HPMC as a potential enhancer of nimodipine biopharmaceutical properties via ball-milled solid dispersions

Accepted Manuscript Title: HPMC as a potential enhancer of nimodipine biopharmaceutical properties via ball-milled solid dispersions Author: Manoela Kl¨uppel Riekes Gislaine Kuminek Gabriela Schneider Rauber Carlos Eduardo Maduro de Campos Adailton Jo˜ao Bortoluzzi Hellen Karine Stulzer PII: DOI: Reference:

S0144-8617(13)00830-8 http://dx.doi.org/doi:10.1016/j.carbpol.2013.08.046 CARP 8036

To appear in: Received date: Revised date: Accepted date:

6-5-2013 12-8-2013 18-8-2013

Please cite this article as: Riekes, M. K., Kuminek, G., Rauber, G. S., Campos, C. E. M., Bortoluzzi, A. J., & Stulzer, H. K., HPMC as a potential enhancer of nimodipine biopharmaceutical properties via ball-milled solid dispersions, Carbohydrate Polymers (2013), http://dx.doi.org/10.1016/j.carbpol.2013.08.046 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

HPMC as a potential enhancer of nimodipine biopharmaceutical properties via ball-milled solid dispersions Manoela Klüppel Riekes1, Gislaine Kuminek1, Gabriela Schneider Rauber1, Carlos Eduardo Maduro de Campos2, Adailton João Bortoluzzi3, Hellen Karine Stulzer1* 1

ip t

Programa de Pós-Graduação em Farmácia, Universidade Federal de Santa Catarina, Florianópolis, 88040-970, Brazil 2 Programa de Pós-Graduação em Física, Universidade Federal de Santa Catarina, Florianópolis, 88040-970, Brazil 3 Programa de Pós-Graduação em Química, Universidade Federal de Santa Catarina, Florianópolis, 88040-970, Brazil

Ac ce p

te

d

M

an

us

cr

* corresponding author:  [email protected],  +55 (48) 3721-4585,  + 55 (48) 3721-9542

E-mail addresses of other authors: Manoela Klüppel Riekes: [email protected] Gislaine Kuminek: [email protected] Gabriela Schneider Rauber: [email protected] Carlos Eduardo Maduro de Campos: [email protected] Adailton João Bortoluzzi: [email protected]

1 Page 1 of 28

ABSTRACT

ip t

The poor solubility of drugs remains one of the most challenging aspects of formulation development. Aiming at improving the biopharmaceutical limitations of the calcium channel blocker nimodipine, the development of solid dispersions is proposed herein. Three different proportions of nimodipine:HPMC were tested and all of them generated amorphous solid dispersions. Improvements of up 318 % in the solubility and a 4-fold increase in the dissolution rate of nimodipine were achieved. Stability studies conducted over 90 days in a desiccator indicated that the initial characteristic of the formulations were maintained. However, at 40 °C/75 % RH recrystallization was observed for solid dispersions with 70 and 80 % of HPMC, whilst the formulation composed of 90 % of the carrier remained amorphous. The increase in the stability observed when the HPMC concentration was increased from 70 to 90 % in the SDs was attributed to the dilution mechanism.

cr

1 2 3 4 5 6 7 8 9 10 11

13

us

12

Keywords: nimodipine, HPMC, solid dispersion, biopharmaceutical properties

an

14 15

M

16 17

21 22 23 24 25 26 27

te

20

Ac ce p

19

d

18

28 29 30 31 32 2 Page 2 of 28

1. INTRODUCTION

34

Together with the permeability, the solubility behavior of an active pharmaceutical ingredient

35

(API) is the limiting step in terms of its oral bioavailability (Leuner & Dressman, 2000),

36

particularly for drugs with low gastrointestinal solubility and high permeability (Vasconcelos,

37

Sarmento & Costa, 2007). Currently, it is estimated that 70 % of the new APIs have poor

38

aqueous solubility (Ku & Dublin, 2010), and unfortunately, around 40 % of newly developed

39

drugs are rejected by the pharmaceutical industry due to their deficient biopharmaceutical

40

properties (Svenson, 2009).

41

Several formulation strategies have been proposed to improve the solubility and dissolution rate

42

of poorly water-soluble APIs and of these the solid dispersion (SD) technique has emerged as a

43

particularly effective approach (Alonzo et al., 2011; Konno, Handa, Alonzo & Taylor, 2008;

44

Newman, Knipp & Zografi, 2012; Vasconcelos, Sarmento & Costa, 2007). This technological

45

tool can be defined as the molecular dispersion of a drug in one or more hydrophilic carriers

46

(Padden et al., 2011; Vasconcelos, Sarmento & Costa, 2007) and its mechanism involves the

47

reduction of the particle size, the enhancement of the wettability of the API in the carrier, the

48

formation of soluble complexes and the obtainment of an amorphous drug (Craig, 2002; Zhong,

49

Zhu, Luo & Su, 2013). In SDs, hydrophilic polymers also play an important role inhibiting the

50

crystallization of the API through decreasing its molecular mobility (Bhugra & Pikal, 2008;

51

Yonemochi, Sano, Yoshihashi & Terada, 2013). In this context, the selection of the ideal carrier

52

is crucial to formulation success (Li et al., 2013).

53

Hydroxypropylmethyl cellulose (HPMC), Fig. 1A, is a non-ionic and water soluble polymer

54

(Rogers, 2009), a mixed cellulose ether (Leuner & Dressman, 2000), which is of great

55

importance as a carrier in drug release systems, including SDs (Asare-Addo, Levina, Rajabi-

56

Siahboomin & Nokhodchi, 2011; Colombo, 1993; Leuner & Dressman, 2000; Pham & Lee,

57

1994). In this regard, HPMC is able to enhance the biopharmaceutical properties of many drugs.

58

Etravirine (Qi et al., 2013), fenofibrate (Kalivoda, Fischbach & Kleinebudde, 2012), mesalazine

59

(Ugandhar, 2012), tacrolimus (Yoshida et al., 2012), raloxifene hydrochloride (Shim et al.,

60

2013) and resveratrol (Wegiel, Mauer, Edgar & Taylor, 2013) are some of the most recent

61

examples. Publications in the literature report that HPMC can control the drug release rate due

62

to its swelling and dissolution properties in aqueous solution, which are probably associated

63

with the formation of soluble complexes between the water-soluble polymeric carrier and the

64

poorly-water-soluble drug (Oh et al., 2012).

65

Nimodipine (NMP), Fig. 1B, is a dihydropyridine calcium channel blocker recommended for

66

patients with subarachnoid hemorrhage (Nguyen, 2004). This compound, which presents low

67

bioavailability (only 13%) and poor solubility in water, belongs to class II in the

68

Biopharmaceutical Classification System (BCS) (Sweetman, 2009), which makes it an ideal

Ac ce p

te

d

M

an

us

cr

ip t

33

3 Page 3 of 28

69

candidate for SDs.

70

Modification I (Mod I) or the metastable form, a racemic compound that exhibits higher

71

solubility in water (360 ± 70 µg L-1), and Modification II (Mod II), the stable polymorph, which

72

is chemically a conglomerate (Grunenberg, Keil & Henck, 1995).

However, as an aggravating factor, it presents two polymorphs;

B RO H3C O OH

OR

H3C

O

OR

OR

O

CH 3 O

O

us

CH 3

O

CH3

O

OR O

H N

cr

A

ip t

73

NO2

O H

n– 2 2

OR

an

74 75

FIGURE 1

76

Some attempts to improve the biopharmaceutical properties of NMP through SDs obtained via

78

classical techniques have been reported (Adinarayana, Rajendran & Rao, 2010; Babu, Prasad &

79

Ramana-Murthy, 2002; Docoslis et al., 2007; Gorajana, Rao & Nee, 2011; Jijun et al., 2011;

80

Kreidel et al., 2012; Kumar, Kumar, Chanderparkash & Singh, 2009; Lu, Wang, Ding & Pan,

81

1995; Papageorgiou et al., 2009; Papaegorgiou, Docoslis, Georgarakis & Bikiaris, 2006;

82

Smikalla & Urbanetz, 2007; Urbanetz, 2006; Urbanetz & Lippold, 2005; Wu, Zhou & Zhang,

83

1998; Yunzhe, Rui, Wenliang & Tang, 2008; Zheng, Yang, Tang & Zheng, 2008). The

84

promising results obtained through these studies were determinant in the choice of NMP as a

85

model drug for the obtainment of novel SDs, prepared via an innovative technique and with a

86

potential carrier.

87

In comparison with other methods used to obtain SDs, high-energy milling has received less

88

attention in the literature (Patterson et al., 2007). However, it can be used to produce amorphous

89

materials (Boldyrev, Shakhtshneider, Burleva & Severtsev, 1994) or to form regions of

90

drug/excipient miscibility (Friedrich, Nada & Bodmeier, 2005). During the milling process,

91

sufficient strain is generated in the solid particles through a combination of impact and attrition,

92

producing structural changes on the crystal and the formation of amorphous regions, particularly

93

on the surface (Balani, Ng, Tan & Chan, 2010; Feng, Rodolfo & Carvajal, 2008; Mallick et al.,

94

2008). Unlike other techniques, milling is environmentally friendly, since it does not require the

95

use of organic solvents. Also, it consists of a relatively simple process which does not require

96

sophisticated equipment and which generally culminates in a decrease in the total experimental

Ac ce p

te

d

M

77

4 Page 4 of 28

97

time and easier scaling up (Barzegar-Jalali et al., 2010; Colombo, Grassi & Grass, 2009;

98

Gaisford, Dennison, Tawfik & Jones, 2010; Zhong, Zhu, Luo & Su, 2013).

99

In this context, the objective of this study was to verify the potential of using HPMC to obtain

100

amorphous and physically stable ball-milled SDs containing NMP. The aim of preparing these

101

formulations was to improve the biopharmaceutical properties of the API.

ip t

102 2. EXPERIMENTAL

104

2.1 MATERIALS

105

NMP was purchased from Chang Zhow ComWin Fine Chemicals (batch 20110305; Jiangsu,

106

China) and HPMC (Methocel K4M®) originating from Dow Chemical Company (Midland,

107

USA) was kindly donated by Colorcon do Brasil LTDA (batch TD 100112N11; Cotia, Brazil).

108

High-performance liquid chromatography (HPLC)-grade acetonitrile and methanol were

109

obtained from J.T. Baker (Phillipsburg, NJ). All other chemicals used were pharmaceutical

110

grade. During the analysis, ultrapure water was obtained using a Milli-Q Gradient System

111

(Millipore, Bedford, MA).

M

an

us

cr

103

112 2.2 METHODS

114

2.2.1 Preparation of ball-milled SDs

115

Firstly, the drug and carrier were accurately weighed to give a final mass of 2.5 g for all of the

116

formulations prepared. SDs composed of 1:9, 2:8 and 3:7 of NMP:HPMC (w/w) were denoted

117

as 1:9 SD, 2:8 SD and 3:7 SD, respectively. Samples were milled in a Spex Dual Mixer Mill

118

8000 D (New Jersey, USA) in 40 mL steel jars for 180 min, at a ball:powder ratio of 4:1 (w/w)

119

and with three steel balls (two with an external diameter of 6.4 mm and one of 12.8 mm).

120

Milling was performed at room temperature and no heating of the samples was noted at the end

121

of the process.

te

Ac ce p

122

d

113

123

2.2.2 Preparation of physical mixtures

124

Physical mixtures were prepared through simple spatulation of drug and carrier, accurately

125

weighed in different proportions. Samples were denoted as 1:9 PM, 2:8 PM and 3:7 PM,

126

corresponding to NMP:HPMC ratios (w/w) of 1:9, 2:8 and 3:7, respectively.

127

5 Page 5 of 28

128

2.2.3 Stability studies

129

The samples were stored in a desiccator under vacuum at 40 °C and with 75 % of relative

130

humidity (NaCl saturated solution). Solid state stability studies on the SDs of NMP were

131

conducted over a period of 90 days.

ip t

132 2.2.4 HPLC analysis

134

The LC analysis was performed through a stability-indicating LC-method previously described

135

in the literature (Riekes et al., 2013) and employing a Shimadzu LC-10A system (Kyoto, Japan)

136

equipped with an LC-10AD pump, DGU-14A degasser, SPD-10AV variable-wavelength

137

detector (set at 235 nm) and an SCL-10AVP system controller unit. The experiments were

138

conducted on a reversed-phase Phenomenex (Torrance, CA) Luna C18 column (250 x 4.6 mm

139

i.d., 5 mm). A security guard holder (C18, 4.0 x 3.0 mm i.d.) was employed to protect the

140

column. The mobile phase was isocratically composed of acetonitrile:methanol:water (55:11:34

141

v/v/v), with a flow rate of 0.5 mL min-1, at 40.0 °C. The samples (at a theoretical concentration

142

of 20 µg mL-1) were filtered through a 0.45 mm membrane filter and a volume of 20 µL was

143

injected. Data acquisition was performed using CLASS-VP software to measure the detected

144

peaks. The content of NMP in SDs was expressed as relative values in relation to the theoretical

145

content of the drug in formulations.

us

an

M

d

te

146

cr

133

2.2.5 Measurement of solubility

148

In order to determine the NMP solubility, an excess of drug in SDs was weighed and added to

149

30 mL of acetate buffer (pH 4.5) containing 0.3 % of sodium lauryl sulfate (SLS), giving a final

150

concentration of 2.0 mg mL-1. Samples were kept under stirring at 150 rpm in a thermostatically

151

controlled water bath (37 ± 2 °C) (Dist, model DI-06) until a constant concentration of NMP

152

was reached. At pre-determined intervals, aliquots were withdrawn (with immediate

153

replacement of fresh solution) and filtered through 0.22 µm filters before taking the readings on

154

a UV/VIS spectrophotometer (Cary 50 BIO, Varian) at 340 nm.

Ac ce p

147

155 156

2.2.6 Solid-state characterization of ball-milled SDs

157

2.2.6.1 X-ray powder diffraction (XRPD)

6 Page 6 of 28

X-ray diffraction patterns were obtained using a ! -! X-ray diffractometer (Xpert Pro Multi-

159

Purpose Diffractometer, PANAlytical) equipped with K! copper radiation (! = 1.5418 Å),

160

operating with a 40 mA current and 45 kV voltage, sample spinner and a Real Time Multiple

161

Strip (RTMS) detector. The measurements were performed at room temperature, scanning at 2!

162

from 5o to 50o, with a 0.03342o step size and 10.16 s step time.

163

During the stability studies, the diffractograms were acquired on a ! -! X-ray diffractometer (D2

164

Phaser, Bruker), operating with K! copper radiation (! = 1.5418 Å), at a current of 10 mA and

165

voltage of 30 kV. Detection was performed on a scintillation counter one-dimensional

166

LYNXEYE detector. The measurements were taken at room temperature, scanning at 2! from

167

5o to 50o, with a 0.091o step size.

us

cr

ip t

158

an

168 2.2.6.2 Thermal analysis

170

Differential scanning calorimetry (DSC) analysis was carried out in triplicate using a Shimadzu

171

calorimeter (DSC-60), operating in a temperature range of 40 – 130 °C. Samples weighing

172

approximately 2 mg were heated in sealed aluminum crucibles (model 201-53090) and scanned

173

at a first scan mode, at a heating rate of 2 °C min-1 and under nitrogen air atmosphere (50 mL

174

min-1). The DSC cell was previously calibrated with indium and zinc.

175

The mass loss of the samples as a function of temperature was determined in triplicate using a

176

Shimadzu thermobalance (TGA-50). Approximately 3 mg of samples were placed in open

177

platinum crucibles which were heated at a heating rate of 20 °C min−1, to temperatures ranging

178

from 40 to 800 °C under nitrogen atmosphere (50 mL min-1). The instrument was preliminarily

179

calibrated with a standard reference of calcium oxalate.

180

The thermal data obtained were processed using TA60 software.

d

te

Ac ce p

181

M

169

182

2.2.6.3 Fourier transform infrared (FTIR) spectroscopy

183

FTIR spectra were acquired on a Shimadzu spectrophotometer (FTIR Prestige), at room

184

temperature, from 4,000 to 400 cm−1, with the collection of 20 scans at a resolution of 4 cm−1.

185

Samples were prepared by mixing 2 % (w/w) of the drug, carrier, physical mixtures and SDs in

186

potassium bromide (KBr).

187

7 Page 7 of 28

2.2.6.4 Scanning electron microscopy (SEM)

189

The samples were mounted on aluminum stubs, sputtered with gold (IC-50 Ion Coater,

190

Shimadzu, Kyoto, Japan) and analyzed using a scanning electron microscope (SSX-550

191

Superscan, Shimadzu, Kyoto, Japan) at an accelerating voltage of 10 or 15 kV with different

192

magnifications.

cr

ip t

188

us

193 2.2.7 In vitro dissolution studies

195

Dissolution studies of the NMP and SDs were performed using the Varian VK 7000 dissolution

196

device and dissolution apparatus II (paddle), according to the specifications of British

197

Pharmacopoeia (British Pharmacopoeia, 2009), which describes the in vitro evaluation of NMP

198

tablets. A known amount of the drug (30 mg) was weighed and submitted to dissolution in 900

199

mL of previously-deaerated acetate buffer (pH 4.5) containing 0.3 % of SLS, kept at 37 ± 0.5 °C

200

and rotated at 75 rpm. At predetermined time intervals (5, 10, 15, 20, 25, 30, 45 and 60 min) 4

201

mL aliquots were withdrawn (with immediate replacement of fresh dissolution medium) and

202

filtered through 0.22 µm filters before readings were taken on a UV/VIS spectrophotometer

203

(Varian UV/VIS CARY) at 340 nm. The correction for the cumulative dilution was calculated.

204

Dissolution experiments were carried out in triplicate and in the absence of light.

205

Dissolution profiles for the NMP and SDs were compared by independent and dependent

206

methods. For the model-independent analysis, the dissolution efficiency (DE) of the drug and

207

SDs was established. In this analysis the area under the dissolution curve at time t was

208

calculated through the trapezoidal rule and expressed as the percentage of the rectangle area

209

described by 100% of dissolution within the same period. The area under the curve was

210

determined using the Image Tool software, version 3.0. In vitro profiles were also investigated

211

by model-dependent methods, and the data were tested to fit first-order, zero-order, Hixson-

212

Crowell and Higuchi models (O’Hara, Dunne, Butler & Denave, 1998; Polli, Rekhi & Shah,

213

1996). The selection of the model-dependent method was based on the best fit obtained,

214

determined by considering the correlation coefficient (r) most similar to 1. The semi-empirical

Ac ce p

te

d

M

an

194

8 Page 8 of 28

215

Korsmeyer-Peppas model was also evaluated correlating drug release to time through a simple

216

exponential equation (Mt/M∞ = k.tn). In this context, Mt/M∞ corresponds to the proportion of

217

drug released at time t, k is the kinetic constant, and it has been proposed that the exponent n is

218

indicative of the release mechanism (Korsmeyer, Gurny, Doelker, Buri & Peppas, 1983).

ip t

219 3. RESULTS AND DISCUSSION

221

3.1 HPLC ANALYSIS

222

The experimental and relative values regarding the contents of NMP in SDs were 100.61 ± 0.43

223

%, 98.98 ± 0.04 % and 99.43 ± 0.35 % for 1:9 SD, 2:8 SD and 3:7 SD, respectively. Also, no

224

degradation peak was observed in the chromatograms analyzed (Appendix A, Fig. A.1),

225

indicating the stability of the drug on employing this technique.

an

us

cr

220

226 3.2 MEASUREMENT OF SOLUBILITY

228

The results obtained for the NMP solubility indicated that improvements were achieved for all

229

of the SDs. Increases of 318.80 ± 1.31 %, 156.14 ± 1.29 % and 102.87 ± 0.99 % were observed

230

for 1:9 SD, 2:8 SD and 3:7 SD, respectively. These data indicate that the solubility of NMP in

231

these SDs was dependent on the carrier concentration, the best result being obtained for the

232

formulation in which the proportion of HPMC was 90 % with 10 % of NMP. Also, the

233

increased solubility of NMP in the presence of HPMC could be due to the formation of soluble

234

complexes between the water-soluble polymeric carrier and poorly soluble drug (Oh et al.,

235

2012).

d

te

Ac ce p

236

M

227

237

3.3 SOLID-STATE CHARACTERIZATION OF BALL-MILLED SDs

238

3.3.1 XRPD

239

X-ray diffraction patterns related to the NMP crystalline phases (Mod I and Mod II) and the raw

240

material, HPMC, physical mixtures and SDs are shown in Fig. 2.

9 Page 9 of 28

3:7 SD

ip t

2:8 SD 1:9 SD

cr



3:7 PM

us

2:8 PM 1:9 PM

an

 

Milled NMP

M



d

NMP raw material Mod II calc

Ac ce p

te



HPMC

0

241 242 243

10

20

Mod I calc 30

40

50

2? FIGURE 2

244

According to Grunenberg, Keil and Henck, NMP presents two crystalline phases: Modification

245

I (Mod I) or the metastable form, a racemic compound that exhibits a higher solubility in water

246

(360 ± 70 µg L-1), and Modification II (Mod II), the stable polymorph, chemically defined as a

247

conglomerate (Grunenberg, Keil & Henck, 1995). These crystalline modifications can be easily

248

distinguished through various solid-state techniques, and the XRPD analysis produced very

249

different diffractograms for these samples, especially at low 2θ angles. The reflection at 6.6 ° is

250

only observed for Mod I, whilst that at 9.3 ° is present exclusively for Mod II (Docoslis

251

et al., 2007; Guo et al., 2011; Riekes et al., 2012; Yang, Ke, Zi, Liu & Yu, 2008). Through these 10 Page 10 of 28

analyses it is possible to affirm that the raw material acquired for the development of the SDs is

253

composed exclusively of the metastable polymorph Mod I.

254

On the other hand, the carrier is completely amorphous, in agreement with previous data

255

reported in literature (Cilurzo, Minghetti, Casiraghi & Montanari, 2002; Oh et al., 2012). For

256

all of the physical mixtures, it is possible to observe the crystalline reflections of the drug

257

overlapping the amorphous halo of the polymer. These data indicate the absence of physical

258

interactions between NMP and HPMC and also verify that amorphous halos related to the SDs

259

are due to the preparation process.

260

In order to determine the drug behavior during milling without a polymeric carrier, NMP was

261

milled for 3 hours. It is extremely important to note that without HPMC the API did not become

262

amorphous. In addition, the initial polymorph Mod I underwent a partial solid-state transition to

263

the less soluble crystalline phase Mod II, since both characteristic reflections at 6.6 ° (Mod I)

264

and at 9.3 ° (Mod II) were observed for this sample. These data demonstrate the importance of

265

adding HPMC to obtain amorphous SDs containing NMP.

266

Also, as can be observed, all of the SDs showed amorphous characteristics, explaining the

267

increase in the solubility of NMP in these formulations. The enhanced solubility is the result of

268

the disordered structure of the amorphous solid. Because of the short-range intermolecular

269

interactions in an amorphous system no lattice energy has to be overcome, whereas in the

270

crystalline material the lattice has to be disrupted in order for it to dissolve (Sathigari,

271

Radhakrishnan, Davis, Parsons & Babu, 2012; Shah, Kakumanu & Bansal, 2006).

cr

us

an

M

d

te

Ac ce p

272

ip t

252

273

3.3.2 Thermal analysis

274

The DSC and thermogravimetric curves for the drug, carrier and SDs are shown in Fig. 3.

275

According to Grunenberg, Keil and Henck NMP polymorphs can be also distinguished by

276

means of DSC, since Mod I shows an endothermic event at 124 ± 1 °C and Mod II melts at 116

277

± 1 °C (Grunenberg, Keil & Henck, 1995). As can be observed, the NMP raw material

278

presented a single melting event at 124.1 °C, confirming its identity as pure Mod I. This data is

279

in agreement with the previous XRPD results. No endothermic event was observed for HPMC,

280

which is consistent with data reported in the literature (Asare-Addo, Levina, Rajabi-Siahboomi

281

& Nokhodchi, 2011). However, discrete endothermic events at 115 and 125 °C were observed

282

for the sample 3:7 SD, indicating a crystallization of NMP into its two polymorphs during the

283

DSC analysis, at elevated temperatures. The absence of crystallization or melting events for the

284

samples 1:9 SD and 2:8 SD, besides indicating their amorphous state, may be due to the lower

285

contents of drug in these samples, which are probably lower than the limits of detection for each 11 Page 11 of 28

polymorph (Riekes et al., 2012). It is very important to mention that the endothermic events

287

found for the sample 3:7 SD does not indicate that this SD is crystalline, since the X-ray

288

analysis verified its amorphous state. In addition, although a single glass transition temperature

289

(Tg) was expected to verify the miscibility of the amorphous phases in the obtained SDs

290

(Palermo, Anderson & Drennen III, 2012), none of the DSC curves showed it. This fact can be

291

explained by the very broad Tg of the heterogeneous HPMC.

ip t

286

292

cr

mW

NMP

us

A

HPMC

an

1:9 SD 2:8 SD

60

TG A

80 90 100 Temperature (\ C)

110

120

130 DrTGA (mg/min)

Ac ce p

B

70

te

50

d

M

3:7 SD

NMP NMP HPMC HPMC 1:9 H1 SD 2:8 H2 SD 3:7 H3 SD

) % ( ss ol t h gi e W

Temperature (\C)

NMP

HPMC 1:9 SD 2:8 SD 3:7 SD

100 293 294

200

300

400 500 600 Temperature (\ C)

700

800

FIGURE 3 12 Page 12 of 28

The thermogravimetric analysis revealed that the NMP was thermally stable up to

296

approximately 250 °C, presenting a single weight loss event (96.2 %) in the temperature range

297

analyzed. The thermal degradation peak, obtained through derivative thermogravimetry,

298

occurred at 347.2 °C. These data are in agreement with previous reports in the literature

299

(Cardoso, Rodrigues, Stulzer, Silva & Matos, 2005). A higher thermal stability was noted for

300

HPMC, also in a single stage of degradation. The thermal degradation occurs at between 335 °C

301

and 425 °C, with a peak temperature of 391 °C. The weight loss for HPMC was of 93.4 %. For

302

the SDs the thermal degradation occurred in two steps (as evidenced by the DrTGA curves), and

303

the relative proportion of the two degradation events is directly proportional to the NMP and

304

HPMC contents of the samples. The first event represents the presence of NMP, whilst the

305

second one is related to HPMC. The weight losses were 94.2 %, 93.7 % and 92.3 %, for 1:9

306

SD, 2:8 SD and 3:7 SD, respectively.

us

cr

ip t

295

an

307 3.3.3 FTIR spectroscopy

309

The FTIR spectra for NMP, HPMC, 1:9 PM and SDs are shown in Fig. 4.

HPMC

Ac ce p

) % ( ec n a ti 1:9 PM m s n a r T

te

NMP

d

M

308

1:9 SD

2:8 SD

3:7 SD 4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000

310 311

800

600

400

Wavenumber (cm-1)

FIGURE 4

13 Page 13 of 28

Characteristic bands for NMP appear at 3,293 cm-1, related to N-H stretching of the polymorph

313

Mod I, at 1,695 cm-1, associated with the carbonyl bond, and at 1,523 cm-1, representing the NO2

314

group (Barmpalexis, Kachrimanis & Georgarakis, 2011; Hu et al., 2003; Papadimitriou,

315

Papageorgiou, Kanaze, Georgarakis & Bikiaris, 2009; Tang, Pikal & Taylor, 2002). In the case

316

of HPMC, bands were observed at 3,443 cm-1 (O-H stretching), at 2,935 cm-1 (C-H stretching),

317

at 1,651 cm-1 (C=O of the glucose unit) and at 1,069 cm-1 (C-O-C group) (Anuar, Wui,

318

Ghodgaonkar & Taib, 2007; Oliveira, Bernardi, Murakami, Mendes & Silva, 2009). In the

319

physical mixture, all of the characteristic bands for the drug and carrier are overlapped,

320

indicating the absence of physical interactions between these compounds. On the other hand,

321

bands related to the amine group of NMP disappeared for all of SDs, confirming the amorphous

322

characteristic of these formulations (Barmpalexis, Kachrimanis & Georgarakis, 2011), as

323

previously observed through XRPD and DSC analysis. Also, although shifts for the bands

324

related to HPMC were not observed, the merging of the O-H band of the carrier with the N-H

325

band of NMP indicates the establishment of hydrogen bonding in SDs. It is important to

326

mention that this kind of interaction between drug and carrier is an additional benefit for the

327

SDs, since they can increase the solid solubility of the drug into the hydrophilic carrier besides

328

acting on inhibiting the crystallization of the drug from a glass solution (Marsac, Shamblin &

329

Taylor, 2006; Van den Mooter, 2012). Cilurzo, Minghetti, Casiraghi and Montanari also

330

observed the disappearance of N-H stretching vibrations for amorphous SDs composed of

331

nifedipine and HPMC (Cilurzo, Minghetti, Casiraghi & Montanari, 2002).

332

te

d

M

an

us

cr

ip t

312

3.3.3 SEM

334

Photomicrographs of the drug, carrier and SDs are shown in Appendix A (Fig. A. 2). Small

335

tabular crystals with a homogeneous particle size distribution can be observed for the NMP raw

336

material. However, particles with undefined shapes are observed for HPMC. From these images

337

it can be verified that the milling promoted considerable changes in the morphology of the NMP

338

and HPMC, generating agglomerates which did not seem to be influenced by the drug:carrier

339

proportion. It is important to mention that crystals of the drug were not observed on the surface

340

of the formulations, confirming its amorphous characteristic in SDs. Also, as no decrease in

341

particle size was noted for 1:9 SD, 2:8 SD and 3:7 SD, it is assumed that the enhancement in

342

NMP solubility is due to the amorphous characteristic, the establishment of hydrogen bonding

343

and the presence of a hydrophilic carrier in SDs, which probably improved the wettability of

344

these formulations.

Ac ce p

333

345

14 Page 14 of 28

346

3.4 IN VITRO DISSOLUTION STUDIES

347

The release rates of the different SDs of NMP in acetate buffer (pH 4.5) containing 0.3 % of

348

SLS and of the pure drug are compared in Fig. 5.

ip t

100

an

us

cr

) % ( 80 se a el er P 60 M N e v it 40 a l u m u 20 C 0 0

10

20

40

50

60

Time (minutes)

M

349 350

d

FIGURE 5

te

351

30

As can be observed, all of the SDs were able to increase the dissolution rate of NMP. These data

353

are in agreement with the enhancement of the drug solubility in these formulations. The

354

formulation 1:9 SD promoted the release of approximately 100 % of NMP in 60 min, which

355

indicates a 3-fold increase in the drug dissolution during this period. Also, 2:8 SD and 3:7 SD

356

presented very similar release profiles for up to 30 min, after which 2:8 SD demonstrated a

357

higher dissolution rate. According to Kogermann and coworkers, this deviation in the

358

dissolution behaviors in the later stages of a dissolution testing could be associated with the

359

formation of physically different diffusion layers around the drug:polymer particles, since it is

360

well known that the dissolution of the drug follows the dissolution of the polymer (Kogermann

361

et al., 2013). The values for the percentage drug release of these SDs were 91.2 ± 5.9 % (2:8

362

SD) and 78.8 ± 1.4 % (3:7 SD) at the end of the assay. These results indicate that the

363

improvement of this biopharmaceutical property was dependent on the drug:carrier ratio, the

364

best results being obtained for the SD with the highest proportion of HPMC.

365

It is also important to mention that the dissolution data for 1:9 SD, 2:8 SD and 3:7 SD were

366

within the acceptance criteria of deviation required by the United States Pharmacopoeia. This

367

means that the relative standard deviation (RSD) values at time intervals of 10 min or less were

Ac ce p

352

15 Page 15 of 28

lower than 20 %, and for longer intervals they were lower than 10 %. The fulfillment of this

369

condition is essential, since a high variability in the results can hinder the identification of

370

trends or effects resulting from changes in the formulation (USP, 2011). Also, achieving the

371

desired deviation range is even more challenging for HPMC-based formulations, since HPMC is

372

known to form hydrogels which slowly erode in aqueous solutions and can result in variability

373

among samples (Kim et al., 2006).

374

Data related to the analysis of the dissolution profiles through independent and dependent

375

methods are shown in Table 1.

376

Table 1. Dissolution efficiency (DE), period in which 50 % release of NMP occurs (t50%) and dissolution

377

rate constant (k) for SDs

cr

t50% (min) 242.73 246.23 318.88

us

DE (%) ± standard deviation 71.5 ± 0.9 59.0 ± 3.6 58.8 ± 3.7

an

Sample 1:9 SD 2:8 SD 3:7 SD

k (min-1) 16.00 12.88 12.09

M

378 379 380 381 378 382

ip t

368

Regarding the dissolution efficiency (DE), SDs increased this value considerably, since for the

384

pure drug the DE was 19.4 ± 0.6 %. Increases in this parameter by up to a factor of four were

385

noted for the formulations obtained in comparison to NMP. The release profiles were also tested

386

through different mathematical models and the best fit was observed for the Higuchi model.

387

According to this model, when exposed to the solvent, both the drug and carrier dissolve at rates

388

proportional to their solubility and diffusion coefficients in the dissolving medium. In this

389

context, the dissolution rate of the minor component may be determined by the component in

390

excess. In turn, this may be applied to solid dispersal systems by arguing that when the drug is

391

present as a minority component its dissolution will be dominated by the dissolution behavior of

392

the carrier (Corrigan, 1986; Craig, 2002; Higuchi, 1967; Higuchi, Mir & Desai, 1965).

393

The Korsmeyer-Peppas model correlates the drug release to time through a simple exponential

394

equation for a drug release fraction of < 0.6. This model has been used to evaluate the drug

395

release from polymeric devices, especially when the release mechanism is unknown or when

396

there is more than one mechanism involved (Korsmeyer, Gurny, Doelker, Buri & Peppas,

397

1983). In this context, n ≤ 0.43 indicates Fickian release and n = 0.85 indicates a purely

398

relaxation-controlled delivery which is referred to as Case II transport. Intermediate values (0.43

399

< n < 0.85) indicate an anomalous behavior, described as a non-Fickian kinetics corresponding

Ac ce p

te

d

383

16 Page 16 of 28

to coupled diffusion and polymer relaxation. Occasionally, values of n > 1 have been observed

401

which are regarded as Super Case II kinetics (Ritger & Peppas, 1987; 1987a).

402

The results obtained through the application of the Korsmeyer-Peppas model show calculated n

403

values of 2.92, 2.2 and 2.3, for 1:9 SD, 2:8 SD and 3:7 SD, respectively, indicating that the

404

NMP was released via Super Case II transport. In the case of this mechanism, it is proposed that

405

the drug release involves diffusion, swelling, relaxation and erosion (Ritger & Peppas, 1987a)

406

of the polymeric component.

ip t

400

cr

407 3.5 STABILITY STUDIES

409

Stability studies on the physical state of the SDs were performed through XRPD, DSC and

410

FTIR spectroscopy. The data obtained are shown in Figures 6 and 7.

an

us

408

411

M

A

d

2:8 SD 40 \C/75 % 1:9 SD 40 \C/75 %

te Ac ce p 0

10

20

3:7 SD 40 \C/75 %

3:7 SD desiccator 2:8 SD desiccator 1:9 SD desiccator

2?

30

40

50

B

3:7 SD 40 \ C/75 % 2:8 SD 40 \ C/75 % 1:9 SD 40 \ C/75 % 3:7 SD desiccator 2:8 SD desiccator 1:9 SD desiccator

50

412

60

70

80

90 100 110 120 Temperature ( \ C)

130

140

150

17 Page 17 of 28

413

FIGURE 6

1:9 SD desiccator

ip t

2:8 SD desiccator

) % ( ec n a ti m s n a r T

cr

3:7 SD desiccator

M

an

us

1:9 SD 40 \ C/75 %

2:8 SD 40 \ C/75 %

3:7 SD 40 \ C/75 %

400

FIGURE 7

te

416

600

Ac ce p

414 415

800

d

4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 Wavenumber (cm-1)

417

As can be observed through XRPD data, all of the SDs remained amorphous for 90 days in a

418

desiccator. No changes were observed in the FTIR spectra, indicating the maintenance of the

419

initial interactions between the drug and carrier. These data suggest that the formulations

420

remained stable under this condition. Also, the crystallization of the sample 3:7 SD during the

421

DSC analysis was still observed and its cause was already discussed in topic 3.3.2.

422

On the other hand, distinct results were obtained when the samples were submitted to more

423

severe conditions of temperature and humidity. At 40 °C/75 % RH, only the sample 1:9 SD

424

remained amorphous, as indicated by the XRPD, DSC and FTIR spectroscopy data. The X-ray

425

diffractograms for 2:8 SD and 3:7 SD clearly indicated the recrystallization of these samples,

426

and the characteristic reflection of Mod II, at 9.3 °, was observed for the formulation 3:7 SD.

427

These data were confirmed by the DSC results and the thermal analysis also revealed the

428

polymorphic mixture of 2:8 SD. The spectroscopy analysis shows the appearance of amine

429

group bands related to crystalline NMP at 3,269 cm-1 for the samples 2:8 SD and 3:7 SD, 18 Page 18 of 28

indicating the presence of the crystalline phase Mod II (Barmpalexis, Kachrimanis &

431

Georgarakis, 2011; Tang, Pikal & Taylor, 2002).

432

Reports in the literature indicate that exposure to high humidity leads to a greater absorption of

433

moisture by the bulk structure of SDs, in addition to surface adsorption, compared to physical

434

mixtures of similar composition. This absorbed water content can induce plasticization and

435

enhance molecular mobility, thereby leading to crystallization (Ahlneck & Zografi, 1990;

436

Sakurai, Sako & Maitani, 2012; Takeuchi, Yasuji, Yamamoto & Kawashima, 2000).

437

It is well known that thermodynamically unstable systems, like amorphous systems, tend to

438

undergo recystallization to their more stable state at any given moment (Serajuddin, 1999).

439

However, various studies have demonstrated that the presence of hydrophilic carriers inhibits

440

the recrystallization of amorphous drugs (Bhugra & Pikal, 2008; Bikiaris, 2011; Leuner &

441

Dressman, 2000; Newman; Knipp & Zografi, 2012; Sethia & Squillante, 2003; Van den Mooter,

442

2012). This inhibition is generally attributed to the interaction between drug and carrier,

443

promoted mainly through hydrogen bonds, and to the dispersion of drug molecules in the

444

polymeric matrix during the preparation process (Leuner & Dressman, 2000). In the case of SDs

445

of NMP, the increase in the stability observed when the HPMC concentration was increased

446

from 70 to 90 % (w/w) was attributed to the dilution mechanism, where the drug was diluted in

447

the polymer and the diffusion pathway for crystallization increased, thereby slowing the process

448

(Bhugra & Pikal, 2008; Konno & Taylor, 2006).

449

te

d

M

an

us

cr

ip t

430

4. CONCLUSIONS

451

SDs composed of NMP and HPMC, in different proportions, were successfully obtained

452

through ball milling and achieved the desired aims. All of the solid-state techniques

453

demonstrated that 1:9 SD, 2:8 SD and 3:7 SD were amorphous and FTIR data verified the

454

establishment of hydrogen bonding between drug and carrier.

455

biopharmaceutical properties of the API was achieved for all formulations, although the best

456

results were attributed to the sample 1:9 SD. This SD was able to improve the drug solubility by

457

up 318 % and the NMP dissolution rate by up to a factor four. In addition to promoting the best

458

performance in terms of these properties, a higher proportion of HPMC in the formulations led

459

to a greater stability of the SDs at 40 °C/75 % RH, due to a dilution mechanism. No changes

460

were noted for the SDs over 90 days of storage in a desiccator, with respect to the physical

461

characteristics. These results verify the potential of HPMC as a polymeric carrier for ball-milled

462

SDs of NMP and highlight the future perspectives for these systems, especially for the sample

Ac ce p

450

The enhancement of the

19 Page 19 of 28

463

1:9 SD, which include the obtainment of solid dosage forms and the determination of their

464

bioavailability compared with commercial formulations.

465 Acknowledgments

467

The authors acknowledge the Brazilian governmental agencies CNPq, FAPESC and CAPES for

468

student scholarships and financial support. Initial XRPD analysis was carried out at the X-ray

469

diffraction Lab (LDRX) of the Physics Department, at the Federal University of Santa Catarina

470

(UFSC). The authors also wish to thank Dr. Milton Domingues Michel from the State

471

University of Ponta Grossa (UEPG) for the SEM measurements and Dr. Siobhan Wiese for

472

revising the manuscript. Thanks are also due to the Consejo Superior de Investigaciones

473

Científicas (CSIC) in Spain, for awarding a license for the use of the Cambridge Structural

474

Database (CSD).

an

us

cr

ip t

466

475 REFERENCES

477 478 479

Adinarayana, G.; Rajendran, A. & Rao, N. K. (2010). Preparation and in vitro evaluation of solid dispersions of nimodipine using PEG 4000 and PVP K30. Journal of Pharmaceutical Research and Health Care,2, 163-169.

480 481

Ahlneck, C. & Zografi, G. (1990). The molecular basis of moisture effects on the physical and chemical stability of drugs in the solid state. International Journal of Pharmaceutics, 62, 87-95.

482 483 484

Alonzo, D. E.; Gao, Y.; Zhou, D.; Mo, H.; Zhang, G. G. Z. & Taylor, L. S. (2011). Dissolution and precipitation behavior of amorphous solid dispersions. Journal of Pharmaceutical Sciences, 100, 33163331.

485 486 487

Anuar, N. K.; Wui, W. T.; Ghodgaonkar, D. K. & Taib, M. T. (2007). Characterization of hydroxypropylmethylcellulose films using microwave non-destructive testing technique. Journal of Pharmaceutical and Biomedical Analysis, 43, 549-557.

488 489 490

Asare-Addo, K.; Levina, M.; Rajabi-Siahboomi, A. R. & Nokhodochi, A. (2011). Effect of ionic strength and pH of dissolution media on theophylline release from hypromellose matrix tablets – Apparatus USP III, simulated fast and fed conditions. Carbohydrate Polymers, 86, 85-93.

491 492 493

Babu, G. V.; Prasad, C. D. S. & Ramana-Murthy, K. V. (2002). Evaluation of modified gum karaya as carrier for the dissolution enhancement of poorly water-soluble drug nimodipine. International Journal of Pharmaceutics, 234, 1-17.

494 495 496

Balani, P. N.; Ng, W. K.; Tan, R. B. H. & Chan, S. Y. (2010). Influence of excipients in comilling on mitigating milling-induced amorphization or structural disorder of crystalline pharmaceutical actives. Journal of Pharmaceutical Sciences, 99, 2462-2474.

497 498 499

Barmpalexis, P.; Kachrimanis, K. & Georgarakis, E. (2011). solid dispersions in the development of a nimodipine floating tablet formulation and optimization by artificial neural networks and genetic programming. European Journal of Pharmaceutics and Biopharmaceutics, 77, 122-131.

500 501 502

Barzegar-Jalali, M.; Valizadeh, H.; Shadbad, M. S.; Adibkia, K.; Mohammadi, G.; Farahani, A.; Arash, Z. & Nokhodchi, A. (2010). Cogrinding as an approach to enhance dissolution rate of a poorly water-soluble drug (glicazide). Powder Technology, 197, 150-158.

503 504

Bhugra, C. & Pikal, M. J. (2008). Role of thermodynamic, molecular, and kinetic factors in crystallization from the amorphous state. Journal of Pharmaceutical Sciences, 97, 1329-1349.

Ac ce p

te

d

M

476

20 Page 20 of 28

Bikiaris, D. N. (2011). Solid dispersions, part II: New strategies in manufacturing methods for dissolution rate enhancement of poorly water-soluble drugs. Expert Opinion on Drug Delivery, 8, 1663-1680.

507 508 509

Boldyrev, V. V.; Shakhtshneider, T. P.; Burleva, L. P. & Severtsev, V. A. (1994). Preparation of the disperse systems of sulfathiazole-polyvinylpyrrolidone by mechanical activation. Drug Development and Industrial Pharmacy, 20, 1103-1113.

510

British Pharmacopoeia. (2009). The Department of Health. London.

511 512 513

Cardoso, T. M.; Rodrigues, P. O.; Stulzer, H. K, Silva, M. A. S. & Matos, J. R. (2005). Physical-chemical characterization and polymorphism determination of two nimodipine samples deriving from distinct laboratories. Drug Development and Industrial Pharmacy, 31, 631-637.

514 515

Cilurzo, F.; Minghetti, P.; Casiraghi, A. & Montanari, L. (2002). Characterization of nifedipine solid dispersions. International Journal of Pharmaceutics, 242, 313-317.

516 517

Colombo, I.; Grassi, G. & Grass, M. (2009). Drug mechanochemical activation. Journal of Pharmaceutical Sciences, 98, 3961-3986.

518 519

Colombo, P. (1993). Swelling-controlled release in hydrogel matrices for oral route. Advanced Drug Delivery Reviews, 11, 37-57.

520 521 522

Corrigan, O.I. (1986). Retardation of polymeric carrier dissolution by dispersed drugs: Factors influencing the dissolution of solid dispersions containing polyethylene glycols. Drug Development and Industrial Pharmacy, 12, 1777–1793.

523 524

Craig, D. Q. M. (2002). The mechanisms of drug release from solid dispersions in water-soluble polymers. International Journal of Pharmaceutics, 231, 131-144.

525 526 527 528

Docoslis, A.; Huszarik, K. L.; Papageorgiou, G. Z.; Bikiaris, D.; Stergiou, A. & Georgarakis, A. (2007). Characterization of the distribution, polymorphism, and stability of nimodipine in its solid dispersions in polyethylene glycol by Micro-Raman spectroscopy and powder X-ray diffraction. The AAPS Journal, 9, E361-E370.

529 530 531

Feng, T.; Rodolfo, P. & Carvajal, M. T. (2008). Process induced disorder in crystalline materials: Differentiating defective crystals from the amorphous form of griseofulvin. Journal of Pharmaceutical Sciences, 97, 3207-3221.

532 533 534

Friedrich, H.; Nada, A. & Bodmeier, R. (2005). Solid state and dissolution rate characterization of coground mixtures of nifedipine and hydrophilic carriers. Drug Development and Industrial Pharmacy, 31, 719-728.

535 536 537

Gaisford, S.; Dennison, M.; Tawfik, M. & Jones, M. D. (2010). Following mechanical activation of salbutamol sulphate during ball-milling with isothermal calorimetry. International Journal of Pharmaceutics, 393, 74-78.

538 539

Gorajana, A.; Rao, N. K. & Nee, W. Y. (2011). Physicochemical characterization and in vitro evaluation of solid dispersions of nimodipine. Asian Journal of Chemistry, 23, 2857-2859.

540 541

Grunenberg, A.; Keil, B. & Henck, J. O. (1995). Polymorphism in binary mixtures, as exemplified by nimodipine. International Journal of Pharmaceutics, 118, 11-21.

542 543 544

Guo, Z.; Ma, M.; Wang, T.; Chang, D.; Jiang, T. & Wang, S. (2011). A kinetic study of the polymorphic transformation of nimodipine and indomethacin during high shear granulation. AAPS PharmSciTech, 12, 610-619.

545 546

Higuchi, W.I. (1967). Diffusional models useful in biopharmaceutics. Journal of Pharmaceutical Sciences, 56, 315–324.

547 548

Higuchi, W.I.; Mir, N.A. & Desai, S.J. (1965). Dissolution rates of polyphase mixtures. Journal of Pharmaceutical Sciences, 54, 1405–1410.

549 550 551

Hu, Y.; Jiang, X.; Ding, Y.; Zhang, L.; Yang, C.; Zhang, J.; Chen, J. & Yang, Y. (2003). Preparation and drug release behaviors of nimodipine-loaded poly(caprolactone)-poly(ethylene oxide)-polylactide amphiphilic copolymer nanoparticles. Biomaterials, 24, 2395-2404.

552 553 554

Jijun, F.; Lishuang, X.; Xiaoli, W.; Shu, Z.; Xiaoguang, T.; Xingna, Z.; Haibing, H. & Xing, T. (2011). Nimodipine (NM) tablets with high dissolution containing NM solid dispersions prepared by hot-melt extrusion. Drug Development and Industrial Pharmacy, 37, 934-944.

Ac ce p

te

d

M

an

us

cr

ip t

505 506

21 Page 21 of 28

Kalivoda, A.; Fischbach, M. & Kleinebudde, P. (2012). Application of mixtures of polymeric carriers for dissolution enhancement of fenofibrate using hot-melt extrusion. International Journal of Pharmaceutics, 429, 58-68.

558 559 560

Kim, E-J.; Chun, M-K.; Jang, J-S.; Lee, I-H.; Lee, K-R & Choi, H-K. (2006). Preparation of a solid dispersion of felodipine using a solvent wetting method. European Journal of Pharmaceutics and Biopharmaceutics, 64, 200-205.

561 562 563

Kogermann, K.; Penkina, A.; Predbannikova, K.; Jeeger, K.; Veski, P.; Rantanen, J. & Naelapää, K. (2013). Dissolution testing of amorphous solid dispersions. International Journal of Pharmaceutics, 444, 40-46.

564 565 566

Konno, H.; Handa, T.; Alonzo, D. E. & Taylor, L. S. (2008). Effect of polymer type on the dissolution profile of amorphous solid dispersions containing felodipine. European Journal of Pharmaceutics and Biopharmaceutics, 70, 493-499.

567 568

Konno H. & Taylor, L. S. (2006). Influence of different polymers on the crystallization tendency of molecularly dispersed amorphous felodipine. Journal of Pharmaceutical Sciences, 95, 2692–2705.

569 570

Korsmeyer, R. W.; Gurny, R.; Doelker, E.; Buri, P. & Peppas, N. A. (1983). Mechanisms of solute release from porous hydrophilic polymers. International Journal of Pharmaceutics, 15, 25-35.

571 572 573

Kreidel, R. N.; Duque, M. D.; Serra, C. H. R.; Velasco, M. V. R.; Baby, A. R.; Kaneko, T. M. & Consiglieri, V. O. (2012). Dissolution enhancement and characterization of nimodipine solid dispersions with Poloxamer 407 or PEG 6000. Journal of Dispersion Science and Technology, 33, 1354-1359.

574 575 576

Ku, M. S. & Dulin, W. (2010). A biopharmaceutical classification-based Right-First-Time formulation approach to reduce human pharmacokinetic variability and project cycle time from First-In-Human to clinical Proof-Of-Concept. Pharmaceutical Development & Technology, 17, 285-302.

577 578 579

Kumar, S.; Kumar, P.; Chanderparkash, A. & Singh, S. K. (2010). Evaluation of some novel techniques for dissolution enhancement of poorly water soluble drug nimodipine. International Journal of PharmTech Research, 2, 950-959.

580 581

Leuner, C. & Dressman, J. (2000). Improving drug solubility for oral delivery using solid dispersions. European Journal of Pharmaceutics and Biopharmaceutics, 50, 47-60.

582 583 584

Li, B.; Konecke, S.; Harich, K.; Wegiel, L.; Taylor, L. S. & Edgar, K. J. (2013). Solid dispersion of quercetin in cellulose derivative matrices influences both solubility and stability. Carbohydrate Polymers, 92, 2033-2040.

585 586

Lu, J.; Wang, M.; Ding, P. & Pan, Z. (1995). Preparation and dissolution of nimodipine-PEG solid dispersion. Chinese Pharmaceutical Journal,30, 23-25.

587 588 589

Mallick, S.; Pattnaik, S.; Swain, K.; De, P. K.; Saha, A.; Ghoshal, G. & Mondal, A. (2008). Formation of physically stable amorphous phase of ibuprofen by solid state milling with kaolin. European Journal of Pharmaceutics and Biopharmaceutics, 68, 346-351.

590 591

Marsac, P. J.; Shamblin, S. L. & Taylor, L. S. (2006). Theoretical and practical approaches for prediction of drug-polymer miscibility and solubility. Pharmaceutical Research, 23, 2417-2426.

592 593

Newman, A.; Knipp, G. & Zografi, G. (2012). Assessing the performance of amorphous solid dispersions. Journal of Pharmaceutical Sciences, 101, 1355-1377.

594 595

Nguyen, H. (2004). Bioequivalence reviews: ANDA 76. In Division of bioequivalence review, U. S. Food and Drug Administration.

596 597

O’Hara, T.; Dunne, A; Butler, J. & Denave, J. A. (1998). Review of methods used to compare dissolution profile data. Pharmaceutical Science and Technology Today, 1, 214-223.

598 599 600

Oh, M. J.; Shim, J. B.; Yoo, H.; Lee, G. Y.; Jo, H.; Jeong, S. M.; Yuk, S. H.; Lee, D. & Khang, G. (2012). The dissolution property of raloxifene HCl solid dispersion using hydroxypropyl methylcellulose. Macromolecular Research, 20, 835-841.

601 602 603

Oliveira, P. R.; Bernardi, L. S.; Murakami, F. S.; Mendes, C. & Silva, M. A. S. (2009). Thermal characterization and compatibility studies of norfloxacin for development of extended release tablets. Journal of Thermal Analysis and Calorimetry, 97, 741-745.

Ac ce p

te

d

M

an

us

cr

ip t

555 556 557

22 Page 22 of 28

Padden, B. E.; Mill, J. M.; Robbins, T.; Zocharski, P. D.; Prasad, L.; Spence, J. K. & La Fountaine, J. (2011). Amorphous solid dispersions as enabling formulations for discovery and early development. American Pharmaceutical Reviews, 1, 66-73.

607 608 609

Palermo, R. N.; Anderson, C. A. & Drennen III, J. K. (2012). Review: Use of thermal, diffraction, and vibrational analytical methods to determine mechanisms of solid dispersion stability. Journal of Pharmaceutical Innovations, 7, 2-12.

610 611 612

Papadimitriou, S.; Papageorgiou, G. Z.; Kanaze, F. I.; Georgarakis, M. & Bikiaris, D. N. (2009). Nanoencapsulation of nimodipine in novel biocompatible poly(propylene-co-butylene succinate) aliphatic copolyesters for sustained release. Nanomaterials, 2009, 1 – 11.

613 614 615 616

Papageorgiou, G. Z.; Bikiaris, D.; Karavas, E.; Politis, S.; Docoslis, A.; Park, Y.; Stergiou, A. & Georgarakis, E. (2006). Effect of physical state and partice size distribution on dissolution enhancement of nimodipine/PEG solid dispersions prepared by melt mixing and solvent evaporation. The AAPS Journal, 8, E623-E631.

617 618 619

Papageorgiou, G. Z.; Docoslis, A.; Georgarakis, M. & Bikiaris, D. (2009). The effect of physical state on the drug dissolution rate: miscibility studies of nimodipine with PVP. Journal of Thermal Analysis and Calorimetry, 95, 903-915.

620 621 622

Patterson, J. E.; James, M. B.; Forster, A. H.; Lancaster, R. W.; Butler, J. M. & Rades, T. (2007). Preparation of glass solutions of three poorly water soluble drugs by spray drying, melt extrusion and ball milling. International Journal of Pharmaceutics, 336, 22-34.

623 624

Pham, A. T. & Lee, P. I. (1994). Probing the mechanisms of drug-release from hydroxypropylmethyl cellulose matrices. Pharmaceutical Research, 11, 1379-1384.

625 626

Polli, J.E.; Rekhi, G.S. & Shah, V.P. (1996). Methods to compare dissolution profiles. Drug Information Journal, 30, 1113-1120.

627 628 629

Qi, S.; Roser, S.; Edler, K. J.; Pigliacelli, C.; Rogerson, M.; Weuts, I.; Van Dycke, F. & Stokbroekx, S. (2013). Insights into the role of polymer-surfactant complexes in drug solubilisation/stabilisation during drug release from solid dispersions. Pharmaceutical Research, 30, 290-302.

630 631 632 633

Riekes, M. K.; Pereira, R. N.; Rauber, G. S.; Cuffini, S. L.; Campos, C. E. M.; Silva, M. A. S. & Stulzer, H. K. (2012). Polymorphism in nimodipine raw materials: Development and validation of a quantitative method through differential scanning calorimetry. Journal of Pharmaceutical and Biomedical Analysis, 70, 188-193.

634 635 636

Riekes, M. K.; Rauber, G. S.; Kuminek, G.; Tagliari, M. P.; Cardoso, S. G. & Stulzer, H. K. (2013). Determination of nimodipine in the presence of its degradation products and overall kinetics through a stability-indicating LC method. Journal of Chromatographic Science, 51, 511-516.

637 638 639

Ritger, P. L. & Peppas, N. A. (1987). A simple equation for description of solute release I. Fickian and non-fickian release from non-swellable devices in the form of slabs, spheres, cylinders or discs. Journal of Controlled Release, 5, 23-36.

640 641

Ritger, P. L. & Peppas, N. A. (1987a). A simple equation for description of solute release II. Fickian and anomalous release from swellable devices. Journal of Controlled Release, 5, 37-42.

642 643

Rogers, T. L. (2009). Hypromellose. In R. C. Rowe, P. J. Sheskey & M. E. Quinn. (6th ed.), Handbook of pharmaceutical excipients (pp. 326-329). London: Pharmaceutical Press.

644 645 646

Sakurai, A.; Sako, K. & Maitani, Y. (2012). Influence of manufacturing factors on physical satbility and solubility of solid dispersions containing a low glass transition temperature drug. Chemical Pharmaceutical Bulletim, 60, 1366-1371.

647 648 649

Sathigari, S. K.; Radhakrishnan, V. K.; Davis, V. A.; Parsons, D. L. & Babu, R. J. (2012). Amorphousstate characterization of efavirenz – Polymer hot-melt extrusion systems for dissolution enhancement. Journal of Pharmaceutical Sciences, 101, 3456-3464.

650 651

Serajuddin, A. T. (1999). Solid dispersion of poorly water-soluble drugs: early promises, subsequent problems, and recent breakthroughs. Journal of Pharmaceutical Sciences, 88, 1058-1066.

652 653

Sethia, S. & Squillante, E. (2003). Solid dispersions: revival with greater possibilities and applications in oral drug delivery. Critical Review on Therapy and Drug Carrier Systems, 20, 215-247.

Ac ce p

te

d

M

an

us

cr

ip t

604 605 606

23 Page 23 of 28

Shah, B,; Kakumanu, V. K. & Bansal, A. K. (2006). Analytical techniques for quantification of amorphous/crystalline phases in pharmaceutical solids. Journal of Pharmaceutical Sciences, 95, 16411665.

657 658 659

Shim, J. B.; Lee, J. K.; Jo, H.; Hwang, J. H.; Jeong, S. M.; Jo, J. I.; Lee, D.; Yuk, S. H. & Khang, G. (2013). Effect of acidifier on the dissolution property of a solid dispersion of raloxifene HCl. Macromolecular Research, 21, 42-48.

660 661 662

Smikalla, M. M. & Urbanetz, N. A. (2007). The influence of povidone K17 on the storage stability of solid dispersions of nimodipine and polyethylene glycol. European Journal of Pharmaceutics and Biopharmaceutics, 66, 106-112.

663 664

Svenson, S. (2009). Dendrimers as versatile platform in drug delivery applications. European Journal of Pharmaceutics and Biopharmaceutics, 71, 445-462.

665

Sweetman, S. C. (2009). The complete drug reference. London: Pharmaceutical Press, pp. 1357-1358.

666 667 668

Takeuchi, H.; Yasuji, T.; Yamamoto, H. & Kawashima, Y. (2000). Temperature- and moisture-induced crystallization of amorphous lactose in composite particles with sodium alginate prepared by spraydrying. Pharmaceutical Development and Technology, 5, 355-363.

669 670 671

Tang, X. C.; Pikal, M. J. & Taylor, L. S. (2002). Spectroscopic investigation of hydrogen bond patterns in crystalline and amorphous phases in dihydropiridine calcium channel blockers. Pharmaceutical Research, 19, 477-483.

672

The United States Pharmacopoeia Monographs. (2011). 34th ed., National Formulary 29.

673 674

Ugandhar, C. (2012). Formation and evaluation of mesalazine solid dispersion. Research Journal of Pharmacy and Technology, 5, 809-812.

675 676 677

Urbanetz, N. A. & Lippold, B. C. (2005). Solid dispersions of nimodipine and polyethylene glycol 2000: Dissolution properties and pysico-chemical characterization. European Journal of Pharmaceutics and Biopharmaceutics, 59, 107-118.

678 679

Urbanetz, N. A. (2006). Stabilization of solid dispersions of nimodipine and polyethylene glycol 2000. European Journal of Pharmaceutical Sciences, 28, 67-76.

680 681

Van den Mooter, G. (2012). The use of amorphous solid dispersions: A formulation strategy to overcome poor solubility and dissolution rate. Drug Discovery Today: Technologies, 9, e79-e85.

682 683

Vasconcelos, T.; Sarmento, B. & Costa, P. (2007). Solid dispersions as strategy to improve oral bioavailability of poor water soluble drugs. Drug Discovery Today, 12, 1068-1075.

684 685 686

Wegiel, L. A.; Mauer, L. J.; Edgar, K. J. & Taylor, L. S. (2013). Crystallization of amorphous solid dispersions of resveratrol during preparation and storage – Impact of different polymers. Journal of Pharmaceutical Sciences, 102, 171-184.

687 688

Wu, W.; Zhou, Q. & Zhang, H. (1998). Preparation and in vitro evaluation of nimodipine solid dispersions. Chinese Pharmaceutical Journal, 33, 29-32.

689 690 691

Yang, X.; Ke, W.; Zi, P.; Liu, F. & Yu, L. (2008). Detecting and identifying the complexation of nimodipine with hydroxypropyl-β-cyclodextrin present in tablets by Raman spectroscopy. Journal of Pharmaceutical Sciences, 97, 2702-2719.

692 693

Yonemochi, E.; Sano, S.; Yoshihashi, Y. & Terada K. (2013). Diffusivity of amorphous drug in solid dispersion. Journal of Thermal Analysis and Calorimetry, in press.

694 695 696

Yoshida, T.; Kurimoto, I.; Yoshihara, K.; Umejima, H.; Ito, N.; Watanabe, S.; Sako, K. & Kikuchi, A. (2012). Aminoalkyl methacrylate copolymers for improving the solubility of tacrolimus. I: Evaluation of solid dispersion formulations. International Journal of Pharmaceutics, 428, 18-24.

697 698

Yunzhe, S.; Rui, Y.; Wenliang, Z. & Tang, X. (2008). Nimodipine semi-solid capsules containing solid dispersion for improving dissolution. International Journal of Pharmaceutics, 359, 144-149.

699 700

Zheng, X.; Yang, R.; Tang, X. & Zheng, L. (2007). Part I: Characterization of solid dispersions of nimodipine prepared by hot-melt extrusion. Drug Development and Industrial Pharmacy, 33, 791-802.

701 702 703

Zhong, L.; Zhu, X.; Luo, X. & Su, W. (2013). Dissolution properties and physical characterization of telmisartan-chitosan solid dispersions prepared by mechanochemical activation. AAPS PharmSciTech, in press.

Ac ce p

te

d

M

an

us

cr

ip t

654 655 656

24 Page 24 of 28

704 705 FIGURE CAPTIONS

707

Fig. 1. (A) Monomeric unit of HPMC, where R represents either -H, -CH3 or –

708

CH2CH(CH3)OH; (B) chemical structure of NMP

709

Fig. 2. Calculated X-ray patterns of Mod I and Mod II and X-ray diffractograms of raw

710

materials, physical mixtures and SDs. The symbol  indicates the reflection at 6.6 °,

711

characteristic of Mod I, whilst  indicates the reflection at 9.3 °, which is present exclusively at

712

Mod II

713

Fig. 3. (A) DSC curves and (B) TGA curves of raw materials and SDs. In the upper right corner

714

of Fig. 3B, derivate thermogravimetric curves (DrTGA) of the samples are presented

715

Fig. 4. FTIR spectra of raw materials, physical mixture and SDs

716

Fig. 5. Release rates of () 1;9 SD, () 2:8 SD, (▲) 3:7 SD and () pure NMP in acetate

717

buffer pH 4.5 containing 0.3 % of SLS

718

Fig. 6. (A) XRPD patterns and (B) DSC curves of SDs after 90 days of stability studies at

719

different conditions. The arrow at the X-ray pattern of 3:7 SD indicates the reflection at 9.3 °,

720

characteristic of Mod II

721

Fig. 7. FTIR spectra of SDs kept 90 days at different conditions. The highlighted areas show the

722

appearance of the amine group bands respective to crystalline NMP

725 726 727 728 729 730 731 732 733

cr

us

an

M

d

te

724

Ac ce p

723

ip t

706

734 735 736 737 738 739 25 Page 25 of 28

740 741 APPENDIX A 0 2 1

5 , 2

NMP

0 0 1 0 8

,0 0

0

5 , -0 0

2

4

6

8

10 12 14 16 18 20 22 24 26 28 30 Minutes

1:9 SD

0 0 1

2

4

6

0 0 1 0 8

0 U6 A m 0 4

U0 A6 m 0 4

0 2

0 2

10 12 14 16 18 20 22 24 26 28 30 Minutes

2:8 SD

an

0 8

8

cr

,5 0

0 2

0

0

2

4

6

8

M

0

ip t

,5 1 U0 A, m1

U0 A6 m 0 4

0

HPMC

0 , 2

us

742

10 12 14 16 18 20 22 24 26 28 30 Minutes

0

2

4

6

8

10 12 14 16 18 20 22 24 26 28 30 Minutes

3:7 SD

0 0 1

d

0 8

te

U0 A6 m 0 4

Ac ce p

0 2 0

743 744 745

0

2

4

6

8

10 12 14 16 18 20 22 24 26 28 30 Minutes

Fig. A. 1. Chromatograms referent to raw materials and SDs

26 Page 26 of 28

NMP

2:8 SD

HPMC

M

an

us

cr

ip t

1:9 SD

746 747 748

Ac ce p

te

d

3:7 SD

Fig. A. 2. Photomicrographs of raw materials and SDs

27 Page 27 of 28

748

HIGHLIGHTS Amorphous ball-milled solid dispersions of nimodipine and HPMC were developed

750

HPMC solid dispersions enhanced the biopharmaceutical properties of the drug

751

Hydrogen bonding was established between nimodipine and HPMC

752

HPMC stabilizes solid dispersions through a dilution mechanism

753

cr

754

ip t

749

Ac ce p

te

d

M

an

us

755

28 Page 28 of 28