International Journal of Pharmaceutics 321 (2006) 27–34
`
`Crystal modification of dipyridamole using different solvents
`and crystallization conditions
`R. Adhiyaman a,1, Sanat Kumar Basu b,∗
`
`a Department of Pharmaceutics, Bapatla College of Pharmacy, Bapatla, Andhra Pradesh, India
`b Division of Pharmaceutics, Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India
`Received 21 March 2005; received in revised form 10 April 2006; accepted 17 April 2006
`Available online 13 May 2006
`
`Abstract
`
`Dipyridamole crystals having different types of habits, improved dissolution rate were prepared by recrystallization from selected solvents, such
`as acetonitrile, benzene and methanol (Method I); crystals have also been made by solvent change using methanolic solution of dipyridamole in
`the presence of 2% solutions of Tween-80, Povidone K30 and polyethylene glycol (PEG) 4000 (Method II). Scanning electron microscopy, X-ray
`powder diffractometry, IR spectrometry and differential scanning calorimetry were used to investigate the physicochemical characteristics of the
`crystals. The comparative dissolution behavior of the newly developed crystals and that of the untreated dipyridamole were also studied. It was
`found that the newly developed crystals were different from each other with respect to physical properties but are chemically identical. The crystals,
`obtained (Method I) from benzene and acetonitrile, produced needle shaped crystals and that obtained from methanol produced rectangular shaped
`crystals. But the crystals obtained (Method II) with the methanolic solution of the drug in the presence of Tween-80, Povidone K30 and PEG-4000
`produced smooth needle shaped crystals. X-ray diffraction spectra and differential scanning calorimetry study of the newly developed crystals,
`clearly indicate that dipyridamole exist in different crystal modification. The dissolution rate of newly developed crystals was found to be greater
`◦
`than the pure drug dipyridamole. Stability studies at 40
`C (75% RH) for 1 month for the modified crystals as well as the pure drug did show some
`changes in the XRD and DSC but not in IR studies.
`© 2006 Elsevier B.V. All rights reserved.
`
`Keywords: Dipyridamole; Recrystallization; Physicochemical characterization
`
`1. Introduction
`
`Different physiological and formulation factors are responsi-
`ble for the bioavailability of drug from the dosage form. One of
`the most important physical factors, which affect the bioavail-
`ability and therapeutic efficacy of drug, is the existence of active
`ingredients in various crystal forms having different internal
`structure and physical properties (Kapoor et al., 1998). The dif-
`ferent crystal form of a drug have different physicochemical
`characteristics, namely crystal shape, crystal size, melting point,
`density, flow properties solubility pattern, dissolution character-
`istics and XRD pattern, though they are chemically identical.
`A physical form having improved dissolution rate and solubil-
`
`∗
`
`Corresponding author.
`E-mail addresses: eskebee@yahoo.com (R. Adhiyaman),
`basusanat kumar@hotmail.com (S.K. Basu).
`1 Present address: J.S.S. College of Pharmacy, The Rocklands, Ootacamund-
`643001, Tamil Nadu, India.
`
`0378-5173/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
`doi:10.1016/j.ijpharm.2006.04.021
`
`ity is useful for improving the bioavailability of a drug (Burt
`and Mitchell, 1980; Watanable et al., 1982). The crystal habit is
`an important variable in pharmaceutical manufacturing, where
`some factors, such as the polarity of crystallization solvent and
`the presence of impurities in the solvent, affect crystallization
`(Chow et al., 1985; Femi-Oyewo and Spring, 1994; Garekani
`et al., 2000). Among them, solvent strongly affects the habit
`of crystalline materials; however, the role-played by solvent
`interactions in enhancing or inhibiting crystal growth is still
`not completely understood (Lahra and Leiserowitz, 2001). The
`drug dipyridamole used herein is practically insoluble in water.
`Its main use in therapy as antiplatelet aggregating and periph-
`eral vasodilating effect is well known. But the water insolubility
`and the poor bioavailability are the limitations of its effec-
`tive use clinically. Keeping this in view, crystal modification
`of dipyridamole has been undertaken to improve dissolution
`and bioavailability. Dipyridamole is a derivative of 1,3,5,7-tetra
`azanaphthalene and used mainly for cardiovascular diseases
`for the above-mentioned purposes. It has been recrystallized
`
`
` P. 1
`
`UT Ex. 2030
`SteadyMed v. United Therapeutics
`IPR2016-00006
`
`

`
`28
`
`R. Adhiyaman, S.K. Basu / International Journal of Pharmaceutics 321 (2006) 27–34
`
`from selected solvents and solvent system. The newly devel-
`oped dipyridamole crystals were characterized by some physic-
`ochemical approaches.
`
`stub (with double side adhesive tape) and coated under vacuum
`with gold in an argon atmosphere prior to observation.
`
`2. Materials and methods
`
`2.1. Materials
`
`Dipyridamole was obtained as generous gift from German
`Remedies (Mumbai, India). The solvents used for the present
`work were acetone, benzene, methanol, obtained from Ran-
`baxy Chemical Laboratories (S.A.S. Nagar, India) and Tween-
`80, Povidone K30 and polyethylene glycol (PEG) 4000 were
`obtained from SDS Chemical Limited (Boisar, India).
`
`2.2. Preparation of dipyridamole crystals
`
`Two different methods used in this study to observe the effect
`of solvents on the development of crystal habits in the changed
`environment are given below.
`
`2.2.1. Method I
`One gram of dipyridamole was dissolved separately in 50 ml
`of selected solvents in a conical flask. The solution was heated at
`the boiling point of the respective solvents and filtered, concen-
`◦
`trated and the solution was left at room temperature (28–30
`C)
`until the solvent was completely evaporated. The crystals were
`further dried under vacuum at room temperature and stored in
`appropriate airtight container for further use.
`
`2.2.2. Method II
`One gram of dipyridamole was dissolved in 40 ml of
`methanol in a conical flask and the solution was heated and fil-
`◦
`tered. The resultant solution was concentrated at 60
`C and then
`◦
`cooled down at room temperature (28–30
`C). The clear solu-
`tion, thus obtained, was rapidly added to equal volume of cold
`◦
`water (5
`C) containing 2% solution of Tween-80, PVP K30 and
`PEG-4000, separately under agitation by means of a glass rod
`◦
`and then left for 1 h at 10–15
`C. The crystals were then recov-
`ered by filtration under vacuum using a sintered glass funnel.
`They were then kept in airtight container for further use.
`
`2.3. Stability studies
`
`One month’s accelerated stability test was carried out for
`each sample after preparation, when the crystals were kept in
`◦
`humidity chambers (75% RH) and at a temperature 40
`C and
`the physicochemical changes of the crystals as observed are
`compared with that of the drug dipyridamole under identical
`conditions. The results are summarized in Figs. 9 (XRD) and
`10 (DSC), respectively.
`
`2.4. Scanning electron microscopy
`
`Electron micrograph of crystals was obtained using a
`scanning electron microscope (JEOL JSM—5200) operating
`between 5 and 24 kV. The specimens were mounted on a metal
`
`2.5. X-ray powder diffraction
`
`The cavity of the metal sample holder of X-ray diffractometer
`was filled with ground sample powder and then smoothed out
`with a spatula. X-ray diffraction pattern of dipyridamole crys-
`tals were obtained using the X-ray diffractometer (Rich Seifert
`Model 3000P) at 30 kV, 30 mA over a range of 10–100 2θ, using
`Cu K␣ radiation wavelength 1.5405 ˚A.
`
`2.6. Infrared spectroscopy
`
`The spectra were recorded on an IR spectrophotometer
`(PERKIN-ELMER USA MODEL—248), after respective sam-
`ples were mixed with dried KBr powder and compressed to a
`12 mm disc by a hydraulic press at 10 tonnes compression for
`30 s.
`
`2.7. Thermal analysis
`
`Differential scanning calorimetry (DSC) of the samples,
`10 mg, was carried out using a thermal analysis system (MET-
`TLER TA 4000 System). Calibration with standard was under-
`taken prior to subjecting the samples, which were heated at
`◦
`C/min in an aluminum pan under a nitrogen atmosphere and
`10
`a similar empty pan was used as the reference. The instrument
`automatically calculated onsets of melting points and enthalpy
`of fusion.
`
`2.8. Dissolution studies
`
`Dipyridamole and its crystals, 25 mg in each case were accu-
`rately weighed and dissolution profile of the drug was deter-
`◦
`mined in a USP Type II Dissolution test apparatus at 37
`C,
`with basket (100 mesh) with a stirring speed of 50 rpm. The
`dissolution medium was 600 ml of phosphate buffer pH 4.0,
`I.P. (Indian Pharmacopoeia). Samples were withdrawn from the
`dissolution vessels at selected time intervals and analyzed for
`dipyridamole content at 285 nm on a UV spectrophotometer
`(BECKMAN-UM-64). The results are shown as the graphical
`plots in Figs. 7 and 8, respectively.
`
`3. Results and discussion
`
`3.1. Morphology of crystals
`
`Fig. 1 shows the scanning electron micrographs (SEM) of
`untreated and recrystallized dipyridamole from different sol-
`vents under solvent evaporation method (Method I). It is clear
`from the figure that the untreated dipyridamole is having small
`irregular needle shaped crystals (Fig. 1d), whereas the crystals
`obtained from acetonitrile is needle shaped (Fig. 1c) and that
`from benzene is rod shaped (Fig. 1b). Recrystallization of dipyri-
`damole from methanol solution with the same method produced
`
`
` P. 2
`
`UT Ex. 2030
`SteadyMed v. United Therapeutics
`IPR2016-00006
`
`

`
`R. Adhiyaman, S.K. Basu / International Journal of Pharmaceutics 321 (2006) 27–34
`
`29
`
`Fig. 2. Scanning electron micrographs of dipyridamole recrystallized from
`methanol with 2% solutions of (a) Tween-80 (SCT); (b) PEG-4000 (SCPEG);
`(c) PVPK30 (SCPVP).
`
`3.2. X-ray diffraction
`
`To obtain information on the physicochemical characteristics
`of the prepared crystals, X-ray powder diffraction measurements
`were conducted.
`XRD spectra for all crystals are presented in Figs. 3 and 4.
`In the powder diffractogram sharp peak at diffraction angle
`(2θ) 30.04, 20.74, 20.81, 12.33, 17.45, 10.25, and 20.93 were
`obtained in case of drug dipyridamole and the modified crys-
`tals obtained from methanol, benzene, acetonitrile, Tween-80,
`PEG-400, PVP K30, respectively. The presences of these sharp
`peaks are clearly evident in the comparative diffractogram pre-
`sented in Figs. 3 and 4 and the data recorded therein. From the
`data recorded, it is clearly evident that there is significant differ-
`ence in the entire diffraction pattern or d-spacing values between
`treated and untreated dipyridamole samples. The intensity of the
`peak in methanol is the highest than that of all other modified
`crystals reported herein. This is probably due to higher crystal
`
`Fig. 1. Scanning electron micrographs of dipyridamole recrystallized from (a)
`methanol, (b) benzene, (c) acetonitrile and (d) untreated dipyridamole.
`
`rectangular needle shaped crystals (Fig. 1a), while using sol-
`vent change method (Method II), the shape of crystals changes
`to fine needles (Fig. 2a–c). The results also showed that the size
`of crystals produced from Methods I and II are somewhat dif-
`ferent from the size of untreated dipyridamole and follows the
`order, i.e. Method I > Method II (compare the magnification of
`the SEM in Figs. 1 and 2). Therefore, it can be concluded that
`cooling rate decreases the crystal size due to incomplete growth
`of large number of small crystals (Garekani et al., 1999).
`
`
` P. 3
`
`UT Ex. 2030
`SteadyMed v. United Therapeutics
`IPR2016-00006
`
`

`
`30
`
`R. Adhiyaman, S.K. Basu / International Journal of Pharmaceutics 321 (2006) 27–34
`
`Fig. 3. X-ray powder diffraction pattern of pure dipyridamole and dipyridamole
`recrystallized from methanol; benzene; acetonitrile.
`
`Fig. 4. X-ray powder diffraction pattern of pure dipyridamole and dipyridamole
`recrystallized from methanol with 2% solutions of Tween-80 (SCT); PEG-4000
`(SCPEG); PVP K30 (SCPVP).
`
`
` P. 4
`
`UT Ex. 2030
`SteadyMed v. United Therapeutics
`IPR2016-00006
`
`

`
`R. Adhiyaman, S.K. Basu / International Journal of Pharmaceutics 321 (2006) 27–34
`
`31
`
`Fig. 6. Differential Scanning Calorimetric thermographs of dipyridamole
`recrystalized from methanol with 2% solution of (a) Tween-80(SCT); (b) PEG-
`4000 (SCPEG); (c) PVP K30 (SCPVP).
`
`perfection in this condition of crystallization (Nokhodchi et al.,
`2003).
`
`3.3. Infrared spectroscopy
`
`The spectra of all modified crystals were identical and the
`main absorption bands of dipyridamole appeared in all of the
`spectra. This indicates that there were no difference between the
`internal structure and conformations of these samples, because
`these were not associated with changes at molecular level.
`
`3.4. Thermal analysis
`
`The DSC data for drug dipyridamole (untreated) and the mod-
`ified crystals are shown in Figs. 5 and 6. It should be noted that
`the DSC thermo grams (Figs. 5 and 6) of all modified crys-
`tals showed only slight variation. However, the modified crystal
`
`Fig. 5. Differential scanning calorimetric thermographs of dipyridamole recrys-
`talized from (a) methanol; (b) benzene; (c) acetonitrile; (d) untreated dipyri-
`damole.
`
`
` P. 5
`
`UT Ex. 2030
`SteadyMed v. United Therapeutics
`IPR2016-00006
`
`

`
`32
`
`R. Adhiyaman, S.K. Basu / International Journal of Pharmaceutics 321 (2006) 27–34
`
`cations were observed during recrystallization of dipyridamole
`under various conditions of the crystallization.
`
`3.5. Dissolution studies
`
`The dissolution profile of dipyridamole and its modified crys-
`tals from different solvents are shown in Figs. 7 and 8, respec-
`tively.
`Recrystallization of the parent drug from various solvents,
`given earlier (Method I), resulted in the increase of the dissolu-
`tion rate of different modified crystals than dipyridamole. Espe-
`cially, crystals obtained from benzene and acetonitrile, show
`higher dissolution rate than untreated dipyridamole because of
`the better crystallinity of the modified crystals in these cases.
`Crystals obtained using only methanol show lower dissolution
`rate than other crystals obtained (Method II). However, it is
`evident that after the addition of Tween-80 and other polymer
`solution, the dissolution rates were increased. This may be due to
`
`Fig. 7. Dissolution profile of pure Dipyridamole and modified crystals obtained
`using various solvents in phosphate buffer pH 4.0. (I.P). (a) Methanol; (b) ben-
`zene; (c) acetonitrile; (d) untreated dipyridamole.
`
`obtained from methanol shows significant changes due to high
`crystal perfection.
`The DSC curve of crystals from SCT (2%, v/v) and methanol
`shows broad exothermic peaks and very slight but insignificant
`variation in transition temperature and a little difference (not
`significant) in enthalpy of fusion. This may be due to oxidation
`or phase transformation. Crystals obtained by using acetoni-
`trile, benzene, SCPVP (2%, w/v) and SCPEG (2%, w/v) show
`a weak endothermic peak and there is no significant variation
`in transition temperature, but significant difference in enthalpy
`of fusion is observed in case of acetonitrile, SCPEG (2%, w/v)
`and SCPVP (2%, w/v) while compared with the thermo gram
`obtained in case of benzene. The appearance of weak endother-
`mic peaks in this case may be due to solvation of the crystals
`(Gordon and Chow, 1992).
`Results from IR spectroscopy, X-ray diffraction analysis and
`DSC taken together led to the conclusion that only habit modifi-
`
`Fig. 8. Dissolution profile of modified crystals of dipyridamole from methanol
`and also from methanol with 2% solutions of PEG-4000, PVP K30, and Tween-
`80 in phosphate buffer pH 4.0. (I.P). (a) Methanol; (b) Tween-80 (SCT); (c)
`PEG-4000 (SCPEG); (d) PVP K30 (SCPVP).
`
`Fig. 9. Comparative X-ray powder diffraction pattern of pure dipyridamole and
`dipyridamole recrystallized from acetonitrile; benzene; methanol and dipyri-
`damole recrystallized from methanol with 2% solutions of PEG-4000 (SCPEG),
`◦
`PVPK30 (SCPVP), Tween-80 (SCT) and kept at elevated temperature (40
`C)
`and 75% RH for one month.
`
`
` P. 6
`
`UT Ex. 2030
`SteadyMed v. United Therapeutics
`IPR2016-00006
`
`

`
`R. Adhiyaman, S.K. Basu / International Journal of Pharmaceutics 321 (2006) 27–34
`
`33
`
`is clearly evident from the DSC thermo grams for all the sam-
`ples including pure dipyridamole under investigation that the
`modified crystals (Methods I and II) showed slight change in
`the value of enthalpy and the heat of fusion. However, the DSC
`curve of crystals from SCT (2%, v/v) and PEG (2%, w/v), very
`weak exothermic peaks were seen in a position significantly
`different from the samples, studied under ambient conditions,
`leading to significant variation in transition temperature and in
`enthalpy of fusion. This may probably be due to oxidation or
`phase transformation under such stress condition. But it is very
`much interesting to note that none of the samples studied under
`such stress condition did show any change in the IR spectrum
`confirming the presence of its chemical identity.
`
`4. Conclusion
`
`In conclusion, it can be said that the crystallization conditions
`and the medium used have major effect on dipyridamole crys-
`tals habit modification under ambient conditions. The crystals
`showed significant changes in the shape, size, melting points,
`dissolution rate, XRD patterns and DSC curves. This suggests
`that the newly developed crystals of dipyridamole under ambi-
`ent conditions exist in different crystalline modification facil-
`itating significantly improved dissolution rate as compared to
`dipyridamole. There are enough references (Dalton et al., 2001;
`el-Yazigi and Sawchuk, 1985) available in the literature wherein
`it has been proved that in vitro dissolution data are good predic-
`tor of in vivo performance in reality. Therefore, it can be safely
`concluded that the improvement obtained in the present study in
`the modified crystals will give better bioavailability and better
`therapeutic activity clinically. But the stability study undertaken
`◦
`C and a relative humidity of 75% shows some physical
`at 40
`changes probably due to some phase transitions but retaining the
`chemical identity. The effect of such changes in reality needs to
`be explored in actual situations, if any.
`
`Acknowledgements
`
`The authors thank Indian Association for the Cultivation of
`Science, Kolkata, India; Bengal Engineering and Science Uni-
`versity, Shibpur, Howrah, India; University Science and Instru-
`mentation Centre, Jadavpur University, Kolkata, India for their
`help during instrumental analysis of samples.
`
`References
`
`Burt, H.M., Mitchell, A.G., 1980. Effect of habit modification on dissolution
`rate. Int. J. Pharm. 5, 239–251.
`Chow, A.H.L., Chow, P.K.K., Wang, Z., Grant, D.G.W., 1985. Modifica-
`tion of acetaminophen crystals; influence of growth in aqueous solutions
`containing.p-aceto oxyacetanilibe on crystal properties. Int. J. Pharm. 23,
`239–258.
`Dalton, J.T., Straughn, A.B., Dickason, D.A., Grandolfi, G.P., 2001. Predictive
`ability of level A in vitro–in vivo correlation for ringcap controlled-release
`acetaminophen tablets. Pharm. Res. 18, 1729–1734.
`el-Yazigi, A., Sawchuk, R.J., 1985. In vitro–in vivo correlation and dissolution
`studies with oral theophylline dosage forms. J. Pharm. Sci. 74, 161–164.
`Femi-Oyewo, M.N., Spring, M.S., 1994. Studies on paracetamol crystals pro-
`duced by growth in aqueous solutions. Int. J. Pharm. 112, 17–28.
`
`Fig. 10. Comparative Differential scanning calorimetric thermographs of pure
`dipyridamole and dipyridamole recrystallized from acetonitrile; benzene;
`methanol and dipyridamole recrystallized from methanol with 2% solutions
`of PEG-4000 (SCPEG), PVPK30 (SCPVP), Tween-80 (SCT) and kept under
`◦
`elevated temperature (40
`C) and 75% RH for one month.
`
`the adsorption of surfactant and polymers on the crystal surface
`(Majumdar et al., 1992).
`
`3.6. Stability studies
`
`The results obtained in the stability test showed slight changes
`in XRD, DSC data for all samples under investigation. XRD
`spectra for all the crystals kept at the elevated temperature
`◦
`(40
`C) are presented in Fig. 9. In the powder diffractogram of
`dipyridamole and the modified crystals, sharp peak at diffraction
`angle (2θ), 20.98, 20.79 and 44.25, respectively were obtained
`incase of drug dipyridamole and crystals from acetonitrile and
`benzene. But in the case of crystals obtained from methanol
`(Method I) and other crystals obtained (Method II), all of them
`showed the sharp peak at diffraction angle (2θ), 44.5. These
`clearly indicate that under the circumstance all retain the same
`state. However, there is significant difference in the d-spacing
`values between the freshly prepared crystals and the crystals
`obtained after storing at elevated temperature including pure
`drug. This is probably due to the existence of different crystal
`habits in the crystalline materials at elevated temperature. The
`DSC data for drug dipyridamole (untreated) and the modified
`crystals kept at elevated temperature are shown in Fig. 10. It
`
`
` P. 7
`
`UT Ex. 2030
`SteadyMed v. United Therapeutics
`IPR2016-00006
`
`

`
`34
`
`R. Adhiyaman, S.K. Basu / International Journal of Pharmaceutics 321 (2006) 27–34
`
`Garekani, H.A., Ford, J.L., Rubinstein, M.H., Rajabi-Siah-boomi, A.P., 1999.
`Formation and compression characteristics of prismatic polyhedral crystal
`and thin plate like crystals of paracetamol. Int. J. Pharm. 187, 77–89.
`Garekani, H.A., Ford, J.L., Rubinstein, M.H., Rajabi-Siah-boomi, A.P., 2000.
`Highly compressible paracetamol; compression properties. Int. J. Pharm.
`208, 101–110.
`Gordon, J.D., Chow, A.H.L., 1992. Modification of phenytoin crystals:
`influence of 3-propanolozymethyl-5,5-diphenly-hydantoin on solution-
`phase crystallization and related crystal properties. Int. J. Pharm. 79,
`171–181.
`
`Kapoor, A., Majumdar, D.K., Yadav, M.R., 1998. Crystal forms of nimesulide
`– a sulfonanilide (non-steriodal anti inflammatory drug). Indian J. Chem.
`37B, 572–575.
`Lahra, M., Leiserowitz, L., 2001. The effect of solvent on crystal growth and
`morphology. Chem. Eng. Sci. 56, 2245–2253.
`Nokhodchi, A., Bolourtchian, W., Dinarvand, R., 2003. Crystal modification of
`phenytoin using different solvents and crystallization conditions 250, 85–
`97.
`Watanable, A., Yamaoka, Y., Takada, K., 1982. Crystal habits and dissolution
`behavior of aspirin. Chem. Pharm. Bull. 30, 2958–2963.
`
`
` P. 8
`
`UT Ex. 2030
`SteadyMed v. United Therapeutics
`IPR2016-00006