polymethyacrylate based microparticulates of insulin for oral delivery: preparation and in vitro...

International Journal of Pharmaceutics 225 (2001) 31–39

Polymethyacrylate based microparticulates of insulin fororal delivery: Preparation and in vitro dissolution stability

in the presence of enzyme inhibitors

Vikas Agarwal, Indra K. Reddy, Mansoor A. Khan *Department of Pharmaceutical Sciences, Texas Tech Uni�ersity HSC, School of Pharmacy, 1300 Coulter, Suite 400, Amarillo,

TX 79106, USA

Received 29 September 2000; received in revised form 24 January 2001; accepted 17 May 2001

Abstract

The purpose of this investigation was to (a) evaluate the coprecipitation technique for preparing microparticulatesof insulin, (b) study the effect of variables such as addition of salts in the precipitating medium and ratio of polymericsolution to volume of precipitating medium on the dissolution and encapsulation efficiency of insulin microparticu-lates, and (c) evaluate the in-vitro enzymatic dissolution stability of insulin microparticulates in the presence ofchicken ovomucoid (CkOVM) and duck ovomucoid (DkOVM) as inhibitors. Insulin dissolved in 0.01 N HCl wasmixed with alcohol USP to get a final concentration of 32% v/v. Eudragit L100, a representative polymethyacrylatepolymer, was then dissolved in this solution which was transferred to a beaker containing cold water withhomogenization to obtain microparticulates. Dissolution studies were carried out in pH 6.8 phosphate buffer usinga 100-ml conversion kit in a standard dissolution assembly. Dissolution stability of microparticulates was evaluatedin the presence of 0.5 �M trypsin and 0.l �M chymotrypsin at various ratios of CkOVM and DkOVM. The resultsindicated that insulin microparticulates could be prepared using the coprecipitation technique with high encapsulationefficiency by proper selection of experimental conditions and amount of polymer. Presence of salts in the precipitatingmedium decreased the dissolution of insulin from the microparticulates. As the ratio of precipitating medium withrespect to the polymeric solution was increased, the encapsulation efficiency increased. In dissolution stabilityexperiments, insulin was not detected in the presence of enzymes alone. When CkOVM and DkOVM wereincorporated, the stability of insulin increased significantly in a concentration dependent fashion. © 2001 ElsevierScience B.V. All rights reserved.

Keywords: Insulin; Microparticulates; Coprecipitates; Inhibitors; Chicken ovomucoid; Duck ovomucoid; Dissolution stability

www.elsevier.com/locate/ijpharm

1. Introduction

Oral delivery of therapeutic proteins is limiteddue to barriers such as enzymatic degradation inthe gastrointestinal tract, low epithelial permeabil-ity and instability under formulation conditions

* Corresponding author. Tel.: +1-806-356-4000x285; fax:+1-806-356-4034.

E-mail address: [email protected] (M.A. Khan).

0378-5173/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.

PII: S0378-5173(01)00740-2

V. Agarwal et al. / International Journal of Pharmaceutics 225 (2001) 31–3932

(Lee et al., 2000). Extensive research literature isavailable that addresses approaches used to over-come these barriers. Among them, the use ofenzyme inhibitors, permeation enhancers and en-teric dosage forms are popular. A synergistic ap-proach to overcome limited oral bioavailability ofproteins has also shown promise (Carino andMathiowitz, 1999).

Among the proteins, insulin has been mostextensively investigated for delivery by the oralroute. Oral delivery of insulin has been attemptedwith the use of sustained release polymeric sys-tems with enzyme inhibitors and/or permeationenhancers. Polymeric systems attempted for in-sulin include enteric coated dosage forms andmicroencapsulation. Microencapsulation is pre-ferred over coated dosage forms due to theirability to yield consistent microparticulates withlimited amount of drug. Microparticulates/micro-spheres of insulin have been prepared with micro-cystalline cellulose (Trenktrog et al., 1996),polyvinyl alcohol (Kimura et al., 1996), and poly-methylacrylates (Morishita et al., 1992a). Some ofthe disadvantages with these approaches includeexposure of the protein to harsh processing condi-tions such as extrusion spheronization, long pro-cessing times to prepare the microparticulates(more than a day), rapid release (within 2 h), andlower encapsulation efficiencies (as low as 40%).The coprecipitation technique involves the precip-itation of freely soluble polymer and drug mixturefrom a solution by addition of a non-solvent(Simonelli et al., 1969; Kislalioglu et al., 1991).Coprecipitates have been formulated as suspen-sions (Dalal and Narurkar, 1991), hard gelatincapsules (Walker et al., 1980) and directly com-pressible tablets (Fassihi et al., 1985). Micropar-ticulate formation of proteins by coprecipitationhas not been addressed adequately. It was ofinterest to see if insulin microparticulates can beprepared by this technique.

The role of ovomucoids in the oral delivery ofinsulin is being investigated in our laboratory.Ovomucoids represent a new class of enzyme in-hibitors derived from the egg white of avian spe-cies. Extensive reviews detailing their source,active domains and mechanism of inhibitory ac-tion can be found elsewhere (Laskowski et al.,

1960). Briefly, avian ovomucoids are present inegg white of avian species and account forroughly 10% of egg white of proteins. Their in-hibitory activity is species dependent and medi-ated by binding to the corresponding enzymethrough their reactive site. Since they inhibit di-gestive enzymes such as bovine trypsin and bovine�-chymotrypsin, they might be useful as absorp-tion modifiers for oral delivery of insulin. Thestability of insulin in the presence of CkOVM andDkOVM has been studied in our laboratory(Agarwal et al., 2000).

At the present time there is no defined methodof selecting the amount of inhibitor in a proteindosage form. This is compounded by the fact thatthe stability of proteins is dependent on it suscep-tibility to different enzymes in the gastrointestinaltract. Alternatively, use of enzyme inhibitors needto be tailored to arrest protein degradationagainst specific degrading enzymes. If the dissolu-tion stability of the protein from the enzyme andinhibitor systems is evaluated, proper selection oftype and amount of inhibitor may be made forincorporation in the bioavailability studies.

The objectives of the present investigation wereto: (1) evaluate the coprecipitation technique forthe preparation of microparticulates of insulin, (2)study the effect of presence of salts in the precipi-tating medium and ratio of precipitating mediumto the polymeric solution on formation of mi-croparticulates, and (3) evaluate the enzymaticdissolution stability of microparticulates in thepresence of inhibitors CkOVM and DkOVM.

2. Materials

Insulin was purchased from Intergen Company,Purchase, NY. Duck ovomucoid was provided byDr Laskowski Jr., Department of Chemistry, Pur-due University, IN. Eudragits, lactose, magne-sium stearate were samples from Rohm Pharma,CHR Hansen, Mahawah, NJ, Whittaker Clarkeand Daniels, South Plainfield, NJ. Talc wasbought from Spectrum Chemical Company, Gar-dena, CA. Trypsin (Type VII, TPCK treated),chicken ovomucoid (Type II-O) and �-chy-motrypsin (TLCK treated) were purchased from

V. Agarwal et al. / International Journal of Pharmaceutics 225 (2001) 31–39 33

Sigma Chemical Company, St. Louis, MO. Alco-hol (USP grade) was purchased from Aaper Co.,Shelbyville, KY. Solvents used were high-perfor-mance liquid chromatography (HPLC) grade. Allother chemicals were of reagent grade and wereused as received. Deionized water filtered using0.2 �M filter under vacuum was used for allexperiments.

3. Methods

3.1. Microencapsulation by coprecipitation

A representative figure of the experimental set-up is shown in Fig. 1. Insulin was dissolved in0.01 N HCl to obtain a concentration of 100

IU/ml. Eight milliliter of this solution was addedto a 17 ml of alcohol USP contained in a beakerunder stirring by a magnetic stirrer rotating at 400rpm. To this solution, 2 gm of Eudragit L100 wasadded over a period of 10 min. The polymericsolution was allowed to stir for additional 5 minto allow the polymer to dissolve completely. Thesolution was then transferred by a peristalticpump from a fixed height to a beaker containing100 ml cold water (4 °C) with homogenization(Pro 250, ProScientific, MD) at 10000 rpm. Ho-mogenization was continued for an additionalminute after the polymeric solution containing thedrug was completely transferred. The suspendedmicroparticulates were separated from the liquidby filtration under vacuum using a Whatman c4filter paper. The microparticulates were trans-

Fig. 1. Apparatus setup for preparation of microparticulates.


ferred to a porcelain dish and allowed to dryovernight in an oven set at 40 °C. They were thenpassed through sieve c40. Aggregates of mi-croparticulates were milled in a mortar and pestlebefore passing through the sieves. The micropar-ticulates retained were then weighed and trans-ferred to a screw capped scintillation vial forfurther use.

To determine the enteric nature of the mi-croparticulates, 25 mg of microparticulates weresoaked in 10 ml of 0.1 N HCl that was equili-brated at 37 °C in a water bath. Following theimmersion of the microparticulates for 2 h asample was taken and analyzed by the HPLCmethod.

The particle size was determined by laser dif-fraction using a particle sizer (LS230, BeckmanCoulter, Miami, FL). The sizing of the micropar-ticulates was determined in a Micro VolumeModel in mineral spirit. The sample was placeddirectly into the module and distribution wascalculated using the Fraunhofer optical model.Data was collected over 60 s and particle size wasexpressed as volume percent mean diameter.

3.2. Yield of microparticulates and drugencapsulation efficiency

The yield of microparticulates was obtained bydividing the theoretical weight of polymer anddrug used by the weight of the microparticulatesobtained. The encapsulation efficiency was deter-mined as follows. Twenty-five milligrams of mi-croparticulates were added to 5 ml of alcoholUSP contained in a 20 ml glass vial. After themicroparticulates dissolved completely, 5 ml ofphosphate buffer pH 6.8 was added to this solu-tion. From this mixture, 0.5 ml was transferred toa HPLC vial, diluted to 1 ml with phosphatebuffer, and analyzed by the HPLC method.

3.3. Addition of salts in the precipitating medium

Salts like calcium chloride, sodium sulfate andtween 80 were added at a concentration of 0.25%w/v in cold water during the microparticulateformation process.

3.4. Ratio of polymeric solution to �olume ofprecipitating medium

The ratio of precipitating medium (cold water)was tested at polymeric solution to precipitatingmedium ratio of 1:2, 1:4 and 1:6.

3.5. Dissolution Studies

In vitro dissolution studies were performed in adissolution apparatus (Vankel VK 600 Van-dekamp Plus, Vankel, Cary, NC) fitted with a100-ml conversion kit. Phosphate buffer USP pH6.8 was used as the dissolution medium. Thebuffer was prepared by mixing 75% of 0.2 Mtribasic sodium phosphate and 25% of 0.1 N HCl.The pH was adjusted to 6.8 with 2 M NaOH or 2M HCl. The temperature of dissolution mediumwas 37 °C and the rotation speed of the paddleswas set to 50 rpm. Microparticulates (125 mg)equivalent to 50 IU of insulin were filled in a size00 capsule and transferred to the prewarmed dis-solution medium. Samples (2 ml) were withdrawnevery hour upto 6 h and the volume was replacedimmediately by fresh phosphate buffer. Sampleswere analyzed by the HPLC method.

3.6. In �itro dissolution stability in the presenceof enzyme inhibitors

The dissolution set up was the same as aboveexcept for the following modifications. The cap-sule contained microparticulates of insulin withvarious amounts of chicken and duck ovomucoid.The dissolution medium contained 0.5 �M trypsinfor capsules containing CkOVM and 0.1 �M �-chymotrypsin for capsules containing DkOVM.Samples withdrawn (2 ml) were immediatelytreated with cold 1% v/v TFA/pH 6.8 buffer (2ml) to stop the enzymatic activity. The sampleswere maintained at 8 °C in the autosamplerthrough the duration of the analysis.

3.7. Insulin analysis

Insulin analysis was performed by a gradientRP-HPLC method. Compounds were separatedon a C18 Vydac 218MS54 column (4.6×250 mm)


with a pore size of 300 A� and particle size of 5�m. The mobile phase consisted of 0.05% v/vTFA/water (A) and 0.05% v/v TFA/acetonitrile(B). The gradient conditions were 27% B for 4min and 27–36 % B in the next 11 min at a flowrate of 1 ml/min. The wavelength of detection was210 nm. Under these conditions, the retentiontime of insulin was 10.8 min.

4. Results and discussion

4.1. Microencapsulation by coprecipitation

The coprecipitation technique involves dissolv-ing the polymer and drug in an organic solventand then adding a non-solvent to precipitate thedrug and polymer. During the precipitation pro-cess, the polymer particles encapsulate the drug.Coprecipitates have been successfully prepared fordrug polymer combinations of ibuprofen/EudragitS100 (Khan et al., 1995), indomethacin/mixture ofEudragit RS100 and RL100 (Karnachi et al.,1995) and Ketprofen and Eudragit S100 (Khan etal., 1996). In these studies, alcohol was used as asolvent for dissolving polymer and drug. Coldwater was used as a non-solvent for precipitation.The drug to polymer ratio used was as high as10:1 and the particle size obtained was less than800 �m.

The approximate range of intestinal transit of adosage form in small intestine is between 3 and 6h (Davis et al., 1986). The objective of this workwas to evaluate coprecipitation technique forpreparing microparticulates of insulin at low drugconcentration for sustained release over 6 h. Ex-tensive preliminary experiments were done to de-termine the effect of various formulation andprocess factors mentioned earlier. These factorswere evaluated with respect to percentage yield ofmicroparticulates, drug encapsulation efficiencyand dissolution studies.

Our preliminary experiments indicate that in-sulin remains in solution with the polymer in 32%v/v mixtures of 0.01 N HCl and alcohol. How-ever, below 32% v/v 0.01 N HCl, insulin is in theform of suspension in the polymeric solution. Thestirring time of 15 min was fixed after initial

Fig. 2. Representative dissolution profile of insulin microcap-sules (insmc) at pH 6.8.

experiments. It is important that the polymer beadded slowly over a period of 10 min to avoid theformation of clumps. The additional 5 min ofstirring time helps in the preparation of a clearpolymeric solution containing the drug. The mag-netic stirrer was used during mixing to avoidexposing the protein to high shear rates involvedin the homogenizer. The addition of polymericsolution to the cold water was done with the helpof a peristaltic pump to control the rate of addi-tion. Precipitation of polymer microparticulatescould be achieved by stirring with a homogenizeror a magnetic stirrer. The microparticulates ob-tained while precipitating with a magnetic stirrerhad a tendency to agglomerate and stop the stir-rer midway during the precipitation process. Thisproblem could be averted by the use of homoge-nizer. A representative dissolution profile of abatch is shown in Fig. 2. The encapsulation effi-ciency for this batch was 95�2% and volume ofprecipitating medium was 100 ml (ratio of 1:4with respect to the polymer solution). This profilehas been used for to evaluate the effect ofvariables.

Insulin was not detectable in the sample ana-lyzed from the supernatant of microparticulatessoaked in 0.1 N HCl for 2 h at 37 °C. Thisreveals that insulin is not present on the surface ofthe microparticulates and the microparticulatesare enteric in nature.

The particle size distribution of the micropartic-ulates was normal as indicated by a typical plot


Fig. 3. Particle size distribution of microparticulates.

shown in Fig. 3. The average diameter of themicroparticulates was 358.1. The range of particlesize was between 184.6–541.6 �M.

4.2. Microparticulate formation efficiency

The yield of microparticulates obtained wasonly about 80%. The loss may be attributed to thepolymeric solution sticking to the glass beakerused in transferring the solution to the precipitat-ing medium. The encapsulation efficiency deter-mined from an average of three runs was95�2.0%. The chromatogram revealed the for-mation of desamido insulin that eluted at 11.8min. The proportion of the peak area with respectto the insulin peak area was close to 5%. Theproportion of buffer used in alcohol is critical foraccurate determination of encapsulationefficiency.

4.3. Effect of salts in the precipitating medium

The addition of salts and surfactant in theprecipitating medium was done with an intentionto decrease agglomerate formation. In the mi-croparticulate formation, electrolytes such as cal-cium chloride, sodium sulfate and surfactant suchtween 80 were used at 0.25% w/v concentration inthe precipitating medium. Comparison of dissolu-tion profiles is shown in Fig. 4. As shown in thefigure, both calcium chloride and sodium sulfateare slowing the release of insulin to some extent.This may be due to the increased likelihood ofpartial neutralization of the negative charge ofinsulin in phosphate buffer which is at a higherpH (pH 6.8) than the pH for the isoelectric point

of insulin (pH 5.5). Similar to floculation ofcharged particles in the presence of oppositelycharged electrolytes, the fraction of insulin mi-croparticulates with increased diameter and lessoverall surface area may be greater. The decreaseddissolution in the presence of surfactant, tween80, may be due to increased encapsulationefficiency.

4.4. Effect of ratio of precipitating medium withrespect to polymeric solution

The ratios of polymeric drug solution versusvolume of precipitating medium investigated inour laboratory were 1:2, 1:4 and 1:6. The drugencapsulation efficiency was effected by the ratio.The range of encapsulation efficiency varied from91.83 to 103.6%. The ratio of water volume topolymeric drug solution is critical for the forma-

Fig. 4. Effect of salts in the precipitating medium on thedissolution of insulin microcapsules: control (�), sodium sul-fate 0.25% (�), calcium chloride 0.25% (�) and tween 0.25%(�).


Fig. 5. Degradation of insulin solution (50 IU) in the presenceof trypsin and �-chymotrypsin: control (�), trypsin 0.5 �M(�) and chymotrypsin 0.1 �M (�).

trypsin. The degradation of insulin solution ismaximum in the first hour and slow thereafter(Fig. 5). Insulin (%) remaining at the end of 1 h isclose to 45%. Assuming a linear degradation rateof insulin solution in the first hour, this corre-sponds to a rate of degradation of 0.46 IU/min.The cumulative percentage insulin released frommicroparticulates in the first hour in the absenceof trypsin is close to 60% (Fig. 6). Assuming alinear rate of release in the first hour, this corre-sponds to a rate of release of 0.5 IU/min. Therates of degradation and release are comparable,suggesting that insulin is undergoing spontaneousdegradation following its release. Due to this ex-tensive degradation in the first hour itself, insulinconcentration does not accumulate enough to bedetected even at the end of 6 h.

In the presence of chicken ovomucoid, insulin(%) remaining for absorption increases in a con-centration dependent fashion. From Table 1 it canbe seen that insulin (%) available increases from17.75% at 1:2 ratio of enzyme to inhibitor to24.70% at 1:4 ratio. This represents a substantialimprovement in the percentage of insulin avail-able for absorption.

Stability of insulin in the presence of �-chy-motrypsin is extremely poor. During the course ofdegradation of insulin solution for 6 h, insulinremaining is almost reduced to a negligible value(Fig. 5). Rate of degradation of insulin solution in

tion of microparticulates. Microparticulates canstart forming only when the volume of water ismore than a critical ratio with respect to thevolume of polymeric solution. The ratio estab-lished for 8% ethanolic solution of Eudragit L100in water for appearance of turbidity was reportedas 1:1.38 (Kachrimanis et al., 2000).

4.5. In �itro dissolution stability in the presenceof enzymes

In vitro dissolution data was generated for arepresentative batch whose characteristics are dis-cussed in Section 4.1. Stability of insulin solutionin the presence of trypsin and �-chymotrypsin isshown in Fig. 5. Dissolution stability of insulinreleased from microparticulates in the presence of0.5 �M trypsin and various ratios of CkOVM isshown in Fig. 6. The control dissolution profilesin Figs. 5 and 6 represent the amount of insulin inthe absence of trypsin. For both solution andmicroparticulate formulations, the area under thecurve (AUC) for the dissolution profiles werecalculated to compare the percentage of insulinavailable for absorption in the presence of trypsin.Comparison of the ratio of AUC values for in-sulin microparticulates at the end of 6 h is shownin Table 1. At the end of 6 h the percentage ofinsulin available for absorption from micropartic-ulates is negligible. This may be explained by thekinetics of insulin degradation in the presence of

Fig. 6. Dissolution stability of insulin released from microcap-sules in the presence of trypsin and CkOVM: insmc (�),insmc+ tryp:ckovm 1: 2 (�), ins+ tryp:ckovm 1:4 (�), in-smc+ tryp (�).


Table 1Cumulative amount of insulin available at the end of 6 h in thepresence of 0.5 �M trypsin and 0.1 �M chymotrypsin

Formulation Ratio (%)AUC (% h)

Solution (sol)600Sol (no enzymes)261.33Sol+trypsin (T) 43.55

Sol+chymotrypsin 62.24 10.37(CT)

Microparticulates479.36Control (no

enzymes)Beyond detection Beyond detectionMC+Tlimit limit85.08 17.75MC+T+CkOVM

(1:2)MC+T+CkOVM 118.45 24.70

(1:4)

Microparticulates479.36MC (no enzymes)

MC+CT Beyond detection Beyond detectionlimitlimit23.3MC+CT+ 111.71

DkOVM(1: 1)

MC+CT+ 190.81 39.40DkOVM (1:2)

MC+CT+ 202.95 42.3DkOVM (1:4)

modifier. The absorption modifier may be anagent that increases permeability of the proteinunder study or an enzyme inhibitor that improvesstability of the protein in the gastrointestinaltract. An enzyme inhibitor may be required toimprove bioavailability of the protein even if per-meation enhancement is the goal. There are manyreports of bioavailability studies of insulin withenzyme inhibitors in various animal species likerats (McPhillips et al., 1997) and dogs (Ziv et al.,1994). The amounts of enzyme inhibitors usedhave been selected without performing in vitrostability studies in the presence of pancreatic en-zymes. Since variations exist in concentration ofpancreatic enzymes with regards to the speciesunder study, it may be helpful to evaluate thestability before hand. In vitro dissolution stabilitymay serve as an effective screening tool to evalu-ate the effectiveness of various concentrations ofinhibitor in the presence of enzymes. This willallow incorporation of practical amounts of en-zyme inhibitor targeted towards inhibition of pan-creatic enzymes. In vitro dissolution stability of aprotein in the presence of enzymes has not beenreported. Single time point measurements havebeen used in some studies to evaluate the in vitroefficacy of inhibitors against specific enzymes(Morishita et al., 1992b; Bernkop-Schnurch et al.,1997). The parameter used in our study, percent-age of protein available for absorption, may serve

the first hour is 0.75 IU/min while the rate ofdissolution remains the same. Insulin solutionavailable for absorption was calculated to be only10.37% as shown in Table 1. Due to this reason,insulin released from microparticulates is exten-sively degraded and not detectable in the presenceof �-chymotrypsin alone (Fig. 7). The reason maybe same as explained above. In the presence ofDkOVM, there is a considerable improvement instability of insulin released from microparticulatesat the end of 6 h. Cumulative percentage ofinsulin remaining in the presence of various ratiosof �-chymotrypsin and DkOVM is shown in Fig.7. As shown in Table 1, the ratio of insulinremaining for absorption steadily increased from23.3% in the presence of 1:1 concentration ofenzyme to inhibitor to 42.3% at 1:4 ratio.

Oral delivery of proteins will be extremelydifficult without the use of an absorption

Fig. 7. Dissolution stability of insulin released from microcap-sules in the presence of �-chymotrypsin and DkOVM: insmc(�), insmc+ctryp:dkovm 1:1 (�), insmc+ctryp:dkovm 1:2(�), insmc+ctryp:dkovm 1:4 (�), insmc+ctryp (�).


as a better indicator for evaluating the efficacy ofinhibitors because it incorporates the completedissolution profile in the calculation.

5. Conclusions

Microparticulates of insulin have been preparedby the coprecipitation technique with appropriatecombination of factors and choice of polymer.Among the factors studied, the addition of salts inthe precipitating medium and ratio of polymericsolution to volume of precipitating medium hadan effect on the dissolution of insulin and encap-sulation efficiency. Dissolution stability studiesindicates that the percentage of insulin remainingfor absorption increased significantly in the pres-ence of CkOVM and DkOVM. Dissolution stabil-ity studies may serve as valuable indicators forjudicious choice and concentration of the in-hibitor for oral protein dosage forms.

Acknowledgements

Dean Nelson and Dr Smith are acknowledgedfor their support in the establishment of Centerfor Drug Delivery and Formulation Develop-ment. We acknowledge the timely help of Ed Hoffat Beckman Coulter for particle size analysis.

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