Serum albumin beads: an injectable, biodegradable system for the sustained release of drugs.

Biologically active compounds were entrapped in cross-linked serum albumin microbeads. Injection of these drug-impregnated beads into rabbits produced no adverse immunological reactions. Sustained release (20 days) of progesterone was demonstrated in vivo.

Binding of quinidine to plasma proteins in normal subjects and in patients with hyperlipoproteinemias.

Quinidine in the plasma binds to various lipoproteins as well as to albumin. With the use of computer simulations the effect of varying the serum level of each of the lipoproteins as well as albumin was examined. The results suggested that the overall binding of quinidine is relatively insensitive to changes in any one serum protein provided the others are present in normal concentrations. Evaluation of the degree of protein binding of quinidine in plasma from normal subjects and patients with types IV and IIa hyperlipoproteinemias supported the computer predictions. The degree of protein binding in plasma from normal subjects and patients with types IV and IIA hyperlipoproteinemias were 71.2% +/- 11.4%, 75.9% +/- 8.7%, and 69.9% +/- 15.0%, respectively. The quinidine binding in plasma of patients with hyperlipoproteinemias did not differ statistically from that in the normal subjects.

Tight binding affinities determined from thermodynamic linkage to protons by titration calorimetry.

A general titration calorimetry method is described that can be used to determine the affinity of tight binding interactions with proteins. The method is based on the thermodynamic linkage between ligand binding and coupled protonation reactions. The protons linked to a given ligand-binding reaction are measured by titration calorimetry, and integration of the resulting data set yields the pH dependence of the binding affinity based on thermodynamic relationships developed elsewhere. When the pH dependence of the binding affinity is combined with the absolute affinity determined independently at a pH at which the affinity can be conveniently measured, the absolute binding affinity over the entire pH range is determined. The method is well suited for determining high-affinity binding interactions of protein antigens with antibodies, but is applicable to any macromolecular ligand-binding reaction that is coupled to protonation.

Evaluation of antiproliferative agents using a cell-culture model.

Chinese hamster ovarian (CHO) cells in culture were used to evaluate the relative antiproliferative potential of drugs. These agents have been used to improve the clinical response after glaucoma filtering surgery. The following drugs were evaluated: 5-fluorouracil (5-FU) as the benchmark, 5-fluorouridine (FUR), 5-fluorodeoxyuridine (FUDR), 5'-deoxy-5-fluorouridine (DFUR), bleomycin, and cytarabine (ARA-C). In addition to cell counting, a colorimetric assay based on the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to follow growth response. The MTT assay was found to be extremely convenient and an indirect measure of cell activity, offering an alternate or addition to a measure of cell number. All agents tested were shown to inhibit cellular proliferation. Dose-response curves for each agent indicate the following absolute potency: FUDR greater than FUR greater than ARA-C greater than 5-FU = bleomycin greater than DFUR. Besides absolute potency, an evaluation of the effects of equivalent inhibitory concentrations of each drug on growth rate was assessed. Several agents affected the proliferation rate patterns differently. Based on these studies, it is suggested that the in vitro model can identify potential agents through an assessment of their overall activity profile in CHO cells, which includes not only their potency based on dose response, but their onset of activity, duration of effect, and potential for toxicity.

Influence of cardiopulmonary bypass on nitroglycerin clearance.

The effect of cardiopulmonary bypass on the clearance of nitroglycerin (NTG) was studied in seven patients scheduled for coronary artery bypass graft surgery. Intravenous NTG was administered through nonadsorbing tubing at a starting dosage of 5-10 micrograms/min and was adjusted as needed. Blood samples were obtained from the radial artery and antecubital vein before bypass and from the arterial outlet of the oxygenator during bypass at least 30 minutes apart during a constant dosage or at least 30 minutes after a dosage change. Serum concentrations were analyzed for NTG by gas chromatography. Venous NTG concentrations were always lower than concurrent arterial concentrations, with an average arteriovenous extraction of 67.2%. Serum concentrations of NTG were generally within the range associated with a therapeutic response in congestive heart failure patients. Consistent with other reports, NTG concentrations varied widely among patients and considerable intrasubject fluctuations in drug concentrations were seen. The mean +/- SD apparent clearance of NTG before bypass of 0.044 +/- 0.02 L/kg/min increased 20% to 0.052 +/- 0.02 L/kg/min during bypass (P = .05). These results suggest that cardiopulmonary bypass increases the clearance of NTG; however, the magnitude appears to be small and only partially explains the reported increased dosage needed during cardiopulmonary bypass.

Kinetics of the efflux of 3H-norepinephrine from rabbit irides.

The pigment cell dependent accumulation of 3H(-)-norepinephrine (1 mumol/l) was studied in rabbit irides. Albino irides served as controls. Neuronal uptake, deamination and O-methylation of 3H(-)-norepinephrine were reduced by desipramine, pargyline and U-0521, respectively. Both albino and pigmented irides accumulated the catecholamine from the incubation medium. At 37.5 degrees C and 120 min of incubation, tissue/medium ratios for pigmented and albino irides were 10.70 and 4.30 ml/g respectively. At 0 degrees C, the accumulation of 3H(-)-norepinephrine in the albino iris was abolished, while the pigmented iris accumulated significant amounts. The tissue/medium ratio was approximately 3 ml/g. When irides were loaded with 1 mumol/l of 3H(-)-norepinephrine for 60 min the drug distributed into four different compartments. Most of the pigment cell dependent binding was associated with compartments III and the "bound fraction". The amount bound in these compartments was 73% of total accumulation. In pigmented irides obtained from reserpine-pretreated rabbits the total binding was reduced to 64% of that in pigmented control irides. The half times for efflux from pigmented iris were 1.2 min, 10.7 min and 94.8 min, respectively, for compartments I, II, and III. Irides were loaded with 3H(-)-norepinephrine in normal physiological salt solution and rates of efflux were studied in Ca2+-free medium. In either albino or pigmented iris, the late efflux declined more slowly compared to that from the control irides. When pigmented irides were loaded with a low concentration of 3H(-)-norepinephrine, the displacement of the labeled amine was equal by each stereoisomer of norepinephrine. Results are discussed in relationship to the pigment cell-dependent uptake of the catecholamine.

Prediction of solubility in nonideal multicomponent systems using the UNIFAC group contribution model.

There is a need to identify suitable blends of solvents to dissolve drugs. Empirical approaches, such as trial-and-error and response surface, require several solubility measurements. In this study the UNIFAC method was used to predict solubility in highly nonideal multicomponent systems in which only the solute enthalpy of fusion and melting point must by measured. UNIFAC combines a group contribution approach with the UNIQUAC model for activity coefficients. Parameters characterizing interactions among constituent groups of a molecule have been previously determined from binary vapor pressure data. These tabulated group parameters are used to predict activity coefficients for newly synthesized compounds. These coefficients, together with the ideal solubility, permit a prediction of solubility. The solubility of 4-hexylresorcinol in ethyl acetate, ethyl myristate, and hexane mixtures was both measured and calculated using UNIFAC. The predicted solubilities were within 10% of the experimental solubilities for all but 3 of 21 mixtures. Since the method accounted for positive and negative deviations from ideality in a hydrogen-bonding system of molecules having different sizes, it shows great potential for use in pharmacy.

Dielectric analysis in the characterization of amorphous pharmaceutical solids. 1. Molecular mobility in poly(vinylpyrrolidone)-water systems in the glassy state.

The effect of water on the relaxation behavior below the glass transition temperature (beta-relaxation) of an amorphous powder, poly(vinylpyrrolidone) (PVP, MW 30,000), was studied by subjecting the sample to dielectric analysis in the frequency range from 20 Hz to 20 kHz. The material stored at 0% relative humidity (RH) (containing 0.05% w/w H2O) exhibited a frequency dependent second-order beta-relaxation (T beta = -56 degrees C at 500 Hz). The peak frequency-temperature data could be fitted to the Arrhenius equation, yielding an activation energy (Ea) of 36.5 kJ mol-1. Water was found to significantly lower T beta, increase the dielectric loss, and increase Ea. The initial decrease in T beta was found to be quite significant, as little as 7% w/w H2O lowering T beta by 26 degrees C, followed by a more gradual decrease. PVP exposed to 69% RH (containing approximately 31% w/w H2O) exhibited T beta at -104 degrees C with an activation energy of 46.3 kJ mol-1. The observations that the beta relaxation was poorly visible when the water content was 0.05% w/w and that the change in Ea was from a low to a high value as the temperature is decreased suggest that thermally activated rotational diffusion of water molecules plays a major role in the beta-relaxation of PVP containing moderate to high water contents. The rate of increase in activation energy as a function of H2O/PVP mole ratio exhibited a minimum at unity, suggesting that water binding to one site on PVP has a distinct effect on the activation energy.

Kinetics of concomitant degradation of tetracycline to epitetracycline, anhydrotetracycline, and epianhydrotetracycline in acid phosphate solution.

The concentrations of tetracycline, epitetracycline, anhydrotetracycline, and epianhydrotetracycline in pH 1.5 phosphate solution were followed as a function of time at four temperatures. Separation and quantification of all four species were accomplished using high-pressure liquid chromatography. Through nonlinear regression analysis, rate constants for the reversible first-order epimerization of tetracycline and anhydrotetracycline and for the first-order dehydration of tetracycline and epitetracycline were obtained. Solutions to the differential equations obtained through Laplace transforms successfully predict concentrations found experimentally. The energy of activation for each reaction step was calculated and ranged from 15 to 27 kcal/mole. The rate constants for tetracycline and epitetracycline dehydration conform with those of earlier studies that used different experimental methods. The study shows that epimerization of tetracycline and anhydrotetracycline can take place at a low pH.

Calorimetric determination of the heat of precipitation of pseudoephedrine racemic compound--its agreement with the heat of solution.

The heat of precipitation of dl-pseudoephedrine was determined by direct calorimetry using a Tronac isoperibolic calorimeter. The precipitation of dl-pseudoephedrine was induced by mixing aqueous solutions of the two enantiomers, namely, d- and l-pseudoephedrine, directly in the calorimeter. The molar heat of precipitation of dl-pseudoephedrine was -2.7 and -3.0 kcal/mol at 25 and 30 degrees C, respectively. The aqueous solubility of dl-pseudoephedrine was determined over a temperature range of 20-40 degrees C. The van't Hoff solubility plot was nonlinear. The apparent heat of solution at saturation was obtained from the solubility data using a nonlinear regression model. A good agreement between the magnitude of the apparent heat of solution at saturation and the heat of precipitation was noticed at both 25 and 30 degrees C.

Quantitative characterization of adsorption isotherms using isothermal microcalorimetry.

The integral heat of adsorption of water vapor on sodium benzoate samples was determined at various partial vapor pressures using a heat conduction microcalorimeter. An equation is presented to describe the calorimetric integral heat response (mJ/g of solid) as a function of relative humidity. This equation, although similar in principle to the well-known BET equation, relates the heat evolved (rather than volume or mass of gas adsorbed) upon adsorption to the partial pressure of the gas. It qualitatively describes the shape of the calorimetric isotherm and quantitatively allows the calculation of "monolayer capacity" or the apparent surface area with water as the adsorbate. The modified BET equation was applied to the calorimetric adsorption data available in the literature. The surface area or the monolayer coverage values of the solid samples used in these studies were calculated from data-fitted parameter estimates. Good agreement was found between Vm or surface area values obtained by the application of the model to the calorimetric data and those reported by the authors using conventional gravimetric or volumetric measurement of adsorption. The model satisfactorily described the experimental calorimetric data of water vapor adsorption on sodium benzoate. The model equation and the use of isothermal microcalorimetry provide a means to obtain the water adsorption surface area of solid materials. The method may also be useful in comparing the surface properties of drugs and excipients obtained by different methods or from different sources. The microcalorimetric method to characterize adsorption is more sensitive and convenient in comparison with some of the conventional techniques.

Prediction of xanthine solubilities using statistical techniques.

Mixture response-surface methodology can be used as a technique to predict solubility in mixed solvent systems. The present report shows that if the intent is to predict solubility in nonideal solutions, mixture response-surface methodology is a better technique than one which assumes a particular mechanism to hold true. This is demonstrated by comparing the predictive ability of the mixture response-surface model with that of an extended Hildebrand approach to nonideal solutions. The nonideal systems are those used by Martin and co-workers involving the solubility of theobromine, caffeine, and theophylline in dioxane-water mixtures.

Interaction of nitroglycerin with human blood components.

Nitroglycerin is rapidly lost from solution when incubated with red blood cells or whole blood. The assumption that the loss is enzymatic in nature may not be true, since no major metabolite is detected during this incubation. Explanation on the basis of a chemical reaction is also difficult, since the products of the chemical hydrolysis of nitroglycerin are the same as the metabolic products. After an initial rapid loss, nitroglycerin disappearance at 37 degrees follows an apparent first-order process in the concentration range of 10--480 ng/ml when incubated with washed red blood cells suspended in normal saline solution. The half-life for the reaction of the apparent first-order phase varies with the initial concentration and increases as the concentration increases (4 min at 10 ng/ml, 52 min at 480 ng/ml), suggesting a mixed kinetic mechanism. Metabolites of nitroglycerin (1,2- and 1,3-dinitroglycerin) react similarly to nitroglycerin in terms of an apparent initial, fast step, a secondary first-order dependence, and concentration-dependent rate effects; however, the rate of the reaction is much slower (t 1/2 = 33 min at 10 ng/ml) for the metabolite. These data suggest the possibility of a physical mechanism for the loss of nitroglycerin. Since the loss to red blood cells can be rapid, it seems that the mechanism should be delineated, and that the rate of disappearance be considered in an analysis of the pharmacodynamics of the drug.

Loss of nitroglycerin from aqueous solution into plastic intravenous delivery systems.

The mechanism of potential loss of nitroglycerin stored in plastic and glass containers was studied from an equilibrium and kinetic approach. Plastic strips equilibrated with dilute aqueous solutions of neat nitroglycerin showed that the drug was lost by absorption. Drug loss was followed by an electron-capture GLC assay. The same assay of control solutions in glass showed no drug loss in 48 hr at pH 5.7. The kinetics of nitroglycerin absorption and desorption were determined using synthesized 14C-labeled drug. Absorption can be quantified using a diffusion model, where the concentration in the aqueous phase falls with time. Curve fitting yielded an average diffusion coefficient in plastic of 2.05 x 10(-9) cm2/sec and a partition coefficient of 104 (plastic-water) at 30 degrees. Temperature-dependence studies of absorption showed that the diffusion coefficient followed an Arrhenius relationship with an energy requirement of 19.6 kcal/mole, whereas effects on the partition coefficient were negligible. Nitroglycerin desorption from plastic disks under sink conditions into water can be quantified by assuming a diffusion model where the concentration at the surface of a plane sheet remains constant. Nonlinear least-squares curve fitting generated a diffusion coefficient of 1.14 x 10(-9) cm2/sec for the desorption process at 30 degrees.

Rate and proposed mechanism of anhydrotetracycline epimerization in acid solution.

The pathway through which the toxic tetracycline degradation product epianhydrotetracycline forms in solution was studied using high-performance liquid chromatography and circular dichroism, taking advantage of the large difference in ellipticity between the reactant and the product at 285 nm. The epimerization of anhydrotetracycline followed a reversible first-order process, and both analytical methods yielded the same rate constants. The rate constants indicate that anhydrotetracycline epimerization is faster than tetracycline epimerization. The equilibrium favored anhydrotetracycline, and the activation energies for the forward and reverse rates were almost the same as those for tetracycline epimerization. The epimerization was catalyzed by phosphate. Activation energies in 0.1 and 1 M phosphate were essentially the same. The equilibrium constants for both anhydrotetracycline and tetracycline favored the natural configuration rather than the epi series. Possible rationalization based on conformational and hydrogen bonding effects is presented.

Tritiated naltrexone binding in plasma from several species and tissue distribution in mice.

The binding of 15,16,-3H-naltrexone in human, monkey, dog, guinea pig, rat, and mouse plasma was investigated over a range of concentrations, including predicted therapeutic levels. Studies using equilibrium dialysis at 37 degrees indicate that the extent of binding is independent of naltrexone concentration over the concentration range of 1-500 ng/ml for dog plasma and of 0.1-500 ng/ml for human, monkey, guinea pig, rat, and mouse plasma. The extent of naltrexone binding in plasma is similar in the six species studied, the range being from 20% bound in rat plasma to 26% in plasma from beagle and mongrel dogs. This relatively low extent of naltrexone binding in plasma is consistent with previous findings of a large apparent volume of distribution of this drug in the dog. To investigate further the distribution of tritiated naltrexone, the tissue levels of radioactivity in mice at 1, 5, and 15 min after intravenous administration of 8-3H-naltrexone were determined. Naltrexone was rapidly distributed from plasma to tissues, with less than 4% of the dose being present in plasma at 1 min after injection.

The influence of cosolvents on the in-vitro percutaneous penetration of diclofenac sodium from a gel system.

The influence of cosolovents on the in-vitro percutaneous penetration of diclofenac sodium from a gel system was studied using a simplex lattice experimental design. Gel formulations were prepared by gelling the vehicle mixture of water, alcohol and propylene glycol with Carbomer 940. The synthetic membrane Durapore and hairless mouse skin were employed as barriers in a Franz-type diffusion cell. It was found that the penetration through the synthetic membrane was well described by the Higuchi model. There existed a better inverse relationship between the penetration rate and the drug solubility in the respective vehicle. It appeared to be a membrane-controlled mechanism when using hairless mouse skin as the barrier. The penetration rates in steady-state for nine formulations were fitted to a polynomial equation based on this simplex lattice method. A three-dimensional plot was constructed in this simplex surface studied. The maximal penetration rate was found to be from the vehicle containing water and ethanol in an exact volume ratio of 3:1 and the minimal penetration rate was observed from the vehicle containing water only.

In-vitro and in-vivo studies of the diclofenac sodium controlled-release matrix tablets.

Controlled release matrix tablets for diclofenac sodium were developed in this study. Five matrix-tablet formulations were prepared by granulating two viscosity grades of HPMC (hydroxylpropylmethylcellulose) in varying ratios with water in the planetary mixer. The in-vitro dissolution tests indicate that all five matrix formulations prolong the release of diclofenac sodium. The main factors controlling drug release were the HPMC viscosity grade and the amount of HPMC used. The larger the amount of high viscosity grade HPMC used, the slower the resultant release rate of diclofenac sodium. There was no significant degradation of diclofenac sodium or change in drug release rate in any of the five formulations during a three-month period of stability testing. The sustained release ability of four formulations was further demonstrated in an in-vivo study in six healthy subjects. There were in-vitro/in-vivo correlations between Cmax, AUC0-14, and the time for 50 or 80% drug to be released.

Studies on the in-vitro percutaneous penetration of indomethacin from gel systems in hairless mice.

The influence of co-solvents on the in-vitro percutaneous penetration of indomethacin from gel systems was studied using a simplex lattice experimental design. Gel formulations were prepared by gelling the vehicle mixture of water, either alcohol or isopropanol and either propylene glycol or PEG 400 with 1% w/w Carbomer 940. Hairless mouse skin was employed as the barrier in a Franz-type diffusion cell. The penetration rates at steady state for seven formulations were fitted to a polynomial equation based on this simple lattice method and a three-dimensional plot was constructed. The formulation having the maximal penetration rate was determined to be the vehicle with a solvent ratio of water: alcohol: propylene glycol equal to 15:33:52, and which possessed a solubility parameter of 15 and a drug solubility of around 10 mg mL-1. When the solubility parameter of the vehicle was > 15, the drug solubility increased. However, the penetration rate decreased with an increasing solubility parameter. For those vehicles with a solubility parameter < 15, both the drug solubility and the penetration rate decreased with a decrease in the solubility parameter. There was shown to be an approximately 20-fold increase in the relative enhancement factor when using both alcohol and isopropanol, but only a threefold increase for both propylene glycol and PEG 400, when compared with water.