Formulation Development, Ex vivo Evaluation and In vivo Antihypertensive study of Losartan Potassium Loaded Nanoproniosomal Gel:

A Novel Vesicular Approach for Transdermal Delivery

M. Sabareesh*¹, J.P. Yanadaiah², K.B. Chandra Sekhar³

¹Research Scholar, Jawaharlal Nehru Technological University, Anantapur, Ananthapuramu,

Andhra Pradesh, India.

²Professor, Dr. K.V. Subba Reddy Institute of Pharmacy, Kurnool, Andhra Pradesh, India.

³Professor, Department of Chemistry, Krishna University, Machilipatnam, Andhra Pradesh, India.

*Corresponding Author E-mail: sabareesh85@gmail.com

ABSTRACT:

The transdermal nanoproniosomal gel of Losartan potassium was prepared to treat hypertension that is efficient to deliver the encapsulated drug over extended periods and to provide better bioavailability. In the present study, the nanoproniosomal gel of Losartan potassium was formulated by Lecithin, Cholesterol, Non-ionic surfactants using the Coacervation-phase separation method. The physical mixture of drug, lecithin, and cholesterol were subjected to compatibility study using FTIR spectroscopy. The prepared nanoproniosomal gels were subjected to various evaluation parameters like the determination of pH and viscosity, vesicle size analysis, rate of spontaneity, entrapment efficiency, zeta potential, ex vivo skin permeation studies, skin irritation test, stability studies and in vivo antihypertensive studies. The physical characterization of nanoproniosomal gels was found to be within the acceptable limits. The ex vivo skin permeation studies showed the cumulative permeation of 47.25 % to 82.49% through the albino rat skin in 24 hrs for all the formulations which indicate the zero-order drug permeation with diffusion, non-fickian release as the possible mechanisms of drug release. Among all formulations, NLPG2 was selected as best formulation because it showed better characteristics than other formulations in several aspects like entrapment efficiency, vesicle size, ex vivo permeation studies, zeta potential, stability studies, and other evaluation parameters. The selected formulation NLPG2 showed the highest transdermal flux (28.67µg/cm²/h), with an enhancement ratio of 2.87 when compared to other formulations. In vivo antihypertensive study revealed that the formulation NLPG2 was successful to regress the rat BP to normal values in experimental hypertensive rats. Finally, it could be concluded that the formulation NLPG2 accentuates the flux of Losartan potassium and is an efficient transdermal therapeutic system for the delivery of a drug. The nanoproniosomal gels are suitable for once a day controlled release formulation.

KEYWORDS: Cholesterol, Lecithin, Losartan Potassium, Permeation studies, Proniosomes, Nonionic surfactants, Transdermal gel.

INTRODUCTION:

The applications of nanotechnology are employed for the development of various nanovesicular carriers with promising features such as protection of the drug from degradation and cleavage, biocompatible or biodegradable to the body. These carriers can act as drug reservoirs to carry both hydrophilic and hydrophobic drugs; which have the affinity for the target site and can able to control the drug release rate. Among them, various carriers such as Liposomes, Niosomes, Ethosomes, Transferosomes, Enzymosomes, Virosomes, Sphingosomes, Archaeosomes, Pharmacosomes, etc.are used but they have some chemical and physical problems such as hydrolysis, oxidation, sedimentation, aggregation, or fusion during storage. Hence, Provesicular concept (Proniosomes) has emerged to solve the stability problems of the conventional vesicular systems^1,2,3.

Proniosomes are nano-sized vesicular structures of dry, free-flowing powder (or) gel with encapsulation of drug in the vesicle that produce multilamellar niosomal dispersion after hydration. They provide the controlled drug release, enhancement of drug penetration and reduce the toxic effects. Proniosomes are the best vesicular carriers for the transdermal route because they act as drug reservoirs for extended periods and improve the skin permeation. They minimize the physical stability problems of niosomes such as aggregation, fusion, and leaking and provide additional convenience in stability, transportation, storage, and dosing^2,4,5,6.

Proniosomal gels are formed by the coacervation phase separation method which involves the admixture of nonionic surfactant, cholesterol with subsequent hydration in aqueous media. They appear as a clear, transparent or translucent semisolid gel texture which makes them stable physically during storage. They have the potential to deliver the drugs through transdermal route. After the application to the skin, the proniosmes are converted into the niosomes in situ by hydration from the skin^7,8,9. This mechanism was shown diagrammatically in the Fig.1.

Figure 1: Niosomes formation from proniosomes by hydration

The gels became more popular and widely used for transdermal application among other dosage forms because of ease of application, and better percutaneous absorption. Gels can reduce physiological stress occurs due to skin flexion, blinking and mucociliary movement and can control the drug release. Hence, proniosomes are commonly prepared in a gel formulation^10,11.

Losartan potassium is an angiotensin II receptor antagonist, used for the treatment of high blood pressure (hypertension) and also used to retard the long term kidney damage in people who are suffering from type 2 diabetes. It is considered as a drug of choice in anti-hypertensive therapy due to its effectiveness and low toxicity. It possesses ideal characteristics such as a low molecular weight which is 423 (below 600 daltons), smaller dose range (25-50 mg), short plasma half-life (2 h), and poor oral bioavailability (approximately 33%) for suitability as a transdermal formulation. Hence, it was selected in the nanoproniosomal formulation to provide the controlled drug delivery across the intact skin for the effective treatment of hypertension^11,12,13.

The main objective of the study was to formulate the nanoproniosomal gel of losartan potassium and to deliver the drug through the transdermal route by eliminating the first-pass metabolism and to enhance the permeability of drug through the skin and also to provide better bioavailability.

MATERIALS AND METHODS:

Materials:

Losartan Potassium was obtained as a gift sample from Vijayasri Chemicals, Hyderabad. Cholesterol, Soya Lecithin, Span 20, Span 80, Tween 20, Tween 80 were obtained from Himedia Laboratories Pvt. Ltd, Mumbai. Methylprednisolone acetate (MPA) injection (Depo-Medrol^TM) manufactured by Pfizer was purchased from a medical shop. All other chemicals and reagents used were of research-grade.

Methods:

Standard curve of the pure drug:

It was carried out by preparing the stock solution by dissolving 100 mg of the drug in 100 ml phosphate buffer pH 7.4 to obtain a final concentration of 1 mg/ml. Then serial dilutions were made to prepare diverse sample solutions of concentrations ranging from 2-12 μg/ml. The solutions were analyzed by using the UV spectroscopic method at an absorption maximum of 234 nm against the blank^14-17.

Drug-Excipient compatibility study:

Fourier Transform-Infrared Spectroscopy (FT-IR spectroscopy) was done to investigate and predict the physicochemical interaction between drug and formulation components^11,18,19.

Preparation of Nanoproniosomal Gel:

It was prepared by the Coacervation-phase separation method. In this method, precisely weighed amount of the nonionic surfactants, drug, cholesterol, and lecithin were added to the wide mouth glass vial with a tightly screwed cap. After mixing all these ingredients, ethanol was added to it and mixed up thoroughly. Heat the above solution until the cholesterol, lecithin, and drug was completely dissolved in surfactant until the appearance of a clear gel. To this clear solution like gel, then add phosphate buffer (it acts as an aqueous phase) and was gently heated until a clear solution obtained, later cooled down while mixing it with glass rod, that results in the formation of nanoproniosomal gel^2,7,20,21. The formulation was shown in Table 1.

Table 1: Formulation table of Nanoproniosomal gel of Losartan potassium

Ingredients	Formulation code (in mg)
Ingredients	NLPG1	NLPG2	NLPG3	NLPG4	NLPG5	NLPG6	NLPG7	NLPG8	NLPG9	NLPG10
Losartan Potassium	25	25	25	25	25	25	25	25	25	25
Lecithin	100	100	100	100	100	100	100	100	100	100
Cholesterol	100	100	100	100	100	100	100	100	100	100
Span 20	1000	--	--	--	500	500	500	--	--	--
Span 80	--	1000	--	--	500	--	--	500	500	--
Tween 20	--	--	1000	--	--	500	--	500	--	500
Tween 80	--	--	--	1000	--	--	500	--	500	500

Evaluation of Nanoproniosomal Gel:

The pH of the proniosomal gel formulations was performed in triplicate by the calibrated digital pH meter²².

Viscosity:

The viscosity of the formulations was measured in triplicate by using Brookfield Viscometer (DV-E)²².

Vesicle size determination (Microscopic evaluation):

The proniosomal gel (100mg) was hydrated with PBS (10ml) in a small test tube by manual shaking and the resulting niosomes were observed using optical microscope at 100 X to determine the size of vesicles^8,21,23.

Rate of spontaneity:

Spontaneity can be defined as the number of niosomes formed spontaneously upon hydration of proniosomes for a period of 15-20m. The specified quantity (20mg) of proniosomal gel was dispersed in saline water in a clean stoppered glass container and warmed a little for the development of niosomes. A drop of this solution was placed on the Neubauer's chamber to count the number of vesicles (niosomes)^8,20,24.

Entrapment efficiency:

To assess the stacking limit of the proniosomal frameworks for Losartan potassium, the proniosomal gel (100mg) was scattered in refined water and warmed a little for the development of niosomes. This dispersion was centrifuged at 18000rpm for 40m at 5^°C (Remi CPR-24 axis). The supernatant portion was utilized for the assurance of free medication at 234 nm spectrophotometrically^8,24.

The rate of entrapment efficiency was determined from

% Encapsulation Efficiency= [1-(Unencapsulated drug/Total drug)] x 100

Ethical clearance approval:

Ethical clearance was obtained from the Institutional Animal Ethical Committee (IAEC) for the handling of experimental animals for evaluation tests such as Ex Vivo Skin permeation studies, Skin irritation studies, and In Vivo antihypertensive studies. The protocol of the animal study was approved by IAEC, Protocol Number: SVCP/IAEC/II-012/2019-20 dt 18.11.19. The experiment was conducted according to the guidelines of CPCSEA (Committee for the purpose of control and supervision of experiments on animals).

Ex Vivo Skin permeation studies:

These studies were performed by using a modified Franz-diffusion cell. It has a receptor compartment with a volume of approximately 60ml and a surface area of 3.14 sq cm. In this study, Albino rat weighing 150-200g was selected and sacrificed using anaesthetic ether, and approximately 4 to 5 Sq cm of the full thickness of the skin was excised from the shaved abdomen site, and was incorporated between the donor and receptor compartments. A precise quantity of proniosomal gel was placed above the skin towards the donor compartment and the receptor compartment was filled with phosphate saline buffer pH 7.4. The heat was provided to maintain the temperature at 37±0.5°C by using a thermostatic hot plate fitted to a magnetic stirrer. The receptor diffusion medium was stirred by a Teflon-coated magnetic bead placed inside a receptor compartment. At appropriate time intervals, samples were withdrawn and were analyzed spectrophotometrically at 234nm^{8,11,19,20,21,24}.

Scanning electron microscopy:

It was carried out to evaluate the morphological characteristics of the niosomes which were formed upon hydration of proniosomal formulations. In this study, a precise quantity of proniosomal gel was diluted with phosphate buffer pH 7.4 in a glass test tube and the formed vesicles were examined by SEM^19,21,23.

Zeta potential:

Zeta potential is a measure of net charge on the surface of niosomes. It was determined by using HORIBA SZ- 100 Zeta meter^19,20,25.

Skin irritation studies:

The skin irritation test was done on healthy albino rats weighing between 160 to 180g. In this study, 0.5g of proniosomal gel was applied evenly to the surface of the previously shaven area of rat skin approximately 1”x1” (2.54 x 2.54cm) square. Then animals were returned to their cages. After 72 h, the test sites were washed with tap water and Erythema and Edema were observed. This study was performed on one rat initially (Initial skin irritation test) and later conducted on two rats (confirmatory skin irritation test)^5,21,26,27.

Stability studies:

The stability study was conducted as per the ICH guidelines and it was carried out to determine the drug degradation from proniosomal gel during the storage period. In this study, the prepared formulations were stored at Refrigeration Temperature (4-8°C), Room Temperature (25±2°C), Oven (45±2°C) for 45 days. At different time intervals, samples were withdrawn and were analyzed for various evaluation parameters^2,3,8,21.

Permeation kinetic studies:

Permeability coefficient (P):

It is the capability of drug permeation through the semi-permeable membrane in µg/cm²/h. It was calculated from the slope of the plot of percentage of drug transported versus time,

P = slope × Vd/S

Where,

Vd = Volume of solution in the donor compartment

S = Surface area of membrane

Flux (J): It is defined as the amount of substance flowing through a unit cross-sectional barrier in unit time. It was calculated by,

J = P × CD

Where,

CD = Concentration of solution in the donor compartment

P = permeability

Enhancement ratio (E_r):

It is an important parameter used to determine the effect of permeation enhancer on diffusion and permeability characteristics of the drug substance. It was calculated by,^28,29

Permeability coefficient of formulation

Enhancement ratio = –––––––––––––––––––––––––

Permeability coefficient of control

In vivo antihypertensive studies:

Healthy male Albino Wistar rats were (weighing approximately 250±25g) selected for this study, and all the animals were healthy during the study. The dose for the rats was determined based on the body weight and surface area ratio. Thirty rats were taken and divided into five groups (Group A to E) each carrying six rats. Group A was considered as control and hypertension was induced in other rats (Group B to E) by injecting MPA (20mg/kg/week) subcutaneously for two weeks. Treatments given to each group were indicated in Table 2.

Then, selected formulation NLPG2 was applied evenly to the surface of the previously shaven area of rat skin and rats were placed in the animal holder and had free access to water and food. Then BP from the tail was recorded at predetermined time intervals up to 24 h using rat blood pressure (BP) instrument (Biopac system, USA). The device consists of a scanner, a tail-cuff, an animal holder attached to the main instrument having a digital BP display panel^30,31.

Statistical analysis: The data was statistically analyzed by using a one-way analysis of variance. A Dunette multiple comparison test and paired t-test using GRAPHPAD INSTAT 3 software (Graph-Pad Software Inc.) were used to test the different formulations and the level of significance was taken as p < 0.05.

RESULTS AND DISCUSSION:

Preparation of standard curve:

The standard curve of the pure drug was constructed by using phosphate buffer pH 7.4. The slope of the given data was found to be 0.0727 and regression was found to be R² = 0.9946. The present analytical method suggests that the drug obeyed the Beer's lamberts linearity principles within limits of 2 to 12 (µg/ml).

Drug-excipient compatibility study:

FT-IR Spectra of all excipients do not show any significant changes in the functional group and fingerprint region, indicating no interaction between pure drug and excipients.

Physicochemical characterization of proniosomal gel:

The Losartan potassium nanoproniosomal gel was characterized for physicochemical properties and the results were given in Table 3. All the prepared formulations showed good physicochemical characteristics and are within limits.

Table 3: Physicochemical characterization

Formulation code	pH*	*Vesicle size (µm)**	Rate of spontaneity*	Entrapment efficiency %*	Viscosity (cps)*
NLPG-1	6.8±0.12	10.5±2.37	16±0.21	62.62±0.24	9840±1.63
NLPG-2	6.9±0.03	4.8±0.52	14±2.53	80.86±0.78	10410±0.81
NLPG-3	6.7±0.02	21.1±1.55	10±0.22	55.23±0.56	10224±1.24
NLPG-4	6.8±0.15	19.4±3.18	11±2.25	60.25±0.78	9190±1.24
NLPG-5	6.8±0.20	11.6±2.04	7±3.26	48.64±0.65	9380±3.26
NLPG-6	7.0±0.01	19.7±2.75	8±0.31	42.54±0.98	10340±1.47
NLPG-7	7.1±0.23	17.5±1.56	10±5.52	56.69±0.65	8529±2.05
NLPG-8	6.8±0.07	19.8±2.52	11±3.60	51.45±0.65	10091±0.81
NLPG-9	6.8±0.14	16.4±4.50	7±4.21	42.98±0.36	10511±0.81
NLPG-10	6.9±0.25	21.9±3.75	18±1.55	56.24±0.65	10220±1.63

*Average of three values

± Standard deviation

Microscopic evaluation:

The microscopic evaluations of the formulation shown that the formulations are in good physical shape and size. The microscopic images were shown in Fig.2 and 3.

Figure 2: Microscopic image of NLPG-2 (Before and after hydration) formed from spans

Figure 3: Microscopic image of NLPG-3 (Before and after hydration) formed from tweens

Ex Vivo Skin Permeation Studies:

The skin permeation data of the formulations were shown in Fig.4. The percentage drug permeation of NLPG-2 was found to be the highest among all, around 82.49 % than other formulations. The decrease in vesicle size due to nanoproniosomal technology may be the reason for the increase in drug diffusion rate and extent.

Figure 4: Ex Vivo skin permeation study chart

Permeation data analysis:

The permeability parameters such as permeation coefficient, flux, enhancement ratio were significantly increased in nanoproniosomal formulations. The kinetic permeation data was shown in Table 4. Among all formulations, NLPG-2 was shown good physicochemical characteristics, better skin permeation, and permeation kinetics. Hence, it was selected as the best formulation for further studies.

Table 4: Ex Vivo Permeation kinetics

S. No	Formulation Code	Permeation coefficient (P) (µg/cm²/h)*	Flux (J) (µg/cm²/h)*	Enhancement ratio (E_r)
1	Control	2.25±0.02	10.12±0.12	-
2	NLPG-1	4.86±0.11	22.47±0.17	2.16
3	NLPG-2	7.33±0.08	28.67±0.04	2.87
4	NLPG-3	3.58±0.14	13.31±0.33	1.59
5	NLPG-4	3.67±0.05	14.88±0.25	1.63
6	NLPG-5	4.08±0.13	18.25±0.57	1.78
7	NLPG-6	3.86±0.21	16.10±0.48	1.71
8	NLPG-7	3.92±0.07	16.32±0.21	1.74
9	NLPG-8	5.75±0.04	24.18±0.33	2.55
10	NLPG-9	3.74±0.18	15.74±0.42	1.46
11	NLPG-10	6.15±0.15	26.80±0.26	2.73

*Average of three values

± Standard deviations

Scanning Electron Microscopy:

The best formulation NLPG-2 was selected for the Scanning Electron Microscopic analysis. It showed good surface morphology. It was shown in Fig.5.

Figure 5: Scanning Electron microscopic image of best formulation NLPG-2

Vesicle size and Zeta potential analysis:

The particle size and the PI value of the best formulation were determined as 48.7 nm and 1.698 (which indicates very broad distribution). The Zeta potential of the best formulation was found to be -80.8 mV and reported as excellent stability. The vesicle size and zeta potential graphs were shown in Fig.6 and 7.

Figure 6: Vesicle size of nanoproniosomal gel of Losartan potassium

Figure 7: Zeta-Potential of nanoproniosomal gel of Losartan potassium

Skin Irritation Studies:

The results shown no changes like Edema and Erythema. Hence, the formulation passed the skin irritation test and is safe to be used on Human skin. The skin irritation test images were shown in the Fig.9.

Figure 9: Skin irritation test

Stability Studies:

Physical appearance and homogeneity were found to be good during the storage period. The Stability studies indicated that the formulation NLPG-2 is highly stable at different time intervals and different temperatures. Hence, the formulation passed the stability studies. The stability data was shown in Table 5.

Table 5: Stability data

Storage period	4⁰C*
Storage period	Vesicle Size	Entrapment efficiency	Viscosity	pH
15 days	4.8±0.12	80.2±0.35	10525±0.81	6.9±0.10
30 days	4.8±0.10	79.7±0.36	10552±0.80	6.9±0.03
45days	4.6±0.14	78.6±0.65	10586±0.84	6.9±0.05
Storage period	25⁰C*
Storage period	Vesicle Size	Entrapment efficiency	Viscosity	pH
15 days	4.8±0.02	80.5±0.25	10502±0.80	6.9±0.02
30 days	4.8±0.11	80.1±0.23	10536±0.71	6.9±0.08
45days	4.7±0.15	79.6±0.56	10558±0.87	6.9±0.04
Storage period	40⁰C*
Storage period	Vesicle Size	Entrapment efficiency	Viscosity	pH
15 days	4.8±0.05	80.6±0.38	10414±0.68	6.9±0.11
30 days	4.7±0.13	80.5±0.49	10425±0.81	6.9±0.13
45days	4.6±0.14	80.2±0.47	10465±0.81	6.9±0.07

*Average of three values

± Standard deviations

Table 6: Influence of proniosomal gel formulation of Losartan potassium on mean BP in MPA induced hypertensive rats

S. No.	Group	Treatments	Mean BP (mm Hg)*			% Reduction in BP*
S. No.	Group	Treatments	Pre-treatment	Post- MPA treatment	Post Proniosomal gel treatment	% Reduction in BP*
1	A	Control	122.15 ± 12.12	--	--	--
2	B	Only MPA	121.06 ± 5.24	160.56 ± 10.28	--	--
3	C	MPA + placebo NLPG2	123.20 ± 15.73	163.28 ± 16.45	163.11 ± 10.31	0.10 ± 0.01
4	D	MPA + NLPG2	122.58 ± 10.48	162.85 ± 11.42	121.70 ± 9.73	25.26 ± 1.75
5	E	MPA+ Marketed tablet	121.69 ± 7.94	161.64 ± 11.43	144.84 ± 7.67	10.39 ± 1.62

*Average of six values

± Standard deviation

In vivo antihypertensive studies:

In this study, hypertension was successfully induced in the normal healthy rats by injecting MPA for two weeks and they remained hypertensive for 72 h after stopping the MPA injection. The Proniosomal gel formulation NLPG2 was found to decrease the BP significantly (p < 0.001) in proximity to the normal value and it was maintained for 24 h (Table.6). This indicates that the drug was permeated and constantly reaches the systemic circulation up to 24 h in rats. However, post-treatment BP values in the treatment group (D) were comparable with the control group (A). On comparing the effects of all the systems, the percentage reduction in mean rat BP by NLPG2 and Placebo NLPG2 was 25.26% and 0.10 %, respectively (Table.6). NLPG2 was successful in reverting the rat BP to normal values. The above results suggest that the developed formulation NLPG2 holds promise for the management of hypertension which needs to be validated by clinical trials.

CONCLUSION:

The nanoproniosomal gel of Losartan potassium was formulated to improve the bio-availability and permeation of the drug. Various formulations were prepared by the coacervation phase separation method. Among them, NLPG-2 showed better characteristics than other formulations in several aspects like entrapment efficiency, vesicle size, ex vivo permeation studies, skin irritation studies, stability studies, in vivo antihypertensive studies, and other evaluation parameters. Hence, the formulation NLPG2 was considered as the best formulation and is suitable for controlled release once a day formulation for the treatment of hypertension. These nanoproniosomal carrier systems hold a promising future for effective transdermal delivery of bioactive agents and other problematic drug molecules for various diseases and disorders. In the future, it has a wide range of applications and opportunities for researchers to experiment with a variety of drugs to study their systemic and local effects.

ACKNOWLEDGEMENT:

We are grateful to the Chairman, Management and Principal of Sree Vidyanikethan College of Pharmacy, Tirupati for providing all the necessary facilities and lab facilities to carry out the research work.

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Received on 03.04.2020 Modified on 15.05.2020

Research J. Pharm. and Tech 2021; 14(3):1423-1430.

DOI: 10.5958/0974-360X.2021.00254.7

S. No.	Group	Treatments	No. of rats in group	Measurement of BP at different time intervals (h)
1	A	Control	6	0, 1, 2, 3, 4, 6, 8, 10, 12, 24
2	B	Only MPA	6	0, 1, 2, 3, 4, 6, 8, 10, 12, 24
3	C	MPA + placebo NLPG2	6	0, 1, 2, 3, 4, 6, 8, 10, 12, 24
4	D	MPA + NLPG2	6	0, 1, 2, 3, 4, 6, 8, 10, 12, 24
5	E	MPA + Marketed tablet	6	0, 1, 2, 3, 4, 6, 8, 10, 12, 24