European Journal of Pharmaceutical Sciences 38 (2009) 489-497
`
`C~t~ht~ i i~t~ a~aii ~ bi ~a~ S~i~ D i
`
`Europea n J ournal o f Ph a rm a ceu ti cal Sciences
`
`ournal homepage:ww w e ls e~ier (cid:128)omllo~ate
`
`Solid state characterization of the anti-HIV drug TMC114: Interconversion of
`amorphous TMC114, TMC114 ethanolate and hydrate
`
`Elke Van Gyseghem a, 1, Sigrid Stokbroekx b, 1, Hector Novoa de Armas b, Jules Dickens b,
`Marc Vanstockemc, Lieven Baertc, Jan Rosierc, Laurent Schuellerc, Guy Van den Mootera,*
`
`Laboratorium voor Farmacotechnologie en Biofarmacie, Katholieke Universiteit Leuven, Campus Gasthuisberg, O&N II, Herestraat 49 bus 921, B-3000 Leuven, Belgium
`Pharmaceutical Sciences, Johnson &Johnson Pharmaceutical Research & Development, A Division of Janssen Pharmaceutica N. IL, Tumhoutseweg 30, B-2340 Beerse, Belgium
`Chem-Pharm Development, Tibotec BVBA, Gen. De Wittelaan L11B 3, B-2800 Mechelen, Belgium
`
`ARTICLE
`
`INFO
`
`ABSTRACT
`
`Article history:
`Received 3 July 2009
`Received in revised form
`14 September 2009
`Accepted 18 September 2009
`Available online 24 September 2009
`
`Keywords:
`TMC114 (Darunavir)
`Solvate/hydrate
`Amorphous form
`Solid state characterization
`Channel solvates
`
`The interconversion of the ethanolate, hydrate and amorphous form of TMC114 ((3-[(4-amino-
`benzenesulfonyl)-isobutyl-amino]-l-benzyl-2-hydroxypropyl)-carbamic acid hexahydrofuro-
`[2,3-b]furan-3-yl ester) in open conditions was characterized. TMCll4 hydrate and ethanolate
`form isostructural channel solvates. The crystal structure of TMC114 was obtained from single crystal
`X-ray diffraction, confirming that it is a channel solvate. Ethanol and water can exchange with one
`another. TMC114 ethanolate converts into TMC114 hydrate at moderate or high relative humidity (RH)
`at 25°C, and it converts back into the ethanolate in ethanol atmosphere. The hydration level of the
`hydrate is determined by the environmental humidity. TMC114 hydrate collapses to the amorphous
`product when water is removed by drying at low RH or increasing temperature. TMC114 ethanolate
`becomes amorphous at elevated temperature in a dry environment below the desolvation temperature.
`Amorphous TMC114 obtained by dehydrating the hydrate during storage at room temperature/<5% RH,
`by increasing the temperature, or via desolvating the ethanolate by heating, converts into the hydrate
`at moderate or high RH at ambient conditions, and into TMC114 ethanolate in an ethanol atmosphere.
`Under ambient conditions, TMC114 ethanolate may convert into the hydrate, whereas the opposite
`will not occur under these conditions. The amorphous form, prepared by melting-quenching shows a
`limited water uptake. Whereas TMC114 ethanolate is stable in the commercialised drug product, special
`conditions can trigger its conversion.
`
`© 2009 Elsevier B.V. All rights reserved.
`
`1. Introduction
`
`TMC114 (PrezistaTM, Darunavir) (Pauwels, 2006; D’Avolio
`et al., 2007; McCoy, 2007; Martinez-Cajas and Wainberg,
`2007; Ghosh et al., 2007; Kovalevsky et al., 2006) or
`(3-[(4-amino-benzenesulfonyl)-isobutyl-amino]-l-benzyl-2-
`hydroxypropyl)-carbamic acid hexahydrofuro-[2,3-b]furan-3-yl
`ester is a key component of many salvage therapies in multi-
`experienced patients of the human immunodeficiency virus (HIV)
`(D’Avolio et al., 2007; Yeni, 2006). The compound was licensed in
`June 2006 in the United States (D’Avolio et al., 2007; Martinez-
`Cajas and Wainberg, 2007) and in February 2007 in the European
`Union (D’Avolio et al., 2007). TMC114 is a next-generation, syn-
`thetic non-peptidic protease inhibitor (PI) (Pauwels, 2006; McCoy,
`
`* Corresponding author. Tel.: +32 16 330304; fax: +32 16 330305.
`E-mail address: Guy.VandenMooter@pharm.kuleuven.be (G. Van den Mooter).
`1 Both authors contributed equally to the paper.
`
`0928-0987/$ - see front matter © 2009 Elsevier B.V. All rights reserved.
`doi: 10.1016/j.ej ps.2009.09.013
`
`2007). Its chemical structure, comprising a bicyclic tetrahydrofu-
`ran (bis-THF) P2 and a p-aminosulfonamide P2~ ligand, is given in
`Fig. 1 (Pauwels, 2006; McCoy, 2007; Ghosh et al., 2007; Kovalevsky
`et al., 2006). TMCll4 is an oral drug given at a dose of 600rag
`twice a day combined with low-dose ritonavir (NorvirTM, 100 mg
`twice a day) (D’Avolio et al., 2007; Yeni, 2006; Hammer et al.,
`2006).
`TMC114 shows pseudo-polymorphic behavior (solvatomor-
`phism) as it exists as TMC114 ethanolate and hydrate crystals;
`the non-solvated form is amorphous. Polymorphism is the ability
`of a substance to exist as two or more crystalline phases, having
`different arrangements andJor conformations of the molecules in
`their crystal lattice (Grant, 1999; Haleblian and McCrone, 1969;
`Haleblian, 1975; Threlfall, 1995). Pseudo-polymorphs are crys-
`talline phases formed when solvent molecules are present in the
`crystal lattice; if the solvent is water the molecular compounds
`are called hydrates. The propensity of a molecule to form solvates
`is related to molecular structure, hydrogen bonding patterns and
`crystal packing. HydratesJsolvates are classified based upon their
`
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`E. Van Gyseghem et al. / European Journal of Pharmaceutical Sciences ?,8 (2009) 489-497
`
`i
`
`Fig. 1. Chemical structure of TMC114 or (3-[(4-amino-benzenesulfonyl)-isobutyl-
`amino]-l-benzyl-2-hydroxypropyl)-carbamic acid hexahydrofuro-[2,3-b]furan-3-
`yl ester.
`
`structural characteristics. This paper shows evidence that TMC114
`ethanolate and hydrate are isostructural solvates, belonging to
`the class of channel solvates/hydrates: solvent or water molecules
`are contained in lattice channels and lie next to other solvent or
`water molecules of adjoining unit cells along an axis of the lat-
`tice, forming "channels" through the crystal (Morris, 1999). Size
`and chemical environment of the channel determine what ldnd of
`solvent molecule can be included and what ldnd of interactions
`occur. The crystal structure of TMC114 ethanolate was obtained
`from single crystal X-ray diffraction, as an ultimate confirmation
`that this compound is a channel solvate. The aqueous solubility of
`TMC114 ethanolate and hydrate are 16 and 10 mg/100 ml, respec-
`tively.
`Pseudo-polymorphic behavior is a commonly acknowledged
`phenomenon in literature, e.g. for lorazepam (US Patent, 2002), car-
`bamazepine (Kaneniwa et al., 1984; Morris, 1999), azithromycin
`and fenoprophen (Cui, 2007), topiramate sodium (Stuart, 2004),
`ampicillin (Brittain et al., 1988; Ivashldv, 1973), theophyllin (Byrn,
`1982), cromolin sodium (Cox et al., 1971), L-lysine monohy-
`drochloride (Bandyopadhyay et al., 1998), since transitions can
`occur during processing, handling and storage (Morris, 1999)
`and influence bioavailability (Kaneniwa et al., 1984) and solubil-
`ity.
`It is essential to gain an understanding of the dehydration
`(desolvation)/hydration (solvation) mechanism and ldnetics. An
`important characteristic of some channel hydrates is the ability
`to take up additional moisture in the channels when exposed to
`high humidity (Morris, 1999; Cox et al., 1971). In instances where
`the solvent serves to stabilize the lattice, the process of desolvation
`may produce a change in lattice parameters, resulting in the forma-
`tion of either a new crystal form or an amorphous form. TMC114
`solvates collapse when solvent or water is removed and becomes
`amorphous.
`This paper focuses on the interconversion process between
`TMC114 ethanolate, TMC114 hydrate and amorphous TMC114 in
`open conditions through application of thermogravimetric anal-
`ysis (TGA), infrared (IR) spectroscopy, (modulated) differential
`scanning calorimetry ((M)DSC), X-ray powder diffraction (XRPD),
`and dynamic vapor sorption (DVS). We also describe the crys-
`tal structure of the TMC114 ethanolate. These data provide an
`experiment-based reasoning for the structural bases of the TMC114
`solvates and the amorphous form and distinguish the conditions at
`which conversion takes place.
`
`2. Materials and methods
`
`2.1. Materials
`
`TMC114 hydrate and TMC114 ethanolate were obtained from
`Tibotec (Mechelen, Belgium).
`Indium, sapphire, nickel and alumel (TA Instruments, Leather-
`head, OK), octadecane puriss, p.a. standard for GC (Fluka
`Chemie, Buchs, Switzerland), lead (LGC, Laboratory of the Gov-
`ernment Chemist, Teddington, OK) and tin (Acros Organics,
`Geel, Belgium), nitrogen 5.5 (Messer Belgium N.V., Machelen,
`Belgium), lithium chloride and sodium chloride (Fisher Sci-
`entific, Loughborough-Leicestershire, OK), magnesium chloride
`(Sigma-Aldrich, Gillingham-Dorset, OK), dichloromethane analyt-
`ical reagent grade and acetonitrile far UV, HPLC gradient grade
`(ACN; Fisher Scientific, Leicestershire, OK), methanol, for HPLC
`(Acros Organics), potassium dihydrogen orthophosphate, AnalaR
`(BDH Laboratory Supplies, Poole, England), potassium dihydrogen
`phosphate Analytical Reagent (Riedel-de Haen, Seelze, Germany),
`potassium dihydrogen phosphate pro analysi (Merck, Darmstadt,
`Germany), hydrochloric acid 1 mol/1 1N, Titrinorm ready to use
`(VWRInternational, Fontenay sous Bois, France), sodium dodecyl
`sulphate Ph. Eur. (Merck) and Natrii laurilsulfas Ph. Eur. (SLS; Alpha
`Pharma, Nazareth, Belgium), Milli-Q water obtained with a Veolia
`purification system (Veolia Water systems, High Wycombe, OK),
`and pressurized helium gas (Messer Belgium N.V.) were applied.
`
`2.2. Methods
`
`2.2.1. Thermogravimetric analysis (TGA )
`The compound was weighed into non-hermetic aluminum sam-
`pie pans. The TG curve was recorded on a TA Instruments Hi-Res
`TGA 2950 thermogravimeter (TA Instruments, New Castle, DE, USA)
`using a 20 °C/min heating rate in the range between room temper-
`ature (RT) and either 300 °C or when the sample showed a weight
`loss of 80% (w/w) due to decomposition, applying a resolution fac-
`tor of four.
`The three-point temperature calibration was achieved using a
`thermometer for ambient temperature calibration and the Curie
`temperature of nickel and alumel. Weight calibration was achieved
`using calibration weight.
`
`2.2.2. Fourier transform infrared spectrometry (FT-IR) with
`micro-attenuated total reflectance (MicroATR)
`To avoid conversion between the physical forms samples were
`analyzed using a suitable MicroATR accessory. 32 IR scans were
`recorded in a Nicolet Magna 560 FT-IR spectrophotometer (Nicolet,
`Thermo Nicolet Corporation, Madison, WI, USA) equipped with a
`mid-IR sensitive deuterated triglycine sulphate detector with KBr
`windows, a Ge on KBr beam splitter and a Harrick Split Pea/Si crystal
`MicroATR accessory, using a I cm 1 resolution in the wavenumber
`
`range of 4000 to 400 cm 1, and applying baseline correction.
`
`2.2.3. Differential scanning calorimetry
`2.2.3.1. Differential scanning calorimetry (DSC). Ca. 3 mg of com-
`pound was exactly weighed in standard non-hermetic aluminum
`TA Instruments sample pans (TA Instruments, Leatherhead, UK).
`The DSC curve was recorded on a TA Instruments Q1000 MTDSC
`equipped with an RCS unit under a nitrogen flow at a heating rate
`of 10 °C/min between 25 °C and a variable final temperature. The
`three-point temperature calibration was achieved using octade-
`cane, indium and lead. Indium was used for heat flow calibration.
`
`2.2.3.2. Modulated differential scanning calorimetry (MDSC).
`3-6mg of the compound was transferred into standard non-
`
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`E. Van Gyseghem et al. / European Journal of Pharmaceutical Sciences 38 (2009) 489-497
`
`491
`
`hermetic aluminum TA Instruments sample pans. The DSC curve
`was recorded on a TA Instruments Q1000 MTDSC equipped with an
`RCS cooling unit using mode T4P and a nitrogen flow of 50 ml/min.
`After the sample was equilibrated at -40°C, it was submitted
`to a heating ramp of 2 °C/rain modulated with 0.32°C every 60 s
`between -40 °C and a variable final temperature.
`The enthalpic response was calibrated with an indium standard
`and the temperature scale was calibrated with octadecane, indium
`and tin. The heat capacity signal was calibrated by comparing the
`response of a sapphire disk with the equivalent literature value at
`80 °C. Validation of temperature, enthalpy and heat capacity was
`carried out using the same standard materials.
`
`2.2.4. X-ray powder diffraction (XRPD)
`XRPD analyses were carried out on an X’Pert PRO MPD diffrac-
`tometer PW3050/60 with PW3040 generator (PANalytical, Almelo,
`the Netherlands), equipped with an X’Celerator detector using Cu
`K~I radiation ()~ = 1.541874A). The instrument was used in the
`Bragg-Brentano geometry. The generator voltage was set at 45 kV
`and the current to 40 mA. Samples were measured in continu-
`ous scan mode in the range of 3°<20<53° with a step size of
`0.016?5°/step and counting time of 10.16 s/step. The incident beam
`path contained a programmable divergence slit (9.5 ram), a Soller
`slit (0.04 rad), a beam mask (10 ram), an anti-scatter slit (2°) and a
`beam knife. The diffracted beam path had a long anti-scatter shield,
`a Soller slit (0.04 rad), and a 0.02-ram Ni filter.
`For the XRPD experiments under controlled temperature and
`humidity, an Anton Paar temperature chamber (TFK450) and a
`humidity chamber (THC) were used. Samples were measured in
`continuous scan mode in the range of 3° < 2(? < 35° with a step size
`of0.0167°/step and counting time of 29.8 s/step. The incident beam
`path contained a programmable divergence slit (5 ram), a Soller
`slit (0.04 rad), a beam mask (15 ram), an anti-scatter slit (1°). The
`diffracted beam path had a long anti-scatter shield, a Soller slit
`(0.04 tad), a 0.02-ram Ni filter and the X’Celerator detector.
`
`2.2.5. Single crystal X-ray diffraction
`Crystals suitable for X-ray diffraction were grown by slow evap-
`oration from an acetone/ethanol (1:1) (Acros Organics) solution.
`Measurements were carried out using a Siemens P4 four-circle
`diffractometer with graphite monochromated Cu Kc~1 radiation
`(Siemens, Karlsruhe, Germany). The intensity data were collected
`using ~o-20 scans, with ~o scan width equal to the low range
`plus the high range plus the separation between the Kc~l and
`Kc~2 positions; 3670 reflections were measured (3.71° < 0< 69.07°,
`-1 <h<11, -20<k<1, -1 <1<23), of which 3467 unique (merg-
`ing R=0.035) and F2_>2cr(F)2 for 3670 reflections which were
`retained in all calculations. Empirical absorption correction, via ¢
`scan was applied (North et al., 1968), the linear absorption coef-
`ficient obtained was 1.340 mm 1. Three standard reflections were
`monitored every 100 reflections (intensity decay: 6%). The struc-
`ture was solved by direct methods and Fourier synthesis. Non-H
`atoms were refined anisotropically by full-matrix least-squares
`techniques. H atoms were calculated geometrically and included in
`the refinement, but were restrained to ride on their parent atoms.
`The isotropic displacement parameters of the H atoms were fixed
`to 1.3 times Ueq of their parent atoms. The Flack parameter for the
`absolute configuration determination was -0.03(2). The program
`used for data collection, data reduction and cell refinement was
`XSCANS (X-ray Single Crystal Analysis System, Version 2.2, 1996).
`The program applied to solve structures was SIR92 (Altomare et
`al., 1993). The program used to refine structure was SHELXL97
`(Sheldrick, 1997). For molecular graphics DIAMOND (Bergerhoff,
`2005) was applied. Software used to prepare material for publi-
`cation was PLATON (Spek, 2003). Detailed crystallographic data
`
`Table 1
`X-ray crystallographic data for TMC114 ethanolate.
`
`Empirical formula c29 H43N3 08 S!
`Formula weigh~ 593.72
`c~s~al size, ram 0.56 ~0i38 ~ 0-24
`Crystal system Orthorhombic
`Space group
`P 212i 2i
`aiA
`9.9882(6)
`biA
`!6.6!97(8)
`
`90
`
`90
`90
`v, A~ Z 3i58.~(3)~ 4
`~a~, Mg/m} !.2~8
`Wavelength, ~ i.5~i8~
`Tempera~ure~ K 293(!)
`
`The~a ~ ~ 69.07
`Limiting indiceS{ Ii !~!!
`@20~1
`~1~23
`
`~
`
`.... Reflections collected .......................................................... 36?0 .............................
`!ndependen~ reflections
`346z
`Data/restrain~siparame~ers 3670/0/376
`~oodness4of-fit on F21.os3
`Fina! R indices [!> 2~(1)] R ~ 0.04, wR~ 0.!!8
`
`Largest difference h01e, eA
`
`3
`
`4027
`
`have been deposited at the Cambridge Crystallographic Data Cen-
`tre (CCDC) and are available on request. The crystallographic and
`experimental data for this compound are summarized in Table 1.
`
`2.2.6. Dynamic vapor sorption (DVS)
`TMC114 hydrate was stored in a dynamic vapor sorption model
`DVS-l-chamber (Surface Measurement Systems, London, UK) for
`1 h at 25, 40, 60 and 80 °C under dry N2. Subsequently, the dried
`sample was each time subjected during 2 h to 75% RH at 25 °C.
`
`2.2.Z Preparation of amorphous product
`Quench-cooled samples were obtained by melting TMC114
`hydrate or ethanolate on a Kofler hot bar (Wagner & Munz,
`Miinchen, Germany), after which the molten product was placed
`on a cold (-20 °C) metal block resulting in a glassy product.
`Alternatively, TMC114 ethanolate was dissolved in
`dichloromethane under magnetic stirring in a solid sub-
`stance:solvent ratio of 5:95 (w/v). The solution was spray-dried
`with a Biichi Mini Spray Dryer B-191 (Biichi, Flawil, Switzerland),
`applying an inlet temperature of 40 °C, a flow control of 8001N/h
`pressurized air, an aspirator setting of 100%, and a peristaltic pump
`setting at 40%. Afterwards, the resulting powder was post-dried
`in a Christ Alpha (Medizinischer Apparatebau, Osterode/Harz,
`Germany) vacuum dryer.
`
`3. Results and discussion
`
`3.1. Stability studies
`
`The influence of RH and temperature on the physical form of
`TMC114 was tested by storing TMC114 ethanolate and hydrate in
`open containers under different conditions, after which the sam-
`ples were analyzed with TGA, IR, DSC and XRPD. These results are
`summarized in Table 2a and b, respectively. Figs. 2 and 3 character-
`ize untreated and quench-cooled TMC114 ethanolate and TMC114
`hydrate to enable drawing conclusions from Table 2a and b.
`
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`
`

`
`492
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`E. Van Gyseghem et al. / European Journal of Pharmaceutical Sciences 38 (2009) 489-497
`
`Table 2
`Physical stability of (a) TMC114 ethanolate (34 days) and (b) TMC114 hydrate (14 days) stored at different temperature and %RH conditions and examined by TGA, IR, DSC
`and XRPD (E: ethanolate, H: hydrate, A: amorphous, E/A: mixture of ethanolate and amorphous material, A/H: mixture of amorphous material and hydrate; NS = no extra
`signal; RT = room temperature).
`
`Condition TGA (~ weight !0SS)!R
`
`DSC
`
`c0nc!usi0n
`
`RT-105 ?C
`
`105-225 ?C
`
`E
`5i3
`2A
`0 d~Y
`E
`4.5
`2.2
`RT/<5%Pd-f
`H
`RTt56%RH
`0i2
`6A
`H
`0.!
`6.6
`RT/75%RH
`501C 1.13.7 E/A
`H
`6.!
`0.!
`40~C/75%RH
`
`!04i6
`~03!
`73i3
`732
`t0ti7
`72i9
`
`TGA
`
`RT~!00~C
`
`!00~225 ~C
`
`IR
`
`Condition
`
`0 day
`
`RT/~5% RH
`RT/56% RH
`RT~75% RH
`50~C
`4OIC/75% RH
`
`Extra (~C)
`
`NS
`NS
`NS
`NS
`76
`NS
`
`DSC
`
`Max (~C)
`
`E
`E
`H
`H
`E/A
`H
`
`Conclusion
`
`A
`H
`H
`A
`H
`
`0i2
`7!i2
`A A/H
`0i!
`0i3
`H
`H 7!.0
`57 03
`H H 71~5
`6i20A
`02
`0! A/H
`A
`76-~
`5i80i~ H H 70A
`
`H
`
`Fig. 2, summarizing DSC-thermograms of untreated TMC114
`hydrate and TMC114 ethanolate using hermetic and non-hermetic
`sample pans, shows a broad endothermic signal for TMC114
`ethanolate (at ca. 101.7 °C, AH= 96Jig) caused by the evaporation
`of ethanol, collapsing and liquefying of the product. The desol-
`vation temperature is higher than the boiling point of the pure
`solvent suggesting that removal of ethanol is somewhat kinetically
`hindered. For TMC114 hydrate, the endothermic signal is posi-
`tioned at ca. 75.0°C (AH= 101Jig), caused by the evaporation of
`water, collapsing and liquefying of the product. The graphs show
`the characteristics of channel desolvation, namely an early onset of
`desolvation and a continuous solvent loss up to a certain temper-
`ature. The relative broad endotherms for desolvation observed for
`TMC114 ethanolate and TMC114 hydrate are additional evidence
`for the presence of ethanol and water molecules in the channels of
`the crystals.
`
`Fig. 3 shows the total, reversing and non-reversing heat flow
`of the quench-cooled ethanolate and hydrate. The reversing heat
`flow shows a clear change in heat capacity (ACp, glass transition Tg)
`in the temperature range from 30 to ?0 °C. The Tg for the quench-
`cooled ethanolate is 4? ° C and for the quench-cooled hydrate 49 ° C.
`The TGA weight change and its first derivative of TMC114
`ethanolate and TMC114 hydrate (data not shown) allowed con-
`cluding that evaporation of the solvent occurs in multiple stages
`up to about 230°C. TMC114 ethanolate mainly looses its weight
`above 80 ° C, indicating its relative stability. The weight loss (in total
`?%) is due to the evaporation of ethanol. TMC114 hydrate’s weight
`loss (in total 6%) starts immediately after increasing the temper-
`ature with a maximal weight loss around ?5 °C. The relative ease
`of dehydration can be caused by the ability of the smaller water
`molecules to migrate through the channels much more easily than
`the more bulky ethanol molecules.
`
`0,0
`
`25
`~o up
`
`50
`
`7"5
`
`100
`
`t25
`
`Temperature ("C)
`
`Uriiversal V3,gA TA Inslurmerit.~
`
`(non-hermetic) and TMC114 ethanolate (hermetic).
`
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`E. Van Gyseghem et al. / European Journal of Pharmaceutical Sciences 38 (2009) 489-497
`
`493
`
`Fig. 3. MDSC heat flow curves of the quench-cooled fractions, from top to bottom: total heat flow of the quench-cooled ethanolate and the quench-cooled hydrate, non-
`
`reversing heat flow of the quench-cooled ethanolate and the quench-cooled hydrate and the reversing heat flow of the quench-cooled ethanolate and the quench-cooled
`
`hydrate.
`
`For TMC1 !4 ethanolate (Table 2a) stored at RT/56% RH, RT/75%
`RH, and 40°C/75% RH an endothermic signal at ca. 73°C was
`observed with DSC, implying the conversion to the hydrate. This
`was confirmed with IR and TGA, the latter showing a higher weight
`loss at the lower temperature interval at those conditions. This
`suggests that the ethanolate is somewhat unstable at high % RH
`and exchanges ethanol with water. Ethanol is pushed out of the
`channels by water. The ethanolate is sensitive to increased tem-
`perature and partially desolvates. For the ethanolate form stored
`at 40 °C/75% RH, the exchange of the ethanol present in the drug
`substance with the water present in the environment was also con-
`firmed using GC head space analysis. The amount of ethanol in the
`fraction after these storage conditions was 60 ppm, which is consid-
`ered as not significant. Therefore, it was concluded that the solvent
`exchange is complete and the sample is converted to the hydrate.
`This was also confirmed by TGA. A weight loss of ±6.9% up to 90 °C
`is due to the evaporation of the water present in the sample (chan-
`nel hydrate). At 50 °C a small amount of amorphous product was
`detected with IR (further evidence in Section 3.2. Phase transition
`studies upon annealing). However, the DSC results (Table 2a) show
`the presence of the hydrate form (signal at ?6 °C). Most probably
`the small amount of amorphous material had already converted
`into the hydrate before the DSC measurement was performed.
`TMC1 !4 hydrate (Table 2b) changes into an amorphous anhy-
`drous product after storage in dry conditions, i.e. RT/<5% RH and
`50 ° C. The TGA results show higher weight losses for samples stored
`at high relative humidity (RT/56% RH, RT/?5% RH and 40°C/75%
`RH). Therefore the % RH strongly determines the amount of water
`present in the hydrate.
`
`3.2. Phase transition studies in nitrogen (dry) atmosphere, in
`ethanol atmosphere, upon heating, upon annealing, and as a
`function of the drying temperature
`
`TMC114 hydrate was placed in a non-hermetic sample pan in
`an MDSC at 25 °C and exposed to a N2 flow of 50 ml/min for 3 h,
`after which the M DS C measurement was started immediately. Fig. 4
`compares the reversing and total heat flow signals of untreated and
`dried TMC114 hydrate. The total heat flow of the untreated hydrate
`showed a large endothermic signal (at 66.2 °C, AH> 100J/g), the
`
`treated hydrate only a very small one (at 69.9 °C, AH< 4J/g). This
`decrease in AH of the treated sample indicates dehydration of the
`product. The reversing heat flow of the treated TMC114 hydrate
`showed a clear change in heat capacity corresponding to a Tg of
`71 °C, confirming the conversion into amorphous material when
`nitrogen is flushed through the sample.
`The transition from crystalline to amorphous of TMC114
`hydrate during drying with N2 was confirmed with XRPD equipped
`with a controlled environmental hot-stage at 25 °C as a function of
`time (Fig. 5). During the drying process a gradual decrease in the
`sample crystallinity was observed. Complete conversion into X-ray
`amorphous material occurred at the end of the experiment.
`TMC1 !4 hydrate and amorphous TMC1 !4 (obtained by dehy-
`drating the hydrate at 50 °C under reduced pressure during 3 h)
`were placed in a desiccator filled with ethanol at 50 °C for 2 days.
`The DSC curves (data not shown) showed an endothermic signal
`at 102.9°C for the treated hydrate and at 98.! °C for the treated
`amorphous form indicating that storage of TMC114 hydrate and
`amorphous TMC114 in an ethanol atmosphere causes in both cases
`conversion into the ethanolate form.
`Fig. 6 summarizes the XRPD diffractograms of untreated and
`quench-cooled TMC114 ethanolate and TMC114 hydrate at RT.
`The angular positions for the majority of the diffraction peaks
`of TMC114 ethanolate and TMC114 hydrate were in close cor-
`respondence, suggesting they are isostructural (see for instance
`characteristics peaks at ?.!°, !1.3°, !3.8°, and !6.?° (28)). This
`implies that the three-dimensional arrangement of the host
`TMC1 !4 molecules has a common structural motif (conformation
`and packing) and that the included solvent molecules occupy com-
`mon crystallographic sites. Quench-cooling of TMC114 ethanolate
`and TMC1 !4 hydrate both make the crystal structure to collapse,
`resulting in amorphous material.
`Removal of solvent from TMC114 hydrate and TMC114
`ethanolate upon heating was monitored by hot-stage XRPD with
`the samples heated at 10 °C/min. The sample is flushed with nitro-
`gen during the experiment. Diffraction patterns were measured at
`fixed temperatures between 25 and !05 °C (TMC114 ethanolate;
`Fig. ?a), and between 25 and 80°C and at 25 °C (after heating)
`(TMC1 !4 hydrate; Fig. ?b), respectively. The changes in the diffrac-
`tion patterns of the ethanolate and hydrate upon heating were
`
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`494
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`E. Van Gyseghem et al. / European Journal of Pharmaceutical Sdences 38 (2009) 489-497
`
`Exo Up
`
`Temperature
`
`Fig. 4. MDSC curves of TMC114 hydrate stored in an open DSC sample pan at 25 °C in a nitrogen flow of 50 ml/min for 3 h, overlay from top to bottom: reversing and total
`heat flow of dried TMC114 hydrate, and reversing and total heat flow of TMC114 hydrate.
`
`comparable, indicating an identical mechanism of solvent removal.
`A small shift in peak positions to lower 20-values could be observed
`in both cases at the characteristic diffraction peaks 7.1°, 11.3°, 13.8°
`and 16.7° (20). In contrast to the ethanolate ofTMC114, the hydrate
`lost its water already at lower temperatures. This was revealed by
`diffraction peaks becoming broader and less intense and a more
`pronounced amorphous halo. For the ethanolate the transforma-
`tion into amorphous material started only at temperatures close to
`100 °C.
`The crystal structure determination of TMC114 showed that
`the compound crystallizes as a (1:1) ethanol solvate. The abso-
`lute configuration of the TMC114 molecule in the crystal is: 3R.
`3aS. 6aR. 11S. 19R (Fig. 8). The crystal structure is stabilized by
`several intramolecular and intermolecular hydrogen bonds and
`weal{ interactions (Table 3). The ethanol molecule links two host
`molecules of TMC114 by two hydrogen bonds of the type O-H,,, O
`(O39,, ,O29: 2.852(4)A. 020 ,, ,O39: 2.955(4)A. There is also an
`
`intermolecular hydrogen bond of the type N-H,, ,O: (N36,, ,O39:
`3.199(5)A) linking the TMC114 molecules one to the other. These
`hydrogen bonds form an infinite three-dimensional network in the
`crystal. The crystal structure shows straight tunnels running along
`the [ 1 0 0] crystallographic direction (Fig. 9). The ethanol molecules
`are located in these channels. The calculations of the possible voids
`(pockets) in the crystal lattice, using the program PLATON (X-ray
`Single Crystal Analysis System, Version 2.2, 1996), show in these
`channels a total potential solvent accessible volume of 43.9 A3. This
`volume is in agreement with the volume of a hydrogen bonded
`water molecule (40A3), and a strong experimental evidence that
`the crystal structure of the TMC114 ethanolate is a channel sol-
`vate that contains voids to accommodate water molecules. These
`pockets along the crystal channels could contain water, and they
`play a key role in the formation of the hydrate under high rela-
`tive humidity conditions, as suggested by other physico-chemical
`techniques.
`
`6000
`
`200~-
`
`Fig. S. X-ray diffractograms, obtained after drying TMC114 hydrate in N2 atmosphere. Overlay from bottom to top: TMC114 hydrate, TMC114 hydrate dried in N2 flow at
`25 °C for 4, 8, 12, 60, 150 min and obtained sample without N2 flow.
`
`2Tbeta
`
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`E. Van Gyseghem et al. / European Journal of Pharmaceutical Sciences 38 (2009) 489-497
`
`495
`
`"!
`
`~ IO00G,
`
`~000,
`
`4000’
`
`200~’
`
`Fig. 6. X-ray difffactograms at RT, overlay from top to bottom: TMC114 ethanolate, TMC114 hydrate, quench-cooled TMC114 hydrate and quench-cooled TMC114 ethanolate.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`3’0
`
`35
`
`40
`
`45
`
`50
`2Theta (")
`
`(a) ~ .~oooo
`
`~ 15000
`
`10000
`
`80’00"
`
`60:00,
`
`4000-
`
`29:00-
`
`Fig. 7. X-ray diffractograms obtained after the hot-stage experiment. Overlay from bottom to top: (a) TMC114 ethanolate at 25, 50, 80, 85, 90, 95, 100, and 105 °C, and (b)
`TMC114 hydrate at 25, 40, 50, 60, 70, 80 °C, and at 25 °C after heating.
`
`35
`
`2Theta
`
`Lupin Ex. 1006 (Page 7 of 9)
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`
`Fig. 8. Oak ridge thermal ellipsoid plot (ORTEP) showing the conformation of the
`TMC114 ethanolate molecule in the crystalline state and its atomic numbering
`scheme. Displacement ellipsoids are drawn at 50% probability level for non-H atoms.
`The hydrogen bond linking the ethanol molecule to the host TMC114 molecule is
`denoted by dashed lines.
`
`Ethanol and/or water are present in lattice channels, next to
`solvent/water molecules in adjacent unit cells along an axis of the
`lattice, forming channels through the crystals. In case of channel
`solvates/hydrates dehydration begins at the "ends" of the crys-
`tal and continues towards the centre along the channels. As the
`temperature increases so does the probability of losing the first
`solvent or water molecules on the surface of the channel ends. The
`loss of these solvent or water molecules leaves a channel for the
`next and sets up a thermodynamic gradient in the same direction
`(Morris, 1999). Factors that influence the desolvation reaction are
`(a) the size of the tunnels in the crystal packing arrangement, (b)
`the strength of H bonding between the compound and (c) its sol-
`vent of crystallization. The solvent serves to stabilize the lattice
`as the process of desolvation produces a change in lattice parame-
`ters, resulting in the formation of an amorphous form. As shown by
`
`Table 3
`lntramolecular and intermolecular hydrogen bonds in the TMC114 ethanolate crys-
`tal structure.
`
`020-H20. 039 0.82 2;! 72.955(4)!62
`b
`N3B~H36Ai. iO8 086 2i37
`3i065(4)!38
`N36~H36B.. 039 0.86 2453.i99(5)i45
`b
`c
`039~H3~-029 0~82 2~04
`2~852~4)
`! 74
`C4~H~A. 07 0.97 248
`2.8!8~4)~00
`d
`2.85~(4)~03
`C!!~H! ! .08 0.98 246
`C12~Ht2B~ ~ .O20 0~97 2~53
`2~888(4)t02
`C!9~H!9-028 0-98 2533165(3)~22
`C21~H21A. ~iN10 0.97 Z54Z932~4)t04
`C23~H23B. ~ .029 0-97 24~ 2904(4)~ ~
`C3!~H3! .020
`0.93 2;57
`e
`3.379(4)!46
`
`Symmetry operations:
`~x 1/2,~+1/2, z+2.
`b x+1,y+1/2, Z+1/2+1.
`c x,y 1/2, Z+1/2+1.
`d X+ 1,y, Z.
`
`~ x,y+l/2, Z+1/2+1.
`
`Fig. 9. Unit cell view showing the packing of the TMC114 ethanolate molecules
`down crystallographic axis a. The hydrogen bonds are denoted by dashed lines and
`the ethanol molecules are depicted as ball and stick models.
`
`hot-stage XRPD, upon heating, amorphous material was obtained
`for the TMC114 ethanolate and hydrate.
`To follow the influence of drying below the deso