Comparative In vivo Evaluation of Propranolol hydrochloride

following Oral and Transdermal administration in Rabbits

 

V. Sai Kishore,* T. E. Gopala Krishna Murthy and C. Mayuren

Bapatla College of Pharmacy, Bapatla-522101

*Corresponding Author E-mail: voiceofsaikishore@yahoo.com

ABSTRACT:

 

INTRODUCTION:

 

Propranolol hydrochloride is a β-blocker widely used in the treatment of angina pectoris, cardiac arrhythmias and hypertension¹. It is subjected to extensive and highly variable hepatic first pass metabolism following oral administration, with a reported systemic bioavailability between 15 and 23%². Prolonged release formulations may reduce the dosing frequency but the bioavailability of Propranolol hydrochloride from these formulations is only 40–60% of that from a conventional tablet^3-5. This has been attributed to the slower absorption in the gastrointestinal tract coupled with an extensive first pass effect⁶. Hence, in the present study, the in vivo performance of orally and transdermally administered Propranolol hydrochloride was evaluated in rabbits in a randomized cross-over design. Membrane-moderated transdermal systems of Propranolol hydrochloride were selected for the transdermal administration based on the invitro studies⁷.

 

The pharmacokinetic parameters of both oral and transdermal formulation analysis were compared statistically to reveal the relative bioavailability and to justify relative rate of extensive first pass metabolism of Propranolol hydrochloride.

 

MATERIALS AND METHODS:

Propranolol hydrochloride (gift sample from Natco Pharm. Pvt. Ltd. Hyderabad), cellulose acetate (S.D Fines chemicals Ltd. Mumbai), Ethyl acetate (Qualigens), Dibutyl phthalate (Ranbaxy Laboratories), Sodium carboxy methyl cellulose (S.D Fines chemicals Ltd. Mumbai), Poly ethylene glycol-6000(S.D Fines chemicals Ltd. Mumbai) were of pharmaceutical grade and obtained commercially.

 

Animals: New Zealand male rabbits (1.2 - 1.8 kg) maintained at 25 + 1^oC were used for the study. The animals were housed in stainless steel metabolic cages and provided standard diet and water ad libitum.

 

Preparation of membrane-moderated transdermal systems:

Membrane-moderated transdermal systems of Propranolol hydrochloride were prepared by incorporating the drug reservoir within a shallow compartment moulded from a drug-impermeable backing membrane and 2% w/v cellulose acetate rate-controlling membrane casted with ethyl acetate-methanol (8:2)employing dibutyl phthalate (40% w/w of dry polymer) as plasticizer .

 

Solvent evaporation technique was employed for the preparation of cellulose acetate films. The polymer solutions were prepared by dissolving the polymer (2% w/w cellulose acetate) in 20 ml of ethyl acetate-methanol (8:2). Dibutyl phthalate at a concentration of 40% w/w of the polymer was used as a plasticizer. 20 ml of the polymer solution was poured in a Petri plate (9.4 cm diameter) placed on a horizontal flat surface. The rate of evaporation was controlled by inverting a funnel over the petri plate. After 24 hours the dried films were taken out and stored in a desiccator. A circular  silicon rubber ring with an internal  diameter of 2.5cm and a thickness of 3mm was fixed on to a backing membrane (an unperforated adhesive strip ;supplied by Johnson and Johnson Limited ,Mumbai). This serves as a compartment for drug reservoir.

 

Drug reservoir gels were formulated as per the composition given in Table 1. The required quantities of polymer was weighed and transferred separately into a mortar. It was triturated with 5 ml of water. Specified amount of Propranolol hydrochloride, methyl paraben, and propyl paraben were weighed accurately and dissolved in glycerin. The resulting drug solution was incorporated into the polymer dispersion slowly with continuous trituration to obtain a gel. The gel was transferred in to a measuring cylinder and the volume was made up to 20 ml with distilled water. 1 g of medicated gel was taken into the compartment as a drug reservoir. Cellulose acetate membrane of known thickness was fixed on the ring with glue to form a membrane moderated therapeutic systems.

 

In-Vivo Evaluation:

Prior approval by Institutional animals ethics committee was obtained for conduction of experiments (Ref: IAEC/I-3/BCOP/2007-2008).

 

Pharmacokinetic evaluation:

The pharmacokinetic performance of Propranolol hydrochloride following oral and transdermal administration was studied in a randomized cross over design in rabbits. Animals were fasted 24 hrs prior to the administration of the drug formulation, but had free access to water. One day before the experiment, hair on the abdominal area was clipped by applying depilatory for 10 min and washed with distilled water. On the day of experiment the animals were anaesthetized with urethane (1 gm/kg, i.p.). Following anaesthesia, animals were secured in a supine position.

Subject selection:

Twelve   healthy rabbits with a mean age of 10 ± 2 weeks and with a mean body weight of 3 ± 0.2 kg included in the above investigation for oral versus transdermal formulations of Propranolol hydrochloride.

 

Study design:

The study was of a non-blinded, open-label design .Subjects were fasted for at least 24 hrs prior to timing of dose. One day before the experiment, hair on the abdominal area was clipped by applying depilatory for 10 min and washed with distill water. Overnight-fasted rabbits, whose hair had been removed prior to the experiment, were divided into two treatment groups (n = 6 per group) as follows:

(1) Group I (Propranolol hydrochloride solution (5 ml) containing 4 mg of drug)

(2) Group II (Transdermal formulation containing 4 mg of drug)

 

Table 1 : Reservoir of the Propranolol hydrochloride membrane controlled Transdermal systems

 

 

Table 2: Summary of the Chromatographic and Mass spectrometric conditions

Chromatographic and Mass spectrometric conditions
HPLC	PERKIN ELMER
Mass spectrometer	API 2000
Ion source	Heated nebulizer
Polarity	Positive ion mode
Detection ions
Propranolol Metoprolol	260.200 amu (parent), 56.0 amu (product) 268.200 amu (parent), 116.20 amu (product)
Column	Vertisep-BDS, 5µ, 4.6xl50mm, C18
Mobile phase	0.1% Formic acid: methanol : acetonitrile (pH: 6.0 ± 0.1) (10:45:45)
Flow rate	lml/min
Retention time	Propranolol    1.6 to 2.4 minutes Metoprolol   1.5 to 2.3 minutes
Run time	3.00 minutes
Curtain Gas (CUR)	10.0 PSI
Nebulizer Current (NC)	3.0V
Ion Spray Voltage (IS)	5500V
Temperature (TEM)	550 °C

 

Blood sampling:

About 1 ml of blood samples were drawn at 0 (before drug administration),  0.5, 1.0, 2.0, 3.0, 4.0 and 6.0 hrs after oral administration and 2, 4, 6, 8, 12, 16, 20, 22,24,26,28,30 and 32 hrs after administration of transdermal formulation involved in the study at a dose equivalent to 4  mg of Propranolol hydrochloride. Blood sample volume was replaced by administration of isotonic saline.  Blood samples were collected into heparinized tubes and centrifugation at 3000 rpm for 10 min and plasma samples were stored at –20°C until analysis by a known LC-MS/MS method⁸.

 

Estimation of Propranolol hydrochloride in plasma

The estimation of Propranolol hydrochloride in plasma was carried out by LC-MS/MS method. A summary of the chromatographic and mass spectrometric conditions are shown in Table 2 and Table 3.

 

Table 3: Summary of the MRM parameters and

Parameter	Propranolol	Metoprolol
Declustering Potential (DP)	33.0	60.0
Entrance Potential	10.0	10.0
Collision Energy (CE)	36.0	36.0

 

Standard solutions:

Standard stock solutions of Propranolol hydrochloride (100 mg/mL) were prepared by dissolving an accurately weighed sample in methanol. The standard solutions used to construct the calibration curve were prepared by adding known amounts of Propranolol hydrochloride (2.012 ng/ml to 402.366 ng/ml) to blank plasma. Analyte concentrations of stock dilutions of standard Propranolol hydrochloride solution with plasma were shown in Table 4.

 

Extraction procedure:

Step 1: Blank, calibration curve standards and the subject samples were withdrawn from the deep freezer and allowed them to thaw. The thawed samples were vortexed to ensure complete mixing of the contents. To 0.5 ml of plasma sample in a ria vial, 50µl of Metoprolol (1µg/ml) was added. To plasma blank and pre-dose (0.0hr), 50 ul of 60%methanol in water solution was added. The samples were vortexed to ensure complete mixing of contents

 

Step 2: Approximately 3 ml of ethyl acetate solution was added and centrifuged for 10 minutes at approximately 4000 rpm at 20°C and the supernatant (organic layer) was transferred into another ria vial.   The organic layer was evaporated under a stream of nitrogen gas at 45°C. The residue was reconstituted with 0.25 ml of reconstitution solution and vortexed. The samples were transferred in auto-injector vials and were loaded in to auto sampler.  20 ul of sample was injected onto LC-MS/MS system.

 

Calibration curve:

Analyte concentrations of stock dilutions of standard Propranolol hydrochloride solution with plasma were shown in Table 4. A calibration curve (Figure:1) was obtained by plotting peak area ratios of  Propranolol to Metoprolol  (y-axis) against Propranolol concentration (x-axis).

 

Determination of Pharmacokinetic Parameters:

Various pharmacokinetic parameters such as peak plasma concentration (C_max), time at which peak occurred (T_max), area under the curve (AUC), elimination rate constant (K_el), biological half-life (t_½) and mean residence time (MRT) were calculated using the noncompartmental pharmacokinetics data analysis software PK Solutions 2.0™(Summit Research Services, Montrose, CO, USA).

 

 

Table4: Analyte Concentrations of Stock Dilutions of Standard Propranolol hydrochloride Solution with Plasma

S. no	Sample name	Analyte concentration (ng/ml)	Analyte peak area	IS Peak Area	Area Ratio	Calculated Concentration (ng/ml)	Accuracy (%)
1	Aqueous mixture	N/A	125661	116069	1.08	87.168	N/A
2	Plasma blank	0	0	0	0	N/A	N/A
3	Blank+ISTD	0	0	85038	0	N/A	N/A
4	CC1	2.012	2196	90727	0.02	1.997	99.23
5	CC2	4.024	4798	95821	0.05	4.077	101.33
6	CC3	10.060	11343	91483	0.12	10.026	99.66
7	CC4	30.177	35748	93281	0.38	30.887	102.35
8	CC5	80.472	94468	93587	1.01	81.275	101.00
9	CC6	150.887	175097	92885	1.89	151.741	100.57
10	CC7	301.775	299210	79548	3.76	302.725	100.31
11	CC8	402.366	430049	90027	4.78	384.441	95.55

Table 5: Statistical treatment of pharmacokinetic parameters (Mean ± S.D.) of Propranolol hydrochloride obtained with oral and transdermal formulations

Pharmacokinetic parameter	Oral solution	Transdermal formulation	Calculated value of ‘t’
Cmax (ng/ml)	28.9 ± 0.43	24.82 ± 0.25	12.53***
MRT (h)	3.3 ± 0.015	17.0 ± 0.024	338.40***
t1/2 (h)	2.028 ± 0.02	3.15 ± 0.01	8.96***
Kel (h-1)	0.34 ± 0.004	0.22 ± 0.003	6.70***
Ka (h-1)	2.12 ± 0.01	0.68 ± 0.01	85.68***
AUC0- (ng h/ml)	100 ± 1.23	614.9.± 1.36	146.40***

Values are presented in Mean ± SD (n = 6); *p<0.05, ** p<0.01,*** p<0.001

 

Table 6: Percent ß-Blockade after oral and transdermal administration of Propranolol hydrochloride in rabbits.

Formulation	Percent ß-blockade
	1h	4h	6h	8h	12h	16h	20h	24h
Oral	98.6±6.8	54.2±4.8	24.4 ±2.6	---	---	---	---	---
Transdermal	----	52.2±4.8	74.2 ±4.2	98.6±3.8	98.2±3.6	97.8±3.8	97.4±3.2	96.8±3.5

Figure 1: Calibration Curve for Estimation of Propranolol hydrochloride in Plasma

 

Figure 2: Plasma Concentration-Time Curves of Propranolol hydrochloride following oral Administration

 

Figure 3: Plasma Concentration-Time Curve of Propranolol hydrochloride following Transdermal Administration

 

Statistical analysis of the pharmacokinetic parameters:

The pharmacokinetic parameters of the tested formulations were statistically analyzed using paired sample’s t-test for normal distributed results of C_max, K_a, AUC_0-24 and AUC_0-α values. All tests were performed at 0.001 level of significance.

 

Pharmacodynamic evaluation:

Pharmacodynamic performance in animals following oral and transdermal administration of Propranolol hydrochloride was assessed by the percent ß –blockade⁹. The animals were fasted for 24 h prior to the administration of drug formulations but had free access to water. Animals were anaesthetized with urethane (1 g/kg, i.p.). Following anaesthesia normal electrocardiogram for all the animals was recorded. Before administering the drug formulations, isoprenaline (2 mg/kg, i.v.) in normal saline was administered into the marginal ear vein. Electrocardiogram was recorded immediately and these cardiograms served as control. Animals were left for one hour and the drug formulations were administered as described in pharmacokinetic studies. Electrocardiogram was recorded after 1, 2, 4 and 6 h in case of oral and 4, 8, 12, 20 and 24 h in case of transdermal post-administration, isoprenaline (2 mg/kg, i.v.) response was recorded as before. Each animal was used as its own control. The percent ß –blockade was determined by taking the difference in the response of isoprenaline at 0 time and at the specified time intervals post-administration of drug formulations.

 

RESULTS AND DISCUSSION:

Plasma Propranolol hydrochloride concentrations following oral and transdermal administration at different times were calculated and are shown in Fig 1 and 2. Pharmacokinetic parameters such as absorption rate constant, elimination rate constant, half life, AUC, AUMC and MRT were calculated from the plot of time versus plasma concentration and reported in Table 5 and subjected to statistical analysis. The results indicated that the parameters significantly differed following transdermal administration, compared to oral administration.

 

The results from the oral administration of Propranolol hydrochloride indicated the maximum plasma concentration (C_max) 28.9± 0.43 ng/ml at 1 hrs (t_max) while transdermal administration exhibited the steady state concentration of 24.82 ± 0.25 ng/ml after an initial lag time 12 hrs. The oral administration of Propranolol hydrochloride resulted in a low and quite variable AUC of 100 ± 1.23 ng.hr/ml, where as the transdermal resulted in AUC of 614.9± 1.36 ng.hr/ml. The MRT of transdermal administration (17.0 ± 0.024 hrs) was found to be more than oral administration(3.3 ± 0.015 hrs). The results indicated that the parameters significantly differed following transdermal administration, compared to oral administration. The concentration of Propranolol hydrochloride in plasma was found to be stabilized and maintained in a narrow range over the study period up to 24 hrs for transdermal formulation where as the concentration was decreased rapidly up on oral administration. The maximum plasma concentration (C_max) was attained at 1 hrs after oral administration (4 mg) and it was observed after 12 h upon application of transdermal formulation of same dose.

 

The mean residence time (MRT) was found to be increased significantly (p<0.001) for transdermal application on comparison with oral administration. Though both the formulations containing an equivalent amount of Propranolol hydrochloride (4 mg), the AUC_0-∞ values observed with transdermal formulations (p<0.001) was found to be six fold than that of oral solution. The low t_max and high C_max values following oral administration was due to rapid absorption from the gastro intestinal tract, in contrast the low C_max and prolonged t_max after transdermal administration was due to barrier properties of skin that leads to accumulation of drug into skin tissues in intial stages followed by continuous delivery into the systemic circulation.

 

Thus, the transdermal drug delivery system of Propranolol hydrochloride indicated an enhancement in bioavailability significantly over oral formulations. The observed significant increase in bioavailability may be due to the reduced extensive first pass metabolism of Propranolol hydrochloride up on transdermal administration.

 

The results of the present study concluded that the route of administration has significant influence on bioavailability of Propranolol hydrochloride. Transdermal administration may influence the bioavailability by hindering the extensive first pass metabolism by organ such as intestine and liver.

 

The percent ß-blockade following oral and transdermal administration was given in Table 6. In agreement with pharmacokinetic data, maximum ß -blockade was obtained at 1 h after oral administration and decreased by 80% after 6 h. In the case of transdermal administration, a steady state ß-blockade.was observed after 12.0 h and was prolonged over a period of 24 h. The in vivo pharmacokinetic studies revealed that the transdermal formulation of Propranolol hydrochloride exhibited controlled release and absorption kinetics over longer periods of time which in turn the plasma concentrations was maintained in a narrow range over longer periods of time and leading to the conclusion that of Propranolol hydrochloride transdermal formulations are suitable for once a day administration.

 

REFERENCES:

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8.       D.Siluk, D E Mager, N Gorinch, D Abernethy, I W Wainer.HPLC- atmospheric pressure chemical ionization mass spectrometric method for enantioselective determination of R,S-propranolol and R,S-hyocy amine in human plasma. J. Chromatogr 2007; 859: 213-21.

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Received on 12.07.2010        Modified on 24.07.2010

Accepted on 01.08.2010        © AJRC All right reserved