Physical Properties of Solid Molecular Dispersions of Indomethacin with Poly(vinylpyrrolidone) and Poly(vinylpyrrolidone-co-vinyl-acetate) in Relation to Indomethacin Crystallization

Purpose. To measure solid-state features of amorphous molecular dispersions of indomethacin and various molecular weight grades of poly(vinylpyrrolidone), PVP, and poly(vinylpyrrolidone-co-vinylacetate), PVP/VA, in relation to isothermal crystallization of indomethacin at 30°C Methods. The glass transition temperatures (Tg) of molecular dispersions were measured using differential scanning calorimetry (DSC). FT-IR spectroscopy was used to investigate possible differences in interactions between indomethacin and polymer in the various dispersions. The enthalpy relaxation of 5% w/w and 30% w/w polymer dispersions was determined following various aging times. Quantitative isothermal crystallization studies were carried out with pure indomethacin and 5% w/w polymers in drug as physical mixtures and molecular dispersions. Results. All coprecipitated mixtures exhibited a single glass transition temperature. All polymers interacted with indomethacin in the solid state through hydrogen bonding and in the process eliminated the hydrogen bonding associated with the carboxylic acid dimers of indomethacin. Molecular mobility at 16.5°C below Tg was reduced relative to indomethacin alone, at the 5% w/w and 30% w/w polymer level. No crystallization of indomethacin at 30°C was observed in any of the 5% w/w polymer molecular dispersions over a period of 20 weeks. Indomethacin alone and in physical mixtures with various polymers completely crystallized to the form at this level within 2 weeks. Conclusions. The major basis for crystal inhibition of indomethacin at 30°C at the 5% w/w polymer level in molecular dispersions is not related to polymer molecular weight and to the glass transition temperature, and is more likely related to the ability to hydrogen bond with indomethacin and to inhibit the formation of carboxylic acid dimers that are required for nucleation and growth to the crystal form of indomethacin.     

Synthesis and Cytotoxic Evaluation of a Series of γ-Substituted γ-Aryloxymethyl-α-methylene-γ-butyrolactones Against Cancer Cells

Purpose. The main objective of this investigation was to explore thecytotoxic structure-activity relationships of -substituted -aryloxymethyl--methylene--butyrolactones against cancer cells. Methods. The target compounds were synthesized in two stepscommencing with aryl-OH which was treated with a bromomethyl ketonefollowed by the Reformatsky-type condensation. Results. Seven types of -methylene--butyrolactones were evaluatedin vitro against 60 human cancer cell lines derived from nine cancercell types. The average values of log G₅₀ indicated that for thearylportion, potencies of these -methylene--butyrolactones are in adecreasing order of quinolin-2(1H)-one (or 2-hydroxyquinoline, 21,–5.89) > quinoline (19, –5.79) > 2-methylquinoline (20, –5.69)> 8-hydroxyquinoline (17, –5.64) > 2-naphthalene (16, –5.59)> benzene (15, –4.90). The same order was obtained for both log TGIand log LC₅₀. However, for the -substituent, the potencies are in adecreasing order of biphenyl (16f–21f) > phenyl and4-substituted phenyl (16b-e–21b-e) > methyl (16a–21a). Conclusions. Unlike cardiovascular activities of -methylene--butyrolactones in which a -methyl substituent is necessary for vasorelaxingeffect while a phenyl or a halogen-substituted phenyl is prefer for theantiplatelet activities, a -biphenyl substituent proved to be the bestfor their cytotoxicities against various cancer cell lines tested.     

Confocal Laser Scanning Microscopic Visualization of the Transport of Dextrans After Nasal Administration to Rats: Effects of Absorption Enhancers

Purpose. To visualize the transport pathway(s) of high molecular weight model compounds across rat nasal epithelium in vivousing confocal laser scanning microscopy. Furthermore, the influence of nasal absorption enhancers (randomly methylated -cyclodextrin and sodium taurodihydrofusidate) on this transport was studied. Methods. Fluorescein isothiocyanate (FITC)-labelled dextrans with a molecular weight of 3,000 or 10,000 Da were administered intranasally to rats. Fifteen minutes after administration the tissue was fixed with Bouin. The nasal septum was surgically removed and stained with Evans Blue protein stain or DiIC18(5) lipid stain prior to visualization with the confocal laser scanning microscope. Results. Transport of FITC-dextran 3,000 across nasal epithelium occurred via the paracellular pathway. Endocytosis of FITC-dextran 3,000 was also shown. In the presence of randomly methylated -cyclodextrin 2% (w/v) similar transport pathways for FITC-dextran 3,000 were observed. With sodium taurodihydrofusidate 1% (w/v) the transport route was also paracellular with endocytosis, but cells were swollen and mucus was extruded into the nasal cavity. For FITC-dextran 10,000 hardly any transport was observed without enhancer, or after co-administration with randomly methylated -cyclodextrin 2% (w/v). Co-administration with sodium taurodihydrofusidate 1% (w/v) resulted in paracellular transport of FITC-dextran 10,000, but morphological changes, i.e. swelling of cells and mucus extrusion, were observed. Conclusions. Confocal laser scanning microscopy is a suitable approach to visualize the transport pathways of high molecular weight hydrophilic compounds across nasal epithelium, and to study the effects of absorption enhancers on drug transport and cell morphology.     

The Effect of Benzyl Alcohol on Recombinant Human Interferon-γ

Purpose. The goal of this study was to investigate the conformational change and aggregation of recombinant human interferon-gamma (rhIFN-) as a result of interaction between benzyl alcohol and the protein. The effects of buffer concentration, buffer species, ionic strength, rhIFN- and benzyl alcohol concentrations on the dynamics of the interaction in liquid formulations were also examined. Methods. The effect of benzyl alcohol on the secondary and tertiary structure of rhIFN- in succinate and acetate buffers was studied using far-UV and near-UV circular dichroism spectrophotometry, respectively. Dynamic light scattering was employed to detect aggregate formation due to the interaction of benzyl alcohol with rhIFN-. Results. The addition of benzyl alcohol at 0.9% (w/v) in various liquid rhIFN- formulations induced changes in circular dichroism (CD) spectra of the protein in the near-UV region, while the CD spectra in the far-UV region remained unaltered. There were gradual decreases in ellipticity with time throughout the near-UV CD spectra. The decreases in near-UV ellipticity induced by benzyl alcohol were accompanied by the formation of high molecular weight aggregates as measured by dynamic light scattering. Loss in near-UV ellipticity was accelerated at lower protein concentration and by increasing buffer or benzyl alcohol concentration. It was also faster in succinate than in acetate buffer. Formulation ionic strength did not affect the CD spectral changes in both the near- and far-UV regions. Conclusions. Interaction between benzyl alcohol and rhIFN- is formulation dependent. Protein concentration, buffer species, buffer concentration, and preservative concentration play a significant role in determining the extent of the interaction and consequently the stability of the product.     

Characterization of Pharmacodynamic Recession Slopes for Direct and Indirect Response Models

Direct pharmacologic effects are known to recede over time with largely linear slopes (Levy's k · m product, J. Pharm. Sci. 53: 342, 1964) and indirect responses have similar behavior. Pharmacodynamic slope properties were examined mathematically for the Hill function with monoexponential drug disposition and simulations were carried out for other pharmacokinetic functions. Both types of pharmacodynamic profiles exhibit a single terminal inflection point (fp) when drug concentrations exceed the EC₅₀ (that concentration causing one-half maximum effect, E_max ). For direct effects it was found that C_fp (the drug concentration at fp) =EC₅₀ , the determinants of inflection time were identified, and Slope_fp = –_zE_max /4 where _z is the terminal disposition slope and is the Hill coefficient. These characteristics were explored for the four basic indirect response models which also exhibit recession profiles with slight sigmoidity and a single terminal inflection point at higher doses. The drug concentration at inflection C_fp is IC₅₀ or SC₅₀ (drug concentrations causing half-maximal inhibition or stimulation), while the inflection response (R_fp ) attains constant values at larger doses. Indirect Response Models I, III, and IV have nearly linear return slopes for a wide range of doses which are governed by the disposition slope _z of the drug, loss constant k_out of the response, maximum inhibition (I_max ) or stimulation (S_max ) factors, and a unique fractional constant (01). Model II exhibits more complex behavior with recession slopes which are less likely to be parallel for various doses. Most indirect responses are expected to show nearly linear recession slopes which are parallel for moderate to large doses and mainly governed by an identical combination of pharmacokinetic (_z ), system (k_out ), and dynamic capacity factors (I_max or S_max ).     

Studies on the effects ofl(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) on 4-aminophenol-induced nephrotoxicity in the Fischer 344 rat

4-Aminophenol (para-aminophenol; PAP) causes selective necrosis to the S₃ segment of the proximal tubule in experimental animals. The mechanism of PAP nephrotoxicity has not been fully elucidated, although it has been suggested to involve glutathione (GSH)-dependentS-conjugation followed by processing by the enzyme -glutamyl transpeptidase (GT) to the corresponding cysteineS-conjugate. This proposed toxicity mechanism was probed further by administering L-(S,5S)--amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125), a potent GT inhibitor, to Fischer 344 (F344) rats before treatment with PAP (100 mg/kg). AT-125 pretreatment did not appear to protect against PAP-induced nephrotoxicity as assessed by renal histopathology, clinical chemistry and proton nuclear magnetic resonance (¹H NMR) spectroscopy of urine. These data suggest that renal GT activity is not a prerequisite for PAP nephrotoxicity and that the generation of a cysteineS-conjugate is not a unique requirement for the induction of PAP nephrotoxicity.     

Dependence of the Molecular Mobility and Protein Stability of Freeze-Dried γ-Globulin Formulations on the Molecular Weight of Dextran

Purpose. The effect of the molecular weight of dextran on the molecular mobility and protein stability of freeze-dried serum -globulin (BGG) formulations was studied. The stabilizing effect of higher molecular weight dextran is discussed in relation to the molecular mobility of the formulations. Methods. The molecular mobility of freeze-dried BGG formulations containing dextrans of various molecular weights was determined based on the free induction decay of dextran and water protons measured by proton NMR. The protein stability of the formulations was determined at temperatures ranging from 20 to 70°C by size exclusion chromatography. Results. Changes in the molecular mobility of freeze-dried formulations that occurred at temperatures below the glass transition temperature could be detected as the molecular mobility-changing temperature (T_mc), at which dextran protons started to exhibit a Lorentzian relaxation decay due to higher mobility in addition to a Gaussian relaxation decay. T_mc increased as the molecular weight of dextran increased. The proportion of dextran protons which exhibited the higher mobility relaxation process (P_hm) at temperatures above T_mc decreased as the molecular weight of dextran increased. Protein stability was closely related to molecular mobility. The temperature dependence of the denaturation rate changed at around T_mc, and denaturation in the microscopically liquidized state decreased as P_hm decreased with increasing molecular weight of dextran. Conclusions. The effect of the molecular weight of dextran on the protein stability of freeze-dried BGG formulations could be explained in terms of the parameters obtained by ¹H-NMR such as T_mc and P_hm. These parameters appear to be useful in preformulation and stability prediction of freeze-dried formulations.     

Prediction of Glass Transition Temperature of Freeze-Dried Formulations by Molecular Dynamics Simulation

Purpose. To examine whether the glass transition temperature (T_g) of freeze-dried formulations containing polymer excipients can be accurately predicted by molecular dynamics simulation using software currently available on the market. Molecular dynamics simulations were carried out for isomaltodecaose, a fragment of dextran, and -glucose, the repeated unit of dextran, in the presence or absence of water molecules. Estimated values of T_g were compared with experimental values obtained by differential scanning calorimetry (DSC). Methods. Isothermal-isobaric molecular dynamics simulations (NPTMD) and isothermal molecular dynamics simulations at a constant volume (NVTMD) were carried out using the software package DISCOVER (Material Studio) with the Polymer Consortium Force Field. Mean-squared displacement and radial distribution function were calculated. Results. NVTMD using the values of density obtained by NPTMD provided the diffusivity of glucose-ring oxygen and water oxygen in amorphous -glucose and isomaltodecaose, which exhibited a discontinuity in temperature dependence due to glass transition. T_g was estimated to be approximately 400K and 500K for pure amorphous -glucose and isomaltodecaose, respectively, and in the presence of one water molecule per glucose unit, T_g was 340K and 360K, respectively. Estimated T_g values were higher than experimentally determined values because of the very fast cooling rates in the simulations. However, decreases in T_g on hydration and increases in T_g associated with larger fragment size could be demonstrated. Conclusions. The results indicate that molecular dynamics simulation is a useful method for investigating the effects of hydration and molecular weight on the T_g of lyophilized formulations containing polymer excipients, although the relationship between cooling rates and T_g must first be elucidated to predict T_g vales observed by DSC measurement. January 16     

Transport Characteristics of Peptidomimetics. Effect of the Pyrrolinone Bioisostere on Transport Across Caco-2 Cell Monolayers

Purpose. To compare the permeation characteristics of amide bond-containing HIV-1 protease inhibitors and their pyrrolinone-containing counterparts across Caco-2 cell monolayers, a model of the intestinal mucosa. Methods. Transepithelial transport and cellular uptake of three pairs of amide bond-containing and pyrrolinone-based peptidomimetics were assessed in the presence and absence of cyclosporin A using the Caco-2 cell culture model. The potential of the peptidomimetics to interact with biological membranes was estimated by IAM chromatography. Results. In the absence of cyclosporin A, apical (AP) to basolateral (BL) flux of all compounds studied was less than the flux determined in the opposite direction (i.e., BL-to-AP). The ratio of the apparent permeability coefficients (P_app) calculated for the BL-to-AP and AP-to-BL transport (P_BLAP/P_APBL) varied between 1.7 and 36.2. When individual pairs were compared, P_BLAP/P_APBL ratios of the pyrrolinone-containing compounds were 1.5 to 11.5 times greater than those determined for the amide bond-containing analogs. Addition of 25 M cyclosporin A to the transport buffer reduced the P_BLAP /P_APBL ratios for all protease inhibitors to a value close to unity. Under these conditions, the amide bond-containing peptidomimetics were at least 1.6 to 2.8 times more able to permeate Caco-2 cell monolayers than were the pyrrolinone-containing compounds. The intrinsic uptake characteristics into Caco-2 cells determined in the presence of 25 M cyclosporin A were slightly greater for the amide bond-containing protease inhibitors than for the pyrrolinone-containing analogs. These uptake results are consistent with the transepithelial transport results determined across this in vitro model of the intestinal mucosa. Conclusions. The amide bond-containing and pyrrolinone-based peptidomimetics are substrates for apically polarized efflux systems present in Caco-2 cell monolayers. The intrinsic permeabilities of the amide bond-containing protease inhibitors are slightly greater than the intrinsic permeabilities of the pyrrolinone-based analogs through Caco-2 cell monolayers.     

Effect of ASTA Z 7557 (INN mafosfamide) a precursor of 4-hydroxy-cyclophosphamide on human T-lymphocytes' Fc-receptors and immunoregulatory functions

Summary The precursor ASTA Z 7557 of the in vivo active metabolite 4-hydroxy-cyclophosphamide (4OH-Cy) of cyclophosphamide (Cy) was tested for selective effects on human T-lymphocytes' Fc-receptor expession and certain immunoregulatory functions. It has been found that ASTA Z 7557 does not alter the expression of Fc-F or Fc-I-receptor on the T-cell membrane nor does it exhibit differential toxicity for either T-cell sub-population. PWM-induced B-cell proliferation is inhibited by ASTA Z 7557, whereas B-cells' Ig-synthesis as well as ConA-driven T-cell proliferation is only blocked with higher doses of the reagent. Final concentrations above 1 g/ml may therefore abrogate ConA-induction of suppressor T-lymphocytes. Already ConA-activated suppressor cells of PWM-driven B-cell cultures, however, are not inhibited even with high doses of ASTA Z 7557.     

Interactions between oligonucleotides and cationic polymers investigated by fluorescence correlation spectroscopy

Purpose. To evaluate whether fluorescence correlation spectroscopy (FCS) can be used to characterize the complexation between oligonucleotides and cationic polymers. Methods. The features of the complexes between rhodamine labeled oligonucleotides (Rh-ONs) and poly(2-dimethylamino)ethyl methacrylate (pDMAEMA), poly(ethylene glycol)-poly(ethyleneimine) (pEG-pEI), and diaminobutane-dendrimer-(NH₂)₆₄ (DAB₆₄) were characterized by light scattering, electrophoretic mobility, electrophoresis, and FCS. Results. At low polymer/Rh-ON ratios, a decrease of the fluorescence of the Rh-ONs was observed on binding of the Rh-ONs to all cationic polymers. This was explained by the creation of multimolecular complexes in which the Rh-labels quench each other. The multimolecular complexes, which are highly fluorescent as they carry a number of Rh-ONs, resulted in high fluorescence peaks in the fluorescence fluctuation profile as measured by FCS. For pDMAEMA and DAB₆₄, at higher polymer/Rh-ON ratios the fluorescence of the polyplexes increased, caused by the formation of monomolecular complexes, which consist of only one Rh-ON per polymer. In the case of pEG-pEI, the fluorescence stayed constant when the polymer/Rh-ON ratio increased, so multimolecular polyplexes remained. FCS confirmed these results as the high fluorescence peaks disappeared in case of pDMAEMA/Rh-ON and DAB₆₄/Rh-ON dispersions, but remained present for pEG-pEI/Rh-ON dispersions. Conclusions. FCS seems applicable for study of the interactions between ONs and different types of cationic polymers.     

Preformulation Stability Studies of the New Dipeptide Angiotensin-Converting Enzyme Inhibitor RS-10029

The degradation kinetics, products, and mechanisms of RS-10029 (2), 2-[2-[(l-carboxylic acid)-3-phenylpropyl]amino-l-oxopropyl] 6,7-dimethoxy- 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (S,S,S), in aqueous solutions from pH 1 to pH 13 were studied at 50, 60, and 80°C. Pseudo-first-order kinetics were obtained throughout the entire pH range studied, and the log(rate)-pH profile reflected four kinetic processes (k _o, k_o, k_o, and k _OH) as well as the three pk _a's of 2. Excellent mass balance (>96%) was obtained for the four major products 3–6 throughout the entire pH range studied even though four other minor products can be detected by high-performance liquid chromatography (HPLC). At pH 8.0 and below, intramolecular aminolysis leading to diketopiperazine (DKP) 5 accounted for greater than 65% of the neutral or water-catalyzed (k _o and k_o) processes. Amide hydrolysis leading to products 3 and 4 and epimerization of DKP 5 to the (R,S,S) diastereomer 6 accounted for the remaining 35% of the neutral or water catalyzed processes. At pH values above 8.0, DKP 5 formation begins to decrease as the amide hydrolysis increases so that both mechanisms account for the neutral or water-catalyzed k_o process. Above pH 11.0 amide hydrolysis dominates and is responsible for the specific base-catalyzed (k _OH) process. The four minor products detected by HPLC are two diastereomers (7 and 8) of 2 and the two diastereomers (9 and 10) of the DKP 5. The stability results between 2 and its ester prodrug (1) are compared.     

Biodegradable PLGA Microspheres Loaded with Ganciclovir for Intraocular Administration. Encapsulation Technique,in Vitro Release Profiles,and Sterilization Process

Purpose. The purpose of this work was to obtain a sterilized formulation consisting of biodegradable microspheres of poly (DL-lactide-co-glycolide) (PLGA) for intraocular sustained release of ganciclovir. Methods. Microspheres were prepared using a dispersion of ganciclovir in fluorosilicone oil (FSiO) that was further dispersed in an acetone solution of PLGA [50/50 and inherent viscosity 0.41 dl/g], and emulsified in silicone oil with a surfactant. Once prepared, the formulation was exposed with an effective radiation dose of 2.5 megarads. The release rate data of ganciclovir from the sterilized and nonsterilized batches were compared using the similarity factor (f₂). Results. The dispersion of the drug in FSiO contributed to achieving a drug payload of up to 95% of the theoretical in the 300-500 m microspheres. Ten mg released ganciclovir in vitroat 1.3 g/h for the first 21 days, but decreased to 0.2 g/h from day 25 until the end of the release study (42 days). No significant differences in the amounts of encapsulated drug (=0.05) were observed between the sterilized and nonsterilized microspheres. Furthermore, dissolution profiles of formulations behaved similarly before and after gamma radiation exposure. Conclusions. The technique of microsphere preparation described resulted in high ganciclovir loading (95%) and prolonged drug release. The ganciclovir formulation behaved similarly before and after the sterilization process.     

A Novel Non-Viral Vector for DNA Delivery Based on Low Molecular Weight,Branched Polyethylenimine: Effect of Molecular Weight on Transfection Efficiency and Cytotoxicity

Purpose. Low molecular weight branched polyethylenimine (LMW-PEI) was synthesized and studied as a DNA carrier for gene delivery with regard to physico-chemical properties, cytotoxicity, and transfection efficiency. Methods. The architecture of LMW-PEI, synthesized by acid catalyzed ring-opening polymerization of aziridine was characterized by size exclusion chromatography in combination with laser light scattering and ¹³C-NMR-spectroscopy. In vitro cytotoxic effects were quantified by LDH and MTT assay and visualized by transmission electron microscopy. The potential for transgene expression was monitored in ECV304 cells using luciferase driven by a SV40 promoter as reporter gene system. Results. LMW-PEI (Mw 11900 D) with a low degree of branching was synthesized as a DNA carrier for gene delivery. In contrast to high molecular weight polyethylenimines (HMW-PEI; Mw l616OOO D), the polymer described here showed a different degree of branching and was less cytotoxic in a broad range of concentrations. As demonstrated by transmission electron microscopy the LMW-PEI formed only small aggregates which were efficiently taken up by different cells in the presence of serum, most likely by an endocytic pathway. LMW-PEI yielded transfection efficiencies measured via expression of the reporter gene luciferase which were up to two orders of magnitude higher than those obtained with HMW-PEI. The reporter gene expression was concentration dependent, but in contrast to lipofection independent of serum addition. Conclusions. The LMW-PEI described here is a new, highly efficient, and non-cytotoxic vector with a favorable efficiency/toxicity profile for gene therapeutic applications.     

Accelerated Degradation of Poly(ε-caprolactone) by Organic Amines

The solid-state degradation of poly(-caprolactone) catalyzed by primary, secondary and tertiary alkylamines was investigated. The degradation process was monitored by weight loss and molecular weight change measured by gel permeation chromatography. Degradation studies were conducted at 37°C in methanol solutions of the alkylamines. Primary alkylamines caused rapid weight loss (i.e., ~90% weight loss in 30 days) that depended on alkylamine concentration, molar ratio of alkylamine to poly(-caprolactone) monomer and alkyl chain length. The secondary alkylamines caused less rapid polymer weight loss (i.e., ~90%) weight loss within 80 days). One tertiary alkylamine (N,N-diisopropylethylamine) showed little catalytic effect while a bicyclic tertiary alkylamine (quinuclidine) was about as catalytic as the primary alkylamines. The degradation products isolated when primary alkylamines were used include both esters and amides indicating that nucleophilic attack by the alkylamines competed with the amine-catalyzed methanolysis reaction. Only ester moieties could be identified in the products from reactions containing secondary and tertiary alkylamines, which indicated that they acted as nucleophilic catalysts. All of the primary alkylamines reduced poly(-caprolactone) molecular weight from about 25,000 to 10,000 within 10 days after which the molecular weight of the remaining solid leveled off even though weight loss continued.     

β2-Adrenergic Receptor Genotype-Related Changes in cAMP Levels in Peripheral Blood Mononuclear Cells After Multiple-Dose Oral Procaterol

Purpose. To evaluate the ₂-adrenergic receptor (₂AR) genotype frequency in the Japanese population and the relationship between ₂AR genotype at amino acid position 16 (₂AR-16) and desensitization to ₂-agonist ex vivo. Methods. The ₂AR genotypes at amino acid positions 16, 27, and 164 of 92 healthy Japanese subjects were determined by polymerase chain reaction-restriction fragment-length polymorphism. The relationship between the ₂AR-16 genotype and the desensitization to ₂-agonist was examined in 10 male subjects ex vivo. Procaterol tablet (HCl salt, 50g, Meptin®) was given orally for 5 days, and peripheral blood was obtained before and after 5 days of consecutive medications followed by the assessment of the intracellular cAMP levels in peripheral blood mononuclear cells after incubation with or without procaterol hydrochloride (0-1000 ng/mL). Results. Allele frequency was Arg16:Gly16 = 46%:54%, Gln27:Glu27 = 92%:8%, and Thr164:Ile164 = 100%:0%, respectively. The cAMP levels were increased by incubation with procaterol hydrochloride, and the increase was suppressed after 5 days of consecutive medications. The suppression was more significant in the homozygote for Gly16 than the homozygote for Arg16. Conclusions. The desensitization to ₂-agonist was associated more frequently with the mutation at ₂AR-16 (Gly16).     

Design and Evaluation of an Osmotic Pump Tablet (OPT) for Chlorpromazine Using (SBE)7m-β-CD

Purpose. The purpose of this study was to develop a controlled-porosity osmotic pump tablet (OPT) which exhibits pH-independent release profiles for a basic drug using a sulfobutyl ether--cyclodextrin, (SBE)_7m--CD, which acts as both a solubilizer and as an osmotic agent. Methods. Chlorpromazine free base (CLP) was chosen as a model drug for this study. The release of CLP from osmotic pump tablets was studied in vitro. In vivo absorption of CLP from the OPT was evaluated in male beagle dogs. Results. The CLP release profile from an OPT prepared from a core tablet composed of a 1:10 molar ratio of CLP to (SBE)_7m--CD was pH-independent, and was controlled by modulating the membrane thickness of the OPT. Another cyclodextrin, hydroxypropyl--cyclodextrin (HP--CD), and a sugar mixture of lactose and fructose resulted in pH-dependent release at the same molar ratio. An in vivo absorption study in dogs with an OPT containing (SBE)_7m--CD correlated very well with the in vitro release profiles using the Japanese Pharmacopoeia dissolution method. Conclusions. In addition to serving as a solubilizer and osmotic agent, (SBE)_7m--CD can also serve as the controlling agent for pH independent release of CLP from OPTs. This system successfully modified the in vivo input rate of CLP without compromising oral bioavailability.     

Isolation and Characterization of a Monomethioninesulfoxide Variant of Interferon α-2b

Purpose. To isolate and characterize a monomethioninesulfoxide variant of the commercially available therapeutic protein interferon -2b. Methods. The methionine (Met)-oxidized variant was isolated by reverse-phase high performance liquid chromatography and characterized by SDS-PAGE, peptide mapping and mass spectrometric analysis of the trypsin/V8-generated peptide fragments. The biological and immunological activities of the isolated variant were also evaluated. Results. The rHuIFN -2b variant was found to contain a Met sulfoxide residue at position 111 of the rHuIFN -2b molecule. The far-UV CD spectra showed a slight loss of -helical content and an increase in the -sheet contribution. The CD spectra indicate that both chromatographic conditions and Met oxidation contribute to the observed secondary structure changes. Both interferon -2b main component and its methionine-oxidized variant showed different reactivity to monoclonal antibodies employed in immunoassays for the protein. Conclusions. A monomethioninesulfoxide rHuIFN -2b variant was found to be present in the rHuIFN -2b bulk drug substance in solution. The Met¹¹¹ residue was identified as Met sulfoxide by comparative tryptic/V8 mapping and mass spectrometric analysis. Nevertheless, the oxidation of the Met¹¹¹ residue did not seem to have a detectable effect on the biological activity of the molecule.     

Subject-by-Formulation Interaction in Determinations of Individual Bioequivalence: Bias and Prevalence

Purpose. 1. To determine properties of the estimated variance component for the subject-by-formulation interaction (² _D) in investigations of individual bioequivalence (IBE), and 2. to evaluate the prevalence of interactions in replicate-design studies published by FDA. Methods. Four-period crossover studies evaluating IBE were simulated repeatedly. Generally, the true bioequivalence of the two formulations, including ² _D= 0, was assumed, ² _D was then estimated in a linear mixed-effect model by restricted maximum likelihood (REML). The same method was applied for estimating ² _D for the data sets of FDA. Results. 1._D estimated by REML was positively biased. The bias and dispersion of the estimated _Dincreased approximately linearly with the estimated within-subject standard deviation for the reference formulation (_WR). Only a small proportion of the estimated _D exceeded the estimated _WR. 2. Distributions of the estimated _D were evaluated. At _WR = 0.30, a level of estimated _D= 0.15 was exceeded, by random chance, with a probability of about 25%. 3. Importantly, the behaviour of the ² _D values estimated from the FDA data sets was similar to that exhibited by the simulated estimates of ² _D which were generated under the conditions of true bioequivalence. Conclusions. 1. _D estimated by REML is biased; the bias increases proportionately with the estimated _WR. Consequently, exceeding a fixed level of _D (e.g., 0.15) does not indicate substantial interaction. 2. The data sets of FDA are compatible with the hypothesis of ² _D = 0. Consequently, they do not demonstrate the prevalence of subject-by-formulation interaction. Therefore, it could be sufficient and reasonable to evaluate bioequivalence from 2-period crossover studies.     

Molecular Mobility of Protein in Lyophilized Formulations Linked to the Molecular Mobility of Polymer Excipients,as Determined by High Resolution 13C Solid-State NMR

Purpose. The mobility of protein molecules in lyophilized protein formulations was compared with that of excipient molecules based on the spin-lattice relaxation time (T₁) of each molecule determined by high resolution ¹³C solid-state NMR. The relationship between molecular mobility and protein stability is discussed. Methods. Protein aggregation of lyophilized bovine serum --globulin (BGG) formulation containing dextran was measured by size exclusion chromatography. The T₁ of the BGG carbonyl carbon and dextran methin carbon in the formulation was determined by high resolution ¹³C NMR, and subsequently used to calculate the correlation time (_C) of each carbon. The spin-spin relaxation time (T₂) of BGG and dextran protons was measured by pulsed NMR spectrometry, and the critical temperature of appearance of Lorentzian relaxation due to liquid BGG and dextran protons (T_mc) was determined. Results. The _C of dextran methin carbon in BGG-dextran formulations exhibited a linear temperature dependence according to the Adam-Gibbs-Vogel equation at lower temperatures, and a nonlinear temperature dependence described by the Vogel-Tamman-Fulcher equation at higher temperatures. The temperature at which molecular motion of dextran changed was consistent with the T_mc. The _C of BGG carbonyl carbon exhibited a similar temperature dependence to the _C of the dextran methin carbon and substantially decreased at temperatures above T_mc in the presence of dextran. The temperature dependence of BGG aggregation could be described by the Williams-Landel-Ferry equation even at temperatures 20°C lower than T_mc. Conclusions. High resolution ¹³C solid-state NMR indicated that the molecular motion of BGG was enhanced above T_mc in association with the increased global segmental motion of dextran molecules.