- Email: [email protected]
Design and Evaluation of Two-Phase Partition–Dissolution Method and Its Use in Evaluating Artemisinin Tablets
+
+
Design and Evaluation of Two-Phase Partition−Dissolution Method and Its Use in Evaluating Artemisinin Tablets NGO THU HOA
AND
R. KINGETX
Received March 6, 1996, from the Laboratory of Galenical and Clinical Pharmacy, Catholic University Leuven, Onderwijs en Navorsing, Gasthuisberg, 3000 Leuven, Belgium. Final revised manuscript received June 17, 1996. Accepted for publication July 29, 1996X. Abstract 0 A method is described for the assessment of the dissolution behavior of solid dosage forms with a high content of a very water insoluble drug. In this method the dissolved drug is continuously extracted from the aqueous dissolution medium into an organic phase. The reported data on the dissolution of artemisinin show that this method guarantees sink conditions in the aqueous dissolution medium during the total duration of the experiment.
Introduction There is no doubt that the determination of dissolution rates is an important tool in the development, evaluation, and control of solid dosage forms. In the design of a reliable in vitro dissolution test model that can positively characterize the role of the dissolution process on drug bioavailability, it is essential that the model be capable of simulating test conditions that are likely to prevail during the in vivo dissolution process. One such condition is the maintenance of sink conditions during in vitro dissolution; i.e., the drug concentration in the dissolution medium does not exceed 1020% of solubility. Slightly and very slightly soluble drugs give rise to difficulties in this respect; with highly dosed drugs the problems become even more severe. A large number of authors have attempted different methods to achieve this objective.1 The types of systems employed for maintaining sink conditions are (a) large fixed fluid volume, (b) multiple phase (partition method), and (c) continuous flow (through flow cell). Studies on the bioavailability of drugs from a given dosage form revealed that in many situations various dosage forms with the same content of the active compound did not give the same therapeutic effect.1 So, control of the bioavailability of drugs is a major requirement in drug production, especially for drugs of very low water solubility. Artemisinin, a sesquiterpene lactone with an endoperoxide bridge, isolated from the Chinese traditional medicinal plant Artemisia annua L., Asteraceae, has received considerable attention in the past decade because of its antimalarial activity.2-6 It is a rapidly acting blood schizontocide with good activity against chloroquine resistant and multidrug resistant strains of Plasmodium parasites. It is effective in the treatment of cerebral malaria. Tablets and capsules, containing 250 mg of artemisinin per unit, are commercially available. It has been demonstrated that when the official dissolution methods are used, sink conditions do not prevail during the duration of the test for some artemisinin formulations7 tested. The objective of this study was the development of a suitable dissolution method for artemisinin solid oral dosage forms. By using an organic phase into which the drug under investigation would almost completely partition, Gibaldi and Feldman8 were able to continuously remove solute from the dissolution medium. Because of its simplicity of design and X
Abstract published in Advance ACS Abstracts, September 1, 1996.
1060 / Journal of Pharmaceutical Sciences Vol. 85, No. 10, October 1996
Figure 1sSchematic of the dissolution test system: (1) water bath; (2) thermostat; (3) round-bottom vessel, 1 L; (4) shaft; (5) mixing blade; (6) paddle; (7) glass cylinder; (8) filter paper; (9) disintegration apparatus.
its ability to guarantee sink conditions, this method was chosen as a basis for further application on solid dosage forms containing a high dose of a water insoluble drug.
Experimental Section MaterialssArtemisinin (ART) with a purity of 99.87% was received from Mediplantex (Hanoi, Vietnam); sodium lauryl sulphate (SLS), sodium dioctyl sulfosuccinate 33% (m/m) (Na DOSS), glycerin, and magnesium stearate were European Pharmacopeia grade and purchased from Merck (Darmstadt, Germany); octanol, cyclohexane, ethyl acetate, chloroform, and sodium hydroxide were reagent grade, also from Merck; polysorbate 80, gelatin, lactose 200 mesh, micronized lactose (Microtose), and sodium starch glycolate (Explotab) were commercial products and received respectively from ICI-Europe (Everberg, Belgium), PB-Gelatines (Vilvoorde, Belgium), DMV (Veghel, The Netherlands), Meggle (Reitmehing, Germany), and Mendell (New York). Description of ApparatussThe design of the devised dissolution apparatus is shown in Figure 1. The apparatus is based on the assembly of the USP rotating paddle method. The main modification consists in the position of the material to be investigated, which is placed in a vertically mounted glass cylinder. This glass cylinder has the same dimensions as those used in the USP disintegration apparatus. The bottom is closed with thin filter paper (Bollore Technologies, Scaer, France, Standard Quality: NHE 12S). Positioned in the aqueous dissolution medium, the glass cylinder is moved up and down 30 times/min by the motor of the disintegration apparatus. Onto the shaft of the paddle a second two-blade mixing stirrer is attached and operated 2 cm above the paddle. General Procedure for Dissolution StudiessDissolution characteristics of artemisinin formulations were evaluated using the described dissolution apparatus. The volume of the organic phase was 200 mL and of the aqueous 800 mL of demineralized water. Artemisinin is a neutral compound unaffected by pH; therefore, water was used as dissolution fluid. Prior to the dissolution experiment, the aqueous and organic layers were equilibrated for 15 min. The paddle was positioned at the interface of the two liquids. The rotation
S0022-3549(96)00115-3 CCC: $12.00
© 1996, American Chemical Society and American Pharmaceutical Association
+
+
Table 1sComposition of Four Very Fast Dissolving Experimental Formulations
Table 2sComposition of Tablets Quantity (mg)
Formula (mg) Compound
A
B
C
D
Artemisinin powder (<0.25 mm) Tween 80 Sodium lauryl sulfate Sodium dioctyl sulfosuccinate Micronized lactose
250 30
250
250
250
30 30
ART powder (<0.25 mm) Lactose 200 mesh Na DOSS 33% (m/m) Sodium starch glycolate Magnesium stearate
M7
M8
M9
200 60 20 5 1
200 40 5 25 1
200 60 20 25 3
30 Quantity (%; m/m)
speed was kept at 100 rpm and the temperature of the dissolution liquid at 37 °C ( 0.5 °C. During the dissolution study 5 mL samples were taken periodically from the aqueous and/or organic layer and replaced with the same volume of solvent. Samples were determined for artemisinin dissolved after appropriate dilution with 96% ethanol, and the cumulative amount of drug released at time ti was calculated from the sample assays using eq 1:
mg dissolved ti )
Asti 1% A1cm
x)i-1
fVv +
Asti
∑A x)2
1% 1cm
fVs
(1)
1% where Asti is the absorbance of the sample at time ti, A1cm denotes the absorbance coefficient, f is the dilution factor, Vv is the volume of dissolution medium in the vessel, and Vs denotes the sample volume taken at time ti. The results reported are mean values of three experimental runs. Artemisinin AssaysConcentrations of artemisinin were measured according to the method described by Zhao and Zeng,8 in which artemisinin is transformed into a UV absorbing compound, with an absorbance maximum at 290 nm, by heating the solution with sodium hydroxide. After appropriate dilution and heating for 30 min at 50 °C with 0.2% sodium hydroxide, the concentration of artemisinin was determined spectrophotometrically at 290 nm using an absorbance coef1% ficient A1cm of 593. Sensitivity was 0.03 mg/mL. Interference of excipients could be excluded by comparison of the UV spectrum of the sample with that obtained for pure artemisinin. Solubility DeterminationsThe solubility of artemisinin in different media has been determined by shaking 10 mL of the liquid with an excess of artemisinin powder during 48 h at 37 °C. After saturation, the solution was filtered immediately through filter paper (5971/2, Schleicher & Schuell) and the artemisinin concentration determined. Preparation of Very Fast Dissolving Experimental FormulationssTwo possibilities to maximize the dissolution rate of artemisinin powder were employed: wetting with a surfactant and diluting the drug with a large amount of hydrophilic diluent. The compositions of the experimental formulations are listed in Table 1. Artemisinin powder was intensively mixed with the surfactant solution in 50% ethanol or with an aqueous solution of lactose. The wet mass was dried at room temperature and used as such. Tablet PreparationsFor a very hydrophobic compound such as artemisinin, wet granulation was chosen for the preparation of tablets. The compositions are listed in Table 2. Artemisinin powder was sieved on a 0.25 mm sieve and mixed with lactose and Na DOSS for 10 min in a small mixer (Philips Chopper Shaker HR 1392) with a rectangular mixing blade. The binder solution containing the glycerin and gelatin was added slowly, and the wetted mass was thoroughly mixed for 5 min until granules were formed. The granulated mass was dried in an oven at 40 °C and then mixed with sodium starch glycolate and magnesium stearate. An instrumented tableting machine (KORSCH MP1, Berlin, Germany) was used for compression allowing the adjustment of compression forces in order to produce tablets with similar crushing strength (5.0 kP). Test Condition VariablessOctanol-cyclohexane (1:1), ethyl acetate, and chloroform were tested as potential organic phases. The high density of chloroform made it impossible to immerse the powder or tablet in the aqueous phase by placing it on the bottom of the round-bottom dissolution vessel. Therefore several attempts were used to keep the tablet in the aqueous phase only. First the solid
Gelatin Glycerin
M7
M8
M9
0.7 0.13
0.7 0.13
1.8 0.45
Table 3sSolubility (mg/mL) of Artemisinin in Different Organic Solvents at 37 °C (n ) 3) Octanol
Cyclohexane
Ethyl Acetate
Chloroform
28.1 ± 2.17
3.56 ± 0.84
116.00 ± 23.17
> 500
Figure 2sAmount (expressed as percentage of solubility) of ART, present in the aqueous phase after release from dissolving model A: 9 ) ethyl acetate−water (mean ± 2.04); 2 ) octanol−cyclohexane−water (mean ± 3.56). dosage form was encased in a paper filter bag, which was fastened to the shaft of the rotating paddle and hung in the aqueous phase. To maximize the liquid exchange through the filter bag, a stainless steel cylinder was inserted inside the filter bag. Various rotational speeds, 50, 100, 130 and 150 rpm, were applied to the paddle; speeds of 130 and 150 rpm disrupt the interfacial layers excessively.
Results and Discussion Optimization of Experimental ConditionssA very fast dissolving model (formula A), containing solely artemisinin powder mixed with approximately 11% polysorbate 80, was used to verify test conditions (nature of organic phase, rotational speed, and so on) required to ensure that sink conditions are maintained with all formulations. Experiments published in the literature1,8 describe the advantages of the use of an organic phase as sink for the drug dissolved. The solubility of artemisinin in different organic solvents (Table 3) was determined in order to evaluate their dissolving ability for artemisinin. The results shown in Figure 2 demonstrate that the following experimental conditions are not capable of maintaining sink conditions in the aqueous phase with octanolcyclohexane or ethyl acetate as organic sink: temperature, 37 °C ( 0.5 °C; ratio of organic phase to water, 2:8; total volume of dissolution medium, 1 L; rotational speed, 100 rpm.
Journal of Pharmaceutical Sciences / 1061 Vol. 85, No. 10, October 1996
+
Chloroform is a very good solvent for artemisinin (Table 3), but because of its high density, it becomes impossible to immerse the powder or the tablet in the water phase by placing it on the bottom of the dissolution vessel. Therefore, the original experimental setup described by Gibaldi and Feldman8 needed some modifications. A water permeable paper filter bag, hanging in the upper aqueous layer, was used to keep the product in contact with the water and away from the chloroform phase. At a speed of 100 rpm and with the dosage form inside the filter bag, sink conditions in the water phase were always maintained, but dissolution proceeded very slowly. Therefore different agitating conditions of the dosage form were investigated (see test condition variables). Since the results remained unsatisfactory, the filter bag was replaced by a glass cylinder with dimensions identical to those prescribed for the glass cylinders of the USP disintegration apparatus. The bottom of this glass cylinder was covered with filter paper, and the dosage form was put inside. This cylindrical device was attached to the shaft of the disintegration apparatus and was raised and lowered through the water phase by the motor at a speed of 30 times/min. Figure 1 illustrates this system of dissolution testing. Control of the Sink Conditions in the Water Phase of the Dissolution System Water-ChloroformsGibaldi and Feldman8 tested the dissolution rate of benzoic acid and salicylic acid by the partition method using octanol-cyclohexane as a reservoir and showed that sink conditions were present if the total dose of drug to be studied represents less than 20% of its solubility in the selected volume of organic phase. The data of Figure 2 prove that, in the case of artemisinin, sink conditions could not be obtained in the initial phase of the dissolution test when octanol, cyclohexane, or ethyl acetate was used, although the total amount of artemisinin present is below the 20% limit stipulated by these authors. The equations they derived prove that, in the presence of sink conditions, dissolution processes follow the same kinetics in both organic and aqueous phases on the condition that the rate of transfer from the aqueous dissolution medium into the organic phase exceeds the release rate in the aqueous phase. For the adjustment of the experimental conditions, the ratio of these two rates is at least as important as the total amount of drug present in the dosage form. Gibaldi and Feldman used a nondisintegrating flat-faced tablet of pure drug to obtain zero-order kinetics for the dissolution of benzoic acid and salicylic acid. However, with artemisinin, this experiment takes too much time when a nondisintegrating tablet is used. Instead, a highly concentrated solution of artemisinin in ethanol (250 mg dissolved in 25 mL of 95% ethanol) was pumped at a constant rate of 30 mg of artemisinin/h into the water phase. This rate will supply a total weight of 180 mg of artemisinin into the aqueous phase after 6 h while 90 mg is sufficient to saturate the aqueous phase based on the water volume used. According to the equations of Gibaldi and Feldman,8 the rate of appearance of artemisinin in the chloroform phase should also follow zero-order kinetics, if sink conditions in the water phase are maintained during the duration of the experiment. The data displayed in Figure 3 prove that the artemisinin appearance rate in the chloroform layer was constant with a constant supply of artemisinin into the aqueous phase. With normal tablets, the dissolution rate will not necessarily be zero order, since many formulation and technological factors can cause different types of dissolution kinetics from different tablet formulations. The amount of artemisinin released in the aqueous phase will necessarily not be constant as in the case of a nondisintegrating flat-faced tablet or the 1062 / Journal of Pharmaceutical Sciences Vol. 85, No. 10, October 1996
+
Figure 3sDissolution profile of ART in the chloroform layer under zero-order drug supply in the aqueous phase.
Figure 4sCumulative percentage in the chloroform layer of ART (mean ± 8.3), released from experimental formulations in a water−chloroform system; formula A ) 9, B ) 2, C ) 1, D ) b.
Figure 5sAmount (expressed as percentage of solubility) of ART (mean ± 4.3) in the aqueous layer, released from experimental formulations in a water− chloroform system; formula A ) 9, B ) 2, C ) 1, D ) b.
pumping of an ethanol solution at constant speed. If the release would take place according to first-order kinetics, the rate of change of the amount of drug remaining in the tablet (A) and dissolved in the aqueous phase (B) and the organic phase (C) may be expressed by the following equations:
dB/dt ) k1A - k2B
(2)
dA/dt ) -k1A
(3)
dC/dt ) k2B
(4)
+
+
Figure 6sDissolution profile of different tablets; formula M7 ) squares, M8 ) triangles, M9 ) circles; open symbols ) values in chloroform layer (mean ± 8.4), solid symbols ) values in water phase (mean ± 1.2).
If experimental conditions are such as to maintain sink conditions in the aqueous phase, a very small amount of artemisinin will be found in the water phase. This amount may be considered essentially constant so that the stationary state or steady state approximation10 can be assumed. This gives
trations in the aqueous phase were always largely below the 10-20% limit.
dB/dt ≈ 0 and k1A ) k2B
The partition method using chloroform as a reservoir for the amount of drug dissolved, described in this report, seems to be an excellent method for the determination of the rate of dissolution of artemisinin from solid oral dosage forms. The results obtained during our study of the optimization11 of the formulation of artemisinin tablets confirm this conclusion. In addition, the method could guarantee sink conditions for all very fast dissolving experimental formulations tested. In fact, the existence of a solid oral dosage formulation with a dissolution rate faster than that of formulation A is most improbable.
and, according to eqs 3 and 4,
-dA/dt ) dC/dt The rate of decrease of the amount of artemisinin in the tablet can be considered almost identical to the rate of appearance of artemisinin in the organic phase, which can correctly reflect the release rate from the tablet into the aqueous phase. To validate the partition method using chloroform as an organic sink, the system was experimentally tested using very fast dissolving experimental formulations (Table 1). Figure 4 shows the cumulative percentage of artemisinin in the chloroform layer. The amount present in the aqueous phase is illustrated in Figure 5. Formulation A had a very high dissolution rate: about 90 mg of artemisinin dissolved in the water phase and passed into the chloroform phase in the first hour and about 170 mg after 2 h, while the solubility of artemisinin in 800 mL of water is only 90.4 mg at 37 °C and a saturation time of 28 h is required. The most important finding is that during the first hour the artemisinin concentration in the aqueous phase is below 20% of the solubility with the very rapidly dissolving formulation A and always less than 10% with formulations B, C, and D. From the second hour on, the concentration of artemisinin in the water phase decreases very fast and sink conditions no longer pose any problem. Dissolution of TabletssThe dissolution profiles of different tablets obtained with the described method are presented in Figure 6. When tested with the classical paddle method or the flow-through cell, sink conditions7 were not present in the dissolution medium. From the data displayed in the lower part of Figure 6 it can be concluded that artemisinin concen-
Conclusion
References and Notes 1. Banakar, U. V. Pharmaceutical dissolution testing, 1st ed.; Marcel Dekker Inc.: New York, 1992. 2. Bruce, L. J.; Chwatt. Br. Med. J. 1982, 284, 767-768. 3. China cooperative Research Group on Qinghaosu and its derivatives as antimalarials, Clinical studies on the treatment of malaria with qinghaosu. J. Tradit. Chin. Med. 1982, 2, 45-50. 4. Dutta, G. P.; Bajpai, R.; Vishwakarma, R. A. Chemotherapy 1989, 35, 200. 5. Hien, T. T.; White, N. J. Lancet 1993, 341, 603-608. 6. Woerdenbag, H. J.; Pras, N.; Van Uden, W.; Wallaart, T. E.; Beekman, A. C.; Lugt, C. B. Pharm. World. Sci. 1994, 16, 1691807. 7. Ngo, T. H.; Michoel, A.; Kinget, R. Int. J. Pharm., accepted. 8. Gibaldi, M.; Feldman, S. J. Pharm. Sci. 1967, 56, 1238-1242. 9. Zhao, S. S.; Zeng, M. Y. Planta Med. 1985, 3, 233-237. 10. Frost, A. A.; Pearson, R. G. Kinetics and mechanism, 2nd ed.; John Wiley & Sons Inc.: New York, 1961. 11. Ngo, T. H.; Kinget, R. Int. J. Pharm., submitted.
JS960115U
Journal of Pharmaceutical Sciences / 1063 Vol. 85, No. 10, October 1996
+
Design and Evaluation of Two-Phase Partition−Dissolution Method and Its Use in Evaluating Artemisinin Tablets NGO THU HOA
AND
R. KINGETX
Received March 6, 1996, from the Laboratory of Galenical and Clinical Pharmacy, Catholic University Leuven, Onderwijs en Navorsing, Gasthuisberg, 3000 Leuven, Belgium. Final revised manuscript received June 17, 1996. Accepted for publication July 29, 1996X. Abstract 0 A method is described for the assessment of the dissolution behavior of solid dosage forms with a high content of a very water insoluble drug. In this method the dissolved drug is continuously extracted from the aqueous dissolution medium into an organic phase. The reported data on the dissolution of artemisinin show that this method guarantees sink conditions in the aqueous dissolution medium during the total duration of the experiment.
Introduction There is no doubt that the determination of dissolution rates is an important tool in the development, evaluation, and control of solid dosage forms. In the design of a reliable in vitro dissolution test model that can positively characterize the role of the dissolution process on drug bioavailability, it is essential that the model be capable of simulating test conditions that are likely to prevail during the in vivo dissolution process. One such condition is the maintenance of sink conditions during in vitro dissolution; i.e., the drug concentration in the dissolution medium does not exceed 1020% of solubility. Slightly and very slightly soluble drugs give rise to difficulties in this respect; with highly dosed drugs the problems become even more severe. A large number of authors have attempted different methods to achieve this objective.1 The types of systems employed for maintaining sink conditions are (a) large fixed fluid volume, (b) multiple phase (partition method), and (c) continuous flow (through flow cell). Studies on the bioavailability of drugs from a given dosage form revealed that in many situations various dosage forms with the same content of the active compound did not give the same therapeutic effect.1 So, control of the bioavailability of drugs is a major requirement in drug production, especially for drugs of very low water solubility. Artemisinin, a sesquiterpene lactone with an endoperoxide bridge, isolated from the Chinese traditional medicinal plant Artemisia annua L., Asteraceae, has received considerable attention in the past decade because of its antimalarial activity.2-6 It is a rapidly acting blood schizontocide with good activity against chloroquine resistant and multidrug resistant strains of Plasmodium parasites. It is effective in the treatment of cerebral malaria. Tablets and capsules, containing 250 mg of artemisinin per unit, are commercially available. It has been demonstrated that when the official dissolution methods are used, sink conditions do not prevail during the duration of the test for some artemisinin formulations7 tested. The objective of this study was the development of a suitable dissolution method for artemisinin solid oral dosage forms. By using an organic phase into which the drug under investigation would almost completely partition, Gibaldi and Feldman8 were able to continuously remove solute from the dissolution medium. Because of its simplicity of design and X
Abstract published in Advance ACS Abstracts, September 1, 1996.
1060 / Journal of Pharmaceutical Sciences Vol. 85, No. 10, October 1996
Figure 1sSchematic of the dissolution test system: (1) water bath; (2) thermostat; (3) round-bottom vessel, 1 L; (4) shaft; (5) mixing blade; (6) paddle; (7) glass cylinder; (8) filter paper; (9) disintegration apparatus.
its ability to guarantee sink conditions, this method was chosen as a basis for further application on solid dosage forms containing a high dose of a water insoluble drug.
Experimental Section MaterialssArtemisinin (ART) with a purity of 99.87% was received from Mediplantex (Hanoi, Vietnam); sodium lauryl sulphate (SLS), sodium dioctyl sulfosuccinate 33% (m/m) (Na DOSS), glycerin, and magnesium stearate were European Pharmacopeia grade and purchased from Merck (Darmstadt, Germany); octanol, cyclohexane, ethyl acetate, chloroform, and sodium hydroxide were reagent grade, also from Merck; polysorbate 80, gelatin, lactose 200 mesh, micronized lactose (Microtose), and sodium starch glycolate (Explotab) were commercial products and received respectively from ICI-Europe (Everberg, Belgium), PB-Gelatines (Vilvoorde, Belgium), DMV (Veghel, The Netherlands), Meggle (Reitmehing, Germany), and Mendell (New York). Description of ApparatussThe design of the devised dissolution apparatus is shown in Figure 1. The apparatus is based on the assembly of the USP rotating paddle method. The main modification consists in the position of the material to be investigated, which is placed in a vertically mounted glass cylinder. This glass cylinder has the same dimensions as those used in the USP disintegration apparatus. The bottom is closed with thin filter paper (Bollore Technologies, Scaer, France, Standard Quality: NHE 12S). Positioned in the aqueous dissolution medium, the glass cylinder is moved up and down 30 times/min by the motor of the disintegration apparatus. Onto the shaft of the paddle a second two-blade mixing stirrer is attached and operated 2 cm above the paddle. General Procedure for Dissolution StudiessDissolution characteristics of artemisinin formulations were evaluated using the described dissolution apparatus. The volume of the organic phase was 200 mL and of the aqueous 800 mL of demineralized water. Artemisinin is a neutral compound unaffected by pH; therefore, water was used as dissolution fluid. Prior to the dissolution experiment, the aqueous and organic layers were equilibrated for 15 min. The paddle was positioned at the interface of the two liquids. The rotation
S0022-3549(96)00115-3 CCC: $12.00
© 1996, American Chemical Society and American Pharmaceutical Association
+
+
Table 1sComposition of Four Very Fast Dissolving Experimental Formulations
Table 2sComposition of Tablets Quantity (mg)
Formula (mg) Compound
A
B
C
D
Artemisinin powder (<0.25 mm) Tween 80 Sodium lauryl sulfate Sodium dioctyl sulfosuccinate Micronized lactose
250 30
250
250
250
30 30
ART powder (<0.25 mm) Lactose 200 mesh Na DOSS 33% (m/m) Sodium starch glycolate Magnesium stearate
M7
M8
M9
200 60 20 5 1
200 40 5 25 1
200 60 20 25 3
30 Quantity (%; m/m)
speed was kept at 100 rpm and the temperature of the dissolution liquid at 37 °C ( 0.5 °C. During the dissolution study 5 mL samples were taken periodically from the aqueous and/or organic layer and replaced with the same volume of solvent. Samples were determined for artemisinin dissolved after appropriate dilution with 96% ethanol, and the cumulative amount of drug released at time ti was calculated from the sample assays using eq 1:
mg dissolved ti )
Asti 1% A1cm
x)i-1
fVv +
Asti
∑A x)2
1% 1cm
fVs
(1)
1% where Asti is the absorbance of the sample at time ti, A1cm denotes the absorbance coefficient, f is the dilution factor, Vv is the volume of dissolution medium in the vessel, and Vs denotes the sample volume taken at time ti. The results reported are mean values of three experimental runs. Artemisinin AssaysConcentrations of artemisinin were measured according to the method described by Zhao and Zeng,8 in which artemisinin is transformed into a UV absorbing compound, with an absorbance maximum at 290 nm, by heating the solution with sodium hydroxide. After appropriate dilution and heating for 30 min at 50 °C with 0.2% sodium hydroxide, the concentration of artemisinin was determined spectrophotometrically at 290 nm using an absorbance coef1% ficient A1cm of 593. Sensitivity was 0.03 mg/mL. Interference of excipients could be excluded by comparison of the UV spectrum of the sample with that obtained for pure artemisinin. Solubility DeterminationsThe solubility of artemisinin in different media has been determined by shaking 10 mL of the liquid with an excess of artemisinin powder during 48 h at 37 °C. After saturation, the solution was filtered immediately through filter paper (5971/2, Schleicher & Schuell) and the artemisinin concentration determined. Preparation of Very Fast Dissolving Experimental FormulationssTwo possibilities to maximize the dissolution rate of artemisinin powder were employed: wetting with a surfactant and diluting the drug with a large amount of hydrophilic diluent. The compositions of the experimental formulations are listed in Table 1. Artemisinin powder was intensively mixed with the surfactant solution in 50% ethanol or with an aqueous solution of lactose. The wet mass was dried at room temperature and used as such. Tablet PreparationsFor a very hydrophobic compound such as artemisinin, wet granulation was chosen for the preparation of tablets. The compositions are listed in Table 2. Artemisinin powder was sieved on a 0.25 mm sieve and mixed with lactose and Na DOSS for 10 min in a small mixer (Philips Chopper Shaker HR 1392) with a rectangular mixing blade. The binder solution containing the glycerin and gelatin was added slowly, and the wetted mass was thoroughly mixed for 5 min until granules were formed. The granulated mass was dried in an oven at 40 °C and then mixed with sodium starch glycolate and magnesium stearate. An instrumented tableting machine (KORSCH MP1, Berlin, Germany) was used for compression allowing the adjustment of compression forces in order to produce tablets with similar crushing strength (5.0 kP). Test Condition VariablessOctanol-cyclohexane (1:1), ethyl acetate, and chloroform were tested as potential organic phases. The high density of chloroform made it impossible to immerse the powder or tablet in the aqueous phase by placing it on the bottom of the round-bottom dissolution vessel. Therefore several attempts were used to keep the tablet in the aqueous phase only. First the solid
Gelatin Glycerin
M7
M8
M9
0.7 0.13
0.7 0.13
1.8 0.45
Table 3sSolubility (mg/mL) of Artemisinin in Different Organic Solvents at 37 °C (n ) 3) Octanol
Cyclohexane
Ethyl Acetate
Chloroform
28.1 ± 2.17
3.56 ± 0.84
116.00 ± 23.17
> 500
Figure 2sAmount (expressed as percentage of solubility) of ART, present in the aqueous phase after release from dissolving model A: 9 ) ethyl acetate−water (mean ± 2.04); 2 ) octanol−cyclohexane−water (mean ± 3.56). dosage form was encased in a paper filter bag, which was fastened to the shaft of the rotating paddle and hung in the aqueous phase. To maximize the liquid exchange through the filter bag, a stainless steel cylinder was inserted inside the filter bag. Various rotational speeds, 50, 100, 130 and 150 rpm, were applied to the paddle; speeds of 130 and 150 rpm disrupt the interfacial layers excessively.
Results and Discussion Optimization of Experimental ConditionssA very fast dissolving model (formula A), containing solely artemisinin powder mixed with approximately 11% polysorbate 80, was used to verify test conditions (nature of organic phase, rotational speed, and so on) required to ensure that sink conditions are maintained with all formulations. Experiments published in the literature1,8 describe the advantages of the use of an organic phase as sink for the drug dissolved. The solubility of artemisinin in different organic solvents (Table 3) was determined in order to evaluate their dissolving ability for artemisinin. The results shown in Figure 2 demonstrate that the following experimental conditions are not capable of maintaining sink conditions in the aqueous phase with octanolcyclohexane or ethyl acetate as organic sink: temperature, 37 °C ( 0.5 °C; ratio of organic phase to water, 2:8; total volume of dissolution medium, 1 L; rotational speed, 100 rpm.
Journal of Pharmaceutical Sciences / 1061 Vol. 85, No. 10, October 1996
+
Chloroform is a very good solvent for artemisinin (Table 3), but because of its high density, it becomes impossible to immerse the powder or the tablet in the water phase by placing it on the bottom of the dissolution vessel. Therefore, the original experimental setup described by Gibaldi and Feldman8 needed some modifications. A water permeable paper filter bag, hanging in the upper aqueous layer, was used to keep the product in contact with the water and away from the chloroform phase. At a speed of 100 rpm and with the dosage form inside the filter bag, sink conditions in the water phase were always maintained, but dissolution proceeded very slowly. Therefore different agitating conditions of the dosage form were investigated (see test condition variables). Since the results remained unsatisfactory, the filter bag was replaced by a glass cylinder with dimensions identical to those prescribed for the glass cylinders of the USP disintegration apparatus. The bottom of this glass cylinder was covered with filter paper, and the dosage form was put inside. This cylindrical device was attached to the shaft of the disintegration apparatus and was raised and lowered through the water phase by the motor at a speed of 30 times/min. Figure 1 illustrates this system of dissolution testing. Control of the Sink Conditions in the Water Phase of the Dissolution System Water-ChloroformsGibaldi and Feldman8 tested the dissolution rate of benzoic acid and salicylic acid by the partition method using octanol-cyclohexane as a reservoir and showed that sink conditions were present if the total dose of drug to be studied represents less than 20% of its solubility in the selected volume of organic phase. The data of Figure 2 prove that, in the case of artemisinin, sink conditions could not be obtained in the initial phase of the dissolution test when octanol, cyclohexane, or ethyl acetate was used, although the total amount of artemisinin present is below the 20% limit stipulated by these authors. The equations they derived prove that, in the presence of sink conditions, dissolution processes follow the same kinetics in both organic and aqueous phases on the condition that the rate of transfer from the aqueous dissolution medium into the organic phase exceeds the release rate in the aqueous phase. For the adjustment of the experimental conditions, the ratio of these two rates is at least as important as the total amount of drug present in the dosage form. Gibaldi and Feldman used a nondisintegrating flat-faced tablet of pure drug to obtain zero-order kinetics for the dissolution of benzoic acid and salicylic acid. However, with artemisinin, this experiment takes too much time when a nondisintegrating tablet is used. Instead, a highly concentrated solution of artemisinin in ethanol (250 mg dissolved in 25 mL of 95% ethanol) was pumped at a constant rate of 30 mg of artemisinin/h into the water phase. This rate will supply a total weight of 180 mg of artemisinin into the aqueous phase after 6 h while 90 mg is sufficient to saturate the aqueous phase based on the water volume used. According to the equations of Gibaldi and Feldman,8 the rate of appearance of artemisinin in the chloroform phase should also follow zero-order kinetics, if sink conditions in the water phase are maintained during the duration of the experiment. The data displayed in Figure 3 prove that the artemisinin appearance rate in the chloroform layer was constant with a constant supply of artemisinin into the aqueous phase. With normal tablets, the dissolution rate will not necessarily be zero order, since many formulation and technological factors can cause different types of dissolution kinetics from different tablet formulations. The amount of artemisinin released in the aqueous phase will necessarily not be constant as in the case of a nondisintegrating flat-faced tablet or the 1062 / Journal of Pharmaceutical Sciences Vol. 85, No. 10, October 1996
+
Figure 3sDissolution profile of ART in the chloroform layer under zero-order drug supply in the aqueous phase.
Figure 4sCumulative percentage in the chloroform layer of ART (mean ± 8.3), released from experimental formulations in a water−chloroform system; formula A ) 9, B ) 2, C ) 1, D ) b.
Figure 5sAmount (expressed as percentage of solubility) of ART (mean ± 4.3) in the aqueous layer, released from experimental formulations in a water− chloroform system; formula A ) 9, B ) 2, C ) 1, D ) b.
pumping of an ethanol solution at constant speed. If the release would take place according to first-order kinetics, the rate of change of the amount of drug remaining in the tablet (A) and dissolved in the aqueous phase (B) and the organic phase (C) may be expressed by the following equations:
dB/dt ) k1A - k2B
(2)
dA/dt ) -k1A
(3)
dC/dt ) k2B
(4)
+
+
Figure 6sDissolution profile of different tablets; formula M7 ) squares, M8 ) triangles, M9 ) circles; open symbols ) values in chloroform layer (mean ± 8.4), solid symbols ) values in water phase (mean ± 1.2).
If experimental conditions are such as to maintain sink conditions in the aqueous phase, a very small amount of artemisinin will be found in the water phase. This amount may be considered essentially constant so that the stationary state or steady state approximation10 can be assumed. This gives
trations in the aqueous phase were always largely below the 10-20% limit.
dB/dt ≈ 0 and k1A ) k2B
The partition method using chloroform as a reservoir for the amount of drug dissolved, described in this report, seems to be an excellent method for the determination of the rate of dissolution of artemisinin from solid oral dosage forms. The results obtained during our study of the optimization11 of the formulation of artemisinin tablets confirm this conclusion. In addition, the method could guarantee sink conditions for all very fast dissolving experimental formulations tested. In fact, the existence of a solid oral dosage formulation with a dissolution rate faster than that of formulation A is most improbable.
and, according to eqs 3 and 4,
-dA/dt ) dC/dt The rate of decrease of the amount of artemisinin in the tablet can be considered almost identical to the rate of appearance of artemisinin in the organic phase, which can correctly reflect the release rate from the tablet into the aqueous phase. To validate the partition method using chloroform as an organic sink, the system was experimentally tested using very fast dissolving experimental formulations (Table 1). Figure 4 shows the cumulative percentage of artemisinin in the chloroform layer. The amount present in the aqueous phase is illustrated in Figure 5. Formulation A had a very high dissolution rate: about 90 mg of artemisinin dissolved in the water phase and passed into the chloroform phase in the first hour and about 170 mg after 2 h, while the solubility of artemisinin in 800 mL of water is only 90.4 mg at 37 °C and a saturation time of 28 h is required. The most important finding is that during the first hour the artemisinin concentration in the aqueous phase is below 20% of the solubility with the very rapidly dissolving formulation A and always less than 10% with formulations B, C, and D. From the second hour on, the concentration of artemisinin in the water phase decreases very fast and sink conditions no longer pose any problem. Dissolution of TabletssThe dissolution profiles of different tablets obtained with the described method are presented in Figure 6. When tested with the classical paddle method or the flow-through cell, sink conditions7 were not present in the dissolution medium. From the data displayed in the lower part of Figure 6 it can be concluded that artemisinin concen-
Conclusion
References and Notes 1. Banakar, U. V. Pharmaceutical dissolution testing, 1st ed.; Marcel Dekker Inc.: New York, 1992. 2. Bruce, L. J.; Chwatt. Br. Med. J. 1982, 284, 767-768. 3. China cooperative Research Group on Qinghaosu and its derivatives as antimalarials, Clinical studies on the treatment of malaria with qinghaosu. J. Tradit. Chin. Med. 1982, 2, 45-50. 4. Dutta, G. P.; Bajpai, R.; Vishwakarma, R. A. Chemotherapy 1989, 35, 200. 5. Hien, T. T.; White, N. J. Lancet 1993, 341, 603-608. 6. Woerdenbag, H. J.; Pras, N.; Van Uden, W.; Wallaart, T. E.; Beekman, A. C.; Lugt, C. B. Pharm. World. Sci. 1994, 16, 1691807. 7. Ngo, T. H.; Michoel, A.; Kinget, R. Int. J. Pharm., accepted. 8. Gibaldi, M.; Feldman, S. J. Pharm. Sci. 1967, 56, 1238-1242. 9. Zhao, S. S.; Zeng, M. Y. Planta Med. 1985, 3, 233-237. 10. Frost, A. A.; Pearson, R. G. Kinetics and mechanism, 2nd ed.; John Wiley & Sons Inc.: New York, 1961. 11. Ngo, T. H.; Kinget, R. Int. J. Pharm., submitted.
JS960115U
Journal of Pharmaceutical Sciences / 1063 Vol. 85, No. 10, October 1996