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The authors review various oral drug delivery systems that have been explored to increase patient compliance for insulin.
Diabetes mellitus is a common disease and its complications are responsible for excess morbidity and mortality, loss of independence, and reduced quality of life (1, 2). Among the major causes of disablement and early death are ischemic heart disease, retinopathy, nephropathy, peripheral vascular disease, and neuropathy (3). Diabetes mellitus is a serious pathologic condition that is responsible for major healthcare problems worldwide and costing billions of dollars annually.
The American Diabetes Association recently recommended an etiological classification of diabetes (4). Diabetes resulting from a deficiency of insulin secretion is classified as type I diabetes. Type I diabetes normally occurs in childhood, has relatively acute onset, and requires insulin for survival.
Diabetes that results from a resistance to insulin (with or without concomitant insulin secretory defect) is classified as type II diabetes. Type II diabetes usually occurs later in life, has an insidious onset, and may or may not require exogenous insulin treatment. Although the majority of diabetes cases falls into one of these two categories, there are several other forms of diabetes, which cannot be classified as type I or type II diabetes. These other specific types include cases arising from genetic defects of the β cell (e.g., maturity onset diabetes of the young (MODY] genes, mitochondrial DNA mutations); genetic defects in insulin action; drug-, chemical-, or disease-induced pancreatic damage; and endocrinopathies. Gestational diabetes mellitus, defined as any degree of glucose intolerance with onset or first recognition during pregnancy, comprises the fourth general category in the revised diagnostic criteria for diabetes (4).
The defect in insulin secretion in type I diabetes may result from one of several different mechanisms. The most common is autoimmune destruction of insulin-producing β cells of the pancreas. Evidence of an immune process occurring in most cases of type I diabetes is the presence of antibodies to islet cells antigens. Such cases are referred to as autoimmune or type IA diabetes. In a minority of cases of type 1 diabetes, evidence of such an autoimmune etiology of the insulin secretory defect is absent. Such cases are referred to as idiopathic or type IB diabetes. This group does not include cases for which a specific cause of β-cell destruction is known (e.g., neoplasia, cystic fibrosis), which are classified under "other specific types" (5).
Development of insulin
Nearly 100 years have passed since Von Mering and Minkowski first demonstrated that pancreatectomized dogs exhibited signs and symptoms characteristic of diabetes mellitus (6). Shortly thereafter, Banting and Best used pancreatic extracts to reverse these symptoms in patients suffering from severe diabetes (7). Insulin replacement therapy has been used in the clinical management of diabetes mellitus for more than 84 years. It is used as a first-line agent in type I diabetes and sometimes in the treatment of type II diabetes where oral hypoglycemic agents combined with diet and exercise fail to achieve appropriate metabolic control (8).
Insulin is a small protein with a molecular weight (MW) of 5808 in humans. It contains 51 amino acids arranged in two chains (A and B) linked by disulfide bridges. There are species differences in amino acids of both chains. Within the β cells, insulin precursor is produced by DNA and RNA-directed synthesis. Proinsulin, a long single-chain protein molecule, is processed within the Golgi apparatus and packaged into granules, where it is hydrolysed into insulin and a residual connecting segment called the C-peptide is formed by removal of four amino acids. Insulin and C-peptides are secreted in equimolar amounts in response to all insulin secretagogues. A small quantity of unprocessed or partially hydrolyzed proinsulin is released as well. Granules within the (3 cells store insulin in the form of crystals consisting of two atoms of zinc and six molecules of insulin (7).
For 60 years, animal pancreas was the only source from which insulin could be produced in sufficient quantities to cover therapeutic needs (9). During the first decade of the "insulin era," only an acid solution of an impure form of hormone was available for subcutaneous (s.c.) injection (1). The introduction of zinc crystallization in 1934 as well as the development of recrystallization methods made it possible to crystallize insulin. The introduction of analytical methods such as disk electrophoresis and gel filtration made it possible to detect the presence of a significant amount of impurities in proteins even in recrystallized insulin. The use of chromatographic purification reduced the impurities to less than 0.5% and immunologic complications of insulin treatment were essentially eliminated. During the late 1970s, the advances in recombinant DNA technology made it possible to biosynthetically produce human insulin. In the 1980s, the first human received injections of human insulin combined from A and B chains expanded separately in Escherichia coli with chemically prepared genes.
In 1982, biosynthetic insulin became the first marketed human healthcare product derived from rDNA technology. This novel technology opened new ways for the development of insulin analogues. However, the pharmacokinetics following s.c. injection of rapid-, intermediate-, and long-acting preparation does not match the profile of physiological insulin secretion. The peak absorption of regular, short-acting human insulin occurs from two to four hours after the injection and usually persists for several hours, but it does not provide the early and quick rise in plasma insulin concentration required to prevent physiological postprandial hyperglycemia after a meal. The prolonged-acting formulation is intended to maintain the basal insulin levels to control blood glucose between meals and during the night when one cannot deliver insulin at a constant and reproducible low-level rate that characterizes normal insulin secretion. These shortcomings of the conventional preparation make it virtually impossible to achieve normoglycemia.
In spite of newer, more potent and highly purified insulins, development of human insulin, change of once-daily injection to twice-daily insulin therapy, and the introduction of portable insulin infusion pumps, diabetes is still a high-risk disease and is far from being controlled. The present mode of insulin administration is by the s.c. route by which insulin is presented to the body in a nonphysiological manner. The s.c. administration of insulin has many challenges. The major drawbacks of this method are local discomfort, inconvenience of multiple injections, and occasional hypoglycemia as a result of overdose. Because of these problems, novel approaches for insulin delivery are being explored, including oral, transdermal, nasal, rectal, pulmonary, uterine, and ocular delivery as well as s.c. implants. Delivery options that use dermal, nasal, and oral approaches have been explored (12–14). This review describes various oral insulin delivery systems.
Inhaled insulin appears to be suitable for patients with diabetes because of its high bioavailability and a pharmacokinetic profile that mimics the time kinetics of insulin secretion after a meal. Clinical studies were conducted among a small number of patients with type I or type II diabetes who had been treated with s.c. insulin. Inhaled insulin was given three times daily, just before meals, and was combined with a bedtime s.c. injection of long-acting insulin (1). In patients with type I or type II diabetes, the metabolic control achieved with inhaled insulin was similar to that obtained with a s.c. insulin regimen. Tolerance of inhaled insulin was good, and treatment satisfaction was better than that with the s.c. regimen. Insulin inhalation appears to be an interesting way of insulin delivery for elderly patients with diabetes. However, no studies have been conducted in elderly patients with diabetes to assess this route's acceptability, convenience, and ease of use in this particular population. In addition, it is necessary to conduct pharmacokinetic studies in the elderly because lung aging might reduce the bioavailability of inhaled insulin (10). Although Pfizer (New York) launched Exubra in 2006, it has been reported that it is not successful. Of the several inhaled insulin devices that are in various stages of development, the Exubera formulation was the first to be approved for use in the United States and in Europe (11).
Pulmonary delivery has emerged as the most feasible option thus far, but oral delivery is the ultimate goal. Oral insulin delivery must protect insulin from proteolytic degradation in the stomach and the upper portion of the small intestine. In addition, the absorption of insulin from the gut must be enhanced. The absorption of insulin is very poor because of the hydrophilic nature of the big molecule. Basic problems of insulin stability in the gut and absorption from the gastrointestinal tract still must be resolved (15). To achieve gastrointestinal absorption, Morishita et al. evaluated whether oligoarginine, a cell-penetrating peptide (CPP), can improve intestinal absorption of insulin in rats (16). Peptides composed of six [R(6)], eight [R(8)] and 10 [R(10)] residues of arginine were used as the CPP. No insulin absorption was observed following administration of insulin solution alone. However, insulin absorption increased dramatically after co-administration of the D-form of R(6), D-R(6), and the L-form of R(6), L-R(6), in a dose-dependent manner. The effects on insulin absorption were more pronounced for D-R(6) than for L-R(6). Among oligoarginines composed of 6, 8, or 10 arginine residues, D-R(8) showed the strongest enhancing effects on insulin intestinal absorption.
Physiological insulin secreted by the pancreas enters portal circulation and inhibits hepatic glucose production. It undergoes metabolism in the liver to a significant extent (~50%). The ratio of plasma insulin in portal circulation versus that in peripheral circulation is two. The physiological hypoglycemic effect of insulin is a result of the absence of hepatic glucose production that is enhanced by an increase in glucose use caused by lower insulin levels in peripheral circulation. When insulin is injected subcutaneously, the plasma insulin concentration in portal and in peripheral circulation is almost equal. The hypoglycemic effect of insulin is a result of its action peripheral tissues. Oral delivery of insulin can mimic the physiological fate of insulin and may provide better glucose homeostasis. This type of delivery will also lessen incidences of peripheral hyperinsulinemia, which is linked to neuropathy and retinoendopathy (17).
In an attempt to prevent degradation of insulin in the stomach, Lowman et al. loaded insulin into poly(methacrylic-g-ethylene glycol) microspheres and administered them orally to healthy and diabetic Wistar rats (18). In the acidic pH of the stomach, insulin was protected from proteolytic degradation because the gels were unswollen as a result of the formation of intermolecular polymer complex. In the basic and neutral environment of intestines, the complexes dissociated, thereby leading to gel swelling and insulin release.
Table I: Apparent permeability coefficient of insulin across various segments of gastrointestinal tract.*
Copolymer networks of poly(methacrylic acid) grafted with poly(ethylene glycol) (PEG) exhibited reversible, pH-dependent swelling behavior as a result of the formation of interpolymer complexes between protonated pendant acid groups and the etheric groups on the graft chains (19). Gels containing equimolar amounts of metha acrylic acid/ethylene glycol (MAA/EG) exhibited the lowest degree of swelling at low pH as a result of increased complexation. The pH of the swelling solution affected the average network mesh size. The in vitro release of insulin from poly(MAA-g-EG) gels containing PEG grafts of MW 1000 Da indicated a significant release of insulin as the gel decomplexed. The results of in vitro studies have showed that insulin release rates could be controlled by appropriate adjustment of the structure of the gels.
Stimuli sensitive terpolymers of butyl methacrylate and acrylic acid (pH sensitive) of various MW with N-isoporopyl acrylamide/butylmethacrylate/acrylic acid feed mole ratio of 85/5/10 were used to modulate release of insulin from pH-sensitive polymeric beads. Protein drug loading from an aqueous medium into the beads was achieved by preparing a 7 or 10% (w/v) polymer solution with 0.2% (w/v) insulin at low pH and below the lower critical solution temperature (LCST) of the polymer (pH 2.0 and 4 °C) and then dropping the solution into an oil bath above the LCST of the solution (35 °C). This loading procedure maintained protein stability while achieving high loading efficiency (between 90 and 95%) in the beads. Insulinrelease studies from beads prepared from terpolymers of the same composition but increasing M W were performed at pH 2.0 and pH 7.4, at 37 °C. There was negligible loss of insulin at pH 2.0 from the beads, indicating no burst effect. At pH 7.4, insulin release was seen from all the beads and the release rate was a function of the MW of the polymer (20, 21).
Reis et al. investigated alginate microparticles produced by emulsification-internal gelation as a promising carrier for insulin delivery (22). The alginate solution containing insulin protein was dispersed into a water immiscible phase. Gelation was triggered in situ by instantaneous release of ionic calcium from carbonate complex via gentle pH adjustment. Particle size could be controlled through the emulsification parameters, yielding spherical insulin-loaded microparticles. The recovery process was optimized, which improved yield, and ensured removal of residual oil from the particle surface. The optimum recovery strategy consisted in successive washing with a mixture of acetone-hexane-isopropanol coupled with centrifugation. This strategy led to small spherical particles with an encapsulation efficiency of 80% and a recovery yield of around 70%. In vitro release studies showed that alginate was not able to suppress insulin release in acidic media. However, this strategy preserved the secondary structure of insulin. Particles had a mean size lower than the critical diameter necessary to be orally absorbed through the intestinal mucosa followed by their passage to systemic circulation and thus can be considered as a promising technology for insulin delivery.
Microspheres. Insulin-loaded chitosan microspheres were administered orally to male Wistar rats and intestinal absorption was evaluated by measuring the plasma insulin levels and hypoglycemic effects (23). A marked absorption of insulin and a corresponding decrease in plasma glucose levels was observed following the oral administration of capsules that contained 20 IU of insulin and sodium glycocholate, as compared with the capsules containing only lactose or only 20 IU of insulin. The hypoglycemic effect started from 8 h after the administration of chitosan capsules when the capsules entered the colon. These findings suggested that chitosan capsules maybe useful carriers for the colon-specific delivery of peptides including insulin.
Ubaidulla et al. synthesized chitosan phthalate polymer and prepared microspheres containing insulin with the emulsion phase-separation technique (24). The in vitro release behavior of the microspheres was investigated under pH 2.0 and pH 7.4. The degree of phthalate substitution in the synthesized polymer was 20%. The prepared microspheres were spherical with an average diameter 46.34 μm. The insulin-loading capacity in the microspheres was 62%. Chitosan phthalate microspheres protected the insulin from gastric enzymes degradation, thereby enhancing the oral stability of insulin. The encapsulated insulin was quickly released in a phosphate buffer saline (pH 7.4), whereas only a small amount of insulin was released under acidic condition (0.1N HCl at pH 2.0). This result could be attributed to the fact that under acidic conditions, carboxylic groups present in the system existed in nonionized form and were poorly hydrophilic. However, in alkaline conditions, they existed in ionized form and are considerably hydrophilic. Results suggested that chitosan phthalate microspheres may be used as a potential carrier for oral insulin delivery.
Qi et al. prepared and characterized insulin enteric micro-spheres (EMS) of HPMC using a multiple emulsion solvent evaporation method (25). The preparation and characteristics of insulin EMS were studied and the gastrointestinal absorption enhancement of insulin by coadministering EMS with sodium N-(8-(2-hydroxybenzoyl] amino) caprylate (SNAC) was determined. The hypoglycemic effects of these microspheres were studied by orally administrating the insulin EMS and SNAC to rats. The particle size of EMS (o(1)/o(2)) and EMS (w/o/w) was about 500 and 30 μm, respectively, and drug loading was 7 and 3%, respectively. After being incubated with 18 μg/mL pepsin solution (pH 1) at 37 °C, only 20% of insulin in EMS (o(1)/o(2)) was digested within 4 h, and 60% of the insulin in EMS (w/o/w) was digested within 1 h. In hydrochloric acid solution (pH 1.2), EMS (o(1)/o(2)) had less drug dissolution than EMS (w/o/w). In phosphate buffer solution (pH 6.8), the entire drug release time of EMS (o(1)/o(2)) and EMS (w/o/w) was 75 and 10 min, respectively. After orally administered with SNAC, EMS (o(1)/o(2)) decreased the blood glucose level of rats remarkably and maintained the hypoglycemic effect for 4 h. EMS (w/o/w) had just a weakly hypoglycemic effect. Results showed that the characteristic-optimized EMS (i.e., EMS (o(1)/o(2)) incorporating SNAC) could enhance insulin absorption significantly in the gastrointestinal tract by taking advantage of both protection from enzyme degradation and improvement of drug permeability.
Senthil et al. attempted to target insulin delivery system to the upper region of the small intestine (26). Insulin-loaded Eudragit (Röhm Pharma Polymers, Darmstadt, Germany) (L-100) microspheres containing protease inhibitor and absorption enhancers were prepared by a solvent evaporation technique. The effect of these microspheres upon the relative hypoglycemia (RH) in white diabetic albino rats was studied in comparison with that produced after s.c. injection of bovine insulin solution. The incorporation of aprotinin and bile salts in microspheres produced prolonged and significant reduction of the blood glucose level when compared with insulin alone and insulin and bile salts.
Biodegradable microparticles also have been prepared with alginate using a piezoelectric ejection process (27). Lectin (wheat germ agglutinin, WGA) was conjugated to alginate microparticles to take advantage of the protective effects of alginate microparticles and the mucoadhesive properties of WGA for improved oral delivery of insulin. Their specific interaction with model mucin was determined by pig mucin immobilized surface plasmon resonance (SPR) biosensor and in vitro adsorption studies. In vitro experiments in the mucin solution showed that the conjugated WGA enhanced the interaction about three times. The hypoglycemic effects of alginate and WGA-conjugated alginate microparticles were examined after oral administration in streptozotocin-induced diabetic rats. In vivo studies with diabetic rats showed that the blood glucose level was lowest when alginate-WGA microparticles were orally administered. The absorption of insulin from alginate-WGA microparticles was sufficient to drop the glucose level of blood.
Insulin-entrapped liposomes cause dose-dependent hypoglycemia. Choudhari et al. prepared liposomes with varying composition by two methods: solvent evaporation hydration and solvent spherule evaporation (28). Liposomes containing lecithin 100 mg, cholesterol 20 mg, insulin 150 units, and Tween 1% v/v were found to be most effective. The effect of insulin-liposome was prolonged in diabetes-induced rabbits than that of normal rabbits. The pharmacodynamics of the insulin-liposome system was comparable with the action of 1 U/kg of insulin administered subcutaneously.
Coated liposomes. In another study, insulin liposomes were prepared by reversed phase separation and coated with chitosan of various molecular weights and concentrations (29). Chitosan coating was carried out by incubation of the liposomal suspensions with the chitosan solution. These chitosan-coated liposomes were administered to mice perorally and their hypoglycemic efficacy was determined. The insulin liposomes coated by 0.2% chitosan (MW 1000 kDa) showed maximum hypoglycemic efficacy. The minimum blood-glucose level and the hypoglycemic effect lasted for 4 h. Chitosan-coated liposomes also reduced tryptic digestion on insulin and enhanced enteral absorption of insulin. The molecular weights and concentrations of chitosan had significant effect on hypoglycemic efficacy of chitosan-coated insulin liposomes after oral administration to healthy mice.
In another study, insulin was bound covalently to the outer surface of multilamellar liposomes loaded with spin label (30). Electron spin resonance controlled encapsulation of the label tempocholine-nitroxide within the aqueous phases of liposomes. The binding of insulin was performed using the Carlsson's heterobifunctional reagent: N-succinimidyl 3-(2-pyridyldithio) propionate. The coupling method resulted in efficient attachment of 2.64.10(-4) mole of insulin per mole of phospholipid. Liposome coupled insulin retained its antigenic specificity as proved by radio immune assay.
DepoFoam (Pacira Pharmaceuticals, Parsippany, NJ) technology consists of novel multivesicular liposomes characterized by their unique structure of multiple nonconcentric aqueous chambers surrounded by a network of lipid membranes. Ye et al. demonstrated that DepoFoam technology can be used to develop sustained-release formulations of insulin with high loading (31). The data showed these formulations had a number of advantages, including high drug loading, high encapsulation efficiency, low content of free drug in the suspension, little chemical change in the drug caused by the formulation process, narrow particle-size distribution, and spherical particle morphology.
Wu et al. reported that the stability and absorption of insulin-liposomes double-coated by chitosan (CH) and chitosan-EDTA conjugates (CEC) was superior to that of the insulin-liposomes coated either by CH or by CEC (32). The protection of insulin against peptic and tryptic digestion was studied with HPLC after oral administration to rats. Liposomes protected insulin against the digestion of pepsin, trypsin, and gastrointestinal contents. In glucose tolerance test in normal rats, as compared with phosphate buffer solution control group, the insulin-liposomes coated by CH and CEC could reduce the glucose-induced peak of hyperglycemia. The reduction of the insulin-liposomes double-coated by CH and CEC was superior to that of other insulin-liposomes. When administered intragastrically to normal rats, the insulin liposomes coated by CH and CEC reduced glucose levels measured after an overnight fast. The hypoglycemic effect of the insulin liposomes double-coated by CH and CEC was superior to that of other insulin liposomes, and the dosage of 50 mU/kg decreased by 45.98% of initial blood glucose level after 1 h. As compared with s.c. injection, the relative pharmacological bioavailability was 17.02%, which was calculated by area under the curve of a glucose level versus time profile after oral administration of the insulin-liposomes double-coated by CH and CEC to rats. The serum insulin concentration-time curve best fit the one-compartment open model. As compared with s.c. injection, the relative bioavailability was 8.91% calculated by the area under the curve of serum insulin concentration versus time profile after oral administration of the insulin-liposomes double-coated by CH and CEC to rats.
Zhang et al. reported that liposomes promote the oral absorption of insulin becasuse of specific site combination on the gastrointestinal tract cell membrane (33). The authors prepared lectin-modified liposomes containing insulin and evaluated the potential of these modified colloidal carriers for oral administration. Wheat germ agglutinin (WGA), tomato lectin (TL), or Ulex europaeus agglutinin 1 (UEA1) were conjugated by coupling their amino groups to carbodiimide-activated carboxylic groups of N-glutaryl-phosphatidylethanolamine (N-glut-PE). Insulin liposomes dispersions were prepared with the reverse-phase evaporation technique and modified with the lectin-N-glut-PE conjugates. The hypoglycemic effect of these liposomes was observed in mice. The pharmacological bioavailability of insulin liposomes modified with WGA, TL, and UEA1 were 21.40, 16.71, and 8.38%, respectively, in comparison with abdominal cavity injection of insulin. After oral administration of the insulin liposomes modified with WGA, TL, and UEA1 to rats, the relative pharmacological bioavailabilities were 8.47,7.29, and 4.85%, the relative bioavailabilities were 9.12,7.89, and 5.37%, respectively, in comparison with s.c. injection of insulin. In the two cases, no remarkable hypoglycemic effects were observed with the conventional insulin liposomes.
Ramadas et al. developed an oral formulation based on liposome encapsulated alginate-chitosan gel capsules for insulin delivery (34). Liposome encapsulation helped to increase the encapsulation efficiency of insulin in alginate-chitosan capsules. This formulation bypassed the acidic medium in stomach and delivered insulin in the neutral environment of the intestine with increased drug absorption and bioavailability. The administration of this formulation was found to reduce blood glucose levels when tested in diabetic rats.
In a study conducted by Attivi et al., insulin nanoparticles were prepared by a water-in-oil-in-water emulsification and evaporation method (35). The polymers used for the encapsulation were blends of biodegradable poly-epsilon-caprolactone (PCL) and nonbiodegradable polymer (Eudragit RS). Poly(alkyl cyanoacrylate) nanocapsules also have been successfully used for oral administration of insulin in diabetic rats. The nanoparticles were characterized by measuring the amount of entrapped insulin, the particle size, the polydispersity of the obtained particles, the zeta potential, and the amount of insulin released after 7 h. The corresponding quantity of entrapped insulin was 25 IU per 100 mg of polymer, and the particle size was 350 nm with a polydispersity of 0.21. The quantity of released insulin was 4.8 IU per 100 mg of polymer after 7 h, and the zeta potential was +44 mV. Bhum-kar et al. prepared gold nanoparticles using various concentrations of chitosan (from 0.01% w/vto 1% w/v) (36). Varying concentrations of chitosan used for the synthesis of gold nanoparticles demonstrated that the nanoparticles obtained at higher chitosan concentrations (>0.1% w/v) were stable and showed no signs of aggregation. The nanoparticles also showed long-term stability in terms of aggregation for about 6 months. Insulin loading of 53% was obtained and found to be stable after loading. Blood glucose lowering at the end of 2 h following administration of insulin-loaded gold nanoparticles to diabetic rats was 30.41 and 20.27% for oral (50 IU/kg) and nasal (10 IU/kg) administration, respectively. Serum gold level studies have demonstrated significant improvement in the uptake of chitosan-reduced gold nanoparticles.
Damge et al. prepared insulin-loaded nanospheres by polymerization of isobutyl cyanoacrylate (IBCA) in an acidic medium (37). These nanospheres displayed a mean size of 145 nm and an association rate of 1 U of insulin per milligram of polymer. These nanospheres were dispersed in an oily medium (Miglyol 812) containing surfactant (Polox-amer 188 and deoxycholic acid) and evaluated for in vitro and in vivo degradation. No degradation due to proteolytic enzyme was observed in vitro. When these nanospheres (100 U per kilogram of body weight) were administered perorally in streptozotocin-induced diabetic rats, a 50% decrease in fasted glucose levels from the second hour up to 10-13 days was observed. This effect was shorter (2 days) or absent when nanospheres were dispersed in water. Using 14 C-labeled nanospheres loaded with (125I) insulin, it was found that nanospheres increased the uptake of (125I) insulin or its metabolites in the gastrointestinal tract, blood, and liver while the excretion was delayed when compared to (125I) insulin nonassociated to nanospheres.
In another study, Damge et al. used nanoparticles prepared with a blend of a biodegradable polyester(poly(-epsilon-capro-lactone)) and a polycationic non-biodegradable acrylic polymer (Eudragit RS) as a drug carrier for oral administration of insulin (38). The rate of encapsulation of insulin was around 96%. The therapeutic efficiency of oral insulin nanoparticles (25, 50, and 100 IU/kg) in diabetic rats and the intestinal uptake of fluorescein isothiocyanate (FITC)-labelled insulin were studied. When administered orally by force-feeding to diabetic rats, insulin nanoparticles decreased fasted glycemia in a dose-dependant manner with a maximal effect observed with 100 IU/kg. These insulin nanoparticles also increased serum insulin levels and improved the glycemic response to an oral glucose challenge for a prolonged period of time.
Chalasani et al. developed a vitamin B12 (VB12) nanoparticles system to enhance the uptake capacity of both nanoparticles and VB12 transport to deliver orally effective insulin (39). Nanoparticles were prepared using various molecular-weight dextrans and epichlorohydrin as a cross-linker by an emulsion method. Nanoparticle surface was modified with succinic anhydride and conjugated with amino VB12 derivatives of carbamate linkage. VB12 attachment was confirmed by IR, XPS analysis, and was quantified by HPLC (4.0 to 4.4% w/w of nanoparticles). The preformed nanoparticle conjugates (Zave = 160–250 nm; polydisperse) were loaded with 2,3, and 4% w/w insulin, and the entrapment was 45–70%. Nanoparticulate conjugates protected 65–83% of entrapped insulin against in vitro gut proteases. In vitro release studies exhibited an initial burst followed by diffusion-controlled first-order kinetics with 75–95% release within 48 h. After oral administration of these carriers (20 IU/kg), 70–75% reduction in plasma glucose was found in 5 h, reached basal levels in 8-10 h, and a prolonged second phase was found until 54 h.
In a study by Lin et al., nanoparticles composed of chitosan and poly(gamma-glutamic acid) were prepared by a simple ionic-gelation method for oral insulin delivery (40). After insulin loading, the nanoparticles remained spherical and the insulin-release profiles were significantly affected by their stability in distinct pH environments. The in vivo results clearly indicated that the insulin-loaded nanoparticles could effectively reduce the blood-glucose level in a diabetic rat model.
Cui et al. investigated the preparation of PLGA nanoparticles (PNP) and PLGA-Hp55 nanoparticles (PHNP) as potential drug carriers for oral insulin delivery (41). The nanoparticles were prepared by a modified emulsion solvent diffusion method in water, and their physicochemical characteristics, drug release in vitro and hypoglycemic effects in diabetic rats were evaluated. The mean particle sizes of the PNP and PHNP were 150 and 169 nm, respectively. The drug recoveries of the nanoparticles were 50.30 ± 3.1 and 65.41 ± 2.3%, respectively. The initial release of insulin from the nanoparticles in simulated gastric fluid over 1 h was 50.46 ± 6.31 and 19.77 ± 3.15%, respectively. The relative bio availability of PNP and PHNP compared with s.c. injection (1 IU/kg) in diabetic rats was 3.68 ± 0.29 and 6.27 ± 0.42%, respectively. The results showed that the use of insulin-loaded PHNP was an effective method of reducing serum glucose levels.
Sarmento et al. prepared a nanoparticulate insulin delivery system by complexation of dextran sulfate (DS) and chitosan in aqueous solution (42). Parameters of the formulation such as the final mass of polysaccharides, the mass ratio of the two polysaccharides, pH of polysaccharides solution, and insulin theoretical loading were identified as the modulating factors of nanoparticle physical properties. Particles with a mean diameter of 500 nm and a zeta potential of approximately –15 mV were produced under optimal conditions of DS:chitosan mass ratio of 1.5:1 at pH 4.8. Nanoparticles showed spherical shape, uniform size, and good shelf-life stability. Polysaccharides complexation was confirmed by differential scanning calorimetry and Fourier transformed infrared spectroscopy. An association efficiency of 85% was obtained. Insulin release at pH below 5.2 was almost prevented up to 24 h and at pH 6.8, and the release was characterized by a controlled profile. This result suggested that the release of insulin was ruled by a dissociation mechanism and DS–chitosan nanoparticles acted as pH-sensitive delivery systems. Furthermore, the released insulin entirely maintained its immunogenic bioactivity evaluated by ELISA, confirming that this new formulation shows promising properties towards the development of an oral delivery system for insulin.
An aqueous nanoparticulate delivery system containing oppositely charged polymers polyethylenimine (PEI) and DS with zinc as a stabilizer was developed by Tiyaboonchai et al. (43). The pH of PEI solutions, the weight ratio of the two polymers, and zinc sulfate concentrations affected the particle size of the nanoparticles. Spherical particles of 250 nm mean diameter with a zeta potential of approximately +30 mV were produced under optimal conditions. Insulin could be loaded up to 90% in these nanoparticles. Circular dichroism spectra showed no significant conformational changes compared with free insulin under optimized formulation conditions. In contrast to rapid release of insulin in vitro, the hypoglycemic activity in streptozotocin-induced diabetic rats was prolonged. This system offered a number of advantages, including ease of manufacturing under mild preparation conditions, completely aqueous processing conditions, use of biocompatible polymers, ability to control particle size, a high level of drug entrapment, and an ability to preserve secondary structure and biological activity of protein.
Simon et al. prepared nanosized insulin-complexes based on amine modified comb-like polyesters (44). Protection of insulin in nanocomplexes from enzymatic degradation was investigated. The interaction with enterocyte-like Caco-2 cells in terms of cytotoxicity, transport through and uptake in the cell layers was evaluated by measuring transepithelial electrical resistance (TEER), release of lactate dehydrogenase (LDH), and insulin transport. The protection capacity of the nanocomplexes and their interaction with Caco-2 cells varied strongly as a function of lactide-grafting (hydrophobicity). With increasing lactide-grafting (P(26)P(26)-1(LL)P(26)-2(LL)) Nanocomplexes protected as much as 95% of the insulin against degradation by trypsin.
Colon-targeted delivery systems
Proteolytic enzymes in the stomach degrade insulin, but in intestines peptidase activity is low and drainage into lymph is maximized. Researchers are exploring colon-specific delivery for insulin. To achieve colon specific delivery of insulin, Hideyuki et al. prepared azopolymer-coated pellets containing fluorescein isothiocyanate dextran (FD-4) (45). In vitro drug-release experiments were carried out according to Japanese Pharmacopoeia XII (rotating basket method). The release of FD-4 from the pellets in phosphate buffered saline was very small. However, the release of FD-4 was markedly increased in the presence of rat ceacal contents. The pharmacodynamic studies of the azopolymer-coated pellets containing these peptides with camostat mesilate (protease inhibitor) were carried out by measuring the hypoglycemic effects. A slight decrease in plasma-glucose levels was observed following the oral administration of these pellets containing 12.5 IU of insulin compared with the same dose of insulin solution. The authors concluded that azopolymer-coated pellets with protease inhibitor might be useful carriers for the colon-specific delivery of insulin.
Yakugaku Zasshi developed two types of microcapsular devices containing new acrylate-based nanogels with a specific solute-permeability for the delayed- or thermosensitive-release of peptide drugs (46). A nanogel-particle of acrylic terpolymer, ethyl acrylate-methyl methacrylate-2-hydroxy-ethyl methacrylate, was newly synthesized by emulsion polymerization to construct delayed-release microcapsules. The insulin-loaded lactose particles were spray coated with the acrylic terpolymers. These microcapsules showed a pH-in-dependent delayed-release profile. Oral administration of the microcapsules with the lag time of 6 h to beagle dogs resulted in significantly reduced blood glucose concentration, leading to colon-specific insulin delivery with pharmacological availability of 5%. Meanwhile, poly(N-isopropylcarylamide) (p(NIPAAm)] nanogel-particles with a reversible temperature-dependent swelling property were prepared by dispersion polymerization to fabricate microcapsular membranes with thermosensitively changeable permeability. The microcapsules constructed by coating of drug-loaded CaCO3 particles with a blend mixture of the p(NIPAAm) nanogels and ethylcellulose pseudo-latex exhibited an on-off positively thermosensitive drug-release; the release rate was remarkably enhanced at higher temperatures possibly due to the formation of voids through the shrinkage of p(NIPAAm) nanogels in the membrane. A possible application of this type of microcapsules can be found in externally temperature-activated pulsatile peptide delivery.
Mucoadhesive system. A biologically adhesive delivery system offers important advantage over conventional drug delivery systems. Whitehead et al. (47) described a novel method of delivering insulin into systemic circulation by mucoadhesive intestinal patches. Intestinal patches localize insulin near the mucosa and protected it from proteolytic degradation. In vitro experiments confirmed the secure adhesion of patches to the intestine and the release of insulin from them. In vivo experiments performed via jejunal administration showed that intestinal insulin patches induced dose-dependent hypoglycemia in normal rats. These studies revealed that reduction in blood glucose levels were comparable with those induced by s.c. injections.
The engineered polymer microspheres made of erodible polymer display strong adhesive interactions with gastrointestinal mucus and cellular lining and can traverse both the mucosal epithelium and the follicle associated epithelium covering the lymphoid tissue of Peyer's patches. Alginate, a natural polymer recovered from seaweed is being developed as a nanoparticle for the delivery of insulin without being destroyed in the stomach. It has in addition, several other properties that have enabled it to be used as a matrix for entrapment and for the delivery of a variety of proteins such as insulin and cells. These properties include: a relatively inert aqueous environment within the matrix, a mild room temperature encapsulation process free of organic solvents, a high gel porosity that allows for high diffusion rates of macromolecules, the ability to control this porosity with simple coating procedures, and dissolution and biodegradation of the system under normal physiological conditions (48).
Thiolated chitosan insulin tablets. The efficacy of orally administered insulin has also been improved using thiolated chitosan. 2-Iminothiolane was covalently linked to chitosan and the resulting chitosan-TBA (chitosan-4-thiobutylamidine) conjugate exhibited 453.5 ± 64.1 micromol thiol groups per gram polymer (49). Two enzyme inhibitors Bowman-Birk-Inhibi-tor (BBI) and elastatinal were covalently linked to chitosan. Chitosan-TBA conjugate (5 mg), insulin (2.75 mg), the permeation mediator reduced glutathione (0.75 mg), and the two inhibitor conjugates (in each case 0.75 mg) were compressed to make chitosan-TBA-insulin tablets. Control tablets were also prepared using chitosan and insulin. Chitosan-TBA-insulin tablets showed a controlled release of insulin over 8 h. In vitro mucoadhesion studies showed that the mucoadhesive/cohe-sive properties of chitosan were at least 60-fold improved by the immobilization of thiol groups on the polymer. After oral administration of chitosan-TBA-insulin tablets to non-diabetic rats, the blood glucose level decreased significantly for 24 h. In contrast, neither control tablets nor insulin given in solution showed a comparable effect. These results concluded that the combination of chitosan-TBA, chitosan-enzyme-inhibitor conjugates and reduced glutathione could constitute a promising strategy for the oral administration of insulin.
Dorkoosh et al. developed novel peroral peptide drug delivery systems based on superporous hydrogel (SPH) and SPH composite (SPHC) (50). The authors studied the release of insulin from SPH and SPHC polymers. In addition, the stability of insulin during the release and the integrity of insulin in the polymeric matrix of SPHC was investigated. The release studies revealed that insulin was released almost completely from the polymers. SPH was more porous than SPHC, therefore, release of pep tides was faster from SPH. FTIR studies demonstrated that no covalent binding occurred between insulin and the polymeric SPHC matrix. The release profile of insulin was time controlled. There was an initial lag time of 10–15 min, followed by burst release during which more than 80% of insulin was released within 30–45 min.
Kavimandan et al. developed hydrogels as a delivery vehicle for insulin-transferrin conjugate (51). In this work, electrospray ionization mass spectrometry (ESI–MS) was used to study the modification of insulin during its reaction with transferrin. The stability of the conjugated insulin to enzymatic degradation was also studied. ESI-MS studies confirmed the site-specific modifications of insulin. The transferrin conjugation of insulin was also shown to increase the stability of insulin to enzymatic degradation.
A liquid formula that can be easily dispersed in water to produce particles named "Nanocubicles" was developed by Chung et al. (52). These nanocubicles containing insulin were administered to fasted streptozotocin induced diabetic rats. For comparison, an aqueous solution of insulin in water was also administered. Nanocubicles without insulin and insulin in phosphate buffer saline (PBS) were administered as controls. Blood glucose concentration and insulin concentration were measured 1, 2, 3, 4, and 6 h after the administration of the insulin formulations. In vitro experiments showed that the particles were taken up by the Caco-2 cells at a high ratio. It was observed in these studies that the serum glucose concentration was controlled for more than 6 h after oral insulin administration but returned to the basal concentration in 3 h when 1 IU/kg of insulin was injected intravenously.
Cilek et al. (53) prepared microemulsions using Labrafil M 1944 CS, Phospholipon 90 G (lecithin), absolute alcohol and bi-distilled water. Aprotinin (2500 KIU/g) was added as the enzyme inhibitor to the formulation. Upon the administration of intragastric recombinant human DNA (rDNA) insulin solution to nondiabetic rats no significant change in blood-glucose level was observed. The microemulsions of rDNA insulin and aqueous solution (200 IU/kg) were administered intragastri-cally by a canulla to diabetic and nondiabetic rats. Therefore, the hypoglycemic effect of s.c. rDNA insulin solution, micro-emulsion containing rDNA insulin (IME) and microemulsion containing insulin and aprotinin (IMEA) were analyzed in diabetic rats. The area above the plasma-glucose levels time curves (AAC), minimum glucose concentration (Cmin ) and time to Cmin (tmin) were derived from the plasma glucose profiles. IME and IMEA caused approximately 30% decrease in plasma glucose levels. The highest AAC value was obtained when IMEA was administered to rats. Thus aprotinin an enzyme inhibitor can increase the bioavailability of insulin.
Phase diagrams containing the microemulsion region were constructed for pseudo-ternary systems composed of polyglycerol fatty acid ester/cosurfactant/Captex 300/water (54). It was necessary to add ethanol, 1-propanol and 1-butanol as cosurfactant to produce microemulsions. Results demonstrated that microemulsions were formed when polyglycerol fatty acid esters with hydrophile-lipophile balances (HLBs) between 8 and 13 (e.g., MO500, MO750, SO750, and ML310) were used. Microemulsions were thermodynamically stable for long periods. Further, several microemulsion formulations had enough acid-protection efficiency.
Ma et al. developed a stable self-emulsifying formulation for the oral delivery of insulin (55). This formulation enabled changes in barrier properties of Caco-2 monolayers, as referred by transepithelial electrical resistance (TEER) and apparent permeability coefficients P(app) of the paracellu-lar marker ranitidine (20-fold greater than control) but not transcellular marker propranolol, suggesting that the opening of tight junctions was involved. In diabetic beagle dogs, the bioavailability of this formulation was as much as 15.2% at a dose of 2.5 IU/kg in comparison with the hypoglycemic effect of native insulin (0.5 IU/kg) delivered by s.c. injection.
Ritschel et al. reported the gastrointestinal absorption of insulin from microemulsions (56). The routes of administration were peroral, intralumenal, or rectal. The experiments were carried out in dogs, rabbits, and rats. An absorption model for pep tides using microemulsions as delivery systems was presented.
A peroral dosage form was developed to deliver insulin to the colon (57). Microemulsions with insulin were gelled using Cab-O-Sil (Cabot Corp., Boston, MA), and filled into gelatin capsules pretreated with formaldehyde vapor. The capsules were coated with Eudragit NE 30 D, Eudragit S100, and cellulose acetate phthalate polymers. In vitro dissolution profiles of the capsule coating, using sodium salicylate as the marker, showed that dissolution of the capsule began at 4 h, at pH 5.5, and was completed at 8 h. An in vivo crossover study in beagle dogs was carried out using i.v. insulin, p.o. insulin micro-emulsion, and colonic release capsule dosage forms without insulin (CRC) were used as controls, a colonic release capsule dosage form with insulin (CRI) and additionally with sodium laurylsulfate (CRIL) or aprotinin (CRIA) as sorption promoter and enzyme inhibitor, respectively. The reduction in blood glucose concentration levels was measured and results were interpreted in terms of pharmacological availability. The pharmacological availability is the ratio of the area under the baseline curve, expressed as percent glucose reduction from baseline versus time of the peroral dosage forms to intravenous insulin administration, corrected for body weight and dose size. The PA for the peroral microemulsion, CRC, CRI, CRIL, and CRIA were 2.1,0.4, 5.0,2.7 and 6.2%, respectively. The release of Insulin occurred throughout the GI tract, with the exception of the stomach.
Human red blood cells have been developed as oral carrier systems for human insulin. In a study by Al-Achi et al., male Wistar rats were made diabetic by a single intraperitoneal injection of streptozocin (100 mg/kg) (58). Rats received orally one of the following (100 U, 2 mL): an insulin solution, a ghosts-insulin suspension, a vesicles-insulin suspension, a liposomes-ghosts-insulin suspension, or a liposomes-vesicles-insulin suspension. Free-carrier suspensions or sodium chloride solution (0.9%) were given orally as controls. Blood glucose concentration was determined just before administration and at 1,2,3,4, 5,6, and 7 h postadministration. Results showed that all treatment groups, except liposomes-ghosts-insulin, were significantly different statistically from their respective controls (i.e., the free carriers).
Niosomes (non-ionic surfactant vesicles)
Niosomes of sorbitan monoesters (Span 20, 40, 60, and 80) were prepared using the film-hydration method without soni-cation (59). Span 80 did not form niosomes in the absence of a sufficient amount of cholesterol. The size of vesicles depended upon the molar ratio or charge incorporation. The amount of insulin released in simulated intestinal fluid from Span 40 and 60 was lower than Span 20 and 80 vesicles. Vesicles containing Span 60 showed the highest protection of insulin against proteolytic enzymes and good stability in the presence of sodium deoxycholate and storage temperatures.
Solid lipid microparticles
Solid lipid insulin-loaded microparticles were produced by a solvent-in-water emulsion-diffusion technique, using iso-butyric acid as the solvent phase, glyceryl monostearate or cetyl palmitate as lipid, soya lecithin and taurodeoxycholate as emulsifiers (60). Isobutyric acid was used a result of its high insulin-solubilization capacity. Solid lipid microparticles of spherical shape were prepared by simple dilution of the emulsion with water. The process was conducted at 50 °C using a high-shear homogenizer. Insulin encapsulation efficiency of about 80% was achieved. The in vitro release of insulin from the microparticles was very low with an initial burst effect of 20% of the dose. After treatment of the solid lipid microparticles with pepsin solution, an insulin loss of about 24% of the total insulin was observed.
Modifying the chemical structure of a peptide or protein is another approach to enhance bio availability by increasing its stability against possible enzymatic degradation or its membrane permeation. However, this approach is more applicable to peptides rather than proteins because of the structural complexity of proteins. For example, substitution of D-amino acids for L-amino acids in the primary structure can improve the enzymatic stability of peptides. A diacyl derivative of insulin maintains its biological activity and also increases absorption from the intestine (1).
Commercial interest in oral delivery of insulin
Several companies have attempted drug delivery systems for proteins, including insulin. Although a commercial product is not yet available on the market, the current state of research spans from proof-of-concept studies to late-stage clinical studies (61).
The Orasome technology (Endorex Corp., Chicago, IL) involves encapsulation of proteins in liposomes. These lipo-somes are rendered resistant to harsh conditions of the gastrointestinal tract such as exposure to acidic pH, bile salts, and detergents by polymerization (62,63). Drugs are absorbed by the uptake of intact liposomes and are released in the tissues of the body.
Researchers at Emisphere Technology (New York, NY) are working with non-Acylated α-amino acids as carriers for the oral delivery of macromolecules (64, 65). They claim that upon oral administration of the carrier with the protein, the carrier forms a noncovalent association with the conformation of the protein that has a higher transport rate when compared with the physiological conformation. This complex dissociates after crossing the cell membrane.
The M2 system (Nobex Corp., Research Triangle Park, NC) is based on the attachment of low molecular weight polymers to specific sites in the protein. These polymer conjugates have been reported to improve stability and absorption when compared with performance of native protein (66).
Limitations of oral delivery of insulin
There are several barriers associated with the oral delivery of insulin:
Enzymatic degradation of insulin. Upon ingestion, insulin is subjected to acid catalyzed degradation in the stomach, luminal degradation in intestine, and intracellular degradation. The pancreatic enzymes that degrade insulin are trypsin and α-chymotrypsin (17). The cytosolic enzyme that degrades insulin is insulin-degrading enzyme (IDE) (67). Insulin is not subjected to enzymatic degradation by brush-border enzymes. The rate of degradation of insulin also depends on its associated state in solution. Insulin is a monomer at lower concentrations (<0.1 μM) and dimerizes in a pH range of 4-8 at higher concentrations. At concentrations >2 mM, the hexamer is formed at neutral pH. The associated state affects the rate of degradation of insulin. In the presence of bile salts, the rate of degradation may increase close to six times (68).
Intestinal transport of insulin. Evidence of active transport for insulin was negative (69). Morpho-cytochemical and biochemical evidence for insulin absorption was demonstrated in rat gastrointestinal tract (70, 71). This result was achieved by direct instillation of a solution of insulin into various parts of the gastrointestinal tract, followed by visualization with gold markers and immunoassay of the insulin in blood. No evidence exists for the transport of insulin by the paracellular route. Researchers found that insulin is absorbed to the apical plasma membrane and is internalized by endocytosis. The presence of insulin receptors has been demonstrated in enterocytes on both the apical and baso-lateral sides (72–74). Permeability studies of insulin across isolated segments of the gastrointestinal tract have been performed with an aim to evaluate the apparent permeability coefficient of insulin. The in vitro permeability studies also serve as screening tools to test the efficacy of absorption modifiers. Insulin permeability across the gastrointestinal tract has been studied by using isolated segments of various regions of the intestine. Table I lists the apparent permae-abilities coefficients of various regions of the gastrointestinal tract. These differences are attributed to the histological differences between various sites (75).
Dosage-form stability issues. The activity of proteins depends on the three-dimensional molecular structure of the protein. The dosage-form development of proteins may expose them to harsh conditions that may alter their structure. This will have implications in the efficacy and immunogenic response to the proteins. During dosage-form development, proteins might be subjected to physical and chemical degradation. The stability of insulin preparations has been documented in detail (76), and research data on solid-state stability of proteins in dosage forms have been reviewed recently (77). Proteins must be characterized for change in conformation, size, shape, surface properties, and bioactivity upon formulation processing. Changes in conformation, size, shape can be observed by use of spectrophotometric techniques, X-ray diffraction, differential scanning calorimetery, light scattering, electrophoresis, and gel filtration (78). Changes in surface properties can be detected with electrophoretic and chromatographic techniques, and changes in the bioactivity of proteins can be observed with bioavailability studies. The interference by formulation excipients also may be a factor when selecting the characterization technique (79). Size-exclusion chromatography with reverse-phase high-performance liquid chromatography was used to determine the formation of covalent insulin dimmers with trace amounts of high molecular weight transformation products after microencapsulating insulin in a mixture of poly(DL-lactide-co-glycolide) and poly(l-lactide) (80). Differential scanning calorimetry was used to differentiate denaturation endotherms of amorphous and crystalline insulin (81). X-ray diffractograms of insulin have been obtained with mixtures of lactose and mannitol to evaluate the effect of spray-drying on the crystalline changes of insulin (82).
Attempts have been made to achieve oral insulin delivery using various systems. It has been proved that the insulin is subjected to acid catalyzed degradation in stomach, luminal degradation in intestine, and intracellular degradation. Scientists have been able to protect the insulin delivery systems from acidic environment of the stomach and target it to the intestine. The maximum bioavailability of the insulin has been reported to be very low because of the poor absorption of insulin from the intestine. The magnitude of apparent permeability of insulin has been reported to be more in the jejunem and ileum than in other parts of the intestine. Researchers have tried to increase the absorption of insulin from the intestine using absorption enhancers such as aprotinin (protease inhibitor), tween, oligoarginine, sodium glycochol-ate, deoxycholic acid, and taurodeoxycholate.
Researchers have prepared microspheres, liposomes, mi-croemulsions, niosomes, nanocubicles, and so forth for the oral delivery of insulin. Pharmacodynamic studies of the insulin from the delivery systems have been successfully carried out in streptozotocin- or alloxan-induced diabetic mice or rats or other animals. The loading of insulin in microparticulate delivery system was the highest. Chitosan-coated microparticles protected insulin from the gastric environment of the body and released it in intestinal pH. Chitosan-coated liposomes have also been reported to have excellent hypoglycemic effects. Limitations to the delivery of insulin have not resulted in fruitful results to date and there is still a need to prepare newer delivery systems, which can produce dose-dependent and reproducible effects in addition to increased bioavailability.
S. Dhawan* is a senior manager of advanced drug delivery and research at Panacea Biotech and a faculty member, Rishi Kapil and Deepak Kapoor are senior research fellows all at UGC-CAS Pharmaceutical Science, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, INDIA, email@example.com. Sunny Chopra is a senior reesearch fellow in the department of pharmaceutical sciences at Jamia Hamdard University, New Delhi, India. *To whom all correspondence should be addressed.
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