Oral delivery of insulin-loaded nanoparticles in diabetic rabbits and in sheep

Transcription

1 Czech J. Anim. Sci., 59, 2014 (6): Originl Pper Orl delivery of insulin-loded nnoprticles in dietic rits nd in sheep S. Oly 1, M. Khorvsh 1, H.R. Rhmni 1, S. Esmeilkhnin 2, B. Oly 3, H. Sdri 4,5 1 Deprtment of Animl Science, Isfhn University of Technology, Isfhn, Irn 2 Isfhn Reserch Center for Agriculture nd Nturl Resources, Isfhn, Irn 3 Deprtment of Physiology, Isfhn University of Medicl Sciences, Isfhn, Irn 4 Institute of Animl Science, Physiology nd Hygiene Unit, University of Bonn, Bonn, Germny 5 Deprtment of Clinicl Science, Fculty of Veterinry Medicine, University of Triz, Triz, Irn ABSTRACT: The ojective of this study ws to produce Eudrgit RS 100 microprticles (ERS) contining insulin (ILNP), nd to evlute the potentil of these nnoprticles s drug crrier for orl dministrtion in lloxn-induced dietic rits nd in sheep. After orl dministrtion of ILNP to dietic rits, significnt hypoglycemic effect ws oserved nd this effect lsted for 2 dys. Concentrtions of lood glucose were significntly decresed in ILNP-treted sheep compred to those in control nd ERS groups on dy 5 of the experiment. Orlly ILNP-treted sheep exhiited higher concentrtions of progesterone compred to control nd ERS in ll smpling times. Sheep in the ILNP group hd lower concentrtions of cortisol thn the nimls in the control group on dys 1 nd 3. However, other lood hormone prmeters were not ffected y the tretments. The results show tht encpsultion of insulin into nnoprticles llows the preservtion of its iologicl ctivity when going through digestive system of rit nd sheep to the lood strem. Keywords: dietes; insulin encpsultion; drug delivery; rumen-ypss; glycemi INTRODUCTION Dietes mellitus is reltively common disese in which the ody does not produce enough insulin, produces no insulin, or hs cells tht do not respond properly to the insulin. Even though, there re severl wys the ptients cn get the necessry insulin their ody needs to function properly, needles nd syringes remin the most common wy to tke insulin. However, injection hs mny disdvntges, including eing pinful nd inconvenient if the ptient hs to use it in pulic plce, s well s dosge errors cn occur esily. Experts in this re re trying to find other wys of how to deliver insulin (Bu et l., 2008; Chturvedi et l., 2013). In this regrd, the esiest wy to control lood sugr for ptients would e n effective orl insulin mediction. A possile wy to chieve this would e protection of insulin from the stomch nd elimintion of intestines when going through the digestive system to the lood strem (Cournrie et l., 2002; Rekh nd Shrm, 2009; Mundrgi et l., 2011, ). During lte gesttion nd in erly lcttion, mternl insulin sensitivity is reduced to ensure tht n dequte supply of nutrients is provided to the foetus nd to mmmry tissues (McDowell et l., 1987; Sno et l., 1993). Therefore, the periprturient period in cttle is chrcterized y incresed concentrtions of lood glucgon, growth hormone, non-esterified ftty cid nd et hydroxyutyrte, nd decresed concentrtions of lood insulin nd liver glycogen (Vzquez-Anon et l., 1994). These chnges re lso oserved in induced or spontneous heptic lipidosis nd ketosis. In ddition to elimintion of negtive energy lnce, sucutneous or intrvenous injection of insulin with concomitnt infusion of dextrose hs een suggested for treting heptic lipidosis to mintin helth nd improve performnce (Fleming, Supported y the Isfhn University of Technology, Irn. 251

2 Originl Pper Czech J. Anim. Sci., 59, 2014 (6): ). In ddition, Hyirli et l. (2002) concluded tht low dose of slow-relese insulin (0.14 IU/kg of ody weight) could e considered for prophylctic use ginst heptic lipidosis nd ketosis. However, to our knowledge, no dvnced rumen ypss delivery systems for insulin hve een investigted until now, sed on the physiologicl fctors in the rumen such s ph, microflor, nd ruminl fermenttion. Nnomterils such s nnoprticles, nnocpsules, micelles, micro/nnoemulsions, liposomes, polymersomes, nd nnoporous mterils re used for drug delivery systems (Fn et l., 2006; Akhlesh et l., 2008). It hs een shown tht microencpsultion of smll peptides in polymeric system is useful delivery system for the orl dministrtion in humn. It is le to protect the peptide ginst chemicl nd/or enzymtic degrdtion, nd my enle the retention of therpeutic properties following orl dministrtion (Trpni et l., 2007). Eudrgit RS 100 (ERS), which is used s coting mteril in microencpsultion, is insolule t physiologicl ph ut undergoes swelling in queous medium (Pigntello et l., 2002). This copolymer is commonly used for producing enteric coted tlets nd preprtion of controlled-relese drug forms (Trpni et l., 2007; Mundrgi et l., 2011). To our knowledge, no dvnced rumen ypss delivery systems for insulin hve een investigted in ruminnts. Therefore, the im of this study ws to encpsulte insulin in ERS microprticles, nd to evlute this insulin delivery system for orl dministrtion in dietic rits nd in sheep nd its effects on selected lood prmeters. In ddition, the in vivo rumen ypss properties of the encpsulted insulin were evluted in fistulted rm. MATERIAL AND METHODS Mterils. Commercilly ville regulr humn insulin ws otined from Exir Phrmceuticl Co. (Tehrn, Irn). The ERS ws kindly provided y Evonik Röhm GmH (Dmstdt, Germny). Polyvinyl lcohol (PVA, 87 hydrolyzed, MW: ) ws otined from Sigm-Aldrich (St. Louis, USA). Preprtion of nnoprticles. The preprtion of nnoprticles ws crried out y the multiple emulsion technique previously descried y Hoffrt et l. (2002). Briefly, 1 ml of n queous solution of insulin (1000 IU) ws first emulsified y sonifiction (Misonix Sonictor, model XL ) (Misonix, Inc., Frmingdle, USA) for 30 s in methylene chloride (10 ml) contining 250 mg of polymer (ERS). The resulting wter-in-oil emulsion ws therefter poured into 40 ml of polyvinyl lcohol queous solution (0.1%) nd sonicted for 1 min, involving the formtion of the second wter-in-oilin-wter emulsion. After evportion of methylene chloride under reduced pressure, the nnoprticles were isolted y centrifugtion ( g, 20 min). Chrcteriztion of nnoprticles. The men dimeter of nnoprticles ws determined y tomic force microscopy (Brucker Nno GmH, Berlin, Germny). The mount of insulin entrpped within polymeric nnoprticles ws determined y high performnce liquid chromtogrphy (HPLC) (Jsco 807-IT; JASCO, Tokyo, Jpn) (Moslemi et l., 2003). Insulin ssocition efficiency. The ssocition efficiency of insulin ws determined indirectly. The mount of insulin entrpped into nnoprticles ws clculted y the difference etween the totl mount used to prepre the systems nd the mount of insulin tht remined in the superntnt. Insulin concentrtion in the superntnt ws determined y HPLC. AE = Totl mount of insulin Free insulin in superntnt 100 Totl mount of insulin In vivo studies of insulin-loded nnoprticles (ILNP) in dietic rits. Five dult mle white rits weighing 2 kg on verge were used. Animls were housed in stinless steel cging with slotted floors ( cm), with controlled environmentl conditions of temperture (20 ± 2 C), humidity (45 65%), t 12 : 12 light-drkness cycle. Cge pns were clened three times per week. The rits were fed stndrd diet nd provided tp wter d liitum. In order to exclude effects in the nimls y differences of feed intke, mounts of feed were given ccording to mounts took y nimls hving lesser intke during one week efore the experiment. The niml group hving lesser intke ws used s control (CTR, n = 2) nd nimls treted with ILNP (n = 3) were pir-fed. Animl protocols were in complince with the ccepted stndrds of niml cre nd were pproved y the Isfhn University of Technology Animl Cre nd Use Committee. Blood glucose levels were mesured in overnightfsted rits using glucometer (Accu-chek Sensor; Roche Dignostics, Mnnheim, Germny) during one week efore the experiment. It ws estlished tht they hd norml lood glucose level (122.7 ± 13.4 mg/dl) t the strt of the experiment. 252

3 Czech J. Anim. Sci., 59, 2014 (6): Originl Pper Dietes in rits ws induced y single injection of lloxn monohydrte into mrginl er vein t dose of 150 mg/kg ody weight. After one week, overnight-fsted rits hd fsted lood glucose greter thn 210 mg/dl nd were considered s dietic. In order to disply the iologicl efficcy of ILNP fter orl dministrtion, single dministrtion of 20 mg ILNP (i.e. 80 times more thn lood insulin levels) ws given intrgstriclly y gvge needle to overnight-fsted (with free ccess to wter) dietic rits. The glycemi of the experimentl rits ws mesured in lood smpled from the til vein prior to nd 24 h fter dministrtion of nnoprticles. In vivo studies of ILNP in sheep. Fifteen Irnin Neini ewes (ody weight 28 kg) were used. Animls were llocted in three groups per five ewes. One group without tretment ws control (CTR), two tretment groups were fed with one single dose of 1 g ILNP or ERS for 5 dys, respectively. During the experimentl period, ll ewes hd free ccess to wter; however, the mounts of feed (lflf hy) were given ccording to mounts took y nimls hving lesser intke during dpttion period (one week efore the experiment). The niml group hving lesser intke ws used s CTR. Both the CTR nd treted nimls were pir-fed, in order to rule out the nutrition-relted effects on lood prmeters, which would llow conclusions to e drwn regrding the efficcy of the ILNP. Blood glucose levels were mesured using glucometer (Accu-chek Sensor; Roche Dignostics, Mnnheim, Germny) on dy 5. In ddition, lood ws smpled from the jugulr vein using vcutiner tues t the eginning of the experiment (dy 1), nd on dys 3 nd 5. All tues were immeditely plced in wet ice nd within 1 h centrifuged t 1850 g for 20 min. The plsm ws otined nd frozen ( 20 C) until nlyses. Plsm concentrtions of estrdiol (kit IS ), progesterone (kit IS ), nd cortisol (kit SO ) were mesured using chemiluminescent immunossy method y LIAISON nlyzer (DiSorin, Sluggi, Itly). Concentrtions of insulin nd insulin-like growth fctor I (IGFI) were mesured using rdioimmunossy with commercil kits (Dignostic System Lortories, Wester, USA) nd n utomtic gmm counter (BioSource Interntionl, Cmrillo, USA). Rumen-ypss property of ILNP in sheep. The nylon g technique ws used to evlute the ypss of ILNP from digestion in rm. The 1.5 g of ILNP ws plced into 4 nylon gs. The nylon gs were incuted in the rumen for 24 h. After the removl of the gs from the rumen, the gs were wshed in cold wter until rinses were cler nd dried t 50 C for 24 h. The residul mount of ILNP retined in the nylon g ws determined y weight chnges of the incuted smples. This experiment ws repeted two times. Sttisticl nlysis. The dt were nlyzed using the MIXED procedure of SAS (Sttisticl Anlysis System, Version 9.1, 2001), using repeted mesures with first-order utoregressive covrince structure in time. The model included tretment, time, nd interction of tretment nd time s fixed effects. Animl ws used s the repeted suject. Results re presented s the mens nd the ccompnying stndrd error of the men. Significnce ws declred t P < RESULTS Structure nd chrcteriztion of nnoprticles. The ILNP showed homogeneous size distriution with men dimeter of 450 nm ( nm; Figure 1) nd high encpsultion efficiency (85%). Effects of orl dministrtion of ILNP on glycemi in dietic rits. As illustrted in Figure 2, ILNP dministered orlly y gvge in dietic overnight fsted rits significntly reduced glycemi y 40 units nd this effect mintined for 2 dys. After orl dministrtion, no differences were in plsm concentrtions of glucose etween the groups. Effects of orl dministrtion of ILNP on lood prmeters in sheep. As shown in Figure 3, lood glucose ws significntly (P < 0.05) decresed in ILNP-treted group compred to CTR nd Figure 1. Atomic force microscopy imge of insulin-loded nnoprticles (ILNP) ILNP show homogeneous size distriution with men dimeter of 450 nm ( nm) 253

4 Originl Pper Czech J. Anim. Sci., 59, 2014 (6): Blood glucose (mg/dl) c * CTR ILNP c Blood glucose (mg/dl) Pre-tretment Post-tretment CTR ERS ILNP Tretments Figure 2. Blood concentrtions of glucose (mens ± SEM, mg/dl) fter orl dministrtion of insulin-loded nnoprticles (ILNP) in dietic overnight fsted rits c differences (P < 0.05) etween pre- nd post-tretment (24 h) periods *significnt (P < 0.05) difference etween control (CTR) nd ILNP during pre-tretment period ERS groups on dy 5. In ll smpling times, lood concentrtions of insulin, IGFI, estrdiol, nd progesterone were not ffected y the tretments (Tle 1). However, lood concentrtions of progesterone were higher in ILNP group compred with CTR nd ERS groups in ll smpling times. Animls in the ILNP group hd lower (P < 0.05) concentrtions of cortisol thn did nimls in the CTR group on dys 1 nd 3 (Figure 4). Figure 3. Blood concentrtions of glucose (mens ± SEM, mg/dl) fter orl dministrtion of insulin-loded nnoprticles (ILNP), control (CTR), nd Eudrgit RS 100 microprticles (ERS) in sheep, differences (P < 0.05) etween tretment groups In ddition, the mount of ILNP plced in the nylon gs remined unchnged fter ruminl incution. DISCUSSION Eudrgit RS 100 is copolymer (ethyl crylte, methyl methcrylte, nd low content of methcrylic cid ester with quternry mmonium groups). The mmonium groups mke the polymers permele. Tle 1. Blood concentrtions of selected hormones (mens ± SEM) fter orl dministrtion of insulin-loded nnoprticles (ILNP), control (CTR), nd Eudrgit RS 100 microprticles (ERS) in sheep on dys 1, 3, nd 5 of the experiment Trit nd group Smpling time dy 1 dy 3 dy 5 Insulin (μiu/ml) CTR ± ± ± 17.1 ERS ± ± ± 15.6 ILNP ± ± ± 11.3 IGFI (ng/ml) CTR 62.6 ± ± ± 8.96 ERS 66.0 ± ± ± 9.49 ILNP 68.0 ± ± ± 4.91 Estrdiol (ng/dl) CTR 0.24 ± ± ± 0.03 ERS 0.23 ± ± ± 0.03 ILNP 0.19 ± ± ± 0.01 Progesterone (ng/dl) CTR 1.90 ± ± ± 0.58 ERS 1.72 ± ± ± 0.69 ILNP 2.82 ± ± ± 0.77 IGFI = insulin-like growth fctor I 254

5 Czech J. Anim. Sci., 59, 2014 (6): Originl Pper Blood cortisol (μg/dl) CTR ERS ILNP This polymer, which swells in queous medium, is commonly used for the enteric coting of tlets nd the preprtion of controlled-relese drug forms (Trpni et l., 2007; Mundrgi et l., 2011). The men dimeters confirmed tht the ILNP produced in the current study were nnoprticles in the rnge of nm. The first experiment of this study shows tht encpsultion of insulin into nnoprticles llows the preservtion of its iologicl ctivity when going through digestive system to the lood strem, s reflected y 40 units decreses in lood glucose levels in the dietic rit model. Considerle point in this reserch is tht of using insulin ssocited to nnoprticles t much lower dosge (lmost 2500 times) thn tht in the previous studies (e.g. Dmge et l., 2007). Tking dvntge of this first experiment, it ws decided to study whether the ILNP re resistnt to the rumen fluid, nd the delivery of the ILNP t the low ph of the omsum to intestinl sorption. The unchnged mount of ILNP in the nylon gs fter ruminl incution longside with significnt decrese in lood glucose using the ILNP clerly show tht the polymer hs efficiently protected the insulin from proteolytic degrdtion in the gstroenteric trct of sheep. The decresed levels of lood glucose occurred without chnges in lood insulin concentrtions mong the tretments. It seems tht orl dministrtion of the ILNP my hve exerted negtive-feedck effect on the c Dy 1 Dy 3 Dy 5 Smpling time Figure 4. Blood concentrtions of cortisol (mens ± SEM, μg/dl) fter orl dministrtion of insulin-loded nnoprticles (ILNP), control (CTR), nd Eudrgit RS 100 microprticles (ERS) in sheep on dys 1, 3, nd 5 of the experiment c differences (P < 0.05) etween tretment groups pncres nd reduced endogenous insulin relese, resulting in comprle concentrtions of insulin in treted (ILNP) nd CTR groups. An interesting finding of the present study ws the decresed lood cortisol in the treted nimls with the ILNP compred to tht in the CTR group. The mechnisms y which ILNP decresed lood cortisol remin uncler. However, it is known tht cortisol stimultes gluconeogenesis (formtion, in the liver, of glucose from certin mino cids, glycerol, lctte nd/or propionte) nd therey increses lood glucose concentrtions. Therefore, we ssume tht decresed lood glucose concentrtion in nimls treted with ILNP compred to the CTR group t lest in prt hs een ssocited with decresed circulting cortisol levels. Decreses in steroid clernce y decresing heptic monooxygenses cytochrome P450 2C (CYP2C) nd cytochrome P450 3A (CYP3A) ctivities vi insulin signlling hs een proposed. Lemley et l. (2009) reported dose-dependent decrese in the ctivities of CYP2C nd CYP3A fter chllenging heptocytes with incresing physiologicl concentrtions of insulin. In the present study, lood concentrtions of progesterone were higher in ILNP compred to CTR nd ERS in ll smpling times. It seems thus tht numericlly higher concentrtions of progesterone in the ewes treted with ILNP my e, in prt, ffected y decresed CYP2C nd CYP3A ctivities. A portion of the erly emryonic nd fetl loss my e ccounted for y indequte concentrtions of progesterone due to incresed rtes of steroid inctivtion, deficiencies in lutel function, or comintion of oth (Inskeep nd Diley, 2005). Therefore, the effect of ILNP on heptic CYP2C nd CYP3A ctivities s well s the resulting iologicl hlf-life of progesterone merits more investigtion. CONCLUSION In conclusion, the present results show tht encpsultion of insulin into nnoprticles llows the preservtion of its iologicl ctivity when going through digestive system of rit nd sheep to the lood strem. The effects of ILNP to prevent heptic lipidosis nd ketosis especilly during the first month postprtum in high producing nimls merit more investigtion in future studies. Acknowledgement. The uthors would like to express their pprecition to Evonik Röhm GmH (Dmstdt, Germny) for kind providing Eudrgit RS

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