Kazuhiro Morimoto

Oral delivery of insulin by using surface coating liposomes: Improvement of stability of insulin in GI tract

The potency of surface coating liposomes with some materials was investigated for oral delivery of peptide drugs. In vitro release of insulin, a model peptide, from liposomes in the bile salts solution was markedly reduced by coating the surface with the sugar chain portion of mucin (Mucin-Lip) or polyethyleneglycol (PEG-Lip). Encapsulation of insulin into Mucin-Lip and PEG-Lip completely suppressed the degradation of insulin in the intestinal fluid, whereas uncoated liposomes suppressed it only partially. These results demonstrated that surface coating liposomes with PEG or mucin gained resistance against digestion by bile salts and increased the stability in the GI tract. When insulin was orally administered to rats as either a solution or non-charged liposome ((N)-Lip), no hypoglycemic effect was observed. Administration of insulin encapsulated in positively charged liposome ((+)-Lip) caused the rapid decrease in the plasma glucose level which recovered to the control level within 3 h. In contrast, PEG-Lip and Mucin-Lip caused a gradual decrease in the glucose level after administration. The hypoglycemic effect by PEG-Lip lasted for much longer duration than that of uncoated liposomes. The slow release of insulin from the surface coating liposomes achieved the longer duration of oral hypoglycemic activity. Consequently, the surface coating should be the potential way to add desirable functions to the liposome for oral drug delivery.

Effects of Viscous Hyaluronate–Sodium Solutions on the Nasal Absorption of Vasopressin and An Analogue

The effects of viscous solutions of hyaluronate-sodium of various average molecular weights (MW) on the nasal absorption of vasopressin (AVP) and its analogue, l-deamino-8-D-arginine vasopressin (l-d-8-DAVP), were examined in rats. Solutions of hyaluronate with MW greater than 3 × 105 daltons enhanced the nasal absorption of AVP; solutions of MW 5.5 × 104 daltons were not effective. The enhancing effects on the nasal absorption of AVP and l-d-8-DAVP were dependent on the concentration in the range of 0–1.5% (w/v) hyaluronate (MW 1.4 × 106 daltons). The nasal absorption of AVP was increased with this solution at lower pH. Bioavailabilities after nasal administration of AVP and l-d-8-DAVP in hyaluronate solutions (MW 1.4 × 106 and 2 × 106 daltons) increased more than 2- and 1.6-fold as compared to nasal administration of AVP and 1-d-8-DAVP in buffer solutions (pH 7.0), respectively. Hyaluronate solution (MW 1.4 × 106 daltons) did not affect the ciliary beat frequency of rabbit nasal mucosal membranes in vitro. Therefore, hyaluronate solution may be useful as a vehicle for nasal delivery of AVP and l-d-8-DAVP.

Effects of proteolytic enzyme inhibitors on nasal absorption of salmon calcitonin in rats

Abstract Proteolytic enzyme inhibitors were examined as absorption enhancers for the nasal absorption of salmon calcitonin (SCT) in rats. Bestatin, diprotinin, leupeptin, aprotinin, soybean trypsin inhibitor and camostat mesilate were used as enzyme inhibitors. The nasal absorption of SCT was evaluated by measuring its hypocalcemic effects. The peptidase activities of rat nasal mucosal tissue were high and found to be in the following order: leucine aminopeptidase (2.72 nmol/min per mg protein) > dipeptidyl aminopeptidase (1.84 nmol/min per mg protein) > cathepsin (650 pmol/min per mg protein) > trypsin.(4.61 pmol/min per mg protein). Nasal administration of SCT (10 IU/kg, pH 7.0) showed low pharmacological availability (3.2%). Coadministration with bestatin (aminopeptidase inhibitor, 0.0 1– 1 mM) or diprotinin A (dipeptidyl peptidase inhibitor, 0.1-1 mM) did not change the hypocalcemic effects. Coadministration with aprotinin (trypsin inhibitor, 10 3 -10 4 KIU/ml), camostat mesilate (aminopeptidase and trypsin inhibitor, 0.1 – 10 mM) or leupeptin (trypsin and cathepsin B inhibitor, 0.1 – 1 mM) enhanced the hypocalcemic effects and, thus, the nasal absorption of SCT. The hypocalcemic effects of SCT at various pH values (pH 4.0, 7.0 and 8.0) with or without aprotinin were the highest at pH 4.0. The pharmacological availabilities after nasal administration of SCT (10 IU/kg) at pH 4.0 and 7.0 were increased from 5.4 to 7.5% and from 3.2 to 6.9% by aprotinin (10 4 KIU/ml), respectively. Therefore, inhibitors which have a trypsin inhibitory activity are useful for enhancing nasal absorption of SCT.

Effects of proteolytic enzyme inhibitors on the nasal absorption of vasopressin and an analogue.

Proteolytic enzyme inhibitors were examined as absorption enhancers for the nasal delivery of vasopressin (AVP) and desmopressin (l-d-8-DAVP) in rats. Aprotinin, soybean trypsin inhibitor, and camostat mesilate were used as enzyme inhibitors. The nasal absorption of AVP and l-d-8-DAVP was evaluated by measuring its antidiuretic effect. Nasal administration of AVP (0.005 IU/kg) or l-d-8-DAVP alone (2.5 ng/kg) produced a small antidiuretic effect. Coadministration with aprotinin (1000 and 10000 KIU/kg) or soybean trypsin inhibitor (1.25 and 6.25 mM) did not change the antidiuretic effect. However, coadministration with camostat mesilate (1 to 50 mM) significantly increased the antidiuretic effect and, thus, the nasal absorption of AVP and l-d-8-DAVP. The activities of aminopeptidase, cathepsin-B, and trypsin in the nasal mucosal tissue of rats were 7 nmol/min/mg protein, 0.7 nmol/min/mg protein, and 4.6 pmol/min/mg protein, respectively. Aprotinin and soybean trypsin inhibitor inhibited only the trypsin activity, whereas camostat mesilate inhibited aminopeptidase and trypsin activities. Aprotinin (MW 6500) and soybean trypsin inhibitor (MW 8000), with relatively high molecular weights, may not permeate into the nasal mucosal tissue. In contrast, camostat mesilate is slowly absorbed (8%/hr) and could inhibit the proteolytic activity in the nasal mucosa, resulting in enhanced nasal absoprtion of AVP and l-d-8-DAVP.

Effects of Proteolytic Enzyme Inhibitors as Absorption Enhancers on the Transdermal Iontophoretic Delivery of Calcitonin in Rats

Abstract— The effects of proteolytic enzyme inhibitors, aprotinin, soybean trypsin inhibitor and camostat mesilate as absorption enhancers on the transdermal iontophoretic delivery of salmon calcitonin (SCT) have been examined in rats. The dermal absorption of SCT was evaluated with hypocalcaemic effect. Application of SCT (12·5 int. units/rat) onto abdominal skin did not produce any hypocalcaemic effect. This produced a small hypocalcaemic effect with cationic iontophoresis (drug phase, anode; reference phase, cathode; high frequency pulses of 1 V at 10 kHz, 2h). Furthermore, camostat mesilate (1 Mm) and aprotinin (106 int. units mL−1) enhanced the hypocalcaemic effects on the application of SCT with iontophoresis. These hypocalcaemic effects were highest with the pH 4·0 preparation compared with those of the pH 5·5, pH 7·0 and pH 8·0 preparations. However, soybean trypsin inhibitor did not change the hypocalcaemic effects. This was because the soybean trypsin inhibitor is a relatively high molecular weight peptide (mol. wt 8000) and an anion at used pH, and therefore was not absorbed through rat skins with cation iontophoresis.

Evaluation of Polyvinyl Alcohol Hydrogel as a Sustained-Release Vehicle for Rectal Administration of Indomethacin

In order to evaluate an indomethacin poly vinyl alcohol (PVA) hydrogel for rectal administration, the in vitro release characteristics of indomethacin from the hydrogel and indomethacin plasma concentrations after rectal administration were examined. The PVA hydrogel containing indomethacin was prepared by a low-temperature crystallization method. The release of indomethacin from the PVA hydrogel agreed with the Fickian diffusion model for 10 hr. Rectal administration of indomethacin hydrogels to rats yielded high indomethacin plasma concentrations, without producing a sharp peak, and a sustained-release effect. In dogs, the indomethacin hydrogel produced a similar sustained-release effect; however, the indomethacin plasma concentration was relatively low compared with that of an indomethacin suppository.

Evaluation of permeability enhancement of hydrophilic compounds and macromolecular compounds by bile salts through rabbit corneas in-vitro

The effect of bile salts, sodium taurocholate (TC‐Na) and sodium taurodeoxycholate (TDC‐Na), on the permeability of hydrophilic compounds and macromolecular compounds through the rabbit cornea in‐vitro was examined. 6‐Carboxyfluorescein and glutathione were used as low molecular weight hydrophilic model compounds and FITC‐dextran (mol. wt 4000) and insulin were used as relatively macromolecular model compounds. TC‐Na (2 and 10 mM) marginally increased the corneal permeabilities to hydrophilic compounds and macromolecular compounds. TDC‐Na (2 and 10 mM) markedly increased the corneal permeabilities of these compounds.

Enhancing Effects of Unsaturated Fatty Acids with Various Structures on the Permeation of Indomethacin through Rat Skin

Effects of straight‐chain, cis‐type, unsaturated fatty acids with various structures (alkyl chain lengths, numbers of double bonds, position of double bonds and cis‐ and trans‐positional isomers) on the skin permeation of indomethacin were examined by using rat skins in‐vitro. Furthermore, the disordering degrees of the intercellular lipid domain in the stratum corneum, which were treated with preparations of unsaturated fatty acids, were measured by the Fourier transform infrared (FT‐IR) method using excised rabbit ear skins.

Effects of proteolytic enzyme inhibitors of enhancement of transdermal iontophoretic delivery of vasopressin and an analogue in rats

Abstract The effects of the proteolytic enzyme inhibitors, aprotinin, soybean trypsin inhibitor and camostat mesilate, as absorption enhancers of transdermal iontophoretic delivery of vasopressin (AVP) and its analogue, 1-deamino-8- d -arginine vasopressin (1-d-8-DAVP) were examined in rats. The dermal absorption of AVP and 1-d-8-DAVP was determined to induce an antidiuretic effect. Administration of AVP (0.5 IU/rat) or 1-d-8-DAVP (0.1 μg/rat) on abdominal skin (available diffusion area 3.14 cm 2 ) produced no antidiuretic effect. This resulted in a slight antidiuretic influence with iontophoresis (drug phase, anode; reference phase, cathode; constant current of 1 mA 3.14 cm 2 ). Moreover, the antidiuretic effect was further enhanced on application of iontophoresis and camostat mesilate (1–50 mM). However, aprotinin and soybean trypsin inhibitor had no influence on the antidiuretic effect. The activities of aminopeptidase, cathepsin B and trypsin in dermal tissue were determined to be 1.7 nmol/min per mg protein, 0.5 nmol/min per mg protein and 2.4 pmol/min per mg protein, respectively. Camostat mesilate significantly inhibited the activities of aminopeptidase and trypsin, whilst aprotinin and soybean trypsin inhibitor resulted in considerable inhibition of the activity of trypsin. Aprotinin (Mol. Wt 6500) and soybean trypsin inhibitor (Mol. Wt 8000), both having a relatively high molecular weight, may not permeate into the dermal tissue. In contrast, a small degree of absorption of camostat mesilate (about 3% 2 h) with iontophoresis might inhibit the proteolytic enzyme activity in dermal tissue and hence might enhance the dermal absorption of AVP and 1-d-8-DAVP.

Influence of absorption enhancers (bile salts) and the preservative (benzalkonium chloride) on mucociliary function and permeation barrier function in rabbit tracheas

Effects of bile salts and benzalkonium chloride on the ciliary beat activity (frequency) and the mucociliary transport rate of tracheas, and permeabilities of 6-carboxyfluorescein (6-CF; Mw 376) and FITC-dextrans (FD-4; Mw 4300) as hydrophilic compounds through trachea were investigated for intratracheal delivery of drugs. Ciliary beat activity was measured by an in vitro photoelectric method by using excised rabbit tracheas. Transport rate was determined by an in vivo endoscopic method in rabbit tracheas. Permeation test was carried out by using excised rabbit tracheas. The ciliary beat activities and mucociliary transport rates were not significantly affected by 1, 10 and 20 mM sodium glycocholate, while they were significantly decreased by 1 and 10 mM sodium taurodeoxycholate and immediately halted after application of 20 mM taurodeoxycholate. They were not significantly affected by 0.02% of benzalkonium chloride, whereas they were completely halted by 0.05% of benzalkonium chloride. The apparent permeability coefficients (Papp) of 6-CF and FD-4 were 2.35x10(-7) and 2.22x10(-8) cm/s, respectively. One mmol of glycocholate did not significantly increase the Papp of 6-CF and FD-4, while, 10 mM of glycocholate and 1 and 10 mM of taurodeoxycholate significantly increased the Papp. Benzalkonium chloride (0.01%) did not significantly increase the Papp of 6-CF, whereas 0.03% significantly increased the Papp of 6-CF. Benzalkonium chloride (0.01 and 0.03%) slightly increased the Papp of FD-4. Therefore, sodium glycocholate may be a safe and useful absorption enhancer for intratracheal delivery of drugs.