Journal of Drug Targeting
`
`ISSN: 1061-186X (Print) 1029-2330 (Online) Journal homepage: http://www.tandfonline.com/loi/idrt20
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`Absorption Enhancing Effect of Labrasol on the
`Intestinal Absorption of Insulin in Rats
`
`Sudarat Eaimtrakarn, Y.V. Rama Prasad, Tomoya Ohno, Takahiro Konishi,
`Yukako Yoshikawa, Nobuhito Shibata & Kanji Takada
`
`To cite this article: Sudarat Eaimtrakarn, Y.V. Rama Prasad, Tomoya Ohno, Takahiro Konishi,
`Yukako Yoshikawa, Nobuhito Shibata & Kanji Takada (2002) Absorption Enhancing Effect of
`Labrasol on the Intestinal Absorption of Insulin in Rats, Journal of Drug Targeting, 10:3, 255-260,
`DOI: 10.1080/10611860290022688
`To link to this article: https://doi.org/10.1080/10611860290022688
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`Published online: 30 Sep 2008.
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`Journal of Drug Targeting, 2002 Vol. 10 (3), pp. 255–260
`
`Absorption Enhancing Effect of Labrasol on the Intestinal
`Absorption of Insulin in Rats
`
`SUDARAT EAIMTRAKARN, Y.V. RAMA PRASAD, TOMOYA OHNO, TAKAHIRO KONISHI, YUKAKO YOSHIKAWA,
`NOBUHITO SHIBATA and KANJI TAKADA*
`
`Department of Pharmacokinetics, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8414, Japan
`
`(Received 22 May 2001; Revised 4 October 2001)
`
`The oral absorption enhancing effect of Labrasole has been studied in rats using insulin as a model
`peptide/protein drug. Insulin solution was prepared by dissolving insulin in pH 7.4 buffer followed by
`the addition of Labrasol. The insulin concentration was 50.0 IU/ml. The test insulin/Labrasol solution
`was administered to the jejunum, ileum and ascending colon of rats at 10.0 IU/kg. After administration,
`blood samples were collected for 5 h and serum glucose levels and insulin levels were measured. In
`another group of rats, insulin solution was injected intravenously at 1.0 IU/kg, and both serum glucose
`and insulin levels were measured. The pharmacological availability of insulin from Labrasol solution
`was found to be 3.9, 8.9 and 9.1% following jejunal, ileal and colonic administrations, respectively, by
`comparing the serum glucose level vs. time profiles obtained after intestinal and i.v. administrations. By
`comparing the serum insulin levels vs. time profiles, the bioavailability of insulin was found to be 0.25
`and 0.20% for intra-ileum and colonic administrations, respectively. The hypoglycemic effect of insulin
`after intra-ileum administration showed a dose-dependency in the insulin dose range from 10.0 to
`1.0 IU/kg. These results suggest the absorption enhancing effect of Labrasol on the intestinal absorption
`of insulin in rats.
`
`Keywords: Enhancement; Insulin; Intestinal absorption; Labrasol; Rat
`
`INTRODUCTION
`
`To obtain the pharmacological activity of peptide/protein
`drugs after oral administration,
`two barriers,
`i.e.
`hydrolysis in the gastrointestinal (GI) tract and low
`membrane permeability, must be conquered. Oral
`administration of protein drugs results in destruction of
`biological activity of the protein drugs, which explains
`why typical oral bioavailabilities (BA) of such drugs are
`usually less than 1% (Pauletti et al., 1996). For insulin, the
`oral BA is 0.6% (Scot-Moncrieff et al., 1994). To develop
`an oral protein delivery system, many studies have been
`performed including the use of protease inhibitors
`(Morishita et al., 1992; Yamamoto et al., 1994) and
`absorption enhancers (Morishita et al., 1993; Amino et al.,
`1998; Utoguchi et al., 1998). On the other hand, Takeuchi
`et al. (1996) designed a bioadhesive polymer modified
`liposomal preparation to prevent the hydrolysis of insulin
`by the proteolytic enzymes. Tozaki et al. (1997) used a
`Chitosan capsule to deliver and absorb insulin from the
`colon where the hydrolytic enzyme activity is lower than
`
`In addition, water-in-oil-in-water
`intestine.
`the small
`(W/O/W) double emulsion systems were shown to be
`effective dosage forms for the enteral delivery of insulin,
`because they can protect
`insulin against proteolysis
`(Matsuzawa et al., 1995; Cunha et al., 1997; Morishita
`et al., 1998; Suzuki et al., 1998). However, usefulness is
`limited in developing an oral insulin preparation because
`of
`insufficient pharmacological availability (PA). To
`increase the PA of insulin, Onuki et al. (2000) found out a
`highly purified docosahexaenoic acid for
`the rectal
`absorption of
`insulin, where high PA, 25.4%, was
`achieved.
`We have been studying a self-microemulsifying drug
`delivery system (SMEDDS) for the improvement of oral
`BA of extremely hydrophobic drugs such as cyclosporine
`(Takada et al., 1988; 1989). SMEDDS is a useful
`technology to increase the BA of hydrophobic drugs and
`soft capsule preparations using this technology are now
`available commercially for
`the treatment of
`renal
`transplant and AIDS patients (Charman, 2000). On the
`other hand, we gave a new focus to SMEDDS, especially
`
`*Corresponding author. Tel.: +81-075-595-4625. Fax: +85-075-595-4751. E-mail: takada@mb.kyoto-phu.ac.jp
`
`ISSN 1061-186X print/ISSN 1029-2330 online q 2002 Taylor & Francis Ltd
`DOI: 10.1080/10611860290022688
`
`

`

`256
`
`S. EAIMTRAKARN et al.
`
`on the surfactant, one of the components of SMEDDS, and
`have been studying the improvement of poorly-absorbable
`drugs such as gentamicin (GM) and glycyrrhizin (GZ),
`where surfactant, Labrasole, was used as a component of
`SMEDDS. Labrasol showed a strong absorption enhan-
`cing effect on them (Shibata et al., 2000; Hu et al., 2001).
`By formulating GM or GZ in the mixture of Labrasol and
`water, the BA was increased from 0.6 to 4.6% for GZ and
`from 0 to 54% for GM. Based on the absorption enhancing
`effect of Labrasol, we have studied this surfactant for the
`absorption enhancement of other representative drugs
`having low oral BA, i.e. peptide/protein drugs.
`In this study, the effect of Labrasol on the absorption of
`insulin, a model peptide/protein drug,
`from the rat
`intestine was studied to confirm the absorption enhancing
`effect of Labrasol on peptide/protein drugs.
`
`MATERIALS AND METHODS
`
`Materials
`
`Crystalline porcine insulin (26.1 IU/mg) and glucose B-
`Test kit were purchased from Wako Pure Chemical
`Industries (Osaka, Japan). Labrasol (Gattefosee´, Gennee-
`villiers Cedex, France) was kindly supplied through CBC
`(Tokyo, Japan). Insulin EIA kit (DAINABOT Tokyo,
`Japan) was obtained from Nacalai Tesque (Kyoto, Japan).
`Male Wistar rats were obtained from Nippon Nousan
`(Yokohama, Japan). All other materials used were of
`reagent grade and were used as received.
`
`Preparation of Insulin Solution
`
`Insulin, 5.73 mg (150.0 IU), was dissolved in 0.3 ml of
`0.5N HCl, and 0.85 ml of phosphate buffered saline (PBS),
`pH 7.4, was added immediately to the solution. Thereafter,
`1.5 ml of Labrasol was added and the pH value was
`adjusted to pH 7.4 by the addition of 0.1N NaOH as
`required. The final insulin concentration was 50.0 IU/ml.
`
`In Vivo Absorption Experiments
`
`Male Wistar rats, 355 ^ 30 g; were fasted at least 12 h
`before absorption experiment. All animal experiments
`were carried out in accordance with the Guidelines for
`Animal Experimentation, Kyoto Pharmaceutical Univer-
`sity. After abdominal incision, the insulin test solution,
`200 ml/kg, was injected either into the jejunum, ileum or
`ascending colon of rats under anesthesia with sodium
`pentobarbital, 50 mg/kg. At 5 min before drug adminis-
`tration, blank blood sample, 200 ml, was obtained from the
`left jugular vein. Subsequent blood samples, 200 ml, were
`obtained at 0.5, 1, 2, 3, 4 and 5 h after administration. For
`i.v. administration, an insulin solution, 1.0 IU/ml, was
`prepared by dissolving insulin in 0.5N HCl and pH 7.4
`phosphate buffer and was injected into the right jugular
`vein, 1.0 IU/kg. After injection, blood samples were
`
`obtained at 2, 5, 10, 20, and 40 min and thereafter 1 and
`1.5 h from the left jugular vein. Serum samples were
`obtained by centrifuging at 10,000g for 10 min. All these
`serum samples were immediately frozen in a deep freezer
`at 2 808C until analysis.
`The serum glucose level was determined using a
`glucose B-Test kit. Post-dose levels of the serum glucose
`were expressed as the percentage of the pre-dose level.
`The percentage change in the serum glucose levels was
`taken as the percentage of the post-dose levels subtracted
`from 100. The cumulative percentage change in the serum
`glucose level was calculated by summing the areas above
`the serum glucose levels in the percentage change vs. time
`curves for 0 – 5 h (AAC) using trapezoidal method. The
`
`PA was calculated from the equation, PA (cid:136) (cid:133)AACoral £
`Dosei:v:(cid:134)=(cid:133)AACi:v: £ Doseoral(cid:134):
`
`The serum insulin levels were measured by an enzyme
`immunoassay (EIA) method using an IMX System
`(DAINABOT, Tokyo, Japan). The basal endogenous
`insulin levels were measured to be zero in all the animals.
`The area under the insulin level vs. time curves for 0 – 5 h
`(AUC insulin) was determined using traperzoidal rule. The
`oral bioavailability (BA) was calculated from the
`BA (cid:136) (cid:133)AUCoral £ Dosei:v:(cid:134)=(cid:133)AUCi:v: £
`equation,
`
`Doseoral(cid:134):
`
`STATISTICS
`
`All values are expressed as their mean ^ SE. Statistical
`differences were assumed to be reproducible when p ,
`0:05 (Student’s unpaired t-test).
`
`RESULTS
`
`The effect of Labrasol on the pharmacological activity,
`hypoglycemic effect, of insulin was at first studied after
`administration of insulin/Labrasol solution to different
`intestinal sites, i.e. the jejunum, ileum and ascending
`colon and the results are shown in Fig. 1, where Labrasol
`solution without insulin was administered to the ileum as a
`control study. After administration of insulin/Labrasol
`solution, 10.0 IU/kg, into the rat jejunum, hypoglycemic
`effect was induced and the lowest glucose level appeared
`at 1 h. The maximum decrease in serum glucose level was
`about 37% as compared to the pre-dose level. When the
`same insulin/Labrasol solution was administered to the
`ileum or colon at the same dose, marked hypoglycemic
`effect was obtained. The maximum decrease in serum
`glucose levels were also observed at 1 h after adminis-
`tration and were decreased to 28:2 ^ 5:4% for ileum and
`29:8 ^ 4:7% for colon administrations, respectively as
`compared to pre-dose level. To ascertain the absorption
`enhancing effect of Labrasol, insulin solution that did not
`contain Labrasol was administered into the rat ileum,
`10.0 IU/kg,
`the hypoglycemic effect was just a little
`observed. Table I shows pharmacokinetic parameters
`
`

`

`LABRASOL ENHANCES INSULIN ABSORPTION
`
`257
`
`the fasted
`the experiments were performed under
`condition, the pre-dose insulin levels were measured to
`be zero. After intestinal administrations, serum insulin
`levels reached to their maximum levels, 461 ^ 105
`(ileum) and 413 ^ 89 mIU/ml (colon) at 30 min and
`thereafter declined rapidly. By comparing to the serum
`insulin level vs. time profile obtained after i.v. injection of
`insulin solution to rats, 1.0 IU/kg, the BA of insulin after
`intra-ileum and colon administrations were calculated to
`be 0.25 and 0.20%, respectively.
`The dose-dependency of the hypoglycemic effect of
`oral
`insulin/Labrasol solution was studied after intra-
`ileum administration to rats by decreasing the dose from
`10.0 to 5.0 and 1.0 IU/kg and the results are shown in Fig.
`3. By decreasing the insulin dose, the hypoglycemic effect
`of insulin decreased. By comparing to the mean AAC
`value obtained after i.v. injection to rats, the PA values
`were 10.4 and 9.4% for 5.0 and 1.0 IU/kg dose,
`respectively. The pharmacological effect of oral insulin/-
`Labrasol solution was dose-dependent, because AAC
`decreased in accordance with the decrease of oral insulin
`dose.
`
`FIGURE 1 Effect of intestinal administration of insulin/Labrasol
`solution on rat serum glucose levels. Serum glucose levels were measured
`after administration of insulin/Labrasol solution or insulin solution. As a
`control experiment, Labrasol solution was administered into the rat
`ileum. W: control (Labrasol solution); †: i.v. injection of insulin/Labrasol
`solution (10.0 IU/kg); O:
`ileum administration of
`insulin/Labrasol
`solution (10.0 IU/kg); A:
`ileum administration of
`insulin solution
`(10.0 IU/kg); V: colonic administration of insulin/Labrasol solution
`(10.0 IU/kg); B: jejunum administration of insulin/Labrasol solution
`(10.0 IU/kg).
`
`obtained after administration of insulin/Labrasol solution
`or insulin solution to jejunum, ileum and colon, and as i.v.
`solution in rats. The area above the serum glucose levels
`(AAC) as the % change vs. time plot (Fig. 1) shows no
`significant difference between ileal and colonic adminis-
`tration. However, significant difference was observed in
`AAC value between jejunum and the other
`two
`administration sites. To determine the PA of insulin from
`enteral Labrasol solution, insulin solution was injected
`intravenously to the other group of rats at 1.0 IU/kg and
`the serum glucose levels were also measured (Fig. 1). By
`comparing to the mean AAC value obtained after i.v.
`injection with that obtained after intestinal administration
`of insulin/Labrasol solution, the PA of oral insulin/Lab-
`rasol solution was determined and the results are shown in
`Table I. The PA of insulin administered into the jejunum,
`ileum and colon were 3.9, 8.9 and 9.1%, respectively.
`To confirm the absorption of insulin in intact form,
`serum insulin levels were measured after intra-ileal and
`colonic administrations of
`insulin/Labrasol solution,
`10.0 IU/kg, and the results are shown in Fig. 2. As all
`
`DISCUSSION
`
`For the oral delivery of peptide/protein drugs, there are
`two barriers, i.e. hydrolytic degradation in the GI tract and
`poor membrane permeability. To conquer these barriers,
`we have developed a new oral protein delivery system,
`gastro-intestinal mucoadhesive patch system, GI-MAPS
`(Eaimtrakarn et al., 2001). The delivery efficiency was
`studied with a recombinant human granulocyte colony-
`stimulating factor (G-CSF) without any strong absorption
`enhancer.
`Instead, a surfactant, polyoxyethylated,
`60 mmol, caster oil derivative (HCO-60e), and an organic
`acid, citric acid, were formulated along with G-CSF. After
`oral administration of GI-MAPS to beagle dogs,
`the
`maximum total blood leukocyte count increased by 170%
`as compared to the pre-dose level and the calculated PA of
`oral G-CSF was 23% as compared to the value obtained
`after i.v. administration of the same dose of G-CSF
`(Eaimtrakarn et al., 2001). However, in the case of oral
`insulin delivery system, many studies have been focused
`on the absorption enhancing technology because of its
`
`TABLE I Pharmacokinetic parameters of insulin following intestinal administration of Labrasol solution and i.v. administration of insulin solution
`
`Administration route
`
`Labrasol
`
`Dose (IU/kg)
`
`AAC (% h)
`
`AUCinsulin (mIU h/ml)
`
`PA (%)
`
`BA (%)
`
`87.1 ^ 21.1
`3.9
`+
`10
`ND
`0.280 ^ 0.047*
`200.5 ^ 10.3
`+
`10
`8.9
`46.21 ^ 8.1
`2
`2.6
`10
`ND
`117.3 ^ 11.6
`10.4
`+
`5
`ND
`21.2 ^ 6.3
`9.4
`+
`1
`ND
`0.338 ^ 0.067*
`205.2 ^ 17.4
`+
`Colon
`10
`9.1
`13.469 ^ 1.353**
`225.3 ^ 17.0
`2
`100
`I.v.
`1
`or (cid:133)0 ! 1:5 h(cid:134)(cid:3)(cid:3)
`AAC: the area above the serum glucose level vs. time curve (cid:133)0 ! 5 h(cid:134); AUC: the area under the serum insulin level vs. time curve (cid:133)0 ! 5 h(cid:134)(cid:3)
`pharmacological availability and equals to (AACGI/DoseGI)/(AACi.v./Dosei.v.) £ 100, where GI means gastrointestinal administration and i.v. means intraveneous injection of
`
`ND
`0.20
`ND
`ND
`ND
`0.25
`100
`
`; PA(%) is
`
`Jejunum
`Ileum
`
`insulin; ND: Data were not obtained.
`
`

`

`258
`
`S. EAIMTRAKARN et al.
`
`surfactant and co-solvent. Among them, surfactant plays
`the most important role on the dissolution of hydrophobic
`drugs. We found out a strong absorption enhancing effect
`of Labrasol, which has been used as a main component of
`SMEDDS, on the intestinal absorption of GM. GM is a
`representative extremely hydrophilic poorly-absorbable
`drug. By formulating GM into Labrasol solution, the BA
`was increased from 0 to 54% (Hu et al., 2001). Banerjee
`et al. (2000) showed that nonionic block copolymers
`synthesized from ethylene oxide and propylene oxide
`(CRL-1605) had an inhibitory activity on intestinal P-gp.
`Nerurkar et al. (1996) also showed that surfactants like
`Cremophor EL and Polysorbate 80 had inhibitory effects
`on P-gp and enhanced the BA of peptides. The surfactant
`used in this study was Labrasol
`that has a high
`hydrophilic – lipophilic balance (HLB) value,
`i.e. 14.
`Labrasol, caprylocaproyl macrogolglycerides,
`is the
`mixture of monoesters, diesters and triesters of glycerol
`and monoesters and diesters of macrogols with a mean
`relative molecular mass between 200 and 400 and is
`registered in European Pharmacopoeia. According to our
`study on the absorption enhancing mechanism of Labrasol
`using Ussing chamber method, Labrasol enhanced the
`apical-to-basolateral transport of GM by decreasing the
`efflux of GM. In addition, with the addition of verapamil,
`a P-gp inhibitor, GM efflux was decreased. As insulin is a
`macromolecular compound and has high water solubility,
`it might not be a substrate of P-gp. Therefore, Labrasol did
`not inhibit the efflux of insulin from the enterocytes but it
`merely enhanced the absorption of insulin. However, the
`absorption enhancing mechanism of Labrasol on insulin is
`yet to be clarified.
`Barichello et al. (1999) showed an enhanced rectal
`absorption of insulin loaded in Pluronice F-127 gels
`where unsaturated fatty acids like eicosapentaenoic acid,
`docosahexaenoic acid and oleic acid were used as
`absorption enhancers. High BAs, 36.3 – 38.7%, were
`achieved with the preparations and good correlation was
`obtained between the relative hypoglycemic effects and
`the insulin BA, where BA was obtained by comparing the
`AUCs after oral and subcutaneous (sc) administrations.
`However, good correlation between BA of oral insulin and
`degree of hypoglycemic effect was not obtained in our
`study. According to the report of Hoffman and Ziv (1997),
`the pharmacodynamics of insulin is dependent on various
`factors, i.e. administration route, liver function and glucose
`concentration etc. In addition, biochemical and physiologi-
`cal processes occurring at different sites affect
`the
`hypoglycemic effect of insulin. Especially,
`the insulin
`receptors are expressed on the plasma membrane of the liver
`parenchymal cells. After oral administration of insulin,
`absorbed insulin encounters hepatic first-pass effect and the
`remaining insulin enters into the systemic circulation.
`Therefore, portal insulin level might be well correlated with
`the hypoglycemic activity of oral insulin. These complicated
`pharmacokinetic and pharmacodynamic characteristics of
`insulin might explain the observed discrepancy between the
`hypoglycemic effect and BA of insulin.
`
`FIGURE 2 Serum insulin concentration vs. time profiles after intra-
`ileum and colonic administrations of
`insulin/Labrasol solution,
`10.0 IU/0.2 ml/kg, in comparison with that (inserted figure) obtained
`after i.v. injection of insulin solution, 1.0 IU/kg. W: i.v. injection of insulin
`saline solution; †:
`intra-ileum administration of
`insulin/Labrasol
`solution; O: intra-colonic administration of insulin/Labrasol solution.
`
`poor membrane permeability. Recently, Onuki et al.
`(2000) showed a strong absorption enhancing effect of
`highly purified docosahexaenoic acid on the rectal
`absorption of insulin, where a W/O/W multiple emulsion
`system was used and high BA, 25.4%, was achieved.
`On the other hand, SMEDDS has attracted interest of
`many scientists because of high improvement of oral BA
`of extremely hydrophobic drugs such as cyclosporine and
`HIV-protease inhibitor, saquinavir (Mahalati et al., 1999).
`In the case of these extremely hydrophobic drugs, the
`dissolution process is the rate-limiting step for
`the
`absorption of drug after oral administration. Therefore,
`SMEDDS was developed to solve the dissolution problem
`of these water-insoluble drugs. However, these hydro-
`phobic drugs were shown to be substrates of intestinal
`efflux pump such as P-glycoprotein (P-gp) (Asperen et al.,
`1998). SMEDDS is defined as a formulation to produce
`thermodynamically stable microemulsions upon dilution
`and is usually composed of three components, i.e. lipid,
`
`FIGURE 3 Effect of insulin dose on the rat serum glucose levels vs.
`time profiles after intra-ileum administration of insulin/Labrasol solution:
`(B) 1.0, (O) 5.0 and (†) 10.0 IU/kg.
`
`

`

`LABRASOL ENHANCES INSULIN ABSORPTION
`
`259
`
`There are many reports on the intestinal absorption
`enhancers for insulin. However, the administered volume
`of the insulin test solution has not been clearly described.
`By comparing the physiological condition of the GI tract
`between rat and human, the average intestinal diameter is
`approximately 5 cm for human and 5 mm for rat that is
`approximately one tenth (Kararli 1995). By considering
`the volume of the luminal space, the volume in rats is
`about one hundredth smaller than human. In the case of
`the treatment of human patients, only several enteric
`capsules can be accepted at each administration time.
`Enteric capsule is more useful than colon delivery capsule,
`because colon delivery capsule is not
`in the market.
`Therefore, we focused on the absorption of insulin from
`the ileum in this study. Size 1 capsule can accommodate
`approximately 0.5 ml of insulin/Labrasol solution. If a
`patient
`takes three capsules,
`the total volume of
`insulin/Labrasol solution is 1.5 ml. By scaling down the
`volume of the insulin/Labrasol solution to rats, the volume
`should be less than 15 ml. When the administered volume
`of insulin preparation is large, so-called solvent-drag
`effect would occur and increased BA would be explained
`in part by this mechanism. To exclude such an artificial
`effect, we administered insulin/Labrasol solution with
`small volume. The PA of insulin from Labrasol solution
`was lower than that reported by Onuki et al. (2000).
`However, we cannot estimate the volume of
`their
`preparation for human patient, because the administered
`volume of their test solution was not described. By simply
`extrapolating the volume of insulin/Labrasol solution for
`human patient,
`the clinical dose of
`insulin/Labrasol
`solution would be more than three capsules. We are now
`developing a new oral protein/peptide delivery system, i.e.
`GI-MAPS. GI-MAPS is a patch system and composed of
`three layers,
`i.e. pH-sensitive adhesive layer, water-
`insoluble base membrane and drug carrying layer.
`Insulin/Labrasol solution can be filled inside GI-MAPS.
`After the surface pH-sensitive layer dissolves in the small
`intestine, GI-MAPS becomes adhesive to the intestinal
`absorptive membrane and forms a closed space between
`the water-insoluble base membrane and the intestinal
`absorptive membrane. Therefore, the dilution of insulin/-
`Labrasol solution would not occur. Now, we are studying
`the GI-MAPS containing insulin/Labrasol solution and the
`result will be published in the next report.
`
`CONCLUSIONS
`
`A new absorption enhancer, Labrasol, has been found out
`for
`the oral administration of
`insulin in rats. By
`formulating Labrasol into insulin solution, both the PA
`and BA of insulin were increased after ileal and colonic
`administrations. Pharmacological availabilities were 8.9
`and 9.1% for
`intra-ileum and colon administrations,
`respectively. BAs of insulin were 0.25 and 0.20% after
`intra-ileum and colon administrations, respectively. As the
`administration volume of insulin/Labrasol solution was so
`
`small, 200 ml/kg, this formulation would be useful for the
`oral insulin therapy with GI-MAPS.
`
`Acknowledgements
`
`This research was supported in part by the Bioventure
`Developing Program of
`the Ministry of Education,
`Culture, Sports, Science and Technology of Japan, and
`Venture SME-University Research Promotion Program of
`Japan Society for the Promotion of Science.
`
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