Urokinase: Stability Studies in Solution and Lyophilized Formulations

Urokinase - a serine protease - is used clinically as a thrombolytic agent to dissolve blood clots. Low molecular weight Urokinase (33,000 dalton) isolated from human kidney cells using tissue culture techniques was used in the stability studies. Quantitative determination of Urokinase was accomplished by either amidolytic or fibrinolytic activity assay methods. The degradation of Urokinase in solution at 55 °C follows pseudo-first order kinetics at several pH values. The pH range for maximum stability has been determined to be about 6 to 7.

The stability of Urokinase is very sensitive to the quantity of residual moisture in the lyophilized formulation. Rubber stoppers used as closures for the glass vials were identified as a major source of moisture. The loss of activity from freeze dried formulations was inversely related to the specific activity of tissue culture derived Urokinase. Similar relationship was also observed for the adsorption of Urokinase from 5% dextrose diluent to the surface of polyvinyl chloride large volume parenteral diluent bags. Initial degradation rates (zero order) for freeze dried urokinase formulations with and without the addition of human serum albumin (HSA) as a stabilizer determined at 50, 40 and 30 °C demonstrated that loss of urokinase followed the Arrhenius relationship with an apparent energy of activation (Ea) of 15 kcal per mol. The addition of HSA resulted in an increase in stability by about a factor of four. However, the apparent Ea for the formulations with and without HSA was not significantly different as evident from parallel slopes in the Arrhenius plots.     

Evaluation of Cholecystokinin (CCK-8) Peptide Thermal Stability for Use as Radiopharmaceutical by Means Isothermal and Nonisothermal Approaches

ABSTRACT

The purpose of this research was to study thethermal stability of cholecystokinin octapeptide (CCK-8) in aqueous solution at pH 12 and ionic strength 0.01M, which were kept as constants, by using isothermal and nonisothermal methods.

The isothermal decomposition of CCK-8 was investigated as a function of temperature (40°C to 70°C). Nonisothermal stability studies were performed using a linear increasing temperature program. Two different nonisothermal studies were carried out at 0.25°K and 0.5°K per hour, and the temperature interval varied from 40°C to 82°C.

The degradation of CCK-8 followed first-order kinetics, obeying the Arrhenius equation in the experimental temperature range. This indicated that the degradation mechanism of CCK-8 could be the equal within the temperature range studied. The nonisothermal approach resulted in activation energy (Ea) and shelf-life (t_90%) values that agree well with those obtained by the isothermal method. The level of uncertainty in the estimates of t_90% and Ea values is determined mainly by the extent of drug degradation and temperature change during the experiment. Therefore, nonisothermal experiments save time, labor and materials (i.e. the amount of drugs necessary to conduct the experiment) compared to the classic isothermal experiments, if they are performed using a suitable experimental design and a precise analytical method.     

Effect of primary drying temperature on process efficiency and product performance of lyophilized Ertapenam sodium

Abstract

The present work aimed to investigate the impact of primary drying temperature on lyophilization process efficiency and product performance of lyophilized Ertapenam sodium (EPM). Phase behavior of EPM formulation (200?mg/mL) using differential scanning calorimetry (DSC) and freeze drying microscopy (FDM) showed Tg′ at ?28.3?°C (onset) and Tc at ?25.0?°C (onset), respectively. The formulation was freeze dried at different product temperature (Tp) during primary drying, using (a) conservative cycle (CC) where the maximum Tp (?31.9?°C) <Tg′, (b) aggressive cycle 01 (AC01) where the maximum Tp (?24.8?°C) >Tg′, and (c) AC02 where the maximum Tp (?21.0?°C) >Tc. The drying kinetics revealed that the sublimation rate was increased from 0.128?g/h/vial in CC to 0.159 and 0.182?g/h/vial in AC01 and AC02, respectively. This ultimately reduced the primary drying time of 208?min in CC to 145?min in AC01 and to 103?minutes in AC02. Morphological evaluation of cake using scanning electron microscopy (SEM) and texture analysis revealed that AC01 lead to induction of microcollapse, whereas AC02 resulted in collapsed cake. Furthermore, the microcollapsed formulations showed similar physicochemical stability to CC formulation, whereas collapsed cake showed significant degradation of EPM and increased degradation on stress stability. The study highlights that primary drying with microcollapse can be utilized to improve the process efficiency without compromising product quality of amorphous EPM.     

Physicochemical Properties of CWJ-a-5, a New Antitumor 3-Arylisoquinoline Derivative

ABSTRACT

The compound CWJ-a-5 [1-(4-methylpiperazinyl)-3-phenylisoquinoline hydrochloride] is a novel 3-arylisoquinoline derivative which has exhibited potent antitumor activity. As part of an effort to develop a useful formulation for clinical evaluation of this compound, the aqueous stability of CWJ-a-5 as a function of pH, ionic strength, and temperature, as well as its various physicochemical properties, have been examined. The pK_a value obtained by potentiometric titration in methanol–water mixtures was 3.61, at 25°C. The aqueous solubility and the apparent partition coefficient of CWJ-a-5 over the pH range 2.08–9.88 were consistent with those expected of a weak acid of similar pK_a value. The degradation of CWJ-a-5 was found to follow apparent first-order kinetics. The pH–rate profiles generated at 80°C were accounted for by acid-catalyzed degradation at low pH and base-catalyzed degradation at high pH. The activation energy was determined as 22.12 kcal/mol for the degradation of CWJ-a-5 in a pH 2.92 solution with a constant ionic strength of 0.2. Increasing the ionic strength up to 0.9 led to a higher degradation rate constant at pH 2.92. However, CWJ-a-5 was very stable even in a pH 2.92 solution, and its shelf-life was calculated to be 2.03 years at 25°C from the Arrhenius plot.     

Development of a Lyophilized Formulation for (R,R)-Formoterol (L)-Tartrate

(R,R)-formoterol is a β-agonist for inhalation. Aqueous instability suggested the need for a reconstitutable lyophilized dosage form. The objective of these studies was to devise a stable, rapid-dissolving, therapeutically compatible dosage form. The effects of diluents and residual moisture on the stability of thermally stressed formoterol formulations were investigated. Drug and various excipients (acetate, lactose, and mannitol) were lyophilized and placed in humidity chambers (0 to 90% relative humidity) at 25 to 50°C. Stability was characterized by time-dependent changes using HPLC, pH, and XRD. Residual moistures were determined by Karl Fisher methods. Regression models were developed to quantify the effects of formulation and environmental variation on drug stability. Solid-state instability was observed as a function of high residual moisture and diluent type. Although the residual moistures in mannitol formulations were typically below 1%, the degradation rate (50°C) varied from 2 to 10 mcg/day, which was 1.3- to 20-fold high than observed for lactose formulations under the same relative humidity conditions. At high relative humidity, the presence of acetate significantly increased the degradation rate (p < 0.04). The critical residual moisture content for lactose formulations was 3%. The amount of lactose was optimized by evaluating the degradation over the temperature range 25 to 50°C. Mannitol and acetate were shown to be unsuitable excipients, and an optimal lactose amount was 50 mg for vials containing 50 mcg of drug.     

Accelerated Stability Testing of Proteins and Peptides: pH-Stability Profile of Insulinotropin Using Traditional Arrhenius and Non-Linear Fitting Analysis

Abstract

The stability of insulinotropin was evaluated as a function of pH and temperature in the present study so as to predict stability at ambient and subambient temperatures. For traditional Arrhenius analysis, there is generally a loss of statistical information for the estimates of the energy of activation (E_a) and the frequency factor (A) due to the use of two separate regression steps. Therefore, a comparison was made between traditional Arrhenius analysis and a non-linear fitting approach utilizing

log (C/C₀) = -0.434 t A exp(-E_aRT)

where C is the concentration, 0 denotes the initial value, t is time, T is temperature, and R is the gas constant. A good agreement was found in the estimates by the two methods; however, the nonlinear method did provide an improvement in confidence limits under some conditions. Arrhenius plots for the degradation of the peptide appear to obey the Arrhenius equation from 5 to 50°C under alkaline and acidic conditions; however, some deviation was observed at neutral pH. The analysis predicts that at 25°C, maximal stability for the peptide is at approximately pH 8. Both methods predict 10% loss of potency in 4-5 months at pH 7 and 25°C, which appears to be reasonably consistent with ongoing real time studies.     

Formulation and Evaluation of Epidermal Growth Factor in Pluronic F-127 Gel

Abstract

Pluronic® F-127 gel was evaluated as a potential topical vehicle for epidermal growth factor delivery. The chemical stability of the polypeptide within the gel matrix was investigated using HPLC. Thermal stability studies were performed on the base gel formulation. Modifications to the formulation were made to improve physical characteristics and chemical stability. Humectants and antioxidants were investigated as potential formulation additives and the microbial status of the product was also evaluated. A formulation containing 10% propylene glycol as humectant showed both physical and chemical stability for 3 months when stored in a refrigerator. Kinetic studies were performed on thermal stability data obtained and apparent degradation rate constants were calculated. The degradation of the epidermal growth factor within the formulation appears to follow Arrhenius kinetic, with an apparent energy of activation of 93 kJ. per mole.     

Identification of Factors Affecting Preservative Efficacy and Chemical Stability of Lamivudine Oral Solution Through Statistical Experimental Design

Abstract

To identify factors affecting the chemical stability and preservative efficacy of lamivudine oral liquid formulations, an optimization study using a central composite design was performed. In this design, five factors, each at three levels, were investigated: pH (4.5, 5.5, and 73, sucrose (5%, 20%. and 50% w/v), propylene glycol (0% 2%, and 5% w/v), glycerin (4% 8%, and 12% w/v). and EDTA (0.100. 0.175, and 0.250 mg/mL). All formulations contained a constant concentration of lamivudine, parabens, and artificial strawberry and banana flavors. All formulations were evaluated for preservative effectiveness against USP and BP standards and for chemical stability at 30°C and 40°C for three months. All formulations were effective against bacteria and yeasts, but indicated reduced preservative effectiveness against the mold Aspergillus niger. Preservative effectiveness improved with increasing pH (4.5 to 7.5) and to a lesser extent with increasing EDTA concentration (0.100 to 0.250 mg/mL). Increasing glycerin concentration (4% to 12% w/v) slightly decreased preservative effectiveness. Over the concentration ranges tested, no change in preservative effectiveness was noted with concentration changes in sucrose or propylene glycol. The pH was the main factor influencing the chemical stability of the drug and preservatives in this study. Lamivudine chemical stability increased with increasing pH from 4.5 to 7.5. Methyl and propylparaben showed extensive degradation at pH 7.5.     

Physicochemical properties of CWJ-a-5, a new antitumor 3-arylisoquinoline derivative

The compound CWJ-a-5 [1-(4-methylpiperazinyl)-3-phenylisoquinoline hydrochloride] is a novel 3-arylisoquinoline derivative which has exhibited potent antitumor activity. As part of an effort to develop a useful formulation for clinical evaluation of this compound, the aqueous stability of CWJ-a-5 as a function of pH, ionic strength, and temperature, as well as its various physicochemical properties, have been examined. The pKa value obtained by potentiometric titration in methanol-water mixtures was 3.61, at 25 degrees C. The aqueous solubility and the apparent partition coefficient of CWJ-a-5 over the pH range 2.08-9.88 were consistent with those expected of a weak acid of similar pKa value. The degradation of CWJ-a-5 was found to follow apparent first-order kinetics. The pH-rate profiles generated at 80 degrees C were accounted for by acid-catalyzed degradation at low pH and base-catalyzed degradation at high pH. The activation energy was determined as 22.12 kcal/mol for the degradation of CWJ-a-5 in a pH 2.92 solution with a constant ionic strength of 0.2. Increasing the ionic strength up to 0.9 led to a higher degradation rate constant at pH 2.92. However, CWJ-a-5 was very stable even in a pH 2.92 solution, and its shelf-life was calculated to be 2.03 years at 25 degrees C from the Arrhenius plot.     

Stability Prediction of cefazolin sodium and Cephaloridine in solid state

Abstract

The stability of commercially-available solid pharmaceutical preparations containing cephaloridine and cefazolin sodium was evaluated with an accelerated isothermal degradation method at three different temperatures (37°, 45° and 60°C). A specific and sensitive differential pulse polarographic method was used for cephalosporin determination. Data obtained from high-temperature studies were processed using Arrhenius relation to predict shelf-life. The greater thermal stability of cephaloridine than cefazolin sodium was found, in contrast to what can be deduced from official monographs. Differential scanning calorimetry and X-ray diffraction were used to characterize the solid state of cephalosporin antibiotics.     

Development of a lyophilized formulation for (R,R)-formoterol (L)-tartrate

(R,R)-formoterol is a beta-agonist for inhalation. Aqueous instability suggested the need for a reconstitutable lyophilized dosage form. The objective of these studies was to devise a stable, rapid-dissolving, therapeutically compatible dosage form. The effects of diluents and residual moisture on the stability of thermally stressed formoterol formulations were investigated. Drug and various excipients (acetate, lactose, and mannitol) were lyophilized and placed in humidity chambers (0 to 90% relative humidity) at 25 to 50 degrees C. Stability was characterized by time-dependent changes using HPLC, pH, and XRD. Residual moisture were determined by Karl Fisher methods. Regression models were developed to quantify the effects of formulation and environmental variation on drug stability. Solid-state instability was observed as a function of high residual moisture and diluent type. Although the residual moisture in mannitol formulations were typically below 1%, the degradation rate (50 degrees C) varied from 2 to 10 mcg/day, which was 1.3- to 20-fold high than observed for lactose formulations under the same relative humidity conditions. At high relative humidity, the presence of acetate significantly increased the degradation rate (p < 0.04). The critical residual moisture content for lactose formulations was 3%. The amount of lactose was optimized by evaluating the degradation over the temperature range 25 to 50 degrees C. Mannitol and acetate were shown to be unsuitable excipients, and an optimal lactose amount was 50 mg for vials containing 50 mcg of drug.     

Assessment of Degree of Disorder (Amorphicity) of Lyophilized Formulations of Growth Hormone Using Isothermal Microcalorimetry

When determining the degree of disorder of a lyophilized cake of a protein, it is important to use an appropriate analytical technique. Differential scanning calorimetry (DSC) and X‐ray powder diffraction (XRPD) are the most commonly used thermo‐analytical techniques for characterizing freeze‐dried protein formulations. Unfortunately, these methods are unable to detect solid‐state disorder at levels < 10%. Also, interpretation of DSC results for freeze‐dried protein formulations can be difficult, as a result of the more complex thermal events occurring with this technique. For example, proteins can inhibit the thermally induced recrystallization of the lyophilized cake, resulting in potential misinterpretation of DSC degree of disorder results. The aim of this investigation was to study the use of isothermal microcalorimetry (IMC) in the assessment of degree of solid‐state disorder (amorphicity) of lyophilized formulations of proteins. For this purpose, two formulations of growth hormone were prepared by lyophilization. These formulations consisted of the same amounts of protein, mannitol, glycine, and phosphate buffer, but differed in the freeze‐drying procedure. After lyophilization, the recrystallization of the samples was studied using IMC at 25°C under different relative humidities (58–75%). The effect of available surface area was studied by determining the heat of recrystallization (Q) of the samples before and after disintegration of the cakes. The results showed that, in contrast to DSC, IMC allowed detection of the recrystallization event in the formulations. Although both formulations were completely disordered and indistinguishable according to XRPD method, IMC revealed that formulation B had a different solid‐sate structure than formulation A. This difference was the result of differences in the freeze‐drying parameters, demonstrating the importance of choosing appropriate analytical methodology.     

In Vitro Evaluation of Albumin Microspheres Containing Actinomycin D

Abstract

Albumin microspheres containing actinomycin D were prepared by the heat stabilization method. The compata-bility of the drug with magnetite and the optimum stability of the drug in different pH were studied. Drug loaded albumin microspheres containing magnetite showed good magnetic response. Release of the drug was slow and continued for 7 days exhibiting sustained release property. The difference as regards to the size, shape, drug content and release rate from freshly prepared and freeze dried drug loaded albumin microspheres was negligible.     

Degradation Kinetics of Fluorouracil-Acetic-Acid-Dextran Conjugate in Aqueous Solution

ABSTRACT

The degradation kinetics of fluorouracil-acetic-acid-dextran conjugate (FUAC-dextran) was investigated in various buffer solutions with different pH value and physiological saline solution at 60°C and 37°C, respectively. The hydrolytic reaction displayed pseudo-first-order degradation kinetics. Hydrolytic rate constant obtained was the function of pH value and independent of species of buffering agents. The smallest rate constant was observed at pH round 3.00. The activation energy of the hydrolytic reaction was estimated from Arrhenius equation as 88.73 ± 6.00 kJ·mol^?1. The special base catalytic degradation of the conjugate was observed from acidic to slight alkaline condition and the special base catalytic rate constants were calculated. The conjugate was more stable in physiological saline than that in buffer solution at pH 7.00 or 9.00 at 37°C. The results revealed that the conjugate was stable in acidic condition and will degrade in alkaline condition.     

潜催化剂动力学参数的研究

对于热固性树脂，适宜的室温稳定性和较快的固化速度是十分重要的为此希望体系有较高的活化能（Ｅａ）和熵因子（Ａ）用Ｐ─苯醌和适当的硫化物合成了１５个烷基硫翁盐．用双环氧树脂Ｃｙ１７９和潜催化剂，通过凝胶化实验考察了硫翁盐的活性，并用动力学参数对之进行了分析讨论     

Formulation for a novel inhaled peptide therapeutic for idiopathic pulmonary fibrosis

A caveolin-1 scaffolding domain, CSP7, is a newly developed peptide for the treatment of idiopathic pulmonary fibrosis. To develop a CSP7 formulation for further use we have obtained, characterized and compared a number of lyophilized formulations of CSP7 trifluoroacetate with DPBS and in combination with excipients (mannitol and lactose at molar ratios 1:5, 70 and 140). CSP7 trifluoroacetate was stable (>95%) in solution at 5 and 25?°C for up to 48?h and tolerated at least 5 freeze/thaw cycles. Lyophilized cakes of CSP7 trifluoroacetate with excipients were stable (>96%) for up to 4?weeks at room temperature (RT), and retained more than 98% of the CSP7 trifluoroacetate in the solution at 8?h after reconstitution at RT. The lyophilized CSP7 formulations were stable for up to 10?months at 5?°C protected from moisture. Exposure of the lyophilized cakes of CSP7 to 75% relative humidity (RH) resulted in an increase in the absorbed moisture, promoted crystallization of the excipients and induced reversible formation of CSP7 aggregates. Increased molar ratio of mannitol slightly affected formation of the aggregates. In contrast, lactose significantly decreased (up to 20 times) aggregate formation with apparent saturation at the molar ratio of 1:70. The possible mechanisms of stabilization of CSP7 trifluoroacetate in solid state by lactose include physical state of the bulking agent and the interactions between lactose and CSP7 trifluoroacetate (e.g. formation of a Schiff base with the N-terminal amino group of CSP7). Finally, CSP7 trifluoroacetate exhibited excellent stability during nebulization of formulations containing mannitol or lactose.     

Comparative Evaluation of Direct Compression Ampicillin Formulations

Direct compression formulations were developed for ampicillin using methyl vinyl ether/maleic anhydride copolymer (I) and vinyl acetate/crotonic acid copolymer (II) as binders. A comparison was made between these formulations and wet granulation method using gelatin as binder regarding the chemical stability of ampicillin as a function of relative humidity (55 to 90%) and temperature (40 to 75°C). Polymer I showed least moisture uptake followed by polymer II and gelatin. The mechanism suggested here involves moisture uptake and dissolution followed by chemical decomposition. The temperature had lesser effect on stability because of low activation energies but an Arrhenius relationship was established for three formulations studied. It was concluded that formulation using polymer I gives the most ideal combination of physiochemical properties for the direct compression of ampicillin in solid dosage forms.     

Effect of Additives on Stability of Etoposide in PLGA Microspheres

The purpose of this article was to determine the shelf life of etoposide in poly(lacticco-glycolic acid) (PLGA) microspheres prepared with and without additives (i.e., tricaprin and isopropyl myristic acid ester [IPM]). The microspheres were prepared by a single-emulsion solvent extraction technique with and without 25% w/w additive. The batches of microspheres were subjected to an accelerated stability study at two elevated temperatures (70°C and 80°C or 80°C and 90°C). Samples were taken at 7, 14, 21, 28, and 35 days for estimation of drug content by high-performance liquid chromatography (HPLC). The drug stability in the microspheres was determined by plotting the log percentage drug remaining versus time to obtain the degradation rate constant k of etoposide at the measured temperature. This degradation rate constant was then used in the Arrhenius equation to obtain the activation energy of etoposide, which was utilized to determine the shelf life of the microspheres at room temperature. The results showed that all three microsphere formulations had good long-term stability at room temperature (6.62-8.86 years at 25°C). The plain microspheres were shown to possess a shelf life of 6.62 years, and the IPM and tricaprin were the most stable with shelf lives of 8.25 and 8.86 years, respectively.     

Study on the rate of decomposition of amoxicillin in solid state using high-performance liquid chromatography

Abstract

Amoxicillin sodium salt degradation in solid state relies on a sequential reaction consisting of two pseudo-first-order processes. Amoxicillin trihydrate, now used in pharmaceutical formulations, is significantly more stable than sodium amoxicillin. It degrades according to Prout-Tompkins model. We studied the stability of amoxicillin at temperatures of 37°, 50°, 80°, 90°, 100° y 110° C. HPLC was chosen as the analytical method. Amoxicillin and its decomposition products are separated by reversed-phase (C18) HPLC with gradient elution.