- Email: [email protected]
Synthesis and comparative skin permeability of atenolol and propranolol esters
J. DRUG DEL. SCI. TECH., 15 (2) 187-190 2005
Synthesis and comparative skin permeability of atenolol and propranolol esters B. Anroop1, B. Ghosh2*, V. Parcha1, A. Kumar1, J. Khanam3 Department of Pharmaceutical Sciences, SBS (PG) Institute of Biomedical Sciences & Research, Balawala, Dehradun (UA), India 2 KLE Society’s College of Pharmacy, II Block, Rajajinagar, Bangalore, 560010 Karnataka, India 3 Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India *Correspondence: [email protected]
1
The prodrug approach is one of the methods which have been evaluated for improving the systemic delivery of pharmacologically active compounds. ln the present study, we have synthesized ester prodrugs of atenolol and propranolol by adding alkyl side chains to the β-hydroxy function of the drug with the objective of enhancing their lipophilicity. In vitro skin permeability study in excised mice skin showed that the prodrugs permeate freely through the skin than the respective drug moieties and significant flux enhancement (6.2-fold) was observed in both cases. In the case of atenolol, the highest permeability coefficient was shown by capriate (C10 side chain) where as in propranolol the highest permeability was recorded with caproate ester (C6 side chain). The difference in permeability may be attributed to their difference in the intrinsic lipophilicities. Key words: Transdermal delivery – Ester – Atenolol – Propranolol.
An epidemiological survey has revealed that 15-25% of the adult population of most countries have elevated blood pressure and normalization of blood pressure plays a primary role in the prevention of cardiovascular mortality and morbidity [1]. β-blockers atenolol and propranolol are among the popular drugs used for maintenance of blood pressure. However, both the drugs suffer the disadvantage of low and variable oral bioavailability due to first-pass metabolism [2]. The transdermal delivery route has been demonstrated to be capable of avoiding hepatic first-pass metabolism and hence various attempts have been taken to develop the cardiovascular drugs in the transdermal dosage forms. It has been shown that some β-blockers can permeate adequately through the skin when delivered from specially constituted vehicles containing enhancer molecules [3]. Similarly, the prodrug approach represents an alternative and promising method of enhancing the skin permeability of drugs by increasing the lipophilicity [4, 5]. Attempts had already been made to enhance the skin permeability of propranolol by esterification of its β-hydroxyl function [6, 7]. In the present study, we have synthesized and evaluated the skin permeability of capric and caproic esters of atenolol and propranolol to examine their suitability for transdermal development.
II. METHODS 1. Synthesis of esters
For atenolol caproate, the reaction was carried out in the fol1owing way. Atenolol (1 mM) dissolved in hot dry benzene (100 ml) was refluxed for 20 h with capric acid (1 mM) and few drops of concentrated sulphuric acid under anhydrous conditions [8]. The reaction was monitered by TLC using solvent system dioxane:acetonitrile:methanol:conc. (25%) ammonia (60:36:5:4) and visualized using Dragandroffʼs reagent. After completion of the reaction, benzene was removed. To the residue thus obtained was added water, extracted with ether and washed with 1% sodium carbonate. The ethereal layer was dried over anhydrous sodium sulphate and concentrated to obtain atenolol caproate as gummy solid (50% yield). Other esters, atenolol capriate, propranolol caproate and propranolol capriate, were synthesized similarly (Figure 1). OH
Dry benzene
R-OCH2-CH-CH2-NHCH (CH3)2 + R’COOH (I)
(II)
R
I. EXPERIMENTAL 1. Materials
Conc. H2SO4
OCOR’ R-OCH2-CH-CH2-NHCH (CH3)2
(a)
R’ CH3 (CH2)4-
CH2CONH2
(b)
Propranolol hydrochloride (Natco Pharmaceuticals, Hyderabad, India) and atenolol (Dabur, Ghaziabad, India) were received as gift samples. Capric acid and caproic acid were purchased from Loba Chemie, Mumbai, India. Octanol was procured from Merck-Schuchardt, Germany. All other chemicals and reagents used were of analytical grade.
CH3 (CH2)8CH2CONH2
(c)
CH3 (CH2)4-
(d)
CH3 (CH2)8-
Figure 1 - Schematic representation.
2. Animals
2. IR and NMR studies
Swiss albino mice, 6-12-week-old, weighing 28-32 g, were obtained from the Institute Animal House (approved by CPCSEA, Chennai, India). Animals were maintained under normal conditions and had free access to food and water.
IR spectra were recorded using Jasco FT/IR-5300 in nujol. H-NMR (CDCl3) spectra were recorded on Brucker (200 MHz) using TMS as internal standard.
1
187
J. DRUG DEL. SCI. TECH., 15 (2) 187-190 2005
Synthesis and comparative skin permeability of atenolol and propranolol esters B. Anroop, B. Ghosh, V. Parcha, A. Kumar, J. Khanam
2.1. Atenolol caproate (III a) IR Vmax: nujol: 3340, 3160 (CONH), 2940 (=CH), 1735 (COOR), 1625 (C=O amide) and 1235 cm-1 (arylether). 1HNMR CDCl3 δ: 7.38 (2H, d, J = 8.75 HzArH), 7.05 (2H, d, J = 8.75 Hz ArH, 4.92-4.97 (3H brd hump NH, NH2) 4.53 (1H, m, -OCH2CHCOOR), 4.12 (2H,s,-OCH2), 3.59 (2H, s, -CH2C6H5), 3.03 (1H, dd, J = 12.5, 5.0 Hz CH (OCOR)CH2a), 2.81 (1H, dd, J = 12.5, 5.0 CH (OCOR)CH2b), 3.14-2.65 (1H, m, NH CH (CH3)2), 1.25 (6H, d, J = 6.25 Hz NH CH (CH3)2), 2.68 (2H, dt, J = 6.2, 7.4 Hz OCO-CH2-(CH2)3-CH3), 2.48 (2H, m, OCOCH2 CH2 (CH2)2 CH3), 1.38 (4H, m, OCO(CH2)2 (CH2)2 CH3) and 0.89 (3H, t, J = 7.5 Hz, OCO(CH2)4 CH3).
for esters it was done in octanol. The partitioning was done by intermittent shaking of equal volume of both the phases for 24 h to ensure the attainment of equilibrium. In both cases, the aqueous solution is suitably diluted and determined spectrophotometrically for drug content. The partition coefficient was determined from the equation:
2.2. Atenolol capriate (III b) Gummy solid (56%) IR Vmax: nujol: 3340, 3160 (CONH), 2940 (=CH), 1735 (COOR), 1625 (C=O amide) and 1235 cm-l (arylether). lH-NMR CDCl3 δ: 0.88 (3H, t, J = 7.5 Hz OCO (CH2)8 CH3), 1.01-1.56 (12H, m, OCO (CH2)2 (CH2)6 CH3), 2.45 (2H, m, OCO CH2 CH2 (CH2)6 CH3) and 2.68 (2H, dt, J = 6.2, 7.4 Hz, OCO CH2 (CH2)7 CH3).
5.1 Skin membrane preparation Swiss albino mice were sacrificed by snapping the spinal cord at the neck. Full thickness skin was obtained from the dorsal surface. Adhering fat and visceral debris were removed from the under surface with tweezers and used immediately. Removal of the surface hair was not attempted to avoid the accidental scratching of stratum corneum.
Ko/w = Co/Cw where Co is the concentration of drug or ester in octanol and Cw is the concentration of drug or ester in pure water.
5. Permeability study
2.3. Propanolol caproate (III c) White amorphous solid m.p.156-158°C (60%) IR Vmax: 3440 (NH), 1625, 1680 (C=C) aromatic), 1735 cm-l (COOR). 1 H-NMR CDCl3 δ: 1.49-1.52 (6H, d, J = 6.6 Hz, -NH CH (CH3)2), 3.48 (2H, brs, CHOAcCH2NH), 3.54 (1H, M, NH CH (CH3)2), 4.41 (2H, d, J = 4 Hz OCH2 CHOAc), 6.81 (1H, d, J = 7.7 Hz ArH), 7.35 (1H, J = 8.1 Hz ArH), 7.48 (3H, m, ArH), 7.80 (1H, brd, J = 7.0 Hz ArH), 8.18 (1H, d, J = 7.2 Hz, ArH), 10.01 (broad hump NH2), 0.89 (3H, t, J = 7.5 Hz OCO(CH2)4 CH3). 1.01-1.56 (4H, m, OCO(CH2)2 (CH2)4 CH3). 2.45 (2H, m, OCOCH2 CH2 (CH2)2 CH3) and 2.63 (2H, dt, J = 6.5, 73 Hz, OCOCH2 (CH2)4 CH3).
5.2. Diffusion study The in vitro permeation studies were performed using a vertical type diffusion cell (Neutron Scientific, Kolkata) according to the procedure of Ghosh et al. [10]. The excised skin was mounted between the half cells with the dermis in contact with the receptor fluid, 0.9% NaCl, and was equilibrated for 1 h. The receiving chamber had a volume of 50 ml and the area available for diffusion was about 1.74 cm2. The donor cell was covered with an aluminium foil to prevent the evaporation of vehicle. However, the skin was not occluded. The drug activity was maintained uniform in matching experiments by loading equivalent amounts of the drug and esters (2 ml, 0.086 M for atenolol and its derivatives, 0.055M for hydrochloride salt of propranolol and its ester analogs in the donor cell). For all esters, solvent used in the donor cell was chloroform. Atenolol was delivered from methanolic solution. In the case of propranolol hydrochloride, solvent was water. Temperature was maintained at 37 ± 0.5°C. The receiver fluid (10 ml) was withdrawn at regular intervals and replaced with fresh 37°C normal saline to maintain the sink condition and assayed after suitable dilution using the standard curves. For atenolol standard, curve was drawn at 226 nm (sensitivity 0.1mcg) and propranolol was measured at 293 nm (sensitivity 0.1 mcg). All experiments were done in triplicate and the values are expressed as mean ± SE.
2.4. Propanolol capriate (III d) White amorphous solid m.p.176-178°C (69%), IR Vmax: 3440 (NH), 1625, 1680 (C=C), 1735 (COOR) cm-1 1H-NMR (CDCl3) δ: 0.89 (3H, t, J = 7.5 Hz OCO (CH2)8 CH3), 1.56 (12H, m, OCOCH2CH2(CH2)6 CH3), 2.48 (1H, m, OCOCH2CH2, (CH2)6 CH3) and 2.69 (2H, dt, J = 6.5, 7.3 Hz, OCOCH2 (CH2)7 CH3).
3. Solubility measurements
A normal equilibrium solubility determination was carried out by the method of Okumara et al. [9]. An excess amount of drug or esters was added and dissolved in measured amount of distilled water in a glass vial to get a saturated solution. The system was stirred for 24 h at 25°C and kept at rest for 1 h to assist the attainment of equilibrium. The solution was then filtered through G-4 sintered glass funnel and after dilution, the solubility of each drug and esters was determined spectrophotometrically (Systronics double beam, India) at the suitable wavelength.
6. Data analysis
Cumulative amount of drugs permeated per unit skin surface area was plotted against time and the slope of the linear portion of the plot was estimated as the steady-state flux (Jss). Permeability coefficient (Kp) was calculated [11] by using the equation:
4. Measurement of partition coefficient
Kp = Jss/Cv
The partition coefficient of drugs and esters was determined in mutually saturated (at 25°C for 24 h) 1-octanol-water system. For drugs the standard solutions were made in water, whereas
where Cv is the total donor concentration of the solute. Statistica1 analysis was performed by Studentʼs t-test. 188
Synthesis and comparative skin permeability of atenolol and propranolol esters B. Anroop, B. Ghosh, V. Parcha, A. Kumar, J. Khanam
J. DRUG DEL. SCI. TECH., 15 (2) 187-190 2005
improving lipophilicity. As expected, capriate ester showed higher permeation than the caproate ester. Designed to compare the skin permeability, our study was conducted with two esters only. So there is a possibility that atenolol esters of higher fatty acids than C10 would show better permeation.
III. RESUL TS AND DISCUSSION
Propranolol was largely studied as a model drug to explore the different aspects of drug delivery system [12-18]. Ester prodrugs of propranolol were first developed with the objective of enhancing its oral bioavailability [19]. It was observed that the prodrugs were relatively stable to chemical attack but they undergo hydrolysis in the liver homogenate, a criteria desirable in the prodrugs. In vitro skin permeability study of some prodrugs have shown that caproyl ester of propranolol has very high permeation (permeability coefficient fourty times higher to that of hydrochloride salt) in hairless mice skin [7].The present study is a comparison of skin permeability of atenolol and propranolol hydrochloride with their respective analogs. Figures 2 and 3 show the permeation profile of the esters in comparison to pure drugs (propranolol hydrochloride and atenolol). In the case of propranolol, the caproate ester showed higher permeation than capriate ester. The permeability coefficient of the capropyl ester was found to be 4.125 x 10-2 (Table I), ten times higher than that reported by Ahmed et al. The difference in permeability coefficient may be due to the species variation in skin. In the case of atenolol, the highest permeation was found in capriate ester (Kp = 2.075 x 10-2). The maximum flux enhancement was 6.2 times for both drugs (observed in atenolol capriate and propranolol caproate, respectively). On a quantitative basis, propranolol esters were found to be more permeable than atenolol. These may be due to the fact that, because of good lipid solubility, propranolol hydrochloride (log k - 0.5714) has a greater acceptability to skin. The addition of the alkyl side chain to β hydroxy moieties further increases its lipophilicity that accelerates its passage through stratum corneum. However, its passage through aqueous layers of skin may be affected by this change and after reaching the optimum value, the rate is likely to have an inverse relationship with further increase in lipophilicity. It is established that permeation rate is not a linear function of either lipophilicity or hydrophilicity [20]. The partition coefficient of propranolol hydrochloride was experimentally determined and found to be 0.278. In the present study, flux enhancements (6.2-fold) was estimated with respect to propranolol hydrochloride, a moiety with lower lipophilicity. So, it appears that hydrochloride salt achieved high flux value because of its ease of permeation through the aqueous layer of skin and comparative flux enhancement of propranolol caproate was less than the value reported by Ahmed et al. In atenolol, maximum permeation was found with capriate ester. The drug itself is much less lipophilic than propranolol (Ko/w 0.216) and hence provides greater scope for increasing permeation by
Figure 2 - Comparison of permeation profiles of propranolol and esters (each data represents the mean ± SE of three experiments). For comparison, purpose flux values of ester were represented in terms of drug equivalents.
Figure 3 - Comparison of permeation profiles of atenolol and esters (each data represents the mean ± SE of three experiments). For comparison, purpose flux values of ester were represented in terms of drug equivalents. Table II - Solubility and partition coefficient of drug and esters at 25°C (mean ± SE, n = 3). Drug/ester
Table I - Permeability parameters of the drugs and prodrugs. Drug/ester
Propranolol HCl Propranolol caproate Propranolol capriate
Atenolol Atenolol caproate Atenolol capriate
Molecular weight
Steadystate flux Jss (µmol/ cm2/h)
Permeability coeff. Kp x 103 (cm/h)
Flux enhancement in fold
295.84 393.96 450.14 266.34 364.46 420.60
0.3641 2.2685 1.3323 0.2875 0.3299 1.7849
6.62 41.25 24.22 3.344 3.838 20.758
6.23 3.66 1.15 6.21
Propranolol HCl Propranolol caproate Propranolol capriate
Atenolol Atenolol caproate Atenolol capriate
Solubility (mg/ml) Donor
Receptor
53.12a±0.114 > 1000b > 1000b 265c±2.165 > 1000b > 1000b
51.36±0.416 0.780±0.003 0.711±0.007 21.72±0.650 3.828±0.007 0.286±0.006
Log k1
-0.5714±0.003
1.798±0.019 1.842±0.007 -0.6655±0.002
1.0958±0.003 1.4842±0.016
Donor fluid is water. Donor fluid is chloroform. c Donor fluid is methanol. k1 is the partition coefficient between octanol and water at 25°C. a b
189
J. DRUG DEL. SCI. TECH., 15 (2) 187-190 2005
Synthesis and comparative skin permeability of atenolol and propranolol esters B. Anroop, B. Ghosh, V. Parcha, A. Kumar, J. Khanam
REFERENCES 1. 2.
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Australian therapeutic trial in mild hypertension. - Reports on the management committee. - Lancet, 1, 1261, 1980. BRIAN B.H., ROBERT J.L. - Catecholamines sympathomimetic drugs adrenergic receptor antagonist. - In: G.H. Goel, B.M. Perry Eds., Goodman and Gillman’s The Pharmacological Basis of Therapeutics, 9th ed., McGrawhill, New York, 1996, p. 1716. REDDY L.H., GHOSH B. - Enhancer aided in vitro permeation of atenolol and prazosin hydrochloride through mice skin. - Ind. J. Exp. Biol., 39, 47, 2001. WILLIAM J.R., KENNETH B.S. - Prediction of transdermal flux of prodrugs of 5-fluorouracil, theophylline and 6-mercaptopurine with a series/parallel model. - J. Pharm. Sci., 89, 1415, 2000. TOMOHIRO H., KAKUJI T. - Binding of prednisolone and its ester prodrugs in the skin. - Pharm. Res, 14, 197, 1997. AHMED S., lMAI T., OTAGIRl M. - Stereoselective hydrolysis and penetration of propranolol prodrugs, In vitro evaluation using hairless mouse skin. - J. Pharm. Sci., 84, 877, 1995. AHMED S., lMAI T., OTAGIRl M.- Evaluation of stereoselective transport and concurrent cutaneous hydrolysis of several ester, Mechanism of stereoselective permeation. - Pharm. Res., 13, 1524, 1996. BRlAN S.F., ANTONY J.H., PETER W.G., AUSTIN R.T. - Vogel’s Textbook of Organic Chemistry, 5th ed., ELBS, Longman, London, 1994, p. 1076. OKUMARA M., SUGIBA YASHI K., OGA WA K., MORlMOTO Y. - Skin permeability of water soluble drugs. - Chem. Pharm. Bull., 37, 1404, 1989. GHOSH B., REDDY L.H., KULKARANI R.V., KHANAM J. Comparison of skin permeability of drugs in mice and human cadaver skin. - Ind. J. Exp. Biol., 38, 42, 2000. ELIAS D.M., WILLIAMS M.L., MALONEY M.E., BONIFAS J.A., BROWN B.E., GRAYSON S., EPSTEIN E.H.- Stratum corneum lipids in disorders of cornification. Steroid sulfate and cholesterol sulfate in normal desquamation and the pathogenesis of recessive x-linked ichthyosis. - J. Clin. Invest., 74, 1414, 1984. GUY R.H., HADGRAFT J. - Selection of propranolol hydrochloride and diazepam skin absorption in vitro. II. Drug vehicle and enhancer penetration kinetics. - J. Pharm. Sci., 81, 330, 1992.
14. 15. 16. 17. 18. 19.
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MAITANI Y., EGROS A.C., OBATA Y., NAGAI T. - Prediction of skin permeability of diclofenac and propranolol from theoretical partition coefficients determined from cohesion parameters. - J. Pharm. Sci., 82, 416, 1993. RAJESH K., PANDIT J.K. - Carboxymethyl cellulose based transdermal drug delivery system for propranolol. - J. Pharm. Pharmacol., 48, 366, 1996. VERMA P.R.P., SUNIL S.I. - Transdermal delivery of prorranolol using mixed grades of Eudragit, Design and in vitro and in vivo evaluation. - Drug Dev. Ind. Pharm., 26, 471, 2000. KRISHNA R., PANDIT J.K. - Comparative bioavailability of propranolol following oral intravenous and transdermal administration in rabbits. - Biopharm. Drug Dispos., 14, 785, 1993. RENUKA B., BABU R.J. - Effect of fasting and stress on transdermal delivery of propranolol in rats. - Pharmazie, 49, 75, 1994. BHATNAGAR S., VYAS S.P. - Organogel based systems for transdermal delivery of propranolol. - J. Microencapsul., 11, 431, 1994. SHAMEEM M., IMAI T., OTAGIRl M. - An in vitro and in vivo correlative approach to the evaluation of ester prodrugs to improve oral delivery of propranolol. - J. Pharm. Pharmacol., 45, 246, 1993. GHOSH B., REDDY L.H. - Effect of physiochemical parameters on skin permeability of antihypertensives. - Ind. J. Exp. Biol., 39, 710, 2001.
ACKNOWLEDGEMENTS The authors wish to thank Professor B.K Razdan, Additional director, S.B.S. (PG) Institute of Biomedical Sciences & Research, Balawala, Dehradun, India, for providing the facilities to carryout the research and for his generous help.
MANUSCRIPT Received 14 April 2004, accepted for publication 5 November 2004.
190
Synthesis and comparative skin permeability of atenolol and propranolol esters B. Anroop1, B. Ghosh2*, V. Parcha1, A. Kumar1, J. Khanam3 Department of Pharmaceutical Sciences, SBS (PG) Institute of Biomedical Sciences & Research, Balawala, Dehradun (UA), India 2 KLE Society’s College of Pharmacy, II Block, Rajajinagar, Bangalore, 560010 Karnataka, India 3 Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India *Correspondence: [email protected]
1
The prodrug approach is one of the methods which have been evaluated for improving the systemic delivery of pharmacologically active compounds. ln the present study, we have synthesized ester prodrugs of atenolol and propranolol by adding alkyl side chains to the β-hydroxy function of the drug with the objective of enhancing their lipophilicity. In vitro skin permeability study in excised mice skin showed that the prodrugs permeate freely through the skin than the respective drug moieties and significant flux enhancement (6.2-fold) was observed in both cases. In the case of atenolol, the highest permeability coefficient was shown by capriate (C10 side chain) where as in propranolol the highest permeability was recorded with caproate ester (C6 side chain). The difference in permeability may be attributed to their difference in the intrinsic lipophilicities. Key words: Transdermal delivery – Ester – Atenolol – Propranolol.
An epidemiological survey has revealed that 15-25% of the adult population of most countries have elevated blood pressure and normalization of blood pressure plays a primary role in the prevention of cardiovascular mortality and morbidity [1]. β-blockers atenolol and propranolol are among the popular drugs used for maintenance of blood pressure. However, both the drugs suffer the disadvantage of low and variable oral bioavailability due to first-pass metabolism [2]. The transdermal delivery route has been demonstrated to be capable of avoiding hepatic first-pass metabolism and hence various attempts have been taken to develop the cardiovascular drugs in the transdermal dosage forms. It has been shown that some β-blockers can permeate adequately through the skin when delivered from specially constituted vehicles containing enhancer molecules [3]. Similarly, the prodrug approach represents an alternative and promising method of enhancing the skin permeability of drugs by increasing the lipophilicity [4, 5]. Attempts had already been made to enhance the skin permeability of propranolol by esterification of its β-hydroxyl function [6, 7]. In the present study, we have synthesized and evaluated the skin permeability of capric and caproic esters of atenolol and propranolol to examine their suitability for transdermal development.
II. METHODS 1. Synthesis of esters
For atenolol caproate, the reaction was carried out in the fol1owing way. Atenolol (1 mM) dissolved in hot dry benzene (100 ml) was refluxed for 20 h with capric acid (1 mM) and few drops of concentrated sulphuric acid under anhydrous conditions [8]. The reaction was monitered by TLC using solvent system dioxane:acetonitrile:methanol:conc. (25%) ammonia (60:36:5:4) and visualized using Dragandroffʼs reagent. After completion of the reaction, benzene was removed. To the residue thus obtained was added water, extracted with ether and washed with 1% sodium carbonate. The ethereal layer was dried over anhydrous sodium sulphate and concentrated to obtain atenolol caproate as gummy solid (50% yield). Other esters, atenolol capriate, propranolol caproate and propranolol capriate, were synthesized similarly (Figure 1). OH
Dry benzene
R-OCH2-CH-CH2-NHCH (CH3)2 + R’COOH (I)
(II)
R
I. EXPERIMENTAL 1. Materials
Conc. H2SO4
OCOR’ R-OCH2-CH-CH2-NHCH (CH3)2
(a)
R’ CH3 (CH2)4-
CH2CONH2
(b)
Propranolol hydrochloride (Natco Pharmaceuticals, Hyderabad, India) and atenolol (Dabur, Ghaziabad, India) were received as gift samples. Capric acid and caproic acid were purchased from Loba Chemie, Mumbai, India. Octanol was procured from Merck-Schuchardt, Germany. All other chemicals and reagents used were of analytical grade.
CH3 (CH2)8CH2CONH2
(c)
CH3 (CH2)4-
(d)
CH3 (CH2)8-
Figure 1 - Schematic representation.
2. Animals
2. IR and NMR studies
Swiss albino mice, 6-12-week-old, weighing 28-32 g, were obtained from the Institute Animal House (approved by CPCSEA, Chennai, India). Animals were maintained under normal conditions and had free access to food and water.
IR spectra were recorded using Jasco FT/IR-5300 in nujol. H-NMR (CDCl3) spectra were recorded on Brucker (200 MHz) using TMS as internal standard.
1
187
J. DRUG DEL. SCI. TECH., 15 (2) 187-190 2005
Synthesis and comparative skin permeability of atenolol and propranolol esters B. Anroop, B. Ghosh, V. Parcha, A. Kumar, J. Khanam
2.1. Atenolol caproate (III a) IR Vmax: nujol: 3340, 3160 (CONH), 2940 (=CH), 1735 (COOR), 1625 (C=O amide) and 1235 cm-1 (arylether). 1HNMR CDCl3 δ: 7.38 (2H, d, J = 8.75 HzArH), 7.05 (2H, d, J = 8.75 Hz ArH, 4.92-4.97 (3H brd hump NH, NH2) 4.53 (1H, m, -OCH2CHCOOR), 4.12 (2H,s,-OCH2), 3.59 (2H, s, -CH2C6H5), 3.03 (1H, dd, J = 12.5, 5.0 Hz CH (OCOR)CH2a), 2.81 (1H, dd, J = 12.5, 5.0 CH (OCOR)CH2b), 3.14-2.65 (1H, m, NH CH (CH3)2), 1.25 (6H, d, J = 6.25 Hz NH CH (CH3)2), 2.68 (2H, dt, J = 6.2, 7.4 Hz OCO-CH2-(CH2)3-CH3), 2.48 (2H, m, OCOCH2 CH2 (CH2)2 CH3), 1.38 (4H, m, OCO(CH2)2 (CH2)2 CH3) and 0.89 (3H, t, J = 7.5 Hz, OCO(CH2)4 CH3).
for esters it was done in octanol. The partitioning was done by intermittent shaking of equal volume of both the phases for 24 h to ensure the attainment of equilibrium. In both cases, the aqueous solution is suitably diluted and determined spectrophotometrically for drug content. The partition coefficient was determined from the equation:
2.2. Atenolol capriate (III b) Gummy solid (56%) IR Vmax: nujol: 3340, 3160 (CONH), 2940 (=CH), 1735 (COOR), 1625 (C=O amide) and 1235 cm-l (arylether). lH-NMR CDCl3 δ: 0.88 (3H, t, J = 7.5 Hz OCO (CH2)8 CH3), 1.01-1.56 (12H, m, OCO (CH2)2 (CH2)6 CH3), 2.45 (2H, m, OCO CH2 CH2 (CH2)6 CH3) and 2.68 (2H, dt, J = 6.2, 7.4 Hz, OCO CH2 (CH2)7 CH3).
5.1 Skin membrane preparation Swiss albino mice were sacrificed by snapping the spinal cord at the neck. Full thickness skin was obtained from the dorsal surface. Adhering fat and visceral debris were removed from the under surface with tweezers and used immediately. Removal of the surface hair was not attempted to avoid the accidental scratching of stratum corneum.
Ko/w = Co/Cw where Co is the concentration of drug or ester in octanol and Cw is the concentration of drug or ester in pure water.
5. Permeability study
2.3. Propanolol caproate (III c) White amorphous solid m.p.156-158°C (60%) IR Vmax: 3440 (NH), 1625, 1680 (C=C) aromatic), 1735 cm-l (COOR). 1 H-NMR CDCl3 δ: 1.49-1.52 (6H, d, J = 6.6 Hz, -NH CH (CH3)2), 3.48 (2H, brs, CHOAcCH2NH), 3.54 (1H, M, NH CH (CH3)2), 4.41 (2H, d, J = 4 Hz OCH2 CHOAc), 6.81 (1H, d, J = 7.7 Hz ArH), 7.35 (1H, J = 8.1 Hz ArH), 7.48 (3H, m, ArH), 7.80 (1H, brd, J = 7.0 Hz ArH), 8.18 (1H, d, J = 7.2 Hz, ArH), 10.01 (broad hump NH2), 0.89 (3H, t, J = 7.5 Hz OCO(CH2)4 CH3). 1.01-1.56 (4H, m, OCO(CH2)2 (CH2)4 CH3). 2.45 (2H, m, OCOCH2 CH2 (CH2)2 CH3) and 2.63 (2H, dt, J = 6.5, 73 Hz, OCOCH2 (CH2)4 CH3).
5.2. Diffusion study The in vitro permeation studies were performed using a vertical type diffusion cell (Neutron Scientific, Kolkata) according to the procedure of Ghosh et al. [10]. The excised skin was mounted between the half cells with the dermis in contact with the receptor fluid, 0.9% NaCl, and was equilibrated for 1 h. The receiving chamber had a volume of 50 ml and the area available for diffusion was about 1.74 cm2. The donor cell was covered with an aluminium foil to prevent the evaporation of vehicle. However, the skin was not occluded. The drug activity was maintained uniform in matching experiments by loading equivalent amounts of the drug and esters (2 ml, 0.086 M for atenolol and its derivatives, 0.055M for hydrochloride salt of propranolol and its ester analogs in the donor cell). For all esters, solvent used in the donor cell was chloroform. Atenolol was delivered from methanolic solution. In the case of propranolol hydrochloride, solvent was water. Temperature was maintained at 37 ± 0.5°C. The receiver fluid (10 ml) was withdrawn at regular intervals and replaced with fresh 37°C normal saline to maintain the sink condition and assayed after suitable dilution using the standard curves. For atenolol standard, curve was drawn at 226 nm (sensitivity 0.1mcg) and propranolol was measured at 293 nm (sensitivity 0.1 mcg). All experiments were done in triplicate and the values are expressed as mean ± SE.
2.4. Propanolol capriate (III d) White amorphous solid m.p.176-178°C (69%), IR Vmax: 3440 (NH), 1625, 1680 (C=C), 1735 (COOR) cm-1 1H-NMR (CDCl3) δ: 0.89 (3H, t, J = 7.5 Hz OCO (CH2)8 CH3), 1.56 (12H, m, OCOCH2CH2(CH2)6 CH3), 2.48 (1H, m, OCOCH2CH2, (CH2)6 CH3) and 2.69 (2H, dt, J = 6.5, 7.3 Hz, OCOCH2 (CH2)7 CH3).
3. Solubility measurements
A normal equilibrium solubility determination was carried out by the method of Okumara et al. [9]. An excess amount of drug or esters was added and dissolved in measured amount of distilled water in a glass vial to get a saturated solution. The system was stirred for 24 h at 25°C and kept at rest for 1 h to assist the attainment of equilibrium. The solution was then filtered through G-4 sintered glass funnel and after dilution, the solubility of each drug and esters was determined spectrophotometrically (Systronics double beam, India) at the suitable wavelength.
6. Data analysis
Cumulative amount of drugs permeated per unit skin surface area was plotted against time and the slope of the linear portion of the plot was estimated as the steady-state flux (Jss). Permeability coefficient (Kp) was calculated [11] by using the equation:
4. Measurement of partition coefficient
Kp = Jss/Cv
The partition coefficient of drugs and esters was determined in mutually saturated (at 25°C for 24 h) 1-octanol-water system. For drugs the standard solutions were made in water, whereas
where Cv is the total donor concentration of the solute. Statistica1 analysis was performed by Studentʼs t-test. 188
Synthesis and comparative skin permeability of atenolol and propranolol esters B. Anroop, B. Ghosh, V. Parcha, A. Kumar, J. Khanam
J. DRUG DEL. SCI. TECH., 15 (2) 187-190 2005
improving lipophilicity. As expected, capriate ester showed higher permeation than the caproate ester. Designed to compare the skin permeability, our study was conducted with two esters only. So there is a possibility that atenolol esters of higher fatty acids than C10 would show better permeation.
III. RESUL TS AND DISCUSSION
Propranolol was largely studied as a model drug to explore the different aspects of drug delivery system [12-18]. Ester prodrugs of propranolol were first developed with the objective of enhancing its oral bioavailability [19]. It was observed that the prodrugs were relatively stable to chemical attack but they undergo hydrolysis in the liver homogenate, a criteria desirable in the prodrugs. In vitro skin permeability study of some prodrugs have shown that caproyl ester of propranolol has very high permeation (permeability coefficient fourty times higher to that of hydrochloride salt) in hairless mice skin [7].The present study is a comparison of skin permeability of atenolol and propranolol hydrochloride with their respective analogs. Figures 2 and 3 show the permeation profile of the esters in comparison to pure drugs (propranolol hydrochloride and atenolol). In the case of propranolol, the caproate ester showed higher permeation than capriate ester. The permeability coefficient of the capropyl ester was found to be 4.125 x 10-2 (Table I), ten times higher than that reported by Ahmed et al. The difference in permeability coefficient may be due to the species variation in skin. In the case of atenolol, the highest permeation was found in capriate ester (Kp = 2.075 x 10-2). The maximum flux enhancement was 6.2 times for both drugs (observed in atenolol capriate and propranolol caproate, respectively). On a quantitative basis, propranolol esters were found to be more permeable than atenolol. These may be due to the fact that, because of good lipid solubility, propranolol hydrochloride (log k - 0.5714) has a greater acceptability to skin. The addition of the alkyl side chain to β hydroxy moieties further increases its lipophilicity that accelerates its passage through stratum corneum. However, its passage through aqueous layers of skin may be affected by this change and after reaching the optimum value, the rate is likely to have an inverse relationship with further increase in lipophilicity. It is established that permeation rate is not a linear function of either lipophilicity or hydrophilicity [20]. The partition coefficient of propranolol hydrochloride was experimentally determined and found to be 0.278. In the present study, flux enhancements (6.2-fold) was estimated with respect to propranolol hydrochloride, a moiety with lower lipophilicity. So, it appears that hydrochloride salt achieved high flux value because of its ease of permeation through the aqueous layer of skin and comparative flux enhancement of propranolol caproate was less than the value reported by Ahmed et al. In atenolol, maximum permeation was found with capriate ester. The drug itself is much less lipophilic than propranolol (Ko/w 0.216) and hence provides greater scope for increasing permeation by
Figure 2 - Comparison of permeation profiles of propranolol and esters (each data represents the mean ± SE of three experiments). For comparison, purpose flux values of ester were represented in terms of drug equivalents.
Figure 3 - Comparison of permeation profiles of atenolol and esters (each data represents the mean ± SE of three experiments). For comparison, purpose flux values of ester were represented in terms of drug equivalents. Table II - Solubility and partition coefficient of drug and esters at 25°C (mean ± SE, n = 3). Drug/ester
Table I - Permeability parameters of the drugs and prodrugs. Drug/ester
Propranolol HCl Propranolol caproate Propranolol capriate
Atenolol Atenolol caproate Atenolol capriate
Molecular weight
Steadystate flux Jss (µmol/ cm2/h)
Permeability coeff. Kp x 103 (cm/h)
Flux enhancement in fold
295.84 393.96 450.14 266.34 364.46 420.60
0.3641 2.2685 1.3323 0.2875 0.3299 1.7849
6.62 41.25 24.22 3.344 3.838 20.758
6.23 3.66 1.15 6.21
Propranolol HCl Propranolol caproate Propranolol capriate
Atenolol Atenolol caproate Atenolol capriate
Solubility (mg/ml) Donor
Receptor
53.12a±0.114 > 1000b > 1000b 265c±2.165 > 1000b > 1000b
51.36±0.416 0.780±0.003 0.711±0.007 21.72±0.650 3.828±0.007 0.286±0.006
Log k1
-0.5714±0.003
1.798±0.019 1.842±0.007 -0.6655±0.002
1.0958±0.003 1.4842±0.016
Donor fluid is water. Donor fluid is chloroform. c Donor fluid is methanol. k1 is the partition coefficient between octanol and water at 25°C. a b
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J. DRUG DEL. SCI. TECH., 15 (2) 187-190 2005
Synthesis and comparative skin permeability of atenolol and propranolol esters B. Anroop, B. Ghosh, V. Parcha, A. Kumar, J. Khanam
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ACKNOWLEDGEMENTS The authors wish to thank Professor B.K Razdan, Additional director, S.B.S. (PG) Institute of Biomedical Sciences & Research, Balawala, Dehradun, India, for providing the facilities to carryout the research and for his generous help.
MANUSCRIPT Received 14 April 2004, accepted for publication 5 November 2004.
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