Polymorphs of DP-VPA Solid Solutions and Their Physicochemical Properties

Journal Pre-proof Polymorphs of DP-VPA solid solutions and their physicochemical properties Chao Hao, Jian Jin, Jiaying Xiong, Zhengge Yang, Lanchang Gao, Yanqin Ma, BiFeng Liu, Xin Liu, Yin Chen, Guisen Zhang PII:

S0022-3549(20)30176-3

DOI:

https://doi.org/10.1016/j.xphs.2020.03.017

Reference:

XPHS 1908

To appear in:

Journal of Pharmaceutical Sciences

Received Date: 13 January 2020 Revised Date:

6 March 2020

Accepted Date: 23 March 2020

Please cite this article as: Hao C, Jin J, Xiong J, Yang Z, Gao L, Ma Y, Liu BF, Liu X, Chen Y, Zhang G, Polymorphs of DP-VPA solid solutions and their physicochemical properties, Journal of Pharmaceutical Sciences (2020), doi: https://doi.org/10.1016/j.xphs.2020.03.017. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Inc. on behalf of the American Pharmacists Association.

Polymorphs of DP-VPA solid solutions and their physicochemical properties

1 2 3

Chao Haoa, Jian Jinb, Jiaying Xionga, Zhengge Yanga, Lanchang Gaoa, Yanqin

4

Mac, Bi-Feng Liua, Xin Liu a, Yin Chenb,*and Guisen Zhanga,b,*

5 6

a

7

Science and Technology, Huazhong University of Science and Technology, Wuhan

8

430074, China

9

b

Systems Biology Theme, Department of Biomedical Engineering, College of Life

Jiangsu Key Laboratory of Marine Biological Resources and Environment, Jiangsu

10

Key Laboratory of Marine Pharmaceutical Compound Screening, School of Pharmacy,

11

Jiangsu Ocean University, Lianyungang 222005, China

12

c

13

1 Yunhe Road, Xuzhou, Jiangsu 221116, China

14

* Author to whom correspondence should be addressed.

15

*Phone: +86-27-87792235. Fax: +86-27-87792170.

16

*E-Mail: [email protected]. E-Mail: [email protected]

Nhwa Institute of Pharmaceutical Research, Jiangsu Nhwa Pharmaceutical Co., Ltd.,

17 18

Abstract

19

Different solid forms possess various physicochemical properties, which can

20

significantly affect the stability, bioavailability, and manufacturability of the final

21

product.

22

1-stearoyl-2-valproyl-sn-glycero-3-phosphatidylcholine

23

1-palmitoyl-2-valproyl-sn-glycero-3-phosphatidylcholine (DP-VPA-C16), is currently

24

under development as an antiepileptic drug. DP-VPA-C16 and DP-VPA-C18 crystallize

25

together in solid solution forms. The solid forms of DP-VPA solid solution were

26

studied herein. Powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA),

27

differential scanning calorimetry (DSC), attenuated total reflection Fourier transform

28

infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), dynamic

DP-VPA,

a

1

complex (DP-VPA-C18)

of and

1

vapor sorption (DVS) and optical microscopy were used to characterize the different

2

crystalline forms, known as polymorphs. The physicochemical properties, including

3

hygroscopicity, thermodynamic behavior, and relative stability, of each form were

4

investigated. DVS analysis showed that DP-VPA solid solution reduced the

5

hygroscopicity of DP-VPA-C16. The relative humidity stability study revealed that

6

Forms A and B are relatively stable, while Forms A-1, B-1, C and D are highly

7

unstable under natural humidity. Further analysis revealed that Form A transforms into

8

Form B through milling. Given the physicochemical properties of the available

9

physical forms, Form B may be the optimal form for the formulation and development

10

of antiepileptic drugs.

11 12 13 14

Keywords DP-VPA; solid solutions; polymorphs; physicochemical properties Introduction

15

It is well-established that solid drug substance can exist in various forms, and

16

that different solid forms possess unique physicochemical properties, which can

17

significantly affect the relative stability, bioavailability, and manufacturability of the

18

final product1-4. Various methods are used to modify the properties of drugs, including

19

the use of salts5-7, polymorphs8-11, solvates12-14, hydrates15-17, co-crystals18-20, and

20

amorphous compounds21-23. Over the past decade, pharmaceutical scientists have

21

focused their attention on the use of co-crystals, which offer several advantages and

22

are now commonly used in the preformulation stage of drug development. Although

23

pharmaceutical research on co-crystals has been conducted for many years, the

24

definition of co-crystals remains controversial24-26. Dunitz defined the co-crystals in

25

the simplest way, stating that “the word ‘co-crystal’ provides a succinct though

26

possibly inelegant definition of what it is intended to describe, a crystal containing

27

two or more components together.27” Co-crystals encompass molecular compounds, 2

1

molecular complexes, hydrates, solvates, inclusion compounds, channel compounds,

2

clathrates, and other types of multi-component crystals27. Stahly separates co-crystals

3

into stoichiometric and nonstoichiometric multi-component crystals26. Crystalline

4

solid solutions are characterized by different molecular constituents that randomly

5

occupy equivalent crystallographic sites28, 29, and a multi-component composition that

6

is either variable or continuous; molecular solid solutions can be regarded as a special

7

form of co-crystals25.

8

Stoichiometric variation, according to various structural and physicochemical

9

properties, offers an opportunity to control the performance. Most molecular solid

10

solutions are prepared as co-crystals through melt-crystallization, grinding, and

11

recrystallization of solvents, among other methods. Organic solid solutions typically

12

comprise molecules with similar structures and sizes28. Solid forms can be

13

characterized using solid-state techniques such as powder X-ray diffraction (PXRD),

14

differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), infrared

15

spectroscopy (IR), Raman spectroscopy, scanning electron microscopy (SEM),

16

dynamic vapor sorption (DVS), optical microscopy and solid-state nuclear magnetic

17

resonance spectroscopy (ssNMR). For drug development, pharmaceutical co-crystals

18

and

19

multi-molecules are present in a fixed stoichiometric ratio30. Contemporary

20

pharmaceutical co-crystal research is less concerned with nonstoichiometric

21

compounds, and only a few groups of molecules are known to form solid

22

solutions31-35. It is also notable that molecular solid solutions can form solvates and

23

exhibit polymorphism.

structurally

homogeneous

crystalline

solids

are

the

focus,

wherein

24

DP-VPA, which is a phospholipid-valproic acid conjugate also known as

25

SPD421 (Fig. 1), offers promise as an antiepileptic agent36. DP-VPA has been shown

26

to be effective in treating several types of epilepsy including status epilepticus (SE)

27

and acute repetitive seizures in children, as well as migraine prophylaxis and manic

28

depression37-39.

29

glycero-3-phosphatidylcholine (DP-VPA-C18) and 1-palmitoyl-2-valproyl-sn-glycero

DP-VPA

is

a

mixture

3

of

1-stearoyl-2-valproyl-sn-

1

-3-phosphatidylcholine (DP-VPA-C16), with a DP-VPA-C18 to DP-VPA-C16 blend

2

ratio of approximately 88:1236. DP-VPA-C18 and DP-VPA-C16 differ in their

3

pharmacokinetic behavior. Following a single oral administration, DP-VPA-C16

4

exhibits a significantly prolonged half-life in plasma compared to DP-VPA-C18. In

5

addition, the peak concentration of DP-VPA-C18 occurs later than that of DP-VPA-C16.

6

Mixtures of DP-VPA-C18 and DP-VPA-C16 offer greater and more prolonged

7

neuroprotective effects than either compound alone40. Although DP-VPA has been

8

studied for two decades, its crystalline forms have not yet been investigated. In this

9

study, polymorphs of DP-VPA solid solution were prepared and investigated.

10

Fig. 1. Chemical structures of DP-VPA-C16 (A) and DP-VPA-C18 (B).

11

Materials and Methods

12

Materials

13

DP-VPA-C16

and

DP-VPA-C18

were

obtained

from

Jiangsu

Nhwa

14

Pharmaceutical Co., Ltd. (Jiangsu, China), All other reagents were of analytical grade

15

(Shanghai Lingfeng Chemical Reagent Co., Ltd, Shanghai, China), and used without

16

further purification.

17

Preparation of solid solution polymorphs

18

DP-VPA-C16 (0.06 g) and DP-VPA-C18 (0.34 g) were suspended in acetone (10

19

ml) and heated to reflux, and kept refluxing for 30 min. The solution was slowly

20

cooled to room temperature; crystals of Form A were precipitated. The mixture was

21

filtered and dried at 50 ℃ for 4 hours. When Form A was exposed to 25 ℃,70% RH

22

environment for 2 hours, Form A-1 was formed.

23

DP-VPA-C16 (0.6 g) and DP-VPA-C18 (3.4 g) were dissolved in anhydrous ethyl

24

alcohol (4.4 ml) at 20 ℃, the solution was transferred to ice water bath; meanwhile

25

acetone (50 ml) was slowly added drop-wise to the solution while stirring, Form B

26

precipitates from the solution. After addition completed, mixture was stirred for 4

1

another 2 hours, then filtered and placed in a dryer. When Form B was exposed to

2

25 ℃,70% RH environment for 2 hours, Form B-1 was got.

3 4

Form C was obtained by vacuum drying Form A or/and Form B at 78 ℃, 10 mbar for 2~4 hours.

5

Form D was prepared by placing the Form A or/and Form B on a slide glass

6

subjected to a slow heating (10~20 ℃/min) from 25 ℃ to 170 ℃; and then,

7

quickly cooled to room temperature by compressed air.

8

The powder X-ray diffraction (PXRD) Analysis

9

The PXRD patterns were collected at room temperature on Bruker D8 Focus

10

diffractometer (Bruker, Karlsruhe, Germany) using Cu-Kα (λ=1.5406 Å) at 40 kV, 40

11

mA passing through graphite curved crystal monochromator with divergence slit

12

(2.5°), anti-scatter slit (4.0°), and receiving slit (0.15 mm). Samples were subjected to

13

powder X-ray diffraction analysis in continuous mode with a step size of 0.02° and a

14

scan rate of 0.2 s/step over an angular range of 3<2θ<30°. Obtained diffractograms

15

were analyzed with EVA software furnished with the PXRD.

16

Thermogravimetric analysis (TGA)

17

TG analysis was conducted on a Netzsch TG 209F3 equipment (Netzsch, Selb,

18

Germany) under a flow of nitrogen (20 ml/min) at a scan rate of 10 ℃/min from 30

19

to 200 ℃. For TG analysis, typical samples of weighing 4~6 mg were used.

20

Differential scanning calorimetry (DSC)

21

The DSC analysis was performed with a Netzsch DSC 200F3 equipment

22

(Netzsch, Selb, Germany), and the sample undergone a heat-cool-heat process

23

(5 ℃/min from 40 to 170 ℃, then cooled to 40 ℃ at 5 ℃/min, finally 5 ℃/min from

24

40 to 170 ℃ ) under a N2 atmosphere(flow rate of 40 ml/min). For DSC analysis,

25

typical samples of weighing 2~4 mg were used.

5

1

Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR)

2

ATR-FTIR spectra were recorded by a Nicolet 330 (Thermo Nicolet, Waltham,

3

USA) FT-IR spectrophotometer, equipped with versatile Smart iTR attenuated total

4

reflection (ATR) sampling accessory. The spectral range was 4000~600 cm-1 with 64

5

scans for each spectrum and the spectral resolution is 2 cm-1.

6

High Performance Liquid Chromatography (HPLC) Analysis Method

7

The content of each component in DP-VPA was determined by an Agilent 1200

8

HPLC system at a UV detection wavelength of 210 nm using a C18 column (Agilent

9

XDB, 5 μm ×4.6 mm × 150 mm column, Agilent Technologies, Inc., USA). The

10

mobile phase was methol:acetonitrile:H2O ( containing 315 mg ammonium formate in

11

1L)= 85:15:5 (V/V). The flow rate was 1.0 ml/min, and the column temperature is at

12

30℃. The sample diluent is methanol.

13

Related substances quantification of DP-VPA was carried out on an Agilent 1260

14

series HPLC system (Agilent Technologies, Inc., USA), equipped with, quaternary

15

gradient pump, and evaporative light scattering detector (Alltech ELSD 2000ES,

16

USA). A Xtimate C18 column ( 5 µm×250 mm×4.6 mm, Welch Material Inc., USA)

17

was used for all separations at a column temperature of 30 ℃. The elution system

18

consisted of methol:acetonitrile:H2O ( containing 385 mg ammonium acetate in 1 L)=

19

85:15:5 (V/V). The flow-rate was set at 1.0 ml/min and the sample injection volume

20

was 20 μL. ELSD was set to a probe temperature of 70 ℃, and the nebulizer for

21

nitrogen gas was adjusted to 1.8 L/min.

22

Water Determination

23

Karl Fischer method are used for determination of water content. Karl Fischer

24

titration were carried out at 25±1 ℃ with a Karl Fischer titrator V20S from

25

Mettler-Toledo, applying the one-component reagent for Karl Fischer titration

26

(pyridine-free titrating agent). Pure methanol were used as solvent . Sample sizes

6

1

were approximately 300 mg and were analyzed twice each.

2

Residue Solvent Quantification

3

Gas chromatography (GC) analysis was carried out with a Agilent 8860 series

4

GC system (Agilent Technologies, Inc., USA) equipped with a flame ionization

5

detector (FID) and a DB-624 capillary column (60 m×0.32 mm×1.8 µm, Agilent

6

Technologies, Inc., USA ). The GC conditions were as follows: column temperature,

7

40 ℃ to 230 ℃ (8 min hold at 40 ℃, 10 ℃/min from 40 ℃ to 90 ℃, 30 ℃/min from

8

90 ℃ to 230 ℃); injection temperature, 150 ℃; detector temperature, 250 ℃; and

9

nitrogen flow rate, 1 mL/min. The sample injection volume was 1 μL, and the

10

injection mode was split with a split ratio of 1:20.

11

Morphology Analysis

12

The morphology of the particles was examined by scanning electron microscopy

13

(SEM) and optical microscopy. SEM was conducted on a Phenom Pro desktop

14

scanning electron microscope (Phenom, Eindhoven, Netherlands), a sputter coating

15

apparatus was applied to induce electric conductivity on the surface of the samples,

16

and the accelerating voltage was 5 kV. The optical microscope photographs were

17

observed on XPN-203E optical microscopy (Shanghai Changfang Optical Instrument

18

Co., Shanghai, China) equipped with a Tk-C1031EC color TV camera

19

(JVCKENWOOD Co., Beijing, China) and a heating stage.

20

Solid-state milling study

21

Solid samples were milled using a Pulverisette 7 ball mill (Fritsch, Idar,

22

Germany), using 20 ml agate containers with 4 agate ball (Ø 10 mm), shaken at 400

23

rpm for 15 min, 60 min, 120 min, 180 min, 360 min. The ground solids were analyzed

24

immediately by PXRD.

7

1

Dynamic vapor sorption (DVS)

2

DVS experiments were performed on an Intrinsic DVS instrument (SMS Ltd.,

3

London, UK). Samples were studied over a humidity range from 0 to 95% RH at

4

25 ℃. Each humidity step was made if less than 0.02% weight change occurred in 30

5

min, with a maximum hold time of 2 hours.

6

Results and Discussion

7

PXRD Analysis

8

The PXRD patterns of the six polymorphic DP-VPA solid solutions, Forms A,

9

A-1, B, B-1, C and D, were analyzed (Fig. 2). Form A shows characteristic peaks at

10

2θ = 4.7°, 7.1°, 9.5°, 11.9°, 14.3°, 16.7° and 19.1°, while Form A-1 presents two

11

additional characteristic peaks at 2θ = 4.4° and 6.6°, arising on the shoulders of peaks

12

4.7° and 7.1°. Form B shows characteristic peaks at 2θ = 4.7°, 7.1°, 9.2°, 10.1° and

13

12.3°. Similar to Form A-1, Form B-1 has two additional characteristic peaks, at 2θ =

14

4.4° and 6.6°, which arise on the shoulders of the peaks at 4.7° and 7.1°. Form C

15

shows characteristic peaks at 2θ = 3.4° and 13.8°, and Form D has characteristic

16

peaks at 2θ = 5.6°, 8.4° and 11.3°. We also found that the both individual component

17

DP-VPA-C16 and DP-VPA-C18 also have two different crystal forms, which we named

18

DP-VPA-C16 Form A/B and DP-VPA-C18 Form A/B (Figs. s1 and s2). The control

19

experiment results show that the polymorphs of the starting material do not affect the

20

crystal form of the final product (Table s1). Comparing the PXRD patterns of

21

DP-VPA-C16, DP-VPA-C18 and DP-VPA, the characteristic diffraction peaks of

22

DP-VPA-C16 are not seen in the Form A or B patterns of DP-VPA, and DP-VPA and

23

DP-VPA-C18 show similar PXRD patterns (Figs. s1 and s2). Furthermore,

24

high-performance liquid chromatography (HPLC) analysis showed that all of the

25

DP-VPA solid solutions contained DP-VPA-C18 and DP-VPA-C16, at a mass ratio of

26

around 88:12 (Fig. s3). The analysis indicated that DP-VPA-C18 and DP-VPA-C16

27

co-crystallized to form DP-VPA solid solutions; moreover, during the solid solution 8

1

crystallization, DP-VPA-C16 molecules are embed within the crystal lattice of

2

DP-VPA-C18 without dramatically influencing the crystal structure of DP-VPA-C18.

3

This is likely attributable to the similar structure and size of the homologous

4

DP-VPA-C18 and DP-VPA-C16 molecules, and the relatively low ratio of DP-VPA-C16;

5

also, the two missing methylene groups are negligible compared to the long alkyl

6

carbon chain, given the current detection sensitivity of PXRD.

7 8

Fig. 2. The PXRD patterns of the six forms of DP-VPA solid solutions. Thermal Analysis

9

The DSC and TGA curves of the four polymorphs are shown in Figures 3 and 4,

10

respectively. The DSC profiles of Forms A and B show two endothermic events, at

11

80–90 ℃ and at 160–167 ℃, respectively. The main peak may result from partial

12

dehydration and melting, which corresponds with the TGA profiles. It was found that

13

Forms A and B lost weight at temperatures below their melting range. After melting,

14

Forms A and B released approximately 40% and 60% of their total water content,

15

respectively. Karl Fischer titration, residue solvent quantification analysis and related

16

substances quantification analysis provide powerful evidence that the water loss

17

continued to the end of the second endothermic event. Karl Fischer titration show that

18

DP-VPA Form A contains 3.21% water, Form B contains 2.97% water. The residue

19

solvent acetone and ethanol were not detected through GC analysis (Figs. s4 to s6).

20

HPLC quantification analysis indicate that the related substances of the material

21

hardly increased after heating of different forms of DP-VPA (Figs. s7 and s8). The

22

first major endothermic peak provides evidence that the majority of water is present in

23

the form of crystalline hydrates; after the first phase transformation, the remaining

24

water forms lattice channels. The transition states Form A-1 and Form B-1 have

25

almost same DSC curves corresponding to their parent matrix, respectively.

26

The morphological changes in Form A were recorded using hot-stage microscopy

27

from 25 ℃ to 170 ℃ at a heating rate of 20 ℃/min (Fig. 5). The morphological

28

changes showed that, following the initial melting behavior, the sample changed into 9

1

a waxy solid at 100 ℃. Above 100 ℃, the waxy solid continued losing water until the

2

second melting step, while its solid morphology exhibited no significant change.

3

Finally, following the second melting behavior, the sample became liquid. The final

4

melted sample was quickly cooled to room temperature using compressed air, and

5

PXRD characterization confirmed that the cooled sample was Form D. Next, Form D

6

was exposed to natural conditions (60% RH, 25 ℃) for at least 24 hours, and optical

7

microscopy and PXRD were used to confirm the solid forms: Form D transformed to

8

Form A at 6 hours (Fig. 6) and the crystal shape reverted to clusters of flakes at 24

9

hours (Fig. 5). The DSC profile of Form C showed two endothermic peaks, at 68.8 ℃

10

and 80.3 ℃, indicating that Form C likely contains both crystal water and absorption

11

water. The DSC profiles of Form D also exhibit two endothermic peaks at 75.8 ℃

12

and 158.9 ℃. During the cooling process , all the forms of DP-VPA solid solutions

13

show two exothermic peaks at around 107.5 ℃ and 157.3 ℃, this corresponding to

14

the two melting event of them. While during the second heating program, the first

15

phase transition endothermic peak is at about 115 ℃, and the second melting

16

endothermic peak is at about 159.3 ℃. This means that the strictly anhydrous

17

DP-VPA solid solutions have higher first melting point.

18

Fig. 3. The DSC curves of the DP-VPA solid solutions: (a)Form A, (b)Form A-1,

19

(c)Form B, (d)Form B-1, (e)Form C and (f)Form D.

20

Fig. 4. The TG curves of the DP-VPA solid solutions Forms A, B, C and D.

21

Fig. 5. Morphology changes of DP-VPA solid solutions Form A under heat.

22

Fig. 6. The PXRD pattern changes of DP-VPA solid solutions Form D when exposed

23

to natural conditions.

24

ATR-FTIR Analysis

25

Further investigation of Forms A–D was performed using attenuated total

26

reflection Fourier transform infrared spectroscopy (ATR-FTIR; Fig. 7); the results are

27

summarized in Table s2. ATR-FTIR is fast and nondestructive, relatively easy to use 10

1

and, most importantly, requires no sample pretreatment; this constitutes an advantage

2

over normal transmission IR, which could not be used due to the potential for phase

3

transformation 41. Forms A–C exhibited similar spectra, whereas Form D differed in

4

relative absorption intensity and had a wider band width, possibly arising from its low

5

crystallinity. The wide band of absorption at 3,418cm-1 is related to O-H bond

6

vibrations of water molecules in the HO-H…O complex. The absorption intensity of

7

C=O bond vibrations was high, peaking at 1,736 cm-1. The bands observed at 1,079,

8

1,183 and 1,230 cm-1 were attributed to the stretching vibrations of C-O bonds. The

9

band at 1,250 cm-1 resulted from the P=O stretching vibrations. C-N stretching

10 11 12

vibrations of the quaternary ammonium salt yielded a peak at 1,163 cm-1. Fig. 7. ATR-FTIR spectrum of the DP-VPA solid solutions Forms A, B, C and D. SEM Analysis

13

The SEM results (Fig. 8) revealed differing morphologies of the crystals in the

14

DP-VPA solid solutions between Forms A and B. The particles of Form A formed

15

irregular plates, whereas Form B crystals formed ball-like and rose-like aggregates of

16

small plates.

17

Fig. 8. The SEM images of DP-VPA solid solutions Form A in 500×(a) and Form B

18

in 800×(b) magnifications.

19

Solid-state Milling

20

Solid-state milling was conducted to investigate the effect of pressure on the

21

DP-VPA solid solutions. Form A was milled and analyzed immediately using PXRD.

22

As shown in Fig. 9, Form A is unstable under certain pressure conditions. After ball

23

milling for 15 minutes, Form A transformed into Form B. Moreover, following 360

24

minutes of continuous ball milling, no further changes were observed in the

25

diffractograms, which indicates that Form B is relatively stable under the pressure

26

conditions used herein.

11

1

Fig. 9. The PXRD pattern changes of solid solutions Form A after milled for 6 hours.

2 3

Stability Against Relative Humidity Fluctuations

4

The DVS analysis was performed as a preliminary evaluation of the stability of

5

DP-VPA solid solutions against relative humidity (RH) fluctuations (Fig. 10). For

6

DP-VPA-C16, both Forms A and B were stable up to 60% RH; however, they quickly

7

absorbed approximately 14% water at 70% RH. Forms A and B of DP-VPA solid

8

solutions remained relatively stable until 70% RH, with less than 2% of water

9

absorbed. As shown in Figure 10, DP-VPA solid solutions reduced the hygroscopicity

10

of DP-VPA-C16. Form A of the DP-VPA solid solutions showed slightly lower

11

humidity sensitivity than Form B, which may be due to the bigger crystalline particles

12

and lower specific surface area of Form A (Fig. 8). The two individual components,

13

and the DP-VPA solid solutions, were not stable above 75% RH. Forms A and B

14

transformed into gel-like substances above 80% RH. To study the morphological

15

transformation of Form A, it was subjected to a high humidity environment and the

16

crystal particles were soaked in acetone (because the evaporation of acetone removes

17

heat from the environment). As a result, water vapor in the environment condenses on

18

the surface of the crystals. An optical microscope equipped with a color camera was

19

used to record morphological changes. As shown in Figure 11, the condensation of

20

atmospheric water vapor resulted in the destructions of the crystal skeleton of Form A

21

within 3 minutes.

22

Fig. 10. The DVS isotherms for Form A and Form B of individual components and

23

DP-VPA solid solutions.

24

Fig. 11. Morphology changes of DP-VPA solid solutions Form A under the imitated

25

high humidity.

26

Since the DVS isotherm analysis showed that DP-VPA was affected by air

27

humidity, the effect of humidity on the polymorphs was investigated further. Forms A 12

1

and B were exposed to 70% RH (saturated solution of potassium iodide) at 20 ℃ in a

2

sealed humidity container for 2 hours. The stress samples were characterized by

3

PXRD, and Forms A-1 and B-1 were detected. Next, the stress samples were

4

transferred to 60% RH (saturated solution of sodium bromide) at 20 ℃ in a sealed

5

humidity container. PXRD characterization was conducted from 45–240 minutes of

6

the dehydration process. As shown in Figure s9 and s10, the two characteristic peaks

7

at 2θ = 4.4° and 6.6° vanished after Form A-1 and Form B-1 were transferred to 60%

8

RH condition for 6 hours. The experiments described above showed that the crystal

9

skeleton of the co-crystals has no effect at up to 70% RH. Forms C and D were

10

exposed to a natural environment (60% RH, 25℃), and PXRD characterization was

11

conducted between 20 minutes and 24 hours of water absorption to monitor

12

transformation. Form C transformed into Form B after 24 hours exposure to the

13

natural environment (Fig. 12), while Form D transformed to Form A within 6 hours

14

(Fig. 6).

15

Fig. 12. The PXRD pattern changes of DP-VPA solid solutions Form C when exposed

16

to natural conditions.

17

Conclusion

18

In this paper, the unique characteristics of co-crystal DP-VPA solid solutions

19

were reviewed and the physicochemical properties of six solid-form DP-VPA solid

20

solutions were studied. Several solid-state analytical techniques were used to

21

characterize the six crystalline forms of the DP-VPA solid solutions. The study

22

demonstrated that DP-VPA solid solutions can improve the stability of DP-VPA-C16

23

under conditions of fluctuating humidity. Therefore, it may be possible to improve the

24

physical stability of active pharmaceutical ingredients (APIs) through a new

25

crystalline organic solid solution strategy. The results showed that moisture

26

significantly affects DP-VPA solid solutions, and that Forms A-1, B-1, C and D are

27

highly unstable under natural humidity conditions. While Forms A and B of DP-VPA

28

solid solution exhibited similar physicochemical properties, Form B demonstrated 13

1

greater stability under pressure. Depending on the balance between the stability and

2

manufacturability of the available physical forms, Form B of DP-VPA solid solution

3

may be the optimal form for formulation and development of antiepileptic drugs.

4

Acknowledgments

5

Authors are thanks Nhwa Institute of Pharmaceutical Research, Jiangsu Nhwa

6

Pharmaceutical Co., Ltd. for provide samples and HPLC setup assistance for us.

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