Dissolution test results showing biorelevant media made from SIF Powder Original is the same as media prepared using methylene chloride Michael Schaich, Bastian Kloefer, Mathew Leigh : info@biorelevant.com Date: 31 January 13 Document Version: 1.2 Dissolution Summary Dissolution tests of five commercially available drug products were carried out in FaSSIF, FeSSIF and FaSSGF made from SIF Powder Original and in FaSSIF, FeSSIF and FaSSGF prepared using methylene chloride (dichloromethane). The similarity of the dissolution profiles in media produced in the two different ways was assessed statistically using F 2 values as proposed by the FDA. The dissolution profiles of each drug product in biorelevant media made by either method were found to be equivalent. 1. Background The conventional method of preparing biorelevant media involves forming a methylene chloride emulsion and the evaporation of the solvent in a rotavap. SIF Powder Original is an instant powder that makes the three biorelevant media FaSSIF (Fasted State Simulated Intestinal Fluid), FeSSIF (Fed State Simulated Intestinal Fluid) and FaSSGF (Fasted State Simulated Gastric Fluid) by adding the powder to the appropriate buffer (for FaSSIF or FeSSIF) or solution (for FaSSGF). 2. Objective Determine the dissolution profile of five commercially available (immediate release) formulated drugs studied by Galia et al. i using FaSSIF, FeSSIF and FaSSGF prepared using methylene chloride Determine the dissolution profile of five commercially available (immediate release) formulated drugs studied by Galia et al using FaSSIF, FeSSIF, FaSSGF made from SIF Powder Original (www.biorelevant.com) Statistically compare the release profiles of the two methods using the F 2 factor 13 biorelevant.com Page 1 of 15
13 biorelevant.com Page 2 of 15
3. Materials 3.1. Ingredients for making FaSSIF, FeSSIF and FaSSGF from SIF Powder Original Ingredient Batch Number Manufacturer/Supplier SIF Powder Original PHA S 113 1/7 biorelevant.com, UK Sodium dihydrogen phosphate dihydrate 41611195 Carl Roth, Germany Sodium hydroxide BCBC791 Fluka, Germany Sodium chloride 71383 Fluka, Germany Acetic acid glacial 41641 Carl Roth, Germany HCl 1N SZBBV Sigma Aldrich, Germany HCl >32% SZBB95V Sigma Aldrich, Germany Water (deionized) NA NA 3.2. Ingredients for preparing FaSSIF, FeSSIF and FaSSGF using methylene Component Batch Number Manufacturer/Supplier Lecithin (phospholipid) 79637 Lipoid, Germany Sodium taurocholate 71275 Prodotti Chimici, Italy Methylene chloride (Dichloromethane) P7G11117G SDS, France Sodium dihydrogen phosphate dihydrate 41611195 Carl Roth, Germany Sodium hydroxide BCBC791 Fluka, Germany Sodium chloride 71383 Fluka, Germany Acetic acid glacial 41641 Carl Roth, Germany HCl 1N SZBBV Sigma Aldrich, Germany HCl >32% SZBB95V Sigma Aldrich, Germany Water (deionized) NA NA 3.3. Formulated Drugs tested Product Name Drug Batch Number Manufacturer/Supplier Paracetamol tablets 5mg Paracetamol PAK29B21 Boots Pharmaceuticals, UK Lopresor 1mg Metoprolol 162392 Daiichi-Sankyo, Switzerland Nizoral mg Ketoconazole CBL1Z Janssen-Cilag, UK Mefanamin Pfizer 25mg Mefenamic Acid 13129 Pfizer, Switzerland Danazol Capsules 1mg Danazol 174489A Mylan, UK 13 biorelevant.com Page 3 of 15
4. Method 4.1. Making FaSSIF, FeSSIF, FaSSGF from SIF Powder Original The desired biorelevant medium was made by dissolving the appropriate amount of SIF Powder Original in the recommended buffer (for FaSSIF or FeSSIF) or NaCl/HCl solution (for FaSSGF). To make other volumes, use the FaSSIF, FeSSIF, FaSSGF calculator on our website. 4.2. Preparing FaSSIF, FeSSIF and FaSSGF using methylene chloride FaSSIF, FeSSIF and FaSSGF were made using methylene chloride used by Galia et al ii and described by Marques iii. The taurocholate is dissolved in about 5 ml of the blank buffer. After the taurocholate dissolves completely a freshly prepared solution of lecithin in methylene chloride is added. From the resultant emulsion, the methylene chloride is removed using a rotary evaporator ( C for 15 min at 25 mbar followed by 15 minutes at 1 mbar). Finally, the dispersion is made up to a volume of 2 L with the appropriate buffer. 4.3. Content analysis using HPLC-UV The following in house developed HPLC method was used: HPLC-Type: 1 Series, Agilent Eluent A: Water +.1 % formic acid Eluent B: Acetonitrile +.1 % formic acid Column: SunFire C18, 5 x 4.6 mm, 3.5 µm Column temperature: C Flow rate: 1.2 ml/min Standard solution diluent: Acetonitrile/Water 3:1 v/v Detection wavelength used for each drug Drug Paracetamol Metopropolol tartrate Danazol Mefanamic acid Ketoconazole Detection 245nm 222nm 285nm 2nm 2nm 13 biorelevant.com Page 4 of 15
Gradient used: Time (min) Eluent B (%) 1 4. 95 6. 95 6.1 1 8.5 1 The HPLC analyses for drug content were performed on the same sample (n=1) in triplicate. 4.4. Sample preparation for HPLC analysis All samples were removed from the middle of the vessel using a 2 ml syringe. The samples were filtered through a.22 µm PVDF filter after discarding the first µl to prevent the effect of possible adsorbed drug at the filter. 4.5. Dissolution in FaSSIF, FeSSIF and FaSSGF All drug products tested were immediate release formulations. The dissolution profile of each product was determined in a USP 2 apparatus (Pharmatest PTWS3) using the same parameters as described by Galia et al. All studies used 5 ml of degassed FaSSIF, FeSSIF and FaSSGF (made using either SIF Powder Original or preparing using methylene chloride) and were carried out at 37 C±.5 C. Dissolution in each media was carried out in triplicate. The dissolution parameters for each drug product are described in Table 1. Table 1 Dissolution parameters used Drug Rotations Per Minute (RPM) Sample time points (minutes) Paracetamol tablets 1 5, 1, 15,, 3, Metopropolol tartrate tablets 1 5, 1, 15,, 3, Danazol capsules 1 5, 1,, 3,, 9 Mefanamic acid capsules 5 5, 1, 15,, 3, Ketoconazole tablets 1 5, 1, 15,, 3,, 1 The parameters used were selected based on Galia et al. Japanese sinkers were used for both capsule formulations whereas the tablet formulations were dropped into the corresponding vessel. The dissolution profiles of the five drug products in FaSSGF, FaSSIF and FeSSIF made from SIF Powder Original and prepared using methylene chloride (MC) are shown in Figures 1-5 with the F 2 Value stated in the top left of each Figure. The standard deviations are expressed with error bars. 13 biorelevant.com Page 5 of 15
Drug released [%] Figure 1. Dissolution profile of Paracetamol in FaSSIF made from SIF Powder Original and prepared using methylene chloride Paracetamol (Tablets 5mg) 1 8 F2 = 89. 1 3 5 FaSSIF SIF Powder FaSSIF MC Figure 2. Dissolution profile of Paracetamol in FeSSIF made from SIF Powder Original and prepared using methylene chloride Paracetamol (Tablets 5mg) 1 8 F2 = 87.2 1 3 5 FeSSIF SIF Powder FeSSIF MC 13 biorelevant.com Page 6 of 15
Drug released [%] Figure 3. Dissolution profile of Paracetamol in FaSSGF made from SIF Powder Original and prepared using methylene chloride Paracetamol (Tablets 5mg) 1 8 F2 = 82.7 1 3 5 FaSSGF SIF Powder FaSSGF SIF MC Figure 4. Dissolution profile of Metoprolol in FaSSIF made from SIF Powder Original and prepared using methylene chloride Metoprolol (Lopressor 1mg) 1 8 F2 = 97.4 1 3 5 FaSSIF SIF Powder FaSSIF MC 13 biorelevant.com Page 7 of 15
Drug released [%] Figure 5. Dissolution profile of Metoprolol in FeSSIF made from SIF Powder Original and prepared using methylene chloride Metoprolol (Lopressor 1mg) 1 8 F2 = 92.2 1 3 5 FeSSIF SIF Powder FeSSIF MC Figure 6. Dissolution profile of Metoprolol in FaSSGF made from SIF Powder Original and prepared using methylene chloride Metoprolol (Lopressor 1mg) 1 8 F2 = 72.1 1 3 5 FaSSGF SIF Powder FaSSGF MC 13 biorelevant.com Page 8 of 15
Drug released [%] Figure 7. Dissolution profile of Danazol in FaSSIF made from SIF Powder Original and prepared using methylene chloride Danazol (Capsules 1mg) 1 8 F2 = 94.4 1 3 5 FaSSIF SIF Powder FaSSIF MC Figure 8. Dissolution profile of Danazol in FeSSIF made from SIF Powder Original and prepared using methylene chloride Danazol (Capsules 1mg) 1 8 F2 = 93.3 1 3 5 FeSSIF SIF Powder FeSSIF MC 13 biorelevant.com Page 9 of 15
Drug released [%] Figure 9. Dissolution profile of Danazol in FaSSGF made from SIF Powder Original and prepared using methylene chloride Danazol (Capsules 1mg) 1 8 F2 = 1. 1 3 5 FaSSGF SIF Powder FaSSGF MC Figure 1. Dissolution profile of Mefenamic acid in FaSSIF made from SIF Powder Original and prepared using methylene chloride Mefenamic Acid (Mefenamin 25mg) 1 8 F2 = 94.6 1 3 5 FaSSIF SIF Powder FaSSIF MC 13 biorelevant.com Page 1 of 15
Drug released [%] Figure 11. Dissolution profile of Mefenamic acid in FeSSIF made from SIF Powder Original and prepared using methylene chloride Mefenamic Acid (Mefenamin 25mg) 1 8 F2 = 98.2 1 3 5 FeSSIF SIF Powder FeSSIF MC Figure 12. Dissolution profile of Mefenamic acid in FaSSGF made from SIF Powder Original and prepared using methylene chloride Mefenamic Acid (Mefenamin 25mg) 1 8 F2 = 1. 1 3 5 FaSSGF SIF POWDER FaSSGF MC 13 biorelevant.com Page 11 of 15
Drug released [%] Figure 13. Dissolution profile of Ketoconazole in FaSSIF made from SIF Powder Original and prepared using methylene chloride Ketoconazole (Nizoral mg) 1 8 F2 = 98.3 1 3 5 FaSSIF SIF Powder FaSSIF MC Figure 14. Dissolution profile of Ketoconazole in FeSSIF made from SIF Powder Original and prepared using methylene chloride Ketoconazole (Nizoral mg) 1 8 F2 = 76.4 1 3 5 FeSSIF SIF Powder FeSSIF MC 13 biorelevant.com Page 12 of 15
Figure 15. Dissolution profile of Ketoconazole in FaSSGF made from SIF Powder Original and prepared using methylene chloride Ketoconazole (Nizoral mg) 1 8 F2 =.8 1 3 5 FaSSGF SIF Powder FaSSGF MC 5. Results In Table 1 below, the F 2 values comparing the dissolution profiles of each of the five drugs in the same biorelevant media (FaSSIF, FeSSIF or FaSSGF) made from SIF Powder Original and prepared using methylene chloride are presented. The purpose of using the F 2 value iv in this study is to compare the similarity of the two preparation methods. An F 2 value above 5 indicates that two profiles are similar and differ by less than 1% v Table 1 F 2 values for comparing the dissolution profiles of the five drugs in FaSSIF, FeSSIF and FaSSGF made from SIF Powder vs prepared using methylene chloride Drug F 2 value FaSSIF (SIF Powder vs methylene chloride) F 2 value FeSSIF(SIF Powder vs methylene chloride) F 2 value FaSSGF(SIF Powder vs methylene chloride) Paracetamol tablets 89. 87.2 82.7 Metopropolol tartrate tablets 97.4 92.2 72.1 Danazol capsules 94.4 93.3 1. Mefanamic acid capsules 94.6 98.2 1. Ketoconazole tablets 98.3 76.4.8 6. Discussion 13 biorelevant.com Page 13 of 15
6.1. Comparison of the methods From the Figures 1 to 15, it can be seen that the method of making biorelevant media FaSSIF, FeSSIF and FaSSGF does not significantly affect the dissolution of the five drugs tested. SIF Powder Original gave equivalent solubility results compared to the same type of media prepared using methylene chloride. The F 2 values comparing the two different methods were always greater than 5 indicating that there was less than 1% difference between the two methods. 6.2. Paracetamol and Metoprolol tartrate dissolution The dissolution profile in the same type of media was the same, irrespective of whether the FaSSIF, FeSSIF or FaSSGF was made from SIF Powder Original or prepared using methylene chloride. For FaSSIF, FeSSIF and FaSSGF the F 2 values obtained using the two different methods were always much greater than 5, which clearly suggests there was much less than % difference between the two methods for the five formulated drugs tested. The release of paracetamol from the tablet formulation was complete within about minutes in FaSSIF, FeSSIF and FaSSGF made using either method. This complete dissolution shows that this drug is highly water soluble in simulated fasted gastric and intestinal fluids as well as fed state intestinal fluid. Metoprolol tartrate release was slightly slower compared to paracetamol but the dissolution of this salt was complete within 3 minutes in FaSSIF, FeSSIF and FaSSGF. For FaSSIF, FeSSIF and FaSSGF the F 2 values from the two different methods were always greater than 5, which indicates there was less than 1% difference between the two methods for the five formulated drugs tested. 6.3. Danazol and Mefenamic acid dissolution For the dissolution of danazol and mefenamic acid in FaSSIF and FeSSIF, the F 2 values obtained from the two different methods were always greater than 5 which indicates there was less than 1% difference between the two methods for these two formulated drugs. The release for these two drugs in all three biorelevant media after 9 minutes was less than 15%. These results reflected the comparative low water solubility of these two drugs. The three fold higher solubility of danazol in FeSSIF compared to FaSSIF is due to the lipophilic properties of this drug, which has a log P of about 4.5. More of this fatty drug dissolves in the bile salts of the fed state. This is also reflected in vivo by the fact that danazol bioavailability is higher in the fed state vi compared to the fasted state. 13 biorelevant.com Page 14 of 15
6.4. Ketoconazole dissolution For the dissolution of ketoconazole in FaSSIF and FeSSIF the F 2 values obtained from the two different methods were always greater than 5 which indicates there was less than 1% difference between the two methods for the five formulated drugs tested. The release of ketoconazole in FaSSIF (fasted state) after 2 hours was less than 5%. In contrast, the release of ketoconazole was 1% in FaSSGF and about 8% in FeSSIF after 2 hours. This is explained by the fact the drug is a lipophilic base that dissolves in acidic conditions to form a water soluble HCl salt and is also soluble in the fed state. This is why medicines that increase the gastric ph can lower the absorption of this basic drug because less drug goes into solution for absorption in the intestinal fluids. The dissolution profiles in all three media were not affected by the method by which the specific biorelevant media were made. 7. Conclusion The dissolution profiles for the five drug products tested in FaSSIF, FaSSGF or FeSSIF made by either preparation method showed no significant difference. This means for dissolution testing, the three biorelevant media (FaSSGF, FaSSIF and FeSSIF) made from SIF Powder Original and by using methylene chloride were the same. i Galia E, Nicolaides E, Hörter D, Löbenberg R, Reppas C, Dressman JB. Evaluation of various dissolution media for predicting in vivo performance of class I and II drugs. Pharm Res. 1998 May;15(5):698-75. ii Personal communication with Prof. Jennifer Dressman iii Margareth Marques Dissolution Media Simulating Fasted and Fed States Dissolution Technologies, May 4, p16 iv FDA. Guidance for Industry: Dissolution testing of immediate release solid oral dosage form. 1997 v http://www.dissolutiontech.com/dtresour/899art/dissprofile.html vi Sunesen VH, Vedelsdal R, Kristensen HG, Christrup L, Müllertz A. Effect of liquid volume and food intake on the absolute bioavailability of danazol, a poorly soluble drug. Eur J Pharm Sci. 5 Mar; 24(4):297-33 13 biorelevant.com Page 15 of 15