Decreased stability in liposomal suspensions: accelerated loss of p-nitrophenyl acetate.

The goal of this investigation was to determine the reason for the previously reported increase in the rate of hydrolysis of p-nitrophenyl acetate to p-nitrophenol in the presence of positively charged liposomes. When this charge was due to incorporation of stearylamine, the rate of loss increased 5- to 10-fold relative to the control buffers. This rate enhancement was accompanied by formation of N-stearylacetamide, an event which was not previously considered. Similar results were obtained with either L-alpha- or dimyristoyl phosphatidylcholine. When the positive charge on the liposomes was conferred by the cetrimonium ion, however, the acceleration was replaced by a reduction in rate together with the absence of amide formation. Separation of the continuous phases from the liposomes provided media which were kinetically equivalent to the control buffers, indicating that rate enhancement and reduction were both due to the liposomal phases. Increasing the pH produced an increase in ester clearance values due to the stearylamine-containing liposomal phase, which is consistent with the formation of free amine, providing increased aminolysis. Although amide formation was also observed in stearylamine suspensions, the rate of p-nitrophenyl acetate loss was much greater in liposomal suspensions. Accelerated loss in the presence of positively charged liposomes is due to the formation of N-stearylacetamide by reaction with stearylamine and not to the positive charge, a hypothesis disproved by use of cetrimonium ion containing liposomes.

Cefuroxime hydrolysis kinetics and stability predictions in aqueous solution.

Cefuroxime hydrolysis rate constants (k) were determined to predict the degradation rate of cefuroxime in aqueous solution as a function of pH, temperature, and buffer. At constant temperature, the pH-rate expression was: k = kH(aH+) + kS1f1 + kS2f2 + kOH(aOH-), where f1 is the fraction of cefuroxime in the undissociated form and f2 is the anionic fraction, kH and kOH are the catalytic rate constants for hydrogen activity (aH+) and hydroxyl ion activity (aOH-), and kS1 and kS2 are first-order rate constants for spontaneous hydrolysis. Formate, acetate, phosphate, and borate buffers did not catalyze degradation. Temperature dependencies for kH, kS1, kS2, and kOH were described with values for A (pre-exponential term) and E (energy of activation) calculated from k = Ae-E/RT (where R is 1.987 cal/mol-deg and T is absolute temperature). Combining the pH and temperature equations allowed predictions for cefuroxime hydrolysis rates in aqueous solutions at any pH and temperature. Results were validated by predicting the observed rate constants for every experimental condition and also for a reconstituted commercial product stored at 30 degrees C. Maximum stability was observed in the pH-independent region from pH 4 to 7, where the time during which cefuroxime concentration exceeded 90% of its initial concentration at 25 degrees C was 1.2 days. Rate constants employed in predictions were based on stability-indicating HPLC assays. For selected conditions, additional rate constants were calculated from changes in cefuroxime UV absorbance.(ABSTRACT TRUNCATED AT 250 WORDS)

Substituent effects on degradation rates and pathways of cytosine nucleosides.

A previous report on the influence of a 6-methyl substituent on cytosine nucleoside degradation proposed that N-glycosyl hydrolysis predominated over the deamination pathway which was characteristic of the unsubstituted parent compounds. The UV absorption data which led to this hypothesis were not conclusive. Evidence for N-glycosyl hydrolysis was indirect and the product concentration was not quantitated. In the present study, specific HPLC methods were employed to assay four cytosine nucleosides and their corresponding bases, thus allowing comparison of the N-glycosyl hydrolysis rate to the overall rate of loss for each nucleoside. These data indicated that the 6-methyl nucleosides underwent partial or complete hydrolysis to yield their corresponding sugars and 6-methylcytosine, which then deaminated to 6-methyluracil. An increase in the reactivity and a change in the reaction products of the 6-methyl nucleosides were attributed to an alteration in conformation. In addition, the 6-methyl arabinosyl nucleoside reacted much faster than the 6-methyl ribosyl nucleoside, presumably due to 2'-OH participation. Degradation of 5-methyl deoxycytidine was also re-examined since its degradation was previously attributed solely to N-glycosyl hydrolysis. In the present study, simultaneous deamination and hydrolysis were measured, although N-glycosyl hydrolysis was found to predominate.

A nomogram to predict the best biological half-life values for candidates for oral prolonged-release formulations.

It is sometimes possible to maintain plasma concentrations between desired maximum and minimum limits by repetitively administering a drug in an oral prolonged-release formulation when this goal cannot be achieved with a rapid-release formulation. However, this approach does not work with all drugs. The biological half-life value of the drug can be one cause for failure of this approach. Although it is recognized that a half-life may be too long or too short, neither the criteria for determining these values nor the consequences of failing to meet them have been established. The best half-life values for prolonged-release candidates were found by simulating once-a-day and twice-a-day administration of formulations and examining the capacity of these formulations to maintain steady-state plasma concentrations between various selected limits. These observations were used to establish criteria to judge the acceptability of half-lives. Half-life values were considered too long if drugs were self-sustaining and simulations of their rapid-release formulations were also successful. Half-life values were considered too short if minor variability in prolonged-release rates resulted in plasma concentrations above and/or below the selected limits. The actual half-life values that were considered too long or too short depended on the dosing interval and the selected maximum and minimum plasma concentrations. A nomogram was constructed to assess the acceptability of the biological half-life of a candidate for once-a-day or twice-a-day prolonged-release formulations. The nomogram employs the user-selected limits for the desired plasma concentrations to predict whether the half-life of a candidate is (1) too long, (2) too short, or (3) acceptable (i.e., between 1 and 2).

A kinetic oxymoron: concentration-dependent first-order rate constants for hydrolysis of ceftazidime.

The influence of pH, temperature, and buffers on the hydrolysis of 10(-4) M ceftazidime was previously reported. The pH-rate profiles showed that maximum stability occurred in the pH-independent region from 4.5 to 6.5. In the present study, hydrolysis rates of 0.031, 0.14, 0.25, and 0.35 M ceftazidime were measured at 30 and 65 degrees C, pH 5.5-6.2. The data were consistent with beta-lactam hydrolysis and the rapid release of pyridine. The sum of the time-dependent concentrations of ceftazidime and pyridine provided mass balance. Simultaneous nonlinear regression for ceftazidime loss and pyridine formation provided similar rate constants (k) to those determined from first-order plots of ceftazidime loss. Although the loss of ceftazidime was first-order for each initial concentration, the k values increased as the initial concentrations increased. Plots of k versus initial concentration were linear with intercepts similar to the k values for 10(-4) M solutions, thus implying that ceftazidime catalyzed its own degradation. At the pH of these studies ceftazidime exists as a base. The ceftazidime catalytic constant, calculated from the slope of the plot, was similar to that found for the general-base catalyst, HPO4(2-). Therefore, it is feasible that ceftazidime also behaved as a intermolecular general-base catalyst. However, first-order plots exhibited excellent linearity even though the catalyst (ceftazidime) was consumed. This would require that the catalytic moieties on ceftazidime remained relatively constant throughout its hydrolysis. This hypothesis was shown to be consistent with literature reports which indicate that the general-base catalytic groups can remain relatively constant during cephalosporin hydrolysis.

Influence of pH, temperature, and buffers on the kinetics of ceftazidime degradation in aqueous solutions.

First-order rate constants (k) were determined for the hydrolysis of ceftazidime in the pH range of 0.5 to 8.5 at 45, 55, and 65 degrees C by a stability-indicating HPLC assay. In the absence of buffer effects, the pH-rate expression was k = kH1f1(aH+) + kH2f2(aH+) + kH3f3(aH+) + kSf3 + kOHf3(aOH-), where KH and KOH are the catalytic rate constants for the activity of hydrogen (aH+) and hydroxyl (aOH-) ions, respectively, and kS is the rate constant for spontaneous hydrolysis. The fractions of ceftazidime in various stages of dissociation (f1, f2, and f3) were calculated from kinetically determined apparent Ka values of 2.03 x 10(-2) and 4.85 x 10(-5). Catalytic constants (kcat) were calculated for formate, acetate, phosphate, and borate buffers, which accelerated hydrolysis. Each of the rate constants (kH1, kH2, kH3, kS, kOH, and kcat) were described as a function of temperature with calculated A and E values in the Arrhenius equation, kT = Ae-E/RT. Ceftazidime hydrolysis rate constants (k) were calculated as a function of pH, temperature, and buffer by combining the pH-rate expression with the buffer contributions calculated from kcat values and the temperature dependencies. These equations and their parameter values successfully calculated 95 of 104 experimentally determined rate constants with errors of < 10%. Maximum stability was observed in the relatively pH-independent region from 4.5 to 6.5. Hydrolysis rate constants at 30 degrees C were predicted and experimentally verified for four ceftazidime solutions, three of which (pH 4.4 acetate buffer and pH 5.5 and 6.5 phosphate buffers) maintained 90% of their initial concentration for approximately 1.5 days.

Critical analysis of "flip-flop" phenomenon in two-compartment pharmacokinetic model.

Computer simulations were used to examine the effect of first-order absorption on the disposition of one- and two-compartment model drugs. Two-compartment systems that attain a clinically acceptable beta-phase after rapid intravenous injection were perturbed by introduction of drug via first-order absorption. The validity of perceiving such a system as a potential "flip-flop" model was tested by comparing the negative slopes of log-linear plasma-time profiles to known values for ka and beta for various values of ka, k12, k21, and k10. Although most log-linear plots showed excellent correlation coefficients (r2 greater than 0.996), their negative slopes (S) did not represent either ka or beta under various combinations. A similar consideration of the one-compartment model enabled a comparison to be made between the two systems. Maximum negative errors were observed for both one- and two-compartment drugs as ka leads to k2 or beta, respectively. The value for S provided a good estimate of the absorption rate constant, ka, when k2 greater than or equal 2ka (one compartment) or beta greater than or equal 2ka. The elimination rate constant (k2 or beta) could be obtained from S for all one-compartment and some two-compartment drugs when the value of ka was approximately twice that of k2 or beta. Large positive errors also were observed with certain two-compartment drugs where the ratio of the four rate constants apparently linearized a nonlinear plasma profile. Conditions wherein S may be expected to approach beta wherein S approaches ka are clearly defined.

Kinetics and mechanisms of degradation of the antileukemic agent 5-azacytidine in aqueous solutions.

The hydrolytic degradation of 5-azacytidine was studied spectrophotometrically as a function of pH, temperature, and buffer concentration. Loss of drug followed apparent first-order kinetics in the pH region below 3. At pH less than 1,5-azacytosine and 5-azauracil were detected; at higher pH values, drug was lost to products which were essentially nonchromophoric if examined in acidic solutions. The apparent first-order rate constants associated with formation of 5-azacytosine and 5-azauracil from 5-azacytidine are reported. Above pH 2.6, first-order plots for drug degradation are biphasic. Apparent first-order rate constants and coefficients for the biexponential equation are given as a function of pH and buffer concentration. A reaction mechanism consistent with the data is discussed together with problems associated with defining the stability of the drug in aqueous solutions. At 50 degrees, the drug exhibited maximum stability at pH 6.5 in dilute phosphate buffer. Similar solutions were stored at 30 degrees to estimate their useful shelflife. Within 80 min, 6 times 10(-4) M solutions of 5-azacytidine decreased to 90% of original potency based on assumptions related to the proposed mechanisms.

Pharmacokinetic prodrug modeling: in vitro and in vivo kinetics and mechanisms of ancitabine bioconversion to cytarabine.

Conversion rates of the prodrug ancitabine to the antileukemic cytarabine have been measured in vivo (rabbits) and in vitro (in the presence of rabbit blood and human red blood cells, blood, and plasma) using HPLC analyses for the prodrug, drug, and its inactive metabolite, 1-beta-D-arabinosyluracil. These observed pH-dependent in vitro rate constants were consistent with those for chemical hydrolysis determined from controls using Tris buffers. Hydrolysis of ancitabine to cytarabine is chemically, not enzymatically, mediated. The blood concentration-time course for administered compound was described by a two-compartment open model following a rapid intravenous injection of prodrug, drug, or metabolite in each of three rabbits. The in vivo conversion rate constant (kc) following a rapid intravenous prodrug injection was estimated by simultaneous nonlinear regression of ancitabine and cytarabine blood concentration-time courses using equations for two-compartment prodrug and drug with all possible models describing potential conversion sites. The best fit was obtained for the case allowing simultaneous conversion of the prodrug in both central and peripheral compartments to the drug in the central compartment with a common value for kc. The resulting kc value (0.09 h-1, three rabbits) is similar to that for chemical hydrolysis (0.07 h-1) at 38.8 degrees C. Reasons why this agreement is regarded as fortuitous are discussed.

Aqueous conversion kinetics and mechanisms of ancitabine, a prodrug of the antileukemic agent cytarabine.

The kinetics of conversion of the prodrug ancitabine to the anticancer drug cytarabine have been studied in aqueous solutions in the pH range of 1.5-10.7, temperature range of 19.5-80.0 degrees C, ionic strength range of 10(-4) to 1.5, and in the presence of several general-base catalysts. Under all conditions ancitabine was quantitatively converted to cytarabine. The pH-rate profiles were linear with slope = 1 in alkaline pH, becoming pH independent in the region of maximum stability at pH less than or equal to 4, where buffer catalysis was found to be insignificant and kobs approximately equal to (1.12 X 10(11) h-1)-exp [-10121 deg/T]. At 30 degrees C, pH less than or equal to 4, it is calculated that an aqueous ancitabine solution will maintain 90% of its initial concentration for 12 d. A novel method for measuring general-base catalysis in competition with predominating specific-base catalysis and in the presence of secondary salt effects at constant ionic strength was developed. Three mechanisms of hydrolytic prodrug conversion are proposed: nucleophilic hydroxide addition, general base-assisted nucleophilic water attack, and spontaneous water attack.

Theoretical basis for the detection of general-base catalysis in the presence of predominating hydroxide catalysis.

The detection of general-base catalysis in the presence of predominating specific-base catalysis in aqueous buffer solutions is examined for various relationships between k0cat and k0OH, the bimolecular rate constants for general-base and hydroxide-ion attack. The three experimental variables that affect the detection of buffer-base catalysis are the type of buffer, conjugate-acid concentration, and ionic strength. Various buffers used in pharmaceutical kinetic studies are considered, and it is concluded that buffers with high Ka values favor detection. Additionally, high conjugate-acid concentrations and ionic strengths appear to optimize the detection of general-base catalysis.

Influence of pH, temperature and buffers on cefepime degradation kinetics and stability predictions in aqueous solutions.

First-order rate constants (k) were determined for cefepime degradation at 45, 55, 65, and 75 degrees C, pH 0.5 to 8.6, using an HPLC assay. Each pH-rate profile exhibited an inflection between pH 1 and 2. The pH-rate expression was k(pH) = kH1 f1(aH+) + kH2 f2(aH+) + ks + kOH(aOH-), where kH1 and kH2 are the catalytic constants (M(-1) h(-1)) for hydrogen ion activity (aH+), kOH is the catalytic constant for hydroxyl ion activity (aOH-), and ks is the first-order rate constant (h(-1)) for spontaneous degradation. The protonated (f1) and unprotonated (f2) fractions were calculated from the dissociation constant, Ka = (8.32x10(-6))e(5295)/RT where T was absolute temperature (T). Accelerated loss due to formate, acetate, phosphate, and borate buffer catalysis was quantitatively described with the catalytic constant, kGA (M(-1) h(-1)) for the acidic component, [GA], and kGB (M(-1) h(-1) for the basic component, [GB], of each buffer. The temperature dependency for each rate constant was defined with experimentally determined values for A and E and the Arrhenius expression, kT = Ae-E/RT, where kT represented kH1, kH2 , kS, kOH, kGA, or kGB. Degradation rate constants were calculated for all experimental pH, temperature, and buffer conditions by combining the contributions from pH and buffer effects to yield, k = k(pH) + kGA[GA] + kGB[GB]. The calculated k values had <10% error for 103 of the 106 experimentally determined values. Maximum stability was observed in the pH-independent region, 4 to 6. Degradation rate constants were predicted and experimentally verified for cefepime solutions stored at 30 degrees C, pH 4.6 and 5.6. These solutions maintained 90% of their initial concentration (T90) for approximately 2 days.

Stability-indicating colorimetric assay for indicine N-oxide using TLC.

Aliquots of aqueous solutions in which indicine N-oxide may be degraded were mixed with 0.5 M formic acid (1:3) to adjust the pH to approximately 2-4 to quench the reaction and to ensure adequate TLC resolution. Silica-coated aluminum sheets were used to isolate indicine N-oxide by cutting the appropriate region from the chromatogram. By a modification of a known procedure, the silica gel then was treated with an acetic anhydride-diglyme mixture, and the mixture was heated to convert the drug to a pyrrole, which was then coupled with 4-dimethylaminobenzaldehyde to produce a color. The absorbance of the resulting solution was determined at 566 nm, and the apparent molar absorptivity, epsilon, based on the final indicine N-oxide concentration was 6.13 x 10(4). The recovery was approximately 92%, and the assays were readily reproducible with a coefficient of variation of 4.4%.

Simultaneous partitioning and hydrolysis kinetics of amoxicillin and ampicillin.

The kinetics of ampicillin and amoxicillin partitioning with simultaneous acid-catalyzed hydrolysis were studied in a stirred transfer cell containing isobutanol as the extract and aqueous hydrochloric acid (0.1-0.5 N) as the raffinate at 37 degrees. Biexponential data for the concentration in both the raffinate (C1) and the extract (C2) as a function of time were analyzed simultaneously by nonlinear regression to estimate the apparent first-order rate constant for transfer from hydrochloric acid to isobutanol (k '12), the reverse transfer constant (k '21), and the hydrolysis rate constant (k). Agreement between k values determined in the presence of simultaneous partitioning and those determined in the absence of partitioning (k app) verified the nonlinear estimates. Apparent partition coefficients, which represent the values that would be obtained in the absence of hydrolysis K'D = C1 infinity/C2 infinity), were estimated from K'D = k'12/k'21. During terminal monoexponential loss, where C1 approximately equal to Y'e-beta t and C2 approximately equal to Z'e-beta t, the kinetically controlled C2/C1 ratio (r) is described by [K'12/K'21-beta)], which decreases with decreasing kappa values until r approaches K'D. The difference between the terminal concentration ratio, r, and its corresponding partition coefficient, K'D, is a measure of the degree to which kinetic processes control distribution. Both ampicillin and amoxicillin showed kinetic control of the distribution ratios in 0.5 N HCl, where the hydrolysis rate constant was significant relative to the distribution rate constants. Ampicillin had r approximately equal to 1.74 and K'D approximately equal to 0.92; amoxicillin had r approximately equal to 0.95 and K'D approximately equal to 0.65. As the (K'12 + K'21/k ratio increased, the r values approached K'D so that in 0.1 N HCl, r approximately K'D = 0.33 for amoxicillin and r approximately 0.6 and K'D approximately 0.56 for ampicillin. In general, amoxicillin distribution rate constants (K'12 + K'21) were roughly twice those of ampicillin, whereas ampicillin K'D and r values were nearly double those of amoxicillin. Thus, the kinetic and thermodynamic rank orders are opposite. This result may have implications in drug design via molecular modification.

Drug stability in liposomal suspensions: hydrolysis of indomethacin, cyclocytidine, and p-nitrophenyl acetate.

First-order rate constants (kL) for hydrolysis of p-nitrophenyl acetate, cationic cyclocytidine, and anionic indomethacin in the presence of buffered liposomal suspensions of positive, negative, and neutral charge were compared to those determined in the corresponding buffers (kB) using the ratio, Rk = kL/kB. Association between the reactants and the liposomes was evaluated by comparing assays for concentration in the filtrates (CF) with the total concentration in the liposomal suspension (CT) using RC = CF/CT. Liposomes did not influence cyclocytidine hydrolysis rates and no association was observed (Rk congruent to RC congruent to 1). In contrast, indomethacin showed approximately 80% reduction in hydrolysis rate and approximately 80% liposome association value (Rk congruent to 0.2 congruent to RC). In neutral and negatively charged liposomal suspensions, p-nitrophenyl acetate displayed approximately 30% decrease in kB (Rk congruent to 0.7) together with approximately 90% liposomal association (RC congruent to 0.1). However, hydrolysis was greatly accelerated in positively charged liposomal suspensions. Loss was described by a biexponential equation where alpha is the fast and beta is the slow pre-exponential coefficient and alpha/beta/kB = 39:6:1. The observed relationships between hydrolysis rates and reactant-liposome associations are reconciled in terms of the hydrophilicity of the reactants and the potential influence of the liposomes on the expected transition states for the hydrolysis reactions.

Calculation of partition coefficient of an unstable compound using kinetic methods.

A stirred transfer cell containing equal volumes of light liquid paraffin and an aqueous phase at 37 degrees was used to demonstrate the feasibility of calculating the partition coefficient of an unstable compound by kinetic analysis. Cyclohept-2-enone was chosen since it is a neutral molecule and, therefore, should have a pH-independent oil-water partition coefficient, KD. Moreover, this cyclic alpha, beta-unsaturated ketone undergoes hydrogen-ion-catalyzed hydration but is sufficiently stable at neutral pH to determine KD. The model system chosen represents first-order transfer between the aqueous (C1) and organic (C2) phases with simultaneous, reversible, first-order hydration. The transfer constants k'12 and k'21, were determined at 37 degrees in the absence of degradation where asymptotic values for C1 agreed with the observed equilibrium values in nonkinetic partitioning studies. The first-order rate constants for hydration in 0.1 N HCl were determined at 37 degrees in the absence of the organic phase. Partitioning with simultaneous hydration when was studied using 0.1 N HCl and light liquid paraffin. Data were analyzed by nonlinear regression based on the equation for C1 as a function of time. The values for k'12 and k'21 from these experiments were comparable to the estimates obtained under stable conditions. This agreement demonstrates that simultaneous degradation and partitioning can be analyzed for k'12 and k'21, thus permitting calculation of the partition coefficient (i.e., KD = k'12/k'21) that would be observed if the drug were stable.

Thermodynamic dependence of interfacial transfer kinetics of nonionized barbituric acid derivatives in two-phase transfer cell.

A theory was developed to describe interfacial transport kinetics of a series of drug homologs in a two-phase transfer cell. When tested, the theory held true for 5,5-disubstituted barbituric acid derivatives in a preequilibrated octan-1-ol = (pH 5) aqueous buffer system maintained at 37 degrees and stirred symmetrically at 50 and 100 rpm. Theoretical prediction of transfer kinetics was not possible in such a cell if the phases were stirred asymmetrically. For symmetric stirring, successful prediction of the transfer kinetics of any homolog in the series was possible from a knowledge of the partition coefficient and transfer kinetics of the parent compound, the partition coefficient of the homolog, and some easily determined system variables. The viscosity and density of the two phases and the phase-volume ratio were needed to define a system constant dependent on the solute diffusion coefficient, interfacial area, donor phase volume, and the boundary layer thickness for diffusion in the donor phase volume, and the boundary layer thickness for diffusion in the donor phase. A method is described to enable estimation of this constant from a knowledge of the transfer kinetics of the parent compound. The rank order of compounds in terms of their observed first-order transfer rate constants is shown to be dependent on the characteristics of the solvent system and stirring conditions employed, as well as on the physical chemistry of the solutes. The results are discussed in light of previously documented investigations.

An efficient method for computer-aided dosage form design.

It is desirable to have slow-release dosage form to be taken once daily, or at most twice daily, as compared to three or four times in a single day. However, the existing computer-aided dosage form design method requires a large amount of computer time when applied to nonlinear disposition drugs. This large commitment of computer time makes it inconvenient to study the feasibility for prolonged-release products containing such drugs. Instead of evaluating all possible combinations of the amount of dose and release rates that produce acceptable steady-state plasma concentrations, only the contour of the dose-release rate domain needs to be determined. An image boundary tracking method has been used to determine such contours. When combined with several modifications of the numerical solution process, the acceptable dose and release rate constants can be determined efficiently. When this modified boundary tracking method was applied to phenytoin, which exhibits nonlinear disposition, the required computer time was reduced to about 5% of the previous method, making the dosage form feasibility assessment practical.