M E M O
`P h a r m a c e u t i c a l R & D - R a h w a y
`TO: Tim Rhodes
`
`FROM:
`
`Leigh Shultz
`
`CONTRIBUTORS:
`
`Chris Lindemann
`Dina Zhang
`Robert Wenslow
`James Qin
`Peter Dormer
`Leonardo Allain
`Kari Lynn
`Todd Gibson
`Feng Li
`
`SUBJECT: L-000224715 Preformulation Report
`
`DATE: 30 Sep 2002
`
`L-000224715 is a DP-IV inhibitor for the treatment of Type II Diabetes Mellitus. It was approved for
`development as a PCC by SARC in January of 2002 and began Phase I clinical trials in July 2002. This memo
`describes the chemical and physical properties of the compound known to date.
`
`cc: Dept. 854, A. Andrews, J. Armstrong, R. Franklin, J. Givand, K. Hansen, W. Hunke, P. Hurter, D. Ip, D. Kim,
`K. Kube, G. Kwei, G. Lankas, E. Luna, K. Lynn, N. Margaretten, D. Mendenhall, M. Moonis, D. Ostovic, S.
`Palkar, J. Pearson, S. Reynolds, I. Santos, S. Shelukar, C. Starbuck, M. Thien, N. Thornberry, R. Tillyer, E. Tsai,
`S. Vincent, A. Weber, J. Wyvratt, D. Zhang, J. Zimmerman
`
`Merck Exhibit 2148, Page 1
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
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`

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`Table of Contents
`1.0 SUMMARY .................................................................................................................... .......................................3
`
`2.0 DESCRIPTION................................................................................................................ .....................................3
`2.1 NAME, STRUCTURE, FORMULA ........................................................................................................................... 3
`2.2 COLOR, FORM, APPEARANCE.............................................................................................................................. 3
`3.0 TEST SUBSTANCES ............................................................................................................ ...............................3
`
`4.0 PHYSICAL CHARACTERIZATION .................................................................................................. ..............4
`4.1 MICROSCOPY ............................................................................................................................... ....................... 4
`4.2 DIFFERENTIAL SCANNING CALORIMETRY (DSC)................................................................................................ 6
`4.3 THERMOGRAVIMETRIC ANALYSIS (TGA) ........................................................................................................... 7
`4.4 X-RAY POWDER DIFFRACTION (XRPD).............................................................................................................. 8
`4.5 SOLID STATE NMR (SSNMR).................................................................................................................... ........ 9
`4.6 CRYSTAL FORMS/POLYMORPHISM .................................................................................................................... 10
`4.7 HYGROSCOPICITY............................................................................................................................... ............... 13
`4.8 EQUILIBRIUM SOLUBILITY IN AQUEOUS MEDIA................................................................................................ 15
`4.9 EQUILIBRIUM SOLUBILITY IN ORGANIC MEDIA................................................................................................. 15
`4.10 PKA............................................................................................................................... .................................. 16
`4.11 DISSOLUTION AND SOLUTION PROPERTIES...................................................................................................... 16
`16
`4.12 UV/VIS ABSORBANCE SPECTRUM
`5.0 STABILITY.................................................................................................................. .......................................17
`5.1 BULK DRUG STABILITY............................................................................................................................... ...... 17
`5.2 SOLUTION STABILITY............................................................................................................................... ......... 18
`5.3 MODES OF DEGRADATION............................................................................................................................... .. 19
`6.0 ANALYTICAL METHODS......................................................................................................... ......................20
`6.1 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY............................................................................................ 20
`6.2 HPLC/MS/MS................................................................................................................. ................................. 20
`6.3 UV/VIS ABSORBANCE............................................................................................................................... ........ 20
`6.4 SOLID-STATE NMR............................................................................................................................ ............... 21
`7.0 BIOPHARMACEUTICAL PROPERTIES ............................................................................................... .......22
`
`8.0 BULK DRUG FORM/DOSAGE FORM DESIGN CONSIDERATIONS .....................................................24
`
`9.0 MECHANICAL PROPERTIES ...................................................................................................... ..................25
`9.1 BULK DRUG CHARACTERIZATION..................................................................................................................... 25
`
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`1.0 SUMMARY
`
`This memo summarizes the preformulation data available for the single known polymorphic form of the crystalline
`monobasic phosphate salt of L-000224715. The phosphate salt is a stable, high-melting, non-hygroscopic material with
`known isomorphic solvates. It is highly soluble in aqueous media across the physiological pH range and has adequate
`stability in solution below pH 6. The flow properties of the phosphate salt are good, suited to a direct compression
`formulation.
`
`2.0 DESCRIPTION
`
`L-000224715, a DP-IV inhibitor, is being developed for oral administration as a treatment for type II (adult-onset) Diabetes
`Mellitus. This compound is the second DP-IV inhibitor in development at Merck.
`
`2.1 Name, Structure, Formula
`
`L-000224715 has a molecular weight of 407.32 g/mol and a molecular formula of C16H15F6N5O. It is being developed as the
`monobasic phosphate salt, with molecular weight 505.32 g/mol (salt factor 1.24) and molecular formula C16H18F6N5O5P. The
`structure of L-000224715 is shown in Figure 1, below.
`
`F
`
`F
`
`F
`
`NH2
`
`O
`
`N
`
`N
`
`N
`
`N
`
`CF3
`
`2.2 Color, Form, Appearance
`
`Figure 1. Structure of L-000224715
`
`The phosphate salt of L-000224715 is a white, crystalline powder with flake-like individual crystals. The powder shows
`some agglomeration and good flow properties.
`
`3.0 TEST SUBSTANCES
`
`All experiments were performed on lots of phosphate salt provided by Process Research. Stability, solubility, microscopy,
`particle sizing, XRPD, hygroscopicity, and thermal experiments were performed on lot L-000224715-006F006 (A-sheet).
`SEM data were obtained on lot L-000224715-006F007 (D-sheet). Particle size data are also shown for lots 006F003 (A-
`sheet) and 006F009 (A-sheet), which were used for assessing the feasibility of the Xcelodose* . Lots 006 and 007 came
`from the same chemical batch, which was split and released separately. Data obtained from other lots of phosphate salt are
`noted in the text. Both aqueous solubility as a function of pH and the pKa were determined with the crystalline free base,
`L-000224715-000T001.
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`4.0 PHYSICAL CHARACTERIZATION
`
`4.1 Microscopy
`
`Optical microscopy performed on lot L-000224715-006F006 at 200X magnification (Figure 2) reveals a birefringent,
`crystalline material. The primary crystals have a well-defined flake-like morphology; many of the flakes have a hexagonal
`shape. Some agglomeration of the crystals is observed.
`
`Figure 2. Optical microscopic image of L-000224715-006F006 (200X)
`
`SEM images of L-000224715-006F007 are shown in Figure 3. Agglomeration of the individual crystals is evident in these
`images.
`
`Figure 3. SEM images of L-000224715-006F007, showing agglomeration (205X, left; 170X, right)
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`Figure 4 shows particle size data for three lots of L-000224715, obtained using a MicroTrac light-scattering instrument with
`isopropanol as the medium. Lot 006 shows a broad, monomodal particle size distribution with a mean of 79 µm after 30 s of
`sonication (D10 = 23 µm, D95 = 190 µm). Prior to sonication, the mean particle size was 113 µm, with a D10 of 27 µm and a
`D95 of 285 µm. The decrease in both D95 and the mean on sonication indicates the loose nature of the agglomerates shown in
`Figure 3. Lots 003 and 009, used for testing the feasibility of the Xcelodose* , have similar average particle sizes, but show
`less agglomeration, decreasing from mean particle sizes of 46 and 65 µm, respectively, to 42 and 56 µm on sonication.
`
` L-224715
` Sonication = 30s
` Lot 006F003
` Lot 006F006
` Lot 006F009
`
`12
`
`10
`
`8 6 4 2 0
`
`% Frequency
`
`1
`
`10
`Particle Size (um)
`
`100
`
`Figure 4. Comparison of particle size distribution for L-000224715-006F003, -006F006, and 006F009
`
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`4.2 Differential Scanning Calorimetry (DSC)
`
`DSC data for lot 006 are shown in Figure 5. Data were obtained by heating a sample in an open pan at 10 °C/min.
`L-000224715-006F006 shows a single endotherm due to melting at 218.0 °C. The endotherm cannot be reliably quantitated
`due to decomposition immediately following melting. No solid-solid transitions are observed for the delivered material by
`DSC, indicating the presence of a single, high-melting polymorph.
`
`Sample: L-000224715-006F006
`Size: 2.6800 mg
`Method: standard
`
`DSC
`
`File: C:...\L224715\006F006_dsc
`Operator: LS
`Run Date: 8-Oct-02 10:15
`
`216.62°C
`235.2J/g
`
`50
`
`100
`
`150
`Temperature (°C)
`
`200
`
`217.99°C
`250
`
`300
`Universal V2.3C TA Instruments
`
`Figure 5. DSC thermogram of L-000224715-006F006 (open dish)
`
`0
`
`-2
`
`-4
`
`-6
`
`Heat Flow (W/g)
`
`-8
`Exo Up
`
`0
`
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`4.3 Thermogravimetric Analysis (TGA)
`
`A TGA trace for L-000224715-006F006 is shown in Figure 6. A sample heated at 10 °C/min under nitrogen flow shows a
`weight loss of 0.09% up to its melting point.
`
`Sample: L-000224715-006F006
`Size: 16.8330 mg
`Method: standard method
`105
`
`TGA
`
`File: C:...\L224715\006F006_tga
`Operator: LS
`Run Date: 8-Oct-02 10:14
`
`0.09241% weight loss to 218C (melt)
`(0.01555mg)
`
`50
`
`100
`
`150
`Temperature (°C)
`
`200
`
`250
`
`300
`Universal V2.3C TA Instruments
`
`Figure 6. TGA trace of L-000224715-006F006
`
`100
`
`95
`
`90
`
`85
`
`Weight (%)
`
`80
`
`0
`
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`4.4 X-ray Powder Diffraction (XRPD)
`
`The X-ray powder diffraction pattern between 4 and 40° 2 is shown in Figure 7. Numerous sharp reflections are observed
`in this region, indicating the high degree of crystallinity of the phosphate salt.
`
`counts
`
`8000
`
`6000
`
`4000
`
`2000
`
`0
`
`5
`
`L-000224715-006F006
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`°2Theta
`
`Figure 7. XRPD pattern for L-000224715-006F006
`
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`4.5 Solid State NMR (SSNMR)
`
`19F solid-state NMR was used to characterize several lots of L-000224715, since the molecule contains six F atoms. Because
`3 of the F atoms are on an aryl ring and the other 3 F atoms are in a trifluoromethyl group, they have very different chemical
`shifts. This large difference in chemical shift makes spectral phasing difficult if a spin-echo pulse sequence is used;
`therefore, all 19F spectra for L-000224715 were obtained using a standard non-echo pulse sequence. Use of the 19F nuclei for
`characterization will allow SSNMR to be used for characterization of drug-excipient blends and dosage forms, since none of
`the common excipients contain fluorine.
`
`The CF3 region of the 19F SSNMR spectrum of L-000224715-006F006 is shown in Figure 8, below. The most intense peak,
`between 65 and 66 ppm, is present in all of the released batches of the phosphate salt studied to date. Most batches also
`have a shoulder at 63 ppm. The presence of this second resonance indicates that some of the CF 3 groups are in a different
`chemical environment. If there were simply 2 distinct molecules in the unit cell of the crystal, two peaks of equal intensity
`would be expected. However, the intensity of the peak at 63 ppm varies from lot to lot. The origin of this peak will be
`discussed in more detail in Section 4.6.
`
`c
`
`
`
` L-000224715-006F006
`
`Peak intensity varies lot-to-lot
`
`-58
`
`-60
`
`-62
`
`-64
`
`-66
` (ppm)
`
`-68
`
`-70
`
`-72
`
`Figure 8. 19F MAS NMR spectrum of L-000224715-006F006
`
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`4.6 Crystal Forms/Polymorphism
`
`As discussed above in Section 4.5, a variation in the intensity of the 19F SSNMR peak at 63 ppm was observed upon
`examination of the spectra from different lots of crystalline material. 13C NMR spectra of the solids (not shown) confirmed
`the absence of ethanol, the crystallization solvent. In addition, the relaxation of the peak at 63 ppm was measured to be 100
`ms, significantly longer than that of amorphous material, which appears as a broad resonance at 64 ppm with a relaxation
`time of 10 ms. Although the moisture content of the material had been shown to be very low (0.14%) by TGA, it was
`hypothesized that the presence of water in the crystal lattice could produce the shoulder seen in the 19F spectrum. A sample
`of L-000224715-006F006 was placed under a stream of nitrogen at 150 °C for 2 hours, after which the 19F NMR spectrum of
`the material was taken (Figure 9). After drying the material, the shoulder at 63 ppm disappeared, suggesting that its
`presence is an indication of water in the crystal lattice of the phosphate salt. Subsequent experimentation showed that the
`delivered material could be dehydrated under a stream of nitrogen at 40 °C, but simply heating the material under vacuum or
`in air does not cause dehydration.
`
` 006F006
` 006F006 150C N2 flow 2 hours
`
`-50
`
`-55
`
`-60
`
`-65
`
`-70
`
`-75
`
`-80
`
`Figure 9. 19F MAS NMR spectra for hydrated and anhydrous L-000224715-006F006 (CF 3 region)
`X-ray powder diffraction was also used to examine the material produced in the final crystallization step of the Process
`Research synthesis, which was carried out in ethanol/water (Figure 10). The dried material (labeled Anhydrous in Figure
`10) has a distinct XRPD pattern from the hydrated material (note the absence of the peak at 14° and the growth of peaks at 23
`and 25° 2 in the anhydrous material). Material slurried in ethanol ( Ethanol Solvate, Figure 10) also shows new peaks
`(especially 12 and 21° 2 ).
`
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`EtOH Solvate
`
`Hydrate
`
`Anhydrous
`
`6000
`
`4000
`
`2000
`
`counts/sec.
`
`20
`°2
`Figure 10. Comparison of XRPD patterns for the anhydrous, hydrous, and ethanol solvated forms of L-000224715-006F
`
`10
`
`30
`
`40
`
`Exposure of the anhydrous solids to 75% RH for days does not induce rehydration. It is unclear whether the presence of
`water is the result of a small amount of a stoichiometric hydrate impurity in the anhydrous phase or is due to a channel
`hydrate (also called a tunnel hydrate). None of the hydrated material has been observed to have more than 0.25% water by
`KF titration or TG analysis.
`
`After the presence of hydrated and solvated material had been confirmed, the phosphate salt was slurried in a variety of
`solvents to induce solvate formation. The wet cakes produced were examined by XRPD, and changes were observed in
`water, methanol, ethanol, isopropanol, acetonitrile, ethyl acetate (EtOAc), acetone, N,N-dimethylformamide (DMF), and
`1-octanol (Figure 11).
`
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`Figure 11. XRPD patterns for solvates of L-000224715-006F
`
`Although both hydrated and solvated materials have been produced for L-000224715-006F, no other anhydrous polymorphs
`have been observed to date. Amorphous material can be made by lyophilization of an aqueous solution, and this amorphous
`material is physically stable at 20 °C. Small amounts of the lyophilized material will recrystallize when exposed to heat or
`humidity; samples recrystallized within days at 40 °C/75% RH, within minutes on heating in the DSC, and within hours upon
`exposure to ~65% RH on the moisture sorption balance at 40 °C.
`
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`4.7 Hygroscopicity
`
`The hygroscopicity of lot L-000224715-006F006 was determined at 25, 30, and 40 °C (Figure 12a, b, and c) from 5 to 95%
`RH using a dynamic vapor sorption balance (VTI SGA-100). At 25 °C, the solids were not dried prior to the hygroscopicity
`experiment. They gain only 0.27% water by weight over the entire humidity range, but the hysteresis on desorption is
`indicative of a hydrated material. The material is also non-hygroscopic at both 30 and 40 °C, and no stoichiometric hydrates
`were detected at any of the three temperatures.
`(a) Adsorption/Desorption Isotherm, 25 C
`
`Adsorption
`Desorption
`
`0.300
`
`0.250
`
`0.200
`
`0.150
`
`0.100
`
`0.050
`
`0.000
`
`-0.050
`
`-0.100
`
`-0.150
`
`Weight (% change)
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`%RH
`
`60
`
`70
`
`80
`
`90 100
`
`(b) Adsorption/Desorption Isotherm, 30 C
`
`Adsorption
`Desorption
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`%RH
`
`60
`
`70
`
`80
`
`90 100
`
`0.350
`
`0.300
`
`0.250
`
`0.200
`
`0.150
`
`0.100
`
`0.050
`
`0.000
`
`Weight (% change)
`
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`(c) Adsorption/Desorption Isotherm, 40 C
`
`Adsorption
`Desorption
`
`0.350
`
`0.300
`
`0.250
`
`0.200
`
`0.150
`
`0.100
`
`0.050
`
`0.000
`
`Weight (% change)
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`%RH
`
`60
`
`70
`
`80
`
`90
`
`100
`
`Figure 12. Hygroscopicity of L-000224715-006F006 at 25 °C (a), 30 °C (b), and 40 °C (c)
`
`The hygroscopicity of the anhydrous phosphate salt, produced by drying under a stream of nitrogen, was also determined at
`25 °C (Figure 13). Although the material gains more water weight at 95% RH (0.68%) than the hydrated solids, it is still
`non-hygroscopic.
`
`L-000224715 Phosphate Salt Anhydrous Solids
`Adsorption/Desorption Isotherm
`
`Adsorption
`Desorption
`
`0.800
`0.700
`0.600
`0.500
`0.400
`0.300
`0.200
`0.100
`0.000
`-0.100
`
`Weight (% change)
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`%RH
`
`60
`
`70
`
`80
`
`90 100
`
`Figure 13. Hygroscopicity of the anhydrous phosphate salt of L-000224715 at 25 °C
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`4.8 Equilibrium Solubility in Aqueous Media
`
`The solubility of the phosphate salt (lot 006F006) was determined using the shake-flask method at 25 °C. The solubility of
`the salt in water was found to be 107.6 mg/mL L-000224715 with a native pH of 4.25. The solubility in 0.9% NaCl (saline)
`was found to be 117.0 mg/mL L-000224715. Solubility data were also obtained at pH 2.3 (123.5 mg/mL) and 4.0 (118
`mg/mL), but the compound was soluble enough to break 250 mM buffers above pH 4. As a result, solubility data for
`L-000224715 were obtained across the physiological pH range by titration of 50 mg of the crystalline free base,
`L-000224715-000T001, with 10-mol% aliquots of HCl (Figure 14). At pH 8, the solubility was 12 mg/mL; this increased to
`40 mg/mL at pH 7.4 and to more than 45 mg/mL at pH 7, where the entire sample had dissolved.
`Solubility of L-000224715 as a function of pH
`
`7.00
`
`8.00
`pH
`
`9.00
`
`10.00
`
`50.00
`45.00
`40.00
`35.00
`30.00
`25.00
`20.00
`15.00
`10.00
`5.00
`0.00
`6.00
`
`[L-000224715] (mg/mL)
`
`Figure 14. Aqueous solubility of L-000224715 as a function of pH (titration data)
`
`L-000224715 is very soluble across the physiological pH range, and solubility is not expected to limit bioavailability for this
`compound.
`
`4.9 Equilibrium Solubility in Organic Media
`
`The equilibrium solubility of L-000224715-006F006 was also determined in possible granulating solvents (ethanol,
`isopropanol, and 90% mixtures of these in water), and in methanol and acetonitrile (possible HPLC mobile phases). The
`solubility data are shown in Table 1.
`
`Table 1. Solubility of L-000224715-006F006 in organic media
`
`Solvent
`
`methanol
`ethanol
`2-propanol
`90% (v/v) EtOH
`90% (v/v) 2-PrOH
`acetonitrile
`
`Solubility
`(mg L-224715/mL)
`0.33
`0.19
`0.08
`2.78
`2.51
`1.96
`
`The solubility values above are for the solvates of L-000224715-006F in each case; see Section 4.6 above for discussion.
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`4.10 pKa
`
`The pKa of L-000224715-001T001 was determined in water by potentiometric titration. The average value determined, 8.03
`± 0.06, is attributed to the primary amine moiety and is in good agreement with the value of 7.5 determined from the
`solubility titration of the free base (see Section 4.9).
`
`4.11 Dissolution and Solution Properties
`
`Although the phosphate salt of L-000224715 is non-hygroscopic, it is highly soluble in water and dissolves rapidly upon
`contact with water. It also goes into solution rapidly in acidic media such as 0.1 N HCl and 0.1% H3PO4. Water and acidic
`solution are both suitable as a dissolution media for L-000224715-006F.
`
`4.12 UV/Vis Absorbance Spectrum
`
`The UV/Visible spectrum of L-000224715 was determined in 50% (v/v) acetonitrile (Figure 15). Absorption maxima are
`observed at 202 and 267 nm.
`
`UV-Visible Spectrum of L-000224715
`
`267
`
`200
`
`0.195
`
`0.095
`
`-0.005
`
`Absorbance (AU)
`
`190 200 210 220 230 240 250 260 270 280 290 300 310 320
`Wavelength / nm
`
`Figure 15. UV-Visible absorbance spectrum of L-000224715 (50% acetonitrile in water)
`
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`16
`
`Merck Exhibit 2148, Page 16
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`5.0 STABILITY
`
`5.1 Bulk Drug Stability
`
`The bulk stability of L-000224715-006F006 was assessed at 1, 2, 4, 8, and 13 weeks at 5, 25, 40, and 60 °C/amb RH; at 25
`°C/60% RH, 30 °C/65% RH, and 40 °C/75% RH; and at 1, 2, and 4 weeks at 80 °C/amb RH. Stability data are reported as %
`initial claim (Table 2) and as area % (Table 3) relative to samples kept at 20 °C. No degradation was detected in any of the
`samples; missing area percent in Table 3 can be attributed to peaks arising from the blank and diluent.
`
`Table 2. Solid-state stability (% initial claim) of L-000224715-006F006
`
`% Initial Claim L-000224715
`1 wk 2 wks 4 wks 8 wks 13 wks
`99.5
`100.8
`99.6
`100.5
`99.4
`100.9
`100.6
`99.4
`99.7
`99.6
`100.9
`100.1
`99.6
`100.7
`99.9
`101.2
`99.8
`99.8
`101.2
`99.5
`99.0
`100.3
`100.4
`99.1
`102.7
`101.4
`98.3
`99.0
`NT
`NT
`100.8
`102.7
`99.2
`101.1
`99.2
`101.2
`99.5
`97.1
`101.6
`99.6
`
`Table 3. Solid-state stability (relative area %) of L-000224715-006F006
`
`Relative Area % L-000224715
`1 wk 2 wks 4 wks 8 wks 13 wks
`99.0
`99.8
`100.4
`98.3
`99.9
`99.6
`99.9
`101.3
`98.9
`100.1
`99.1
`100.0
`101.4
`100.6
`99.7
`100.4
`100.1
`100.4
`99.8
`99.8
`99.4
`99.9
`100.6
`100.6
`99.8
`99.0
`99.8
`101.6
`NT
`NT
`99.5
`99.8
`100.2
`99.7
`100.3
`99.0
`99.8
`100.1
`100.6
`100.4
`
`Station
`5 °C/amb RH
`25 °C/amb RH
`40 °C/amb RH
`40 °C/75% RH
`60 °C/amb RH
`80 °C/amb RH
`25 °C/60% RH
`30 °C/65% RH
`NT = not tested
`
`Station
`5 °C/amb RH
`25 °C/amb RH
`40 °C/amb RH
`40 °C/75% RH
`60 °C/amb RH
`80 °C/amb RH
`25 °C/60% RH
`30 °C/65% RH
`NT = not tested
`
`The bulk stability of L-000224715-006F006 was also assessed upon exposure to 1, 2, and 4 weeks equivalent of fluorescent
`laboratory light. No loss of % claim, loss of area percent, or degradates were detected versus dark control samples.
`
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`17
`
`Merck Exhibit 2148, Page 17
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`5.2 Solution Stability
`
`The solution stability of L-000224715-006F was assessed in solution at 40 and 80 °C in 20 mM buffers (pH 4, acetate; pH 6
`and 8, phosphate; pH 10, carbonate), 0.01 N HCl, and deionized water at 0.1 mg/mL. Data are reported as area percents
`relative to samples stored at 20 °C (Table 4).
`
`Table 4. Solution thermal stability of L-000224715-006F
`
`Conditions
`
`water
`pH 2
`pH 4
`pH 6
`pH 8
`pH 10
`
`Relative Area % L-224715, 40 °C
`1 wk 2 wk 4 wk
`93.9
`89.8
`75.9
`99.9
`98.1
`100.6
`101.8
`93.2
`94.1
`99.0
`101.6
`98.5
`88.2
`81.6
`60.2
`83.1
`62.9
`36.5
`
`Relative Area % L-224715, 80 °C
`1 wk 2 wk 4 wk
`0.0
`0.0
`0.0
`99.0
`100.3
`95.4
`100.9
`93.6
`90.9
`24.9
`5.0
`0.4
`0.0
`0.0
`0.0
`0.0
`0.0
`0.0
`
`From this initial study, it was observed that L-000224715 is most stable at pH 2 and 4 and that it degrades rapidly at elevated
`temperatures (80 versus 40 °C) and in basic solution. A more extensive solution study was done with L-000224715 in
`solution from pH 1 to pH 10 at 40 °C. Pseudo-first order rate constants were calculated (with units of 1/h) for loss of
`L-000224715 with time and are graphed logarithmically in Figure 16.
`Rate of Degradation vs pH, 40 C
`
`-2.250
`-2.750
`-3.250
`-3.750
`-4.250
`-4.750
`-5.250
`-5.750
`
`log k(obs)
`
`0
`
`2
`
`4
`
`6
`
`pH
`
`8
`
`10
`
`Figure 16. pH-rate profile for degradation of L-000224715 in solution at 40 °C
`
`As a comparison, the half-life for degradation at pH 4 is 13.5 years, at pH 7 is 10 weeks, and at pH 10 is 7.4 days. Acid-
`catalyzed degradation is observed at pHs lower than 4; both thermal and base-catalyzed degradation are observed at high pH.
`The data below pH 4 are cleaner due to a single pathway for degradation. Two modes of degradation are observed at higher
`pH and cause more scatter in the data.
`
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`18
`
`Merck Exhibit 2148, Page 18
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`5.3 Modes of Degradation
`
`The degradation of L-000224715 observed in solution was studied by LC/MS/MS. The proposed degradation pathways
`elucidated in this study are shown in Scheme 1, below.
`
`Scheme 1. Solution degradation pathways for L-000224715
`
`F
`
`F
`
`F
`
`F
`
`F
`
`F
`
`NH2
`
`O
`
`N
`
`L-224715
`
`base
`or
`acid
`
`N
`
`N
`
`N
`
`CF3
`
`O
`
`N
`
`N
`
`N
`
`N
`
`CF3
`
`F
`
`F
`
`F
`
`F
`
`F
`
`F
`
`NH2
`
`O
`
`HN
`
`O
`
`N
`
`N
`
`N
`
`CF3
`
`amide bond cleavage
`degradates
`
`O
`
`N
`
`N
`
`N
`
`N
`
`CF3
`
`elimination (de-amination) degradates
`
`The main path for degradation is hydrolysis of the amide bond, which proceeds by both an acid- and base-catalyzed
`mechanism to produce a carboxylic acid and a free amine, which is UV silent above 220 nm. L-000224715 also degrades
`thermally by an elimination mechanism to produce a mixture of unsaturated products. The thermodynamic product,
`identified by solution 1H NMR (P. Dormer, Process Research), is the trans olefin in conjugation with the aryl ring. The trans
`olefin in conjugation with the amide has been identified as a minor product. The formation of the elimination products is
`accelerated by base, which aids in deprotonation. At very high pH, however (pH 10), hydrolysis predominates.
`
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`19
`
`Merck Exhibit 2148, Page 19
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`6.0 ANALYTICAL METHODS
`
`6.1 High Performance Liquid Chromatography
`
`System:
`Column:
`Mobile phase:
`Flow:
`Detector:
`Temperature:
`Inj. vol:
`Diluent:
`Sample conc.
`Gradient:
`
`Perkin Elmer Series200
`YMC-Pack ODS-AM 5µm 250X4.6mm
`A) 0.1% phosphoric acid B) acetonitrile
`1.0 mL/min
`210 nm
`ambient
`20 µL
`0.1% phosphoric acid
`0.1 mg/mL of L-000224715
`
`Time
`(min)
`0
`2
`13
`16
`25
`27
`28
`36
`
`A
`
`98
`98
`59
`59
`30
`30
`98
`98
`
`B
`
`2
`2
`41
`41
`70
`70
`2
`2
`
`L-000224715 has a retention time of 14.8 ± 0.1 min; the des-fluoro process impurities elute at 14.2 ± 0.1 and 14.4 ± 0.1 min.
`
`6.2 HPLC/MS/MS
`
`Stability samples of L-000224715 containing degradates were analyzed by LC/MS/MS (J. Qin) to determine the degradate
`molecular weights. The LC/MS/MS method is as follows:
`
`Waters 2790
`LC System:
`Symmetry C18 5µm 150X3.9 mm
`Column:
`A) 0.1% formic acid B) acetonitrile
`Mobile phase:
`0.2 mL/min
`Flow:
`Micromass Q-TOF2 (ESI)
`Detector:
`Ionization mode: Positive
`Temperature:
`40 °C
`50 µL
`Inj. vol:
`Sample conc.
`0.1 mg/mL of L-000224715
`Gradient:
`
`A
`
`B
`
`Time
`(min)
`0
`15
`
`95
`10
`
`5
`90
`
`6.3 UV/Vis Absorbance
`
`The absorbance spectrum was obtained in 50% acetonitrile/water (quartz cuvette) on an Agilent 8453 UV/Vis spectrometer
`by Leonardo Allain (PAC). The concentration of the API in solution was 0.0081 mg/mL.
`
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`20
`
`Merck Exhibit 2148, Page 20
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`6.4 Solid-state NMR
`
`Solid-state NMR experiments were performed by Robert Wenslow (Analytical Research) on a Bruker DSX-400
`spectrometer. 19F NMR spectra were obtained using a 4-mm CRAMPS probe and a standard MAS pulse sequence (no echo
`was used). 19F chemical shifts are reported in ppm downfield of CCl3F using Teflon (122 ppm) as a secondary reference.
`
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`21
`
`Merck Exhibit 2148, Page 21
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`7.0 BIOPHARMACEUTICAL PROPERTIES
`
`L-000224715 was dosed to rats in Drug Metabolism (M. Beconi) as a solution of the HCl salt (1.0 mg/mL) in
`ethanol:PEG400:water (2:3:5, v/v/v) for both IV and oral administration. For dosing in rhesus monkeys and beagle dogs,
`solutions of the fumarate salt (2 mg/mL for monkeys, 4 mg/mL for dogs) in saline were used for both IV and oral
`administration. Pharmacokinetic data are shown in Table 5 below.
`
`Table 5. PK data for dosing of L-000224715 in 3 species (RY Drug Metabolism)
`
`Dose
`Single PO (2 mpk)
`
`Single IV (1 mpk)
`
`Parameter
`Tmax (hr)
`%F
`t1/2 (hr)
`
`Rat (n=3)
`Mean (SD)
`1.08 (0.88)
`70
`1.5 (0.2)
`
`Dog (n=2)
`Dog 1 Dog 2
`1.0 0.5
`110
`5.0 4.9
`
`Monkey (n=2)
`Mk 1
` Mk2
`2.0 2.0
`68
`4.8 2.7
`
`Aqueous solutions of the phosphate salt of L-000224715 were dosed to fasted male rats and fasted beagle dogs in
`Biopharmaceutical Chemistry (K. Lynn). Figure 17 shows exposure data in dogs; L-000224715 shows linear
`pharmacokinetics with dose. Likewise, exposure data in the rat is shown in Figure 18. Linear, dose-dependent
`pharmacokinetics were also observed in this species.
`
`L-224715 Phosphoric Acid Salt Solutions Orally Dosed
`To Fasted Beagle Dogs at 2, 10 and 50 mg/kg
`(arithmetic mean ± SD)
`
`2 mpk
`50 mpk
`10 mpk
`
`100
`
`10
`
`1
`
`0.1
`
`L-224715 Plasma Concentration, µM
`
`0.01
`
`0
`
`4
`
`8
`
`12
`
`16
`
`20
`
`24
`
`Time, hours
`Figure 17. Exposure data for L-000224715-006F orally dosed to fasted beagle dogs
`
`Q:\Dept_854\preformulation reports\L-224715 Preformulation Report.doc
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`22
`
`Merck Exhibit 2148, Page 22
`Mylan Pharmaceuticals Inc. v. Merck Sharp & D