MERCK RESEARCH LABORATORIES PROGRESS REPORT
` FEBRUARY 2002
`
`Title: L-221869 (dipeptidyl-peptidase IV)
`Category: Endocrine/Metabolic
`Target Class: dipeptidyl-peptidase IV
`Compound #: 0726
`Disease: noninsulin-dependent diabetes mellitus Dosage Form/Potency: To Be Determined
`Cross Project Function:
`Project Team:
`Dept.: 854 Pharmaceutical Research
`Department Head: Michael Kaufman
`Key Words: L-tartrate hemi-hydrate; solubility; stability; binary mixtures; gelatin; HPMC
`Summary
`The solubility of L-221869 tartrate hemi-hydrate was determined in water, saline, buffers (pH 2, 4, and 6),
`methanol, ethanol, and 2-propanol. Chemical stability of the salt in a dry-filled capsule formulation is being
`assessed by analysis of binary mixtures of the salt with gelatin and HPMC. No instability is noted after 2 weeks, but
`method development for these samples is ongoing to increase the sensitivity of the assay.
`
`Reporting Area: Pharmaceutical Research & Development
`Sub-Group: Pharmaceutical Chemistry Rahway
`Author(s): Shultz, Leigh
`
`1.
`
`Solubility of crystalline L-221869 L-tartrate hemi-hydrate [NB: (60659:146-147, 151)]
`
`The solubility of L-221869 tartrate hemi-hydrate (M. Palucki, NB 72061-54) was determined in water, saline, buffers (pH 2, 4,
`and 6), methanol, ethanol, and 2-propanol. The salt sample was completely soluble in the aliquot of solvent added in all cases
`except ethanol and 2-propanol, so solubility data is not equilibrium data except in those cases. The ethanol and 2-propanol
`samples were equilibrated on the rotator for 18 hours, diluted with 0.1% phosphoric acid, and quantitated by HPLC against
`known standards of the tosylate salt of L-221869. The solubility data are shown in Table 1 below with pH data for the aqueous
`solutions. The undissolved salts in the ethanol and 2-propanol samples were analyzed by optical microscopy; no amorphous
`material or new crystal morphologies were observed.
`
`Solvent
`
`water
`0.9% NaCl
`0.01 N HCl
`20 mM sodium acetate
`20 mM sodium phosphate
`methanol
`ethanol
`2-propanol
`
`Table 1. Solubility of L-221869 L-tartrate hemi-hydrate
`Sol. Salt
`Sol. Parent
`pHinitial
`(mg/mL)
`(mg/mL)
`>23.3
`>16.5
`>13.5
`>9.6
`>20.1
`>14.3
`>9.4
`>6.7
`>10.9
`>7.7
`>15.4
`>10.9
`0.82
`0.59
`0.17
`0.12
`
`6.13
`5.57
`2.08
`3.99
`6.01
`n/a
`n/a
`n/a
`
`pHfinal
`
`3.54
`3.43
`3.23
`3.69
`3.72
`n/a
`n/a
`n/a
`
`2.
`
`Chemical stability of L-221869 L-tartrate hemi-hydrate in binary mixtures with gelatin (type B) and HPMC [NB: (60659:144-
`145, 149, 152-153, 157-159)]
`
`In order to investigate the stability of L-221869 tartrate hemi-hydrate (NB72061-54) in a dry-filled capsule formulation, binary
`mixtures of the salt were prepared with gelatin (lab grade, type B, Fisher lot no. 987519) and with HPMC (USP, 23537, 6 cps).
`Samples of the salt were weighed out, as were samples of gelatin and HPMC, according to the table below. The salt sample was
`ground gently with a mortar and pestle with ca. 85 mg of the excipient; the rest of the excipient was then added, and the mixture
`was ground until uniform.
`
`Mass L-221869
`salt (mg)
`22.6
`22.9
`
`Mass L-221869 parent
`(mg) FB = 0.71(Tar salt)
`16.1
`16.3
`
`Excipient
`
`gelatin
`HPMC
`
`Total mass (exc +
`salt) (mg)
`324.0
`320.9
`
`% w/w drug
`loading
`5.0
`5.1
`
`Merck Exhibit 2152, Page 1
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

` MERCK RESEARCH LABORATORIES PROGRESS REPORT
` FEBRUARY 2002
`
` Page 2 of 6
`
`Samples of the mixture (ca. 20 mg each) were weighed into vials and placed at 5 °C, 40 °C/75% RH, and 80 °C for 1, 2, and 4
`weeks. The samples are analyzed by dissolving each in 9.0 mL of 0.1% phosphoric acid, syringe-filtering, and analyzing on the
`HPLC using the method for the parent compound (L221869_method1). Separate columns are used for gelatin and HPMC
`samples because gelatin tends to be retained on the column after each run. No chemical degradation was evident after 2 weeks
`under any of the experimental conditions, but degradation in the gelatin samples may have been masked by the elution of gelatin
`itself. Method development to circumvent this problem is ongoing.
`
`Title: Dipeptidyl-peptidase IV
`Category: Endocrine/Metabolic
`Target Class: dipeptidyl-peptidase IV
`Compound #:
`Disease: noninsulin-dependent diabetes mellitus Dosage Form/Potency: To Be Determined
`Cross Project Function:
`Reporting Area: Pharmaceutical Research & Development
`Project Team:
`Sub-Group: Pharmaceutical Chemistry Rahway
`Dept.: 854 Pharmaceutical Research
`Author(s): Shultz, Leigh
`Department Head: Michael Kaufman
`Key Words: L-224715; benzenesulfonate; anhydrate form A; chemical stability; L-tartrate; hemi-hydrate; pH; phosphate; solubility;
`binary mixtures; gelatin; HPMC
`
`Summary
`The chemical stability of the benzenesulfonate, tartrate, and phosphate salts is being determined both in the solid
`state and in solution. Two-week data indicate that the benzenesulfonate and tartrate are stable in the bulk under all
`conditions studied; the same is true for the phosphate after one week. All of the salts are most stable in solution
`between pH 2 and 4 and degrade completely in water, pH8 solution, and pH10 solution at 80 °C after 1 week. The
`phosphate salt shows the best stability in water after one week, but more stability data is needed before salt
`selection can occur. The solubility of the 3 salts has been determined in water, 0.01N HCl, and alcohols. All three
`salts are very water-soluble, the phosphate having more than 80-mg/mL parent solubility. All are sparingly soluble
`in 2-propanol. Experiments are ongoing to determine the chemical stability of binary mixtures of each of the three
`salts with capsule materials (gelatin, HPMC). Two-week data are available for the benzenesulfonate and the
`tartrate salts; no degradation has been detected to date. Method development is ongoing to increase the sensitivity
`of these assays.
`
`1.
`
`Bulk and solution chemical stability of L-224715 benzenesulfonate (anhydrate form A) [NB: (60659:142-143, 149-150, 154,
`160)]
`
`The bulk stability of the benzenesulfonate salt (anhydrate form A) of L-224715 has been assessed after 2 weeks at 40 °C/75% RH
`and 80 °C by HPLC analysis of solid samples stored under these conditions. No degradation or significant loss of parent was
`noted under the above conditions. Four-week data (forthcoming) will be needed to confirm this finding.
`
`The solution stability of the benzenesulfonate salt has also been assessed after two weeks at 40 and 80 °C. Like the free base, the
`benzenesulfonate salt is most stable between pH 2 and 4 at all temperatures studied. At pH 2, ca. 7.7 area % parent is lost after 2
`weeks at 80 °C due to hydrolysis of the amide bond, and ca. 9 area % parent is lost after 2 weeks at pH 4 (80 °C) due to both
`hydrolysis and de-amination. Total loss of parent is observed in water, pH 8 buffer, and pH 10 buffer at 80 °C after 1 week. At
`40 °C, the area % degradates (relative to parent) due to hydrolysis and de-amination are similar to those seen for the free base at 1
`and 2 weeks:
`
`Salt of L-224715
`
`Rel Area %
`Hydrolysis
`1wk 2wk 4wk
`
`Rel Area %
`De-amination
`1wk 2wk 4wk
`
`Merck Exhibit 2152, Page 2
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

` MERCK RESEARCH LABORATORIES PROGRESS REPORT
` FEBRUARY 2002
`
` Page 3 of 6
`
`Free Base
`Benzenesulfonate
`
`8.9
`8.3
`
`20.4
`18.2
`
`47.7
`
`3.8
`3.6
`
`11.8
`7.4
`
`28.8
`
`Four-week data (both bulk and solution) for the benzenesulfonate salt are forthcoming.
`
`2.
`
`Bulk and solution chemical stability of L-224715 L-tartrate (hemi-hydrate) [NB: (60659:143, 149, 152, 154)]
`
`The bulk stability of the tartrate salt (hemi-hydrate) of L-224715 has been assessed after 2 weeks at 40 °C/75% RH and 80 °C by
`HPLC analysis of solid samples stored under these conditions. No degradation or significant loss of parent was noted under the
`above conditions. Four-week data (forthcoming) will be needed to confirm this finding.
`
`The solution stability of the tartrate salt has also been assessed after two weeks at 40 and 80 °C. Like the benzenesulfonate salt,
`the tartrate salt is most stable between pH 2 and 4 at all temperatures studied. At pH 2, ca. 2.5 area % parent is lost after 2 weeks
`at 80 °C due to hydrolysis of the amide bond, and ca. 7 area % parent is lost after 2 weeks at pH 4 (80 °C) due to both hydrolysis
`and de-amination. Total loss of parent is observed in water, pH 8 buffer, and pH 10 buffer at 80 °C after 1 week. At 40 °C, the
`area % degradates (relative to parent) observed due to hydrolysis and de-amination are slightly lower than those seen for the
`benzenesulfonate salt at 1 and 2 weeks:
`
`Salt of L-224715
`
`Free Base
`Benzenesulfonate
`L-Tartrate Hemihydrate
`
`Rel Area %
`Hydrolysis
`1wk 2wk 4wk
`8.9
`20.4
`47.7
`8.3
`18.2
`6.3
`13.7
`
`Rel Area %
`De-amination
`1wk 2wk 4wk
`3.8
`11.8
`28.8
`3.6
`7.4
`4.2
`6.1
`
`The amount of degradation observed in solution after 2 weeks is unexpected based on the initial pH of the stability samples (pH =
`5.1 at 0.1 mg/mL salt). After the data was acquired, the pH of the 40-°C stability samples was measured. The pH of the 1-week
`sample was 7.5, and the pH of the 2-week sample was 7.6. To see if the rise in pH upon storage at 40 °C was reproducible, a
`fresh sample of the tartrate salt in water was prepared using the same stock solution used for the stability samples. This fresh
`solution had a pH of 5.1. The solution was placed in at 40 °C for 48 hours, during which time the pH of the solution rose to 7.54.
`HPLC analysis of this solution showed relative area % degradates to be low (hydrolysis, 0.4%; de-amination, 0.6%), indicating
`that the degradation of the tartrate salt observed on stability is the result of the rise in pH, rather than the rise in pH being due to
`degradation. The cause of the pH increase at 40 °C is still under investigation.
`
`Four-week bulk and solution stability data for the tartrate salt are forthcoming.
`
`3.
`
`Bulk and solution chemical stability of L-224715 phosphate [NB: (60659:153, 157, 159)]
`
`The bulk stability of the phosphate salt of L-224715 has been assessed after 1 week at 40 °C/75% RH and 80 °C by HPLC
`analysis of solid samples stored under these conditions. No degradation or significant loss of parent was noted under the above
`conditions. Two- and four-week data (forthcoming) will be needed to confirm this finding.
`
`The solution stability of the phosphate salt has also been assessed after one week at 40 and 80 °C. Like the other salts studied, the
`phosphate salt is most stable between pH 2 and 4 at all temperatures studied. At pH 2, ca. 1 area % parent is lost after 1 week at
`80 °C due to hydrolysis of the amide bond, and ca. 1.5 area % parent is lost after 1 week at pH 4 (80 °C) due to both hydrolysis
`and de-amination. Total loss of parent is observed in water, pH 8 buffer, and pH 10 buffer at 80 °C after 1 week. At 40 °C, the
`area % degradates (relative to parent) observed due to hydrolysis and de-amination are lower than those seen for the
`benzenesulfonate salt after 1 week:
`
`Salt of L-224715
`
`Free Base
`Phosphate
`
`Rel Area %
`Hydrolysis
`1wk 2wk 4wk
`8.9
`20.4
`47.7
`3.8
`
`Rel Area %
`De-amination
`1wk 2wk 4wk
`3.8
`11.8
`28.8
`2.3
`
`Merck Exhibit 2152, Page 3
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

` MERCK RESEARCH LABORATORIES PROGRESS REPORT
` FEBRUARY 2002
`
` Page 4 of 6
`
`Benzenesulfonate
`L-Tartrate Hemihydrate
`
`8.3
`6.3
`
`18.2
`13.7
`
`3.6
`4.2
`
`7.4
`6.1
`
`Two- and four-week stability data (both solution and bulk) will be needed to confirm the stability of the phosphate salt. Given
`that all of the salts of L-224715 show good bulk stability thus far, solution stability data (especially stability in water) will be used
`to differentiate them; stability in water is expected to be important for such a water-soluble salt which may go amorphous in a
`formulation.
`
`4.
`
`Solubility and pH of L-224715 benzenesulfonate [NB: (60659:143, 146-147, 151, 155, 158)]
`
`The solubility of the anhydrate (form A) of the benzenesulfonate salt (K. Hansen, NB 70130-347) of L-224715 was determined in
`water, 0.01N HCl, methanol, ethanol, and 2-propanol. The samples of the salt dissolved immediately in all of the above solvents
`except 2-propanol; this sample was diluted with 0.1% phosphoric acid and analyzed by HPLC against known standards of the free
`base. The solubility data obtained are shown in the table below:
`
`Solvent
`
`Sol. Parent
`Sol. Salt
`(mg/mL)
`(mg/mL)
`>38.4
`>53.3
`water
`>6.3
`>8.7
`0.01 N HCl
`>15.8
`>21.9
`methanol
`>15.0
`>20.8
`ethanol
`3.94
`5.47
`2-propanol
`* volume of solution too low to measure pH
`
`pHinitial
`
`pHfinal
`
`6.13
`2.08
`n/a
`n/a
`n/a
`
`*
`2.16
`n/a
`n/a
`n/a
`
`The pH of the water sample was not obtained due to sample limitations, but the pH of a 10.0-mg/mL solution of the salt was
`measured at 5.75, while the pH of a 1.0 mg/mL solution was 6.66. The pH of a saturated solution should be lower than that of the
`10.0-mg/mL solution.
`
`The solid remaining in the 2-propanol sample was analyzed by microscopy; no amorphous material or morphology changes were
`observed. The solubility values is aqueous solution are likely those of the hemi-hydrate, which forms above 85% RH (C.
`Lindemann, Analytical Research).
`
`5.
`
`Solubility and pH of L-224715 L-tartrate hemi-hydrate [NB: (60659:143, 146-147, 150-151, 155)]
`
`The solubility of the hemi-hydrate of the tartrate salt (K. Hansen, NB 70130-359) of L-224715 was determined in water, 0.01N
`HCl, methanol, ethanol, and 2-propanol. The samples of the salt dissolved immediately in all of the above solvents except
`ethanol and 2-propanol; these samples were diluted with 0.1% phosphoric acid and analyzed by HPLC against known standards
`of the free base. The solubility data obtained are shown in the table below:
`
`Solvent
`
`water
`0.01 N HCl
`methanol
`ethanol
`2-propanol
`
`Sol. Salt
`(mg/mL)
`>11.5
`>10.9
`>27.3
`1.14
`0.093
`
`Sol. Parent
`(mg/mL)
`>8.3
`>7.8
`>19.6
`0.82
`0.067
`
`pHinitial
`
`pHfinal
`
`6.13
`2.08
`n/a
`n/a
`n/a
`
`3.57
`2.98
`n/a
`n/a
`n/a
`
`The pH of a 1.0-mg/mL solution of the salt was 3.98, while the pH of a 0.1-mg/mL solution was measured at 5.1.
`
`The solids remaining in the ethanol and 2-propanol samples were analyzed by microscopy; no amorphous material or morphology
`changes were observed.
`
`6.
`
`Solubility and pH of L-224715 phosphate [NB: (60659:158-159)]
`
`Merck Exhibit 2152, Page 4
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

` MERCK RESEARCH LABORATORIES PROGRESS REPORT
` FEBRUARY 2002
`
` Page 5 of 6
`
`The solubility of the phosphate salt (K. Hansen, NB 70316-25) of L-224715 was determined in water, 0.9% NaCl, 0.01N HCl,
`methanol, ethanol, and 2-propanol. The samples of the salt dissolved immediately in all of the aqueous solvents; the supernatants
`from the alcoholic samples were diluted with 0.1% phosphoric acid and analyzed by HPLC against known standards of the free
`base. The solubility data obtained are shown in the table below:
`
`Solvent
`
`Sol. Parent
`Sol. Salt
`(mg/mL)
`(mg/mL)
`>81, <121
`>100, <150
`water
`>58
`>72
`saline
`>61
`>75
`0.01 N HCl
`0.33
`0.41
`methanol
`0.036
`0.045
`ethanol
`0.079
`0.098
`2-propanol
`* volume of solution too low to measure pH
`
`pHinitial
`
`pHfinal
`
`6.13
`5.57
`2.08
`n/a
`n/a
`n/a
`
`*
`*
`*
`n/a
`n/a
`n/a
`
`The volume of solution resulting from the aqueous samples was in each case too low to measure pH. The pH of a 1.0-mg/mL
`solution of the salt was 5.61, suggesting a monobasic salt.
`
`The solids remaining in the ethanol and 2-propanol samples have not yet been analyzed by microscopy; the higher solubility in 2-
`propanol (with respect to ethanol) may be due to the formation of amorphous material in that solvent.
`
`7.
`
`Solubility of L-224715-000T001 (crystalline free base) [NB: (60659:146-147, 150)]
`
`The solubility of the crystalline free base of L-224715 was determined in PEG 400, glycerol, methanol, ethanol, and 2-propanol.
`The samples of the salt dissolved immediately in methanol and ethanol and slowly in 2-propanol and PEG 400. Heating for
`several minutes with a heat gun was required to dissolve the drug in glycerol. Aliquots of the PEG 400 and glycerol samples
`were diluted with 0.1% phosphoric acid and analyzed by HPLC to determine if any degradation of the drug occurred upon
`dissolution in these media; none was observed. The solubility data obtained are shown in the table below:
`
`Solvent
`
`Sol. Parent
`(mg/mL)
`>18.1
`PEG 400
`~6.4*
`glycerol
`>24.8
`methanol
`>21.8
`ethanol
`>11.1
`2-propanol
` * free base goes into glycerol at this
` conc. when heated.
`
`
`
`8.
`
`Chemical stability of L-224715 benzenesulfonate (anhydrate form A) in binary mixtures with gelatin (type B) and HPMC [NB:
`(60659:144, 149, 152-153, 157-159)]
`
`In order to investigate the stability of L-224715 benzenesulfonate (anhydrate form A, NB70130-347) in a dry-filled capsule
`formulation, binary mixtures of the salt were prepared with gelatin (lab grade, type B, Fisher lot no. 987519) and with HPMC
`(USP, 23537, 6 cps). Samples of the salt were weighed out, as were samples of gelatin and HPMC, according to the table below.
`The salt sample was ground gently with a mortar and pestle with ca. 85 mg of the excipient; the rest of the excipient was then
`added, and the mixture was ground until uniform.
`
`Mass L-224715
`salt (mg)
`22.2
`22.9
`
`Mass L-224715 parent
`(mg) FB = 0.72(Bs salt)
`16.0
`16.5
`
`Excipient
`
`gelatin
`HPMC
`
`Total mass (exc +
`salt) (mg)
`320.3
`319.6
`
`% w/w drug
`loading
`5.0
`5.2
`
`Samples of the mixture (ca. 20 mg each) were weighed into vials and placed at 5 °C, 40 °C/75% RH, and 80 °C for 1, 2, and 4
`weeks. The samples are analyzed by dissolving each in 9.0 mL of 0.1% phosphoric acid, syringe-filtering, and analyzing on the
`
`Merck Exhibit 2152, Page 5
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

` MERCK RESEARCH LABORATORIES PROGRESS REPORT
` FEBRUARY 2002
`
` Page 6 of 6
`
`HPLC using the method for the parent compound (L221869_method1). Separate columns are used for gelatin and HPMC
`samples because gelatin tends to be retained on the column after each run. No chemical degradation was evident after 2 weeks
`under any of the experimental conditions (both gelatin and HPMC), but degradation in the gelatin samples may have been masked
`by the elution of gelatin itself. Method development to circumvent this problem is ongoing.
`
`9.
`
`Chemical stability of L-224715 L-tartrate hemi-hydrate in binary mixtures with gelatin (type B) and HPMC [NB: (60659:147,
`149, 157-159)]
`
`In order to investigate the stability of L-224715 tartrate (hemi-hydrate, NB70130-359) in a dry-filled capsule formulation, binary
`mixtures of the salt were prepared with gelatin (lab grade, type B, Fisher lot no. 987519) and with HPMC (USP, 23537, 6 cps).
`Samples of the salt were weighed out, as were samples of gelatin and HPMC, according to the table below. The salt sample was
`ground gently with a mortar and pestle with ca. 85 mg of the excipient; the rest of the excipient was then added, and the mixture
`was ground until uniform.
`
`Mass L-224715
`salt (mg)
`22.4
`22.9
`
`Mass L-224715 parent
`(mg) FB = 0.719(Tar salt)
`16.1
`16.5
`
`Excipient
`
`gelatin
`HPMC
`
`Total mass (exc +
`salt) (mg)
`327.7
`328.1
`
`% w/w drug
`loading
`4.9
`5.0
`
`Samples of the mixture (ca. 20 mg each) were weighed into vials and placed at 5 °C, 40 °C/75% RH, and 80 °C for 1, 2, and 4
`weeks. The samples are analyzed by dissolving each in 9.0 mL of 0.1% phosphoric acid, syringe-filtering, and analyzing on the
`HPLC using the method for the parent compound (L221869_method1). Separate columns are used for gelatin and HPMC
`samples because gelatin tends to be retained on the column after each run. No chemical degradation was evident after 2 weeks
`under any of the experimental conditions (both gelatin and HPMC), but degradation in the gelatin samples may have been masked
`by the elution of gelatin itself. Method development to circumvent this problem is ongoing.
`
`10.
`
`Chemical stability of L-224715 phosphate in binary mixtures with gelatin (type B) and HPMC [NB: (60659:155, 160)]
`
`In order to investigate the stability of L-224715 phosphate (hemi-hydrate, NB70316-25) in a dry-filled capsule formulation,
`binary mixtures of the salt were prepared with gelatin (lab grade, type B, Fisher lot no. 987519) and with HPMC (USP, 23537, 6
`cps). Samples of the salt were weighed out, as were samples of gelatin and HPMC, according to the table below. The salt
`sample was ground gently with a mortar and pestle with ca. 85 mg of the excipient; the rest of the excipient was then added, and
`the mixture was ground until uniform.
`
`Mass L-224715
`salt (mg)
`21.06
`21.01
`
`Mass L-224715 parent
`(mg) FB = 0.806(PO4 salt)
`16.97
`16.93
`
`Excipient
`
`gelatin
`HPMC
`
`Total mass (exc +
`salt) (mg)
`323.49
`321.06
`
`% w/w drug
`loading
`5.2
`5.3
`
`Samples of the mixture (ca. 20 mg each) were weighed into vials and placed at 5 °C, 40 °C/75% RH, and 80 °C for 1, 2, and 4
`weeks. The samples will be analyzed by dissolving each in 9.0 mL of 0.1% phosphoric acid, syringe-filtering, and analyzing on
`the HPLC using the method for the parent compound (L221869_method1). Separate columns will be used for gelatin and HPMC
`samples because gelatin tends to be retained on the column after each run. Method development to circumvent the co-elution of
`gelatin and degradates is ongoing.
`
`Merck Exhibit 2152, Page 6
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`