Preparation of molecularly imprinted cyclodextrin microspheres.

Molecularly imprinted cyclodextrins (MI-CDs) are prepared by cross-linking CDs in the presence of a template molecule. The binding ability of MI-CDs to the template molecule is specific; therefore, MI-CDs will prove to be useful materials. In this study, we prepared microspheres of MI-CDs (MSs-MI-CD) in a dimethylsulfoxide/poly(dimethylsiloxane) (PDMS) emulsion, using cholesterol as the template molecule. MSs-MI-CD were prepared under various conditions and were evaluated with respect to their morphology, size, and binding ability. MSs-MI-CD prepared at 65 degrees C were in an aggregated form; however, we could prepare separated and uniform MSs-MI-CD at 95 degrees C. The viscosity of PDMS influenced the size of MSs-MI-CD. The mean particle diameters of MSs-MI-CD prepared with 50 and 1000 mm(2)/s PDMS were 146 and 43 microm, respectively. The binding ability of MSs-MI-CD to cholesterol was higher than that of non-imprinted microspheres. Cholesterol imprinting also promoted the binding ability to other steroids; however, the increase in binding ability was most remarkable in the case of cholesterol, suggesting that we successfully introduced the cholesterol-specific binding ability into MSs-MI-CD. The novel MSs-MI-CD preparation method is useful and simple, and it will provide opportunities for further studies on the specific binding ability of MI-CDs.

Effect of application volume of ethanol-isopropyl myristate mixed solvent system on permeation of zidovudine and probenecid through rat skin.

Permeation of zidovudine (AZT) and probenecid from an ethanol-isopropyl myristate (IPM) mixed system through rat skin was studied in a finite system. Several volume sizes of the ethanol-IPM mixed systems containing AZT and probenecid, both as suspensions, were applied on the skin of the hairless rat using a vertical glass cell, and the fractions of the drugs permeated in 8 hr Q%,8 hr were determined. For the systems containing 40% ethanol, the Q%,8 hr value decreased with the reduction of volume of the system applied, and the decreasing profile was similar to that calculated on the assumption that the permeability of the drug does not change with the volume of the sample applied. On the other hand, in the systems containing 10% or 20% ethanol, the Q%,8 hr value showed a maximum when a specific volume of the sample was applied. Therefore, the effect of sample volume on the Q%,8 hr value was different between the 40% ethanol-IPM system and the 10% or 20% ethanol-IPM system. Following pretreatment of the skin with 0.105 ml/cm2 of drug-free 40% ethanol-IPM for 2 hr, several volume sizes of 10% ethanol-IPM systems containing the drugs were applied on the skin to explain why the different profiles were observed in the system containing 10% or 20% ethanol. The results for pretreated skin suggest that the amount of ethanol in the systems with low ethanol concentration and small application volume is too small to exert an effect that enhances permeation of the drugs. In those systems, the integrated effect of ethanol on the skin would be important for the enhancing effect. Total volume, as well as concentration, of an enhancer should be set precisely in designing an efficient transdermal delivery system.

Investigation of the release behavior of diethylhexyl phthalate from the polyvinyl-chloride tubing for intravenous administration.

The release behavior of diethylhexyl phthalate (DEHP) from a polyvinyl-chloride (PVC) tube, which is part of an intravenous administration set, was investigated with the coexistence of polysorbate 80 (Tween 80) in various solutions such as physiological saline (PS), distilled water for injection (DWI) and glucose solution (TZ). The cumulative amount of DEHP released after 5 h was in the following order; PS, DWI > 50% TZ. From a comparison of the amount of released DEHP and the critical micelle concentration (CMC) of various solutions, the lower the CMC of the solution, the higher the amount of DEHP released from the PVC tubing. When the concentration of Tween 80 was kept constant at 1 mg/ml, the cumulative amount of DEHP released with a flow rate 90 ml/h was higher than that at 60 ml/h. These results suggest that the release of DEHP from the PVC tubing is closely correlated with the interaction of Tween 80 and DEHP such as the formation of micelles, the collision of micelles against the surface of the PVC tubing and the diffusion properties of DEHP and or Tween 80 in the liquid medium.

Analysis of skin disposition of flurbiprofen after topical application in hairless rats.

Cutaneous disposition of topically applied flurbiprofen (FP) was evaluated using a new in situ experimental model in hairless rats. A disk-shaped agar gel (3.85 cm in diameter and 0.5 cm in thickness) was subcutaneously implanted in the abdominal region of rats as a drug receptor, and a drug donor cell was subsequently placed above this agar gel. No significant pharmacokinetic modification of FP was observed as a result of this experimental procedure. A bolus injection and a constant intravenous infusion of FP were applied to the rats, followed by an analysis of FP levels in the plasma and agar gels. Using these results, the clearance rate of FP from the systemic circulation to the gel could be calculated. FP (1% gel formulation, 1.0 g/3.14 cm(2)) was then topically applied to the skin of these rats. From these experiments, the amount of FP that migrated from the formulation to the systemic circulation and the amount of FP that migrated directly to the agar gel across the skin, over 10 h, were separately evaluated to be 235.4 and 2.0 microg, respectively. Thus, most of the FP was absorbed into the systemic circulation. The effect of endogeneous vasoactive compounds and penetration enhancers on the FP disposition within skin was also determined. Epinephrine and bradykinin were used as vasoactive compounds that were entrapped in agar gel, and an isopropyl myristate system (IPM system) and a l-menthol-ethanol-glycerin-water system (MEGW system) were used as enhancers in the formulation. Epinephrine enhanced the direct delivery of FP into the agar gel to more than ten times its former level, in spite of the fact that it had no effect on systemic delivery. Bradykinin strengthened systemic delivery slightly, without changing the quantity of FP in the gel. IPM increased only the systemic delivery of FP, as was the case with bradykinin, whereas the MEGW system markedly increased both the blood concentration and the quantity of FP in the gel (13 and 200 times, respectively). This technique has proven to be an effective tool for the quantitative evaluation of cutaneous disposition of a topically applied drug.

Skin disposition of drugs after topical application in hairless rats.

Drug fraction transported from a topical formulation on skin to subcutaneous tissues or muscles is dependent on the physicochemical properties of the entrapped drug. Cutaneous disposition of model drugs, antipyrine (ANP), lidocaine (LC) and piroxicam (PXC) as well as flurbiprofen (FP) was thus evaluated in hairless rats in which an agar gel disc was subcutaneously inserted into the abdominal region as a drug receptor and a drug donor cell was placed above it. Time courses of plasma level and agar gel amount were measured after topical application of 50% ANP, 3% LC, 1% PXC and 1% FP in hydroxypropylcellulose gel. Percutaneous absorption clearance of unionized form, CLab* was proportional to true octanol/water distribution coefficient and the order of FP > PXC > LC > ANP, suggesting that skin permeation of the drug was determined mainly by its distribution from the formulation to the skin barrier. PXC, however, had a relatively low flux compared to the other three drugs, probably due to its high molecular weight and melting point. Migration clearance of unionized form from systemic circulation to the subcutaneous agar gel, CLg* was also influenced by the lipophilicity of drugs. On the other hand, fraction from the formulation to the systemic circulation was in the order of PXC > FP > ANP > LC. This fraction was much higher than the direct migration fraction from the formulation to the subcutaneous agar gel. Factors determining for these fractions are still unclear. A drug having a low lipophilicity and a low protein binding, however, had a tendency to have a great targeting ability to the subcutaneous agar gel. In addition, most of the drug in the agar gel was contributed by the direct flow from formulation, not from the systemic circulation. The present in situ experimental method is a useful tool to evaluate skin disposition of drugs. Detailed understanding of the skin disposition of drugs from several formulations will enable the findings of a good drug and formulation candidates.

Effect of several hydrophilic polymers on the permeation of morphine and salicylic acid through excised hairless rat skin.

Several hydrophilic polymers changed the cumulative amount of morphine (MOR) permeated through excised hairless rat skin from 1% MOR hydrochloride solution containing ethanol and l-menthol at concentrations of 40% and 5%, respectively, as permeation enhancers. Anionic polymers (carboxyvinylpolymer and methylvinylether-maleic anhydride copolymer) in the test solutions decreased the skin permeation of MOR, whereas cationic polymers (polyethyleneimine and chitosan) increased it, compared with that without polymers. Little change, however, was observed by the addition of nonionic polymers (hydroxypropylcellulose and polyethyleneoxide). On the other hand, the cationic and anionic polymers in the test solutions decreased and increased, respectively, the skin permeation of salicylic acid (SA) from the same enhancing system containing sodium salicylate. These opposite results were probably caused by the change in escaping tendency of the drugs from the vehicles, which was due to the drug-polymer interaction. (The escaping tendency has a great effect on the drug partition from the polymer solution to the skin barrier). The effect of hydrophilic polymers on the partition was then evaluated by Donnan membrane theory. The partition of MOR was increased and decreased by the presence of polymers having identical and opposite charge to MOR. The low partition of the drugs to skin may also be caused by low diffusion of the drugs in the polymer solutions. The drug release from the hydrophilic polymer solutions was then measured, and the release rate was found to have decreased in the presence of polymers having opposite charge to MOR and SA. It is suggested that these drug-polymer interactions changed the drug partition to skin thus changing the skin permeation of the drug.

Direct transport of 2',3'-didehydro-3'-deoxythymidine (D4T) and its ester derivatives to the cerebrospinal fluid via the nasal mucous membrane in rats.

We investigated the absorption and transport of 2',3'-didehydro-3'-deoxythymidine (D4T) and its ester prodrugs from the nasal cavity in rats. The absorption of D4T and its acetate (C2-D4T) was rapid and almost complete, although the hemi-succinate (Suc-D4T) was absorbed rather slowly; the plasma concentrations of the prodrug, Suc-D4T, and regenerated D4T remained unchanged throughout the experimental period (180 min). Concentrations in the cerebrospinal fluid (CSF) following intravenous (i.v.) and intranasal (i.n.) administration were also measured. After i.n. administration, drug concentrations were higher in the fraction derived from the subarachnoid space located close to the nasal mucosa than those in the fractions located far from the nasal cavity. This difference was not found following the i.v. administration of the drugs. Following nasal administration, the intact Suc-D4T was found in the CSF at a concentration higher than that of D4T, although transport of the intact prodrug to the CSF was not observed following i.v. administration. These results suggest that direct transport of the drugs from the nasal cavity to the CSF significantly contributes to the higher concentrations in CSF of D4T and/or its ester prodrugs, and indicate the possible value of nasal administration for the treatment of patients with AIDS dementia.

Nasal absorption of 2',3'-didehydro-3'-deoxythymidine (D4T) and its esters in rats.

Nasal absorption of 2',3'-didehydro-3'-deoxythymidine (D4T) and its esters (5'-acetyl D4T: C2-D4T and 5'-hemisuccinyl D4T: Suc-D4T) was investigated in rats. Bioavailability of D4T following intranasal (i.n.) administration was 98.0%, and the elimination from plasma was as rapid as that following intravenous administration of D4T. There seemed to be complete and rapid absorption of D4T from the nasal cavity. The plasma concentration-time profile of D4T following i.n. administration of C2-D4T was almost the same as that after administration of D4T itself. This suggests that C2-D4T was rapidly absorbed from the nasal cavity, and that some amount of dosing C2-D4T was hydrolyzed to D4T before entering the systemic circulation. In contrast, Suc-D4T showed slower absorption in the i.n. administration, and the plasma D4T level was maintained for a long period.

Intestinal absorption and first-pass elimination of 2', 3'-dideoxynucleosides following oral administration in rats.

Intestinal absorption and first-pass elimination of 2',3'-dideoxynucleosides (ddNs), including 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (DDI) and 2',3'-didehydro-3'-deoxythymidine (D4T), following oral administration was investigated in rats. Enzymatic degradation of ddNs in rat intestinal washing and in the intestinal homogenate showed them to be stable in the washing with half lives of more than 140 h, whereas degradation of DDI in the intestinal homogenate was more than ten times as rapid as those of AZT and D4T. Intestinal absorption was studied in three segments of the rat intestine (duodenum, jejunum and colon) using an in situ closed-loop method. The area under plasma ddN concentration curve (AUC) and the residual percent of dose 1 h after dosing indicated a greater absorption of AZT and D4T in the upper intestinal tract than in the colon, very poor absorption of DDI in all segments, and considerable absorption of AZT in the colon. The AUC and the mean residence time (MRT) of ddNs following four different routes (intravenous: i.v., intra portal vein: i.p.v., intra duodenal: i.d. and intra gastric: i.g.) were measured using the in viva multiple sites of input method in rats. AZT and D4T were rapidly absorbed from the gastrointestinal tract and their bioavailability was more than 90%. DDI was less absorbed (33.02%) following i.d. administration compared with AZT and D4T. This poor absorption of DDI was partly attributable to its metabolism in the intestine.

Pulmonary delivery of salmon calcitonin dry powders containing absorption enhancers in rats.

PURPOSE: To evaluate the effects of absorption enhancers in dry powders and in liquids, pulmonary absorption of salmon calcitonin (sCT) in various formulations was measured. METHODS: The dry powder of sCT was prepared by a freeze-drying method with a jet mill. After intratracheal administration of sCT dry powder and liquid (solution) preparations to rats, plasma sCT levels and calcium levels were measured. RESULTS: After intratracheal administration without absorption enhancers, sCT in the dry powder and in the liquid were absorbed nearly to the same degree. Absorption enhancers (oleic acid, lecithin, citric acid, taurocholic acid, dimethyl-beta-cyclodextrin, octyl-beta-D-glucoside) were much more effective in the dry powder than in the solution. The reason may be that the enhancers added to the dry powder dissolved at high concentrations in a trace volume of the fluid lining the alveolar epithelium. CONCLUSIONS: The present results suggest that the pulmonary absorption of peptides and proteins can be greatly improved by formulating them into dry powders with smaller amounts of enhancers than in liquid dosage forms.

Permeability of peptides and proteins in human cultured alveolar A549 cell monolayer.

PURPOSE: The transport of peptides or proteins across the alveolar cell monolayer was studied in vitro in order to elucidate their transport pathway. METHODS: The permeability of 14 peptides or proteins and 6 dextrans with MW 1,000-150,000 was measured in cultured human lung adenocarcinoma A549 cell monolayers at 37 degrees C or 4 degrees C. The stability of the tested peptides and proteins was also evaluated. RESULTS: The permeability coefficients of these macromolecules across the A549 cell monolayer at 37 degrees C ranged from 10(-5) to 10(-7) (cm/sec), and exhibited a good inverse correlation with molecular weight. All macromolecules were stable throughout the transport experiment, and degradation by proteases was minimal. Permeability at 4 degrees C did not differ from that at 37 degrees C. Clear selectivity for direction of transport was not observed. CONCLUSIONS: These results suggested that the tested peptides and proteins appeared to penetrate the A549 cell monolayer via a paracellular route by passive diffusion.

Stability, specific binding activity, and plasma concentration in mice of an oligodeoxynucleotide modified at 5'-terminal with poly(ethylene glycol).

Oligodeoxynucleotide (ODN) composed of 15 nucleotides was modified at 5'-terminal phosphate with hexylamine linker and chemically activated poly(ethylene glycol). This derivative showed improved characteristics in terms of enzymatic stability, binding activity, and in vivo retention in mouse. The data are discussed in comparison with those of corresponding unmodified and phosphorothioate ODNs.

Study on pulmonary delivery of salmon calcitonin in rats: effects of protease inhibitors and absorption enhancers.

Effects of protease inhibitors and absorption enhancers on the absorption of salmon calcitonin (sCT) were evaluated after intratracheal coadministration to rats using the plasma Ca level as an index. Remarkable absorption enhancement could be attained with unsaturated fatty acids such as oleic acid and polyoxyethylene oleyl ether (absorption enhancers) and with chymostatin, bacitracin, potato carboxypeptidase inhibitor and phosphoramidon (protease inhibitors). sCT degrading enzymes had four times higher activity per total protein in membrane fraction of lung homogenates than the activity in cytosol fraction. These enzymes are thought to be serine proteases and metalloenzymes from the in vitro action profile of protease inhibitors. A good correlation between the in vitro activity of protease inhibitors and the in vivo enhancing effect on sCT activity suggested that membrane enzymes are responsible for the inactivation of sCT. Metabolic degradation and low permeability of sCT may be possible barriers to the absorption of sCT.

Nasal absorption of zidovudine and its transport to cerebrospinal fluid in rats.

The nasal absorption of zidovudine (AZT) and its subsequent transport to cerebrospinal fluid (CSF) was examined in rats. Both rapid absorption and a high CSF concentration were observed after the nasal application. Plasma and CSF concentrations of AZT increased when probenecid was coadministered with AZT. Thus, this nasal coadministration of AZT and probenecid could be useful for the treatment of AIDS patients with neuropathies.

Prodrugs of 2',3'-didehydro-3'-deoxythymidine.

Six ester prodrugs of 2',3'-didehydro-3'-deoxythymidine (D4T) were synthesized, and their physicochemical properties were evaluated. Marked differences were observed. All of the prodrugs were chemically stable within the pH range 2-7. Hydrolysis of these esters was observed in all cases for four rat enzyme systems (plasma, liver, duodenum, and kidney), with D4T being regenerated. D4T or the prodrug was administered orally to rats, and the plasma concentrations of D4T and a corresponding prodrug were measured. The half-life of D4T after intravenous administration was 35.9 min. The half-life calculated from the terminal phase and the maximum concentration in plasma following oral administration of D4T were 35.9 min and 48.4 microM, respectively. After oral prodrug administration (with water or olive oil as a solvent), though none of the prodrugs was detected in plasma except for 5'-hemisuccinyl D4T and 5'-hemiglutaryl D4T with olive oil as a solvent, retention time of plasma D4T concentration was extended and the elevated D4T concentration in plasma decreased.

Control of drug release with a combination of prodrug and polymer matrix: antitumor activity and release profiles of 2',3'-diacyl-5-fluoro-2'-deoxyuridine from poly(3-hydroxybutyrate) microspheres.

Drug release was controlled by a combination of prodrug and polymer matrix. Prodrugs of 5-fluoro-2'-deoxyuridine with different physicochemical properties were synthesized by esterification with aliphatic acids (propionate, n-butyrate, and n-pentanoate). Microspheres containing these ester prodrugs were prepared with poly(3-hydroxybutyrate) of three molecular weights (65,000, 135,000, and 450,000). The release rates from the spheres depended on both the lipophilicity of the prodrug and the molecular weight of the polymer. Regardless of the polymer, the relative release rates were propionate greater than butyrate greater than pentanoate. The release of butyrate and pentanoate from the spheres consisting of low-molecular-weight polymer (M(r), 65,000) was faster than that from the spheres of higher molecular weight (M(r), 135,000 or 450,000). A single intraperitoneal injection of spheres of the highest molecular weight polymer containing butyrate or pentanoate resulted in higher antitumor effects against P388 leukemia in mice than did free prodrugs given over a period of five consecutive days. The polymer sphere itself showed low toxicity to and good biocompatibility with mice and rats.

Prodrugs of 2',3'-dideoxyinosine (DDI): improved oral bioavailability via hydrophobic esters.

Five ester prodrugs of 2'3'-dideoxyinosine (DDI) were synthesized for the purpose of improving oral bioavailability. The prodrugs, acetate (C2-DDI), octanoate (C8-DDI), stearate (C18-DDI), benzoate (Bz-DDI), and hemisuccinate (Suc-DDI) were proved to quantitatively regenerate their parent drug by enzymatic hydrolysis. Though the chemical stability of the prodrugs under acidic conditions was not improved, their solubility in water was significantly decreased by esterification, except for Suc-DDI. Bioavailability was evaluated by oral administration to rats. Two hydrophobic prodrugs (C8-DDI and Bz-DDI) showed higher absolute bioavailability (23.5% and 31.0%, respectively) than did DDI (15.2%), though that of C2-DDI (11.5%) and Suc-DDI (4.5%) was poor.

In vivo and in vitro degradation of poly(3-hydroxybutyrate) in rat.

The inflammatory activity and biodegradation of poly(3-hydroxybutyrate) were examined. Poly(3-hydroxybutyrate) sheet did not cause any inflammation in the chorioallantoic membrane of the developing egg. The i.v. injection of 14C-labelled poly(3-hydroxybutyrate) granules showed that 86, 2.5 and 2.4% of the total radioactivity administered were distributed in the liver, spleen and lung, respectively, and the radioactivity decreased slowly but steadily in most tissues examined during 2 month. Crude extracts of rat tissues showed that the activity degraded the poly(3-hydroxybutyrate) granules in vitro.