Effect of counterions on the properties of amorphous atorvastatin salts

European Journal of Pharmaceutical Sciences 44 (2011) 462–470

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Effect of counterions on the properties of amorphous atorvastatin salts Vishal M. Sonje a, Lokesh Kumar b, Vibha Puri b, Gunjan Kohli b, Aditya M. Kaushal c, Arvind K. Bansal b,⇑ a

Department of Pharmaceutical Technology (Formulations), National Institute of Pharmaceutical Education and Research (NIPER), S.A.S. Nagar, Mohali, Punjab 160 062, India Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), S.A.S. Nagar, Mohali, Punjab 160 062, India c Department of Pharmaceutics, University of Minnesota, Minneapolis, MN 55455, USA b

a r t i c l e

i n f o

Article history: Received 21 May 2011 Received in revised form 20 August 2011 Accepted 29 August 2011 Available online 1 September 2011 Keywords: Amorphous Salts Glass transition Counterion Atorvastatin

a b s t r a c t Amorphous systems have gained importance as a tool for addressing delivery challenges of poorly water soluble drugs. A careful assessment of thermodynamic and kinetic behavior of amorphous form is necessary for successful use of amorphous form in drug delivery. The present study was undertaken to evaluate effect of monovalent sodium (Na+; ATV Na), and bivalent calcium (Ca2+; ATV Ca) and magnesium (Mg2+; ATV Mg) counterions on properties of amorphous salts of atorvastatin (ATV) model drug. Amorphous form was generated from crystalline salts of ATV by spray drying, and characterized for glass transition temperature (Tg), fragility and devitrification tendency. In addition, chemical stability of the amorphous salt forms was evaluated. Fragility was studied by calculating activation enthalpy for structural relaxation at Tg, from heating rate dependency of Tg. Density functional theory and relative pKa’s of counterions were evaluated to substantiate trend in glass transition temperature. Tg of salts followed order: ATV Ca > ATV Mg > ATV Na. All salts were fragile to moderately fragile, with D value ranging between 9 and 16. Ease of devitrification followed the order: ATV Na  ATV Mg  ATV Ca, using isothermal crystallization and reduced crystallization temperature method. Chemical stability at 80 °C showed higher degradation of amorphous ATV Ca (5%), while ATV Na and ATV Mg showed degradation of 1–2%. Overall, ATV Ca was better in terms of glass forming ability, higher Tg and physical stability. The study has importance in selection of a suitable amorphous form, during early drug development phase. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction Combinatorial chemistry and high throughput screening have pushed many compounds with poor solubility and dissolution characteristics into the development pipeline, making them challenging to formulate. Therefore, modulation of solubility and/or dissolution rate has become critical for optimization of potential drug molecules (Lipinski, 2000; Lipinski et al., 1997). Salt formation is one of the most preferred approaches to optimize solubility of an ionizable drug candidate (Berge et al., 1977). Alternatively, use of different polymorphs, amorphous materials and/or cocrystal formation, may provide improvements. Salt formation provides a versatile tool for modulation of biopharmaceutical properties, like aqueous solubility. A further enhancement of aqueous solubility can be achieved by generating amorphous form of salts, as they lack lattice interactions, in

Abbreviations: ATV, atorvastatin; ATV Ca, atorvastatin calcium; ATV Mg, atorvastatin magnesium; ATV Na, atorvastatin sodium; Tg, glass transition temperature; DSC, Differential Scanning Calorimetry; HSM, hot stage microscopy; XRPD, X-ray Powder Diffractometry; DFT, density functional theory. ⇑ Corresponding author. Tel.: +91 172 2214682/2126; fax: +91 172 2214692. E-mail address: [email protected] (A.K. Bansal). 0928-0987/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2011.08.023

comparison to their crystalline counterparts (Kaushal et al., 2004). In literature (Tong et al., 2002; Tong and Zografi, 1999; Towler et al., 2008), a combination approach of salt formation and amorphous state has been reported; wherein, the properties of amorphous salts have been shown to be dictated by the nature and type of counterion. Tong et al. studied the effect of counterion on the amorphous salts of Indomethacin and found that the amorphous salts had a much higher Tg than the amorphous indomethacin acid. Furthermore, they established a trend showing the effect of ionic charge density of the counterion on the molecular mobility, and in turn, on the stability of the amorphous systems. It was found that the Tg depended on the ionic density of the counterion, which decreased in the order of Li+ > Na+ > K+ > Rb+ > Cs+. It was hypothesized that higher ionic density of the counterion increased the electrostatic interaction between the drug and the counterion. Smaller sized cations led to stronger interaction. Increase in cation radius decreased the charge density, increased the molecular mobility and consequently decreased the Tg of the amorphous salt (Tong et al., 2002). Guerrieri et al. similarly studied effect of counterion on Tg of procaine salts, and observed that Tg of procaine salts was higher than Tg of procaine free base. Tg varied with the type of counterion, showing an increase with decrease in pKa of corresponding counterion (Guerrieri et al., 2010).

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However, the authors only studied the effect of monovalent ions on Tg. To our knowledge, comparative assessment of effect of monovalent and bivalent counterion has not been reported in literature, till date. In the present work, we have compared the effect of mono and bivalent ions on the glass transition temperature of a model drug. We also compared the fragility and devitrification behavior (under temperature and combined temperature – humidity stress) of different salts. Towler et al. reported a similar behavior, wherein, salts of nicardipine and propranolol were characterized for their Tg and crystallization tendency. Several molecular descriptors were calculated for various counterions and a multivariate analysis was performed to correlate the Tg with various molecular descriptors. The important molecular descriptors which influenced Tg included pKa and electrophilicity index of the counterion. A direct correlation was found between Tg and electrophilicity index. In contrast, Tg was found to decrease with a corresponding increase in the pKa value of the counterion (Towler et al., 2008). Atorvastatin (ATV) was chosen as a model drug for the present study. ATV ([(R-(R⁄,R⁄)]-2-(4-fluorophenyl)-beta, delta-dihydroxy5-(1-methylethyl)-3-phenyl-4-[(phenyl amino) carbonyl]-1H-pyrrole-1-heptanoic acid) is a member of the drug class known as statins. It is a selective, competitive inhibitor of 3-hydroxy methyl glutaryl coenzyme A (HMG CoA) reductase enzyme involved in the conversion of 3-hydroxy methyl glutaryl coenzyme A to mevalonate; a precursor of sterols, including cholesterol (Christians et al., 1998). The chemical structure is shown in Fig. 1, and salt formation occurs at the terminal carboxylic group. Three salt forms, viz., sodium (ATV Na), calcium (ATV Ca) and magnesium (ATV Mg) were chosen to assess the effect of the salt formation on the amorphous drugs salts. Comparative analysis of monovalent sodium and bivalent calcium and magnesium salts was performed to assess their relative effect on the properties of amorphous salts. 2. Materials and methods 2.1. Materials Crystalline form of ATV (ATV Ca from Dr. Reddy’s Laboratories, India; ATV Na and Mg salts from Cadila Pharmaceuticals, Ahmedabad, India) were obtained as gratis samples and used as supplied (chemical purity >99.5% for all the samples). All other chemicals used were of analytical reagent grade. 2.2. Differential Scanning Calorimetry (DSC) Calorimetric response of the samples was measured using DSC (Perkin Elmer, Diamond DSC, USA), operating with PyrisÒ software (version 7). Powder sample (2–8 mg) was weighed into an

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aluminum pan and sealed with pin-holed lid. Heating rates of 10 °C/min was employed under nitrogen purge at 40 ml/min. Glass transition was reported as the onset temperature, while endothermic transitions were reported as the midpoints. Calibration of the instrument was performed using zinc and indium. 2.3. Thermogravimetric analysis (TGA) The mass loss of sample as a function of temperature was determined using MettlerToledo 851e TGA (Mettler Toledo, Switzerland). Samples were placed in open alumina crucibles and heated at a rate of 20 °C/min under nitrogen purge (40 ml/min). 2.4. Hot stage microscopy (HSM) Thermal behavior of the samples was observed under the Leica DMLP polarized microscope (Leica Microsystems Wetzlar GmbH, Germany), equipped with Linkam LTS 350 hot stage. Photomicrographs were acquired using JVS color video camera and analyzed using Linksys32 software. Samples were mounted in silicone oil and heated from 25 to 200 °C at a heating rate of 20 °C/min. 2.5. Optical and polarized light microscopy Particle characteristics were assessed by optical microscopy using a Leica DMLP polarized light microscope (Leica Microsystems, Germany). Photomicrographs were obtained using RICOH XR-X3000D camera (RICOH, Japan). For hot stage microscopy (HSM), samples were mounted in air/silicone oil and heated from 25 to 300 °C. Photographs were processed using LinksysÒ software. 2.6. X-ray Powder Diffractometry (XRPD) XRPD patterns of samples were recorded at room temperature on Bruker’s D8 advance diffractometer (Bruker, Germany) with Cu Ka radiation (1.54 Å), at 40 kV, 40 mA passing through nickel filter. Analysis was performed in a continuous mode with a step size of 0.05° and step time of 1 s over an angular range of 3–40° 2h. Obtained diffractograms were analyzed with DIFFRACplus EVA (version 9.0) diffraction software. 2.7. Preparation of amorphous samples Amorphous salts were generated by three different techniques of melt quenching, rapid evaporation from solution under vacuum (rotavapor) and spray drying. Melt quenching was further done in two ways. Samples were melt quenched in situ in the DSC or prepared outside the DSC instrument (termed ex situ). In the in situ method, a small amount of powder sample was heated to 15 °C

Fig. 1. Chemical structure of Atorvastatin salts, for X = Ca, Mg and Na. Two molecules of ATV combine with one atom of Ca/Mg.

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above the melting temperature at a heating rate of 20 °C/min under nitrogen purge (50 ml/min) and held isothermally for 1 min, followed by rapid cooling to 25 °C at 20 °C/min. In the ex situ method, samples were taken in stainless steel beaker and melted on a hot plate (Sonar Associated Scientific Technologies, India), followed by rapid cooling over ice. For rotavapor evaporation, sample dissolved in methanol were evaporated under vacuum on a rotavapor (R-200, Buchi Labortechnik AG, Switzerland) using a re-circulating water bath (CBN 8-30, Heto, Denmark), at a temperature of 60 °C. For spray drying, sample was dissolved in methanol (3–5% w/v), followed by drying using a laboratory level spray dryer (Labultima, India) with atomization pressure of 0.5–0.7 Kg/cm2, inlet temperature of 70 °C, feed rate of 3 ml/min and aspirator volume of 100 mm/WC. 2.8. Critical cooling rate DSC instrument was first calibrated for ‘tau lag’ onset melting temperatures using a standard of indium at different heating rates. Critical cooling rate was determined by heating the crystalline salts to 20 °C above their melting point, followed by cooling at different predetermined rates (1, 2, 5, 7, 10, 15, and 20 °C/min) to 25 °C. Subsequently, the glassy materials were reheated at 20 °C/min to 10 °C above the respective crystalline melting points. The second heating run was used to determine the amorphous or crystalline nature of the sample by examining the glass transition and crystallization events. 2.9. Fragility determination Fragility was determined by the heating rate dependence of Tg. For this, the amorphous samples were heated to 20 °C above the Tg to erase the thermal history and then cooled to temperatures below the Tg at controlled rates, before being reheated at the same rate to determine the Tg. Cooling and heating rates employed were 5, 10, 15, 20, 25 and 30 °C/min. Experiments were performed in triplicate and the onset of Tg value was determined. 2.10. Devitrification studies Devitrification tendency of amorphous salts was studied using temperature and humidity as the stressors. In isothermal crystallization study, the spray dried ATV salts were stored in sealed aluminum pouches at temperature of Tg20 °C for the respective drug salts, followed by analysis using XRPD after 0, 1, 3, 7, 14 and 30 days. Samples were also separately stored at 25 °C ± 92% RH to accelerate devitrification of calcium salt. Humidity conditions were generated by using saturated potassium nitrate solution in open petridish under ambient temperature. Samples were evaluated for the emergence of crystallinity, at regular intervals for two weeks. 2.11. Chemical stability Samples were kept in aluminum pouches at 80 °C for up to 4 weeks and analyzed by a validated HPLC method at 0, 1, 3, 7, 14 and 30 days. Related substances were determined using Shimadzu HPLC system (Shimadzu, Japan) equipped with a model series SPD-M20A photodiode array detector, a gradient elution pump with degassing device DGU-20A5, a cooling autosampler SIL20AC, a column heater/cooler CTO-10A VP and a system controller CBM-20A. The diode array detector was used for the spectrum extraction, while the detection was performed at 246 nm. Separations were performed using a LichrospherÒ C18 (250  4.6 mm, 5 lm) column. Data was acquired using Class VP data acquisition software, version 6.12 SP1. For assay of ATV salts, chromatographic

separation was achieved using a mobile phase consisting of 1 ml of triethylamine in 1000 ml HPLC grade water (pH 4.0 adjusted with orthophosphoric acid) (mobile phase A) and a mixture of acetonitrile and methanol in 80:20 ratio (mobile phase B) in a gradient mode. 2.12. Intrinsic dissolution rate (IDR) Intrinsic Dissolution Rate (IDR) of crystalline and amorphous ATV salts was determined using the rotating disc apparatus (Electrolab, Mumbai, India). ATV salt (100 mg) was compacted using hydraulic press at a pressure of 800 psi with a dwell time of 15 s, in a 8 mm punch die set, in 100 mM phosphate buffer pH 6.8 with 2% SLS as the dissolution medium. Sample were withdrawn at periodic time intervals, replaced with dissolution medium and analyzed using UV spectroscopy, at a wavelength of 244, 246 and 256 nm for ATV Na, ATV Ca and ATV Mg, respectively. Analysis of disc by XRPD confirmed that no solid-state transition occurred during the compaction. 3. Results 3.1. Characterization of crystalline forms The crystalline forms of ATV salts were characterized for their solid state properties. All samples showed birefringence in polarized light microscope, thus confirming their crystalline nature. ATV Ca showed a broad endotherm from 50 to 110 °C in DSC that was attributed to water loss (corresponding to a weight loss of 3.6% in TgA), thereby confirming the sesquihydrate stoichiometry of ATV Ca. The water loss was followed by melting at 158.3 °C. Similarly, ATV Mg showed an endotherm for water loss in DSC and a step weight loss of 4.6% in TGA from 40 to 90 °C attributed to the sesquihydrate nature of the salt, followed by melting at 190.9 °C. In contrast, ATV Na showed a water loss of 7–8% over a broad range of temperature, with melting being observed at 197.2 °C. All thermal transitions could be visually observed over the same temperature range in the HSM. All the salts were crystalline, as observed by XRPD and birefringence in the polarized light microscopy. ATV Ca had a distinct XRPD pattern composed of sharp peaks. However, ATV Mg and ATV Na showed a relatively diffused XRPD pattern. Comparison of the XRPD diffractograms of all the salts with the reported literature showed that ATV Ca, ATV Na and ATV Mg were Form A (Briggs et al., 1999), Form I (Kroselj et al., 2007) and Form A (Leonard and Miller, 2007) respectively. 3.2. Generation of amorphous forms Amorphous salts were generated by a range of methods like melt quenching, rotary evaporation, and spray drying. However, slight degradation of drug was observed during melt quenching, as confirmed by HPLC. Rotavapor evaporation of the solvent by heat and vacuum also resulted in partially crystalline material as observed from the presence of birefringence in polarized light microscopy. In contrast, spray drying yielded pure amorphous form for all the three ATV salts, which was confirmed by a halo pattern in the XRPD and an absence of birefringence on microscopic observation. 3.3. Characterization of amorphous form Amorphous samples showed an absence of birefringence in the polarized light microscopy, Tg in the heating curve (Fig. 2), and a characteristic halo pattern in the XRPD diffractogram. The residual

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solvent content in all amorphous ATV salts was between 1000 and 2000 ppm, as measured by Head Space GC analysis. Glass transition behavior of the samples was studied using DSC at a heating rate of 10 °C/min. Tg of the samples followed an order of ATV Ca > ATV Mg > ATV Na. 3.4. Critical cooling rate and fragility of amorphous samples Critical cooling rate indicates the ease of the formation of an amorphous state when hot melt is cooled at different rates, and is given by:

qc ¼ ðT m  T n Þ=s1n

ð1Þ

where Tm is the melting point in K, Tn is the temperature under consideration and s1n is the induction time for nucleation. Critical cooling rate determination was conducted to rank order the salts, in terms of their glass forming ability. For this, the melts of ATV salts were subjected to different cooling rates of 1, 2, 5, 7, 10, 15 and 20 °C/min. A successful glass formation was observed at all cooling rates (confirmed by the lack of melting endotherm in the re-heating scan), suggesting that the critical cooling rates for all ATV salts was below 1 °C/min. Therefore, all ATV salts did not differ in their glass forming ability under the experimental conditions studied (Zarzycki, 1991). The activation energy for structural relaxation at Tg determined from heating rate dependence of Tg is used as a measure of

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fragility. Dependence of Tg upon experimental heating rate is attributed to it being a kinetic phenomenon. The extent of change of Tg value, with change in heating rate determines fragility of glass structure. Amorphous samples of all the three salts were heated at different rates (5–30 °C/min) and the onset Tg was recorded. A plot of the natural log of heating rate (°C/min) vs inverse of Tg (K) showed linearity (Fig. 3). The respective Tg values were further used to calculate activation energy (Ea) for structural relaxation, using the following equation:

ln q ¼ Ea =RT

ð2Þ

where q is the heating rate, R is the universal gas constant and T is the temperature of glass transition measured at various heating rates. Value of Ea was maximum for ATV Mg (573.8 KJ/mol), followed by ATV Na (436.5 KJ/mol) and ATV Ca (433.5 KJ/mol). According to this method, the fragility of ATV salts followed the order: ATV Ca > ATV Na > ATV Mg. Fragility is an important property of amorphous materials, that is described based on the temperature dependence of mean relaxation time or viscosity above Tg. Angell proposed a classification of ‘strong’ and ‘fragile’ liquids, based on the viscosity–temperature relationship, known as Angell’s plot. ‘Strong’ liquids are characterized by Arrhenius type relationship, with activation energy independent of temperature. In contrast, ‘fragile’ liquids show a non-linear dependence of viscosity upon temperature with a deviation from Arrhenius behavior, characterized by temperature

Fig. 2. Heating curves of the amorphous ATV salts (a) ATV Ca (b) ATV Mg and (c) ATV Na at 10 °C/min.

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Fig. 3. Plot of ln q (heating rate) vs. 1000/Tg.

dependence of activation energy (Angell, 1985; Angell, 1991). Majority of pharmaceutical amorphous materials demonstrate ‘fragile’ behavior (Crowley and Zografi, 2001). Ratio of Tm/Tg is the simplest way of describing fragility, and a ratio of less than and more than 1.5 signify ‘fragile’ and ‘strong’ glass characteristics, respectively. All salts of ATV showed a Tm/Tg ratio of less than 1.5 (Table 1), thus signifying their fragile behavior. Literature reports have indicated that within homologous series of glass formers, the values of Tm/Tg are sensitive to subtle functional group substitutions that affect intermolecular interactions. These changes though need not necessarily have an effect on the fragility of glass formers (Angell et al., 1978). Herein, substitution of counterion Ca, Mg and Na gave Tm/Tg of 1.03 and 1.22 and 1.27, respectively. Heat capacity jump at Tg is also used to describe fragility, wherein, ‘fragile’ liquids show significantly higher jump as compared to the ‘strong’ liquids. Amorphous Na, Mg and Ca salts of ATV exhibited DCp values of 540, 240 and 250 J/mol/K respectively, indicating the sodium salt to be the most fragile. High DCp values of ATV Na indicate marked changes at molecular mobility above Tg, due to additional degrees of freedom. In contrast, in ATV Ca and Mg salts, the metal ion is joined to two molecules of atorvastatin, thereby showing restricted degree of freedom. However, the result of fragility in terms of jump in Cp, should not be viewed in isolation, as intermolecular hydrogen bonding can also contribute to the overall DCp. In a previous work from our lab (Kaushal and Bansal, 2008), we have reported the impact of intermolecular hydrogen bonding on thermodynamic quantities of amorphous form of structurally related compounds. Rearrangement of molecules involving hydrogen bonding liquids will involve energy for both, local expansion and breaking of hydrogen bonds. This may contribute to the overall DCp value, thus making the interpretation difficult. In the present study, the drug molecule is same, i.e., atorvastatin, and only the counterion is varied. The electrophilicity

Fig. 4. Plot of Log s vs. Tg/T equivalent to Angell’s plot.

index of the counterion apart from affecting the intra-molecular environment, also affects intermolecular interactions (refer Section 4). Contribution of these interactions to the overall DCp value can make interpretation difficult. Value of Kauzmann temperature TK with respect to Tg, as TK/Tg or Tg  TK also provides an information about the fragility of compounds. TK or Kauzmann temperature (temperature at which molecular mobility becomes zero) was also estimated from ‘m’ by following relationship, where mmin is a constant equal to 16 (Tong and Zografi, 1999):

 mmin  TK ¼ Tg 1  m

ð3Þ

A TK/Tg value of 0 indicates strong, while a value of 1 corresponds to fragile behavior. Similarly, Tg  TK value of >50 and <50 K indicate strong and fragile glass behavior respectively. As given in Table 1, TK/Tg values for Ca, Mg and Na were 0.70, 0.79 and 0.74, respectively. TK/Tg indicates departure of glass from Arrhenius behavior, as the material is heated beyond Tg. All the three salt forms exhibited near to fragile glass behavior when compared by the value of TK/Tg. More accurate prediction of fragility can be given by an alternative parameter called Fragility parameter (m).

m¼

d loghsi ; dðT g =TÞ T¼T g

ð4Þ

Eq. (4) can be expressed in terms of the temperature dependent apparent activation energy, Ea, as follows (Bohmer et al., 1993; Moynihan et al., 1974):

Table 1 Thermodynamic parameters for ATV samples. Salt

Tm (K)

Tg (K)b

Tm/Tg

DCp(J/mol K)

TKa(K)

Tc (K)

TK/Tg

Tg  TK (K)

Ea (kJ/mol)

D

m

ATV Ca ATV Mg ATV Na

431.6 463.9 470.2

416.8 (0.60) 380.6 (0.38) 369.3 (1.70)

1.03 1.22 1.27

250 240 540

293.9 302.5 273.7

# 396.5 404.4

0.70 0.79 0.74

122.9 76.8 95.9

433.5 573.8 436.5

15.4 9.4 12.9

54.3 79.0 61.7

#, Not observed. Tm, melting temperature (peak). Tg, glass transition temperature (onset). Tc, recrystallization temperature (peak; 10 °C/min). a Determined from m b Statistically significant difference observed between Tg of all salts (n = 3; P < 0.05)

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Fig. 5. XRPD diffractogram of amorphous ATV salts (at periodic time points after storing at temperature of Tg-20 °C. For ATV Ca, the respective diffractogram for the three salts (a–e, ATV Ca; f–j, ATV Mg; k–o, ATV Na) are 0, 1, 3, 7 days and crystalline salt. ATV Na and ATV Mg salts showed emergence of crystallinity after 1–3 days, whereas ATV Ca remained amorphous even after 7 days.

Fig. 6. Photomicrographs of amorphous ATV salts (200X) stored at 25 °C ± 92% RH for different time periods. ATV Na and Mg showed emergence of crystallinity at 3 days, whereas ATV Ca remained amorphous even after 7 days.

m¼

1 ½Ea ðT g Þ=RT g  2:303

ð5Þ

where Ea(Tg) corresponds to the activation energy and R refers to the gas constant. A large value of m indicates fragile behavior,

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due to rapidly changing dynamics at Tg. The values of temperature dependent apparent activation energy was obtained from heating rate dependence of Tg. Values of 54.3, 79.0 and 61.7 were observed for the fragility parameter (m) for ATV Ca, ATV Mg and ATV Na salts respectively (Gupta et al., 2004). A greater value of m indicates a larger departure from Arrhenius behavior. A strong liquid typically has a m value of 17 (Mauro, 2008). Strength parameter ‘D’ was calculated using following relation with m:

D ¼ ðln 10Þm2min =m  mmin

ð6Þ

where mmin is a constant equal to 16 (Ramos et al., 2004). D values of <10 indicate fragile, whereas >30 indicate strong glass behavior. ATV Ca, Mg and Na gave values of 15.4, 9.4 and 12.9, thus indicating their fragile to moderately fragile nature, like most pharmaceuticals (Crowley and Zografi, 2001). From the plot analogous to Angell plot (Fig. 4), it could be seen that as the temperature is increased above the Tg, sudden fall in viscosity was observed. Also, the molecular mobility rose sharply above or below Tg. Although the fragility pattern among the 3 salts remained almost similar, the fall in viscosity was observed to be most pronounced for ATV Mg. The fragility index of a glass-forming liquid is a critical parameter for a glassy state that reflects the behavior of glass in response to temperature change. Fragile liquids because of rapid fall in viscosity and relaxation times are likely to show a higher value of TK, the zero mobility temperature. However, fragile glasses are also prone to widespread changes in their structural properties by only a small excursion in storage temperature (Bohmer and Angell, 1994). 3.5. Devitrification studies The relative trend in physical stability of the amorphous spray dried salts was studied by isothermal crystallization using XRPD as the analytical tool. Spray dried ATV salts were stored at temperature of Tg20 °C. ATV Ca remained amorphous even after storage for 7 days, while ATV Na and ATV Mg started to crystallize out immediately, as evident from the XRPD diffractogram (Figs. 5 and 6). Alternatively, amorphous samples were also stored at 25 °C/ 92%RH, wherein, ATV Ca did not crystallize after 7 days, while ATV Na and ATV Mg started to crystallize after one day and showed significant re-crystallization in 3 days. This suggests that ATV Ca was more stable towards devitrification on storage at accelerated humidity and temperature condition. Crystallization tendency was also evaluated by determination of reduced crystallization temperature (RCT). RCT represents a normalized measure of how far above Tg a compound must be heated before spontaneous crystallization occurs. It is a useful method to compare the crystallization tendency of the compounds having different Tg (Zhou et al., 2002). Amorphous samples were heated at 1 °C/min and RCT defined as (Tc  Tg)/ (Tm  Tg), was used to the compare crystallization tendencies. RCT of ATV Ca could not be calculated as it did not crystallize during the DSC run, and hence was considered to have the lowest crystallization tendency (Table 2). This correlates well to the observation of the isothermal crystallization study. The RCT of ATV Na was comparable to ATV Mg, suggesting a similar tendency to recrystallize at a temperature above Tg.

the degradation rate of a range of b-lactam antibiotics was enhanced by an order of magnitude for amorphous as opposed to crystalline drugs, even with low sorbed water levels (Pikal et al., 1977). Similarly, Oberholtzer and Brenner demonstrated that the temperature dependent degradation of amorphous cefoxitin sodium, compared to crystalline cefoxitin sodium (Oberholtzer and Brenner, 1979). Therefore, selection of an amorphous form having the highest chemical stability becomes important for subsequent formulation development. Crystalline ATV salts showed lesser decomposition as compared to the amorphous samples, which is in accordance with the established observation of lower stability of amorphous pharmaceuticals, in comparison to their crystalline counterparts. Amorphous ATV Ca and ATV Mg showed a significant increase in the related substances (RS) to 5% and 4% respectively after 30 days at 80 °C, while <1% increase was observed for ATV Na (Fig. 7). This could, however, be due to the concomitant devitrification shown by the ATV Na at the employed storage condition. It should be noted that the temperature of stability studies was less than the Tg of all amorphous ATV salts. 3.7. Intrinsic dissolution rate (IDR) Fig. 8 shows the IDR profiles of amorphous and crystalline ATV salts. A regression value of 0.98–0.99 was obtained for all the amorphous as well as crystalline salts. Dissolution advantage from the amorphous ATV Ca over crystalline form is higher, as compared to that of amorphous ATV Mg and ATV Na, over their crystalline counterparts. However, the overall dissolution profile for both crystalline as well as amorphous ATV Mg is better, as compared to that of ATV Ca and ATV Na (statistically significant at P < 0.05). Amorphous ATV Ca and ATV Mg salt forms showed statistically significant higher dissolution rate, compared to their crystalline counterparts (P < 0.05). In contrast, in case of ATV Na, the dissolution

Table 2 Devitrification tendency using RCT.

*

Compound

Tc (K)*

RCT

ATV Ca ATV Mg ATV Na

– 375.9 361.3

– 0.50 0.52

Temperature in Kelvin (heating rate – 1 °C/min).

3.6. Chemical stability The chemical reactivity of amorphous pharmaceuticals is generally higher as compared to their crystalline counterparts, due to the higher molecular mobility, leading to an enhanced chemical degradation (Yoshioka and Aso, 2007). Pikal et al. showed that

Fig. 7. Plot of % related substances (RS) vs. time (days) for ATV salts on storage at 80 °C.

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properties. Hence, it is of interest to study the molecular parameters that affect Tg in amorphous salts. Eisenberg et al. have described the molecular parameters that can influence Tg in polymers. They describe the importance of intermolecular interactions, whereby the stronger the intermolecular interaction, higher is the Tg. More thermal energy is required to attain the molecular mobility necessary to undergo the transition. Further, they suggest the use of density functional theory (DFT) in explaining the intermolecular interactions (Eisenberg and Shen, 1966). Conceptual DFT theory mainly suggests three energy factors responsible for the intermolecular interactions. The first one is electrostatic factor, which is dominant in ionic and hard molecules. Second factor is covalent bonding which comes from sharing of electrons. The third factor is polarization contribution that arises as a result of instantaneous fluctuation of electron movement. In case of salts, electrostatic contributor is mainly responsible for the intermolecular interactions. Parameters such as hardness, softness, electrophilicity index and chemical potential are calculated within DFT to explain the inter- and intra-molecular interactions. Hardness and softness describe the polarizability of the electronic structure, hardness (g) has been found to be related to Klopman’s frontier molecular orbital theory, and is given by difference between the Ionization potential, I and Electron affinity, A.

g ¼ ½@ 2 E=@N2 m ¼ ½@ l=@Nm ffi I  A

ð7Þ

where E is the system energy, N is the number of electrons, l is the electronic chemical potential, opposite of the electronegativity, and t(r) is the external potential defined by nuclear charges and positions in the system. The chemical potential (l) is the first order derivative of the system energy.

l ¼ ½@E=@Nm  ½I þ A=2 ¼ vm

ð8Þ

where (I + A)/2 is the Mulliken electronegativity vm. Intermolecular interactions involving charge transfer can be characterized by the electrophilicity index (x) and is given by:

x ¼ l2 =2g

Fig. 8. Intrinsic dissolution rate for crystalline and amorphous ATV salts (n = 3).

rates was similar in both amorphous and crystalline form at initial time points, but differ significantly at later periods. According to Kaplan’s classification compounds having IDR below 100 lg/min/ cm2 usually exhibit dissolution rate limited absorption (Kaplan, 1972). None of the ATV salt, in either crystalline or amorphous form, falls in the category of dissolution rate limited drugs. Moreover, no physical transformation was observed in ATV salts during the dissolution study.

4. Discussion 4.1. Interpretation of Tg For amorphous materials, Tg is the most important property, and the storage temperature relative to the Tg can influence physical stability, chemical stability as well as the mechanical

ð9Þ

Towler et al. reported that higher the electrophilicity index of the counterion, higher is the intermolecular interactions, leading to higher Tg value. Electrophilicity index for the counterions in the ATV salts was calculated from the Eq. (9), using reported values of electron affinity and ionization potential for the counterions (Parr et al., 1999). An effort was made to explain the pattern in Tg in ATV salts on the basis of DFT theory (correlating with various molecular descriptors such as hardness, softness, electrophilicity index), ionic density of counterion, pKa of counterion and packing effects in the amorphous state. Table 3 shows the DFT parameters for atorvastatin counterions. The order of electrophilicity index x was ATV Mg > ATV Na > ATV Ca. In addition to intra and intermolecular interactions, structural properties of molecules also affect, in terms of free volume and packing differences (Towler et al., 2008). In case of ATV Na, one molecule of ATV binds with one atom of Na+, while in case of ATV Ca and ATV Mg, two molecules of ATV are associated with

Table 3 Selected molecular descriptors for the studied counterions calculated using DFT. Counterion

A (eV)

I (eV)

g (eV)

S (eV1)

l (eV)

x (eV)

Calcium Magnesium Sodium

0.02 0.81 0.55

6.11 7.65 5.14

6.09 6.84 4.59

0.16 0.15 0.22

3.07 4.23 2.84

0.77 1.31 0.88

A, electron affinity; I, ionization potential; g: hardness; S, softness; l, electronic chemical potential; x, electrophilicity index.

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(vide letter – SR/SO/HS-23/2006) for providing Senior Research Fellowship. Vibha Puri would also like to acknowledge Indian Council for Medical Research (ICMR), Government of India for providing Senior Research Fellowship. References

Fig. 9. Effect of electrophilicity index, pKa and charge on Tg of ATV salts.

one atom of Ca2+ or Mg2+. Given the bulk of one molecule of ATV Ca and ATV Mg is more than a corresponding molecule of ATV Na, the molecular mobility of ATV Ca and ATV Mg would be lesser, thereby giving a higher Tg value. Apart from the above descriptors, Tg is also affected by the pKa of the counterion, with higher Tg value observed for the stronger counterion (Towler et al., 2008). Fig. 9 shows the effect of pKa of the counterion, electrophilicity index x, on Tg of ATV salts. ATV Na exhibited the lowest Tg, though, sodium ion has the highest pKa value (14) amongst the three counterions investigated in the present work. Amongst the bivalent counterions, Ca (pKa:12.7) has a higher pKa compared to Mg (pKa:11.7) (Towler et al., 2008). Their Tg value was also found to follow the same order (ATV Ca: 416.8 K; ATV Mg; 380.6 K; ATV Na: 369.3 K). On the basis of the above hypothesis the order of the Tg is expected to be ATV Ca > ATV Mg > ATV Na, which matches the experimentally observed pattern of Tg in ATV salts. 5. Conclusion The present study investigated the effect of counterion on the three salts of a model drug ATV viz. – ATV Ca, ATV Na and ATV Mg. All the three model compounds showed a very low critical cooling rate (<1 °C/min), indicating their good glass forming ability and ease with which amorphous forms can be obtained. Tg was found to be affected by the packing properties as well as the strength of the counterion. Low D value (9–16) and high m value (54–79) indicated fragile to moderately fragile nature of the ATV glasses. Similarly, the temperature dependency of the molecular mobility (i.e., fragility) suggests the fact that no significant differences exist between the organization of molecules relative to each other. Amorphous ATV Ca showed a better physical stability, but a higher chemical degradation. Overall, ATV Ca was found to be better in terms of glass forming ability, higher Tg and physical stability thereby suggesting its suitability as a candidate for formulation development. Acknowledgements Lokesh Kumar would like to acknowledge Department of Science and Technology (DST), Government of India

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