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An erythrocyte based bioadhesive system for nasal delivery of propranolol
Journal of Controlled Release, 23 ( 1993) 23 I-237 0 1993 Elsevier Science Publishers B.V. All rights reserved
231 016%3659/93/$06.00
COREL 00806
An erythrocyte based bioadhesive system for nasal delivery of propranolol S.P. Vyas, Naresh Talwar, Jayant S. Karajgi and N.K. Jain’ Project Laboratory, Department ofPharmaceuticalSciences, Doctor Harisingh Gour Vishwavidyalaya,Sagar, M.P., India (Received
10 March 1992; accepted in revised form 8 October 1992)
An erythrocyte based bioadhesive system has been developed for controlled systemic delivery of propranolol hydrochloride through the nasal route. Rat erythrocytes were loaded with propranolol HCl by a method based on hypotonic swelling, isotonic resealing and reannealing. The loaded erythrocytes were cross-linked by treating with glutaraldehyde and were characterised in-vitro for drug payload efficiency, propranolol release, drug diffusion through rat intestine, bioadhesion and morphological characteristics. Loaded erythrocytes were found to release propranolol HCl slowly, the release being dependent on the degree of crosslinking. The system was found to possess good bioadhesive properties. In-vivo studies conducted on rats revealed that the developed system maintained constant plasma levels of propranolol for extended periods of time (nearly 10 h). The studies suggest the potential of the system for controlled systemic delivery of propranolol. Key words: Propranolol; Glutaraldehyde treated erythrocytes; Bioadhesion; Nasal; In vitro-in vivo evaluation
Introduction The systemic administration of drugs has been approached by various dosage forms and by various routes. The nasal mucosa is considered to be a potential route for systemic administration of drugs. Nasal administration is easy, protects the drug from hepatic first pass metabolism, meets patient compliance, provides a large surface area with a well developed vascular bed for drug absorption and can be used for sustained Correspondence to: S.P. Vyas, Project Laboratory, Department of Pharmaceutical Sciences, Doctor Harisingh Gour Vishwavidyalaya, Sagar, M.P. 470 003, India. ‘Present address: Principal, College of Pharmacy, Pushp Vihar, New Delhi, India.
and controlled drug delivery. The nasal route also eliminates intersubject variation normally associated with the oral route [ I]. Moreover, nasally administered drugs such as propranolol, alpren0101and metaprenolol have shown blood levels comparable to those by intravenous administration [2,3]. An assortment of drugs have been delivered systemically through the nasal route. Some of these include insulin [ 41, progestational steroids [ 51, clofilium [ 61, testosterone [ 71, and sodium chromoglycate [ 8 1. Another modality of modern drug therapy is the development of bioadhesive drug delivery systems (BDDS). Bioadhesive, or, more appropriately, mucoadhesive systems have been prepared for both oral, and peroral administration
232
[ 9 ] . The nasal mucosa presents an ideal site for BDDS. Various factors which influence the delivery of drugs through the nasal mucosa include surfactants [ 3, lo], dose, pH and osmolarity [ 111, viscosity, particle size and nasal clearance [ 12 1, and drug structure [ 13 1. The mucociliary clearance, which remains a limiting factor, can be excluded either by reducing the viscosity of nasal mucosa or by increasing the applied drug versus nasal mucosa contact i.e., by bioa~esion [ 141. In the past bioadhesive starch microspheres have been utilized for nasal delivery of gentamitin [ 15 1. Recently, propranolol has been administered by magnetic bioadhesive HSA-microspheres [ 16 1. In the present report, we characterise and evaluate glutaraldehyde treated erythrocytes as microcontainers encapsulating propranolol, for its systemic delivery through the nasal route. Erythrocytes have been recognised as carriers for a variety of bioactive agents for their selective delivery to reti~uloendothelial system f 221, for zero order release in circulation, or to function as circulating microprocessors, especially for enzymes I. 17 ]. The use of erythrocytes for bioadhesive nasal tissue application for systemic delivery of propranolol is apparently new and hitherto unreported. In the present study, propranolol was chosen as a model drug due to its well established identity as a candidate for controlled drug delivery by the peroral route. Propranolol hydrochloride, a &blocker, has been studied for nasal absorption and is known to be absorbed fairly well through the nasal mucosa f I 1. Materials and Methods Materials The following materials were used as obtained - Proprauolol HCl (Lupin Labs. Pvt. Ltd., India ) , Giutaraldehyde ( 25% aqueous solution, Fluka, Switzerland ) . All other chemicals were of A.R. grade.
Metals Separation of e~throc~tes~o~
whole blood
Blood was drawn from albino rats by cardiac puncture and was anticoagulated with heparin. The blood was centrifuged at 3000 rpm for 5 min at 4°C (Eltek Sico centrifuge, RC 4 100, India), and the plasma and buffy coat were removed. The separated erythrocytes were washed thrice with isotonic phosphate buffered saline pH 7.4 (PBS; NaCl 150 mmol/l, K*HPO~/~*PO~ mmoI/l) and mixed with sufficient PBS to obtain a hae-• matocrit (Hct) of 50%. Loading of propranolol HCI
The erythrocytes were loaded with proprano101HCl following a previously described procedure based on hypotonic preswelling of cells, with minor modification [ I8 1. To 1 ml of packed erythrocytes obtained by centrifugation, 4 ml of 0.6% sodium chloride solution was added. The swollen cells were collected by ~ent~fugation at 2000 rpm for 5 min and the cell haemolysate ( 100 ,& obtained by lysing normal erythrocytes in water, 1: I ) was layered over the cells. Propranolol HCl solution ( 10 mg/ml in water) was added in 100 ,~laliquots to the swollen cells contained in a stoppered glass tube, and the contents were mixed by gentle inversion. The drug solution was further added to reach the point of lysis. The isotonicity was restored by adding hypertonic saline solution. The cells were incubated at 37 aC for 15 min to allow annealing and finally washed thrice with isotonic PBS to remove any unentrapped drug. An aliquot of drug loaded erythrocytes was used for determination of encapsulation efficiency while the remaining portion was used for further studies. Quantitation ofpropranolol HCl
Drug loaded erythrocyte suspension (1 ml, 30% Hct ) was iysed by adding an equal volume of water. The drug solution so obtained was suitably diluted and assayed on a ~uo~meter (Systronics, India), according to a previously re-
233
ported procedure [ 191, the excitation and emission wavelengths being 295 and 340 nm, respectively. Glutaraldehyde treatment of loaded erythrocytes The propranolol loaded erythrocytes were stabilized by treating with glutaraldehyde. One ml aliquots of loaded cells (30% Hct) were added to live volumes of 0.1, 0.2, 0.3, 0.4 and 0.5% v/ v solutions of glutaraldehyde in isotonic PBS and incubated at 37 “C for 10 min. In another modification cells were incubated for 5, 10 and 20 min with 0.3% glutaraldehyde. After incubation, the suspensions were diluted fifty times with isotonic PBS and centrifuged at 2000 rpm for 5 min. The cells were washed thrice and finally resuspended in isotonic PBS. In vitro characterisation of propranolol loaded erythrocytes In vitro drug release. Loaded erythrocyte suspension (2 ml, 30% Hct), was incubated at 4°C in a stoppered glass tube. Aliquots from the clear supematant were periodically withdrawn and assayed for propranolol content spectrophotometrically at 218 nm on a spectrophotometer (Shimadzu, UV-Vis 150-02, Japan). In vitro haemoglobin leakage. Clear supematants were withdrawn at various time intervals and assayed spectrophotometrically at 540 nm [ 201. The percent haemoglobin release was determined by measuring the absorbance of samples and comparing with that obtained from the same number of normal cells hemolysed in distilled water. In vitro diffusion study. In vitro diffusion of propranolol HCl from glutaraldehyde treated erythrocytes was studied across rat intestine using a Franz diffusion cell. The method followed was essentially as described by Vyas et al. [ 161. A piece of rat intestine was cut open and cleaned by washing with isotonic saline. Accurately known amount of loaded erythrocyte suspension ( 1.0 ml, 30% HCt) was layered over the intes-
tinal piece and kept in a desiccator at 80% RH for 30 min to allow bioadhesion to occur. The segment bearing the adhered erythrocytes was interposed between the donor and receptor compartments of the Franz diffusion cell. The receptor and donor compartments were filled with 25 ml and 10 ml of phosphate buffer (pH 6.8), respectively. The diffusion cell was maintained at 37 + 1 “C. The contents of the receptor compartment were stirred continuously using a magnetic stirrer at 30 rpm. Periodically, 0.5 ml samples were withdrawn from the sampling port with the help of a hypodermic syringe, suitably diluted and assayed for propranolol content spectrophotometrically at 218 nm. A control experiment with propranolol hydrochloride solution was also performed. Bioadhesion. The method described by Ranga Rao and Buri [ 2 1 ] was followed. A freshly cut 5-6 cm long piece of small intestine of rat was obtained and cleaned by washing with isotonic saline. The piece was cut open and the mucosal surface was exposed. Treated erythrocyte suspension (0.2 ml, 30% Hct ) was added evenly on the mucosal surface. The intestinal piece was maintained at 80% RH for 30 min in a desiccator. The piece was taken out and a weighed volume of isotonic PBS was allowed to flow over the intestinal piece at a rate of approximately 5 ml/ min. The perfusate was collected and weighed. The per cent bioadhesion was estimated by the ratio of amount of applied to adhered erythrocytes. Morphological examination and cell counting. Morphological examination was carried out on an optical microscope (Leitz, Biomed, Germany). Counting of cells was performed on a haemocytometer. In vivoperformance evaluation In vivo performance of drug-loaded-treated erythrocytes was carried out in albino rats of either sex weighing about 250 g. Nine rats in three equal groups were used for the study in a cross over design. The rats were anaesthetized with in-
234
travenously given pentobarbitone (50 mg/kg body weight ). For each treatment the amount of propranolol administered was 1 mg. To the first group propranolol hydrochloride solution (0.2 ml) was administered intravenously through the caudal vein (Treatment I ) . The second group of rats received propranolol HCl solution (0.43 ml in 0.9% saline ) nasally (Treatment II ) . To the third group propranolol loaded erythrocyte suspension (0.43 ml; 0.2% glutaraldehyde treated) was administered nasally as described by Hussain et al. [ 1 ] (Treatment III). The suspension was instilled dropwise by a syringe over a period of 10 min to allow bioadhesion to occur. The treatments were crossed over after a wash out period of seven days. The blood samples were collected periodically from the femoral aorta. After centrifugation 0.2 ml of plasma was analysed for propranolol content fluorimetrically as described earlier.
TABLE 1 Encapsulation
parameters
Subject No.
Parameter
I.
Drug entrapment (mg)” Percent cell recovery Mean hemoglobin loss (%)
2. 3.
Value (meanfSD;
‘Milligrams of propranolol of packed erythrocytes.
n=3)
2.32 & 0.23 89f3.5 40+5
HCl encapsulated
per millilitres
Results and Discussion A gentle swelling procedure with minor modifications was followed for the encapsulation of propranolol hydrochloride in erythrocytes. Upon addition of 300 ~1 of aqueous drug solution cells reached the point of lysis. The lysis point was observed microscopically as described previously [ 18 1. The procedure was modified with respect to the use of hypotonic saline solution for swelling of the cells, in place of Hanks’ Balanced Salt Solution (HBSS). The use of HBSS is based on the fact that it helps in maintaining electrolyte balance of the cell, which is essential for cell viability, resulting in excellent circulation survival. The aim of the present investigation was to use erythrocytes as a microcapsular system enclosing drug solution for nasal administration of propranolol. Since the study was not concerned with cell viability and circulation survival, therefore the HBSS was replaced by saline solution. The results of encapsulation procedure are recorded in Table 1. Approximately 2-2.5 mg of propranolol hydrochloride was found to be encapsulated per ml of packed erythrocytes, which
0 0
i
a
lb
2i
HOURS
Fig. 1. In vitro release profile of propranolol HCl from nontreated and glutaraldehyde (v/v: 0,0.5%; l ,0.4%, A, 0.3%; q, 0.2O& A, O.l%)-treated erythrocytes (m, non-treated). Bars at data points indicate t SD.
indicated good encapsulation efficiency (6080%). The encapsulation efficiency was calculated as the ratio of amount of drug actually encapsulated to the amount initially added expressed as percentage. Satisfactory cell recoveries were obtained after loading ( > 80%). Light microscopic examination revealed no difference in the morphology of the loaded cells as compared to the normal cells. The cells were found to be discoid in shape like normal cells. The haemoglobin content of the cells was reduced to 60% of the original content. Propranolol loaded erythrocytes were stabilized with glutaraldehyde. Fig. 1 illustrates the release profiles of propranolol from non-treated and glutaraldehyde-treated cells. Rapid efflux of
235
propranolol was noted from non-treated cells, 50% of the encapsulated drug was lost in 1.O-t 0.2 h and 90% leached out in 3.0 + 0.77 h. Glutaraldehyde treatment of cells stabilized the cells which resulted in a discernible decrease in efflux of the drug. The efflux proportionally decreased with increasing glutaraldehyde concentration. Treatment of cells with 0.1% v/v glutaraldehyde resulted in eftlux of 84 It 3% of the loaded drug in 24 hours while on treatment with 0.5% v/v glutaraldehyde only 402 2% release of the encapsulated propranolol was noted. Similar results were found with respect to time of glutaraldehyde treatment. The cells treated for longer periods allowed lower total drug release as compared to cells treated for shorter duration. The glutaraldehyde treatment of the erythrocytes results in crosslinking of the membrane proteins [ 221. The degree of crosslinking is dependent upon the concentration of glutaraldehyde and time of treatment. Crosslin~ng results in the formation of a membrane through which diffusivity of the drug molecule is decreased with increasing degree of crosslinking. The glutaraldehyde treated erythrocytes were also found to be completely resistant to osmotic shock, as no leakage of haemo~obin was found when cells were added to water (hypotonic) . The in vitro release profiles of haemoglobin are illustrated in Fig. 2. Negligible haemoglobin loss was found from treated cells upon storage for up
to 24 h, while from non-treated cells, 40% of haemoglobin was found to be extracellular. Propranalol-loaded ~utaraldehyde-treated erythrocytes were noted to possess good bioadhesive properties (Table 2 ) . With increasing degree of crosslinking there was a slight decrease in percent bioadhesion. The observation can be correlated to the crosslinking density, increase of which results in reduction in active sites available for mucoadhesion. The findings are in agreement with those reported elsewhere [ 16 1, where similar results have been found in the case of human serum albumin microspheres prepared by denaturation of albumin by varying heat treatment time. Difference in percent bioadhesion was found to be significant when compared for 0.1% and 0.5% glutaraldehyde treatment and treatment times of 5 and 20 min (~~0.05, Student t-test ) . In-vitro across-the-intestine diffusion profiles of propranolol HCI through rat intestine are illustrated in Fig. 3. In the case of control, 95% of the drug had diffused across at the end of 90 min. The drug eluting out of the crosslinked erythrocytes was found to diffuse more slowly. The percent drug diffused was seen to decrease progressively with increase in the degree of crosslinking. The in-vivo performance evaluation of stabilized erythrocyte cells was conducted on three groups of rats in a crossover design. Various derived pharmacokinetic parameters of propranoTABLE 2 Bioadhesion of propranolol loaded erythrocytes Subject No.
1. 2. 3. 4. 5. 6. 7. Fig. 2. In vitro haemoglobin loss from treated (0 ) and nontreated (A ) erythrocytes. Bars at data points indicate + SD.
Glutaraldehyde treatment Treatment
Time
0.1% 0.2% 0.3% 0.4% 0.5% 0.3% 0.3%
10 min 1Omin 10 min 10 min 10 min 5 min 20 min
and Q indicate significantly dent f-test).
l
Bioadhesion (meankSD) (n=3)
(96)
88.5 + 2.32* 87.2 t 1.56 86.6k2.12 s4.9+ 1.12 80.0 f 0.96’ 87.8f3.12” 80.3 f 1.07#
different data (p-z 0.05; Stu-
236
IO0
1
60 0 : i? 5 0 60
i ,-
Fig. 3. In vitro diffusion profile of propranolol across rat intestine from glutaraldehyde (v/v: A, 0.1%; 0, 0.3%; 0, O.S%)-treated erythrocytes (m, control). Bars at data points indicate + SD. TABLE 3 Derived pharmacokinetic treatment I, II and III Subject Treatment No. I.
2. 3.
I-(i.v.) II- ( nasal control) III- (nasal erythrocytes)
parameters of propranolol following
AUCo_, f SE (ngaheml-‘)
C,,, + SE (ng/ml)
t,,, (min)
1920f22 1206f34
IO
1130~54 1133f30
358+20
120
1080+24
101HCl from drug plasma profile following treatments I, II and III are recorded in Table 3. Mean plasma levels of propranolol in rats following treatments I, II and III are shown in Fig. 4. Following treatment I, maximum plasma concentration of propranolol (C,,,) was found to be 1920 + 22 ng/ml which declined gradually. The inter-subject variation in drug levels was found to be insignificant (p< 0.05). Following treatment II, i.e. nasal control, the plasma profile was found to be nearly parallel to the i.v. profile. However, a lower C,,, ( 1206k 34 ng/ml)
Fig. 4. Plasma profile of propranolol in rats (n = 9). Bars at data points indicate f SD. A, treatment I; 0, treatment II; n , treatment III.
was noted. The maximum systemic level of propranolol (C,,,,,) in case of treatment III which involved the nasal administration of stabilized erythrocytes was found to be 358 ? 20 ng/ml at 120 min. The plasma levels were found to be constant on or around the C,,, level for 8-10 h period. The plasma profile indicated controlled release of propranolol from erythrocytes vis-a-vis constant drug absorption through the nasal mucosa. The treatment recorded no primary or secondary maxima that are generally associated with oral administration [ 22 1. The absence of primary and secondary maxima indicated the protection of drug from first pass metabolism. The inter subject variation in drug levels was also found to be insignificant (p< 0.05). The areaunder-the-curve values (AU&,) for nasal administration were found comparable to those of i.v. administration. Moreover, sustained therapeutic levels of propranolol were maintained. It is concluded from the present study that propranolol HCl loaded erythrocytes can be utilized as controlled release bioadhesive delivery system for nasal administration of propranolol. The developed system provided sustained sys-
237
temic levels of propranolol first-pass metabolism.
with elimination
of
Acknowledgements 12
The authors thankfully acknowledge the tinancial assistance provided by the Ministry of Human Resources Development, Govt. of India, N. Delhi. Supply of gift sample of propranolol HCl by M/s. Lupin Lab. Pvt. Ltd. (India) is also gratefully acknowledged.
13
14
References 1
2
3
7
8
9
10
11
A. Hussain, S. Hirai and R. Bawarshi, Nasal absorption of propranolol from different dosage form by rats and dogs. J. Pharm. Sci. 69 (1980) 1411-1413. A.A. Hussain, S. Hirai and R. Bawarshi, Nasal absorption of propranolol in rats. J. Pharm. Sci. 68 (1979) 1196. G.S.M.J.E. Duchateau, J.ZuidemaandF.W.H.M. Markus, Bile salts and intranasal drug absorption. Int. J. Pharm. 31 (1986) 193-199. T. Nagai and Y. Machida, Mucosal adhesive dosage forms. Pharm. Int. 6 (1985) 196-200. Y.W. Chien, Nasal delivery of progestational steroids. Int. J. Pharm. 46 ( 1988) 133-l 40. K.C. Champanale and C.L. Cries, Nasal drug delivery system of a quarternary ammonium compound clofilium tosylate. J. Pharm. Sci. 73 ( 1984) 125 1-I 284. A. Hussain, R. Kimura and C.H. Huang, Nasal absorption of testosterone in rats. J. Pharm. Sci. 73 ( 1984) 1300-1301. A.N. Fisher, K. Brown, S.S. Davis, G.D. Parr and D.A. Smith, The nasal absorption of sodium cromoglycate in albino rat. J. Pharm. Pharmacol. 37 ( 1985) 38-41. M.L. Claus, J.A. Bouwstra, J.J. Tukker and H.E. Junginger, Intestinal transit of bioadhesive microspheres in an in-situ loop in the rat-A comparative study with copolymers and blends based on poly (acrylic acid), J. Controlled Release 13 ( 1990) 5 l-62. S. Hirai, T. Yashiki and H. Mima, Mechanism for the enhancement of the nasal absorption of insulin by surfactants. Int. J. Pharm. 9 ( 198 1) 173- 184. T. Ohwaki, H. Ando, F. Kakimoto, K. Uesugi, S. Wa-
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tanobe, Y. Miyake and M. Kayano, Effects of dose, pH and osmolarity on nasal absorption of secretin in rat-II: histological aspects of the nasal mucosa in relation to the absorption variation due to the effect of pH and osmolarity. J. Pharm. Sci. 76 (1987) 695-698. A.S. Harris, U. Ohlin, S. Lethagen and I.M. Nilsson, Effect of concentration and volume on nasal bioavailability and biological response to desmopressin. J. Pharm. Sci. 77 (1988) 337-339. C. McMartin, L.E.F. Hutchinson, R. Hyde and G.E. Peters, Analysis of structural requirement for the absorption of drugs and macromolecules from the nasal cavity. J. Pharm. Sci. 76 (1987) 539-540. L. Illum, H. Jorgensen, H. Bisgaard, 0. Krogsgard and N. Rossing, Bioadhesive microspheres as a potential nasal drug delivery system. Int. J. Pharm. 39 (1987) 189199. L. Illum, N. Farraj, H. Critchely and S.S. Davis, Nasal administration of gentamicin using a novel microsphere delivery system. Int. J. Pharm. 46 (1988) 261-265. S.P. Vyas, S. Bhatnagar, P.J. Gogoi and N.K. Jain, Preparation and characterization of HSA-propranolol microspheres for nasal administration. Int. J. Pharm. 69 (1991) 5-12. G. Ihler, A. Lantzy, J. Purpura and R.H. Clew, Enzymatic degradation of uric acid by uricase-loaded erythrocytes. J. Clin. Invest. 56 (1975) 595-602. W.N. Field, M.D. Gamble and D.A. Lewis, A comparison of the treatment of thyrodectomized rats with free thyroxine and thyroxine encapsulated in erythrocytes, Int. J. Pharm. 51 (1989) 175-178. B.M. Trivedi, M. Gohel and H. Chawa, Fluorometric determination of propranolol. Ind. J. Pharm. Sci. 48 (1986) 142-143. K.P. Mishra and B.B. Singh, Temperature effects on resealing of electrically hemolysed rabbit erythrocytes, Ind. J. Exp. Biol. 24 (1986) 737-741. K.V. Ranga Rao and P. Buri, A novel in situ method to test polymers and coated microparticles for bioadhesion. Int. J. Pharm. 52 (1989) 265-270. E. Zocchi, M. Touetti, C. Polvani, L. Guide, U. Benatti and A. Deflora, In vivo liver and lung targetting of adriamycin encapsulated in glutaraldehyde treated murine erythrocytes. Biotechnol. Appl. Biochem. 10 ( 1988) 555-562. N.K. Jain, S.U. Naik, B.R. Sainath and S.K. Date, Design and performance evaluation of a novel sustained release capsule. J. Controlled Release 3 ( 1986) 177-l 83.
231 016%3659/93/$06.00
COREL 00806
An erythrocyte based bioadhesive system for nasal delivery of propranolol S.P. Vyas, Naresh Talwar, Jayant S. Karajgi and N.K. Jain’ Project Laboratory, Department ofPharmaceuticalSciences, Doctor Harisingh Gour Vishwavidyalaya,Sagar, M.P., India (Received
10 March 1992; accepted in revised form 8 October 1992)
An erythrocyte based bioadhesive system has been developed for controlled systemic delivery of propranolol hydrochloride through the nasal route. Rat erythrocytes were loaded with propranolol HCl by a method based on hypotonic swelling, isotonic resealing and reannealing. The loaded erythrocytes were cross-linked by treating with glutaraldehyde and were characterised in-vitro for drug payload efficiency, propranolol release, drug diffusion through rat intestine, bioadhesion and morphological characteristics. Loaded erythrocytes were found to release propranolol HCl slowly, the release being dependent on the degree of crosslinking. The system was found to possess good bioadhesive properties. In-vivo studies conducted on rats revealed that the developed system maintained constant plasma levels of propranolol for extended periods of time (nearly 10 h). The studies suggest the potential of the system for controlled systemic delivery of propranolol. Key words: Propranolol; Glutaraldehyde treated erythrocytes; Bioadhesion; Nasal; In vitro-in vivo evaluation
Introduction The systemic administration of drugs has been approached by various dosage forms and by various routes. The nasal mucosa is considered to be a potential route for systemic administration of drugs. Nasal administration is easy, protects the drug from hepatic first pass metabolism, meets patient compliance, provides a large surface area with a well developed vascular bed for drug absorption and can be used for sustained Correspondence to: S.P. Vyas, Project Laboratory, Department of Pharmaceutical Sciences, Doctor Harisingh Gour Vishwavidyalaya, Sagar, M.P. 470 003, India. ‘Present address: Principal, College of Pharmacy, Pushp Vihar, New Delhi, India.
and controlled drug delivery. The nasal route also eliminates intersubject variation normally associated with the oral route [ I]. Moreover, nasally administered drugs such as propranolol, alpren0101and metaprenolol have shown blood levels comparable to those by intravenous administration [2,3]. An assortment of drugs have been delivered systemically through the nasal route. Some of these include insulin [ 41, progestational steroids [ 51, clofilium [ 61, testosterone [ 71, and sodium chromoglycate [ 8 1. Another modality of modern drug therapy is the development of bioadhesive drug delivery systems (BDDS). Bioadhesive, or, more appropriately, mucoadhesive systems have been prepared for both oral, and peroral administration
232
[ 9 ] . The nasal mucosa presents an ideal site for BDDS. Various factors which influence the delivery of drugs through the nasal mucosa include surfactants [ 3, lo], dose, pH and osmolarity [ 111, viscosity, particle size and nasal clearance [ 12 1, and drug structure [ 13 1. The mucociliary clearance, which remains a limiting factor, can be excluded either by reducing the viscosity of nasal mucosa or by increasing the applied drug versus nasal mucosa contact i.e., by bioa~esion [ 141. In the past bioadhesive starch microspheres have been utilized for nasal delivery of gentamitin [ 15 1. Recently, propranolol has been administered by magnetic bioadhesive HSA-microspheres [ 16 1. In the present report, we characterise and evaluate glutaraldehyde treated erythrocytes as microcontainers encapsulating propranolol, for its systemic delivery through the nasal route. Erythrocytes have been recognised as carriers for a variety of bioactive agents for their selective delivery to reti~uloendothelial system f 221, for zero order release in circulation, or to function as circulating microprocessors, especially for enzymes I. 17 ]. The use of erythrocytes for bioadhesive nasal tissue application for systemic delivery of propranolol is apparently new and hitherto unreported. In the present study, propranolol was chosen as a model drug due to its well established identity as a candidate for controlled drug delivery by the peroral route. Propranolol hydrochloride, a &blocker, has been studied for nasal absorption and is known to be absorbed fairly well through the nasal mucosa f I 1. Materials and Methods Materials The following materials were used as obtained - Proprauolol HCl (Lupin Labs. Pvt. Ltd., India ) , Giutaraldehyde ( 25% aqueous solution, Fluka, Switzerland ) . All other chemicals were of A.R. grade.
Metals Separation of e~throc~tes~o~
whole blood
Blood was drawn from albino rats by cardiac puncture and was anticoagulated with heparin. The blood was centrifuged at 3000 rpm for 5 min at 4°C (Eltek Sico centrifuge, RC 4 100, India), and the plasma and buffy coat were removed. The separated erythrocytes were washed thrice with isotonic phosphate buffered saline pH 7.4 (PBS; NaCl 150 mmol/l, K*HPO~/~*PO~ mmoI/l) and mixed with sufficient PBS to obtain a hae-• matocrit (Hct) of 50%. Loading of propranolol HCI
The erythrocytes were loaded with proprano101HCl following a previously described procedure based on hypotonic preswelling of cells, with minor modification [ I8 1. To 1 ml of packed erythrocytes obtained by centrifugation, 4 ml of 0.6% sodium chloride solution was added. The swollen cells were collected by ~ent~fugation at 2000 rpm for 5 min and the cell haemolysate ( 100 ,& obtained by lysing normal erythrocytes in water, 1: I ) was layered over the cells. Propranolol HCl solution ( 10 mg/ml in water) was added in 100 ,~laliquots to the swollen cells contained in a stoppered glass tube, and the contents were mixed by gentle inversion. The drug solution was further added to reach the point of lysis. The isotonicity was restored by adding hypertonic saline solution. The cells were incubated at 37 aC for 15 min to allow annealing and finally washed thrice with isotonic PBS to remove any unentrapped drug. An aliquot of drug loaded erythrocytes was used for determination of encapsulation efficiency while the remaining portion was used for further studies. Quantitation ofpropranolol HCl
Drug loaded erythrocyte suspension (1 ml, 30% Hct ) was iysed by adding an equal volume of water. The drug solution so obtained was suitably diluted and assayed on a ~uo~meter (Systronics, India), according to a previously re-
233
ported procedure [ 191, the excitation and emission wavelengths being 295 and 340 nm, respectively. Glutaraldehyde treatment of loaded erythrocytes The propranolol loaded erythrocytes were stabilized by treating with glutaraldehyde. One ml aliquots of loaded cells (30% Hct) were added to live volumes of 0.1, 0.2, 0.3, 0.4 and 0.5% v/ v solutions of glutaraldehyde in isotonic PBS and incubated at 37 “C for 10 min. In another modification cells were incubated for 5, 10 and 20 min with 0.3% glutaraldehyde. After incubation, the suspensions were diluted fifty times with isotonic PBS and centrifuged at 2000 rpm for 5 min. The cells were washed thrice and finally resuspended in isotonic PBS. In vitro characterisation of propranolol loaded erythrocytes In vitro drug release. Loaded erythrocyte suspension (2 ml, 30% Hct), was incubated at 4°C in a stoppered glass tube. Aliquots from the clear supematant were periodically withdrawn and assayed for propranolol content spectrophotometrically at 218 nm on a spectrophotometer (Shimadzu, UV-Vis 150-02, Japan). In vitro haemoglobin leakage. Clear supematants were withdrawn at various time intervals and assayed spectrophotometrically at 540 nm [ 201. The percent haemoglobin release was determined by measuring the absorbance of samples and comparing with that obtained from the same number of normal cells hemolysed in distilled water. In vitro diffusion study. In vitro diffusion of propranolol HCl from glutaraldehyde treated erythrocytes was studied across rat intestine using a Franz diffusion cell. The method followed was essentially as described by Vyas et al. [ 161. A piece of rat intestine was cut open and cleaned by washing with isotonic saline. Accurately known amount of loaded erythrocyte suspension ( 1.0 ml, 30% HCt) was layered over the intes-
tinal piece and kept in a desiccator at 80% RH for 30 min to allow bioadhesion to occur. The segment bearing the adhered erythrocytes was interposed between the donor and receptor compartments of the Franz diffusion cell. The receptor and donor compartments were filled with 25 ml and 10 ml of phosphate buffer (pH 6.8), respectively. The diffusion cell was maintained at 37 + 1 “C. The contents of the receptor compartment were stirred continuously using a magnetic stirrer at 30 rpm. Periodically, 0.5 ml samples were withdrawn from the sampling port with the help of a hypodermic syringe, suitably diluted and assayed for propranolol content spectrophotometrically at 218 nm. A control experiment with propranolol hydrochloride solution was also performed. Bioadhesion. The method described by Ranga Rao and Buri [ 2 1 ] was followed. A freshly cut 5-6 cm long piece of small intestine of rat was obtained and cleaned by washing with isotonic saline. The piece was cut open and the mucosal surface was exposed. Treated erythrocyte suspension (0.2 ml, 30% Hct ) was added evenly on the mucosal surface. The intestinal piece was maintained at 80% RH for 30 min in a desiccator. The piece was taken out and a weighed volume of isotonic PBS was allowed to flow over the intestinal piece at a rate of approximately 5 ml/ min. The perfusate was collected and weighed. The per cent bioadhesion was estimated by the ratio of amount of applied to adhered erythrocytes. Morphological examination and cell counting. Morphological examination was carried out on an optical microscope (Leitz, Biomed, Germany). Counting of cells was performed on a haemocytometer. In vivoperformance evaluation In vivo performance of drug-loaded-treated erythrocytes was carried out in albino rats of either sex weighing about 250 g. Nine rats in three equal groups were used for the study in a cross over design. The rats were anaesthetized with in-
234
travenously given pentobarbitone (50 mg/kg body weight ). For each treatment the amount of propranolol administered was 1 mg. To the first group propranolol hydrochloride solution (0.2 ml) was administered intravenously through the caudal vein (Treatment I ) . The second group of rats received propranolol HCl solution (0.43 ml in 0.9% saline ) nasally (Treatment II ) . To the third group propranolol loaded erythrocyte suspension (0.43 ml; 0.2% glutaraldehyde treated) was administered nasally as described by Hussain et al. [ 1 ] (Treatment III). The suspension was instilled dropwise by a syringe over a period of 10 min to allow bioadhesion to occur. The treatments were crossed over after a wash out period of seven days. The blood samples were collected periodically from the femoral aorta. After centrifugation 0.2 ml of plasma was analysed for propranolol content fluorimetrically as described earlier.
TABLE 1 Encapsulation
parameters
Subject No.
Parameter
I.
Drug entrapment (mg)” Percent cell recovery Mean hemoglobin loss (%)
2. 3.
Value (meanfSD;
‘Milligrams of propranolol of packed erythrocytes.
n=3)
2.32 & 0.23 89f3.5 40+5
HCl encapsulated
per millilitres
Results and Discussion A gentle swelling procedure with minor modifications was followed for the encapsulation of propranolol hydrochloride in erythrocytes. Upon addition of 300 ~1 of aqueous drug solution cells reached the point of lysis. The lysis point was observed microscopically as described previously [ 18 1. The procedure was modified with respect to the use of hypotonic saline solution for swelling of the cells, in place of Hanks’ Balanced Salt Solution (HBSS). The use of HBSS is based on the fact that it helps in maintaining electrolyte balance of the cell, which is essential for cell viability, resulting in excellent circulation survival. The aim of the present investigation was to use erythrocytes as a microcapsular system enclosing drug solution for nasal administration of propranolol. Since the study was not concerned with cell viability and circulation survival, therefore the HBSS was replaced by saline solution. The results of encapsulation procedure are recorded in Table 1. Approximately 2-2.5 mg of propranolol hydrochloride was found to be encapsulated per ml of packed erythrocytes, which
0 0
i
a
lb
2i
HOURS
Fig. 1. In vitro release profile of propranolol HCl from nontreated and glutaraldehyde (v/v: 0,0.5%; l ,0.4%, A, 0.3%; q, 0.2O& A, O.l%)-treated erythrocytes (m, non-treated). Bars at data points indicate t SD.
indicated good encapsulation efficiency (6080%). The encapsulation efficiency was calculated as the ratio of amount of drug actually encapsulated to the amount initially added expressed as percentage. Satisfactory cell recoveries were obtained after loading ( > 80%). Light microscopic examination revealed no difference in the morphology of the loaded cells as compared to the normal cells. The cells were found to be discoid in shape like normal cells. The haemoglobin content of the cells was reduced to 60% of the original content. Propranolol loaded erythrocytes were stabilized with glutaraldehyde. Fig. 1 illustrates the release profiles of propranolol from non-treated and glutaraldehyde-treated cells. Rapid efflux of
235
propranolol was noted from non-treated cells, 50% of the encapsulated drug was lost in 1.O-t 0.2 h and 90% leached out in 3.0 + 0.77 h. Glutaraldehyde treatment of cells stabilized the cells which resulted in a discernible decrease in efflux of the drug. The efflux proportionally decreased with increasing glutaraldehyde concentration. Treatment of cells with 0.1% v/v glutaraldehyde resulted in eftlux of 84 It 3% of the loaded drug in 24 hours while on treatment with 0.5% v/v glutaraldehyde only 402 2% release of the encapsulated propranolol was noted. Similar results were found with respect to time of glutaraldehyde treatment. The cells treated for longer periods allowed lower total drug release as compared to cells treated for shorter duration. The glutaraldehyde treatment of the erythrocytes results in crosslinking of the membrane proteins [ 221. The degree of crosslinking is dependent upon the concentration of glutaraldehyde and time of treatment. Crosslin~ng results in the formation of a membrane through which diffusivity of the drug molecule is decreased with increasing degree of crosslinking. The glutaraldehyde treated erythrocytes were also found to be completely resistant to osmotic shock, as no leakage of haemo~obin was found when cells were added to water (hypotonic) . The in vitro release profiles of haemoglobin are illustrated in Fig. 2. Negligible haemoglobin loss was found from treated cells upon storage for up
to 24 h, while from non-treated cells, 40% of haemoglobin was found to be extracellular. Propranalol-loaded ~utaraldehyde-treated erythrocytes were noted to possess good bioadhesive properties (Table 2 ) . With increasing degree of crosslinking there was a slight decrease in percent bioadhesion. The observation can be correlated to the crosslinking density, increase of which results in reduction in active sites available for mucoadhesion. The findings are in agreement with those reported elsewhere [ 16 1, where similar results have been found in the case of human serum albumin microspheres prepared by denaturation of albumin by varying heat treatment time. Difference in percent bioadhesion was found to be significant when compared for 0.1% and 0.5% glutaraldehyde treatment and treatment times of 5 and 20 min (~~0.05, Student t-test ) . In-vitro across-the-intestine diffusion profiles of propranolol HCI through rat intestine are illustrated in Fig. 3. In the case of control, 95% of the drug had diffused across at the end of 90 min. The drug eluting out of the crosslinked erythrocytes was found to diffuse more slowly. The percent drug diffused was seen to decrease progressively with increase in the degree of crosslinking. The in-vivo performance evaluation of stabilized erythrocyte cells was conducted on three groups of rats in a crossover design. Various derived pharmacokinetic parameters of propranoTABLE 2 Bioadhesion of propranolol loaded erythrocytes Subject No.
1. 2. 3. 4. 5. 6. 7. Fig. 2. In vitro haemoglobin loss from treated (0 ) and nontreated (A ) erythrocytes. Bars at data points indicate + SD.
Glutaraldehyde treatment Treatment
Time
0.1% 0.2% 0.3% 0.4% 0.5% 0.3% 0.3%
10 min 1Omin 10 min 10 min 10 min 5 min 20 min
and Q indicate significantly dent f-test).
l
Bioadhesion (meankSD) (n=3)
(96)
88.5 + 2.32* 87.2 t 1.56 86.6k2.12 s4.9+ 1.12 80.0 f 0.96’ 87.8f3.12” 80.3 f 1.07#
different data (p-z 0.05; Stu-
236
IO0
1
60 0 : i? 5 0 60
i ,-
Fig. 3. In vitro diffusion profile of propranolol across rat intestine from glutaraldehyde (v/v: A, 0.1%; 0, 0.3%; 0, O.S%)-treated erythrocytes (m, control). Bars at data points indicate + SD. TABLE 3 Derived pharmacokinetic treatment I, II and III Subject Treatment No. I.
2. 3.
I-(i.v.) II- ( nasal control) III- (nasal erythrocytes)
parameters of propranolol following
AUCo_, f SE (ngaheml-‘)
C,,, + SE (ng/ml)
t,,, (min)
1920f22 1206f34
IO
1130~54 1133f30
358+20
120
1080+24
101HCl from drug plasma profile following treatments I, II and III are recorded in Table 3. Mean plasma levels of propranolol in rats following treatments I, II and III are shown in Fig. 4. Following treatment I, maximum plasma concentration of propranolol (C,,,) was found to be 1920 + 22 ng/ml which declined gradually. The inter-subject variation in drug levels was found to be insignificant (p< 0.05). Following treatment II, i.e. nasal control, the plasma profile was found to be nearly parallel to the i.v. profile. However, a lower C,,, ( 1206k 34 ng/ml)
Fig. 4. Plasma profile of propranolol in rats (n = 9). Bars at data points indicate f SD. A, treatment I; 0, treatment II; n , treatment III.
was noted. The maximum systemic level of propranolol (C,,,,,) in case of treatment III which involved the nasal administration of stabilized erythrocytes was found to be 358 ? 20 ng/ml at 120 min. The plasma levels were found to be constant on or around the C,,, level for 8-10 h period. The plasma profile indicated controlled release of propranolol from erythrocytes vis-a-vis constant drug absorption through the nasal mucosa. The treatment recorded no primary or secondary maxima that are generally associated with oral administration [ 22 1. The absence of primary and secondary maxima indicated the protection of drug from first pass metabolism. The inter subject variation in drug levels was also found to be insignificant (p< 0.05). The areaunder-the-curve values (AU&,) for nasal administration were found comparable to those of i.v. administration. Moreover, sustained therapeutic levels of propranolol were maintained. It is concluded from the present study that propranolol HCl loaded erythrocytes can be utilized as controlled release bioadhesive delivery system for nasal administration of propranolol. The developed system provided sustained sys-
237
temic levels of propranolol first-pass metabolism.
with elimination
of
Acknowledgements 12
The authors thankfully acknowledge the tinancial assistance provided by the Ministry of Human Resources Development, Govt. of India, N. Delhi. Supply of gift sample of propranolol HCl by M/s. Lupin Lab. Pvt. Ltd. (India) is also gratefully acknowledged.
13
14
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