Kanji Takada

Synthesis of palmitoyl derivatives of insulin and their biological activities.

In order to improve the lipophilicity of peptides, bovine insulin was chosen for the chemical modification using palmitic acid. The N-hydroxysuccinimide ester of palmitic acid was used for attachment to terminal amino groups, and p-methoxybenzoxycarbonyl azide was used for protection of the glycine-Al amino terminus. We obtained two purified derivatives of insulin, Bl-monopalmitoyl- and Bl,B29-dipalmitoyl-insulin, which were confirmed to be more lipophilic than the parent insulin on high-performance liquid chromatography (HPLC). The hypoglycemic effects of both products were measured in rats after intravenous and intramuscular injections. The mono derivative was more active than the di derivative and produced a longer effect duration than the native insulin after intravenous injection. The derivatives were also shown to be less immunoreactive as judged by an enzyme immunoassay.

Self-dissolving microneedles for the percutaneous absorption of EPO in mice

Erythropoietin (EPO) loaded microneedles were prepared using thread-forming polymer as a base for the percutaneous administration of EPO. The used polymers were dextrin, chondroitin sulfate and albumin. Under room temperature, EPO solution was added to high concentration of polymer solution and microneedles were prepared by forming thread with polypropylene tips. The mean weight of microneedle was 0.59 ± 0.01 mg and length and basal diameter were 3.24 ± 0.16 and 0.55 ± 0.03 mm, respectively. Four microneedles were percutaneously (pc) administered to mice at the EPO dose levels of 100 IU/kg. After administration, blood samples were collected for 24 h and serum EPO levels were measured. Dextrin EPO microneedles were administered both pc and subcutaneously (sc) to mice. Serum EPO levels vs. time profiles showed Cmax of 138.6 ± 16.1 and 146.5 ± 8.0 mIU/ml, respectively. Tmax were 7.5 h. The values of bioavailability (BA) of EPO were 82.1 and 99.4%, respectively. By decreasing the dose from 100 to 50 and 25 IU/kg, dose-dependent serum EPO levels vs. time profiles were not clearly obtained. When chondroitin sulfate and albumin were used as the microneedle base, the serum EPO levels vs. time profiles showed almost the same pattern. Cmax of chondroitin sulfate and albumin microneedles were 96.3 ± 8.8 and 132.2 ± 18.9 mIU/ml, respectively. AUCs were 835.1 and 1098.7 mIU h/ml. Tmax were 8 and 6.8 h. These results suggest the usefulness of microneedles for the percutaneous administration of EPO.

Evaluation of intestinal pressure-controlled colon delivery capsule containing caffeine as a model drug in human volunteers.

The delivery ability of a pressure-controlled colon delivery capsule (PCDC) containing caffeine as a test drug was evaluated after oral administration to healthy male human volunteers. The driving force causing PCDC disintegration in the intestinal tract is the physiological luminal pressure which results from peristalsis. Three kinds of PCDCs having different thickness of a water-insoluble polymer membrane was prepared by coating the inner surface of the gelatin capsules with ethylcellulose (EC). The mean thickness were 40 +/- 1 (SE) for type 1, 44 +/- 1 for type 2 and 50 +/- 1 micron for type 3 PCDC, respectively. Caffeine was dissolved with a suppository base (PEGs 400 and 1000) and the capsules were filled. Doses were 15, 45 or 75 mg. After blank saliva samples were obtained, test preparations were orally administered to the volunteers and saliva samples were collected for 1 min intervals hourly from 1 to 10 h in the fasted state study, and from 1 to 20 h and at 25 h in the fed state study. Caffeine concentrations in the saliva samples were analyzed by HPLC. The maximum salivary caffeine excretion rate increased as the oral caffeine dose increased. The maximum salivary caffeine excretion rate increased predominantly compared to the pre-dose level in 75 mg dose study. Therefore, all following studies were performed with this dose. The first appearance time of caffeine into the saliva, TI, was used as a parameter to estimate the disintegration time of test preparations in the gastrointestinal tract. The mean TI of types 1, 2, and 3 PCDCs were 3.0 +/- 0.4, 4.0 +/- 0.4 and 4.5 +/- 0.3 h, respectively. After oral administration of 75 mg caffeine in pain gelatin capsule as a reference preparation, caffeine appeared in the saliva within 0.5 h. The mean hardness of the PCDCs were 1.05 +/- 0.10 (type 1), 1.55 +/- 0.06 (type 2) and 2.08 +/- 0.15 newton (type 3), respectively. There were good correlations between three parameters: EC coating membrane thickness, hardness and TI (determination coefficient r2 = 0.935 between TI and thickness, r2 = 0.998 between thickness and hardness, r2 = 0.958 between hardness and TI). The effect of food intake on the delivery ability was examined with type 3 PCDCs. Food intake prolonged the mean TI, from 4.5 +/- 0.3 to 7.8 +/- 1.3 h. This increase is thought to be ascribed to prolonged gastric emptying time. Comparison with reported colon arrival times indicates that the type 3 PCDC functions in colon delivery of caffeine and is thought to be applicable to other drugs.

Enhanced intestinal absorption of vancomycin with Labrasol and D-α-tocopheryl PEG 1000 succinate in rats

Vancomycin hydrochloride (VCM) is a glycopeptide antibiotic used for the treatment of infections caused by methicillin-resistant staphylococci. It is water soluble, having a high molecular weight, and poorly absorbed from the gastrointestinal tract. Mixtures of VCM with Labrasol and D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) were prepared to improve oral absorption of VCM. Administration of VCM solution to rat ileum at a dose of 20 mg/kg did not result in detectable plasma VCM concentration. Formulation containing 50% of Labrasol resulted in a Cmax value of 5.86+/-0.97 microg/ml and an AUC(0-6h) value of 16.06+/-1.78 microgh/ml. Addition of TPGS to VCM solution at 12.5% concentration also increased the plasma VCM concentration with a Cmax value of 4.98+/-0.45 microg/ml. But the AUC(0-6 h) (9.87+/-1.90 microgh/ml) was significantly lower than that obtained with Labrasol. The addition of 5.0 and 25.0% TPGS to solutions of VCM containing 50% of Labrasol did not result in any significant increase either in Cmax or AUC(0-6 h) of VCM. Whereas the addition of 12.5% of TPGS has resulted in an increase in Cmax and AUC(0-6 h) by 2.2 and 2.4 times, respectively, suggesting that this concentration of 50% Labrasol and 12.5% TPGS (1:0.25) was optimum for improving intestinal absorption of VCM. A dose dependent decrease in the Cmax and AUC(0-6 h) values was observed when the dose of absorption enhancers was decreased by 50% with formulation containing Labrasol and TPGS in 1:0.25 ratio. The results of the study indicate that formulations containing Labrasol and TPGS improve intestinal absorption of hydrophilic macromolecular drug, VCM.

A novel emulsifier, labrasol, enhances gastrointestinal absorption of gentamicin.

Gentamicin (GM) is an important aminoglycoside antibiotic for the treatment of infections caused by a wide spectrum of aerobic gram-negative bacilli and gram-positive cocci. As a class, the aminoglycosides are poorly absorbed from the gastrointestinal (GI) tract and are commonly used as injectable and topical preparations. This study was aimed at finding the effect of a novel emulsifier, Labrasol, on the absorption of GM from the GI tract of rats. GM formulations were prepared, either as saline solution or as Labrasol microemulsions, and were administrated to rat small intestine and colon. Plasma GM levels following intestinal application were compared to those obtained with intravenous (i.v.) administration. A 5 mg/kg dose of GM preparation containing Labrasol, 1 ml/kg, administrated into colon resulted in the mean AUC of 21.179+/-1.374 microg x h/ml, compared to 7.813+/-0.105 microg x h /ml obtained with i.v. administration of GM, 1 mg/kg. The absolute bioavailability (BA) of the Labrasol preparation was 54.2%. Labrasol facilitates the transmucosal delivery of GM from rat colon by forming microemulsions, and the BA obtained with Labrasol microemulsion was higher than with other surfactants (8.4% for Tween 80 and 3.4% for Transcutol P). Additionally, in vitro permeation studies demonstrated that Labrasol also inhibited the intestinal secretory transport. The effect of Labrasol is ascribed to both (1) enhanced GM absorption from the GI lumen into the systemic circulation and (2) inhibition of efflux of GM from the enterocytes to the GI lumen.

Gastrointestinal mucoadhesive patch system (GI-MAPS) for oral administration of G-CSF, a model protein

A new gastrointestinal mucoadhesive patch system (GI-MAPS) has been designed for the oral delivery of protein drugs. The system consists of four layered films, 3.0 x 3.0 mm2, contained in an enteric capsule. The 40 microm backing layer is made of a water-insoluble polymer, ethyl cellulose (EC). The surface layer is made of an enteric pH-sensitive polymer such as hydroxypropylmethylcellulose phthalate (HP-55), Eudragit L100 or S100 and was coated with an adhesive layer. The middle layer, drug-containing layer. made of cellulose membrane is attached to the EC backing layer by a heating press method. Both drug and pharmaceutical additives including an organic acid, citric acid, and a non-ionic surfactant, polyoxyethylated castor oil derivative (HCO-60), were formulated in the middle layer. The surface layer was attached to the middle layer by an adhesive layer made of carboxyvinyl polymer (Hiviswako 103). Fluorescein (FL), 30mg, was first used as a model drug for oral administration of GI-MAPS having different surface layers in beagle dogs. The plasma FL concentration vs. time profiles demonstrated that the targeting of the systems was obtained, because the Tmax, the time when plasma FL concentrations reaches to its maximum lelev, was 2.33+/-0.82 h for HP-55 system, 3.33+/-0.41 h for Eudragit L100 system and 5.00+/-0.00 h for Eudragit S100 system. The same three kinds of GI-MAPSs containing 125 microg of recombinant human granulocyte colony-stimulating factor (G-CSF) were prepared and orally administered to dogs and the increase in total white blood cell (WBC) counts were measured as the pharmacological index for G-CSF. Comparison with the total increase of WBCs after iv injection of the same amount of G-CSF (125 microg) indicated the pharmacological availabilities (PA) of G-CSF were 23%, 5.5% and 6.0% for Eudragit L100, HP-55 and Eudragit S100 systems. By decreasing the amount of HCO-60 and citric acid, the PA of G-CSF decreased. These results suggest the usefulness of GI-MAPS for the oral administration of proteins.

Development of a Colon Delivery Capsule and the Pharmacological Activity of Recombinant Human Granulocyte Colony‐stimulating Factor (rhG‐CSF) in Beagle Dogs

A peroral dosage form was examined to deliver recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) to the colon in beagle dogs. A new gelatin capsule with its inside surface coated with ethylcellulose was prepared for this purpose. RhG‐CSF was dissolved with propylene glycol and was filled in the capsule. Several kinds of ethylcellulose‐gelatin capsules with an ethylcellulose layer of thickness 46 to 221 mm were used. The capsule was filled with propylene glycol solution containing fluorescein as an absorption marker, castor oil derivative and citric acid. The hardness of the capsule was tested after the gelatin layer was dissolved using a hardness tester and was dependent on the thickness of the ethylcellulose layer of the capsule.

Absorption enhancing effect of Labrasol on the intestinal absorption of insulin in rats

The oral absorption enhancing effect of Labrasol™ 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.

Improvement of large intestinal absorption of insulin by chemical modification with palmitic acid in rats.

Abstract— The intestinal absorption of 125I‐labelled palmitoyl insulin was examined following administration into in‐situ closed large intestinal loops of rats. When mono‐ and dipalmitoyl insulins (Palins‐1 and Palins‐2, respectively) were administered in polyoxyethylene hydrogenated castor oil (HCO 60) micellar system into intestinal loops, a marked increase in plasma radioactivity and a corresponding disappearance of residual radioactivity in the intestinal lumen were observed in the following rank order: Palins‐2 > Palins‐1 > native insulin. In addition, the derivatives were more stable than native insulin in the mucosal tissue homogenates of the large intestine. These results suggest that chemical modification of insulin with palmitic acid may not only increase the lipophilicity of insulin but also reduce its degradation, resulting in the increased transfer of insulin across the large intestinal mucous membrane. The linoleic acid‐HCO 60 mixed micelles system did not have a significant effect on the large intestinal absorption of radioactivity associated with the lipophilic insulin analogues.

Lipophilic Peptides: Synthesis of Lauroyl Thyrotropin-Releasing Hormone and Its Biological Activity

Thyrotropin-releasing hormone (TRH) was derivatized by chemical attachment of lauric acid to the N-terminal pyroglutamyl group. The product was confirmed to be more lipophilic than TRH by high-performance liquid chromatography and measurement of partition coefficients. The central nervous system activity and endocrine activity of the lauroyl derivative were only slightly reduced, to 81 and 64% of the parent TRH, respectively. Lipophilic derivatization may be generally applicable to transmembrane delivery of peptides.