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Oral insulin therapy has great potential benefits over conventional therapy for diabetic patients as well as mimicking the physiological fate of insulin. Here we evaluated the characteristics of insulin and deoxycholate-based synthetic Nα-deoxycholyl-l-lysyl-methylester (DCK) complex, and diabetes correction in pancreatectomized canines after oral administration. After the insulin/DCK complexation was made, the insulin’s folding structure, stability against digestive enzymes, lipophilicity and permeability to Caco-2 monolayer were evaluated in vitro. Diabetic canines were kept under fasting conditions, and Eudragit-coated gelatin capsules containing insulin or insulin/DCK powder were singly or triply administered. Evaluation of glucodynamics, pharmacokinetics, oral glucose tolerance test (OGTT) and reproducibility were carried out. After complexation with DCK, the folding structure of insulin did not become denatured and the resistance against digestive enzymes was powerfully improved. The lipophilicity and permeability of insulin/DCK (coupling ratio up to 1:10) were also highly increased. The insulin/DCK complex, administered orally into diabetic canines at the doses of 21, 42, and 81 IU/kg, reduced the plasma glucose levels by about 28%, 44% and 67%, respectively, while the plasma insulin concentrations increased. During OGTT, insulin/DCK nearly maintained the normoglycemic state in the diabetic canines, whereas the hyperglycemic state of placebo-treated controls was not corrected. During oral administration of insulin/DCK, it repetitively showed similar therapeutic efficacy in diabetic canines for 3 days. The therapeutic efficacy of insulin/DCK was exhibited in its digestive enzyme resistance, deoxycholate-based lipophilicity for enhancing permeability and intact insulin delivery without chemical modification, providing potential oral therapeutic remedy as an alternative to injectable insulin medication.

The need for an efficacious and safe oral anticoagulant that does not require monitoring has been largely unmet. Many efforts have centered on preparing orally available heparin to improve patient compliance. In this study, novel orally active heparin derivatives (LHD), i.e. low molecular weight heparin (LMWH) conjugated with deoxycholic acid (DOCA), were evaluated in vitroand in vivofor their enhancement effect of oral heparin absorption. After oral administration of 10 mg/kg of water-soluble LHD, Ws-LHD1.5 showed optimum oral efficacy and its bioavailability was about 24% in rats. The oral absorption of LHD1.5 was also enhanced by several solubilizers, among which Poloxamer 407 provided the best results. When 5 mg/kg of LHD1.5 with Poloxamer 407 was orally administered to monkeys, the maximum anti-FXa activity in plasma was 0.26 ± 0.04 IU/mL and its bioavailability was 17.4%. In a rat thrombosis model, 5 mg/kg of orally administered LHD1.5 formulated with Poloxamer reduced thrombus formation by 63.9 ± 16.6%, which was higher than the efficacy of clinically used enoxaparin (49.4 ± 17.8% at 100 IU/kg, sc). Considering the oral absorption efficacy and therapeutic effect, the conjugation ratio was optimized as about 1.5 molecules of DOCA per mole of heparin. Therefore, LHD1.5 with Poloxamer 407 can be further formulated as a solid oral anticoagulant drug.

One approach for enhancing intestinal absorption of therapeutic peptides is chemical conjugation to bile acids. In this study, recombinant salmon calcitonin (sCT) was chemically modified by covalent attachment of deoxycholic acid (DOCA). Three different sCT‐DOCA conjugates, namely, sCT‐mono‐DOCA, sCT‐di‐DOCA, and sCT‐tri‐DOCA, were prepared and characterized for their structural and biological properties. The permeability of these conjugates in the gastrointestinal tract was evaluated using Caco‐2 cell monolayers to determine their potential as an oral dosage form. The conjugates were isolated by reversed‐phase fast protein liquid chromatography (FPLC) and confirmed by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Circular dichroism spectra in 50% trifluoroethanol aqueous condition showed the ordered secondary structure of sCT‐DOCA. The biological activities of sCT‐DOCA conjugates were evaluated by cyclic AMP assay using T‐47D cells, and the mean EC50values of sCT, sCT‐mono‐DOCA, and sCT‐di‐DOCA were 0.034, 0.076, and 0.46 nM, respectively. The transport experiments using the Caco‐2 cell monolayer showed that the permeability of sCT‐DOCA conjugates in buffer was not altered from the native sCT. However, the permeability of sCT‐DOCA conjugates was increased up to 2.5 times that of the native sCT when sCT‐DOCA was formulated in 1% dimethylsulfoxide (DMSO) solution. The conjugated DOCA of sCT‐DOCA significantly enhanced the absorption of sCT‐DOCA in the intestinal membrane when sCT‐DOCA conjugates were completely solubilized by DMSO. In conclusion, this study proposes that therapeutic peptides that have poor absorption profiles could potentially be developed into orally active drugs by conjugation with DOCA in the formulation with appropriate solubilizing agents. Drug Dev. Res. 64:129–135, 2005. © 2005 Wiley‐Liss, Inc.

Major limitations in the use of liposomes for oral formulation relate to their physical and chemical instability in the GI tract. In this study, the conjugate (PAA-DSPE) of poly(acrylic acid) and distearoylphosphatidylethanolamine (DSPE) was synthesized, and liposomes were prepared with the mixture of PAA-DSPE and DSPE to improve the stability of liposomes in the GI tract. The prepared PAA-DSPE was characterized by FT-IR and ¹³C-NMR to confirm the coupling between PAA and DSPE. In the elemental analysis, the coupling ratio of PAA and DSPE in PAA-DSPE was calculated as 1:1.73. The average size of a PAA-DSPE/DSPE liposome, measured by dynamic light scattering, was in the range of 300–500 nm, and the minimum average size was 320 nm at 6 mol% of PAA-DSPE. The stability of the prepared liposomes was evaluated in different pH (2, 5, 7.4) solutions, and in different concentrations of bile acid (0, 0.1, 1, 10%) and pancreatin solutions (0, 0.03, 0.3, 3%). The stability of liposomes in different conditions was determined by measuring the fluorescence intensity of 5(6)-CF leaked from liposomes. The amount of the 5(6)-CF leakage from the PAA-DSPE/DSPE liposomes of 6 mol% PAA-DSPE was the lowest in all the cases of acidic, bile, and pancreatin solutions. In conclusion, the optimum amount of PAA-DSPE in liposome was 6 mol%, and PAA-DSPE/DSPE liposomes could improve the stability in the GI tract. Drug Dev. Res. 61:13–18, 2004. © 2004 Wiley-Liss, Inc.

Major limitations in the use of liposomes for oral formulation relate to their physical and chemical instability in the GI tract. In this study, the conjugate (PAA‐DSPE) of poly(acrylic acid) and distearoylphosphatidylethanolamine (DSPE) was synthesized, and liposomes were prepared with the mixture of PAA‐DSPE and DSPE to improve the stability of liposomes in the GI tract. The prepared PAA‐DSPE was characterized by FT‐IR and 13C‐NMR to confirm the coupling between PAA and DSPE. In the elemental analysis, the coupling ratio of PAA and DSPE in PAA‐DSPE was calculated as 1:1.73. The average size of a PAA‐DSPE/DSPE liposome, measured by dynamic light scattering, was in the range of 300–500 nm, and the minimum average size was 320 nm at 6 mol% of PAA‐DSPE. The stability of the prepared liposomes was evaluated in different pH (2, 5, 7.4) solutions, and in different concentrations of bile acid (0, 0.1, 1, 10%) and pancreatin solutions (0, 0.03, 0.3, 3%). The stability of liposomes in different conditions was determined by measuring the fluorescence intensity of 5(6)‐CF leaked from liposomes. The amount of the 5(6)‐CF leakage from the PAA‐DSPE/DSPE liposomes of 6 mol% PAA‐DSPE was the lowest in all the cases of acidic, bile, and pancreatin solutions. In conclusion, the optimum amount of PAA‐DSPE in liposome was 6 mol%, and PAA‐DSPE/DSPE liposomes could improve the stability in the GI tract. Drug Dev. Res. 61:13–18, 2004. © 2004 Wiley‐Liss, Inc.

Previously, poly(L‐lactide) microspheres containing retinoic acid (RA) had been prepared for parenteral administration in order to overcome “acute retinoic acid resistance.” In this study, acute toxicities of RA‐loaded microspheres were investigated in mice after the microspheres were injected intramuscularly. The maximum dose of RA in the microspheres was determined by considering both the injection volume and dispersion of microspheres in the vehicle media, and the dose levels were determined to be 0, 90, 180, and 360 mg RA/kg. Mice were carefully observed for any clinical signs for 14 days, and gross observation of the organs was performed at 14 days. When a dose of 90 mg RA/kg was administered, no severe toxicities were observed; however, with the administration of 180 and 360 mg RA/kg, abnormal appearances of organs, such as discolor, atrophy, hemorrhage, etc., were observed. There were no mortalities or bone fractures observed at any of the doses. In our previous study of cancer treatment using the RA‐loaded microspheres, the growth of head and neck carcinoma in athymic nude mice was successfully inhibited at the dose of 100 mg RA/kg. It was concluded that a dose of approximately 90 mg RA/kg of the RA‐loaded microspheres would be most desirable for cancer therapy. Drug Dev. Res. 57:134–139, 2002. © 2002 Wiley‐Liss, Inc.

Previously, poly(L-lactide) microspheres containing retinoic acid (RA) had been prepared for parenteral administration in order to overcome “acute retinoic acid resistance.” In this study, acute toxicities of RA-loaded microspheres were investigated in mice after the microspheres were injected intramuscularly. The maximum dose of RA in the microspheres was determined by considering both the injection volume and dispersion of microspheres in the vehicle media, and the dose levels were determined to be 0, 90, 180, and 360 mg RA/kg. Mice were carefully observed for any clinical signs for 14 days, and gross observation of the organs was performed at 14 days. When a dose of 90 mg RA/kg was administered, no severe toxicities were observed; however, with the administration of 180 and 360 mg RA/kg, abnormal appearances of organs, such as discolor, atrophy, hemorrhage, etc., were observed. There were no mortalities or bone fractures observed at any of the doses. In our previous study of cancer treatment using the RA-loaded microspheres, the growth of head and neck carcinoma in athymic nude mice was successfully inhibited at the dose of 100 mg RA/kg. It was concluded that a dose of approximately 90 mg RA/kg of the RA-loaded microspheres would be most desirable for cancer therapy. Drug Dev. Res. 57:134–139, 2002. © 2002 Wiley-Liss, Inc.