In previous studies, it was established that chitosan and its quaternized derivatives are potent enhancers of hydrophilic compounds absorption across intestinal epithelia. The aim of this study was to evaluate the application of a new quaternized chitosan, triethyl chitosan (TEC), in pharmaceutical approaches. TEC was synthesized by a one step process via a 22 factorial design to optimize the preparation conditions. In ex vivo experiments, everted rat colon sac was used to determine the effect of TEC on the penetration of hydrophilic compounds of different molecular masses (e.g., sodium fluorescein and brilliant blue) through colonic epithelia in comparison with chitosan at pH 7.4. These studies indicated a significant increase in absorption of sodium fluorescein and brilliant blue in the presence of TEC compared to chitosan. TEC bearing positive charge is able to interact with the tight junctions of colon epithelia and hence increase the permeation of sodium fluorescein and brilliant blue through the tight junctions. This investigation has shown that triethyl chitosan could be used as a penetration enhancer for poorly absorbable compounds in the colon drug delivery system.U ranijim istraživanjima utrđeno je da su kitozan i njegovi kvartenizirani derivati snažni promotori apsorpcije hidrofilnih spojeva kroz intestinalnu sluznicu. Cilj rada bio je evaluirati novi kvartenizirani kitozan, trietil kitozan (TEC ).TEC je sintetiziran u jednom stupnju. U ex vivo eksperimentima na kolonu štakora praćen je učinak tog polimera na penetraciju hidrofilnih spojeva različitih molekulskih masa (fluorescein natrija i briljant plavila). Rezultati su uspoređivani s učinkom kitozana na pH 7,4. Primjećeno je da TEC značajno povećava apsorpciju ispitivanih tvari u odnosu na nemodificirani kitozan. TEC svojim pozitivnim nabojem dolazi u interakciju s epitelom kolona i tako povećava njegovu permeabilnost. Ispitivanja ukazuju da se trietil kitozan može upotrijebiti kao promotor penetracije za spojeve koji se slabo apsorbiraju u kolonu

AHMAD REZA DEHPOUR

ASSAL MIR MOHAMMAD SADEGHI

KHOSROW BAYATI

KOOROUSH SHAMIMI

LEILA MOEZI ELAHEH NAHID

MOHAMMAD REZA AVADI

MORTEZA RAFIEE-TEHRANI

PARISA YOUNESSI

English

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Oral absorption of protein and peptide drugs is hindered by their high molecularmass, hydrophilicity and susceptibility to the proteolytic enzymes in the gastrointestinaltract (1). Therefore, various approaches, such as absorption enhancers, alternative routesand protease inhibitors, have been examined to overcome the delivery problems of thesepeptides and proteins through the gastrointestinal tract (2).Deacetylation of chitin, the second most abundant biopolymer isolated from insects,crustaceans such as crab and shrimp as well as from fungi, leads to poly-(1,4)-D-glu-cosamine or the so called chitosan (3) with excellent biodegradable and biocompatible339Acta Pharm. 54 (2004) 339–345 Original research paperPreparation and ex vivo evaluation of TEC as anabsorption enhancer for poorly absorbable compoundsin colon specific drug deliveryPARISA YOUNESSI1MOHAMMAD REZA AVADI2,3KOOROUSH SHAMIMI4ASSAL MIR MOHAMMAD SADEGHI3LEILA MOEZI4ELAHEH NAHID1KHOSROW BAYATI1,3AHMAD REZA DEHPOUR4MORTEZA RAFIEE-TEHRANI11 Faculty of Pharmacy, Tehran Universityof Medical Sciences, Tehran, Iran2 Faculty of Pharmacy, Shahid BeheshtiUniversity of Medical Sciences, TehranIran3 Hakim Pharmaceutical Company, TehranIran4 Faculty of Medicine, Tehran Universityof Medical Sciences, Tehran, IranReceived April 28, 2004Accepted November 18, 2004In previous studies, it was established that chitosan andits quaternized derivatives are potent enhancers of hy-drophilic compounds absorption across intestinal epithe-lia. The aim of this study was to evaluate the applicationof a new quaternized chitosan, triethyl chitosan (TEC), inpharmaceutical approaches. TEC was synthesized by aone step process via a 22 factorial design to optimize thepreparation conditions. In ex vivo experiments, evertedrat colon sac was used to determine the effect of TEC onthe penetration of hydrophilic compounds of differentmolecular masses (e.g., sodium fluorescein and brilliantblue) through colonic epithelia in comparison with chi-tosan at pH 7.4. These studies indicated a significant in-crease in absorption of sodium fluorescein and brilliantblue in the presence of TEC compared to chitosan. TECbearing positive charge is able to interact with the tightjunctions of colon epithelia and hence increas the perme-ation of sodium fluorescein and brilliant blue throughthe tight junctions. This investigation has shown that tri-ethyl chitosan could be used as a penetration enhancerfor poorly absorbable compounds in the colon drug de-livery system.Keywords: triethyl chitosan, absorption enhancer, colon* Correspondence, e-mail: rafitehr@ams.ac.ircharacteristics. Because of their permeation enhancing effect, enzyme inhibitory capabi-lities, and mucoadhesive properties, chitosan and its derivatives are important excipi-ents for peroral peptide delivery systems. Chitosan acts by opening tight junctions be-tween epithelial cells to allow the paracellular transport of hydrophilic molecules (4).Moreover, there has recently been an increasing interest in targeting peptide andprotein drugs as well as oligonucleotides and vaccines to the colon because of the rela-tively low activity of proteolytic enzymes and their long residence time in colon. In ad-dition, it is more effective to treat colonic diseases such as ulcerative colitis, colorectalcancer and Crohn’s disease with a direct delivery of drugs to the affected area (2).Chitosan is a polycation with an apparent pKa 5.5 and thus, in neutral and basic en-vironments as found in the large intestine, the chitosan molecules will lose their chargeand precipitate from solution (5).However, it has been shown that N-trimethyl chitosan chloride (TMC), a partiallyquaternized derivative of chitosan with superior solubility (6) could be a useful polymerfor the drug delivery system in the large intestine and colon, where a neutral and basicenvironment prevails (7).Different alkylated chitosans have been reported (8, 9). One of the relatively newderivatives of chitosan is N-triethylated chitosan (TEC), which has not yet been intro-duced into pharmaceutical approaches. In this investigation, TEC was synthesized andevaluated as a potential absorption enhancer of hydrophilic compounds including bril-liant blue and sodium fluorescein. The present study is continuation of our in vitro andex vivo studies with different alkylated chitosans as absorption enhancers.EXPERIMENTALMaterialsChitosan (98% deacetylated, viscosity of 1% m/V solution is 264 mPa s) was a giftfrom Primex (Iceland). Ethyl iodide was obtained from Sigma (Austria). Sodium hydrox-ide, N-methyl pyrrolidone (NMP), sodium iodide, sodium fluorescein (Mr 332) and bril-liant blue (Mr 792) were purchased from Merck (Germany). All chemicals and solventswere of pharmaceutical and analytical grade and were used as received.MethodsSynthesis and identification of TEC. – TEC was synthesized as previously described (9).Briefly, chitosan was dispersed in 8 mL of N-methyl pyrrolidone and stirred with a mag-netic stirrer for 4 hours at room temperature. Then, aqueous sodium hydroxide (20%, m/V)solution (1.2 mL), sodium iodide (480 mg) and 3 mL ethyl iodide were added. The mixturewas heated at 60 °C for 6 hours under stirring. The product chitosan–N+(CH2CH3)3I–was precipitated with acetone and separated by centrifugation. To exchange I– with Cl–,the polymer was dissolved in 4 mL of 10% aqueous sodium chloride solution. The polymerwas precipitated with acetone, centrifuged and dried to obtain a white water-solublepowder in quantitative yield.340P. Younessi et al.: Preparation and ex vivo evaluation of TEC as an absorption enhancer for poorly absorbable compounds in colonspecific drug delivery, Acta Pharm. 54 (2004) 339–345.The 1H NMR spectrum was recorded in D2O using a 500 MHz spectrometer (Bru-cker AC500, USA) and the degree of quaternization was calculated. The chemical shift(, ppm) was adjusted with external standard (dioxane in D2O).Ex vivo evaluation. – According to the requirement of the local Ethical Committee,healthy male Wistar rats weighing 200–250 g (three groups of animals, 3 animals in eachgroup), were anesthetized with ether. Animals were kept under 12-h light/dark con-ditions at 22 ± 1 °C, and fasted for about 12 h prior to the experiment. After verificationof the loss of the pain reflex, a midline incision was made and a segment of large intes-tine (about 3.5 cm) was quickly removed and washed gently with Krebs Ringer Bicar-bonate (KRB) solution sodium chloride 118 mmol L–1, sodium hydrogencarbonate 25mmol L–1, potassium dihydrogenphosphate 1.19 mmol L–1, calcium chloride 1.03 mmolL–1, magnesium sulfate 1.2 mmol L–1, glucose 5 mmol L–1and albumin 1% pH = 7.4) andeverted by means of a plastic rod inserted through the lumen. One end of the colon wastied to a needle and the opposite end was tied securely (10, 11). Then, 1 mol sodiumfluorescein or brilliant blue was added to 0.5% TEC in 100 mL KRB, the sac was placedinto the solution and saturated with O2 and CO2 maintained at 37 °C. Control solutionswere prepared without the dissolved TEC polymer. One mL of KRB solution was in-serted with a needle to the inside compartment of the sac (12, 13).Serial samples of an known volume were taken from the inside compartment bymeans of an insulin syringe every 10 minutes for 1.5 or 2 hours and each time, the vol-ume was replaced with fresh solution (12). The absorbed amounts of sodium fluoresceinand brilliant blue were detected by a spectrophotometer NovospecII (UK) at 494 and 634nm, respectively.Statistical analysisThe data are expressed as the mean ± SD. For statistical analysis, the two-way ana-lysis of variance was used and followed by Tukey’s HSD as post-hoc test to compare themeans.RESULTS AND DISCUSSIONA one step synthesis of TEC was carried out (9). The synthesis was based on nu-cleophilic substitution of chitosan’s amine protons with ethyl groups of an ethyl iodidein the presence of sodium iodide and sodium hydroxide in water/NMP medium.It was shown that a strong inorganic base, such as NaOH, was more suitable thanorganic bases (such as amines with similar alkalinity to that of chitosan) to neutralizethe acidity of hydrophilic acid produced by the reaction. Studies have shown that so-dium hydroxide and ethyl iodide are the two most effective reaction variables in thesynthesis of triethyl chitosan (10). Table I shows four reaction conditions in triplicate ba-sed on the experimental design. Optimum amounts of the reactants were found to be37.5 mmol of ethyl iodide (3.0 mL) and 6 mmol NaOH (1.2 mL, 20%) (formulation A4,Table I), as the highest degree of quaternization (66%) was achieved.341P. Younessi et al.: Preparation and ex vivo evaluation of TEC as an absorption enhancer for poorly absorbable compounds in colonspecific drug delivery, Acta Pharm. 54 (2004) 339–345.The 1H NMR spectrum of TEC is shown in Fig. 1. The signal at 1.2 ppm was attri-buted to the CH3 groups of the ethyl substituent, while H2–H6 protons of the polysac-charide backbone superimposed the CH2 groups. The intense band at 4.8 ppm was re-lated to HDO (a proton-exchanged species of D2O with OH and amine). In this region,as observed more clearly from an extended spectrum, three anomeric protons (H1S) ap-peared at 4.23, 4.98 and 5.1 ppm. They were attributed to the mono-N-acetylglucosamineunit, mono and di-N-alkyl substitued glucosamine units, respectively. The integral ofthese signals as well as the CH3 of ethyl groups were used to calculate the degree of qua-ternization (11).A model rat cannulated everted intestine could eliminate the possible variables dueto blood flow and permit serial sampling from the serosal contents (13). Moreover, theeverted intestinal sac has the advantage of simplicity and flexibility (12). Barthe et al.(14) noted that the values explained in caco-2 cell monolayers, which is one of the estab-lished in vitro models for intestinal epithelia, might exaggerate the potential of polymersfor realistic absorption enhancement. The everted intestinal sac model may give morerealistic values for intestinal function (15).Using inverted sacs for absorption studies an O2 and CO2 mixture was bubbled intointestinal mucosa to increase or obtain peristaltic intestinal movement.342P. Younessi et al.: Preparation and ex vivo evaluation of TEC as an absorption enhancer for poorly absorbable compounds in colonspecific drug delivery, Acta Pharm. 54 (2004) 339–345.Table I. The effect of reaction conditions on the degree of quaternizationFormulation NaOH (%, m/m)/C2H5I (mL) Degree of quaternization (%)A1 20.0/1.5 60%A2 10.0/1.5 36%A3 10.0/3.0 46%A4 20.0/3.0 66%Fig. 1. 1H NMR spectrum oftriethyl chitosan chloride (de-gree of quaternization 66%) inD2O.In the present study we have shown that TEC, which has better solubility than chi-tosan and does not need an acidic environment for its activity, has significantly increa-sed the intestinal absorption of both sodium fluorescein and brilliant blue compared tochitosan (Figs. 2a and 2b). TEC, with the involvement of actin filaments of the intestinalepithelium, was able to increase the permeation of the hydrophilic compounds throughthe tight junctions. It has been reported that chitosan induces a redistribution of cyto-skeletal F-actin and the ZO-1 protein, thereby altering the integrity of tight junctions(16). Confocal laser scanning microscopy has also confirmed that chitosan and TMC areable to open the tight junctions to allow the paracellular transport of large hydrophiliccompounds (17, 18).In addition, TEC effectiveness as permeation enhancer is more pronounced in thecase of fluorescein compared to brilliant blue probably due to the lower molecular massof fluorescein (19).CONCLUSIONSIt has been shown that TEC with superior water solubility could be a potential ab-sorption enhancer of hydrophilic compounds, including sodium fluorescein and bril-liant blue, in colon and is promising for colon-specific drug delivery.It has been also concluded that the potential use of TEC can be an important contri-bution to the development of effective delivery systems for hydrophilic compounds suchas peptide drugs, especially in the more neutral and basic environment of the large in-testine and colon where the efficacy of chitosan as an absorption enhancer is not so strong.However, more in vitro studies in caco-2 cell and in vivo experiments are required to con-firm TEC as an enhancing agent.Acknowledgements. – We are grateful to Mrs Partoazar and Mr. Assadi (managing director ofHakim Pharmaceutical Co.) and for the technical assistance of Dr. Shafaroodi.343P. Younessi et al.: Preparation and ex vivo evaluation of TEC as an absorption enhancer for poorly absorbable compounds in colonspecific drug delivery, Acta Pharm. 54 (2004) 339–345.0510152025303540450 20 40 60 80 100 120 140Time (min)Sodiumfluorescein(µg)Fig. 2. a) Sodium fluorescein alone (), added chitosan (), added TEC () (n = 3); b) brilliantblue alone (), added chitosan () added TEC (n = 3). Bars denote SD values.0102030405060700 20 40 60 80 100Time (min)Brilliantblue(µg)REFERENCES1. R. Hejazi and M. Amiji, Chitosan-based gastrointestinal delivery systems, J. Control. Rel. 89 (2003)151–165.2. H. Tazoki, J. Komoiko, C. Toada, T. Maruyama, A. Terabe, T. Suzuki, A. Yamamoto and S. Mu-ranishi, Chitosan capsules for colon-specific drug delivery: improvement of insulin absorptionfrom the rat colon, J. Pharm. Sci. 86 (1997) 1016–1021.3. W. Paul and C. P. Sharma, Chitosan, a drug carrier for 21st century: a review, S.T.P. Pharma Sci.10 (2000) 5–22.4. H. L. Luessen, C. O. Rentel, A. F. 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Pol. 9 (2004) 421–433.S A @ E T A KPriprava i ex vivo evaluacija TEC kao promotora apsorpcije tvari u koloniPARISA YOUNESSI, MOHAMMAD REZA AVADI, KOOROUSH SHAMIMI, ASSAL MIR MOHAMMAD SADEGHI, LEILAMOEZI, ELAHEH NAHID, KHOSROW BAYATI, AHMAD REZA DEHPOUR i MORTEZA RAFIEE-TEHRANIU ranijim istra`ivanjima utvr|eno je da su kitozan i njegovi kvaternizirani derivatisna`ni promotori apsorpcije hidrofilnih spojeva kroz intestinalnu sluznicu. Cilj rada bioje evaluirati novi kvaternizirani kitozan, trietilkitozan (TEC). TEC je sintetiziran u jed-nom stupnju. U ex vivo eksperimentima na kolonu {takora pra}en je u~inak tog polimerana penetraciju hidrofilnih spojeva razli~itih molekulskih masa (fluorescein natrija i bri-ljant plavila). Rezultati su uspore|ivani s u~inkom kitozana pri pH 7,4. Primije}eno je daTEC zna~ajno pove}ava apsorpciju ispitivanih tvari u odnosu na nemodificirani kitozan.TEC svojim pozitivnim nabojem dolazi u interakciju s epitelom kolona i tako pove}avanjegovu permeabilnost. Ispitivanja ukazuju da se trietilkitozan mo`e upotrijebiti kao pro-motor penetracije za spojeve koji se slabo apsorbiraju u kolonu.Klju~ne rije~i: trietilkitozan, promotor apsorpcije, kolonFaculty of Pharmacy, Tehran University of Medical Sciences, Tehran, IranFaculty of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, IranHakim Pharmaceutical Company, Tehran, IranFaculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran345P. Younessi et al.: Preparation and ex vivo evaluation of TEC as an absorption enhancer for poorly absorbable compounds in colonspecific drug delivery, Acta Pharm. 54 (2004) 339–345.

Priprava i ex vivo evaluacija TEC kao promotora apsorpcije tvari u kolonu

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Priprava i ex vivo evaluacija TEC kao promotora apsorpcije tvari u kolonu

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