- Email: [email protected]
Quick detection of nystatine from sustained release dosage forms using containing microbiologically detected dissolution (MDD)
S40
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 2 S ( 2 0 0 7 ) S22–S50
theoretical plate number, microliter sample volume, aqueous media, short analysis time and pH-based, well designable solution circumstances for optimum separation. This last property is based on the protonation constant of the compounds studied and the concomitant Offord’s equation. CZE can therefore be used reciprocally pH-dependent runs can be applied for the determination of protonation constants. Neverthless, the mobility depends not only on the charge, but also, on other factors, we have also found it to be the case in our morphine derivatives. We have therefore aimed at: (1) determining the protonation constants of morphine derivatives by CZE and comparative techniques, (2) separating highly similar compounds on the basis of their pK values, and (3) interpreting the observed discrepancies in terms of solvation parameters. Methods: The protonation constants were determined using potentiometry, UV-pH and NMR-pH titrations under identical conditions. The CZE separations were carried out on an Agilent instrument, in phosphate buffers as background electrolytes with the same ionic strength as in the previous determinations. Result: The CZE-based protonation constants in some cases, have been found to be significantly different from those obtained by classical methods. For morphine pK1 is 8.30 and pK2 is 9.37 by potentiometry, but these values by CZE have been observed as 7.84 and 9.57, respectively. Also, in some cases the Offord’s equation was proved to be inadequate for the prediction of mobility differences. The mobility of quaternary amines always exceeded that of the protonated tertiary amines, despite the fact that the molar mass of the latter is smaller. We have also been able to separate codeine and Nmethylmorphine at low pH, where their masses and charges are exactly identical. Conclusion: The Offord’s equation primarily was devised to predict the mobility of proteins, which restricts its applicability for small molecules. Thus, slight structural alterations (size, shape) can result in barely predictable solvation and migration, even on molecules in different protonation stages. Protonation constants derived from CZE measurements are thus dependent on partly unknown experimental factors, their use is therefore recommended only besides complementary methods, and in cases of poor solubility or limited analyte quantity or impurities in samples, when classical techniques fail. doi:10.1016/j.ejps.2007.05.085 P-42 Combination of film coating and matrix forming effect of Eudragit® copolymers in controlled release minitablets a , G. Ujhelyi b , A. Szabo ´ ´ ´ c , Gy. Marosi c , N. Angyal a,b , A.Z. David a a I. Klebovich , I. Antal a
Semmelweis University, Department of Pharmaceutics, Hungary Sanofi Aventis, Chinoin Co. Ltd., Hungary c Budapest University of Technology and Economics, Organic Chemical Technology Department, Hungary E-mail address: nora.angyal@sanofi-aventis.com (N. Angyal). b
Objective: The aim of the study was to prepare and evaluate multiunit dosage form of minitablets, based on a combination
of Eudragit® RL and RS polymers to achieve sustained drug release. Three series of minitablets were prepared. Coated subunits had three different coating thicknesses with 5%; 10% and 15% polymer content. Uncoated matrix minitablets contained 10%, 15% and 20% amount of polymer. Coated matrix minitablets had the above mentioned various quantities of matrix component and diverse coating thickness. All formulations contained the same amount of active substance, and all had the same qualitative composition. Method: Minitablets with 3 mm diameter were prepared with direct compression technology and fluid-bed bottom spray was used for the coating process. The model drug was 25% of theophylline and Eudragit® RL and RS polymers were applied as film-coating polymers as well as matrix formers. The dissolution of the minitablets was tested in three different test media using the basket method apparatus: 0.1 M HCl of pH 1 and phosphate buffers of pH 4.5 and 6.8. Result: The dissolution profile of the matrix minitablets with 5% coating thickness was close to that of the non-matrix tablets with 10% or 15% coating level. Combining the advantages of matrix formation and the coating technology, it was possible to achieve the desired release profile with smaller amount of polymers in the coating layer, which was balanced by the same polymers in the matrix of the cores. Conclusion: Coating process using Eudragit® may be very complicated because of the sticking tendency of the polymer. In addition, providing the uniform coating thickness is very difficult. Preparing coated matrix minitablets, it was possible to decrease the coating process time making the technology more convenient and to avoid the use of high polymer amounts. Moreover, with coated matrix minitablets, the inhomogeneity of the coating layer may have less significant influence on the dissolution, which makes the process more robust. doi:10.1016/j.ejps.2007.05.086 P-43 Quick detection of nystatine from sustained release dosage forms using containing microbiologically detected dissolution (MDD) a , B. Kocsis b , Sz. Pal ´ ´ a , K. Mayer a , S. Nagy a A. Devay a
Institute of Pharmaceutical Technology and Biopharmacy, Univer´ sity of P´ecs, H-7624, P´ecs, Rokus Str. 2, Hungary b Department of Medical Microbiology and Immunology, University of P´ecs, H-7643, P´ecs, Szigeti Str. 12, Hungary ´ E-mail address: [email protected] (A. Devay). Objective: The dissolution kinetics of pharmacons from different drug delivery systems gives important informations on biopharmaceutical behaviour of the drugs. It is not easy to determine concentration of nystatin by classic UV-spectrophotometry. Developing possibilities of nystatin determination methods are continuing intensively. Classic UV analytical methods in the pharmaceutical practice are not suitable to follow exact changes of antifungal activity of nystatin. Our aim was to find a method suitable for a quick detection analyzing sustained release of the active agent from dosage forms.
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 2 S ( 2 0 0 7 ) S22–S50
Method: Optical density at 304 nm for nystatin was measured by UV–vis spectrophotometer. The test strain was Candida albicans, ATCC 90028. Tablets were produced compressing pellets coated with Eudragit. The dissolution test was performed using Ph. Eur. 4 paddle method. Antifungal activity of samples from dissolution tests of tablets was determined on Silica gel TLC plates by MDD method. Diameter of inhibition spot was determined by analyzing distribution of brightness on TLC plate. Result: Area of inhibition spot on TLC plate vs. nystatin concentration increased in non-linear way. Function of inhibition spot vs. amount of active agent was linearized by logarithmic transformation. Measurement of the active ingredient was executed both by spectrophotometric and MDD test. Comparison of UV detected drug concentration and the concentration calculated by the MDD method revealed close correlation between the techniques. Conclusion: In traditional test systems only the amount of dissolved pharmacon is detected (by UV spectrophotometry or by UV detection after HPLC separation), but changes of antifungal activity in function of time cannot be detected by the traditional methods. Classic biological methods are too consumptive of time. MDD is able to produce exact results quickly analyzing the drug delivery system of nystatin. doi:10.1016/j.ejps.2007.05.087 P-44 Examination of dosage forms containing cefalexin by microbiologically detected dissolution a , B. Kocsis b , Sz. Pal ´ ´ a , K. Mayer a , S. Nagy a A. Devay a
Institute of Pharmaceutical Technology and Biopharmacy, Univer´ sity of P´ecs, H-7624, P´ecs, Rokus Str. 2, Hungary b Department of Medical Microbiology and Immunology, University of P´ecs, H-7643, P´ecs, Szigeti Str. 12, Hungary ´ E-mail address: [email protected] (A. Devay). Objective: The dissolution kinetics of pharmacons from a drug delivery system gives basic informations on biopharmaceutical behaviour of the tested drugs. Microbiological Drug Dissolution (MDD) method is able to follow the kinetics of biological activity of an antibiotic. Cefalexin was used in our experiments as model drug. Dissolution from different cefalexin delivery systems was controlled by MDD in our study. The biological activity of compounds is usually given at definite concentration but nowadays more concentrations are recommended. MDD method follows this guideline. Method: Tablets were produced compressing pellets coated with Eudragit. The dissolution test was carried out according to Ph. Eur paddle method using Erweka DT 700. Amount of dissolved antibiotic was measured on base of UV absorbance at 262 nm. Antimicrobial activity of samples from dissolution test of manufactured tablets was determined by MDD method. Determination of diameter of inhibition spot by analyzing distribution of brightness. Result: Calibration curves for classic UV absorption method and MDD method was determined. The antimicrobial activity of cefalexin was measured by MDD method. Calibration curve
S41
of MDD of cefalexin was linearized by logarithmic transformation. Minimal detectable amounts on TLC plate of cefalexin was determined. Dissolution curves of the active ingredient was presented and compared in 3D function fitted by Weibull distribution. Conclusion: 3D graphs are able to present relationship of dissolution processes between both classic UV and MDD method. Our method is able to study dissolution from drug delivery systems of cefalosporin antibiotics using high sensitive bacterial strains. This method is also suitable in earlier phase of drug research to determine biological activity of the active agent. doi:10.1016/j.ejps.2007.05.088 P-45 Investigation of phosphatidylserine based nanostructures improving their stability in pharmaceutical dosage forms ´ ´ A. Devay, S. Nagy, K. Mayer, Sz. Pal Institute of Pharmaceutical Technology and Biopharmacy, University of P´ecs, Hungary ´ E-mail address: [email protected] (Sz. Pal). Objective: Lipid based nanostructures offer new possibilities in drug delivery. Advanced manufacturing processes are able to adjust various features of these structures thus enhancing the bioavailability of the encapsulated agents. The first lipid structure was discovered in 1961 when Bangham described spherical formations called liposomes. The aim of our experiment was to stabilize these nanostructures optimizing process parameters and the content of liposomes. Evaluation of stability was measured by the size and the polydispersity of these lipid based nanostructures. Method: Phosphatydilserine and lidocaine were dissolved in 96% ethanol. This solution was dried into thin film using a rotary evaporator (Rotavac, Heidolph, Germany) under reduced pressure at 70 ◦ C then purified water was added by automatic burette at the same temperature at a constant speed. After hydration heavy stirring (Ultra Thurrax) was applied. After the formation of liposomes ultrasonication (Realsonic, Hungary) was carried out. As stabilizing agent different amount of cholesterol was added to the structure. Size and size distribution was determined by the method of dynamic light scattering using a Zetasizer Nano S (Malvern Instruments Ltd., United Kingdom) non-invasive back scattering. The characterization of liposomes by their size can reveal important process factors that could be used to optimize the manufacture of these lipid-based microstructures to improve the bioavailability of captured drug. Result: Results were evaluated using softwares Systat version 10.2 and TableCurve3D version 5.01 (Systat Software Inc.). Changes in size and polydispersity of liposomes directly refers to their stability which can be modified applying definite amount of excipients and a proper manufacturing technique. Conclusion: During our experiments we could observe some really important and significant process factors that could be used to optimize the stability of these lipid-based formations to improve the bioavailability of captured drugs. doi:10.1016/j.ejps.2007.05.089
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 2 S ( 2 0 0 7 ) S22–S50
theoretical plate number, microliter sample volume, aqueous media, short analysis time and pH-based, well designable solution circumstances for optimum separation. This last property is based on the protonation constant of the compounds studied and the concomitant Offord’s equation. CZE can therefore be used reciprocally pH-dependent runs can be applied for the determination of protonation constants. Neverthless, the mobility depends not only on the charge, but also, on other factors, we have also found it to be the case in our morphine derivatives. We have therefore aimed at: (1) determining the protonation constants of morphine derivatives by CZE and comparative techniques, (2) separating highly similar compounds on the basis of their pK values, and (3) interpreting the observed discrepancies in terms of solvation parameters. Methods: The protonation constants were determined using potentiometry, UV-pH and NMR-pH titrations under identical conditions. The CZE separations were carried out on an Agilent instrument, in phosphate buffers as background electrolytes with the same ionic strength as in the previous determinations. Result: The CZE-based protonation constants in some cases, have been found to be significantly different from those obtained by classical methods. For morphine pK1 is 8.30 and pK2 is 9.37 by potentiometry, but these values by CZE have been observed as 7.84 and 9.57, respectively. Also, in some cases the Offord’s equation was proved to be inadequate for the prediction of mobility differences. The mobility of quaternary amines always exceeded that of the protonated tertiary amines, despite the fact that the molar mass of the latter is smaller. We have also been able to separate codeine and Nmethylmorphine at low pH, where their masses and charges are exactly identical. Conclusion: The Offord’s equation primarily was devised to predict the mobility of proteins, which restricts its applicability for small molecules. Thus, slight structural alterations (size, shape) can result in barely predictable solvation and migration, even on molecules in different protonation stages. Protonation constants derived from CZE measurements are thus dependent on partly unknown experimental factors, their use is therefore recommended only besides complementary methods, and in cases of poor solubility or limited analyte quantity or impurities in samples, when classical techniques fail. doi:10.1016/j.ejps.2007.05.085 P-42 Combination of film coating and matrix forming effect of Eudragit® copolymers in controlled release minitablets a , G. Ujhelyi b , A. Szabo ´ ´ ´ c , Gy. Marosi c , N. Angyal a,b , A.Z. David a a I. Klebovich , I. Antal a
Semmelweis University, Department of Pharmaceutics, Hungary Sanofi Aventis, Chinoin Co. Ltd., Hungary c Budapest University of Technology and Economics, Organic Chemical Technology Department, Hungary E-mail address: nora.angyal@sanofi-aventis.com (N. Angyal). b
Objective: The aim of the study was to prepare and evaluate multiunit dosage form of minitablets, based on a combination
of Eudragit® RL and RS polymers to achieve sustained drug release. Three series of minitablets were prepared. Coated subunits had three different coating thicknesses with 5%; 10% and 15% polymer content. Uncoated matrix minitablets contained 10%, 15% and 20% amount of polymer. Coated matrix minitablets had the above mentioned various quantities of matrix component and diverse coating thickness. All formulations contained the same amount of active substance, and all had the same qualitative composition. Method: Minitablets with 3 mm diameter were prepared with direct compression technology and fluid-bed bottom spray was used for the coating process. The model drug was 25% of theophylline and Eudragit® RL and RS polymers were applied as film-coating polymers as well as matrix formers. The dissolution of the minitablets was tested in three different test media using the basket method apparatus: 0.1 M HCl of pH 1 and phosphate buffers of pH 4.5 and 6.8. Result: The dissolution profile of the matrix minitablets with 5% coating thickness was close to that of the non-matrix tablets with 10% or 15% coating level. Combining the advantages of matrix formation and the coating technology, it was possible to achieve the desired release profile with smaller amount of polymers in the coating layer, which was balanced by the same polymers in the matrix of the cores. Conclusion: Coating process using Eudragit® may be very complicated because of the sticking tendency of the polymer. In addition, providing the uniform coating thickness is very difficult. Preparing coated matrix minitablets, it was possible to decrease the coating process time making the technology more convenient and to avoid the use of high polymer amounts. Moreover, with coated matrix minitablets, the inhomogeneity of the coating layer may have less significant influence on the dissolution, which makes the process more robust. doi:10.1016/j.ejps.2007.05.086 P-43 Quick detection of nystatine from sustained release dosage forms using containing microbiologically detected dissolution (MDD) a , B. Kocsis b , Sz. Pal ´ ´ a , K. Mayer a , S. Nagy a A. Devay a
Institute of Pharmaceutical Technology and Biopharmacy, Univer´ sity of P´ecs, H-7624, P´ecs, Rokus Str. 2, Hungary b Department of Medical Microbiology and Immunology, University of P´ecs, H-7643, P´ecs, Szigeti Str. 12, Hungary ´ E-mail address: [email protected] (A. Devay). Objective: The dissolution kinetics of pharmacons from different drug delivery systems gives important informations on biopharmaceutical behaviour of the drugs. It is not easy to determine concentration of nystatin by classic UV-spectrophotometry. Developing possibilities of nystatin determination methods are continuing intensively. Classic UV analytical methods in the pharmaceutical practice are not suitable to follow exact changes of antifungal activity of nystatin. Our aim was to find a method suitable for a quick detection analyzing sustained release of the active agent from dosage forms.
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 2 S ( 2 0 0 7 ) S22–S50
Method: Optical density at 304 nm for nystatin was measured by UV–vis spectrophotometer. The test strain was Candida albicans, ATCC 90028. Tablets were produced compressing pellets coated with Eudragit. The dissolution test was performed using Ph. Eur. 4 paddle method. Antifungal activity of samples from dissolution tests of tablets was determined on Silica gel TLC plates by MDD method. Diameter of inhibition spot was determined by analyzing distribution of brightness on TLC plate. Result: Area of inhibition spot on TLC plate vs. nystatin concentration increased in non-linear way. Function of inhibition spot vs. amount of active agent was linearized by logarithmic transformation. Measurement of the active ingredient was executed both by spectrophotometric and MDD test. Comparison of UV detected drug concentration and the concentration calculated by the MDD method revealed close correlation between the techniques. Conclusion: In traditional test systems only the amount of dissolved pharmacon is detected (by UV spectrophotometry or by UV detection after HPLC separation), but changes of antifungal activity in function of time cannot be detected by the traditional methods. Classic biological methods are too consumptive of time. MDD is able to produce exact results quickly analyzing the drug delivery system of nystatin. doi:10.1016/j.ejps.2007.05.087 P-44 Examination of dosage forms containing cefalexin by microbiologically detected dissolution a , B. Kocsis b , Sz. Pal ´ ´ a , K. Mayer a , S. Nagy a A. Devay a
Institute of Pharmaceutical Technology and Biopharmacy, Univer´ sity of P´ecs, H-7624, P´ecs, Rokus Str. 2, Hungary b Department of Medical Microbiology and Immunology, University of P´ecs, H-7643, P´ecs, Szigeti Str. 12, Hungary ´ E-mail address: [email protected] (A. Devay). Objective: The dissolution kinetics of pharmacons from a drug delivery system gives basic informations on biopharmaceutical behaviour of the tested drugs. Microbiological Drug Dissolution (MDD) method is able to follow the kinetics of biological activity of an antibiotic. Cefalexin was used in our experiments as model drug. Dissolution from different cefalexin delivery systems was controlled by MDD in our study. The biological activity of compounds is usually given at definite concentration but nowadays more concentrations are recommended. MDD method follows this guideline. Method: Tablets were produced compressing pellets coated with Eudragit. The dissolution test was carried out according to Ph. Eur paddle method using Erweka DT 700. Amount of dissolved antibiotic was measured on base of UV absorbance at 262 nm. Antimicrobial activity of samples from dissolution test of manufactured tablets was determined by MDD method. Determination of diameter of inhibition spot by analyzing distribution of brightness. Result: Calibration curves for classic UV absorption method and MDD method was determined. The antimicrobial activity of cefalexin was measured by MDD method. Calibration curve
S41
of MDD of cefalexin was linearized by logarithmic transformation. Minimal detectable amounts on TLC plate of cefalexin was determined. Dissolution curves of the active ingredient was presented and compared in 3D function fitted by Weibull distribution. Conclusion: 3D graphs are able to present relationship of dissolution processes between both classic UV and MDD method. Our method is able to study dissolution from drug delivery systems of cefalosporin antibiotics using high sensitive bacterial strains. This method is also suitable in earlier phase of drug research to determine biological activity of the active agent. doi:10.1016/j.ejps.2007.05.088 P-45 Investigation of phosphatidylserine based nanostructures improving their stability in pharmaceutical dosage forms ´ ´ A. Devay, S. Nagy, K. Mayer, Sz. Pal Institute of Pharmaceutical Technology and Biopharmacy, University of P´ecs, Hungary ´ E-mail address: [email protected] (Sz. Pal). Objective: Lipid based nanostructures offer new possibilities in drug delivery. Advanced manufacturing processes are able to adjust various features of these structures thus enhancing the bioavailability of the encapsulated agents. The first lipid structure was discovered in 1961 when Bangham described spherical formations called liposomes. The aim of our experiment was to stabilize these nanostructures optimizing process parameters and the content of liposomes. Evaluation of stability was measured by the size and the polydispersity of these lipid based nanostructures. Method: Phosphatydilserine and lidocaine were dissolved in 96% ethanol. This solution was dried into thin film using a rotary evaporator (Rotavac, Heidolph, Germany) under reduced pressure at 70 ◦ C then purified water was added by automatic burette at the same temperature at a constant speed. After hydration heavy stirring (Ultra Thurrax) was applied. After the formation of liposomes ultrasonication (Realsonic, Hungary) was carried out. As stabilizing agent different amount of cholesterol was added to the structure. Size and size distribution was determined by the method of dynamic light scattering using a Zetasizer Nano S (Malvern Instruments Ltd., United Kingdom) non-invasive back scattering. The characterization of liposomes by their size can reveal important process factors that could be used to optimize the manufacture of these lipid-based microstructures to improve the bioavailability of captured drug. Result: Results were evaluated using softwares Systat version 10.2 and TableCurve3D version 5.01 (Systat Software Inc.). Changes in size and polydispersity of liposomes directly refers to their stability which can be modified applying definite amount of excipients and a proper manufacturing technique. Conclusion: During our experiments we could observe some really important and significant process factors that could be used to optimize the stability of these lipid-based formations to improve the bioavailability of captured drugs. doi:10.1016/j.ejps.2007.05.089