EP 1 567 529 & EP 2 314 591
`EXPERIMENTAL REPORT
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`Example 25: stability and dissolution experiments hydrate vs amorphous
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`In this experiment, the thermal characteristics (by DSC - differential scanning calorimetry) and
`
`powder dissolution are determined for (3R,3aS,6aR)-hexahydrofuro [2,3-b] furan-3-yl (1S,2R)-3-[[(4-
`
`aminophenyl) sulfonyl] (isobutyl) amino]-l-benzyl-2-hydroxypropylcarbamate ("Compound") in the
`
`hydrate crystalline form and in the amorphous form, with reference to storage under defined
`
`conditions.
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`Sample Preparation
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`Compounds to be tested are stored in a closed container at 25°C, in which a relative humidity (RH) of
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`93% is installed by means of a saturated (NH4)H2P04 solution. The same set up is used also at 40°C
`
`(RH>93%). Compounds are stored in the specified conditions for i day, i week, 2 weeks and 1
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`month. Subsequently the samples are analyzed using DSC and powder dissolution.
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`DSC
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`DSC experiments are carried out using a DSC-7 (Perkin-Elmer, Norwalk, CT) using N2 as purge gas.
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`Liquid N2 is used as a coolant. Samples (approximately 5 mg) are analyzed using aluminum hermetic
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`pans (TA Instruments, Brussels, Belgium). Temperature calibration is performed using indium and tin
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`standards, enthalpic calibration is carried out using indium standards. Validation of temperature and
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`enthalpic response is performed using tin and indium standards. All samples are scanned at lO°C/min
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`from 15°C to 140°C.
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`The results are indicated in Table 27. In view of the different solid state nature of Compounds in the
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`samples tested, the temperature characteristics necessarily are also different: the temperature
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`values indicated are the onset of a melting peak (for the crystalline hydrate form) and the Tg (for the
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`amorphous form). Rather than being based on a comparison of the absolute temperature values,
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`conclusions are drawn from comparing the temperature changes depending on the sample history.
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`Dissolution
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`Dissolution (Hanson Dissolution test station SR8 PLUS) is performed according to the rotating paddle
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`method (USP 24) at 37°C and 50 rpm in 1000 ml of O.01N HCI to which was added 0.1% sodium
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`laurylsulfate. The dissolution test was performed on 50.0 mg of Compound (in either form), which
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`was placed between filter paper at the bottom of the vessel. Two ml of the samples are taken a tO.6,
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`12, 20, 30, 60 and 120 minutes and immediately filtered over a O.45pm PVDF membrane filter
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`(Acrodica, USA) into a vial. The samples are analyzed using HPLC. The peak of Compound in the
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`chromatogram is integrated and the concentration of Compound in the samples is calculated using a
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`calibration curve, made of standards of known concentrations.
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`The results are depicted in Figures 27 to 30. Each of these figures is a graph representing the
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`percentage of Compound dissolved (Y-axis) over time (X-axis).
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`Lupin Ex. 1021 (Page 1 of 6)
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`

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`Example 26: dissolution experiments ethanolate vs amorphous
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`In this experiment, the dissolution is tested of(3R,3aS,6aR)-hexahydrofuro [2,3-b] furan-3-yl (1S,2R)-
`
`3-[[(4-aminophenyl) sulfonyl] (isobutyl) amino]-l-benzyl-2-hydroxypropylcarbamate ("Compound")
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`in the ethanolate crystalline form and in the amorphous form.
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`The results are depicted in Figures 31 to 33. In each figure, a graph indicates the percentage of
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`Compound dissolved (Y-axis) over time (X-axis).
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`Figure 31 relates to samples at zero storage time. Samples 1-6 all represent the amorphous form.
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`The mean dissolution percentage of the amorphous form is 75.16% at 120 minutes, whilst for the
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`ethanolate crystalline form this is 99.98%. After 90 minutes, the dissolution level of the ethanolate
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`crystalline form is more or less stable and complete (100%), while the dissolution level of the
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`amorphous form is still increasing after 120 minutes.
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`Figure 32 relates to samples that were subjected to 1 month of storage at 25°C. Samples 1-6 all
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`represent the amorphous form.
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`The mean dissolution percentage of the amorphous form after one month of storage is 81.07%
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`(which is 107.87% compared to the value found at zero storage time). For the ethanolate crystalline
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`form, the dissolution percentage is 97.28% (which is 97.29% as compared to the value found at zero
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`storage time). All these observations are extracted at time point 120 minutes.
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`As at zero storage time, the dissolution level of the ethanolate crystalline form is more or less stable
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`and complete after 90 minutes dissolution (97%), while the dissolution level of the amorphous form
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`is still increasing after 120 minutes.
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`Figure 33 relates to samples that were subjected to 3 months of storage at 25°C. Samples 1-4 all
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`represent the amorphous form.
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`The mean dissolution percentage of the amorphous form after three month of storage is 87.92%
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`(which is 116.99% compared to the value found at zero storage time). For the ethanolate crystalline
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`form, the dissolution percentage is 102.67% (which is 102.69% as compared to the value found at
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`zero storage time). All these observations are extracted at time point 120 minutes.
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`As at zero storage time, the dissolution level of the ethanolate crystalline form is more or less stable
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`and complete after 90 minutes dissolution (103%), while the dissolution level of the amorphous form
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`is still increasing after 120 minutes.
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`Lupin Ex. 1021 (Page 2 of 6)
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`

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`Table 271
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`Thermal characteristics of (3R,3aS,6aR)-hexahydrofuro [2,3-b] furan-3-yl (lS,2R)-3-[[(4-aminophenyl)
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`sulfonyl] (isobutyl) amino]-l-benzyl-2-hydroxypropylcarbamate (crystalline hydrate and amorphous)
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`Sample history T hydrate (°C)
`Untreated
`70.38 (0.23)
`after storage 25°C/93% RH:
`1 day
`69.80 (0.92)
`after storage at 25°C/93% RH:
`1 week
`71.10 (1.09)
`after storage at 25°C/93% RH:
`2 weeks
`70.64 (0.16)
`after storage at 25°C/93% RH:
`1 month
`69.49 (0.21)
`- O.g9
`Zl °C a~ter 1 month oJ: storage at 25°C/93% RH
`after storage 40°C/93% RH:
`1 day
`after storage at 40°C/93% RH:
`1 week
`after storage at 40°C/93% RH:
`2 weeks
`after storage at 40°C/93% RH:
`1 month
`Z~ °C a~ter 1 month oJ: storage at 40C/93% RH
`
`70.37 (1.52)
`68.48 (0.07)
`70.15 (0.82)
`67.77 (0.89)
`- 2.61
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`T amorphous (°C)
`63.87 (3.01)
`63.37 (6.71)
`45.19 (1.19)
`44.45 (0.41)
`n.d.
`>_ -19.42
`49.16 (4.18)
`32.01 (single value)
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`31.83 (3.67)
`32.38 (0.17)
`-31.49
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`1 Each of the reported values is the mean of two measurements. In parentheses the difference between the two
`
`measurements is indicated (with one exception where a single value was available).
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`Lupin Ex. 1021 (Page 3 of 6)
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`

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`Figure 27: dissolution of Compound crystalline hydrate after storage at 25°C/93% RH
`
`day
`week
`Weeks
`
`month
`
`+ 1 day
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`+ I week
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`~2 weeks
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`~ 1 month
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`T|me (rain)
`
`t2o
`
`i40
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`Figure 28: dissolution of amorphous Compound after storage at 25°C/93% RH
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`8O
`
`~> 60
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`© 40
`
`20
`
`0 20 40
`
`60 80
`
`100 120 140
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`Time (rain)
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`Lupin Ex. 1021 (Page 4 of 6)
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`

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`Figure 29: dissolution of Compound crystalline hydrate after storage at 40°C/93% RH
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`lOO
`
`month
`
`+ 1 day
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`+ 1 week
`
`~ 2 weeks
`
`! mon~
`
`0
`
`20
`
`40
`
`60
`
`80
`
`100
`
`120
`
`140
`
`Time
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`Figure 30: dissolution of amorphous Compound after storage at 40°C/93% RH
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`8O
`
`~ 6O
`
`~, 40
`
`2O
`
`0
`
`20
`
`40
`
`60
`
`80
`
`100
`
`120
`
`t40
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`Time (rain)
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`Lupin Ex. 1021 (Page 5 of 6)
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`

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`Figure 31: dissolution profiles of Compound (amorphous form and ethanolate crystalline form)
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`Figure 32: dissolution profiles after 1 month of storage at 25°C)
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`Figure 33: dissolution profiles after 3 months of storage at 25°C)
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`Lupin Ex. 1021 (Page 6 of 6)