A Graphical Method for the Determination of the Mode of Hydrolysis of Biodegradable Polymers

Purpose. To develop a qualitative method for the determination of the mode of scission in the hydrolysis of biodegradable polymers. Methods. The method requires determination of the molar fraction of monomer (m₁ by ¹HNMR or HPLC, and the degree of polymer degradation () determined by ¹HNMR. Results. If the scission of the backbone bonds is completely random, the molar fraction of the monomer must equal the square of the degree of degradation as predicted by Kuhn (1). If the degradation follows an exclusive chain-end unzipping mechanism then, m₁ = . Experimental data falling on the theoretical curves (m₁ vs. ) confirm the corresponding mode of scission. If the data fall between the two curves, it suggests a faster chain-end scission than random scission. When data fall below both of these curves, it suggests the chain-end bonds are less reactive than the internal bonds. Conclusions. The acid catalyzed hydrolysis of a poly(ortho ester) and the base catalyzed hydrolysis of poly(D,L-lactide) (PLA) were by a random scission mechanism, while acid catalyzed hydrolysis of PLA demonstrated faster chain-end scission.     

Human Drug Absorption Kinetics and Comparison to Caco-2 Monolayer Permeabilities

Purpose. This study aims to assess the drug absorption kinetics of three drugs and compare their resulting first-order intestinal permeation rate constants to their Caco-2 monolayer permeabilities. Methods. In vitro dissolution — in vivo absorption analysis was conducted on four formulations of each ranitidine HC1, metoprolol tartrate, and piroxicam to yield apparent and "true human clinical permeation rate constants. Drug permeability coefficients through Caco-2 monolayers were also determined. Results. In vitro dissolution — in vivo absorption analysis revealed different relative and absolute contributions of dissolution and intestinal permeation to overall drug absorption kinetics for various drug formulations and yielded estimates of each drug's true and apparent human intestinal permeation rate constant [k _p = 0.225 hr^–1, 0.609 hr^–l, and 9.00 hr^–1 for ranitidine, metoprolol, and piroxicam, respectively]. A rank order relationship was observed for both the apparent and true permeation rate constant with Caco-2 monolayer permeability. The decrease in the true permeation rate constant relative to the apparent permeation rate constant was most significant (almost three-fold) for the least permeable compound, ranitidine. Conclusions. There were marked differences in the permeation kinetics of ranitidine, metoprolol, and piroxicam. The possibility of an association between absorption kinetics from dosage forms in humans and Caco-2 monolayer permeability may allow for a direct kinetic interpretation of human oral absorption from Caco-2 monolayer permeability values.     

Regional Drug Delivery I: Permeability Characteristics of the Rat 6-Day-Old Air Pouch Model of Inflammation

Purpose. To determine the permeability characteristics of the rat air pouch model of inflammation using permeability extremes within which the NSAIDs S[ + ] ibuprofen, piroxicam and diclofenac could be evaluated. Methods. Permeability was calculated using concentration data obtained following intrapouch and intravenous administration of [³H]-water, [¹⁴C]-urea, [¹⁴C]-inulin and [¹²⁵I]-albumin and compared to similar data obtained for the three NSAIDs. Results. Similar permeability values (5–6.5 ml hr^–1) were obtained for the three NSAIDS which fell between the permeability extremes of the molecular weight markers [³H]-water (9.7 ml hr^–1), [¹⁴C]-urea (6.8 ml hr^–1), [¹⁴C]-inulin (1.0 ml hr^–1) and [¹²⁵I]-albumin (0.6 ml hr^–1). Coadministration of equipotent anti-inflammatory doses of the NSAIDs did not affect local blood flow to the air pouch (as assessed by urea kinetics) but did reduced vascular permeability (as assessed by albumin flux into the pouch). Conclusions. Comparison of the NSAIDs with the permeabilities of the molecular weight markers indicates that a perfusion rate limitation probably exists. Systemic absorption is complete over the first two hours following intrapouch administration of the NSAIDs, therefore albumin flux into the pouch is insufficient to materially affect the permeability of the NSAIDs. However, subsequently (post 5hr) albumin concentration in the pouch rises sufficiently to lower the effective flux of the NSAIDs.     

Stabilization of 10-Hydroxycamptothecin in Poly(lactide-co-glycolide) Microsphere Delivery Vehicles

Purpose. The purpose of this study was to investigate the potential of poly(lactide-co-glycolide) (PLGA) microspheres to stabilize and deliver the analogue of camptothecin, 10-hydroxycamptothecin (10-HCPT). Methods. 10-HCPT was encapsulated in PLGA 50:50 microspheres by using an oil-in-water emulsion-solvent evaporation method. The influence of encapsulation conditions (i.e., polymer molecular weight (M_w), polymer concentration, and carrier solvent composition) on the release of 10-HCPT from microspheres at 37°C under perfect sink conditions was examined. Analysis of the drug stability in the microspheres was performed by two methods:i) by extraction of 10-HCPT from microspheres and ii). by sampling release media before lactone— carboxylate conversion could take place. Results. Microspheres made, of low M_w polymer (inherent viscosity 0.15 dl/g) exhibited more continuous drug release than those prepared from polymers of higher M_w (i.v. = 0.58 and 1.07 dl/g). In addition, a high polymer concentration and the presence of cosolvent in the carrier solution to dissolve 10-HCPT were both necessary in the microsphere preparation in order to eliminate a large initial burst of the released 10-HCPT. An optimal microsphere formulation released 10-HCPT slowly and continuously for over two months with a relatively small initial burst of the released drug. Both analytical methods used to assess the stability of 10-HCPT revealed that the unreleased camptothecin analogue in the microspheres remained in its active lactone form (>95%) over the entire 2-month duration of study. Conclusions. PLGA carriers such as those described here may be clinically useful to stabilize and deliver camptothecins for the treatment of cancer.     

Population Pharmacokinetics of Terfenadine

Purpose. After oral administration of terfenadine, plasma concentrations of the parent drug are usually below the limits of quantitation of conventional analytical methods because of extensive first-pass metabolism. Data are usually reported on the carboxylic acid metabolite (Ml) but there are no published reports of pharmacokinetic parameters for terfenadine itself. The present study was undertaken to evaluate the population pharmacokinetics of terfenadine. Methods. Data from 132 healthy male subjects who participated in several different studies were included in this analysis. After an overnight fast, each subject received a single 120 mg oral dose of terfenadine; blood samples were collected for 72 hours. Terfenadine plasma concentrations were measured using HPLC with mass spectrometry detection and Ml plasma concentrations were measured using HPLC with fluorescence detection. A 2-compartment model was fitted to the terfenadine data using NONMEM; terfenadine and Ml data were also analyzed by noncompartmental methods. Results. Population mean Ka was 2.80 hr^–1, T_lag was 0.33 hr, Cl/F was 4.42 × 10³ 1/hr, V_C/F was 89.8 ×10³1, Q/F was 1.85 ×10³ 1/hr and V_p/F was 29.1 × 10³1. Intersubject CV ranged from 66 to 244% and the residual intrasubject CV was 21%. Based on noncompartmental methods, mean terfenadine C_max was 1.54 ng/ml, T_max was 1.3 hr, t_{1/2 Z} was 15.1 hr, Cl/F was 5.48 × 10³ 1/hr and V_z/F was 119.2 × 10³1. Ml concentrations exceeded terfenadine concentrations by more than 100 fold and showed less intersubject variability. Conclusions. Terfenadine disposition was characterized by a 2-compartment model with large intersubject variability, consistent with its significant first-pass effect.     

HYDRODYNAMIC AND QUASI-ELASTIC LIGHT SCATTERING STUDIES ON THE 12S GLOBULIN FROM RAPESEED*

The 12S globulin, one of the major storage proteins of rapeseeds, has the following physico-chemical constants, as determined by ultracentrifugation, quasi-elastic light scattering measurements and gel chromatography: sedimentation coefficient s⁰_{20, w}= 12.7 times 10^-13 s; diffusion coefficient (quasi-elastic light scattering) D⁰_{20, w}= 3.8 × 10^-7 cm² s^-1; Stokes radius (by quasi-elastic light scattering) R_s= 5.7 nm and (by gel chromatography) R_s= 5.5 nm; partial specific volume (calculated from the amino acid composition) v?= 0.729 mlg^-1; molecular weight M_s, D = 300, 000 daltons, M_{s, Rs}= 294,000 daltons (R_s from the gel chromatography); frictional ratio f/fo = 1.28.     

Characterization and In Vitro Methotrexate Release from Methotrexate/Gelatin Conjugates of Opposite Conjugate Bond Polarity

Purpose. Our laboratory has previously prepared gelatin/methotrexate (MTX) conjugates containing mixed conjugation sites and by-product crosslinking, both of which may alter conjugate effectiveness. In this study, we prepared and evaluated gelatin/MTX conjugates having specific conjugate bond sites and minimal by-product crosslinking. Methods. Opposite polarity conjugates were produced by coupling gelatin having blocked amino groups with MTX (G-MTX) and by coupling MTX having blocked amino groups with gelatin (M-GEL) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl. Amino groups were blocked using citraconic anhydride and deblocked under acidic conditions. Gelatin and MTX contents were determined spectrophotometrically. The stability of each conjugate was determined by evaluating their in vitro release of MTX in isotonic buffer at pH 7.4 and 37°C for 7 days. Results. The G-MTX and M-GEL conjugates contained 21 and 1.2 mole MTX/mole gelatin and released 12 and 17% MTX by 7 days resulting in pseudo-first order release rate constants of 0.76×10^–3 and 1.0×10^–3 hr^–1, respectively. Alternate MTX species ( 10%) were detected during the release study and were attributed to low molecular weight gelatin/MTX fragments and MTX polymers. Conclusions. Gelatin/MTX conjugates having opposite conjugate bond polarities and minimal by-product crosslinking have been produced and slowly released MTX by hydrolytic cleavage indicating good stability for future cell culture studies.     

Production and Characterization of 22 Monoclonal Antibodies Directed Against S 20499, a New Potent 5-HT1A Chiral Agonist: Influence of the Hapten Structure on Specificity and Stereorecognition

Purpose. An immunoconjugate model was proposed to produce stereo-selective monoclonal antibodies (MAbs) for the quantitation of a 5-HT_1A agonist, S 20499. MAbs produced were characterized in terms of stereoselectivity and specificity towards the opposite enantiomer and structural analogs. Methods. The immunogen was formed following the effective addition of a butanoic acid spacer arm between the parent S 20499 structure and bovine serum albumin (BSA). After fusion (modified Köhler and Milstein's procedure), specificity of MAbs was obtained using the Abraham's criteria. Experimental and calculated partition coefficients were determined. Results. Twenty-two hybridoma cell lines were established secreting MAbs (apparent association constants ranging from 1.1 X 10⁸ to 2.8 X 10⁹ M^–1). Several MAbs showed cross-reactivity levels of less than 5% with S 20500 (optical antipode), which could allow a stereospecific assay to be set up. Both chroman and azaspiro moieties were part of the epitopic site. Dealkylation and hydroxylation(s) led to various crossreactivity levels. Four antibody families were described in terms of specificity. Conclusions. This study highlighted the influence of the immunoconjugate construction (coupling site and type of spacer arm) in the immuno-stereospecificity of Abs. The results obtained for two mono-hydroxylated metabolites suggest that the lipophilicity behavior could be a valuable tool for predicting Ab-crossreactivity.     

Polyacetal-diethylstilboestrol: A Polymeric Drug Designed for pH-triggered Activation

Those polymer anticancer-drug conjugates currently undergoing clinical evaluation have a tripartite structure; a water-soluble polymer, an anticancer agent and a pendant linker. To simplify the construct it would be attractive to develop anticancer polymer therapeutics that contain the bioactive agent as an integral part of the polymer backbone. The aim of this study was to utilise the reaction between a divinyl ethers and diols, to synthesise polyacetals incorporating a drug with bis-hydroxyl functionality into the polymer backbone. Degradation of the polymer backbone in the acidic environment of the lysosome or the extracellular fluid of some tumours would then trigger drug release eliminating the need for a biodegradable linker. A tert-polymerisation approach was used to incorporate non-steroidal oestrogen diethylstilboestrol (DES) into the mainchain of water-soluble polyacetals synthesised using as co-monomer PEG of M_w 2900 or 3400 g/mol. When PEG₂₉₀₀ was used the resultant polymer had a M_w of 18,900 g/mol, a M_w/M_n of 1.9 and a DES loading 4.3 wt.%. With PEG₃₄₀₀ the polymer M_w was 43,000 g/mol, M_w/M_n=1.8 and it had a DES loading 4.7 wt.%. ¹H-NMR confirmed the presence of two distinct sets of acetal peaks, which correspond to the two possible mainchain acetals; from PEG at 1.25–1.3(d) and 4.7–4.8(q) ppm and from DES at 1.55–1.6(d) and 5.4–5.5(q) ppm. These were consistent with the acetal signals observed for the non-water-soluble co-polymer DES: tri(ethylene glycol) divinyl ether (TEGDVE) (1:2, M_w=6859 g/mol, M_w/M_n=1.3). When evaluated in vitro, the DES-polyacetal displayed greater cytotoxicity than DES against human and murine tumour cell lines (IC₅₀=48 and 420 μg/ml against MCF-7 human breast cancer cells and IC₅₀=97 and 560 μg/ml against B16F10 murine melanoma cells, respectively). These polymers showed no significant haemolysis at concentrations up to 20 mg/ml confirming suitability for further in vivo evaluation. An enhanced rate of hydrolytic degradation of the polymer backbone was seen at pH 5.5, (65% trans-DES released in 96 h), compared to pH 7.4 (4% trans-DES released in 96 h). These bioresponsive DES-polyacetals tert-polymers are the first water-soluble anticancer polymeric drugs designed for acidic pH-triggered release of a drug incorporated into the polymer mainchain. Their in vitro characteristics suggest further in vivo evaluation is warranted.     

Solubilization of Drugs by Physiological Mixtures of Bile Salts

Purpose. The solubilization of a number of steroids was determined in bile salt simple micelles and a bile salt/phospholipid micellar system to provide a better basis to predict the extent of drug solubilization in vivo. Methods. Excess solid drug was dispersed in taurodeoxycholate or mixed micelle solutions prepared with fixed mole ratios of taurocholate, taurodeoxycholate, taurochenodeoxycholate, glycodeoxycholate, glycocholate, and glycochenodeoxycholate with egg phosphatidylcholine. Drug concentrations were determined from the absorbance following centrifugation. Using NMR spectroscopy, the diffusivities of the simple and mixed micelles were 2 × 10^-6 and 8 × 10^-7 cm²/s, respectively. Results. From the change in the concentration of drug in solution with a change in the lipid concentration, the solubilization ratio (SR) was calculated. The SR and aqueous solubility were used to calculate the micelle/aqueous partition coefficients (K_m/w). K_m/w was correlated with octanol/water partition (P_o/w) for the TDC and mixed micelle data sets with correlation lines of logK_m/w = 0.74logP_o/w + 1.55 (r² = 0.91) and logK_m/w = 0.61 logP_o/w + 2.44 (r² = 0.95), respectively. Conclusions. With such data, a refined, predictive relationship between the in vitro and the in vivo solubilization with additional information concerning the bile salt/lipid concentration in the human intestine appears possible.     

The pH-Partition Profile of the Anti-Ischemic Drug Trimetazidine May Explain Its Reduction of Intracellular Acidosis

Purpose. The anti-ischemic drug trimetazidine (TMZ) acts by a combination of molecular mechanisms which begin to be understood. Thus, it acts in the micromolar range to significantly reduce intracellular acidification during ischemia. To search for a possible physicochemical explanation of this phenomenon, we investigated the transfer mechanisms of the various electrical forms of this dibasic drug. Methods. The transfer characteristics of TMZ were studied by electrochemistry at the water/1,2-dichloroethane interface. Cyclic voltammetry was used to measure the formal transfer potentials of singly and doubly protonated forms of TMZ (noted TH⁺ and TH₂ ²⁺, respectively ) as a function of aqueous pH, and the partition coefficient of neutral TMZ (log P _T) was measured by two-phase titration. Results. log P _T was measured to be 1.04 ± 0.06, and the acid-base dissociation constants in water were deduced to be pK ^w _a1 = 4.54 ± .02 and pK ^w _a2 = 9.14 ± 0.02. The partition coefficients of TH⁺ and TH₂ ²⁺ were found to be respectively log P ⁰ _TH+ = –3.78 ± 0.16 and log P ⁰ _{TH 2} ²⁺= –9.84 ± 0.30, which agrees well with the charge being delocalized on two nitrogen atoms in TH⁺. The pH-partition profile of TMZ was then established in the form of its ionic partition diagram, which showed that the affinity of the ions for the organic phase is pH-dependent and strongly increased by the interfacial potential. Conclusions. This behavior suggests a physiochemical mechanism whereby efflux of protonated TMZ out of an acidified cell is facilitated, in effect exporting protons to extracellular space.     

The effect of molecular weights of microencapsulating polymers on viability of mouse-cloned pancreatic β-cells: biomaterials,osmotic forces and potential applications in diabetes treatment

Introduction: Ideal cell-containing microcapsules should be capable of maintaining cell viability and exhibit significant structural stability to support cellular functionality. To date, such microcapsules remain unavailable; thus, this study used our well-established microencapsulating methods to examine a total of 32 different microencapsulating formulations and correlate polymers’ molecular weights (M_wt) and UDCA addition, with cell viability and microcapsules’ stability, postmicroencapsulation.

Methods: MIN6 mouse-cloned pancreatic β-cells were microencapsulated using control (n?=?16; without UDCA) and test (n?=?16; with UDCA) different polymers. Confocal microscopic imaging, cell viability, and microcapsules’ stability were assessed.

Results: Best cell viability (>50%) was obtained at average M_wt of 50,000?g/mol (poly-l-ornithine), followed by 110,000?g/mol (poly-l-lysine). There was no linear correlation between M_wt and viability. Confocal imagining showed similar microcapsules’ shape and cell distribution among all different polymers’ molecular weights, which suggests that the microencapsulating method was efficient and maintained microcapsules’ uniformity. UDCA addition resulted in enhanced osmotic stability of the microcapsules and improved cell viability, when the formulation contained 1% polylornithine, 1% polyethylene glycol, 20% Eudragit^® NM30D, 1% polytetrafluoroethylene, or 5% pentamethylcyclopentasiloxane.

Conclusions: UDCA addition improved microenvironmental conditions within the microcapsules but this effect was largely dependent on the polymer systems used.     

Are MDCK Cells Transfected with the Human MDR1 Gene a Good Model of the Human Intestinal Mucosa?

Purpose. To investigate whether Madin-Darby canine kidney cells transfected with the human MDR1 gene (MDCK-MDR1) are a good model of the human intestinal mucosa. Methods. P-glycoprotein (P-gp) expression in Caco-2 cells was compared with P-gp expression in MDCK wild- type (MDCK-WT) and MDCK-MDR1 cells using Western blotting methods. The polarized efflux activities of P-gp(s) in MDCK-MDR1 cells, MDCK-WT cells, and Caco-2 cells were compared using digoxin as a substrate. Apparent Michaelis-Menten constants (K _M,V _max) for the efflux of vinblastine in these three cell lines were determined. Apparent inhibition constants (K _I) of known substrates/inhibitors of P-gp were determined by measuring their effects on the efflux of digoxin in Caco-2 or MDCK-MDR1 cell monolayers. Results. MDCK-MDR1 cells expressed higher levels of P-gp compared to Caco-2 and MDCK-WT cells, as estimated by Western blots. Two isoforms of P-gp were expressed in Caco-2 and MDCK cells migrating with molecular weights of 150 kDa and 170 kDa. In MDCK-MDR1 cells, the 150 kDa isoforms appeared to be overexpressed. The MDCK-MDR1 cells exhibited higher polarized efflux of [³H]-digoxin than did Caco-2 and MDCK-WT cells. K _M values of vinblastine in Caco-2, MDCK-WT, and MDCK-MDR1 cells were 89.2 ± 26.1, 24.5 ± 1.1, and 252.8 ± 134.7 M, respectively, whereas V _max values were 1.77 ± 0.22, 0.42 ± 0.01, and 2.43 ± 0.86 pmolcm^–2s^–1, respectively. Known P-gp substrates/inhibitors showed, in general, lower K _I values for inhibition of digoxin efflux in Caco-2 cells than in MDCK-MDR1 cells. Conclusions. These data suggest that the MDCK-MDR1 cells overexpress the 150 kDa isoform of P-gp. MDCK-MDR1 cells are a useful model for screening the P-gp substrate activity of drugs and drug candidates. However, the apparent kinetics constants and affinities of substrates determined in the MDCK-MDR1 cell model may be different than the values obtained in Caco-2 cells. These differences in substrate activity could result from differences in the relative expression levels of total P-gp in Caco-2 and MDCK-MDR1 cells and/or differences in the partitioning of substrates into these two cell membrane bilayers.     

In Vitro and In Situ Permeability of a 'Second Generation' Hydroxypyridinone Oral Iron Chelator: Correlation with Physico-Chemical Properties and Oral Activity

Purpose. The in vitro and in situ transport of CGP 65015 ((+)-3-hydroxy-1-(2-hydroxyethyl) -2-hydroxyphenyl-methyl-1 H-pyridin-4-one), a novel oral iron chelator, is described. The predictive power of these data in assessing intestinal absorption in man is described. Methods. Caco-2 epithelial monolayer and in situ rat jejunum perfusion intestinal permeability models were utilized. In vivo iron excretion and preliminary animal pharmacokinetic experiments were described, lonization constants and octanol/aqueous partition coefficients were measured potentiometrically. Solubilities and intrinsic dissolution rates were determined using standard procedures. Results. Caco-2 cell (P_app 0.25 X 10^–6 cm.s^–1) and rat jejunum (P_w 0.4) permeabilities of CGP 65015 were determined. The log D(pH 7.4) of CGP 65015 was 0.58 and its aqueous solubility was > 0.5 mg.ml^–1 (pH 3–9). The intrinsic dissolution rate of CGP 65015 in USP simulated intestinal fluid was 0.012 mg.min^–1.cm^–2. CGP 65015 promotes iron excretion effectively and dose dependently in animals. Conclusions. Caco-2 and rat intestinal permeabilities predict incomplete oral absorption of CGP 65015 in man. Preliminary rat pharmacokinetics support this. Physico-chemical data are, also, in line and suggest that CGP 65015 may, in addition, be solubility/dissolution rate limited in vivo. Nevertheless, early animal pharmacological data demonstrate that CGP 65015 is a viable oral iron chelator candidate.     

An Experimental Model for Measuring Middle Ear Antimicrobial Drug Penetration in Otitis Media

Bacteria are an important cause of acute otitis media and successful treatment depends on achieving inhibitory or bacteriacidal antimicrobial drug concentrations in the middle ear. To evaluate further otitis media treatment success and failure, we developed a chinchilla model to study antimicrobial drug penetration through the middle ear mucosa. Using quantitative histomorphometry, we measured the middle ear space in 10 chinchillas and found a mean ±SD volume of 2.09 ± 0.08 ml and a mean SD surface area of 14.41 ± 1.48 cm². To measure the apparent rate constant (K _e) of antibiotic elimination from the middle ear, through the middle ear mucosa, an antibiotic solution was inoculated into the middle ear cavity, and samples were aspirated between 1 and 8 hr later. In normal ears, the mean K _e ±SD for amoxicillin was 0.118 ± 0.013 hr^–1, that for a trimethoprim 0.461 ± 0.090 hr^–1, and that for sulfamethoxazole 0.265 ± 0.062 hr^–1. In ears inoculated with type 7F Streptococcus pneumoniae to induce acute otitis media, the K _e ±SD increased for all three drugs (P < 0.05): amoxicillin to 0.286 ± 0.089 hr^–1, trimethoprim to 0.662 ± 0.118 hr^–1, and sulfamethoxazole to 0.411 ± 0.056 hr^–1. These values demonstrate that amoxicillin had the lowest apparent penetration rate constant of the three antibiotics but the greatest increase from normal to infected mucosa (142%). Trimethoprim had the highest apparent penetration rate constant of the three antibiotics but the smallest increase from normal to infected mucosa (44%), while the sulfamethoxazone apparent penetration rate constant increased from normal to infected mucosa by 55%. The K _e for amoxicillin was the same for inoculation volumes of 0.8 and 1.6 ml (P = 0.557) and the same for sampling intervals of 4 and 8 hr (P = 0.054). All three antimicrobial drug concentration–time curves were log-linear, as predicted by Fick's first law of diffusion. In conclusion, this model overcomes the technical limitations of previous models and permits investigation of the many factors that can influence antibiotic penetration into the middle ear and reduce otitis media treatment efficacy.     

In vivo kinetic analysis of covalent binding between N-acetyl-L-cysteine and plasma protein through the formation of mixed disulfide in rats

Purpose. This investigation was undertaken to study the relationship between plasma drug clearance and covalent protein-binding kinetics of N-acetyl-L-cysteine (NAC). Methods. NAC was intravenously administered to rats via a bolus injection or continuous infusion. Plasma concentrations of protein-unbound and total NAC were analyzed using a compartment model, taking into consideration of the protein binding process, and the apparent first-order binding and dissociation rate constants (k_on and k_off) were obtained. Results. Plasma total NAC after a bolus injection showed biphasic elimination with an inflection point at 1 hr. After 1 hr, NAC was largely present in the covalent protein-bound form. During the steady state of the infusion, approximately 30%-40% of plasma NAC bound with protein covalently. The k_on, k_off, and the elimination rate constant of protein-unbound drug (k_e) were 0.23, 0.57, and 4.3 hr^-1. The dissociation half-life of NAC from protein estimated from k_off was in agreement with the elimination half-life of plasma total NAC. This suggests that the dissociation of NAC from protein rate-limited the drug elimination in plasma (k_off < k_e). Conclusion. We demonstrated that plasma total drug clearance is kinetically limited by covalent protein binding. The compartmental model described here is useful for analyzing its kinetics in vivo.     

Hydroxypropyl-β-Cyclodextrin Increases the Aqueous Solubility and Stability of Pilocarpine Prodrugs

Purpose. The effects of 2-hydroxypropyl--cyclodextrin (HP--CD) on the aqueous solubility and stability of two lipophilic bispilocarpine prodrugs were investigated at pH 7.4. Methods. The solubility of prodrugs was studied by phase-solubility method (0–72.5 mM HP--CD). The stability of one of the prodrugs was investigated as a function of temperature (40°C–70°C) and HP--CD concentration (0–72.5 mM). The apparent rate constants (k ₁, k ₂) for degradation of prodrug in 1:1 and 1:2 inclusion complexes and apparent stability constants (K _1:1, K _l:2) were calculated by the curve-fitting method. Results. The phase-solubility diagrams were classified as A_p-type and the apparent stability constants (K _l:l, K _l:2) for 1:1- and 1:2-inclusion complexes were calculated to be 143–815 M^–l and 29–825 M^–1, respectively. The stability of prodrug increased as a function of HP--CD concentration over the studied temperature range. The shelf-life (t _90%, calculated by the Arrhenius equation) of the prodrug in 72.5 mM HP--CD solution increased 5.1-fold and 6.1-fold at 25°C and 4°C, respectively. Conclusions. The solubility of the prodrugs was shown to increase markedly in phase-solubility studies. The degradation rate of prodrug in stability studies was shown to be slower in the l:2-complex than in the l:l-complex and the relative amounts of complex species were found to be dependent on CD concentration.     

Preparation and evaluation of double-walled microparticles prepared with a modified water-in-oil-in-oil-in-water (w1/o/o/w3) method

Abstract

In this study, a modified water-in-oil-in-oil-in-water (w₁/o/o/w₃) method was developed to prepare double-walled microparticles containing ovalbumin (OVA). The microparticles were characterized with respect to their morphology, particle size, encapsulation efficiency, production yield, thermal properties and in vitro drug release. Microscopy observations clearly showed that microparticles have spherical shape and smooth surface. These microparticles were characterized to have double-walled structure, with a cavity in the centre. By using w₁/o/o/w₃ method, a significant decrease in mean particle size and a significant increase in encapsulation efficiency were obtained. The mean particle size and the encapsulation efficiency of double-walled microparticles were also affected by the changing amount of OVA and mass ratio of polymers. Microparticles prepared with two polymers exhibited a significantly lower initial burst release followed by sustained release compared to microparticles made from poly(d,l-lactide-co-glycolide) 50/50 only. It can be concluded that these microparticles can be a potential delivery system for therapeutic proteins.     

藤梨根化学成分的研究

A number of constituents were isolated from the root of Actinidia chinensis Planch. Seven of them were indentified as physcion (emodin-6-methyl ether) (T₁), emodin (frangala-emodin) (T₂), emodin-8-β-D-glucoside (T₃), questin (emodin-8-methyl ether) (T₅), citreorosein (ω-hydroxyemodin) (T₆), emodic acid (T₇) and β-sitosterol (T₄).Comparison of the ¹HNMR data of emodin (T₂) and its 8-glucoside (T₃) shows that the aromatic proton experiences a downfield shift of about 0.5 ppm upon glycosidation of an ortho phenolic group, a fact which can be used to advantage for the allocation of the glycosidic linkage.