. Pharmacokinetic study of niosome encapsulated insulin

Transcription

1 Indian Journal of Experimental Biology Vol. 38, September 2000, pp Pharmacokinetic study of niosome encapsulated insulin G Khaksa, R D'Souza, S Lewis, & N Udupa* Department of Pharmaceutics, College of Pharmaceutical Sciences, Manipal , India Received 11 November1999; revised 24 May 2000 Pharmacokinetic profile and hypoglycemic effect, after intraperitoneal injection of insulin and insulin encapsulated in niosomes were determined in diabetic rats. Niosomes (non-ionic surfactant vesicles) of different doses and different lipid compositions were prepared by lipid layer hydration method. Plasma samples were collected at specified time intervals and plasma concentration of insulin was determined by HPLC. Blood glucose level was estimated spectrophotometrically using commercial glucose assay kit. III vitro release and pharmacokinetic profile of niosomal formulation and free insulin were evaluated. Though there was a slight delay in the ill vitro drug release due to cholesterol content in the niosomes, there was no difference between the two preparations when plasma levels were compared in vivo. Niosomes significantly reduced the blood glucose level in diabetic rats. Fall in blood glucose level was almost 92% of initial value. In case of the niosomal form the half-life of insulin was prolonged by 4-5hr in contrast to 2hr for free drug. Niosomes maintained the plasma insulin level up to 12hr, but free drug was cleared quickly. The area under the plasma concentration-time curve for niosomal forms was, ± 0.99 miu. hr/ml and for free insulin was ± 1.l0rnIU. hr/ml. More than 80% of the drug was successfully encapsulated to give a formulation with sustained release characteristics. Entrapment efficiency increased with increasing lipid concentration and decreased with increasing drug concentration. The results showed that insulin entrapped in niosomes prolongs the existence of drug in the body therefore increasing its therapeutic value. The problems associated with insulin therapy are typical problems confronting delivery of polypeptide and protein drugs. Rapid enzymatic degradation results in short biological half-life when administered orally. In addition membrane permeability is often poor because of the high molecular weight and lack of lipophilicity of these agents. Insulin has been administered by various routes such as oral l, ocular 2, nasae, rectal 4, transdermal 5 and buccal 6. However, the success in terms of reproducibility and bioavailability has been very limited. Consequently, much effort has been dedicated to the formation of alternative effective delivery systems of insulin in the form of niosomal formulation. The concept of the carriers to deliver drugs to target organs and modify drug disposition has been widely discussed and is well documented 7. Majority of such reports have concerned the use of phospholipid vesicles in the formulations. These exhibit certain disadvantages due to their predisposition to oxidative degradation leading to poor stability. Hence the phospholipids must be stored and handled in a nitrogen atmosphere. Many synthetic amphiphiles form vesicles, but as most are ionic and relatively toxic they are generally unsuitable for use as drug carriers. Correspondent author: Fax: , info@mahe.emet.in Vesicle formation by some members of dialkyl polyoxyethylene ether non ionic surfactant series has been reported by many authors 8.\o. Encapsulation of a drug in vesicular structures can be predicted to prolong the existence of the drug in the systemic circulation. Niosomes are unilamellar or multi lamellar vesicles formed on admixture of a non ionic surfactant, cholesterol and dicetylphosphate with subsequent hydration in aqueous media. Niosomes are biodegradable, biocompatible, non-toxic and capable of encapsulating large quantities of material in relatively small volume of vesicles. In this paper the effect of niosomal encapsulation on pharmacokinetic and hypoglycemic effect of insulin in diabetic rats are reported. Materials and Methods Plain bovine insulin injection purified (40 IU/ml) was purchased from Knoll Pharmaceuticals Ltd. Sorbitan monostearate (span 60), cholesterol (CHOL) and dicetylphosphate (DCP) were from Sigma Chemical Co., (St. Louis, MO, USA). Cellulose nitrate filter (0.2I1m was obtained from Sartorius (Germany). Glucose assay kit was purchased from Dr. Reddy's Laboratory, Hyderabad,India. All other chemicals and reagents used were of analytical grade. Preparation and characterization of niosomes-entrapment of insulin niosomes was carried

2 902 INDIAN J EXP BIOL, SEPTEMBER 2000 out by lipid layer hydration methods. Niosomal formulations of different doses and different lipid compositions we~e prepared (Table 1). Lipid ingredients were dissolved in lsml diethyl ether in a SOml round bottom flask. The ether was evaporated under reduced pressure using a rotary evaporator to form a thin film on wall of the flask. Phosphate buffered saline (PBS ph 7.4) containing insulin was added in parts to the dried film with gentle agitation. The mixture was intermittently mixed on a vortex to get a good dispersion. The suspension was maintained at 2 _ 8 C in a refrigerator for 2hr to form rigid lameilar vesicles. Empty niosomes were prepared in the same manner devoid of insulin. The diameter of the niosomes was determined using a calibrated eye-piece micrometer. The entrapment efficiency of insulin in niosome was calculated after determining the concentration of entrapped insulin by measuring the UV absorbance from niosome completely lysed bytriton X-IOO. In vitro release studies-the niosomes remaining after removal of unentrapped drug were filled into a dialysis membrane securely attached to one side of the dialysis tube. The dialysis tube was then immersed in a receptor compartment consisting of 100ml of PBS ph 7.4 with constant stirring at 2S C. Samples were withdrawn at predetermined time intervals and analyzed spectrophotometrically at 220nm. In vivo studies-male Sprague-Dawley rats weighing 220-2S0g, were obtained from Department of Pharmacology, Kasturba Medical College, Manipal, India. Diabetes was induced by intraperitoneal (ip) injection of alloxan ISO mg Ikg after an overnight fast. The rats became diabetic within 48h with blood glucose level exceeding 2S0mgldl. The baseline glucose level in diabetic rats was first determined before administration of the drug. The alloxanized rats were injected intra peritoneally with niosomal formulation and free drug solution under light ether anesthesia. Control rats received the same volume of niosomes devoid of insulin. Blood samples were withdrawn from the orbital plexus at specific time intervals. Plasma was separated and frozen until analysis. Insulin was extracted by the method described below and analyzed by high-performance liquid chromatography. AnaLysis-A reversed-phase high-performance liquid chromatographic (RP-HPLC) method described in our previous publication by Khaksa et al. II was adopted. In brief, a volume of 100fli of plasma sample was pippetted into a test tube and diluted to Iml with phosphate buffered saline ph 7.4, to permit samples to be analyzed under the sample conditions. The sample was extracted with Iml dichloromethane by vortexing for Smin. The tube was theil centrifuged to separate the layers and then immersed in an ice bath. The supernatant layer was decanted and 0.8rul of organic phase was transfelted to another tube. To the organic extract, 130fll of (O.OSN) HCl was added for back extraction. After shaking and centrifugation the organic layer was evaporated under a stream of nitrogen. A volume of 100fli aliquot was injected into a CIS column (ODS, Sflm, 2Scm x 4.6mm, Zorbax, Tokyo, Japan) with mobile phase consisting of a mixture of 0.2M sodium sulphate anhydrous (PH 2.3) and acetonitrile (74/26, v/v). The eluent was monitored with a UV/YIS detector set at 214 nm with a flow rate of 1.2ml/min. Phannacodynamics-Blood glucose was estimated spectrophotometrically using a commercial assay kit. lofll of plasma was used for each assay. Percentage change in glycemia was calculated by the following formula. Change in glycemia (%) = GX-GO/GO x 100 Where the GO and GX are the initial glycemia, and glycemia at O.S to 24hr, respectively. Statistical analysis was done by Student's t - test to compare each group with the respective control group. Phannacokinetic analysis and statistics-area under the plasma concentration-time curve (AUC) was calculated using trapezoidal rule. Test of significance of difference was made using analysis of variance (ANOYA) followed by Student's t-test with P < O.OS as a level of significance. All the experiments were caltied out in triplicate. Results The entrapment efficiency of insulin by niosomes is shown in Table 1. The entrapment efficiency decreased with dose increment, from 85% (401U/rul), to S8 % (200IU/ml). Niosomes prepared with lower dose of insulin and higher lipid content showed better encapsulation. The cumulative percentage of drug released in PBS (PH 7.4) from two different formulations Fl and F2 is shown in Fig.I. For formulation Fl, 41.34% of drug released in 6hr while with F2, 48.10% of drug was released in 6hr. In case of free insulin 8S% of the drug was released in 6hr. The mean diameter of formulated niosomes was in the range of 8-12flm. In alloxanised

3 KHAKSA ETKHAKSA et al..' PHARMACOKINETrC STUDY OF NIOSOME ENCAPSULATED INSULIN 903 Table I-Entrapment efficiency of insulin by niosomes of different compositions Formulation Lipid Molar ' Insulin Entrapment composition ratio (IU/ml) efficiency (%)' FI CHOL : SPAN: DCP 55: 35 : ± 0.061(5) F2 CHOL : SPAN : DCP 47.5 : 47.5 : ± (6) F3 CHOL : SPAN : DCP 55 : 35 : ± (4) F4 CHOL : SPAN : DCP 55 : 35 : ± 0.19 (6) [' Mean value ± S.D. Number of observations in parentheses] t!. 10 OF---~----~----~----r ~ Fig. 1-/11 vitro release profiles of two niosomal formulations of insulin and free insulin in phosphate buffered saline ph 7.4 at 25 C, (n=3) : (. ) free insulin; C-) niosome encapsulated insulin (FI ); (.A.) niosome encapsulated insulin (F2). rats the mean fasting blood glucose level was mgldl. Fig.2 shows mean plasma glucose level vs. time after ip injection of niosomal formulations. The fall in blood glucose level was almost 92% of initial value (data not shown). The mean plasma concentration-time profile of niosome encapsulated insulin in two different formulations and of free insulin in diabetic rats is shown in Fig.3. A summary of the AUC's are presented in Table 2. Significant differences were found in AUC's of encapsulated and free insulin. The AUC's for niosomal formulations was found to be much greater (26.07±0.99 miu.hlml and 32.96±1.02 miu.hr/ml) when compared to free insulin (l1.72±1.02 miu.hlml). Discussion Numerous researchers have demonstrated the effecti veness of non ionic surfactants as carriers of Table 2~omparison of area under the plasma concentration - time curve (AUC) of niosomal insulin (20 IUlkg) with free insulin (5 IUlkg) in diabetic rats [Values are mean ± SE of 5 animals in each group] Formulation N AUC (miu. HI ml) FI ± 0.99' F ± 1.Q2' Free Insulin 5 II. 72 ± 1.02 P < 0.05 compared to free insulin group. (A NOV A followed by Student's t-test) d rugs '. N' IOsomes can entrap so I utes In. a manner analogous to liposomes, are stable in vitro, and can increase the stability of the entrapped drug l4. Various studies have been carried o':lt using liposome encapsulated insulin and reported the enhanced biological response elicited by the encapsulated d rug '. W e h ave use d non.. IOmc su rf actant veslc. I es as a carrier for insulin. The entrapment efficiency increased with increase in the cholesterol content. X ray diffraction methods demonstrated that cholesterol increases the width of the phospholipid bilayer. Cholesterol is also known to abolish the gel to liquid phase transition of niosome systems resulting in niosomes that are less leaky. This effect is evident as niosomes containing higher cholesterol concentration exhibited a slower release pattern. The vesicle size is an important parameter in determining the rate and extent of biodegradation and hence half life of the carriers besides influencing the extent of drug encapsulation. Dicetylphosphate renders negative charge to the bilayer and increases stability by preventing aggregation of the niosomes. The relatively slow release pattern of entrapped drug from vesicles prepared from lipid hydration method indicates enhanced stability of the system. Comparati ve release profile of free and encapsulated insulin indicates that by encapsulation it could be possible to sustain and control the drug release for a longer period of time. The body weight and plasma glucose level of rats were monitored to ensure the induction of diabetes.

4 904 INDIAN J EXP BIOL, SEPTEMBER 2000 After treatment with alloxan, rats showed a slightly.decreased body weignt and a significant increase in the plasma glucose. level than before treatment. The, pharmacokinetics of insulin was studied after the administration of insulin in the free form and niosomal. form using two different compositions. In contrast to the rats treated with ' insulin alone, rats administered with niosomal insulin showed no mortality at the same dose (20IU/kg); implying that insulin might be released from niosomes in a sustained pattern, thus preventing hypoglycemic shock. As expected, intraperitoneal administration of free insulin into diabetic rats did not significantly decrease the blood glucose level. However administration of insulin entrapped in cholesterol/span niosomes maintained a decrease in the plasma glucose levels for at least 12 hr. There was no significant difference between the two niosome types, despite the increased permeability of the niosomes with lower cholesterol content as demonstrated by the increase in-vitro flux, in PBS (PH 7.4). Treat- 120 'i! ~ :s eo '0 >t t.- 60 u ;;, " ~.. ii: a Fig. 2-Hypoglycemic response after intraperitoneal administration of insulin in diabetic rats: (.) free insulin (5 IU/kg); (_) niosomal insulin FI (20 IV/kg); (.A.) niosomal insulin F2 (20 IV/kg); (_ ) empty niosomes. Fig 3----Plasma concentration-time profiles following intraperitoneal administration of insulin in diabetic rats : (. ) free insulin (5 IU/kg); (_ ) niosomal insulin FI (20 IUlkg); (.A.) niosomal insulin F2 (20 IV/kg).

5 KHAKSA ETKHAKSA et at. : PHARMACOKINETIC STUDY OF NIOSOME ENCAPSULATED INSULIN 905 ment with both niosomal forms significantly (P<0.05) reduced the blood glucose level in diabetic rats and maintained the hypoglycemic effect during the experimental period of 2-12hr. This prolonged hypoglycemic response may be due to the formation of a drug depot within the peritoneal cavity of the rat. Since the drug is in the aqueous compartment and encapsulated within the vesicles it is protected from the external environment. The aqueous drug is released during biodegradation of the vesicle and is absorbed to exert its hypoglycemic effect. Progressive degradation of the bilayer contributes to sustained and controlled drug release. Plasma glucose concentration after insulin dosing was expressed as a percentage of the initial concentration. The percentage change in plasma glucose was the percentage of the initial concentration subtracted from 100. futraperitoneal injection of insulin in niosomal form, resulted in an increase in the area under the plasma drug concentration - time curve. This may be because insulin enters the systemic circulation to exert hypoglycemic effect at a controlled rate which is determined by the rate of bioerosion of the non ionic surfactant bilayers. This was in contrast to the narrow peak, hence rapid disappearance of drug from plasma when administered as a free solution. The profile of insulin level is altered in both the niosomal forms, tmax being shifted to a longer time and AVC's being much greater in both the forms. Although both niosomal formulations exhibited a different trend, the difference was not statistically significant. It can be concluded that niosomes are a promising vehicle for drug delivery. Being non ionic they are likely to be less toxic than vesicles produced from ionic amphiphiles. Acknowledgement The authors are thankful to ICMR, New Delhi for financial support for this study. References 1 Cho W Y & Flynn M, Oral delivery of insulin, Lancet, 23 (1989) Chiou G C Y & Chuang C Y, Improvement of systemic absorption of insulin through eyes with absorption enhancers, J Pharm Sci, 78 (1989) Hirari S, Yashiki T & Mina H, Mechanisms for the enhancement of nasal absorption of insulin by surfactants, Int J Pharm, 9 (1981) Ichikawa K, Ohata I, Mitomi M, Kawamura S, Maeno H & Kawata H, Rectal absorption of insulin suppositories in rabbits, J Pharm Pharmacal, 32 (1980) Siddiqui 0, Sun Y, Liu J C & Chien Y W, Facilitated transdermal transport of insulin, J Pharm Sci, 76 (1987) Aungst B J, Rogers N J & Shefter E, Comparison of nasal, rectal, buccal, sublingual and intramuscular insulin efficacy and the effects of bile salt absorption promoter, J Pharm Exp Ther, 244 (1988) Gregoriadis G,Targetting of drugs: Implications in medicine, Lancet, 2 (198 I) Azmin M N, Florence A T, Handjani-Vila R M, Stuart J F B, Vanlerberghe G & Whittaker J S, The effect of non-ionic surfactant vesicle (niosome) entrapment on the absorption and distribution of methotrexate in mice, J Pharm Pharmacal, 37 (1985) Toshimitsu Y, Stembery B & Florence A T, Preparation and properties of vesicles (niosomes) of sorbitan monoesters (span 20, 40, 60 and 800 and sorbitan triestcr (span 85), Int J Pharm, 105 (1994) Baillie A J, Florence A T, Hume L R, Murihead G T & Rogerson A, The preparation and properties of niosome - non-ionic surfactant vesicles, J Pharm Pharmacal, 37 (1985) Khaksa G, Nalini K, Bhat M & Udupa N, High - performance liquid chromatographic determination of insulin in rat and human plasma, Anal Biachem, 260 (1998) Walters K A, Dugard P H & Florence A T, Non-ionic surfactants and gastric mucosal transport of paraquat, J Pharm Phannacal, 33 (1981) Rogerson A, Cummings J, Willmott N & Florence A T, The distribution of doxorubicin in mice following administration in niosomes, J Pharm Pharmacal, 40 (1988) Namdeo A & Jain N K, Niosomes as drug carriers, Ind J Pharm Sci, 58 (1996) Kim A, Yun M, Oh Y, Ahn W & Kim C, Pharmacodynamics of insulin in polyethylene glycol - coated liposomes, Int J Pharm, 180 (1999) Stevenson R W, Patel H M, Parsons J A & Ryman B E, Prolonged hypoglycemic effect in diabetic dogs due to subcutaneous administration of insulin in liposomes, Diabetes, 31 (1982)506.