.	0.29	± S.D.	0.21
Relative Bioavailability (%)	100 %		218.70%
Log Transformed (LN) Relative Bioavailability (%)	100%		124.43%

DISCUSSION:

There is much cause for optimism about the enhanced relative bioavailability of paclitaxel liposomes. When medications are combined with liposomes, the pharmacokinetics of the pharmaceuticals may be significantly altered¹⁰. In the case of rapid release, the pharmacokinetics of the formulation may approach those for the free drug, or they may become almost indistinguishable from the pharmacokinetics of the carrier. This is because the pharmacokinetics of the formulation are dependent on the rate at which the drug is released from the carrier. When compared to free medications, pharmaceuticals that are coupled with liposomes tend to have a more limited distribution, and this distribution may either be directed toward or away from the normal tissues through which the free drug is distributed. Although paclitaxel liposomes and classical liposomes share some of the same pharmacokinetic properties, paclitaxel liposomes have the significant advantage of displaying dose-independent pharmacokinetics, whereas classical liposomes have dose dependent kinetics¹¹. This is a significant improvement over classical liposomes, which have dose dependent kinetics. When compared to the free medication, the altered pharmacokinetics of the preparation might either result in higher or reduced toxicities in certain tissues, depending on the particular liposome formulation that was used and the particular drug that was being administered¹². It is also possible to see either an increase or a reduction in the maximum dosage of a particular medicine that is tolerated. When developing reasonable therapeutic applications for liposome-associated pharmaceuticals, having an in-depth knowledge of the pharmacokinetics of liposomes may be of tremendous assistance in the design process.

CONCLUSION:

Liposomes are nontoxic, flexible, biocompatible, non-immunogenic vesicles which encapsulate drug and protect them from enzymatic degradation, alter pharmacokinetic and pharmacodynamics properties of drug¹³. The pharmacokinetic study, which is still being done, will help to strengthen the assertion. Future human BA/BE^{14,15,16,17,18} research using paclitaxel's improved liposome will be conducted.Thus, by reducing the negative effects of certain extremely strong medications, liposomal formulations can be successfully used to enhance the pharmacokinetics and therapeutic index of a pharmacological ingredient¹⁹. The studies also highlighted the fact that the drug has more targeted delivery as compared to crude drug²⁰. From the above experimental data it can be concluded that this formulations were successfully prepared and can be applied^21,22.

ACKNOWLEDGEMENT:

The authors would like to thank Department of Pharmaceutical Technology, JIS University, Kolkata. For providing necessary facilities for the research work.

CONFLICT OF INTEREST:

There is no conflict of interest amongst the authors.

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Received on 09.02.2023 Modified on 22.06.2023

Research J. Pharm. and Tech 2024; 17(6):2676-2680.

DOI: 10.52711/0974-360X.2024.00419