Usefulness of the Kohlrausch-Williams-Watts Stretched Exponential Function to Describe Protein Aggregation in Lyophilized Formulations and the Temperature Dependence Near the Glass Transition Temperature

Purpose. We studied the feasibility of using the Kohlrausch-Williams-Watts stretched exponential function (KWW equation) to describe protein aggregation in lyophilized formulations during storage. Parameters representing mean aggregation time (_a) and stretched exponential constant (_a) were calculated according to the KWW equation by assuming that the time required for protein molecules to aggregate () varies because of the fact that protein aggregation occurs at a rate that depends on the degree of protein deformation resulting from stresses created during freeze-drying. The temperature dependence of the parameters near the glass transition temperature was examined to discuss the possibility of predicting protein aggregation by accelerated testing. Methods. Protein aggregation in lyophilized bovine serum -globulin (BGG) formulations containing dextran or methylcellulose, at temperatures ranging from 10 to 80°C, was followed by size-exclusion chromatography. Results. Non-exponential BGG aggregation in lyophilized formulations could be described by the KWW equation. The _a and _a parameters changed abruptly around the NMR relaxation-based critical mobility temperature for formulations containing dextran and methylcellulose. In the glassy state, in contrast, the _a parameter of these formulations exhibited continuous temperature dependence. The parameter , as calculated from _a and _a, reflected differences in values between the two excipients. Conclusions. The results indicate that the parameter _a is reflective of physical changes wihtin lyophilized formulations. Within the temperature range, during which no abrupt changes in _a were observed, knowledge regarding the _aand _a parameters allows the rate of protein aggregation to be predicted. The parameter was found to be useful in comparing the protein aggregation behavior of formulations having different _a and _a values.     

Quantitative Structure–Pharmacokinetics Relationships: II. A Mechanistically Based Model to Evaluate the Relationship Between Tissue Distribution Parameters and Compound Lipophilicity

The tissue-to-unbound plasma distribution coefficients (Kpus) of 14 rat tissues after iv administration of nine 5-n-alkyl-5-ethyl barbituric acids, determined in a previous study, were used to identify a model of the relationship between tissue distribution and lipophilicity of the homologs, expressed in terms of their octanol to water partition ratio, P. Based on mechanistic considerations and assumptions, the parameter model was expressed asKpu= f_W,[l + a(nP_l,)P^b], where f_W, is the tissue water content, (nP_l, ) is the binding capacity of the tissue, n is the number of the binding sites, a and b are the parameters of the relationship Ka = aP^b and Ka is the binding association constant of each tissue. The parameter model was linearized and fitted to the predetermined Kpu values, yielding correlation coefficients ranging between .940 and .997. The predictive performance of the parameter model was evaluated using a leave-one-out procedure with subsequent computation of the mean prediction error (ME = measurement of the prediction bias) and the square root of the mean squared prediction error (RMSE = measurement of the prediction accuracy). The ME varied between –22.48 and 61.14%, indicating a slight tendency for overpredicting. The RMSE was between 24.73 and 102% for the individual tissues across the different homologs; and between 28.33 and 85.2% for the individual homologs across the different tissues. The apparently high Kpu prediction errors, when translated through the low sensitivity of the barbiturate whole-body physiologically based pharmacokinetic model, established previously, leads to predicted tissue concentration–time profiles within 5 to 20% of the original ones. Therefore, it is concluded, that the identified mechanistically based model is a good predictor of the tissue-to-unbound Kpus in the rat tissues.     

Development and Use of Enzyme-Linked Immunosorbent Assays (ELISA) for the Detection of Protein Aggregates in Interferon-Alpha (IFN-α) Formulations

Purpose. Protein aggregates are thought to be involved in the immunogenicity of recombinant proteins in humans. To probe human IFN- formulations for the presence of soluble protein aggregates, enzyme-linked immunosorbent assays (ELISA) were developed. Methods. For the detection of IFN--IFN- and HSA-IFN- aggregates, sandwich ELISAs were developed using a monoclonal anti-IFN- antibody as a capture antibody and the same anti-IFN- antibody and an anti-human serum albumin (HSA) antibody (HRP-labeled), respectively. Results. Marketed freeze-dried, HSA-containing IFN- formulations tested in the ELISAs all contained IFN--IFN- and/or HSA-IFN- protein aggregates, although in varying amounts. These aggregates were predominantly IFN- dimers and 1:1 conjugates of HSA with IFN-. Test formulations revealed that aggregation of IFN- was strongly affected by the presence of pharmaceutical excipients, pH of the formulation, lyophilisation procedure, and storage temperature and time. Conclusions. The ELISAs are rapid, highly specific for aggregates in the presence of both IFN- and HSA monomers and allow the direct detection of both types of aggregates in formulations in the nanogram range. The new assays will assist the monitoring of the aggregate-inducing processes during IFN- formulation and storage in an early phase and the development of aggregate-free IFN- formulations.     

A pharmacokinetic model-independent approach for estimating dose required to give desired steady-state trough concentrations of drug in plasma

A maintenance dose designed to give a desired minimum concentration of drug in plasma at steady-state can be determined in a model-independent manner assuming that concentration-time data needed for the calculation are obtained after absorption and distribution are complete. Using a few concentration-time points obtained after the first dose, numerical values of and Z, a parameter consisting of different pharmacokinetic parameters for different models, can be obtained. An administration interval () can be chosen based on . Using the values of , Z, and , a maintenance dose is calculated. This approach will allow calculation of a maintenance dose when drug is present in plasma at the time the first monitored dose is given.     

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.     

Comparative Investigation by Two Analytical Approaches of Enthalpy Relaxation for Glassy Glucose,Sucrose, Maltose,and Trehalose

No HeadingPurpose. In an effort to understand the stability of glassy sugars such as glucose, sucrose, maltose, and trehalose, the molecular mobility below the glass transition temperature (T_g) was investigated by an enthalpy relaxation measurement with differential scanning calorimetry (DSC).Methods. The glassy sample was aged over several days at (T_g – 10) K to (T_g – 30) K, before a DSC heating scan was taken. The relaxed enthalpy (H_relax) was estimated from the endothermic peak area. The enthalpy relaxation time was analyzed from the time course of H_relax using two different approaches; Kohlrausch-Williams-Watts (KWW) and extended Adam-Gibbs (exAG).Results. ^KWW, which is defined as the mean average enthalpy relaxation time in a distribution, and ^eff₀ and ^eff, which correspond to the enthalpy relaxation time of the initial minimum and final maximum cooperative rearrangement region, were estimated by KWW and exAG, respectively. And three activation energies for enthalpy relaxation were calculated from the Arrhenius plot.Conclusions. Although these Es originated from different theoretical backgrounds, almost the same trend was observed for a comparison of the values of the four sugars. The finding that the Es of glassy trehalose were the largest among the four sugars may support the reason that glassy trehalose is an effective stabilizer.     

Inactivation and Aggregation of β-Galactosidase in Lyophilized Formulation Described by Kohlrausch-Williams-Watts Stretched Exponential Function

Purpose. To examine whether the empirical Kohlrausch-Williams-Watts (KWW) equation is applicable not only to protein aggregation but also to protein denaturation in lyophilized formulations. Lyophilized -galactosidase (-GA) formulations containing polyvinylalcohol and methylcellulose were used as model formulations. The possibility of predicting storage stability based on the temperature dependence of the estimated parameters of inactivation/aggregation—time constant () and its distribution () is discussed. Methods. Protein aggregation in lyophilized -GA formulations at 10-70°C and 6-43% relative humidity was determined as a function of time by size exclusion chromatography. Enzyme activity was also determined using 2-nitrophenyl--D-galactopyranoside as a substrate. Results. Inactivation and aggregation of -GA were describable with the empirical KWW equation, regardless of whether the temperature was above or below the NMR relaxation-based critical mobility temperature (T_mc) or whether protein molecules with different degrees of deformation resulting from stresses during lyophilization exist in the formulation. The estimated parameter for protein aggregation decreased rapidly as temperature increased beyond T_mc because the mobility of polymer molecules increased in the initial stages of glass transition. The time required for 10% enzyme to aggregate (t₉₀) calculated from the and parameters exhibited a change in temperature dependence gradient near T_mc. In contrast, t₉₀ for protein inactivation exhibited temperature dependence patterns varying with the excipients. Conclusions. The t₉₀ calculated from the estimated and parameters was found to be a useful parameter for evaluating the stability of lyophilized -GA formulations. The prediction of t₉₀ by extrapolation was possible in the temperature range in which did not rapidly vary with temperature.     

Analysis of the Compression Mechanics of Pharmaceutical Agglomerates of Different Porosity and Composition Using the Adams and Kawakita Equations

Purpose. To analyze the mechanics of some pharmaceuticalagglomerates during uniaxial confined compression by using compressionparameters derived from the Heckel, Kawakita and Adams equations, and tostudy the influence of these compression parameters on the tablet-formingability of agglomerates. Methods. Force and displacement data sampled during in-diecompression of agglomerates was used to calculate compression parametersaccording to the Heckel ( _y ), Kawakita(1/b and a), and Adams (₀)equations. Mechanical strength of single agglomerates as well as the airpermeability and tensile strength of tablets prepared from them were alsodetermined. Results. _y from the Heckelequation did not differ between agglomerates of different porosity. Both1/b and ₀ varied with agglomerate porosityand composition. These two compression parameters were linearly related toeach other. No general correlation was found between 1/b and₀ and the strength of single agglomerates. The twoparameters were related to the intergranular pore structure and tensilestrength of tablets formed from the agglomerates. Conclusions. 1/b and ₀ maybe interpreted as measures of the agglomerate shear strength during uniaxialconfined compression, and as such they may be used as indicators of thetabletting performance of the agglomerates.     

Effects of bucumolol,nadolol and nifenalol on maximum upstroke velocity of action potential in guinea pig papillary muscles

Summary The effects of bucumolol (BUC), nadolol (NAD) and nifenalol (NIF) on contractile forces and on action potentials (APs) were investigated in isolated guinea pig atrial and papillary muscles, respectively. Log 1/ED₄₀ values for the negative inotropic effects of these drugs were 0.097,10 and 0.74 mmol/l in this order. BUC (50 mol/l), NAD (0.5 mmol/l) and NIF (0.2 mmol/l) produced about 60,20 and 20% reduction of V _max at 1 Hz. The frequency-dependent reductions at these and higher concentrations were greatest for BUC, intermediate for NAD and least for NIF. These potencies at certain frequencies were, as a whole, consistent with log P-potency relationship established in our previous papers (Harada et al. 1981; Ban et al. 1985). The reductions of V _max in APs in response to premature stimuli during basic stimuli at the rate of 0.25 or 0.027 Hz decayed exponentially during diastolic intervals (DI). The time constants of these decay processes () estimated by linear and nonlinear regression analyses and by eye were 12.2–9.6 s for BUC (50–100 mol/l) and 2.9–4.8 s for NAD (1–2 mmol/l) and 57–87 ms for NIF (0.2–1 mmol/l). In terms of the molecular weight (MW)-log relationship (Ban et al. 1985), these values are within the 95% fiducial limit for BUC and NAD and deviated from the lower fiducial limit for NIF. The frequency-dependent reductions of V _max by these drugs were explained in terms of a function of and the intercept A_o. Based on the study mady by Cohen et al. (1984) we estimated the distortion of the time course of recovery of V _max from that of sodium channel availability in the presence of drugs. Our computation shows that the relative change of the estimated at the same level of the zero-intercept does, but that of the estimated at different levels does not, reflext exactly that of the time constants of the recovery of sodium channel availability.     

In Vitro Cutaneous Disposition of a Topical Diclofenac Lotion in Human Skin: Effect of a Multi-Dose Regimen

Purpose. This study determines comparative bioavailability of diclofenac sodium lotion compared to an aqueous solution after topical application to viable human skin in vitro. In addition, the difference between a single dose and multiple doses (8 times) was also determined. Methods. An in vitro flow-through diffusion cell system was employed, using radiolabelled diclofenac sodium. Results. Multiple doses of lotion (2 l/cm² and 5 l/cm²) delivered a total of 40.1 ± 17.6 g and 85.6 ± 41.4 g diclofenac, respectively, at 48 h, compared to only 9.4 ± 2.9 g and 35.7 ± 19.0 g absorbed after topical application of diclofenac as an aqueous solution (P < 0.05). A single dose study showed no statistical difference between diclofenac delivered in lotion or an aqueous solution. Over 48 h the total absorption from lotion was 10.2 ± 6.7 g and 26.2 ± 17.6 g (2 l/cm² and 5 l/cm², respectively), compared to 8.3 ± 1.5 g and 12.5 ± 5.7 g from an aqueous solution. Both single doses of lotion and aqueous diclofenac showed decreased diclofenac absorption into the receptor fluid between 12 and 24 h. However, when applied multiple times, absorption from lotion was continually increasing up to 48 h. The total dose accountability ranged from 76.8 ± 8.2% to 110.6 ± 15.1% of the applied dose. Conclusions. Diclofenac lotion exhibited enhanced diclofenac percutaneous absorption rate through human skin (mass, flux and partition coefficient) when applied a multiple number of times and this enhanced absorption was maintained over 48 h. This suggests that a constituent of the lotion (DMSO) will enhance human skin absorption of diclofenac when used in a multi-dose regimen, but not after a single dose.     

Multiple inhibitory mechanisms mediate non-adrenergic non-cholinergic relaxation in the circular muscle of the guinea-pig colon

Summary The mechanisms responsible for nerve-mediated, non-adrenergic, non-cholinergic (NANC) relaxation in mucosa-free circular muscle strips from the proximal colon of the guinea-pig were investigated. Electrical field stimulation (EFS, 1–20 Hz, trains of 5 s duration, 100 V, 0.25 ms pulse width) in the presence of atropine (1 mol/l) and guanethidine (3 mol/l) evoked a triphasic motor response consisting of. (a) a primary relaxation, (b) a rebound contraction and (c) a secondary relaxation. These three responses were abolished by tetrodotoxin (1 mol/l). Both apamin (0.01–0.3 mol/l), a known blocker of low conductance, calcium-activated potassium channels in smooth muscles, and L-nitroarginine (L-NOARG) (1–100 mol/l), a known blocker of nitric oxide (NO) synthase, increased the tone of the strips. Maximum effects on tone were observed with 0.1 mol/l apamin (21 ± 3% of KCl-induced contraction) and 30 mol/l L-NOARG (26 ± 4% of KCl response). The combined administration of 0.1 mol/l apamin and 30 mol/l L-NOARG produced an increase in tone (47 ± 5% of KCl response) that was larger than that produced by either compound alone. Neither apamin (0.1 mol/l) nor L-NOARG (30 mol/l) affected the isoprenaline-induced relaxation.Apamin (0.1 mol/l) depressed, but did not abolish, the primary relaxation to EFS at all frequencies without affecting the secondary relaxation. Apamin also enhanced the rebound contraction at a frequency of 1 Hz. L-NOARG (30 mol/l) depressed, but did not abolish, the primary relaxation to EFS at all frequencies, had no effect on the rebound contraction and inhibited the secondary relaxation evoked at frequencies of 1–5 Hz, but not 10–20 Hz. L-arginine (300 mol/l) reversed the effect of L-NOARG on tone and the inhibitory effect on the EFS-evoked relaxation. In the presence of apamin and L-NOARG, the primary relaxation was suppressed at all frequencies; the secondary relaxation was inhibited at 1–5 Hz and unchanged at 10–20 Hz, as observed with L-NOARG alone. We conclude that three distinct mechanisms mediate the NANC relaxation of the circular muscle of the proximal colon of the guinea-pig in response to EFS. One mechanism can be operationally defined as apamin-sensitive and a second as L-NOARG-sensitive, the latter implying a possible role of NO as an inhibitory transmitter. A third NANC inhibitory mechanism, which is apamin- and L-NOARG-resistant, is also suggested.Correspondence to: C. A. Maggi at the above address     

Development of a Remote Electrode System for Monitoring the Water Content of Materials Inside a Glass Vial

Purpose: This article explores the use of a remote electrode dielectric measurement system to monitor the water content of hydrated ovalbumin inside a glass vial. Methods: The intrinsic dielectric properties of hydrated ovalbumin were characterized first using conventional parallel plate electrodes. The second stage was to simulate a remote electrode measurement by placing nonconductive, nondispersive polyethylene films between the sample and electrodes. Finally, a study on the dielectric measurement of ovalbumin contained in a 10 ml glass vial was undertaken with the electrodes external to the glass vial. Results: The dielectric behavior of hydrated ovalbumin was characterized by charge transfer (i.e., protons) in the hydrogen bonded network of water molecules in the bulk sample. The mechanism was identified as an anomalous low-frequency dispersion and a dielectric loss peak (₃). The dielectric relaxation time, ₃, of the ₃ dispersion was especially sensitive to water content. Moreover, a good correlation (R² = 93%) was observed between relaxation times ₃ obtained from measurements using conventional parallel plate electrodes and the remote electrode system. Conclusions: Dielectric measurements using remote electrodes attached to a glass vial are therefore applicable for the in situ measurement of water content in materials. The application of this technology to the determination of the lyophilization end point is suggested.     

Novel Direct Curve Comparison Metrics for Bioequivalence

Purpose. The object of this work was to devise four new direct curve comparison (DCC) metrics and examine each metric's distribution properties and performance characteristics. Methods. DCC metrics, Cmax, and AUCi were calculated from two bioequivalence studies of three sustained release carbamazepine formulations, where a range of profile similarity was observed. DCC metric values and their confidence intervals were compared to Cmax and AUCi. Results. The DCC metrics , _m, _a, and _s exhibited more favorable distributions than Cmax and AUCi ratios, which were frequently skewed. The DCC metrics performed differently than Cmax and AUCi ratios in profile comparisons due to the nature of the DCC metrics. Unlike Cmax and AUCi, the DCC metrics utilize all data points to directly compare entire profiles. Each DCC metric appears to measure exposure in a single assessment. Possible bioequivalence acceptance criteria are: 1.40, _m 0.35, _a 0.27, and _s 0.102. Conclusions. These DCC metrics, particularly p_m, are promising bioequivalence metrics for exposure.     

New Cationic Lipid Formulations for Gene Transfer

Purpose. To develop appropriate dosage forms of DNA for gene delivery. Methods. 3[N-(N, N dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) was mixed either with Tween 80 alone, or with additional lipid components including castor oil and phosphatidylcholine (PC) or dioleoylphosphatidylethanolamine (DOPE) to make different lipid formulations. The particle size and the physical stability of the formulations upon mixing with plasmid DNA containing the luciferase cDNA were examined using laser light scattering measurement. The transfection activity of the DNA/lipid complexes was tested in presence or absence of serum using a cell culture system. Results. We demonstrated that many favorable properties as a gene carrier could be achieved by formulating DNA into new dosage forms using Tween 80 as the major emulsifier. Compared to the cationic liposomes, these new formulations transfected different cell lines with an equivalent or higher efficiency. Not only are they resistant to serum, but also form stable DNA complexes which could be stored for longer periods of time without losing transfection activity. Conclusions. Cationic lipids formulated into different lipid formulations using Tween 80 as a surfactant appeared to have more favorable physical and biological activities than traditional cationic liposomes as a carrier for gene delivery.     

Use of the Peptide Carrier System to Improve the Intestinal Absorption of L-α-Methyldopa: Carrier Kinetics,Intestinal Permeabilities,and In Vitro Hydrolysis of Dipeptidyl Derivatives of L-α-Methyldopa

Intestinal permeabilities of five dipeptidyl derivatives of L--methyldopa (I) were studied by an in situ intestinal perfusion method. The dipeptides displayed a significant increase in their permeabilities compared to L--methyldopa. The increases ranged from 4 to 20 times. These results suggest that the peptide transport system is less structurally specific than the amino acid transport systems and can be used as an absorption pathway for peptide analogues. The kinetic advantage demonstrated by the dipeptide, L--methyldopa-L-phenylalanine, over the amino acid analogue, L--methyldopa, suggests that the peptide carrier would be a possible route for improving the intestinal absorption of pharmacologically active amino acid analogues. Furthermore, the preliminary results of in vitro hydrolysis studies of selected dipeptidyl derivatives indicate that the peptide carrier system could be used as a base for a prodrug strategy.     

Influence of Hydrodynamics and Particle Size on the Absorption of Felodipine in Labradors

Purpose. To study the influence of GI hydrodynamics and drug particle size on felodipine absorption in the dog. Methods. Labradors fistulated at midjejunum were used to selectively study the influence of hydrodynamics and particle size on the in vivodissolution and absorption of the poorly soluble, lipophilic drug felodipine. A combination of infusion and oral administration of either normal saline or a 5% glucose solution was used to maintain fasted and establish fed state motility patterns, respectively. The absorption characteristics of both a micronized (8 m) and a coarse fraction (125 m) of felodipine were subsequently studied under these two motility patterns. Results. A reduction in particle size led up to an approximate 22-fold increase in maximum plasma concentration and up to an approximate 14-fold increase in area under the curve, with a commensurate decrease in the time at which the maximum plasma concentration occurred. Although the absorption of felodipine from the solution and micronized suspension was not influenced by a change in the hydrodynamics, felodipine was absorbed from the coarse suspension almost twice as well in the fed state as under fasted conditions. Conclusions. Absorption from coarse suspensions of felodipine was sensitive to luminal hydrodynamics, whereas micronized suspensions were not. However, the particle size seems to have a much more important influence on the bioavailability of felodipine than the hydrodynamics per se.     

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.     

Synthesis and Anti-inflammatory Effect of Chalcones and Related Compounds

Purpose. Mast cell and neutrophil degradations are the important players in inflammatory disorders. Combined with potent inhibition of chemical mediators released from mast cells and neutrophil degranulations, it could be a promising anti-inflammatory agent. 2,5-Dihydroxychalcone has been reported as a potent chemical mediator and cyclooxygenase inhibitor. In an effort to continually develop potent anti-inflammatory agents, a novel series of chalcone, 2- and 3-hydroxychalcones, 2,5-dihydroxychalcones and flavanones were continually synthesized to evaluate their inhibitory effects on the activation of mast cells and neutrophils and the inhibitory effect on phlogist-induced hind-paw edema in mice. Methods. A series of chalcones and related compounds were prepared by Claisen-Schmidt condensation of appropriate acetophenones with appropriate aromatic aldehyde and the anti-inflammatory activities of these synthetic compounds were studied on inhibitory effects on the activation of mast cells and neutrophils. Results. Some chalcones showed strong inhibitory effects on the release of -glucuronidase and histamine from rat peritoneal mast cells stimulated with compound 48/80. Almost all chalcones and 4-hydroxyflavanone exhibited potent inhibitory effects on the release of -glucuronidase and lysozyme from rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP). Some chalcones showed potent inhibitory effects on superoxide formation of rat neutrophils stimulated with fMLP/cytochalasin B (CB) or phorbol myristate acetate (PMA). 2,3-Dihydroxy-, 2,5-dihydroxy-4-chloro-, and 2,5-dihydroxychalcone showed remarkable inhibitory effects on hind-paw edema induced by polymyxin B in normal as well as in adrenalectomized mice. Conclusions. These results indicated that the anti-inflammatory effects of these compounds were mediated, at least partly, through the suppression of chemical mediators released from mast cells and neutrophils.     

Protein Aggregates Seem to Play a Key Role Among the Parameters Influencing the Antigenicity of Interferon Alpha (IFN-α) in Normal and Transgenic Mice

Purpose. During long-term treatment of various malignant or viral diseases with IFN- up to 20% of patients develop anti-IFN- antibodies for as yet unknown reasons. Methods. To address this issue, a mouse model using Balb/C mice was established and the relevance of several potentially anti-IFN- antibodies inducing factors was studied. Results. The model revealed that both a higher frequency of injections and a higher dosage of IFN- were more immunogenic and that the route of administration affected the antibody response to IFN-. The intrinsic immunostimulatory activity of IFN- itself also enhanced the immune response. IFN- protein aggregates (IFN--IFN- and human serum albumin (HSA)-IFN- aggregates), which were recently identified in all marketed IFN- products, were significantly more immunogenic than IFN- monomers. These aggregates broke the tolerance against human IFN- monomers in human IFN- transgenic mice. Conclusions. Based on these animal studies it is proposed that the immune response to IFN- in humans is most probably elicited by a combination of several factors among which IFN- protein aggregates seem to play a key role.     

Biliary Excretion of 17β-Estradiol 17β-d-Glucuronide Is Predominantly Mediated by cMOAT/MRP2

Purpose. The mechanism for the biliary excretion of 17-estradiol170-d-glucuronide (E₂17G), a cholestatic metabolite of estradiol, isstill controversial. The purpose of the present study is to examine thetransport of E₂17G across the bile canalicular membrane. Methods. We examined the uptake of [³H]E₂17G by isolatedcanalicular membrane vesicles (CMVs) prepared from Sprague-Dawley (SD)rats and Eisai Hyperbilirubinemic rats (EHBR) whose canalicularmultispecific organic anion transporter/multidrug resistance associatedprotein 2 (cMOAT/MRP2) function is hereditarily defective. Also,in vivo biliary excretion of intravenously administered [³H]E₂17Gwas examined. Results. In CMVs prepared from SD rats, but not from EHBR, amarked ATP-dependent uptake of [³H]E₂17G was observed.Moreover, E₂17G competitively inhibited the ATP-dependent uptake of[³H]2,4-dinitrophenyl-S-glutathione (DNP-SG). In addition, nosignificant inhibitory effect of verapamil (100 M) and PSC-833 (5 M) onthe uptake of [³H]E₂17G was observed. In vivo, the biliary excretionof intravenously administered [³H]E₂17G was severely impaired inEHBR while the biliary excretion of [³H]E₂17G in SD rats wasreduced by administering a cholestatic dose (10 mol/kg) unlabeledE₂17G, but not by PSC-833 (3 mg/kg). Conclusions. The transport of E₂17G across the bile canalicularmembrane is predominantly mediated by cMOAT/MRP2.