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Pharmaceutical solids can often form solvated species which can affect the material’s physical and chemical stability. Carbamazepine (CBZ) has been proven to form a 1:1 solvate with acetone. This report investigates the solvation behavior of CBZ over a wide temperature and acetone concentration range using Dynamic gravimetric Vapor Sorption (DVS). The solvation transition point increased from 62.5% P/P0 at 10°C to 97.8% P/P0 at 30°C, resulting in a 15.9 kJ mol−1 heat of solvation. The desolvation kinetics were studied between 15 and 30°C, indicating CBZ desolvation is a first-order reaction with autocatalysis by the desolvated product and an activation energy of 81.9 kJ mol−1.

Second derivative infrared (IR) spectroscopy can be used as a quick, easy, reproducible, cost-effective, non-destructive tool by which to evaluate the purity and structural integrity of samples of water-soluble proteins from a variety of sources. For this study, second derivative IR spectra were collected at ambient conditions for aqueous (D2O) solutions of seven different commercial samples of the same enzyme, porcine pancreatic elastase (2.0 to 3.8 mg protein/100 µL D2O, pD = 5.4 to 9.1). As with other globular proteins possessing a large fraction of β-structure, the amide I′ region [1700-1620 cm−1] of the second derivative IR spectra for each of the seven elastase samples exhibits a characteristic pair of bands: one of weak intensity appears near 1684 cm−1; the other close to 1633 cm−1is moderate-to-strong. However, one of the seven samples shows a striking decrease in the observed intensities of the amide I′ bands relative to the 1516 cm−1 absorption, along with the appearance of a strong, new band at 1614 cm−1. These intensity disparities strongly suggest that this sample is of much lower quality than the others and clearly has an appreciable proportion of the protein present in a non-native state. In addition, minor differences evident in the position and relative intensity of some individual amide I′ bands among the seven spectra imply that subtle variations exist in the conformation of the peptide backbone of the seven samples. For two of the samples, these small, but reproducible, changes seem to be correlated with marked losses of enzyme activity. Finally, bands outside the amide I′ region may prove useful in assessing sample purity and identifying non-protein contaminants.

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.

Second derivative infrared (IR) spectroscopy can be used as a quick, easy, reproducible, cost-effective, non-destructive tool by which to evaluate the purity and structural integrity of samples of water-soluble proteins from a variety of sources. For this study, second derivative IR spectra were collected at ambient conditions for aqueous (D2O) solutions of seven different commercial samples of the same enzyme, porcine pancreatic elastase (2.0 to 3.8 mg protein/100 microL D2O, pD = 5.4 to 9.1). As with other globular proteins possessing a large fraction of beta-structure, the amide I' region [1700-1620 cm-1] of the second derivative IR spectra for each of the seven elastase samples exhibits a characteristic pair of bands: one of weak intensity appears near 1684 cm-1; the other close to 1633 cm-1 is moderate-to-strong. However, one of the seven samples shows a striking decrease in the observed intensities of the amide I' bands relative to the 1516 cm-1 absorption, along with the appearance of a strong, new band at 1614 cm-1. These intensity disparities strongly suggest that this sample is of much lower quality than the others and clearly has an appreciable proportion of the protein present in a non-native state. In addition, minor differences evident in the position and relative intensity of some individual amide I' bands among the seven spectra imply that subtle variations exist in the conformation of the peptide backbone of the seven samples. For two of the samples, these small, but reproducible, changes seem to be correlated with marked losses of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Reaction conditions for the covalent bonding of 5'-deoxy-5-fluorouridine to serum albumin microbeads by means of a water-soluble carbodiimide were studied. Optimum coupling of dFUR to the microbeads occurred when pure water was used as solvent. There was no significant difference in the bonding efficiency for microbeads prepared at different stirring speeds, and there was a limit to the amount of dFUR that could be bound with increasing reaction time. Yields were low possibly because of competing coupling reactions involving carbodiimide and other reactive groups in the protein. The release of dFUR from dFUR-bound microbeads was slow and biexponential. The fraction of dFUR bound in the interior of the microbeads increased with increasing reaction time.

Fibrinogen is the natural substrate for thrombin, the final proteolytic enzyme of the blood coagulation cascade. Following reaction with thrombin, fibrinogen is converted into fibrin monomers which then spontaneously polymerize into fibrin threads forming the fibrin matrix. This biochemical reaction between fibrinogen and thrombin has been used to produce natural, biodegradable, biocompatible systems for sustained drug delivery. Three potential delivery systems have been explored: 1) fibrin microparticles with drug entrapped are produced by an emulsion process, 2) reaction between fibrinogen adsorbed on suspended drug and thrombin in solution results in the formation of fibrin-coated drug particulates, and 3) fibrin sheets suitable as implants have been cast with drug entrapped. The drug release behavior of both small and large molecular weight substances from these systems was evaluated.

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.

Glutaraldehyde treatment of dexamethasone-containing cylindrical fibrin gels (obtained by the thrombin-induced polymerization of fibrinogen in the presence of the drug) causes cross-linking of the gels and modification of the pore structure. The effect on the release of dexamethasone was assessed by measuring the diffusion coefficient of the drug across treated and untreated gels; diffusion across the treated gels was significantly decreased as compared with untreated gels, but was little affected by the concentration of glutaraldehyde used in the treatment. In biodegradable tests, the treated gels (all concentrations of glutaraldehyde) were resistant to digestion even in the presence of plasmin, but untreated gels were digested, and the digestion rate was accelerated by plasmin. The volume of the gels was progressively reduced as the concentration of glutaraldehyde was increased or the amount of fibrinogen was decreased, but the extent of the reduction did not correlate with the changes in the diffusion coefficient.

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.

The stability of nitroglycerin injection was studied over an eight-week period. Gas-liquid chromatographic assay and thin-layer chromatography were used to study the stability in relation to sorption by rubber and plastic (Viaflex), light and the effect of two diluents (5% dextrose in water and 0.9% sodium chloride for injection). More nitroglycerin was lost from vials with rubber closures, and the loss occurred at a faster rate when compared with nitroglycerin packaged in ampuls. There was no difference in the loss of nitroglycerin from injection packaged in ampuls when stored in the light or in the dark, or in the loss of nitroglycerin from two diluents, 5% dextrose in water and 0.9% sodium chloride for injection, packaged in glass bottles. A 70.4% greater loss of nitroglycerin occurred from Viaflex bags than from glass bottles. Loss of nitroglycerin in this study may have been characterized by reversible first-order kinetics.

Reduction of naltrexone and naloxone with sodium borohydride gave a mixture (85:15) of the 6alpha- and 6beta-hydroxy epimers, alpha- and beta-naltrexol and alpha- and beta-naloxol, respectively. Each pair of epimers was separated by preparative thin-layer chromatography and the physical and spectral properties of each compound were determined. Previous assignments for the configuration of the epimers were verified. A semi-quantitative electron capture gas-liquid chromatographic method was devised for distinguishing either alpha- or beta-naltrexol in the presence of the other and in the presence of large amounts (at least 10-fold greater) of naltrexone. The method was used to determine the approximate weight ratio of beta-naltrexol to naltrexone present in enzymatically hydrolyzed urine samples. It was found that substantially greater quantities of beta-naltrexol and/or its conjugates were excreted in the urine of man, monkey, guinea pig and rabbit after administration of naltrexone, whereas very small quantities were excreted by the mouse, rat and dog. In contrast, just trace amounts of the 6alpha-hydroxy epimer, alpha-naltrexol, were detected in the urine of only 2 of the 7 species that had received naltrexone, i.e., monkey and guinea pig. After administration of 3H-15,16-naltrexone, 1 mg/kg, i.v. to the guinea pig, 25% of the radioactivity found following thin-layer chromatography of the extract of acid-hydrolyzed urine corresponded to beta-naltrexol. In gall bladder bile from the guinea pig, only conjugates of naltrexone and beta-naltrexol were found 2 hours after administration of naltrexone. Following administration of beta-naltrexol, 1 mg/kg, i.v. to guinea pigs only beta-naltrexol and/or its conjugates were detected in urine or bile. However, urine collected after administration of alpha-naltrexol, 1 mg/kg, i.v. to guinea pigs contained alpha-naltrexol and its conjugates, as well as a yet unidentified metabolite.

Recent reports have suggested that cyclic guanosine 3', 5'-monophosphate (cGMP) may mediate drug-induced vascular smooth muscle relaxation. This hypothesis was examined by correlating increases in cGMP formation with vascular smooth muscle relaxation after sensitivities to nitroglycerin and nitroprusside were altered. Both nitroglycerin and nitroprusside produced a concentration-dependent increase in rat aortic cGMP levels, which preceded relaxation. A specific inhibition of nitroglycerin relaxation was accomplished by pretreating helical rat aortic strips with 5.5 X 10(-4) M nitroglycerin for 1 hr (in vitro nitroglycerin tolerance). Both nitroglycerin and nitroprusside were inhibited by pretreating tissues with 10(-5) M methylene blue for 1 hr. Whenever nitroglycerin or nitroprusside relaxation was inhibited, there was a corresponding inability to generate cGMP. Vascular tissues derived from nitroglycerin-tolerant animals (in vivo tolerance) exhibited a profile of cGMP reduction that closely resembled that of the in vitro model. Vascular smooth muscle relaxation induced by 8-bromoguanosine-3', 5'-monophosphoric acid was not inhibited by either in vitro nitroglycerin tolerance or methylene blue, suggesting that the subsequent action of cGMP, once formed, is not impaired. The results of this study support the contention that cGMP generation may be an important step in the promotion of vascular smooth muscle relaxation by nitroglycerin and nitroprusside.

The effects of in vitro nitroglycerin tolerance and methylene blue pretreatment on the ability of nitroglycerin and nitroprusside to promote relaxation and tissue accumulation of cyclic GMP were examined in the carbachol-contracted longitudinal smooth muscle of the guinea-pig ileum. Nitroglycerin and nitroprusside produced concentration-dependent increases in cyclic GMP levels. However, only nitroglycerin increased cyclic GMP levels before the onset of relaxation. Nitroglycerin tolerance produced approximately 200- and 5-fold shifts to the right of nitroglycerin and nitroprusside relaxation curves, respectively. Methylene blue pretreatment produced approximately 5-fold shifts to the right of both nitroglycerin and nitroprusside relaxation curves. Whenever there was an inhibition of nitroglycerin- or nitroprusside-induced relaxation, there was a corresponding reduction of cyclic GMP generation. Methylene blue inhibited the ability of 8-bromoguanosine 3',5'-monophosphoric acid to promote smooth muscle relaxation, suggesting that it also may impair the subsequent actions of cyclic GMP. This study provides the first demonstration of nitroglycerin tolerance in a nonvascular smooth muscle and provides evidence that cyclic GMP mediates the relaxant effects of nitroglycerin in the longitudinal smooth muscle of the guinea-pig ileum. However, because nitroprusside promoted tissue accumulation of cyclic GMP subsequent to relaxation, an exclusive role for cyclic GMP-mediated relaxation for this drug in the longitudinal smooth muscle appears unlikely.