Zezhi Shao

Pulmonary Delivery of Free and Liposomal Insulin

The effects of oligomerization and liposomal entrapment on pulmonary insulin absorption were investigated in rats using an intratracheal instillation method. The results indicated that both dimeric and hexameric insulins can be rapidly absorbed into the systemic circulation, producing a significant hypoglycemic response. Intratracheal instillation of insulin in two different oligomerized states has not resulted in any significant difference in the duration of hypoglycemic effect. However, the initial hypoglycemic response (first 10 min) obtained from intratracheal administration of 25 IU/kg hexameric insulin appears to be slower than that from the 25 IU/kg dimeric insulin, thereby suggesting that hexameric insulin may have a lower permeability coefficient across alveolar epithelium than the dimeric insulin. Intratracheal administration of insulin liposomes (dipalmitoylphosphatidyl choline:cholesterol, 7:2) led to facilitated pulmonary uptake of insulin and enhanced the hypoglycemic effect. Nevertheless, similar insulin uptake and pharmacodynamic response were obtained from both the physical mixture of insulin and blank liposomes and liposomally entrapped insulin.

Cyclodextrins as Nasal Absorption Promoters of Insulin: Mechanistic Evaluations

The safety and effectiveness of cyclodextrins (CD) as nasal absorption promoters of peptide-like macromolecules have been investigated. The relative effectiveness of the cyclodextrins in enhancing insulin nasal absorption was found to be in the descending order of dimethyl-β-cyclodextrin (DMβCD) > α-cyclodextrin (α-CD) > β-cyclodextrin (β-CD), hydroxypropyl-β-cyclodextrin (HPβCD) > γ-cyclodextrin (γ-CD). A direct relationship linking absorption promotion to nasal membrane protein release is evident, which in turn correlates well with nasal membrane phospholipid release. The magnitude of the membrane damaging effects determined by the membrane protein or phospholipid release may provide an accurate, simple, and useful marker for predicting safety of the absorption enhancers. In order to estimate further the magnitude of damage and specificity of cyclodextrin derivatives in solubilizing nasal membrane components, the enzymatic activities of membrane-bound 5′-nucleotidase (5′-ND) and intracellular lactate dehydrogenase (LDH) in the perfusates were also measured. HPβCD at a 5% concentration was found to result in only minimal removal of epithelial membrane proteins as evidenced by a slight increase in 5′-ND and total absence of LDH activity. On the other hand, 5% DMβCD caused extensive removal of the membrane-bound 5′-ND. Moreover, intracellular LDH activity in the perfusate increased almost linearly with time. The cyclodextrins are also capable of dissociating insulin hexamers into smaller aggregates, and this dissociation depends on cyclodextrin structure and concentration. Enhancement of insulin diffusivity across nasal membrane through dissociation may provide an additional mechanism for cyclodextrin promotion of nasal insulin absorption.

Cyclodextrins as Mucosal Absorption Promoters of Insulin. II. Effects of β-Cyclodextrin Derivatives on α-Chymotryptic Degradation and Enteral Absorption of Insulin in Rats

The relative effectiveness of two β-cyclodextrin derivatives, i.e., dimethyl-β-cyclodextrin (DMβCD) and hydroxypropyl-β-cyclodextrin (HPβCD), in enhancing enteral absorption of insulin was evaluated in the lower jejunal/upper ileal segments of the rat by means of an in situ closed loop method. The incorporation of 10% (w/v) DMβCD to a 0.5 mg/ml porcine-zinc insulin solution dramatically increased insulin bioavailability from a negligible value (~0.06%) to 5.63%, when administered enterally at a dose of 20 U/kg. However, addition of 10% (w/v) HPβCD did not improve enteral insulin uptake significantly with a bioavailability of only 0.07%. Similarly, the pharmacodynamic relative efficacy values obtained after the enteral administration of 20 U/kg insulin, 20 U/kg insulin with 10% HPβCD, and 20 U/kg insulin with 10% DMβCD were 0.24%, 0.26%, and 1.75%, respectively. Biodegradation studies of 0.5 mg/ml insulin hexamers by 0.5 µM α-chymotrypsin revealed no inhibitory effect on the enzymatic activity by the two cyclodextrins. On the contrary, the apparent first-order rate constant increased significantly in the presence of 10% DMβCD, suggesting insulin oligomer dissociation by DMβCD. Histopathological examination of the rat intestine was performed to detect tissue damage following enteral administration of the β-cyclodextrin derivatives. Light microscopic inspection indicated no observable tissue damage, thereby arguing direct membrane fluidization as the primary mechanism for enhanced insulin uptake. This study indicates the feasibility of using cyclodextrins as mucosal absorption promoters of proteins and peptide drugs.

Nasal membrane and intracellular protein and enzyme release by bile salts and bile salt-fatty acid mixed micelles : correlation with facilitated drug transport

The effects of four bile salts, one fusidate derivative, and one mixed micellar formulation of bile salt-fatty acid combination on the nasal mucosal protein and enzyme release have been investigated in rats using an in situ nasal perfusion technique. Deoxycholate (NaDC) was found to possess the maximum protein solubilizing activity, followed by taurodihydrofusidate (STDHF), cholate, glycocholate (NaGC), and taurocholate (NaTC) in a descending order. The difference in protein solubilization of NaDC and NaGC was further characterized by the release of 5′-nucleotidase (5′-ND), a membrane-bound enzyme, and lactate dehydrogenase (LDH), an intra-cellular enzyme, in the perfusate. While both NaDC and NaGC caused comparable 5′-ND release from nasal membrane, intracellular LDH release was significantly higher with NaDC. The greater protein and LDH solubilizing effects of NaDC corresponded well with its faster rate of disappearance from the nasal perfusate. Therefore, the dihydroxy bile salt NaDC tends to cause intracellular damage and cell lysis, whereas the trihydroxy bile salt NaGC appears to produce primarily mucosal membrane perturbations. Linoleic acid in the form of soluble mixed micelles with glycocholate caused a further increase in nasal protein release. However, the rate and extent of nasal membrane protein release by the mixed micelles composed of 15 mM glycocholate and 5 mM linoleic acid were significantly lower than those caused by either deoxyholate or STDHF at the same concentrations. Nasal absorption of acyclovir, a non-absorbable hydrophilic model antiviral agent, was found to be enhanced in the presence of conjugated trihydroxy bile salts and bile salt-fatty acid mixed micelles. A nonlinear correlation exists between first-order nasal absorption rate constant and nasal protein release rate.

Effects of formulation variables on nasal epithelial cell integrity: Biochemical evaluations

Abstract The effects of pH, osmolarity, type and concentration of buffers on the nasal mucosal epithelium have been investigated in rats using an in situ nasal perfusion technique. Traditionally, histological approaches which are qualitative and not predicative of nasal mucosal sensitivity, have been used to assess the damage to the nasal mucosa. A biochemical approach has been used in this report to assess irritation to the nasal mucosa which may provide a priori indication of nasal sensitivity to chronic use of nasal formulations. The nasal mucosal irritation may be predicted by determining the amount of total protein and two enzymes, lactate dehydrogenase (LDH, EC 1.1.1.27), a cytosolic enzyme and 5′-nucleotidase (5′-ND, EC 3.1.3.5), a membrane-bound enzyme released during perfusion. To determine the effect of pH on the nasal mucosa, phosphate buffers ranging in pH from 2 to 12 were utilized. Solutions within a pH range of 3–10 caused minimal release of the biochemical markers whereas solutions of pH above 10 caused significant membrane and intracellular enzyme release. Acetate buffers (pH 4.75) at three different concentrations, 0.07, 0.14 and 0.21 M, were used to study the effect of buffer concentration on the nasal mucosal integrity. The results indicate that the alteration to the nasal mucosal cells by buffers is concentration dependent. To study the effect of buffer type, four different buffers, i.e., acetate, adipate, citrate, and phosphate (0.07 M, pH 4.75) were studied. The acetate buffer was found to have the most irritation potential when compared to adipate, citrate, and phosphate buffers. To determine the effects of unionized and ionized species of a buffer, 0.025 M benzoate buffers at pH 3.2 and 5.2 were studied. The results indicate that the unionized species of benzoic acid causes more cellular perturbation than the ionized species. Hypertonic and isotonic sodium chloride solutions caused minimal mucosal cell aberrations while hypotonic solutions caused extensive leakage of LDH. These results along with other results from our laboratory may help in designing well tolerated nasal formulations for chronic use.

The physicochemical properties, plasma enzymatic hydrolysis, and nasal absorption of acyclovir and its 2'-ester prodrugs.

A series of 2′-(O-acyl) derivatives of 9-(2-hydoxyethoxymethyl)guanine (acyclovir) was synthesized by acid anhydride esterification. Aqueous solubilities in isotonic phosphate buffer (pH 7.4), partition coefficients in 1-octanol/phosphate buffer, and hydrolysis kinetics in rat plasma were determined. The ester prodrugs showed consistent increases in lipophilicity with corresponding decreases in aqueous solubility as a function of side-chain length. The bioconversion kinetics of the prodrugs appear to depend on both the apolar and the steric nature of the acyl substituents. When perfused through the rat nasal cavity using the in situ perfusion technique, acyclovir showed no measurable loss from the perfusate. Nasal uptake of acyclovir prodrugs, on the other hand, were moderately improved. Furthermore, the extent of nasal absorption appears to depend on the lipophilicity of the prodrugs in the descending order hexanoate > valerate > pivalate > butyrate. Simultaneous prodrug cleavage by nasal carboxylesterase was also noted in the case of hexanoate.

Differential Effects of Anionic, Cationic, Nonionic, and Physiologic Surfactants on the Dissociation, α-Chymotryptic Degradation, and Enteral Absorption of Insulin Hexamers

Various surfactants were investigated to compare their effects on insulin dissociation, α-chymotryptic degradation, and rat enteral absorption. With a circular dichroism technique, sodium dodecyl sulfate (SDS) at a 5 mM concentration was found to completely dissociate procine-zinc insulin hexamers (0.5 mg/ml) into monomers. The catalytic activity of α-chymotrypsin (0.5 µM) was also abolished by 5 mM SDS. When insulin was injected into the distal jejunum/ proximal ileum segment of the rat, 5 mM SDS greatly enhanced its pharmacological availability, from a negligible value to 2.8%. Being a cationic surfactant, hexadecyl trimethylammonium bromide (CTAB) also efficiently dissociated insulin hexamers at concentrations of 1–5 mM. However, extensive charge–charge interaction was observed below a CTAB concentration of 0.6 mM, leading to insulin precipitation at a molar CTAB:insulin ratio of 1:1 to 2:1. An α-chymotryptic degradation study also revealed near-complete dissociation of insulin hexamers at 1 mM CTAB. Above 1 mM, however, CTAB acted as an enzyme inhibitor, most likely by means of charge repulsion. Enteral absorption studies showed a much lower pharmacological availability, only 0.29%. Nonionic surfactants such as Tween 80 and polyoxyethylene 9 lauryl ether were ineffective in dissociating insulin hexamers. Tween 80, at 5 mM, neither significantly altered the α-chymotryptic degradation pattern nor enhanced the enteral absorption of insulin. The relative effectiveness of different species of bile salts on insulin hexamer dissociation appeared to be similar. Sodium glycocholate at a 30 mM concentration also significantly increased insulin pharmacological availability, to 2.3%. A morphological study did not reveal any significant alteration of the rat intestinal mucosal integrity after exposure to 5 mM SDS for 30 min. The results further emphasize the importance of the degree of insulin aggregation on its enteral transport.

Dissociation of Insulin Oligomers by Bile Salt Micelles and Its Effect on α-Chymotrypsin-Mediated Proteolytic Degradation

Bile salts have been found to be effective absorption promoters of insulin across mucosal barriers, i.e., nasal and gastrointestinal. One of the mechanisms proposed for absorption enhancement is the dissociation of insulin oligomers to monomers, rendering a higher insulin diffusivity. α-Chymotryptic degradation and circular dichroism studies were used to characterize such a transition. When zinc insulin (hexamers) and sodium insulin (dimers) were subjected to α-chymotryptic degradation, a 3.2-fold difference in the apparent first-order rate constants was observed (zinc insulin being slower than sodium insulin), representing the intrinsic difference in the concentration of total associated species in solution (three times). In the presence of a bile salt, sodium glycocholate (NaGC), the rate of degradation of both zinc and sodium insulin increased in an asymptotic manner. A maximum increase of 5.4-fold was observed for zinc insulin at a 30 mM NaGC concentration and a 2.1-fold increase was noted for sodium insulin at 10 mM NaGC, both values being close to the theoretical numbers of 6- and 2-fold as predicted by the complete dissociation of hexamers and dimers to monomers. The result indicates dissociation of insulin oligomers to monomers by bile salt micelles, probably by hydrophobic micellar incorporation of monomeric units. Circular dichroism studies also revealed progressive attenuation of molecular ellipticities at negative maxima of 276, 222, and 212 nm for zinc insulin solution in the presence of NaGC. Therefore, both α-chymotryptic degradation and circular dichroism studies have consistently demonstrated that the bile salts may be capable of dissociating insulin oligomers to monomers, a fact which may play an important role in enhancing insulin bioavailability.

Bile salt-fatty acid mixed micelles as nasal absorption promoters. III: Effects on nasal transport and enzymatic degradation of acyclovir prodrugs

The absorption enhancement and presystemic degradation kinetics of a homologous series of acyclovir 2′-ester prodrugs were investigated in rats using the in situ nasal perfusion technique in the presence of bile salt–fatty acid mixed micells. In vitro incubation studies indicated that nasal perfusate containing a mixed micellar solution generated higher ester-cleaving activity than isotonic phosphate buffer washings. Inhibitor screening and substrate specificity studies demonstrated the enzyme to be most likely carboxylesterase rather than true cholinesterase. The extent of prodrug cleavage by the carboxylesterase appears to correlate well with the substrate li-pophilicity for esters with linear acyl chains. On the other hand, branching of the acyl side chain significantly retards acyclovir pro-drug breakdown. To estimate the nasal epithelial membrane and cytoplasmic damaging effect caused by sodium glycocholate (NaGC)–linoleic acid (15 mM:5 mM) mixed micelles, the release profiles of 5′-nucleotidase (5′-ND), lactate dehydrogenase (LDH), and carboxylesterase in the nasal perfusate were measured as a function of time. The results indicated that the activities of all three enzymes resulting from the mixed micellar solution appeared to be significantly higher than those caused by 15 mM NaGC alone. The apparent nasal absorption rate constants of acyclovir and its butyrate, valerate, pivalate, and hexanoate ester prodrugs in mixed micellar solutions containing an esterase inhibitor (1 mM phenylmethylsulfonyl fluoride) were individually calculated. Without an inhibitor, lengthening of the linear acyl side chain of the prodrug resulted in greatly accelerated degradation coupled with moderate absorption improvement. The solubilities and micellar binding constants of acyclovir prodrugs were also determined. Mixed micelles composed of 15 mM NaGC and 5 mM linoleic acid are incapable of incorporating these esters into the micellar cavity, although NaGC micelle alone can actively solubilize them in a concentration-dependent manner.

Acyclovir permeation enhancement across intestinal and nasal mucosae by bile salt-acylcarnitine mixed micelles.

The purpose of this study was to investigate the absorption enhancement of acyclovir, an antiviral agent, by means of bile salt-acylcarnitine mixed micelles. The specificity, site dependence, palmitoyl-DL-carnitine chloride (PCC) concentration dependence, and effects of absorption promoters on acyclovir absorption via the nasal cavity (N) and four different intestinal segments of the rat, i.e., duodenum (D), upper jejunum (UJ), combined lower jejunum and ileum (LJ), and colon (C) were evaluated. The present study employed the rat in situ nasal and intestinal perfusion techniques and utilized sodium glycocholate (NaGC), three acylcarnitines, and their mixed micelles as potential nasal and intestinal absorption promoters. Acylcarnitines used were DL-octanoylcarnitine chloride (OCC), palmitoyl-DL-carnitine chloride (PCC), and DL-stearoylcarnitine chloride (SCC). All acylcarnitines and NaGC by themselves produced negligible enhancement of acyclovir absorption in the rat intestine, while OCC and SCC were totally ineffective in the nasal cavity. However, the mixed micellar solutions of NaGC with PCC or SCC could significantly increase the mucosal membrane permeability of acyclovir in the colon and nasal cavity. On the other hand, NaGC-OCC mixed micelles slightly increased the absorption of acyclovir by both routes. When a mixed micellar solution of NaGC with PCC was used, the rank order of apparent acyclovir permeability (Papp; cm/sec), corrected for surface area of absorption, was N (10.54 ± 0.62 x 10−5) > D (6.82 ± 0.30 x 10−5) > LJ (2.90 ± 0.08 x 10−5) > C (2.54 ± 0.14 x 10−5) > UJ (2.30 ± 0.22 x 10−5). In contrast, the Papp rank order for acyclovir without any absorption promoter was D (2.49 ± 0.44 x 10−5) > UJ (0.64 ± 0.03 x 10−5) > LJ, C, and N (0). The effect of mixed micellar solutions was synergistic and was much greater than that with single adjuvants probably because of micellar solubilization of acylcarnitines by NaGC. The magnitude of absorption promotion was dependent on the hydrophobicity, i.e., carbon-chain length of the acylcarnitines. The enhanced permeability could be reversed within 60-120 min after removal of the adjuvant from the duodenum, colon, and nasal cavity. These results suggest that bile salt-acylcarnitine mixed micelles can be used as intestinal or nasal mucosal absorption promoters of poorly permeable agents.