The Relationship Between Protein Aggregation and Molecular Mobility Below the Glass Transition Temperature of Lyophilized Formulations Containing a Monoclonal Antibody

Purpose. To find out if the physical instability of a lyophilized dosage form is related to molecular mobility below the glass transition temperature. Further, to explore if the stability data generated at temperatures below the glass transition temperature can be used to predict the stability of a lyophilized solid under recommended storage conditions. Methods. The temperature dependence of relaxation time constant, , was obtained for sucrose and trehalose formulations of the monoclonal antibody (5 mg protein/vial) from enthalpy relaxation studies using differential scanning calorimetry. The non-exponentiality parameter, , in the relaxation behavior was also obtained using dielectric relaxation spectroscopy. Results. For both sucrose and trehalose formulations, the variation in with temperature could be fitted Vogel-Tammann-Fulcher (VTF) equation. The two formulations exhibited difference sensitivities to temperature. Sucrose formulation was more fragile and exhibited a stronger non-Arrhenius behavior compared to trehalose formulation below glass transition. Both formulations exhibited <2% aggregation at t values <10, where t is the time of storage. Conclusions. Since the relaxation times for sucrose and trehalose formulations at 5°C are on the order of 10⁸ and 10⁶ hrs, it is likely that both formulations would undergo very little (<2%) aggregation in a practical time scale under refrigerated conditions.     

Freeze-Concentration Separates Proteins and Polymer Excipients Into Different Amorphous Phases

Purpose. To study the miscibility of proteins and polymer excipients in frozen solutions and freeze-dried solids as protein formulation models. Methods. Thermal profiles of frozen solutions and freeze-dried solids containing various proteins (lysozyme, ovalbumin, BSA), nonionic polymers (Ficoll, polyvinylpyrrolidone [PVP]), and salts were analyzed by differential scanning calorimetry (DSC). The polymer miscibility was determined from the glass transition temperature of maximally freeze-concentrated solute (T_g) and the glass transition temperature of freeze-dried solid (T_g). Results. Frozen Ficoll or PVP 40k solutions showed T_g at –22°C, while protein solutions did not show an apparent T_g. All the protein and nonionic polymer combinations (5% w/w, each) were miscible in frozen solutions and presented single T_gs that rose with increases in the protein ratio. Various salts concentration-dependently lowered the single T_gs of the proteins and Ficoll combinations maintaining the mixed amorphous phase. In contrast, some salts induced the separation of the proteins and PVP combinations into protein-rich and PVP-rich phases among ice crystals. The T_gs of these polymer combinations were jump-shifted to PVP's intrinsic T_g at certain salt concentrations. Freeze-dried solids showed varied polymer miscibilities identical to those in frozen solutions. Conclusions. Freeze-concentration separates some combinations of proteins and nonionic polymers into different amorphous phases in a frozen solution. Controlling the polymer miscibility is important in designing protein formulations.     

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.     

Immobilized Artificial Membrane (lAM)-HPLC for Partition Studies of Neutral and Ionized Acids and Bases in Comparison with the Liposomal Partition System

Purpose. To study the partitioning of model acids ((RS)-warfarin and salicylic acid), and bases (lidocaine, (RS)-propranolol and diazepam), with immobilized artificial membrane (lAM)-HPLC, as compared to partitioning in the standardized phosphatidylcholine liposome/buffer system. Methods. The pH-dependent apparent partition coefficients D were calculated from capacity factors (k_IAM) obtained by IAM-HPLC, using a 11-carboxylundecylphosphocholine column. For lipophilic compounds k_IAM, values were determined with organic modifiers and extrapolation to 100% water phase (k_IAMw) was optimized. Temperature dependence was explored (23 to 45° C), and Gibbs free energy (G), partial molar enthalpy (H) and change in entropy (S) were calculated. Equilibrium dialysis was used for the partitioning studies with the liposome/buffer system. Results. For extrapolation of k_IAMw, linear plots were obtained both with the respective dielectric constants and the mole fractions of the organic modifier. All tested compounds showed a similar pH-D diagram in both systems; however, significant differences were reproducibly found in the pH range of 5 to 8. In all cases, G and H were negative, whereas S values were negative for acids and positive for bases. Conclusions. In both partitioning systems, D values decreased significantly with the change from the neutral to the charged ionization state of the solute. The differences found under physiological conditions, i.e. around pH 7.4, were attributed to nonspecific interactions of the drug with the silica surface of the IAM column.     

A Population Growth Model of Dissolution

Purpose. To develop a new approach for describing drug dissolution which does not require the presuppositions of time continuity and Fick's law of diffusion and which can be applied to both homogeneous and heterogeneous media. Methods. The mass dissolved is considered to be a function of a discrete time index specifying successive 'generations' (n). The recurrence equation: _n+1 = _n + r(l – _n)(1 – _n X ₀/) was derived for the fractions of dose dissolved _n and _{n +1}, between generations n and n + 1, where r is a dimensionless proportionality constant, X ₀ is the dose and is the amount of drug corresponding to the drug's solubility in the dissolution medium. Results. The equation has two steady state solutions, _ss = 1 when (X ₀/) 1 and _ss = /X ₀ when (X ₀/) > 1 and the usual behavior encountered in dissolution studies, i.e, a monotonic exponential increase of _n reaching asymptotically the steady state when either r < /X ₀ < 1 or r < 1 < /X ₀. Good fits were obtained when the model equation was applied to danazol data after appropriate transformation of the time scale to 'generations'. The dissolution process is controlled by the two dimensionless parameters /X ₀ and r, which were found to be analogous to the fundamental parameters dose anddissolution number, respectively. The model was also used for the prediction of fraction of dose absorbed for highly permeable drugs. Conclusions. The model does not rely on diffusion principles and therefore it can be applied under both homogeneous and non-homogeneous conditions. This feature will facilitate the correlation of in vitro dissolution data obtained under homogeneous conditions and in vivo observations adhering to the heterogeneous milieu of the GI tract.     

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.     

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.     

Protection Afforded by Maltosyl-β-cyclodextrin Against α-Chymotrypsin-Catalyzed Hydrolysis of a Luteinizing Hormone-Releasing Hormone Agonist, Buserelin Acetate

Purpose. The present study addresses how maltosyl--cyclodextrin (G₂--CyD) impacts upon the -chymotrypsin-catalyzed hydrolysis of buserelin acetate, an agonist of luteinizing hormone-releasing hormone with emphasis upon the direct effect of G₂--CyD on the activity of the protease. Methods. Kinetic and solubility studies were performed in isotonic phosphate buffer (pH 7.4) at 25°C and 37°C. The interaction of -chymotrypsin with G₂--CyD in the buffer solution was examined by differential scanning calorimetry. Results. G₂--CyD decelerated the -chymotrypsin-catalyzed hydrolysis of buserelin acetate to give the 1–3 tripeptide and the 4–9 hexapeptide fragments. This deceleration can be explained solely by a nonproductive encounter between a complex of the substrate with G₂--CyD and the protease at relatively low CyD concentrations, while the direct inhibitory effect of G₂--CyD on the proteolytic activity made a considerable contribution to the overall deceleration of the hydrolysis at higher CyD concentrations. Calorimetric studies indicate the presence of intermediate states in the thermal unfolding of -chymotrypsin, simultaneously accompanied by the autolysis. By contrast, a two-state thermal unfolding of -chymotrypsin was observed in the presence of G₂--CyD, suggesting reduced proteolytic activity upon binding to G₂--CyD. Conclusions. These results suggest that G₂--CyD at higher concentrations inhibits the proteolytic action of -chymotrypsin through direct interaction with the protease, as well as through the formation of a non-productive complex with the substrate.     

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.     

Antifungal Activity of a New Triterpenoid Glycoside from Pithecellobium racemosum (M.)

Purpose. In a continuation of our search for novel antifungal compounds from higher plants, the standard extract of the bark of Pithecellobium racemosum was found to have good activity against important AIDS-related opportunistic yeasts. Methods. The extract was subjected to bioguided fractionation using silica gel column chromatography which led to purification of triterpene glycosides. The structures of these compounds were determined by a combination of spectroscopic (IR, NMR, HRMS) and chemical methods. Results. Compound 1 is a new glycoside, 3-O[-L-arabinopyranosyl (1 -2)][-L arabinopyranosyl (1 -6)]2-acetoamido-2-deoxy--D-gluco-pyranosyl oleanolic acid and Compound 2 was identified as the known compound 3-O-[-L-arabinopyranosyl (l-2)]-L-arabinopyranosyl (1-6)] 2-acetamido-2-deoxy--D-glucopyranosyl echinocystic acid. Conclusions. Compound 1 is a new glycoside, 3-O-[-L-arabinopyranosyl (1-2)]-L-arabinopyranosyl (l-6)]-2-acetoamido-2-deoxy--D-glucopyranosyl oleanolic acid and exhibits moderate antifungal activity against T. mentogrophytes, C. albicans and S. cerevisiae with MIC values of 6.25, 12.5 and 12.5 g/ml respectively.     

Stabilizing and Solubilizing Effects of Sulfobutyl Ether β-Cyclodextrin on Prostaglandin E1 Analogue

Purpose. Parent cyclodextrins are known to accelerate the degradations such as dehydration and isomerization of E-type prostaglandins in neutral and alkaline solutions. The objective of this study was to attempt the stabilization and solubilization of E₁-type prostaglandin analogue in aqueous solution by biocompatible cyclodextrin derivatives. Methods. The interaction of an E₁-type prostaglandin, methyl 7-[(1R,2R,3R)-3-hydroxy-2-[(E)-(3S)-3-hydroxy-4-(m-methoxymethylphenyl)1-butenyl]-5-oxocyclopentyl]-5-thiaheptanoate (MEester) with cyclodextrins (CyDs) was studied by spectroscopies and the solubility method. The degradation of MEester was monitored by high-performance liquid chromatography. Results. ¹H-nuclear magnetic resonance spectroscopic studies indicated that MEester forms 1:1 inclusion complexes with -, -, and -CyDs in solutions, where -CyD interacts with the -side chain containing methyl ester moiety of the drug, whereas - and -CyDs preferentially include around the five-membered ring and both side chains of the drug. Parent -CyD and hydrophilic derivatives, such as 2-hydoxypropyl-- and --CyDs, sulfobutyl ether -CyD (SBE--CyD) and maltosyl -CyD showed higher solubilizing abilities against MEester over parent - and -CyDs. SBE--CyD and 2,6-dimethyl--CyD (DM--CyD) significantly decelerated the degradation of MEester, particularly the base-catalyzed dehydration, in neutral and alkaline solutions, whereas other CyDs accelerated the degradation. The acid-catalyzed degradation of MEester (pH < 3) was decelerated by the addition of CyDs, especially -CyD. Conclusions. SBE--CyD with low hemolytic activity and low toxicity is useful as a pharmaceutical carrier for the preparation of injectable MEester, because of its higher stabilizing and solubilizing effects on MEester. Furthermore, SBE--CyD can be useful as a stabilizing agent for drugs, that are subject to base-catalyzed degradations, probably because of the electric repulsion between anionic charges of the sulfobutyl moiety and catalytic anionic species such as hydroxide ion.     

Proofs of the Structure of Lipid Coated Nanoparticles (SMBV™) Used as Drug Carriers

Purpose. Supramolecular Biovectors (SMBV) consist of cross-linkedcationic nanoparticles surrounded by a lipid membrane. Thepurpose was to study the structure of the lipid membrane and tocharacterise its interaction with the nanoparticles in order to differentiateSMBV from other polymer/lipid associations. Methods. The interaction of lipids with the nanoparticle surface wasstudied using zeta potential, Fluorescence Energy Transfer (FET) andFluorescence Microscopy. SMBV were compared to liposomes andmixtures nanoparticles/liposomes. Finally the structure of SMBVwas visualised by Electron Microscopy. Results. Zeta potential measurements showed that lipids on SMBVhad a pronounced shielding effect on the surface charge. This was notthe case for mixtures of nanoparticles and liposomes. FET experimentsconfirmed these results indicating that, for SMBV, the lipids aremuch closer to the nanoparticle surface. SMBV Fluorescence microscopyon model microparticles showed a lipid crown on SMBV thatwas confirmed by electron microscopy on SMBV nanoparticles. Conclusions. Results show that in case of SMBV lipids are stronglyadsorbed on the polysaccharide core surface probably due to ionic/hydrophobicinteractions. The resulting supramolecular structure is aspherical cationic polysaccharide particle surrounded by a phospholipid/cholesterol layer.     

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.     

Disposition Kinetics of α-Tocopherol in Apolipoprotein B Knockout Mice

Purpose. We examined the effects of apolipoprotein B (apoB) on the disposition kinetics of -tocopherol by using apoB knockout mice. Methods. The concentrations of -tocopherol in plasma and tissues were measured by gas chromatography-mass spectrometry. Results. In apob (–/–) mice, the endogenous levels of -tocopherol in plasma and tissues (except liver) were significantly lower, and the liver concentration was significantly higher than those in wild-type mice. After single i.v. administration of -tocopherol (25 mg/kg), the area under the plasma concentration-time curve (AUC) and the distribution volume at steady state were significantly decreased, whereas the total clearance of -tocopherol was significantly increased in apob (–/–) vs. wild-type mice. -Tocopherol was highly distributed to the liver, compared with other tissues. After an oral administration of -tocopherol (100 mg/kg), the intestinal absorption of -tocopherol was very low in apoB knockout mice, as the value of AUC_0-32h for apob (–/–) mice (17.7 ± 8.3 g h/mL) was significantly less than that for apob (+/+) wild-type mice (96.5 ± 15.8 g h/mL, mean ± SD of five experiments, p < 0.01). The biliary excretion of -tocopherol was significantly greater in apob (+/–) mice than in apob (+/+) mice. Conclusions. These results show that apoB plays a role in hepatic secretion and intestinal absorption of -tocopherol.     

Antagonists that differentiate between α2A- and α2D-adrenoceptors

Four antagonists were examined for their ability to differentiate _2A from the orthologous _2Dadrenoceptors. The antagonists were (2S,12bS) 1, 3-di-methylspiro(1, 3, 4, 5, 6, 6, 7,12b-octahydro-2H-benzo[b]furo[2,3-a]quinolizine)-2,4-pyrimidin-2-one (MK 912), 2-[2-(methoxy-1, 4-benzodioxanyl)imidazoline (RX 821002), efaroxan and benoxathian. The ₂-autoreceptors in rabbit brain cortex were chosen as _2A- and the a₂-autoreceptors in guinea-pig brain cortex as _2D-adrenoceptors. Slices of the brain cortex were preincubated with ³H-noradrenaline and then superfused and stimulated electrically by brief pulse trains (4 pulses, 100 Hz) that led to little, if any, ₂-autoinhibition. 5-Bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK 14,304) was used as an ₂-adrenoceptor agonist.UK 14, 304 decreased the stimulation-evoked overflow of tritium. The antagonists shifted the concentration-inhibition curve of UK 14, 304 to the right in an apparently competitive manner. Dissociation constants of the antagonists were calculated from the shifts. MK 912, RX 821002 and efaroxan had markedly higher affinity for (guinea-pig) _2D-adrenoceptors (pK _d values 10.0, 9.7 and 9.1, respectively) than for (rabbit) _2A-adrenoceptors (pK _d 8.9, 8.2 and 7.6, respectively). Benoxathian had higher affinity for _2A- (pK _d 7.4) than for _2D-adrenoceptors (pK _d 6.9). Ratios calculated from the K _d values of the four compounds differentiated between _2A and _2D up to 100 fold. It is concluded that MK 912, RX 821002, efaroxan and benoxathian are antagonists with high power to differentiate _2A- from _2D-adrenoceptors.     

Influence of polymorphism on the surface energetics of salmeterol xinafoate crystallized from supercritical fluids

Purpose. To characterize the surface thermodynamic properties of two polymorphic forms (I and II) of salmeterol xinafoate (SX) prepared from supercritical fluids and a commercial micronized SX (form I) sample (MSX). Methods. Inverse gas chromatographic analysis was conducted on the SX samples at 30, 40, 50, and 60°C using the following probes at infinite dilution: nonpolar probes (NPs; alkane C5-C9 series); and polar probes (PPs; i.e., dichloromethane, chloroform, acetone, ethyl acetate, diethyl ether, and tetrahydrofuran). Surface thermodynamic parameters of adsorption and Hansen solubility parameters were calculated from the retention times of the probes. Results. The free energies of adsorption (-G_A) of the three samples obtained at various temperatures follow this order: SX-II > MSX SX-I for the NPs; and SX-II > MSX > SX-I for the PPs. For both NPs and PPs, SX-II exhibits a less negative enthalpy of adsorption (H_A) and a much less negative entropy of adsorption (S_A) than MSX and SX-I, suggesting that the high -G_A of SX-II is contributed by a considerably reduced entropy loss. The dispersive component of surface free energy (_s ^D) is the highest for MSX but the lowest for SX-II at all temperatures studied, whereas the specific component of surface free energy of adsorption (-G_A ^SP) is higher for SX-II than for SX-I. That SX-II displays the highest -G_A for the NP but the lowest _s ^D of all the SX samples may be explained by the additional -G_A change associated with an increased mobility of the probe molecules on the less stable and more disordered SX-II surface. The acid and base parameters, K_A and K_D, that were derived from H_A ^SP reveal significant differences in the relative acid and base properties among the samples. The calculated Hansen solubility parameters (_D, _P, and _H) indicate that the surface of SX-II is the most polar and most energetic of all the three samples in terms of specific interactions (mostly hydrogen bonding). Conclusions. The metastable SX-II polymorph possesses a higher surface free energy, higher surface entropy, and a more polar surface than the stable SX-I polymorph.     

Pharmacokinetics of sulfametopyrazine (Kelfizine W) effects of a single oral dose

Summary The pharmacokinetics of sulfametopyrazine were studied for seven days after a single oral dose of 2 g. in healthy volunteers in order to establish its chemotherapeutic value. — The appearance and disappearance of the drug in the plasma were evaluated both for compounds with a free amino group and for total sulphonamides. The half-life and absorption, distribution, elimination and excretion coefficients were calculated, as well as the concentrations in plasma water and interstitial fluid. The estimated drug concentrations in the urine agreed with those calculated from the excretion coefficients. — In all subjects at the end of the seventh day the concentrations in all body compartments of active compounds exceeded the minimum required for a therapeutic effect. The highest concentrations found in the urine were always significantly lower than the drug's basal solubility at pH 5, thus excluding any risk of crystalluria.Glossary of symbols total binding capacity of plasma proteins for SMP - Specific gravity of blood ( Bl), and interstitial fluid (IF) - minimum inhibitory concentration for bacterial growth. Evaluation of against E. coli or other pathogenic bacteria in a medium free of antagonists [29] - ratio of dose interval to half-life - dose interval - safety factor. Proportionality constant between andc _min for a therapeutic efficacy of 95 per cent - fraction of the administered drug absorbed from the depot (gastrointestinal tract etc.) - distribution coefficient with respect to the drug concentration in blood plasma (ml/g) - D* initial dose of the drug - D maintenance dose - M molecular weight of the drug (280) - G weight of subject (kg) - F area between time axis and concentration curve (plotted c' values) - t _50% apparent biological half-life - w ¹ water content of plasma (ml/ml) - p protein concentration of plasma (pBl), or interstitial fluid (pIF) (g/l) - c _IF concentration in the interstitial fluid - C ₀ concentration in plasma at zero time after i.v. administration - c ₁ ⁰ concentration in plasma after oral absorption extrapolated to zero time - c ₁ concentration in plasma water of the drug with free amino function - c _min minimum inhibitory concentration needed in plasma water (minimum therapeutic concentration) - k ₀₁ rate constant for absorption - k ₁ rate constant for absorption determined at timet; (similarlyk₂) - K total elimination coefficient - k _el rate constant for elimination - k _F rate constant for formation of metabolites - k _D excretion coefficient of SMP with free amino function - k _U coefficient of metabolite excretion - D ₀ quantity of SMP in the body at time zero - D _B quantity of SMP in the body at timet - D _U quantity of SMP excreted in the urine at timet - M _F quantity of metabolites formed at timet - M _B quantity of metabolites present in the body at timet - M _U quantity of metabolites excreted in the urine at timet - K dissociation constant for the sulphonamide-protein complex - notation for quantities related to drug concentrations in plasma, e.g. c (corresponding term without refer to plasma water)     

Localization of Purine Metabolizing Enzymes in Bovine Brain Microvessel Endothelial Cells: An Enzymatic Blood-Brain Barrier for Dideoxynucleosides?

Purpose. The specific activities of the purine and pyrimidine metabolizing enzymes, purine nucleoside phosphorylase (PNP), adenosine deaminase (ADA) and cytidine deaminase (CDA) were determined in bovine brain microvessel endothelial cells (BBMECs), whole cerebral tissue and erythrocytes. In addition, the substrate specificities (K_m and V_max) of purified calf spleen PNP for inosine and 2,3-dideoxyinosine (ddl) and of purified calf intestinal ADA for 2,3-dideoxyadenosine (ddA), 6-chloro-2,3-dideoxypurine (6-Cl-ddP), and 2--fluoro-2,3-dideoxyadenosine (F-ddA) have been explored. Methods. BBMECs were isolated from bovine cerebral cortex by a two step enzymatic dispersion treatment followed by centrifugation over 50% Percoll density gradients. Activities of alkaline phosphatase, -glutamyl transpeptidase, ADA, PNP and CDA were determined in various tissue homogenates (cerebral cortex, BBMECs and erythrocytes). Enzyme kinetic studies were also conducted using commercially available enzymes and several nucleoside analogs of interest. Results. The activities of ADA and PNP were 42-fold and 247-fold higher in the cerebral microvessels than in the cerebral cortex, respectively, while there was no detectable CDA activity in the microvessel fraction and very little overall activity in the cortex. Conclusions. ADA and PNP may serve as an enzymatic blood-brain barrier for some of the anti-HIV dideoxynucleosides. Simulations of brain availability for ddl, ddA, 6-Cl-ddP, and F-ddA demonstrated that the quantitative significance of enzyme localization may vary dramatically, however, depending on the membrane permeability of the drug and its bioconversion rate constant within the endothelial cell.     

Effects of deuterium substitution on the chronotropic responses to some sympathomimetic amines in the isolated rat atria

Summary In spontaneously beating rat atria the potencies for the chronotropic effects of the following deuterated phenylethylamine derivatives were higher than the potencies of the corresponding non-substituted (protio-) amines: ,,d₂--phenylethylamine; ,,,-d₄-p-tyramine; ,,,-d₄-m-tyramine; ,,-d₃-p-octopamine. In contrast, ,,-d₃-noradrenaline and ,,-d₃-m-octopamine were equipotent with the corresponding protio-amines. Experiments performed in atria depleted of endogenous noradrenaline by pretreatment with reserpine and in atria exposed to the monoamine oxidase (MAO) inhibitor pargyline indicated: a. p-octopamine had both direct and indirect effects, but the chronotropic responses to p-octopamine in tissues with normal MAO activity depended mostly on the direct action of the amine; deuterium substitution enhanced the indirect component of action of p-octopamine; b. m-octopamine possessed considerable indirect effects while d₃-m-octopamine behaved as an amine of direct action. The substitution of deuterium for hydrogens in the -carbon of the alkyl-side chain of phenylethylamines decreases the rate of deamination by MAO. Therefore, the results obtained with all the amines, except for m-octopamine and ,,p-d₃-m-octopamine, could be interpreted in terms of the direct, indirect or mixed action of those compounds and/or of the influence that MAO activity has on the chronotropic responses to these amines. The results obtained with protio-and deuterio-m-octopamine suggested that deuterium substitution, either at the - or the -carbon, can alter some other mechanisms in addition to the enzymatic deamination.Career Investigator on leave of absence from the Consejo de Investigaciones Cientificas y Técnicas, ININFA, Junín 956, 5°P, RA-1113 Buenos Aires, Argentina Send offprint requests to S. M. Celuch     

The Solution Conformations of Lidocaine Analogues

IR and ¹H NMR studies in CDCl₃ and CCl₄ of a series of tertiary aminoxylidides with the amino group in the 2 to 6 position of the acyl chain are described. Lidocaine, diethylaminoaceto-2,6-xylidide, forms an intramolecular five-membered ring hydrogen-bonded monomer at all concentrations in both solvents. -Diethyl-amino-propiono-2,6-xylidide forms an intramolecular six-membered ring hydrogen-bonded monomer in CDCl₃ and CCl₄ but a trans intermolecularly associated species is the major form present at high concentrations in CCl₄. The longer-chain homologues are mixtures of nonassociated trans and cis monomers at low concentrations but associated trans forms predominate at high concentrations. Evidence for the presence of a hydrogen-bonded seven-membered ring intramolecular monomer in CDCl₃ for -diethylaminobutyro-2,6-xylidide is presented. The relationship between the molecular conformation and the partition coefficient is discussed.