Scholarly article on topic 'Hydroxypropyl Methylcellulose as a Novel Tool for Isothermal Solution Crystallization of Micronized Paracetamol'

Hydroxypropyl Methylcellulose as a Novel Tool for Isothermal Solution Crystallization of Micronized Paracetamol Academic research paper on "Chemical engineering"

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Academic research paper on topic "Hydroxypropyl Methylcellulose as a Novel Tool for Isothermal Solution Crystallization of Micronized Paracetamol"

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HPMC as a novel tool for isothermal solution crystallisation of micronized Paracetamol

Nuno Miguel Reis, Zizheng K Liu, Cassilda M Reis, and Malcolm Mackley

Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/cg4009637 • Publication Date (Web): 23 May 2014

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10 11 12

20 21 22

HPMC as a novel tool for isothermal solution crystallisation of micronized

Paracetamol

Nuno M Reisa,b\ Zizheng K Liua, Cassilda M Reisa, and Malcolm R Mackleya

aDepartment of Chemical Engineering and Biotechnology, University of Cambridge, New Museums

Site, Pembroke Street, Cambridge CB2 3RA, UK bDepartment of Chemical Engineering, Loughborough University, Loughborough LE11 3TU, UK

! Corresponding author: Phone +44 (0) 1509 222 505; Fax +44 (0) 1509 223 923; e-mail n.m.reis@lboro.ac.uk

ACS Paragon Plus Environment 1

2 HPMC as a novel tool for isothermal solution crystallisation of micronized

3 Paracetamol

6 Nuno M Reis , Zizheng K Liua, Cassilda M Reisa, and Malcolm R Mackleya

7 aDepartment of Chemical Engineering and Biotechnology, University of Cambridge, New Museums

Site, Pembroke Street, Cambridge CB2 3RA, UK

cooling crystallisation of Paracetamol in water was carried out in the presence of 0.1-0.8% w/w

11 Department of Chemical Engineering, Loughborough University, Loughborough LE11 3TU, UK

14 Abstract

16 Pulmonary inhalation is increasingly being selected as a preferred route for the delivery of both

18 small and large drug macromolecules for the treatment of a range of pathologies. The direct !0 crystallisation of micronized powders, in particular Paracetamol remains difficult, as it requires the

21 ability to work in high solution supersaturations where agglomeration, wall crusting and

23 heterogeneous nucleation hinders the control of crystal size and crystal size distribution. Polymer

24 additives are recognised to help driving the production of a given polymorph or controlling crystal

26 shape by means of adsorption on crystal surface. With the aim of exploiting the polymer-control

28 nucleation and growth of crystals for enhanced direct crystallisation of micronized powders, batch

31 hydroxypropyl methylcellulose (HPMC). In the presence of polymer the onset of nucleation was

33 delayed and extended beyond the cooling time of the solution, resulting in an isothermal cooling

34 crystallisation and the production of micronized Paracetamol with a mean crystal size D50, in the

36 range of 15-20 ^.m and an improved crystal size distribution. Equally, the rate generation of

38 solution cloudiness was reduced by over 3-fold for the highest HPMC concentration tested, with no

41 inhibition by HPMC is unknown, however a modification of crystal's shape observed upon the

43 addition of HPMC to the solution suggested it might be related to mass transfer limitations and

46 technique can potentially be used for direct crystallisation of other micronized drugs.

detectable impact on final product yield. The mechanisms for nucleation delay and growth

inter-molecular hydrogen bounding between the large HPMC and the small drug molecules. This

Keywords: Paracetamol, micronized, cooling crystallisation, isothermal crystallisation, HPMC

+ Corresponding author: Phone +44 (0) 1509 222 505; Fax +44 (0) 1509 223 923; e-mail n.m.reis@lboro.ac.uk

2 Introduction

4 Pulmonary inhalation is being increasingly selected as a preferred route for the delivery of both

6 small and large drug macromolecules for the treatment of a range of pathologies. The fast

9 2016, benefiting from the remarkable development in drug formulation and inhalation device

10 11 12

14 and grinding techniques for size reduction of crystals. These methods apply high energy that result

16 in a final product with broad size distribution, limited crystallinity and poor flowing properties with

'' limited dispersability. An alternative technique that is in increasing adoption by industry is spray

19 drying.3 It allows producing micronized APIs of desirable size however particles are amorphous and

21 have a larger tendency to re-crystallise or degrade. It is therefore recognised that direct

expanding dry powder inhalers (DPI) market is expected to reach over US$13bn per annum by 2016, benefiting from the remarkable development in drug formulation and inhalation device designs mainly in recent years.1 Dispatch such technological advancement, the production of micronized (~1-10 ^m) active pharmaceutical ingredients (APIs) still relies on traditional milling

crystallisation of micronized crystals solution is advantageous, however producing small crystal

24 sizes requires operation with the high supersaturations at which the control of agglomeration, wall

26 crusting, crystal size and crystal size distribution (CSD) becomes extremely challenging.

28 Crystallisation is an important process in the chemical, pharmaceutical, biotechnological and allied

30 industries, as it is used extensively for separation and purification of organic fine chemicals or APIs

31 and production of microsized APIs for drug delivery.5 Important product characteristics such as

33 crystal size, CSD, and crystal morphology are determined by the operating conditions during the

35 crystallization process, which include: supersaturation, temperature profiling, the presence of

36 additives, air-water interface, anti-solvent addition rate, fluid mixing, residence time, materials of

38 crystalliser, and agglomeration/breakage phenomena.

40 Stagnant crystallisation experiments have shown that polymers and other additives can help driving

42 the production of a given polymorph,6-9 reducing the crystal size10 or controlling the crystal shape.9

The list of polymers tested is extensive and includes both hydrophilic and hydrophobic powders,

45 such as ethylcellulose, methylcellulose (CM), polyethylene glycol (PEG), polyvinyl alcohol (PVA),

47 agar, gelatine, polyvinyl pyrrolidone (PVP), poly(ethylene oxide) (PEO), corn starch, carrageenan,

polyethylene (PE), polyvinyl chloride (PVC), polymethyl methacrylate (PMMA) and

50 hydroxylpropyl methylcellulose (HPMC) to name a few.3, 8-12 Generally, crystallisation additives

52 are effective in producing polymorphs of pharmaceuticals while avoiding others by encouraging the

53 growth of a desirable form or disrupting the growth of the other form.8, 9 13, 14 Modification of

55 crystal shape occurs as the polymer deposition on the crystal surface inhibits the growth of specific

57 surfaces of the crystal. Reduction of particle size by means of long molecular weight additives was

only marginally reported in the literature, and exception is perhaps the work of Femi-Oyewo and

reduced to less than 10 ^m upon presence of polymer additives HPMC, polyvinylpyrrolidone (PVP

There is also very few studies touching the effect of organic polymers in the kinetics of

2 Spring , although a recent work by Xie et al. showed that salbutamol sulphate crystal size was

5 K25), lecithin and Span 85 during anti-solvent water-ethanol crystallisation.

9 crystallisation. An exception is the work of Raghavan et al.15 who have studied anti-solvent

11 crystallisation of hydrocortisone acetate (HA) in the presence of four different polymers (HPMC,

12 PVP, MC and PEG400). In the absence of solution additive, the nucleation of HA was observed to

14 be spontaneous; however in the presence of a polymer the nucleation was delayed by several hours

16 and a level of growth inhibition of HA was observed. The mechanism of nucleation retardation was

18 explained by the authors in terms of association of ha with the polymer through hydrogen bonding

19 whilst the growth inhibition was related to the adsorption of polymer to the surface of the crystal.

21 HPMC is a natural polymer extensively used in the pharmaceutical industry as a tableting ingredient

23 and as a binder16 because of its regulatory approval status. Steckel et al.2 have shown that HPMC

can act as a stabilising hydrocolloid during the production of micronized fluticasone-17-propionate

26 by spray drying, resulting in one order of magnitude reduction in particle size.

28 In this work small concentrations of HPMC were used for manipulating the kinetics of cooling

30 crystallisation of Paracetamol, which offered a new level of control over the nucleation and crystal

32 growth under well mixed conditions and allow reduced crystal size and improved CSDs. The mean

34 crystal size, csd and crystal morphology have been experimentally evaluated for a concentration of

35 HPMC in range of 0 - 0.8% w/w using standard powder characterisation techniques.

38 Experimental section

40 Materials

42 Paracetamol or acetaminophen (CAS number, 103-90-2, min 99% purity) and HPMC (CAS 900443

44 65-3, typical MW = 10,000) were supplied by Sigma-Aldrich, Inc (Dorset, UK).

46 Crystallisation procedure

48 Paracetamol was crystallised in water by dissolving a given amount of Paracetamol at 70 oC

50 corresponding to a saturated solution at 65 °C, and rapidly cooling it to room temperature (20 °C).

53 the presence of 0-0.8% w/w of HPMC (percentage of mass of HPMC per mass of water), giving a

55 maximum drug to polymer ratio of 6.25:1 in weight or 413:1 in molar basis. The crystallisation

58 = d corresponding to a working volume of 60 ml (Figure 1). The solution was magnetically stirred

For each experiment, 3.0 grams of Paracetamol were dissolved in 60 grams of deionised water in

vessel consisted of 42 mm internal diameter, d, glass-jacketed unbaffled tank with a liquid height, h

10 11 12

20 21 22

at 70 °C for 1 hour to ensure complete dissolution of Paracetamol, by circulating hot water (HAAK, Fisons DCS B3, Pacific Diagnostic, Inc. USA). The maximum working volume of the tank was 83 ml, and all experiments were done in the presence of a free surface. Mixing was provided using an 18 mm length magnetic stirrer at 300 rpm, controlled by a stirrer plate (KIKA, Labortechnik, Janke & Kunkel Gmbh & Co KG. UK).

The Paracetamol solubility estimated from Granberg&Rasmuson19 for working temperature of 65 °C and 0% HPMC was -48.3 grams per kilogram of water, i.e. -2.9 grams per 60 ml of water. The cooling crystallisation was started by quickly switching circulating hot water in a 3-way valve to chilled water at 20 ± 0.1 °C from a refrigerated/cooling circulator (Grant LTD20, Cambridge, UK), which induced a rapid temperature quenching in the crystallisation vessel. The experiments were continued for about 45 minutes from the start of the cooling process, which was found sufficient to complete of crystals growth and fully deplete supersaturation in the solution. The supersaturation ratio of pure paracetamol solution defined as S = C/C* was estimated as equal to 3.8 based on the solubility data for pure paracetamol reported by Granberg&Rasmuson.19 The initial paracetamol concentration remained constant for all HPCM concentrations tested. The crystallisation vessel was equipped with a thermocouple (TME 2050, k-type) and a reflectance fibre optical probe for on-line monitoring of the temperature and turbidity of crystallisation solution, respectively (Figure 1). Details of the optical probe are given in section 2.3. At the end of each crystallisation run, the slurry was vacuum filtered through a 0.2 ^m cut-off PTFE membrane (Whatman Inc, USA) and the crystals washed with 3*1.5 ml of deionised water at room temperature. Paracetamol crystals were then dried in a desiccator for about 12 hours after which the dry weight was determined in an analytical balance. The crystal size and CSD were then characterised using an automated optical microscopy system, and crystal morphology analysed by Scanning Electron Microscopy (SEM). Crystal system was identified using powder X-ray diffraction (XRD) and the thermal behaviour using Differential Scanning Calorimetry (DSC) techniques.

Monitoring of solution turbidity

Solution cloudiness or turbidity was monitored real-time using a reflectance optical fibre probe. The optical probe (FCR 7UV200, Avantes, Eerbeek, Netherlands) consisted of 6 x 200 ^m diameter optical fibres carrying the light from a deuterium halogen light source and a 7th fibre in the core of the probe delivering the scattered light to a spectrometer connected to a PC, which was controlled by Avasoft (Avantes, Eerbeek, Netherlands). The probe was inserted though the lid of the crystalliser at a certain angle to avoid direct reflection from the walls of the crystalliser. The crystalliser was fully covered with aluminium foil to impede interference of environment light. The

2 reflected light spectrum was continuously scanned through the experimental time, t, with a CCD

spectrometer using a proper integration time. An integration function was then defined to monitor

w=615nm

R(t) = {IJt) (1)

w=585nm

normalised for each experiment by assuming 100 % light transmittance at time zero, i.e. T(t=0) = 1

5 the total reflected light, Iw or the photon counts in the spectrum for a wavelength range, w, between

7 585 and 615 nm, where crystal particles were found to scatter light strongly:

12 The relative solution transmittance, T(t) defined as the fraction of incident light that passed through

14 the solution in the monitored wavelength range w, was then calculated as T(t) = R(t)/R(t=0) and

17 and 0% transmittance towards the end of the crystallisation, i.e. T(t=x>) = 0.

20 Crystal characterisation

22 The mean crystal size and CSDs of Paracetamol powder were determined using an automatic

23 optical system, the Morphologi G3 (Malvern Instruments, Malvern, UK). Optical microscopy

25 offered a more robust CSD method for sizing Paracetamol crystals in comparison with laser

27 diffraction measurements because it offers the possibility of analysing the sample in the dispersed

28 powder form using compressed air at 1.5 barg and avoided sonication process for dispersion of

30 crystals, which has shown to result in the partial dissolution or agglomeration of Paracetamol

32 crystals (results not shown). Paracetamol is very prone to agglomeration, therefore CSD analysis is

33 especially difficult. Nevertheless, the Morphologi G3 system allows the reproducible, automatic

35 analysis of a large number of particles or crystals and the possibility of using different types of

37 filters to manually discard large agglomerates resulting from crystal caking during the sampling and

38 powder drying process.

40 For CSD analysis using Morphologi G3 1.5 mg of dry powder were loaded in 20 ^m thick

42 aluminium foil and dispersed into the clean, glass surface of the microscope using compressed air at

43 1.5 barg. The G3 software measured the projected area of the crystals/particles and assigned it the

45 equivalent diameter of a circle, CE, through a standard operating procedure (SOP). The crystal size

47 computed this way was based on CE3, which would only fit laser scattering measurements for

48 perfectly spherical particles. A typical run comprised a sample of 5,000-10,000 crystals to ensure

50 the statistical significance of the acquired CSD. The reproducibility of CDS measurements was

52 found very high upon SOP optimisation (results not shown).

53 For SEM of the micronized powders, the dried crystals were spread over a surface and dried

55 overnight in He atmosphere. The samples were then coated with a 20 nm thick gold coating and

57 scanned at 5 Kev in FEI Philips XL30 FEGSEM equipped with an Oxford Instruments INCA EDX

58 system running a 30 mm2 SiLi thin window pentafet EDX detector. 60

2 The effect of HPMC on the thermal behaviour above ambient of dry Paracetamol powder was

recorded using a Perkin-Elmer Pyris 1 scanning differential calorimeter. For that purpose,

performed, intercalated by 1 min holding time.

5 approximately 5 mg of sample were heated at 10 °C/min from 40 to 180 °C in perforated crimped

7 aluminium pans while being purged with dry nitrogen. The heating and cooling cycles were

10 For analysis of the effect of HPMC on crystal system, powder X-ray diffractions in dried powder

12 were acquired at room temperature on a Philips PW1820 diffractometer using Copper-k-alpha

14 radiation (tube operated at 40 kV, 40 mA), a 6-6 goniometer, automatic divergence and 0.2 mm

15 receiving slits, a silicon secondary monochromator, and a scintillation counter. The XRD trace was

17 scanned in 2-6 from 5° to 60°, at a rate of 0.050° of 26 per 2 seconds. The powder samples were

19 prepared as flat surfaces in aluminium sample holders.

21 Results and discussion

23 Effect of HPMC on the kinetics of Paracetamol crystallisation

25 Paracetamol was crystallised in water in a small batch stirred glass vessel containing a reduced

27 HPMC concentration, by quickly dropping the temperature of a slightly undersaturated solution

from 70 oC to 20 oC. Turbidity was on-line monitored during the cooling process using an optical

30 microprobe immersed in the solution, which allowed sensitive detection of the onset of nucleation

32 and quantitative detection of the rate of generation of cloudiness, which is ultimately linked to the

35 transmittance (the lower the transmittance the higher the solution cloudiness) for a selected number

37 of HPMC concentrations. The HPMC concentrations tested in this study were 0.0, 0.1, 0.3, 0.4, 0.7

generation and growth of crystals. Figure 2 shows the variation of solution temperature and relative

and 0.8% w/w but only a selection of these is shown in Figure 2 for simplicity in clarity of data

40 presented. The temperature cooling profile was highly reproducible in the whole set of experiments,

42 therefore only one cooling curve is shown in Figure 2. HPMC effectively delayed the onset of

45 the plots. With pure Paracetamol, a shower of crystals was detected within 1-2 minutes, and 90% of

47 the solution cloudiness generated within the following minute, and crystal growth was completed

nucleation and reduced the rate of generation of cloudiness in the solution as can be confirmed from

within 5 minutes from the beginning of the cooling process, which compares well with the cooling

50 time of the solution in the vessel. In the presence of HPMC, the time of onset of nucleation

52 extended to up to 9 minutes depending on the HPMC concentration, so well beyond the cooling

time of the solution in the vessel. This ultimately means that the crystallisation in the presence of

55 HPMC occurs entirely under full isothermal conditions, at which the solution supersaturation is at

57 its maximum and therefore a shower of crystals beneficial for the production of micronized powders

59 is expected. The ability of running solution crystallisations in full isothermal conditions as shown in

crystal growth rates, however some quantitative information was extracted in order to quantify the

2 this manuscript is novel and might find broad applications in the direct crystallisation of micronized

6 The turbidity profiles in Figure 2 did not allow the full deconvolution of crystal nucleation and

9 effect of polymer additive in the overall batch cooling crystallisation kinetics. For that purpose, an

11 arbitrary induction time, tind and crystal growth completion time, tG were defined corresponding to

12 the time required to give a 20% and 80% decrease, respectively, in solution transmittance. For this

14 analysis the whole range of HPMC concentrations tested was considered. Equally, a maximum

16 generated cloudiness rate, Rc, was calculated from the initial gradient of relative solution

17 absorbance versus cooling time, which is summarised in Figure 3a. During the initial stage of

19 crystal growth, when submicron nuclei grow to a size to be optically detected and therefore the

21 concentration of crystals is proportional to the absorbance of the solution, the increase in solution

22 absorbance (defined as -log10 of transmittance) is essentially related to the increase in the

24 concentration of particles or the formation of new crystals, therefore Rc should capture the relative

26 effect of polymer additive concentration on kinetics of crystal nucleation. Figure 3a showed that tind

27 increased by up to 5.4-fold and tG by up to 9.5-fold upon the addition of HPCM to the solution, with

29 the maximum effect being observed at the highest HPMC concentration tested of 0.8% w/w.

31 Equally, Rc decreased up to 73.4% with increasing HPMC concentration, being the inhibitory effect

32 more noticeable at low HPMC concentrations. This represents a major effect of polymer additive in

34 respect to the overall kinetics of crystallisation, from where a major reduction in crystal size could

36 be expected.

38 Effect of HPMC on mean crystal size and CSD

40 A common motivation for controlling crystallisation processes in pharmaceutical industries is the

42 production of uniform particle sizes with a given mean crystal size. Therefore, the Paracetamol

43 powder produced in the presence of different HPMC concentrations has been characterised in

45 respect to particle size using the Morphologi G3 system, which applies compressed air for

47 dispersing dry powders on a glass microscope slide and advanced imaged analysis to determine a

48 circle equivalent diameter, CE of the crystals. This allowed confirming that the presence of HPMC

50 in the initial crystallisation solution resulted in a significant reduction in the mean crystal size, D50

52 and an improvement in CSD as shown in Figures 4a and 4b. With pure Paracetamol the D50

53 obtained was 39.6 um. This was per si considerably smaller than the mean sizes reported by other

55 authors for batch cooling crystallisation from solution. For example, Chew et at.17 and Fujiwara et

56 5 18

57 at. reported a D50 in the range of 100-250 um for batch crystallisers, and Zarkadas & Sirkar

59 produced crystal sizes with 50-150 um in a continuous hollow fibre device. The smaller crystal

2 sizes obtained in this study are related to the large surface area-to-volume ratio of the glass vessel

5 nucleation rates that cannot be mimicked in larger crystallisers. Nevertheless, the CSD in Figure 4a

7 was broad as can be seen from be great deviation of the values represented by percentile 10, D10 =

used in the crystallisation runs, which delivered high cooling rates for high supersaturation and

15.5 ^m and percentile 90, D90 = 62.1 ^m. This could be associated with the non-isothermal

use of HPMC concentrations above 0.1% w/w resulted in no further improvement in particle size

10 conditions in the crystallisation vessel at the time of onset of solution cloudiness. The addition of

12 0.1% w/w of HPMC resulted in D50 decreasing from 39.6 to 15.4 ^.m and the volume-based CSD

14 becoming remarkably sharper, with D10 = 8.3 ^m and D90 = 22.6 ^m. The frequency-based CSD in

15 Figure 4b confirmed a larger number of fines in the dried powder crystallised in the presence of

17 0.1% w/w HPMC, and smaller number of large crystals. The dashed vertical line represents the

19 smallest crystal size that could be detected in with the used setup with Morphologi G3. That

20 significant decrease in the mean crystal size was confirmed by SEM images in Figures 4c and 4d

22 (note the different scales in the SEM microphotographs).

24 The effect of varying initial concentrations of HPMC on particle size distribution in respect to

26 percentile 10, D10, percentile 50, D50 and percentile 90, D90 is fully summarised in Figure 3b. The

29 reduction or CSD, and it appeared that the presence of a very small concentration of HPMC in the

31 solution was sufficient to provide a good level of particle size control.

33 Another aspect that contributed to smaller crystal sizes was the fact that Paracetamol solubility

35 decreases very quickly with HPMC content, as shown in supplementary data (Figure S1). For a

36 0.1% mass ratio of HPMC to Paracetamol (corresponding to approximately 0.03% w/w of HPMC

38 in respect to mass of solution) the solubility measured at 30 oC dropped by 40%. This means that in

40 the presence of HPMC the crystallisation is isothermal but also supersaturation is higher than for a

41 pure Paracetamol system. This should in theory return higher crystallisation yields, and in respect to

43 particle size control this represents enhanced supersaturation in the presence of HPMC which

45 favours production of smaller crystals and more fines.

47 Overall and based on the turbimetry data, it is not possible to identify the main mechanic leading to

50 appears linked to a higher supersaturation and tighter supersaturation control resulting from the

52 delay of onset of nucleation, higher number of fines and reduction in Paracetamol solubility.

54 Effect of HPMC on crystal morphology

56 A number of recent studies mainly performed at static conditions have linked the use of organic

58 polymer additives with crystal polymorphism as reviewed in the Introduction section. A given

59 crystal form can be inhibited upon the presence of polymer or "impurities", therefore polymer

a significant reduction in crystal size and improvement in CSD in the presence of HPMC, but it

2 additives can induce selective production of a given polymorph.6-9 Equally, other studies have

34 reported a modification of crystal habit upon the addition of polymer additives.8,9 Mainly with

5 isentropic crystals like Paracetamol,20 polymers can present higher selectivity to bind given faces a

7 crystal, so selectively inhibiting the growth on a given direction. In order to test for possible effects

10 thermal behaviours and X-ray diffraction patterns. Optical and electronic microscopic analysis of

12 the crystals showed that crystals shape changed to an elongated-prismatic shape upon the addition

of HPMC additive on crystal structure, Paracetamol powders were characterised regarding their

of polymer. The DSC and XRD data in Figures 5 and 5b, respectively, confirmed that the

Paracetamol samples crystallised with 0% and 0.1% w/w of HPMC compared in Figure 4 agreed

15 crystalline product obtained with pure Paracetamol and 0.1% w/w of HPMC were in fact from the

17 same crystal form I, i.e. monoclinic Paracetamol. The experimental XRD patterns of the two

20 well with XRD data of e.g. Martino et at.21 and Nichols and Frampton22 by showing peaks unique

22 to the monoclinic form I such as 26 = 12.025, 15.425 and 26.475. Also, the melting point

24 (endothermic) at 164 °C and two crystallisation (exothermic) points at 88-89°C and 135-139 °C

25 shown by DSC analysis in Figure 5b also confirmed that the two samples were of the same crystal

27 form. This confirmed that the detected difference in kinetics of crystallisation is not due to the

29 production of a different polymorph.

31 Detailed SEM analysis to all Paracetamol samples revealed a side effect with the use of HPMC

34 or holes on the crystal surface. Figure 6 shows cavities up to 100-200 nanometers in diameter that

36 become more prominent as more HPMC was added to the solution. The reason behind the

39 interactions of HPMC and Paracetamol, with HPMC adsorbing the crystal surface as suggested by

41 Thompson et at. for Metacetamol (a small molecule with -OH groups). Using AFM and SEM,

44 to 15 nm steps interspersed with holes in the presence of Metacetamol.

46 Effect of HPMC on final product yield

concentrations of 0.3% w/w and above, with extended agglomeration and a large number of cavities

formation of these cavities is unknown, however it is suspect to be linked to the intermolecular

they have shown surface features in pure Paracetamol crystals between 1 and 20 nm, that changed

The product yield calculated in terms of dried powder weight was found independent of the HPMC

50 concentration used, and in average 59.5 ± 1.62% as shown in Figure 3c. This was slightly lower

52 than the theoretical yield expected from pure Paracetamol (74.1%) based on the solubility data of

53 Granberg&Rasmuson.19 Note that the addition of HPMC to the solution can only increase the

55 theoretical yield up to a maximum of 3.6% assuming all HPMC added to the solution ends up in the

57 dried powder. Based on the solubility data shown in Figure S1 the addition of HPMC to the

crystallisation solution should have resulted in higher yield than for pure Paracetamol, as HPMC

2 was found to decrease significantly the solubility of Paracetamol in water. This might be related to

the dissolution of some material during the sample washing or to surface poisoning. Femi-Oyewo &

the surface of the crystals. This data herein reported shows that under well mixed conditions the

have proposed different hypothesis spanning from inhibition of heterogeneous nucleation to

the possible impact of HPMC on the nucleation and/or growth of Paracetamol crystals.

5 Spring10 reported 91% reduction on yield of Paracetamol crystallised in the presence of HPMC

7 under static conditions, which was shown to be related to the irreversible adsorption of polymer of

10 adsorption of polymer to the surface of the crystals is reversible however it cannot entirely

12 overcome surface poisoning that leads to the terminal size of the crystal.

14 Remarks on the effect of HPMC on kinetics of Paracetamol crystallisation

16 The mechanics for delaying the onset of nucleation and reducing the rate of solution cloudiness in

18 cooling crystallisation of Paracetamol crystals by HPMC is still unknown, however recent studies

21 increased mass transfer resistances both related to the hydrogen bounding between the polymer and

23 Paracetamol molecules. This section offers an overview to those important mechanisms that support

27 Heterogeneous nucleation is thermodynamically more favourable and is known to be related to the

28 presence of interfaces, such as crystalliser's wall, impeller or air-waste free surface. Zarkadas

30 &Sirkar have found that a combination of homogeneous and heterogeneous nucleation is possible

32 in a cooling crystallisation process of Paracetamol in water at high supersaturation ratios, i.e. S =3

33 and above, which was the case in this present study. In order to validate this hypothesis a further set

35 of experiments was carried out in a combination of experimental conditions that were recognised to

37 promote heterogeneous nucleation, such is the case of stirring, glass wall material and air-water

38 interface, as summarised in Table 1. For each crystallisation run, the time required to detect the

40 onset of solution cloudiness was noted (in these set of experiments the induction time was

42 determined using naked eye). It was observed that the addition of 0.1 % w/w HPMC has similar

43 effect in respect to the delay on induction time to the removal of free surface during the

45 crystallisation vessels, resulting in delayed induction time from 4.0 to 9-10 min. This is likely to be

47 associated with the fact that HPMC reduced the frequency of collisions between the solute particles,

48 which, in turn, led to the delay of nucleation. The rate for primary nucleation is linearly

50 proportional to the diffusion coefficient4 of Paracetamol, having a molecular weight of 151.2 g/mol.

52 When the diffusion is sufficiently rapid the nucleation is not diffusion controlled. This is normally

53 the case at high values of S, and explains the very short induction time obtained with pure

55 Paracetamol (run 1 in Table 1). Nevertheless, with the presence of HPMC in the crystallisation

57 solution the nucleation became increasingly limited as more polymer additive is added to the

58 solution This was explained by ZarkaHac&Sirkar and Tian et at

solution. This was explained by Zarkadas&Sirkar and Tian et al. based on inter-molecular

2 hydrogen bonding between Paracetamol and the polymer additive. HPMC has both a hydrogen

5 bonding ability and network association with drug molecules as concluded from FT-IR results of

7 Tian et al.24 Similar conclusion was obtained by Sahoo et al.25 using Raman spectroscopy. The last

10 from hydroxyl OH- groups present in both polymer additive and drug molecules. Raghavan et al.15

12 has also reported strong effect of cellulose polymers on morphology of hydrocortisone acetate. This

bonding donor and acceptor groups on its ring structure (Figure S2), therefore showing a high H-

have identified intra-molecular hydrogen bounding between HPMC and Ciprofloxacin, resulting

means HPMC effectively can work as a "molecular transporter" therefore limiting both the

15 diffusivity of drug molecules and reducing the impact probability for the formation of nuclei

17 through collisions.

19 The production of smaller crystals and the reported morphological changes could also be explained

21 based on several growth-inhibition hypothesis, such as i) mass transfer limitations, ii) impurities

22 deposition,25iii) polymer adsorption on the surface of the crystals, or iv) a combination of those.

24 During growth some impurities can adsorb to the surface of the crystals "blocking" the active

26 growth sites (sometimes only blocking growth of a given face of the crystal) or entirely suppresses

27 crystal growth.26 HPMC was previously shown capable of adsorbing the surface of Paracetamol

29 crystals.9 This has been linked to a reduction in the growth rate of steps when compared to pure

31 Paracetamol. Using AFM and SEM, Thompson et at. showed that metacetamol changed the

32 growth steps of Paracetamol and disrupting the crystal lattice by adsorbing to the surface and

34 inducing the formation of defects or cavities in the crystal surface.

36 A parallel observation from Table 1 is the relevant effect of mixing and interfaces on the

38 crystallisation of Paracetamol in respect to induction time in the presence of 0.10% w/w HPMC.

41 induction time approximately doubled in the absence of headspace in the crystallisation vessel.

43 Interfaces are known for creating temperature gradients therefore being preferential zones for

46 (rows 6 and 7), however this is believed to be related to the lower cooling rate in unstirred vessel.

49 Conclusions

51 Batch cooling crystallisation experiments of Paracetamol in water in the presence 0.1-0.8% w/w of

53 HPMC revealed the ability of the natural polymer additive modifying the overall kinetics of

54 crystallisation by slowing the rate of generation of cloudiness in the solution and delaying the onset

56 of nucleation beyond the cooling time of a crystalliser. This allowed direct production of

58 micronized Paracetamol crystals from solution, with a D50 in the range of 15-20 ^m and sharp CDS,

59 which was linked to the isothermal crystallisation conditions in the presence of HPMC and

Although it is difficult to separate the effect of interfaces from that of mixing, it was observed that

nucleation. On the other hand, the removal of agitation resulted in further increased induction times

2 reduction of Paracetamol solubility in the presence of polymer. The observed habit change induced

by the presence of HPMC suggested the polymer acted by inhibiting growth on specific surfaces of

HPMC adsorbed to surface of the crystals disrupting the hydrogen bounding network. This

Acknowledgment

5 the crystals, as previously reported for other crystallisation systems. The high density of cavities

7 observed on the surface of the crystals for higher HPMC concentrations strongly suggests that

10 technique can potentially be used for enhancing direct crystallisation of other micronized drugs for

12 pulmonary drug delivery.

17 The authors are grateful to Dr Jeremy Skepper from Multi Imaging Centre, Cambridge, for help

18 with SEM; Dr Eric Rees and Dr Joanna Stasiak for help with XRD; Zlatko Saracevic for help with

20 DSC analysis, and Henrietta Adomako at Loughborough University for assistance with Paracetamol

22 solubility experiments. NMR is grateful to Fundacao para a Ciencia e Tecnologia, FCT

24 (sfrh/Bpd/26904/2005) and European Commission Marie Curie ief programme (project

25 221768) for financial support.

28 Supporting Information Available

30 Solubility data for Paracetamol in the presence of different mass ratios of HPMC, and chemical

32 structure of Paracetamol and HPMC molecules. This information is available free of charge via the

34 Internet at http://pubs.acs.org/.

36 References

38 (1) Pulmonary Drug Delivery Systems: Technologies and Global Markets; Rockville, MD, USA,

39 2012; p. 222.

41 (2) Steckel, H. International Journal of Pharmaceutics 2003, 258, 65-75.

42 (3) Martin, G. P.; Zeng, X. M. Pharmaceutical composition for pulmonary delivery.

43 W0/2001/058425, 2001.

(4) Garside, J.; Mersmann, A.; Nyvlt, J. Measurement of crystal growth and nucleation rates; 2nd

46 editio.; IChemE: Rugby, UK, 2002.

47 (5) Fujiwara, M.; Nagy, Z. K.; Chew, J. W.; Braatz, R. D. Journal of Process Control 2005, 15,

48 493-504.

50 (6) Grzesiak, A. L.; Matzger, A. J. Crystal growth & design 2008, 8, 347-350.

51 (7) Price, C. P.; Grzesiak, A. L.; Matzger, A. J. Journal of the American Chemical Society 2005,

52 127, 5512-7.

(8) Lang, M.; Grzesiak, A. L.; Matzger, A. J. Journal of the American Chemical Society 2002,

55 124, 14834-5.

56 (9) Capes, J. S.; Cameron, R. E. CrystEngComm 2007, 9, 84.

57 (10) Femi-Oyewo, M. N.; Spring, M. S. International Journal of Pharmaceutics 1994, 112, 17-28.

(11) Pawar, A.; Paradkar, A. R.; Kadam, S. S.; Mahadik, K. R. Indian Journal of Pharmaceutical Sciences 2007, 69, 658-664.

(12) Xie, S.; Poornachary, S. K.; Chow, P. S.; Tan, R. B. H. Crystal Growth & Design 2010, 10, 3363-3371.

(13) Davey, R. J.; Blagden, N.; Potts, G. D.; Docherty, R. Journal of American Chemical Society 1997, 7, 1767-1772.

(14) Gu, C.-H.; Chatterjee, K.; Young, V.; Grant, D. J. Journal of Crystal Growth 2002, 235, 471481.

(15) Raghavan, S. L.; Trividic, a; Davis, a F.; Hadgraft, J. International journal of pharmaceutics 2001,212, 213-21.

(16) Weiner, M. L.; Kotkoskie, L. A. Excipient Toxicity and Safety (Drugs and the Pharmaceutical Sciences); Marcel Dekker: New York, USA, 2000.

(17) Chew, C. M.; Ristic, R. I.; Dennehy, R. D.; De Yoreo, J. J. Crystal Growth & Design 2004, 4, 1045-1052.

(18) Zarkadas, D. M.; Sirkar, K. K. Industrial & Engineering Chemistry Research 2007, 46, 29282935.

(19) Granberg, R. A.; Rasmuson, A. C. Journal of Chemical & Engineering Data 2000, 45, 478483.

(20) Heng, J. Y. Y.; Williams, D. R. Langmuir: the ACS journal of surfaces and colloids 2006, 22, 6905-9.

(21) Martino, P. Di; Guyot-Hermann, A.-M.; Conflant, P.; Drache, M.; Guyot, J.-C. International Journal of Pharmaceutics 1996, 128, 1-8.

(22) Nichols, G.; Frampton, C. S. Journal of pharmaceutical sciences 1998, 87, 684-93.

(23) Thompson, C.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Wilkinson, M. J. International journal of pharmaceutics 2004, 280, 137-150.

(24) Tian, F.; Baldursdottir, S.; Rantanen, J. Molecular Phamaceuticals 2009, 6, 202-210.

(25) Sahoo, S.; Chakraborti, C. K.; Behera, P. K. International Journal of Applied Pharmaceuticals 2012, 4.

(26) Myerson, A. Handbook of Industrial Crystallization; Myerson, A., Ed.; Butterworth Heinemann: Boston, USA, 2001.

(27) Mullin, J.W. Industrial Crystallization. Butterworth- Heinemann: London, 1993.

10 11 12

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Tables

Table 1. Induction times detected by naked eye, Tw for batch cooling crystallisation of Paracetamol with HPMC under different nucleation conditions

Run Wall material Water-air interface Mixing HPMC concentration (w/w) tind' (min)

1 Glass Yes Yes 0.00% 1.7

2 Glass Yes Yes 0.10% 4.0

3 Glass No Yes 0.10% 9.5

4 LLDPE Yes Yes 0.10% 4.4

5 LLDPE No Yes 0.10% 10.5

6 LLDPE Yes No 0.10% 15.2

7 LLDPE No No 0.10% 20.5

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Figures list

thecmocpyple.

optical probe

light source

spectrometer

magnetic plate

cold water

Figure 1. Experimental setup used in the batch cooling crystallisation experiments.

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20 1.2

8 0.8 C

_£0 CD DC

10 20 30

cooling time [min]

Figure 2. Real-time profiles temperature and relative solution transmittance (or turbidity) in the vessel during batch cooling crystallisation. For clarity, only HPMC concentrations of 0%, 0.1%, 0.4% and 0.8% w/w were shown.

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[a.u./min] 0.04

- 0.01

0.00 0.20 0.40 0.60 0.80 1.00 HPMC concentration [gHPMC/100 g solvent]

0.00 0.20 0.40 0.60 0.80 1.00 HPMC concentration [gHPMC/100 g solvent] (b)

0.00 0.20 0.40 0.60 0.80 1.00 HPMC concentration [gHPMC/100 g solvent]

10 11 12

20 21 22

Figure 3. Effect of initial HPMC concentration on (a) induction time, tinci crystal growth time, tG and maximum rate of generation solution cloudiness, Rc as determined from turbidity plots in Figure 2; (b) crystal size distribution of dried crystals, and (c) final product yield, for 45 min of cooling. For more details calculating tind, tG and Rc in (a) see text. The product yield expected from the solubility data of Granberg&Rasmuson19 was 73.5% w/w, which is plotted in (c) as a guideline.

0.1% w/w HPMC

0% w/w HPMC „ 0 8

0.1% w/w HPMC

® 0.2 a>

0.1 1 10 100 1000 Equivalent diameter of crystal, CE [|im]

<o w/w HPMC

0.1 1 10 100 1000 Equivalent diameter of crystal, CE [|im]

Figure 4. Crystal size distribution for Paracetamol crystallised in 0% w/w and 0.1% w/w of HPMC based on (a) volume and (b) frequency. SEM images of Paracetamol samples crystallised in (c) 0% w/w HPMC and (d) 0.1% w/w HPMC. Note the different scale bars used in (c) and (d).

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"05 c Q)

0% w/w HPMC

0.1% w/w HPMC

_LjJjjJl Iu^Ju^JAW^

15 25 35 45

0% w/w HPMC

0.1% w/w HPMC

50 80 110 140 170

Temperature [°C] (b)

Figure 5. (a) XRD patterns and (b) DSCs for Paracetamol crystallised in 0% w/w and 0.1% w/w of HPMC.

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Figure 6. SEM images of Paracetamol crystallised in the presence of (a) 0.1% w/w, (b) 0.3% w/w, (c) 0.7% w/w and (d) 0.8% w/w of HPMC showing microporosity in crystal's surface.

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For Table of Contents Only

HPMC as a novel tool for isothermal solution crystallisation of micronized

Paracetamol

Nuno M Reisabi, Zizheng K Liua, Cassilda M Reisa, and Malcolm R Mackleya

aDepartment of Chemical Engineering and Biotechnology, University of Cambridge, New Museums

Site, Pembroke Street, Cambridge CB2 3RA, UK bDepartment of Chemical Engineering, Loughborough University, Loughborough LE11 3TU, UK

1.2 1.0

0.8 ^ 06

"o 0.4

0.2 0.0

0% w/W HPMC

.1 1 10 100 1000 Equivalent diameter crystal [|jm]

Micronized Paracetamol crystals were directly produced from solution crystallisation in water by quickly dropping the temperature in the presence of 0.1-0.8% w/w of HPMC. The polymer reduced the solubility of Paracetamol and delayed the unset of nucleation beyond the cooling time of the vessel, consequently allowing to produce fine crystals with sharp CSDs.

: Corresponding author: Phone +44 (0) 1509 222 505; Fax +44 (0) 1509 223 923; e-mail n.m.reis@lboro.ac.uk