Analysis of known crystals to design polymorph prediction strategies.

Computational analysis of known crystals was used to provide insight into polymorph prediction strategies. It was hypothesized that intermolecular crystalline interactions are inadequately accounted for in current computational strategies. The QEq method of partial charge assignments was applied to the crystal milieu (CMc) and to the isolated molecule (IMc). For 90 known crystal structures, these two partial charging methods showed that the unit cell energy was almost always lower (more stable) with CMc partial charge assignments. In simulations on a model drug, the CMc charging re-ranked the unit cell energies of possible polymorphs in a way that aided the identification of physically reasonable packings more easily than IMc. Decomposing the unit cell energy for known crystals showed that the cohesive IMc deviation from CMc was dominant. On the other hand, for simulations, the corresponding IMc conformational component of unit cell energy dominates when conformational adaptive strategies (CA) are used; this was not apparent with rigid-body simulations (RB). Furthermore, evidence is presented that polymorph strategies that use unit cell energy for optimizing are especially prone to this undesired conformational bias. Suggestions are made for polymorph strategies that might enhance the probability of identifying physically relevant polymorphs in the future.

Fabrication of polymeric scaffolds with a controlled distribution of pores.

The design of tissue engineering scaffolds must take into account many factors including successful vascularisation and the growth of cells. Research has looked at refining scaffold architecture to promote more directed growth of tissues through well-defined anisotropy in the pore structure. In many cases it is also desirable to incorporate therapeutic ingredients, such as growth factors, into the scaffold so that their release occurs as the scaffold degrades. Therefore, scaffold fabrication techniques must be found to precisely control, not only the overall porosity of scaffolds, but also the pore size, shape and spatial distribution. This work describes the use of a regularly shaped porogen, sugar spheres, to manufacture polymeric scaffolds. Results show that pre-assembling the spheres created scaffolds with a constant porosity of 60%, but with varying pores sizes from 200-800 microm, leading to a variation in the surface area and likely degradation rate of the scaffolds. Employing different polymer impregnation techniques tailored the number of pores present with a diameter of less than 100 microm to suit different functions, and altering the packing structure of the sugar spheres created scaffolds with novel layered porosity. Replacing sugar spheres with sugar strands formed scaffolds with pores aligned in one direction.

pKa determinations by using a HPLC equipped with DAD as a flow injection apparatus.

A semi-automated method to determine pKa values spectrophotometrically is described. The method uses the capabilities of a HPLC equipped with a diode array detector (DAD) as a flow injection apparatus. The advantages are low sample consumption, rapid sample throughput, high sensitivity, and precision. Experimental pKa values obtained for two model compounds, benzoic acid (approximately 4.0) and 2-aminopyridine (approximately 6.8), are consistent with literature values. Constant ionic strength was maintained for a wide pH range. Solubilized samples in non-aqueous solvents were also investigated. The weakening in pKa values, often seen when using non-aqueous solvents, was small (0.04-0.40 pH units) compared to conventional methods.

Circulation time and body distribution of 14C-labeled amino-modified polystyrene nanoparticles in mice.

Commercially available amino-modified polystyrene particles of size range 100-1,000 nm were radioactively labeled with [14C]-formaldehyde. A study of the circulation time and body distribution of these particles was carried out in mice. The animals were sacrificed at 1-30 min after intravenous administration of the particles. The blood and organ (liver, spleen, lung) profiles of particles were determined by measuring their radioactivity by means of liquid scintillation counting. In general, larger particles were eliminated from blood faster than smaller particles. The blood elimination half-life ranged from 1.36 to 4.92 min. The particles were mainly taken up by the liver with larger particles being taken up faster than the smaller particles. At 30 min after injection, 60% of the administered 100 nm particles were present in the liver, whereas 85% of the 100 nm particles were found in the liver. Accumulation in the spleen was 1-3% of the total number administered in the entire size range. At 1 min after injection, less than 3% of the total dose administered was present in the lungs and this value decreased rapidly to less than 1% at 2 min. The only exception occurred for 100 nm, where 2.35% was present in the lungs at 2 min after injection.

Synthesis of the cholesteryl ester prodrugs cholesteryl ibuprofen and cholesteryl flufenamate and their formulation into phospholipid microemulsions.

Phospholipid micoremulsions have been suggested as a drug-delivery system for hydrophobic compounds. In this study hydrophobicity was achieved by derivatizing with cholesterol. Cholesteryl ibuprofen (3) and cholesteryl flufenamate (4) were synthesized. 3 was isolated as an amorphous, white solid with a melting range of 114-120 degrees C. 4 was isolated as a crystalline, white solid with a melting range of 145-148 degrees C. The proposed structures of 3 and 4 were supported by IR, NMR, MS, and organic microanalysis. Phospholipid:cholesteryl ester microemulsions were prepared by the addition of a 1-propanol solution of the cholesteryl ester, other lipids, and phospholipid to a rapidly mixing KCl/KBr solution. The hydrophobic phase was modified by the addition of cholesteryl oleate or triolein to study the effect of the fluidity of the hydrophobic core on the formation of the microemulsions. The results indicated that a molar ratio of 75:25 and a total lipid concentration of 60 mg/mL consistently gave microemulsions with a mean size of 100-150 nm. In addition, the formation of eutectic mixtures of 3 and 4 with cholesteryl oleate were determined to be 16% (w/w) for 3 and 12% (w/w) for 4; melting points were 35.2 and 45.2 degrees C, respectively. The solubilities of 3 and 4 in triolein were determined to be 13.2% (w/w) and 11.5% (w/w), respectively. Other investigators have shown that if the core of a phospholipid:cholesteryl estermicroemulsion exists in a liquid state at physiologic temperature, the turnover of the cholesteryl esters from these microemulsions occurs at a faster rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Immobilization of active urokinase on albumin microspheres: use of a chemical dehydrant and process monitoring.

A method of immobilizing urokinase on albumin microspheres has been developed. Laser scattering, which was used to follow particle size from the initial emulsification stage to the final aqueous resuspension of the microsphere stage, showed that particle coalescence and crosslinking were critical parameters in manufacturing the microspheres. Chemical dehydration with 2,2-dimethoxypropane was used to convert an albumin emulsion into an albumin suspension and to reduce coalescence. An optimal amount of dehydrant produced 0.3-micron particles which resisted a 50 degrees C temperature challenge. Since oil/glutaraldehyde emulsion resulted in large particles with no urokinase activity, the cross-linking concentration of glutaraldehyde was reduced by solubilizing 25% (w/v) glutaraldehyde in the oil phase with n-propanol. A concentration of 0.015% (v/v) glutaraldehyde effectively immobilized urokinase and stabilized albumin microspheres. Amidolytic activity using the specific chromogenic substrate for urokinase, S-2444, showed that enzyme activity could be retained during this glutaraldehyde cross-linking.

The relationship of salicylate lipophilicity to rectal insulin absorption enhancement and relative lymphatic uptake.

The sodium salts of the 3,5-dichloro, 3,5-dibromo-, 3,5-diiodo-, and 5-methoxy- analogs of salicylic acid have been evaluated as enhancers of rectal insulin absorption. A relationship was found between adjuvant potency and relative lipophilicity. Maximal adjuvant activity was obtained with 0.1 M 3,5-dichlorosalicylate. Higher concentrations (0.15 M) of 3,5-diiodosalicylate produced a decline in adjuvant activity, which may be associated with extraction of specific cellular proteins. This may indicate the existence of an optimal salicylate lipophilicity for adjuvant efficacy. Relative adjuvant activity was found to be related to the lymph:plasma absorption ratio of [125I]insulin. Lymphatic uptake of insulin was not related to lymph flow rate.

Resolving multiple overlapping calorimetric transitions by use of a microcomputer: studies on erythrocyte membranes.

Buffer changes and certain drugs cause temperature shifts, amplitude changes, and transition broadening in the differential scanning calorimetric (DSC) analysis of erythrocyte membranes. However, it has been difficult to interpret and quantitate these shifts and changes because the scans are composed of multiple overlapping transitions and because more than one transition may be simultaneously affected. An empirical approach has been developed by using Gaussian modeling to resolve these calorimetric transitions. Data analysis was carried out on a microcomputer using a nonlinear regression program (PCNONLIN) to fit the data scans. These results show that changes in the calorimetric scans of erythrocyte membranes due to alterations in the buffer environment, such as pH and osmolarity, can be resolved by fitting the data scans with the proposed mathematical model and optimizing the resolution parameters with PCNONLIN. In addition, resolution uncovered hidden characteristics that may not have been readily evident. Under certain conditions, for example, apparent transition shifts were shown to actually be amplitude changes and transition broadening. Determination of the limitations and validity of this method was accomplished with simulation studies. This technique offers a simple means for fitting overlapping DSC transitions by use of a commercially available nonlinear regression program that can be run on a microcomputer.

Predicting coordinated lipid biosynthesis: application to the surfactant-accommodated epidermis.

Factorialized correlation analysis is proposed as a method for predicting the coordination of multiple enzyme pathways. The approach can be used potentially to find new relationships and to predict relationships that have been established in other tissues. However, careful tracer studies are needed to verify the cause-and-effect relationships between precursor and products. In this study, guinea pigs that were chronically treated with an anionic, a nonionic and a cationic surfactant passed through an irritation stage to a clinical state that appeared normal. The method was used to examine binary coordination of lipid biosynthesis in the epidermis by using a factorialized table of regression coefficients. Coordinated lipid relationships that have been reported in other tissues were predicted between sphingomyelin and cholesterol, as well as between phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine. A new inverse relationship was found between triglycerides and both sphingomyelin and cholesterol, using this method. These data are discussed with respect to a membrane fluidization model for the accommodated state.

The reversibility of absorption promoter interaction with red blood cell membranes studied with differential scanning calorimetry.

Absorption promoters, or adjuvants, are used to enhance the gastrointestinal absorption of poorly absorbed drugs such as macromolecules. In the present work, adjuvant-membrane interactions have been studied by differential scanning calorimetry (DSC) using red blood cell (RBC) membranes as model membrane. These interactions caused temperature shifts, amplitude changes, and broadening of the RBC transitions. Because more than one transition may be simultaneously affected by a given adjuvant, complex overlappings occur. Gaussian modeling and nonlinear regression analysis, therefore, were used to resolve these transitions. A correlation, which may serve as an indicator of adjuvant potency, was found between adjuvant concentration and induced transition temperature shifts. Further, these shifts recovered to baseline after successive washings with buffer (for most adjuvants). Sodium lauryl sulfate induced transition alterations, however, never recovered. Thus the DSC might be useful in monitoring reversible adjuvant-membrane interactions.

The influence of concentration of two salicylate derivatives on rectal insulin absorption enhancement.

3,5-Diiodosalicylate sodium (DIS), a highly lipophilic salicylate, was evaluated against 5-methoxysalicylate sodium (MS) as a potential adjuvant absorption promoter for rectal insulin delivery. Comparative blood glucose measurements were made using the two adjuvants under identical conditions as promoters of rectal insulin absorption in rats. Concentrations of DIS greater than and including 0.1 M produced an unexpected, progressive decrease in adjuvant activity as determined by a decline in observed hypoglycaemic response. This was not due to formation of an insulin-DIS complex. The adjuvant MS produced a classical, sigmoidal log-dose response curve. Possible reasons for the occurrence of the DIS optimum phenomenon are discussed as well as are the observed differences in adjuvant potency of these agents in a propylene glycol-containing vehicle.

A relationship between diffusional transport in lipid membranes and a lipophilic-eutectic parameter.

A theoretical model has been developed to relate passive diffusional transport with a parameter termed the lipophilic-eutectic coefficient, Le. Based on this parameter it is proposed that the lipophilicity of a substance can be estimated. The method is based on the melting point lowering of a pure substance in the presence of an impurity of similar structure. Cholesterol was chosen as a model biological lipid because it is a neutral lipid that is found in relatively high concentrations in a variety of membranes. Transdermal absorption in humans and intestinal absorption in the rat both show a high degree of correlation with respect to Le. For our studies, Le appears to be as satisfactory as the partition coefficient for estimating lipophilicity and its determination is less demanding analytically.

The pharmacodynamic response of the basal skin potential to quinidine gluconate.

The basal skin potential (BSP) was explored as an indirect means of continuously monitoring the cardiac response of quinidine gluconate. A method was developed to follow the BSP using a high impedance, recording polygraph and nonpolarizing calomel electrodes. Intravenous administration of quinidine gluconate caused time dependent changes in the BSP. Further studies showed that blood levels during the elimination phase and the QT interval of the electrocardiogram (ECG) over their entire time period were highly correlated with the BSP. Optimal correlation with the QT interval occurred when the BSP curve was shifted to earlier times by approximately 10 minutes, reflecting possible differences in the accessibility or mechanism of the respective pharmacologic compartments. Further application of the BSP for pharmacodynamic monitoring will require electrode refinements and an increased understanding of its mechanism of action.

Percutaneous absorption: a new physicochemical predictive model for maximum human in vivo penetration rates.

A diffusion model for stratum corneum-limited percutaneous absorption based on the interaction of the diffusate with the stratum corneum was derived. Two types of interactions were proposed, ion-dipole and lipid-lipid, based on current knowledge of the stratum corneum and on irreversible thermodynamic arguments. The resulting flux equations predict a linear dependence of flux on the dipole moment and ln X of the diffusates , where X is the mole fraction solubility. These flux equations were tested on 21 different diffusates whose human percutaneous absorption rates in vivo had been previously determined. A solubility method was used to classify the interaction pathway for each diffusate . Correlation of the maximum absorption rate for the lipid and polar pathways give correlation coefficients of 0.946 and 0.998, respectively. It is believed that these studies provide a starting point for the ultimate goal of percutaneous absorption research: to be able to bypass in vivo and in vitro studies and to predict absorption solely on the basis of the physicochemical properties of the diffusates .

Guinea pig ear as a new model for in vivo percutaneous absorption.

A new animal model for in vivo percutaneous absorption utilizing the hairless, relatively thick skin of the guinea pig ear is proposed. Topical absorption studies were carried out with [14C]hydrocortisone and [14C]testosterone. Systemic studies were also conducted to correct for incomplete urinary excretion. In addition, a single stratum corneum correction factor was developed from published data to enable the guinea pig ear skin to be directly compared with human forearm skin. A comparison of human percutaneous absorption with the corrected guinea pig ear absorption shows a high correlation for both hydrocortisone and testosterone. The effects of ambient changes in relative humidity are also discussed with respect to in vivo percutaneous absorption.

General method for calculation of hydrogen-ion concentration in multicomponent acid-base mixtures.

A generalized method for the rapid evaluation of complicated ionic equilibria in terms of the hydrogen-ion concentration was developed. The method was based on the derivation of a single general equation that could be used to evaluate any mixture. A tableau method also was developed which allowed calculation of the numerical solution to the general equation without computer analysis or graphical or intuitive approximations. Examples illustrating the utility of the method are presented. These examples include a mixture of barbital, citric acid, boric acid, monobasic sodium phosphate, and sodium hydroxide. Calculated hydrogen-ion concentrations showed good agreement with experimental values for simple and complex solutions. The major advantages of the method are its simplicity and the obtainment of numerical solutions without initial approximations in the calculations. However, activity corrections are not included in the calculations.

Separation of penicillin G potassium and its degradation products using high-pressure liquid chromatography.

A high-pressure liquid chromatographic technique was developed for the separation of penicillin G potassium and several of its decomposition products. The method utilized a buffered acetonitrile-phosphate mobile phase on a reversed-phase C18 column. Separation of penicillin G potassium and six degradation products was attained within 25 min.

Rotating-disk method for determining cutaneous metabolism.

Since design and evaluation of topical dosage forms should account for both skin permeation and cutaneous metabolism, an in vitro system was formulated to determine the metabolic component of viable guinea pig skin utilizing the diffusion layer property of the rotating disk. The drug investigated was vidarabine, an antiviral agent, which was rapidly metabolized to 9-beta-D-arabinofuranosylhypoxanthine. The aqueous diffusion coefficient of the drug was determined by the capillary cell method. The rotating-disk system was standardized using benzoic acid. The dorsal skin of a guinea pig was removed after shaving, and the epidermal section was excised by a keratome. After the section was mounted on the stainless steel disk with a tissue adhesive, the preparation was immersed in a 10-ml beaker containing 5 ml of drug solution at 37 degrees. At suitable intervals, samples were withdrawn, separated by TLC, and assayed by liquid scintillation. The enzyme rate constant was 1.54 x 10(-1) sec-1.

Kinetics of drug transport and concurrent metabolism in human cell cultures I: theory.

A method for evaluating the passive permeability and single-step metabolism of drugs in suspension cultures of mammalian cells was formulated assuming linear kinetics. It was assumed that the metabolizing enzymes are driven by endogenous substrates present in steady-state quantities. The presence of the drug in radiolabeled tracer quantities was assumed to cause only a small perturbation from the endogenous steady-state operating point. The time course solutions for the drug and its metabolite are given in terms of macroscopic constants, and their physical interpretations are given in terms of metabolic and transport parameters.