Crystal Habit, Characterization and Pharmacological Activity of Various Crystal Forms of Arteether

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provided by Elsevier - Publisher Connector Acta Pharmaceutica Sinica B 2011;1(2):129–135

Institute of Materia Medica, Chinese Academy of Medical Sciences Chinese Pharmaceutical Association

Acta Pharmaceutica Sinica B

www.elsevier.com/locate/apsb www.sciencedirect.com

ORIGINAL ARTICLE Crystal habit, characterization and pharmacological activity of various crystal forms of arteether

Renu Chadhaa,n, Sushma Guptaa, Geeta Shuklab

aInstitute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India bDepartment of Microbiology, Panjab University, Chandigarh 160014, India

Received 7 March 2011; revised 28 March 2011; accepted 30 March 2011

KEY WORDS Abstract The aim of this study was to investigate and characterize different crystal forms of arteether, a rapidly acting, highly potent antimalarial drug for the treatment of acute Plasmodium Arteether; Polymorphism; falciparum malaria. Six different crystal forms were prepared utilizing various polar and non-polar Solubility; solvents. Scanning electron microscopy (SEM) revealed differences in the surface characteristics of Antimalarial activity the six forms from those of a commercial sample. Differential scanning calorimetry (DSC) revealed the absence of a desolvation endotherm indicating that the forms were neither hydrates nor solvates. Powder X-ray diffraction (PXRD) patterns of the forms showed much weaker major peaks than in the commercial sample indicating them to be less crystalline. Solubility and dissolution studies showed that the most amorphous form was the most soluble and possessed the highest antimalarial activity.

nCorresponding author. Tel.: þ91 9316015096. E-mail address: [email protected] (Renu Chadha).

Peer review under responsibility of Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association. doi:10.1016/j.apsb.2011.06.010 130 Renu Chadha et al.

1. Introduction (156.6 1CwithDH of 25.45 J/g). A mass of 2–8 mg was placed in the aluminum pan, covered with a lid and sealed. DSC curves The majority of drugs are marketed as solid dosage forms were obtained under a nitrogen purge of 50 mL/min at a heating containing drug in a crystalline state1.Theformationofthis rate of 10 1C/min over the temperature range of 50–350 1C. crystalline state is subject to a phenomenon known as polymorphism whereby different crystal forms are produced depend- 2 2.2.2. Scanning electron microscopy ing on solvent, temperature and crystallization conditions . Thus, Photomicrographs of the different forms of arteether were the crystal habit of a drug is an important variable in pharma- obtained using a Jeol JSM-6100 scanning electron microscope. ceutical manufacturing. A number of basic physicochemical 3 Samples were mounted on a metal stub with an adhesive and properties such as solubility , dissolution rate and melting coated with gold under vacuum. behavior, and certain micromeritic properties or performance characteristics such as tablet compressibility, mechanical strength4 and powder flow depend on the crystal habit of a particular 2.2.3. X-ray powder diffraction drug5. The search for the crystal form with the best combination Powder diffraction patterns were recorded on an X-ray of solubility and stability6–8 with all its associated intellectual diffractometer (XPERT-PRO, PANalytical, Netherlands) with property issues9 is therefore of crucial importance in the devel- copper as tube anode. Diffractograms were recorded using opment of a new chemical entity and one of the most active areas voltage 40 kV, 35 mA, angular range 5 and fixed divergence of pharmaceutical research10. slit. Care was taken to avoid changes in crystal form during The physicochemical properties of a particular crystal form sample preparation. Samples (approximately 200 mg) were have an important effect on how it is processed into a drug loaded into the sample holder taking care to avoid any product. For instance, the solubility affects the dissolution preferred orientation of the crystals. rate and possibly the mass transport of a drug and therefore is an important factor in how the active drug is absorbed from 2.2.4. Fourier transform infrared spectrometry its dosage form11–13. Those who are involved in the industrial FT-IR spectra were obtained using a Mode Spectrum RXI production and marketing of a product line need to know not FT-IR spectrometer (Perkin Elmer, England) over the range only the exact nature of the material being processed but also 400–4000/cm. Dry KBr (50 mg) was finely ground in an agate its long-term stability and how its chemical and physical mortar after which the sample (1–2 mg) was added and gently properties are affected by its crystal form10. The aim of the mixed. A manual press was used to form pellets. present work was to investigate the different crystal forms of arteether, a poorly soluble drug (17 mg/mL at room tempera- 2.2.5. Dissolution study ture) used to treat cerebral malaria and as a second line 7 1 14–16 Dissolution studies were carried out for 4 h at 37 0.5 C treatment for chloroquine resistant malaria . Various using phosphate buffer (pH 7.0):methanol (2:1) (500 mL) as crystal forms were characterized in terms of their solubility dissolution media in a paddle apparatus-12 equipped with and dissolution behavior in order to select the form with paddles rotating at 50 rpm. After introducing an appropriate optimum physicochemical properties and in vivo pharmacolo- amount of sample, aliquots were withdrawn after 5, 10, 15, 20, gical activity. 25, 30, 45, 60, 90, 120, 150, 180, 240 and 300 min and analyzed by UV spectrometry at 240 nm using a calibration curve of 2. Materials and methods arteether in methanol:phosphate buffer (pH 7.0) (2:1). Each dissolution study was performed in duplicate batches. Arteether was generously provided by IPCA Laboratories, Mumbai, India. Analytical grade methanol, ethanol, dichlor- 2.2.6. Aqueous solubility determination omethane, DMSO, toluene and THF were obtained from An MSW-275 shaker (Macro Scientific Works, New Delhi) Sigma Aldrich, USA, and used as received. was used to measure aqueous solubility of the different forms of arteether. Solubility studies were performed by adding 2.1. Preparation of crystal forms 50 mg sample and methanol:phosphate buffer (pH 7.0) (2:1) and shaking at 37 1C for 24 h. Aliquots were removed, filtered Crystal forms I and VI were prepared by recrystallizing through a 0.45 mm membrane filter and analyzed by UV arteether from hot saturated solutions of dichloromethane spectrometry at 240 nm as described above. (DCM) and toluene, respectively. Forms II and III were prepared by adding hot water as antisolvent to saturated 2.2.7. Solution calorimetry solutions of drug in THF and methanol, respectively. Form IV Enthalpy of solution (DHsol) of the various crystal forms was was obtained by rotary evaporation of a solution of drug in determined in phosphate buffer (pH 7):acetonitrile (3:1) using ethanol at 80 1C under vacuum. Form V was prepared by an isoperibol solution calorimeter model 4300 (Calorimetry recrystallizing the drug from a mixture of DMSO:H2O (70:30). Science Corporation, Utah, USA). This is a semi-adiabatic calorimeter with temperature resolution after noise reduction 2.2. Characterization close to 1 mK corresponding to a heat resolution of 1–4 mJ in a 25 mL buffer (pH 7) reaction vessel. Details are given in our 2.2.1. Differential scanning calorimetry previous papers17,18. The performance of the system was DSC thermograms were obtained using a DSC Q20 (TA Instru- validated by showing that DHsol of potassium chloride ments-Waters LLC, USA). The calorimeter was calibrated for (17.301 kJ/mol) in triply distilled water was in good agreement temperature and heat flow using the melting point of pure indium with the known value of 17.322 kJ/mol. The precision of an Investigation and characterization of various crystal forms of arteether 131 individual measurement was better than 70.03 kJ/mol for (Fig. 1) indicating that none of the forms were a solvate. All three consecutive experiments. forms exhibited a broad melting endotherm between 62 and 83 1C with significant differences in their onsets and peaks and 2.3. In vivo studies a broad exotherm between 152 and 165 1C corresponding to decomposition. The positions of the decomposition peaks and Activity against infection with Plasmodium berghei (NK 65) was used as a measure of antimalarial activity in vivo. Experi- ments were performed according to the guidelines of the Committee for Control and Supervision of Experiments on Animals and approved by the Institutional Animal Ethics Committee, Panjab University, Chandigarh. BALB/c mice (age 4–5 weeks, weight 16–20 g) were provided with a standard pellet diet and water ad libitum prior to experiments. Animals were divided into 8 groups (n¼6 per group) and challenged with an intraperitoneal (ip) injection of 106 P. berghei infected red blood cells (RBCs) on day 1. They were then treated with a single dose of arteether (6 mg/kg) suspended in 100 mL 0.5% carboxymethylcellulose (CMC) or vehicle (control) twice a day on day 1 and for 7 days thereafter. The mean percent parasitemia (infected RBCs 100/total no. of RBCs) was mea- sured every alternate day for up to 30 days. On each occasion, a tail blood smear was taken, fixed in methanol and stained with Giemsa stain for counting of at least 500 cells.

2.4. Statistical analysis

Data are expressed as mean7standard deviation (SD). Para- sitemia of arteether was assessed by one-way ANOVA followed by Tukey’s test using Jandel Sigma Stat version 2.0. Differences were considered signiﬁcant at Po0.05.

3. Results and discussion

3.1. Differential scanning calorimetry

DSC was used to conﬁrm the identity of the various crystal forms and that there were no transformations during sample preparation. Thermochemical parameters are given in Table 1. The commercial drug sample showed a melting endotherm at 83.24 1C followed by a decomposition exotherm at 160.32 1C. Figure 1 DSC thermograms of the various crystal forms of DSC scans of the various forms showed the absence of a arteether: (a) commercial sample, (b) form I, (c) form II, (d) form desolvation peak in the initial region of the thermograms III, (e) form IV, (f) form V and (g) form VI.

Table 1 Thermochemical parameters (DSC), solubilities, heats of solution and FT-IR peaks of various crystal forms of arteether.

Sample Onset of Peak Enthalpy of Decomposition Enthalpy of Solubility Heat of FT-IR peaks temp. (1C) temp. fusion (J/g) temp. (1C) decomposition (mg/ solution (1C) (J/g) 10 mL) (kJ/mol) C–O–C C–H stretch stretch (cm1) (cm1)

Commercial 81.61 83.24 60.65 160.32 469.6 1.73 0.33 1192.9 2949.3 sample Form I 68.62 78.91 50.34 159.31 511.4 3.49 2.36 1202.4 2937.6 Form II 70.52 81.71 50.12 158.77 481.8 3.12 2.18 1207.3 2937.5 Form III 76.90 82.37 39.05 163.93 468.0 2.28 1.92 1205.8 2947.9 Form IV 80.81 84.42 58.12 156.11 514.3 2.19 1.18 1199.2 2946.7 Form V 63.28 70.47 19.70 157.84 499.1 4.54 5.04 1203.4 2937.0 Form VI 82.13 83.87 65.34 163.41 543.4 2.07 1.06 1202.6 2937.9 132 Renu Chadha et al. their enthalpies of decomposition were significantly different. with different external shapes but the same internal structure. A broad endotherm with little enthalpy of fusion was observed These differences in external shape include variation in size, the for form V suggesting it was the least crystalline form. relative development of certain faces and the kind and the number of faces. SEM of the six forms of arteether revealed distinct 3.2. Scanning electron microscopy morphological features (Fig. 2). The SEM of the commercial sample displayed prismatic crystals with thin flat sheets or flakes Differences in crystal habit normally arise due to differences in the peeling off the larger mass. Form I showed elongated fine environmental conditions of crystal growth, which lead to crystals crystalline plates resembling plumes. Form II showed pointed

Figure 2 SEM images of the various crystal forms of arteether.

Figure 3 PXRD of the various crystal forms of arteether: (a) commercial sample, (b) form I, (c) form II, (d) form III, (e) form IV, (f) form V and (g) form VI. Investigation and characterization of various crystal forms of arteether 133

needles and a ﬁlliform habit with thin pointed plates. Form III also showed plates with angular edges. Form IV displayed long, slender needle-like crystals. Form V showed an unorganized mass with no discernible crystallinity. Form VI displayed a reticulated habit with a lattice or network of small crystals. Rel. Int. (%/count)

3.3. Powder X-ray diffraction ) 1 y

Form VI (2 PXRD revealed that all forms were crystalline to various extents. There was no signiﬁcant difference in the position of the peak corresponding to 100% relative intensity (Fig. 3) but the relative intensity of the various major peaks (Table 2) indicates that all forms were less crystalline than the commercial sample. Rel. Int. (%/count) Crystallinity decreased in the following order: commercial sam- ple4form VI4form IV4form III4form II–form I4form V. ) 1 y Form V (2 3.4. Fourier transform infrared spectroscopy (FT-IR)

The various forms of arteether showed reproducible differences in the number and the position of absorption bands Rel. Int. (%/count) in the FT-IR spectra (Table 1) with average band shifts of 2–15/cm. Signiﬁcant differences in the regions 3000–2800/cm )

1 and 1000–1250/cm (Fig. 4) attributed to variation in the y

Form IV (2 arrangement of asymmetric C–H and C–O–C stretching vibrations indicate differences in intermolecular interactions in these regions. Rel. Int. (%/count) ) 1 y Form III (2 Rel. Int. (%/count) ) 1 y Form II (2 Rel. Int. (%/count) ) 1 y Form I (2 Rel. Int. (%/count) Relative intensity in terms of the number of counts of some major peaks in the PXRD of the various crystal forms of arteether. ) 1 y

9.0859.2378 39.047/15,766 9.2109 100/4.03,861 9.9883 100/2.95,271 1.57/4,642 9.1618 9.9632 100/89,670 9.210 4.55/4,076 9.9883 100/1.78,654 9.2255 1.57/2,676Figure 10.0275 100/1.97,257 9.2204 4.14/8,168 4 100/13,531FT-IR 9.1609 9.9879 46.67/6,315 100/57,548 spectra 9.9684 6.16/3.545 of the various crystal forms of arteether: Arteether (2 18.322318.4839 6.02/24,297 15.11/61,004 18.502 19.01 9.02/26,622 14.88/1,574 17.6262 18.4307 5.43/4,872 14.25/12,773 18.5023 19.7008 9.02/7,324 0.19/853 17.6533 18.4753 3.31/8,335 19.19/27,832 17.6776 18.468 36.86/4,987 17.24/2332 17.573 18.451 7.80/4,488 13.48/7,755

Table 2 (a) commercial sample, (b) form I, (c) form II, (d) form III, (e) form IV, (f) form V and (g) form VI. 134 Renu Chadha et al.

3.5. Solution calorimetry 3.6. Solubility studies

All forms exhibited exothermic dissolution behavior. Calcu- The solubility study shows that the most crystalline form lated values of DHsol are given in Table 1. DHsol of form V was (commercial sample) was the least soluble (1.73 mg/10 mL) and found to be the most negative (5.04 kJ/mol) indicating that the least crystalline form (form V) was the most soluble maximum ease of molecular release from the lattice and lowest (4.54 mg/10 mL) with a 2.62-fold increase in solubility (Table 2). crystallinity. DHsol of the commercial sample was found to be The order of increasing solubility is commercial sample4form the least negative (0.33 kJ/mol) indicating minimum ease of VI4form IV4form III4form II4form I4form V. release from the lattice (i.e. the largest amount of energy to break the lattice) and highest crystallinity. 3.7. Dissolution studies

The dissolution curves of the different forms showed differences in the rate of dissolution consistent with the orders of crystallinity, DHsol and solubility. The results of dissolution studies are presented in graphical form as mean percentage release vs. time (Figs. 5 and 6). The highest dissolution rate was found for the most soluble form (form V).

3.8. Antimalarial activity

It is clear from the data presented in Table 3 that arteether does not prevent mortality. However, it was found to increase survival time (15–19 days) compared to control (10 days) due to signiﬁcant reductions (Po0.001) in parasitemia. At the end of treatment (day 8), percent parasitemia (Table 3)wasinthe following order: control4commercial sample4form VI4form IV4form III4form II4form I4form V. Mortality at 30 days Figure 5 Dissolution proﬁles of the various crystal forms of was in the following order: control4form VI–commercial sam- arteether. ple4form IV4form II–form III4form V–form I.

Figure 6 Percent parasitemia observed in P. berghei infected mice at various times after treatment with the various crystal forms of arteether (std drug¼commercial sample; tests 1–6¼forms I–VI).

Table 3 Antimalarial activity of the various crystal forms of arteether against P. berghi infection in mice.

S. no. Group Treatment Mean % parasitemiaa % Mortality at 30 days (n¼6) on day 8 PI

1 Control group 0.5% CMC solution 59.6873.57 100 2 Commercial sample Arteether(6 mg/kg) 40.3972.03 63.3 3 Test group 1 Form I 18.5671.43 16.7 4 Test group 2 Form II 20.5570.55 33.3 5 Test group 3 Form III 25.3172.70 33.3 6 Test group 4 Form IV 30.5672.03 50.0 7 Test group 5 Form V 16.5671.43 16.7 8 Test group 6 Form VI 36.2973.25 63.7

aValues are expressed as mean7SD (n¼6); t: no. of days; PI: post-inoculation. Investigation and characterization of various crystal forms of arteether 135

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