US20220235063A1

US20220235063A1 - Morphic forms of marizomib and uses thereof

Info

Publication number: US20220235063A1
Application number: US17/459,531
Authority: US
Inventors: Robert Mansfield
Original assignee: Celgene Research And Development I Ulc Formerly Known Triphase Research And Development I Corp AS; 1096271 BC ULC; Celgene International II SARL
Current assignee: 1096271 BC ULC; Celgene International II SARL
Priority date: 2016-08-19
Filing date: 2021-08-27
Publication date: 2022-07-28
Also published as: JP2022101588A; US20210017185A1; WO2018033631A9; WO2018033631A1; EP3500576A1; US10703760B2; US20190185482A1; JP2019524894A; US11136332B2

Abstract

The present invention relates to polymorphic forms of marizomib (e.g., Morphic Form I). The morphic forms can be used alone and in pharmaceutical compositions for the treatment of disease.

Description

RELATED APPLICATIONS

This application claims priority to, and benefit of, U.S. Provisional Patent Application No. 62/377,156, filed Aug. 19, 2016, the contents of which are incorporated by reference in their entirety.

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Marizomib is a proteasome inhibitor capable of inhibiting all three domains of the proteasome (i.e., the chymotrypsin-like (CT-L); caspase-like (C-L) and trypsin-like (T-L) domains). Accordingly, marizomib can be useful for treating diseases such as cancer. Thus, there is a need for pure, stable morphic forms of marizomib that can be used for administration to subjects in need thereof. The present disclosure teaches stable and pure morphic forms of marizomib.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides morphic Form I of marizomib, characterized by an X-ray powder diffraction pattern including peaks at about 7.2, 14.5, and 36.7°2θ using Cu Kα radiation.
In another aspect, the present disclosure provides a pharmaceutical composition comprising a morphic Form of marizomib (e.g., Form I) and a pharmaceutically acceptable carrier.
In another aspect, the present disclosure provides a method of treating a disease comprising administering to a subject in need thereof an effective amount of a morphic Form of marizomib (e.g., morphic Form I).
In another aspect, the present disclosure provides a morphic Form of marizomib (e.g., morphic Form I) for use in the treatment of a disease.
In another aspect, the present disclosure provides a morphic Form of marizomib (e.g., morphic Form I) for inhibiting a protease.
In another aspect, the present disclosure provides the use of a morphic form of marizomib (e.g., morphic Form I) in the manufacture of a medicament for the treatment of a disease.
In another aspect, the present disclosure provides a method of preparing a morphic form of marizomib (e.g., morphic Form I) comprising recrystallizing marizomib from a solvent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an XRPD spectrum of a Sample 1 of Morphic Form I of marizomib.

FIG. 2 shows an XRPD spectrum of a Sample 2 of Morphic Form I of marizomib.

FIG. 3 shows an XRPD spectrum of a Sample 3 of Morphic Form I of marizomib.

FIG. 4 shows an overlay of all three XRPD spectra of

Samples

1, 2 and 3 of Form I of marizomib.

FIG. 5 shows a plot of variable temperature XRPD spectra of Sample 1 of Morphic Form I of marizomib.

FIG. 6 shows a plot of variable temperature XRPD spectra of Sample 3 of Morphic Form I of marizomib.

FIG. 7 shows an overlay of two XRPD spectra of marizomib after four years of storage at 2-8° C.

FIG. 8 shows an overlay of five XRPD spectra contrasting the XRPD spectra of Form I of marizomib with amorphous forms of marizomib.

FIG. 9 shows an XRPD spectrum of a Pawley fitting procedure for Sample 1 of Morphic Form I of marizomib.

FIG. 10 shows an XRPD spectrum of a Pawley fitting procedure for Sample 3 of Morphic Form I of marizomib.

FIG. 11 shows an overlay of XRPD spectra from a polymorph screen.

FIG. 12 shows an overlay of XRPD spectra from a polymorph screen.

FIG. 13 shows a plot of a thermogravimetric analysis of Sample 1 of Morphic Form I of marizomib.

FIG. 14 shows a plot of a thermogravimetric analysis of Sample 3 of Morphic Form I of marizomib.

FIG. 15 shows a differential scanning calorimetry plot of Sample 1 of Morphic Form I of marizomib at 2° C. per minute.

FIG. 16 shows a differential scanning calorimetry plot of Sample 1 of Morphic Form I of marizomib at 5° C. per minute.

FIG. 17 shows a differential scanning calorimetry plot of Sample 1 of Morphic Form I of marizomib at 10° C. per minute.

FIG. 18 shows a differential scanning calorimetry plot of Sample 1 of Morphic Form I of marizomib at 50° C. per minute.

FIG. 19 shows a differential scanning calorimetry plot of Sample 3 of Morphic Form I of marizomib at 2° C. per minute.

FIG. 20 shows a differential scanning calorimetry plot of Sample 3 of Morphic Form I of marizomib at 5° C. per minute.

FIG. 21 shows a differential scanning calorimetry plot of Sample 3 of Morphic Form I of marizomib at 10° C. per minute.

FIG. 22 shows a differential scanning calorimetry plot of Sample 3 of Morphic Form I of marizomib at 50° C. per minute.

FIG. 23 shows a ¹HNMR spectrum for both Sample 1 and Sample 3 of marizomib.

FIG. 24 is an HPLC plot of Sample 1 of marizomib.

FIG. 25 is an HPLC plot of Sample 3 of marizomib.

FIG. 26 is an XRPD plot of marizomib before and after attempted hydrolysis.

FIG. 27 is a 1HNMR plot of marizomib before and after attempted hydrolysis.

FIG. 28 is an HPLC plot of marizomib isolated after a hydrolysis attempt.

FIG. 29 is an HPLC plot of marizomib isolated from ethyl acetate

FIG. 30 is an HPLC plot of marizomib isolated from ethanol.

FIG. 31 is a polarized light microscope image of Morphic Form I of Sample 1 of marizomib.

FIG. 32 is a polarized light microscope image of Morphic Form I of Sample 3 of marizomib.

FIG. 33 is a photo of Sample 1 of Morphic Form I of marizomib at 25° C.

FIG. 34 is a photo of Sample 1 of Morphic Form I of marizomib at 150° C.

FIG. 35 is a photo of Sample 1 of Morphic Form I of marizomib at 160° C.

FIG. 36 is a photo of Sample 1 of Morphic Form I of marizomib at 160° C. after five minutes.

FIG. 37 is a photo of Sample 3 of Morphic Form I of marizomib at 25° C.

FIG. 38 is a photo of Sample 3 of Morphic Form I of marizomib at 150° C.

FIG. 39 is a photo of Sample 3 of Morphic Form I of marizomib at 160° C.

FIG. 40 is a photo of Sample 3 of Morphic Form I of marizomib at 160° C. after five minutes.

FIG. 41 is an overlay of an XRPD pattern of marizomib with solids from the 2-pyrrolidone experiment described in Example 12.

FIG. 42 is a thermal analysis of solids from-pyrrolidone experiment described in Example 12.

FIG. 43 is an Overlay of an XRPD pattern of marizomib with solids from the melamine experiment described in Example 12.

FIG. 44 is a thermal analysis of solids the melamine experiment described in Example 12.

FIG. 45 is an overlay of XRPD patterns of marizomib with the solids of the melamine 1:1 and 1:3 experiments described in Example 12.

FIG. 46 is an overlay of XRPD patterns of marizomib with solids from the cytosine experiments described in Example 12.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to morphic Forms (e.g., Form I) of marizomib. The morphic Forms can be solvates or hydrates.
As defined herein, “pure” is understood to mean that a compound is uniform in chemical makeup. It is understood that a pure compound does not contain molecules of another chemical makeup in an appreciable amount, e.g., less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some instances, the purity is measured excluding any solvent (e.g., organic solvent such as ethyl acetate, and inorganic solvent such as water). Purity can be measured by a number of techniques known in the art, for example HPLC.
In some embodiments, the morphic forms of marizomib are substantially pure. For example, the morphic forms described herein (e.g., morphic Form I) can be greater than about 90% pure, greater than about 91% pure, greater than about 92% pure, greater than about 93% pure, greater than about 94% pure, greater than about 95% pure, greater than about 96% pure, greater than about 97% pure, greater than about 98% pure, greater than about 99% pure, or greater than about 99.9% pure.
As defined herein, “stable” or “stability” relates to the ability of a compound to remain pure for a period of time. A stable compound can be one that maintains its purity (e.g., does not have molecules of an undesired chemical formula) despite extended storage (e.g., greater than one month, greater than six months, or greater than a year). A stable compound can also be a compound that remains pure despite conditions such as high temperature or humidity.
In some embodiments, the morphic forms of marizomib described herein retain their purity for extended periods of time (i.e., are stable). In some embodiments, the morphic Forms described herein (e.g., morphic Form I) can retain at least 99% purity for one month, for two months, for three months, for four months, for five months, for six months, for 12 months, for 24 months, for 36 months, for 48 months, or for 60 months or more.
Without wishing to be bound by theory, chemical compounds are known to exist in multiple different polymorphic forms. Accordingly, one of skill in the art would expect that marizomib can potentially exist in multiple different morphic forms, in addition to the amorphous form. Surprisingly, the present disclosure teaches that marizomib exists primarily in one morphic form (i.e., morphic Form I). The present disclosure teaches that morphic Form I is particularly stable.

Morphic Form I of Marizomib

Marizomib is a proteasome inhibitor with the structure:
In some embodiments, morphic Form I of marizomib is characterized by an X-ray diffraction pattern including peaks at about 7.2, 14.5, and 36.7°2θ using Cu Kα radiation. In some embodiments, morphic Form I is further characterized by X-ray powder diffraction peaks at about 18.1, 19.6, and 20.8°2θ using Cu Kα radiation. In some embodiments, morphic Form I of marizomib is further characterized by X-ray powder diffraction peaks at about 16.3, 19.8, and 20.5°2θ using Cu Kα radiation. In some embodiments, morphic Form I of marizomib is further characterized by X-ray powder diffraction peaks at about 15.2, 21.5, and 22.3°2θ using Cu Kα radiation. In some embodiments, morphic Form I of marizomib is further characterized by X-ray powder diffraction peaks at about 14.7, 29.2, and 30.0°2θ using Cu Kα radiation. In some embodiments, morphic Form I of marizomib is further characterized by X-ray powder diffraction peaks at about 8.2, 14.8, and 27.7°2θ using Cu Kα radiation. In some embodiments, morphic Form I of marizomib is free of solvent (e.g., water or organic solvent).
In some embodiments, morphic Form I of marizomib is further characterized by an X-ray powder diffraction pattern substantially similar to that set forth in FIG. 1. In some embodiments, morphic Form I of marizomib is further characterized by an X-ray powder diffraction pattern substantially similar to that set forth in FIG. 2. In some embodiments, morphic Form I of marizomib is further characterized by an X-ray powder diffraction pattern substantially similar to that set forth in FIG. 3. In some embodiments, morphic Form I of marizomib is further characterized by a degradation event at about 175° C. measured by thermogravimetric analysis.
In some embodiments, morphic Form I of marizomib is further characterized by two exotherms at about 150-180° C. as measured by differential scanning calorimetry at a rate of about 2° C. per minute. For example, morphic Form I can be characterized by two exotherms at about 155-175° C.
In some embodiments, morphic Form I of marizomib is characterized by an endotherm at about 168.5° C. and an exotherm at about 173-183° C., as measured by differential scanning calorimetry at a rate of about 5° C. per minute.
In some embodiments, morphic Form I of marizomib is characterized by two endotherms at about 175.5° C. and 180.6° C. and an exotherm at about 183-193° C.; or characterized by two endotherms at about 171.2° C. and 178.6° C. and an exotherm at about 183-193° C., as measured by differential scanning calorimetry at a rate of about 10° C. per minute.
In some embodiments, morphic Form I of marizomib is characterized by two endotherms at about 186.5° C. and 192.6° C. and an exotherm at about 193-205° C.; or characterized by two endotherms at about 180.9° C. and 191.6° C. and an exotherm at about 193-205° C. as measured by differential scanning calorimetry at a rate of about 50° C. per minute.
In some embodiments, morphic Form I of marizomib is characterized by melting and/or degradation at about 160-175° C. as measured by hot stage microscopy.
In some embodiments, morphic Form I of marizomib is at least about 98% pure as measured by HPLC. For example, the morphic Form can be at least about 99.1% pure as measured by HPLC.
As set forth in the present disclosure, three unique samples of morphic Form I of marizomib were characterized, Sample 1, Sample 2, and Sample 3. Samples 1 and 2 are not micronized, whereas Sample 3 was micronized. X-ray powder diffraction spectra measurements for Samples 1, 2 and 3 are given in Tables 1, 2 and 3, respectively. As set forth herein, both the micronized and the non-micronized samples are the same morphic form (i.e., morphic Form I).

TABLE 1

XRPD: Form I Sample 1

	Pos.	Rel. Int.
	[°2θ]	[%]

	7.2	100.0
	11.4	0.1
	14.0	0.1
	14.5	6.4
	17.7	0.2
	18.0	2.0
	19.1	0.1
	19.6	0.9
	20.8	0.4
	21.8	0.2
	22.3	0.2
	23.0	0.3
	25.4	0.2
	26.6	0.2
	26.9	0.2
	27.6	0.3
	29.2	0.7
	36.7	4.3
	37.5	0.3

TABLE 2

XRPD: Form I Sample 2

	Pos.	Rel. Int.
	[°2θ]	[%]

	7.2	100.0
	11.5	0.3
	14.0	0.2
	14.5	5.2
	17.7	0.2
	18.1	2.6
	19.1	0.3
	19.6	2.2
	20.8	0.7
	22.3	0.4
	23.1	0.4
	25.5	0.4
	26.6	0.5
	26.9	0.4
	27.7	0.5
	29.2	0.6
	36.7	4.9
	37.5	0.5

TABLE 3

XRPD: Frm I Sample 3

	Pos.	Rel. Int.
	[°2θ]	[%]

	7.2	100.0
	8.2	4.2
	10.2	2.4
	11.4	2.2
	13.7	2.0
	14.5	7.7
	14.7	5.6
	14.8	3.6
	15.2	6.0
	16.3	9.3
	18.1	18.0
	19.1	3.2
	19.6	16.3
	19.8	11.9
	20.5	6.6
	20.8	5.6
	21.5	6.2
	22.1	2.8
	22.3	6.3
	23.0	2.6
	23.9	3.4
	25.4	2.7
	26.5	3.4
	26.9	2.5
	27.7	4.3
	28.8	3.3
	29.2	4.6
	29.5	3.4
	30.0	4.5
	32.3	3.3
	36.7	4.6

FIG. 1 shows an XRPD spectrum of Sample 1 of morphic Form I of marizomib. As set forth in FIG. 1, the XRPD spectrum includes peaks at about 7.2, 14.5, and 36.7°2θ. FIG. 2 shows an XRPD spectrum of Sample 2 of morphic Form I of marizomib. As set forth in FIG. 2, the XRPD spectrum includes peaks at about 7.2, 14.5, and 36.7°2θ. FIG. 3 shows an XRPD spectrum of Sample 3 of morphic Form I of marizomib. As set forth in FIG. 3, the XRPD spectrum includes peaks at about 7.2, 14.5, and 36.7°2θ.
FIG. 4 shows an overlay of the three XRPD spectra of FIGS. 1, 2 and 3 (i.e., Samples 1, 2 and 3). As shown in FIG. 4, there can be slight differences between the XRPD spectra of even the same morphic form (i.e., morphic Form I) of marizomib. For example, certain peaks may be more or less pronounced (e.g., smaller or larger) in one spectrum compared with another.
As shown in the tables and figures of the present application, not all values for peaks (pos. °2θ) are identical for different lots of the polymorphs of the present application. An artisan of ordinary skill will understand that even different lots of the same polymorphic forms can produce slightly different characterization data, while not being appreciably different. For instance, slight variations in the calibration of the instruments used to perform a given measurement, or minor fluctuations in relative humidity between measurements can give rise to data that displays slight differences between samples. One of ordinary skill in the art will thus be able to, for instance, calibrate his or her instruments and take repeated measurements in order to minimize any discrepancies between signals to properly characterize the polymorphs of the present application. However, despite some minor variation in the batch-to-batch differences, polymorphs of the present application are identified and characterized by their characteristic peaks, such as those described above (e.g., about 7.2, 14.5, and 36.7°2θ, 0.2°2θ).
In some embodiments, differences in XRPD spectra (e.g., resolution) can be attributed to preferred orientation. Without wishing to be bound by theory, in XRPD it can be desirable to have a sample in which particles are oriented randomly (e.g., a powder). However, it can be difficult or in some cases impossible to achieve truly random particle orientations in practice. As particle size increases, the randomness of particle orientation can decrease, leading to increased challenges with achieving a preferred orientation.
Without wishing to be bound by theory, larger particles that do not exhibit random orientations can result in XRPD spectra in which some peaks are either diminished in intensity or in some embodiments missing altogether. Accordingly, in some embodiments micronized samples (e.g., Sample 3) can facilitate more random orientation of particles and thus a more accurate XRPD spectrum. In some embodiments a micronized sample with more randomness in particle orientation results in an XRPD spectrum with more peaks. In contrast, samples with larger particles (e.g., Sample 1, Sample 2) can lead to XRPD spectra with less pronounced peaks and in some embodiments fewer peaks.
As set forth in FIG. 5, morphic Form I of marizomib is stable even at elevated temperatures. FIG. 5 shows five unique XRPD spectra of sample 1 of morphic Form I of marizomib. The lowest trace (505) shows an XRPD spectrum at 25° C. Above that trace is the same sample at 100° C. (510). Shown above is another trace at 150° C., (515) then at 160° C. (520), and finally the highest trace shows an XRPD spectrum of the sample after five minutes at 160° C. (525).
Additionally, FIG. 6 shows five unique XRPD spectra of sample 3 of morphic Form I of marizomib at different temperatures. The lowest (605) is an XRPD spectrum of Sample 3 at 25° C., and also shown are traces at 100° C. (610), 150° C. (615), 160° C. (620), and 160° C. after five minutes (625). As set forth in FIGS. 5 and 6, only one morphic form (i.e., Form I) of marizomib was observed even as the temperature increased. Without wishing to be bound by theory, this suggests that Form I is the major morphic Form of marizomib. Without wishing to be bound by theory, this also suggests that Form I is the most stable morphic form of marizomib.
Without wishing to be bound by theory, the results of FIGS. 5 and 6 are further supported by FIG. 7. FIG. 7 shows an overlay of the same sample of morphic Form I of marizomib initially (705) and after storage for four years at 2-8° C. (710). The XRPD spectra are substantially identical, demonstrating that morphic Form I is stable at low temperatures for extended periods of time.
FIG. 8 shows an overlay of five XRPD spectra of marizomib. Form I of marizomib is shown in trace 805, whereas marizomib that has been co-spray dried with polymers are shown in traces 810, 815, 820, and 825. Trace 810 shows an XRPD spectrum of marizomib that has been co-spray dried with hydroxypropyl methylcellulose. Trace 815 shows an XRPD spectrum of marizomib that has been co-spray dried with polyvinylpyrrolidone. Trace 820 shows an XRPD spectrum of marizomib that has been co-spray dried with hydroxypropyl methylcellulose acetate succinate-M. Trace 825 shows an XRPD spectrum of marizomib that has been co-spray dried with hydroxypropyl methylcellulose acetate succinate-L.
Pawley fitting is a process in which observed peaks in a powder pattern can be fitted without a structural model but at 20 values constrained by the size and symmetry of the unit cell. In some embodiments, it can be a useful precursor to Rietveld fitting and can give an indication of the “best fit possible” from an eventual structural refinement. This fitting method was applied to XRPD diffractograms of the micronized and non-micronized supplied materials, Sample 3 and Sample 1, respectively.
Marizomib crystallizes in the monoclinic space group, P21, with these unit cell parameters:

- a=10.57 Å, b=24.49 Å c=12.63 Å and β=108.34°

Without wishing to be bound by theory, fitting the two samples showed good correlation between calculated and observed profiles, with only very slight differences in peak shape and intensity. Sample 1 exhibited strong preferred orientation (crystals were of lath morphology) as opposed to the micronized Sample 3. As such, without wishing to be bound by theory, the micronized sample (Sample 3) appeared to display numerous peaks that were not visible in the non-micronized sample (Sample 1).
Without wishing to be bound by theory, the average particle size of particles of Sample 1 (i.e., non-micronized) was about 150 μm to about 300 μm, compared with an average sample size of about 1 μm to about 20 μm for the micronized Sample 3. Without wishing to be bound by theory, the smaller particle size in Sample 3 reduces technical challenges associated with preferred orientation and allows for more accurate representation of peaks. For example, Sample 1, Sample 2, and Sample 3 all show an XRPD peak at about 36.7°2θ, at intensities of 4.3, 4.9, and 4.6, respectively. However, the peak appears larger in Samples 1 and 2 because of the preferred orientation of the sample.
The Pawley fitting procedure can enable visualization of the presence or absence of a different phase. The peak at about 8°2θ, on the micronized sample (i.e., Sample 3) diffractogram was found to be also present in the non-micronized sample (Sample 1) diffractogram, as seen in the FIGS. 9-10. FIG. 9 shows an XRPD spectrum of a Pawley fitting procedure for Sample 1 of Morphic Form I of marizomib. FIG. 10 shows an XRPD spectrum of a Pawley fitting procedure for Sample 3 of Morphic Form I of marizomib. Without wishing to be bound by theory, these results suggest that the majority of the material from these two batches (i.e., Sample 1 and Sample 3) is representative of the single crystal data and any subtle difference between them is attributable to crystal morphology inducing preferred orientation.
FIGS. 11 and 12 show a overlays of XRPD spectra of the solids isolated from the polymorph screen set forth in Example 2 using the various solvents used in the screen. A solid was not obtained for two solvents (DMF and DMA) since marizomib remained in solution and did not precipitate, thus no XRPD pattern for DMF or DMA was obtained. The solvents are listed next to the corresponding spectra.
FIG. 13 shows a plot of a thermogravimetric analysis of Sample 1 of morphic Form I of marizomib. As shown in FIG. 13, no weight loss was observed until about 175° C. Without wishing to be bound by theory, this weight loss can be due to sample degradation.
Similarly, FIG. 14 shows a plot of a thermogravimetric analysis of sample 3 of morphic Form I of marizomib. As shown in FIG. 14, no weight loss was observed until about 175° C. Without wishing to be bound by theory, this weight loss can be due to sample degradation.
FIG. 15 shows a differential scanning calorimetry plot of Sample 1 of Morphic Form I of marizomib at 2° C. per minute. As shown in FIG. 15, no endotherm was observed. Two exotherms were observed between about 150-180° C. The first exotherm was smaller than the second exotherm. Without wishing to be bound by theory, the exotherms could be due to sample degradation.
FIG. 16 shows a differential scanning calorimetry plot of Sample 1 of Morphic Form I of marizomib at 5° C. per minute. As shown in FIG. 16, an endotherm was observed at 169.6° C. (5.5. J/g) followed by a sharp exotherm at 173-183° C. Without wishing to be bound by theory, the exotherm could be due to sample degradation.
FIG. 17 shows a differential scanning calorimetry plot of Sample 1 of Morphic Form I of marizomib at 10° C. per minute. As shown in FIG. 17, a small endotherm was observed at 175.5° C. (7.1 J/g) and 180.6° C. (0.6 J/g). The two endotherms were followed by an exotherm at about 183-193° C. Without wishing to be bound by theory, the exotherm could be due to sample degradation.
FIG. 18 shows a differential scanning calorimetry plot of Sample 1 of Morphic Form I of marizomib at 50° C. per minute. As shown in FIG. 18, two small endotherms were observed at about 186.5° C. (12/8 J/g) and about 192.6° C. (18.9 J/g). The two endotherms were followed by an exotherm at about 193-205° C. Without wishing to be bound by theory, this could be due to sample degradation.
FIG. 19 shows a differential scanning calorimetry plot of Sample 3 of Morphic Form I of marizomib at 2° C. per minute. As shown in FIG. 19, no endotherms were observed. Two exotherms were observed at about 155-175° C. Without wishing to be bound by theory, this could be due to sample degradation.
FIG. 20 shows a differential scanning calorimetry plot of Sample 3 of Morphic Form I of marizomib at 5° C. per minute. As shown in FIG. 20, a small endotherm was observed at 167.5° C. (0.4 J/g) followed by a sharp exotherm at about 173-183° C. Without wishing to be bound by theory, this could be due to sample degradation.
FIG. 21 shows a differential scanning calorimetry plot of Sample 3 of Morphic Form I of marizomib at 10° C. per minute. As shown in FIG. 21, two small endotherms were observed at about 171.2° C. (6.1 J/g) and about 178.6° C. (2.0 J/g), followed by an exotherm at about 183-193° C. Without wishing to be bound by theory, this could be due to sample degradation.
FIG. 22 shows a differential scanning calorimetry plot of Sample 3 of Morphic Form I of marizomib at 50° C. per minute. As shown in FIG. 22, two small endotherms were observed at about 180.9° C. (8.3 J/g) and 191.6° C. (19.0 J/g). The two small endotherms were followed by an exotherm at about 193-205° C. Without wishing to be bound by theory, this could be due to sample degradation.
As set forth in FIGS. 15-22, as the heating rate of the DSC experiments increased, a shift in onset temperature as well as a variation in enthalpy was observed. Without wishing to be bound by theory, these fluctuations observed in the temperature onset and enthalpy can be due to combination of melt and degradation of the sample. In other words, without wishing to be bound by theory, a pure melt of a crystalline, non-solvated morphic form is a thermodynamic event, so that changes in heating rate and/or amount of input material will not significantly affect the onset temperature of the melt. In contrast, sample degradation is a kinetic event, so that changes in the experimental parameters (amount of input material, heating rate, type of pan used, i.e. sealed or unsealed) can cause a shift in the onset temperature of that event.
Without wishing to be bound by theory, based on the results of DSC analysis of Samples 1 and 3, the changes observed in the main endothermic event can be due to both thermodynamic and kinetic factors, i.e. both melt and degradation of the sample.
Without wishing to be bound by theory, the results of the DSC and TG analysis of morphic Form I is consistent with the results set forth in FIGS. 5 and 6. FIGS. 5 and 6 demonstrate that Sample 1 and 3 remained as Form I throughout the increase in temperature from 25° C. to 160° C. Discoloration of the sample was observed at about 160° C. from white to yellow, as shown in FIGS. 33-36. However, after about five minutes to enable equilibration at about 160° C., the sample was observed to melt and degrade simultaneously.
Without wishing to be bound by theory, this result is further supported by the outcome from the hot stage microscopy experiments, in which crystals from Sample 1 were subjected to heating under air at ˜ 30° C./min. No changes were observed between 30 and 150° C. Between 160-175° C. changes were observed which captured the melt of the crystals and degradation of the sample (in form of discoloration) occurring substantially simultaneously.
FIG. 23 shows a ¹HNMR spectrum for both Sample 1 (2305) and Sample 3 (2310) of marizomib. As shown in FIG. 23, both spectra are consistent with the structure of marizomib.
FIG. 24 is an HPLC plot of Sample 1 of marizomib. As shown in FIG. 24, the purity of Sample 1 was at least 98.2%.
FIG. 25 is an HPLC plot of Sample 3 of marizomib. As shown in FIG. 25, the purity of Sample 3 was at least 99.1%.
FIG. 31 is a polarized light microscope image of Morphic Form I of Sample 1 of marizomib. As shown in FIG. 31, particles of Sample 1 had a lath morphology with particle size of 150 μm up to 300 μm in length.
FIG. 32 is a polarized light microscope image of Morphic Form I of Sample 3 of marizomib. As shown in FIG. 32, there is no lath morphology in the micronized sample. The samples are in some embodiments more sphere-like.
FIG. 33 is a photo of Sample 1 of Morphic Form I of marizomib during the variable temperature XRPD analysis at 25° C. FIG. 34 is a photo of Sample 1 of Morphic Form I of marizomib during the variable temperature XRPD analysis at 150° C. FIG. 35 is a photo of Sample 1 of Morphic Form I of marizomib at during the variable temperature XRPD analysis 160° C. Discoloration of the sample, from white to yellow, was observed at about 160° C.
FIG. 36 is a photo of Sample 1 of Morphic Form I of marizomib at during the variable temperature XRPD analysis 160° C. after five minutes. As shown in FIG. 36, the sample appears to be both melting and degrading. Without wishing to be bound by theory, this result is consistent with the TG and DSC analysis of Sample 1, which suggested that the morphic Form I begins to melt and/or degrade at about 160° C.
Sample 3 of morphic Form I showed similar characteristics to Sample 1. FIG. 37 is a photo of Sample 3 of Morphic Form I of marizomib at 25° C. FIG. 38 is a photo of Sample 3 of Morphic Form I of marizomib at 150° C. FIG. 39 is a photo of Sample 3 of Morphic Form I of marizomib at 160° C. FIG. 40 is a photo of Sample 3 of Morphic Form I of marizomib at 160° C. after five minutes. As shown in FIGS. 37-40, Sample 3 had properties similar to Sample 1. For example, both samples exhibited discoloration at about 160° C. and melting and/or degradation after holding for about five minutes at about 160° C.
A fourth sample of marizomib, Form I (“Sample 4”) was subjected to various conditions to attempt to prepare a different morphic form beyond Form I. The indexing parameters of Sample 4 are given below in Table-4:

TABLE 4

Parameters for Sample 4 Indexing

Bravais Type	Primitive Monoclinic

a [Å]	10.594
b [Å]	24.507
c [Å]	12.661
α [deg]	90
β [deg]	108.53
γ [deg]	90
Volume [Å³/cell]	3, 116.3
Chiral Contents?	Chiral
Extinction Symbol	P	1 2₁1
Space Group(s)	P2₁(4)

Attempted Hydrolysis of Marizomib

Without wishing to be bound by theory, it was hypothesized that marizomib could be hydrolyzed upon exposure to water. The results are given in Example 1. FIG. 26 is an overlay of three XRPD plots of Sample 1 of marizomib before (2615) and after attempted hydrolysis with water (2610) and methanol (2605). FIG. 27 is a ¹HNMR plot of Sample 1 of marizomib before (2710) and after (2705) attempted hydrolysis. FIG. 28 is an HPLC plot of Sample 1 of marizomib isolated after a hydrolysis attempt with water. The samples were matured between 50° C. and room temperature heat-cool cycles (8 h per cycle) for 96 h. As shown in FIG. 28, the purity of the sample is reduced from about 99% to about 75% after four days. FIG. 29 is an HPLC plot of Sample 1 of marizomib isolated from ethyl acetate. FIG. 30 is an HPLC plot of Sample 1 of marizomib isolated from ethanol. As shown in FIG. 28, there was observed a new peak at a retention time of about 4.729 minutes. Without wishing to be bound by theory, this peak is understood to represent the hydrolysis product, as set forth in Scheme 1, below:
However, as shown in FIGS. 26 and 27, the spectral characterization before and after attempted hydrolysis was substantially similar. Without wishing to be bound by theory, it is proposed that the hydrolysed product crystallizes in the same morphic form as the starting material (Form I). Alternatively, without wishing to be bound by theory, it is proposed that the hydrolysis product (above) is more soluble in aqueous media and does not crystallize under the same conditions as marizomib itself. In other words, XRPD could not be used to distinguish between morphic Form I and the hydrolysis product.

Methods of Preparing Morphic Form I

Morphic Form I of marizomib can be prepared by a number of methods as set forth herein. In one embodiment, marizomib is dissolved in a solvent (e.g., acetone) and crystallization is initiated by addition of an anti-solvent (e.g., heptane).
In some embodiments, the solvent is selected from the group consisting of n-heptane, ethyl acetate, methyl-isobutyl ketone, 2-propanol, acetone, chloroform, dimethyl sulfoxide, tert-butyl methyl ether, anisole, cumene, methyl ethyl ketone, isopropyl acetate, dimethylformamide, toluene, tetrahydrofuran, dichloromethane, acetonitrile, nitromethane, ethanol, and dimethylacetamide.
Without wishing to be bound by theory, a morphic form of a compound (e.g., marizomib) does not have to fully dissolve to convert to a new polymorphic form of the compound in an anti-solvent. In some embodiments, anti-solvents can lead to new polymorphs. Without wishing to be bound by theory, even if an anti-solvent is incapable of dissolving 1/50 its volume of marizomib, a small yet appreciable amount of marizomib can still be dissolved in the anti-solvent. Without wishing to be bound by theory, as a small amount of drug (e.g., marizomib) dissolves and then precipitates, the solvent characteristics can lead to a new form (in some embodiments called a seed) in the precipitate. As the drug (e.g., marizomib) dissolves and precipitates to the new form, over time or near-complete or in some embodiments complete conversion to a new form can be observed. Without wishing to be bound by theory, such interconversion was not observed following the morphic form screen set forth herein. That is, only morphic Form I was observed when exposing Form I to both solvents and anti-solvents.

Methods of Using Morphic Form I

In some embodiments, morphic Form I of the present disclosure can be used for the treatment of a disease. For instance, morphic Form I can be used in the manufacture of a medicament for the treatment of a disease. Additionally, the present disclosure contemplates a pharmaceutical composition comprising morphic Form I and a pharmaceutically acceptable carrier.
In some embodiments, the disease is cancer or a neoplastic disease. In some embodiments, the neoplastic disease treated by the morphic Forms disclosed herein may be a cancer selected from breast cancer, sarcoma, leukemia, ovarian cancer, uretal cancer, bladder cancer, prostate cancer, colon cancer, rectal cancer, stomach cancer, lung cancer, lymphoma, multiple myeloma, pancreatic cancer, liver cancer, kidney cancer, endocrine cancer, skin cancer, melanoma, angioma, and brain or central nervous system (CNS) cancer. In one embodiment, the neoplastic disease is a multiple myeloma.

Salt and Co-Crystal Screen

As set forth in Example 12 and Table 18, approximately 117 screening experiments were conducted using 46 coformers/counterions targeting salts and cocrystal of marizomib (Sample 4). Experiments were conducted at various stoichiometric ratios using standard crystallization techniques, including cooling, evaporation, anti-solvent addition, reaction crystallization, slurry at ambient and elevated temperature, solvent assisted grinding or a combination of these techniques. Solids isolated from screening experiments were analyzed by XRPD and/or ¹HNMR and compared to the known XRPD pattern of marizomib and representative XRPD patterns of the coformer/counterion.
Cocrystal screening of marizomib was conducted using primarily pharmaceutically acceptable coformers containing a diverse range of complementary functional groups including carboxylic acids, amino acids, amines, sulfonamides and amides that could potentially form supramolecular heterosynthons that could compete with the 2-point recognition amide-amide interaction present within the crystal structure of marizomib. However, solids isolated from cocrystal screening experiments were primarily consistent with marizomib, coformer, or mixtures of marizomib and coformer, indicating cocrystal formation did not occur under the tested experimental conditions. The detailed conditions and observations of all attempted cocrystal formation experiments and the results from PLM and XRPD analysis are summarized in Table 18.
Additional coformers such as pyroglutamic acid, 2-pyrrolidone and cytosine that contain secondary amides groups similar to marizomib were explored. Additionally, coformers that contain the 2-aminopyridine moiety e.g. adenine and melamine, were also included in screening.
Unique peaks in the presence of marizomib and/or coformer were identified from experiments involving marizomib with several coformers including 2-pyrrolidone (FIG. 41), melamine (FIG. 43, FIG. 45), and cytosine (FIG. 46).
A mixture of marizomib and a unique material (FIG. 41), as evidenced by the two additional diffraction peaks at 14.1 and 15.5°2θ, was crystallized after slow evaporation of a hazy solution produced from a temperature cycling (50° C. to RT) experiment in acetone containing equimolar amounts of marizomib and 2-pyrrolidone. Trace 4101 shows marizomib Sample 4. Trace 4102 shows a trace of marizomib and 2-pyrrolidone (1:1) after slow evaporation after temperature cycles between 50° C. and room temperature (4 cycles). The proton NMR spectrum of solids isolated from this experiment showed marizomib: 2-pyrrolidone in approximate 1:1.2 mole ratio. Thermal analysis of solids isolated from this experiment is presented in FIG. 42. Trace 4201 shows the heat flow (W/g). Trace 4202 shows the weight percent of the sample. A sharp endotherm was observed at 32.8° C. (peak maximum), likely attributed to the melting of residual 2-pyrrolidone, followed by broad endothermic events at 91.4° C. and 131.3° C. (peak maxima) in the DSC data. By TGA, a weight loss of 5% was observed upon heating between 27° C. and 92° and a change in the slope after approximately 120° C. likely due to decomposition was seen. The thermal behavior for this material was different from that observed for marizomib Sample 4 used as starting material. As noted herein, marizomib Sample 4 exhibits a negligible weight loss prior to decomposition after approximately 150° C. and no thermal events are observed in the DSC data up to approximately 166° C., where an exotherm is observed followed by a small endotherm at approximately 175° C.
Grinding marizomib in a mortar and pestle in the presence of 2-pyrrolidone also produced the same unique peaks observed in the solution based experiment, in addition to peaks which were characteristic of marizomib. A third experiment was conducted in which marizomib was dissolved in 2-pyrrolidone at 50° C., cooled to RT and evaporated. However, no solids were produced and the clear solution remained.
A suspension containing equimolar amounts of marizomib and melamine in DMSO:H₂O (1:1) was slurried at 50° C. for approximately 2 days then at RT for 6 days and produced a mixture of marizomib and a unique material based on XRPD. The acquired proton NMR spectrum of the material from the experiment showed marizomib:melamine in approximate 1:1.6 mole ratio. Additional peaks, not present in the proton NMR spectrum of marizomib Sample 4 used as starting material were also observed, suggesting possible degradation of the marizomib. DMSO and water were also detected in the spectrum. An overlay of XRPD patterns is shown in FIG. 43. Trace 4301 shows a melamine reference. Trace 4302 shows the mixture of marizomib and melamine (1:1) slurry. Trace 4303 shows a trace of marizomib Sample 4 for reference. Thermal analysis of solids isolated from this experiment is presented in FIG. 44. Trace 4401 shows the weight percent of the sample. Trace 4402 shows the heat flow (W/g). Multiple endotherms are observed at 97.5° C., 107.9° C., and 124.1° C. (peak maxima). Note: no thermal events are observed in the DSC data for marizomib up to approximately 166° C., where an exotherm is observed followed by a small endotherm at approximately 175° C. Melamine is reported to have a melting point around 345° C. The TGA thermogram shows continuous weight loss with heating. Approximately 10% weight loss is observed between ˜30° C. and 104° C. associated with the broad endotherm at 97.5° C. (peak max.) in the DSC data.
A second experiment containing marizomib and an excess of melamine (1:3 mole ratio) was performed in an attempt to isolate the unique material as a single crystalline phase. The sample was slurried overnight at 60° C. then at RT for 1 day and produced a mixture of marizomib and melamine with unique peaks (FIG. 45). Trace 4501 shows a melamine reference. Trace 4502 shows marizomib:melamine (1:3) after slurry at 60° C. in DMSO:H₂O (1:1) and then room temperature for 1 day. Trace 4503 shows marizomib:melamine (1:1) after slurry at 50° C. in DMSO:H₂O (1:1) for two days and then room temperature for 6 days. Trace 4504 shows marizomib Sample 4 for reference.
A suspension containing marizomib and an excess of cytosine (1:3 mole ratio) in dioxane:water was slurried at 60° C. overnight and produced a light yellow clear solution. After stirring at RT for 1 day, solids precipitated. Based on XRPD, the isolated solids were composed of a mixture of unique peaks and marizomib suggesting the presence of a secondary unidentified phase (FIG. 46). Trace 4601 shows an XPRD pattern of cytosine. Trace 4602 shows a pattern of marizomib:cytosine (1:3) after slurry at 60° C. overnight and then at room temperature for 1 day. Trace 4603 shows an XRPD spectrum of marizomib Sample 4. Proton NMR analysis of the solids from the experiment revealed primarily cytosine with trace marizomib and minor additional peaks. Dioxane was also observed. The data suggest no cocrystal formation occurred with cytosine and that the unique peaks are possibly attributable to a form of cytosine.
In a few cases, additional unidentified peaks were observed as a mixture with marizomib and/or coformer/counterion, and were likely due to partial degradation.
Based on the chemical structure of marizomib, the formation of stable salts of the compound was anticipated to be unlikely (predicted pKa˜−1.4), but the use of strong acids was included within the screen to evaluate possible salt formation. Salt screening attempts with sulfonic acids and mineral acids produced marizomib, mixtures of marizomib and counterion, or discolored solutions suggesting possible degradation (Example 13, Table 19). No crystalline salt of marizomib was produced in this study.

Pharmaceutical Compositions

In some embodiments, the present disclosure relates to a pharmaceutical composition comprising physiologically acceptable surface active agents, carriers, diluents, excipients, smoothing agents, suspension agents, film forming substances, and coating assistants, or a combination thereof, and morphic Form I. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990), which is incorporated herein by reference in its entirety. Preservatives, stabilizers, dyes, sweeteners, fragrances, flavoring agents, and the like may be provided in the pharmaceutical composition. For example, sodium benzoate, ascorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. In addition, antioxidants and suspending agents may be used. In various embodiments, alcohols, esters, sulfated aliphatic alcohols, and the like may be used as surface active agents; sucrose, glucose, lactose, starch, crystallized cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium methasilicate aluminate, synthetic aluminum silicate, calcium carbonate, sodium acid carbonate, calcium hydrogen phosphate, calcium carboxymethyl cellulose, and the like may be used as excipients; magnesium stearate, talc, hardened oil and the like may be used as smoothing agents; coconut oil, olive oil, sesame oil, peanut oil, soya may be used as suspension agents or lubricants; cellulose acetate phthalate as a derivative of a carbohydrate such as cellulose or sugar, or methylacetate-methacrylate copolymer as a derivative of polyvinyl may be used as suspension agents; and plasticizers such as ester phthalates and the like may be used as suspension agents.
Unless otherwise defined, a “solvent” of marizomib is a substance that can dissolve at least 1/50 its volume of marizomib at 50° C. or below. Unless otherwise defined, an “anti-solvent” is any substance that fails to dissolve at least 1/50 its volume of marizomib at 50° C.
The term “pharmaceutical composition” refers to morphic Form I in combination with other chemical components, such as diluents or carriers. The pharmaceutical composition facilitates administration of the compound (e.g., a morphic Form such as morphic Form I) to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, oral, injection, aerosol, parenteral, and topical administration. Pharmaceutical compositions can also be obtained by reacting compounds (e.g., a morphic Form such as morphic Form I) with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
The term “carrier” defines a chemical compound that facilitates the incorporation of a compound (e.g., morphic Form I) into cells or tissues. For example dimethyl sulfoxide (DMSO) is a commonly utilized carrier as it facilitates the uptake of many organic compounds into the cells or tissues of an organism.
The term “diluent” defines chemical compounds diluted in water that will dissolve the compound of interest (e.g., a morphic Form such as morphic Form I) as well as stabilize the biologically active form of the compound. Salts dissolved in buffered solutions are utilized as diluents in the art. One commonly used buffered solution is phosphate buffered saline because it mimics the salt conditions of human blood. Since buffer salts can control the pH of a solution at low concentrations, a buffered diluent rarely modifies the biological activity of a compound.
The term “physiologically acceptable” defines a carrier or diluent that does not abrogate the biological activity and properties of the compound.
The pharmaceutical compositions described herein can be administered to a human patient per se, or in pharmaceutical compositions where they are mixed with suitable carriers or excipient(s). Techniques for formulation and administration of the compounds of the instant application may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 18th edition, 1990.
Suitable routes of administration may, for example, include oral, rectal, transmucosal, topical, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections. The morphic Forms (e.g., morphic Form I) can also be administered in sustained or controlled release dosage forms, including depot injections, osmotic pumps, pills, transdermal (including electrotransport) patches, and the like, for prolonged and/or timed, pulsed administration at a predetermined rate.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tabletting processes.
Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds (e.g., a morphic Form such as morphic Form I) into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art; e.g., in Remington's Pharmaceutical Sciences, above.
Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, and the like. In addition, if desired, the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like. Physiologically compatible buffers include, but are not limited to, Hanks's solution, Ringer's solution, or physiological saline buffer. If desired, absorption enhancing preparations (for example, liposomes), may be utilized.
For transmucosal administration, penetrants appropriate to the barrier to be permeated may be used in the formulation.
Pharmaceutical formulations for parenteral administration, e.g., by bolus injection or continuous infusion, include aqueous solutions of the active compounds (e.g., a morphic Form such as morphic Form I) in water-soluble form. Additionally, suspensions of the active compounds (e.g., a morphic Form such as morphic Form I) may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or other organic oils such as soybean, grapefruit or almond oils, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds (e.g., a morphic Form such as morphic Form I) to allow for the preparation of highly concentrated solutions. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
For oral administration, the compounds (e.g., a morphic Form such as morphic Form I) can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the morphic Forms (e.g., morphic Form I) to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by combining the morphic Forms (e.g., morphic Form I) with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the morphic Forms (e.g., morphic Form I) may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds (e.g., a morphic Form such as morphic Form I) for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound (e.g., a morphic Form such as morphic Form I) and a suitable powder base such as lactose or starch.
Further disclosed herein are various pharmaceutical compositions well known in the pharmaceutical art for uses that include intraocular, intranasal, and intraauricular delivery. Suitable penetrants for these uses are generally known in the art. Pharmaceutical compositions for intraocular delivery include aqueous ophthalmic solutions of the morphic Forms (e.g., morphic Form I) in water-soluble form, such as eyedrops, or in gellan gum (Shedden et al., Clin. Ther., 23(3):440-50 (2001)) or hydrogels (Mayer et al., Ophthalmologica, 210(2):101-3 (1996)); ophthalmic ointments; ophthalmic suspensions, such as microparticulates, drug-containing small polymeric particles that are suspended in a liquid carrier medium (Joshi, A., J. Ocul. Pharmacol., 10(1):29-45 (1994)), lipid-soluble formulations (Alm et al., Prog. Clin. Biol. Res., 312:447-58 (1989)), and microspheres (Mordenti, Toxicol. Sci., 52(1):101-6 (1999)); and ocular inserts. All of the above-mentioned references, are incorporated herein by reference in their entireties. Such suitable pharmaceutical formulations are most often and preferably formulated to be sterile, isotonic and buffered for stability and comfort. Pharmaceutical compositions for intranasal delivery may also include drops and sprays often prepared to simulate in many respects nasal secretions to ensure maintenance of normal ciliary action. As disclosed in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990), which is incorporated herein by reference in its entirety, and well-known to those skilled in the art, suitable formulations are most often and preferably isotonic, slightly buffered to maintain a pH of 5.5 to 6.5, and most often and preferably include antimicrobial preservatives and appropriate drug stabilizers. Pharmaceutical formulations for intraauricular delivery include suspensions and ointments for topical application in the ear. Common solvents for such aural formulations include glycerin and water.
The morphic Forms (e.g., morphic Form I) may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the morphic Forms (e.g., morphic Form I) may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds (e.g., a morphic Form such as morphic Form I) may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
In some embodiments, a suitable pharmaceutical carrier may be a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. A common cosolvent system used is the VPD co-solvent system, which is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of POLYSORBATE 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
Alternatively, other delivery systems for hydrophobic pharmaceutical compounds (e.g., a morphic Form such as morphic Form I) may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds (e.g., a morphic Form such as morphic Form I) may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external micro-environment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. The liposome may be coated with a tissue-specific antibody. The liposomes will be targeted to and taken up selectively by the desired organ. Alternatively, small hydrophobic organic molecules may be directly administered intracellularly.
Additional therapeutic or diagnostic agents may be incorporated into the pharmaceutical compositions. Alternatively or additionally, pharmaceutical compositions may be combined with other compositions that contain other therapeutic or diagnostic agents.

Methods of Administration

The morphic Forms (e.g., morphic Form I) and pharmaceutical compositions comprising the same may be administered to the patient by any suitable means. Non-limiting examples of methods of administration include, among others, (a) administration though oral pathways, which administration includes administration in capsule, tablet, granule, spray, syrup, or other such forms; (b) administration through non-oral pathways such as rectal, vaginal, intraurethral, intraocular, intranasal, or intraauricular, which administration includes administration as an aqueous suspension, an oily preparation or the like or as a drip, spray, suppository, salve, ointment or the like; (c) administration via injection, subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, intraorbitally, intracapsularly, intraspinally, intrasternally, or the like, including infusion pump delivery; (d) administration locally such as by injection directly in the renal or cardiac area, e.g., by depot implantation; as well as (e) administration topically; as deemed appropriate by those of skill in the art for bringing the compound of the invention (e.g., a morphic Form such as morphic Form I) into contact with living tissue.
Pharmaceutical compositions suitable for administration include compositions where the active ingredients are contained in an amount effective to achieve its intended purpose. The therapeutically effective amount of the compounds disclosed herein (e.g., a morphic Form such as morphic Form I) required as a dose will depend on the route of administration, the type of animal, including human, being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize. More specifically, a therapeutically effective amount means an amount of compound (e.g., a morphic Form such as morphic Form I) effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight and mammalian species treated, the particular compounds employed (e.g., a morphic Form such as morphic Form I), and the specific use for which these compounds are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine pharmacological methods. Typically, human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the compositions identified by the present methods using established pharmacological methods.
In non-human animal studies, applications of potential products are commenced at higher dosage levels, with dosage being decreased until the desired effect is no longer achieved or adverse side effects disappear. The dosage may range broadly, depending upon the desired affects and the therapeutic indication. Typically, dosages may be between about 10 microgram/kg and 100 mg/kg body weight, preferably between about 100 microgram/kg and 10 mg/kg body weight. Alternatively dosages may be based and calculated upon the surface area of the patient, as understood by those of skill in the art.
The exact formulation, route of administration and dosage for the pharmaceutical compositions of the present invention can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl et al. 1975, in “The Pharmacological Basis of Therapeutics”, which is hereby incorporated herein by reference in its entirety, with particular reference to Ch. 1, p. 1). Typically, the dose range of the composition administered to the patient can be from about 0.5 to 1000 mg/kg of the patient's body weight. The dosage may be a single one or a series of two or more given in the course of one or more days, as is needed by the patient. In instances where human dosages for compounds (e.g., a morphic Form such as morphic Form I) have been established for at least some condition, the present invention will use those same dosages, or dosages that are between about 0.1% and 500%, more preferably between about 25% and 250% of the established human dosage. Where no human dosage is established, as will be the case for newly-discovered pharmaceutical compounds, a suitable human dosage can be inferred from ED₅₀or ID50 values, or other appropriate values derived from in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals.
It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
Although the exact dosage will be determined on a drug-by-drug basis, in most cases, some generalizations regarding the dosage can be made. The daily dosage regimen for an adult human patient may be, for example, an oral dose of between 0.1 mg and 2000 mg of each active ingredient, preferably between 1 mg and 500 mg, e.g. 5 to 200 mg. In other embodiments, an intravenous, subcutaneous, or intramuscular dose of each active ingredient of between 0.01 mg and 100 mg, preferably between 0.1 mg and 60 mg, e.g. 1 to 40 mg is used. In cases of administration of a pharmaceutically acceptable salt, dosages may be calculated as the free base. In some embodiments, the composition is administered 1 to 4 times per day. Alternatively the compositions of the invention may be administered by continuous intravenous infusion, preferably at a dose of each active ingredient up to 1000 mg per day. As will be understood by those of skill in the art, in certain situations it may be necessary to administer the compounds disclosed herein (e.g., a morphic Form such as morphic Form I) in amounts that exceed, or even far exceed, the above-stated, preferred dosage range in order to effectively and aggressively treat particularly aggressive diseases or infections. In some embodiments, the compounds (e.g., a morphic Form such as morphic Form I) will be administered for a period of continuous therapy, for example for a week or more, or for months or years.
Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound (e.g., a morphic Form such as morphic Form I) but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
Dosage intervals can also be determined using MEC value. Compositions should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.
In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.
The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
Compounds disclosed herein (e.g., a morphic Form such as morphic Form I) can be evaluated for efficacy and toxicity using known methods. For example, the toxicology of a particular compound, or of a subset of the compounds, sharing certain chemical moieties, may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans. Alternatively, the toxicity of particular compounds (e.g., a morphic Form such as morphic Form I) in an animal model, such as mice, rats, rabbits, or monkeys, may be determined using known methods. The efficacy of a particular compound may be established using several recognized methods, such as in vitro methods, animal models, or human clinical trials. Recognized in vitro models exist for nearly every class of condition, including but not limited to cancer, cardiovascular disease, and various immune dysfunction. Similarly, acceptable animal models may be used to establish efficacy of chemicals to treat such conditions. When selecting a model to determine efficacy, the skilled artisan can be guided by the state of the art to choose an appropriate model, dose, and route of administration, and regime. Of course, human clinical trials can also be used to determine the efficacy of a compound (e.g., a morphic Form such as morphic Form I) in humans.
The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

EXAMPLES

The disclosure is further illustrated by the following examples, which are not to be construed as limiting this disclosure in scope or spirit to the specific procedures herein described. It is to be understood that the examples are provided to illustrate certain embodiments and that no limitation to the scope of the disclosure is intended thereby. It is to be further understood that resort may be had to various other embodiments, modifications, and equivalents thereof which may suggest themselves to those skilled in the art without departing from the spirit of the present disclosure and/or scope of the appended claims.
Abbreviations used herein are given below:


	Acronym	Meaning

	ca.	Approximately
	DMA	Dimethylacetamide
	DMF	Dimethylformamide
	DSC	Differential Scanning Calorimetry
	H₂O	Water
	¹H-NMR	Proton Nuclear Magnetic Resonance
	HPLC	High Performance Liquid Chromatography
	ID	Identification
	MeCN	Acetonitrile
	MEK	Methyl Ethyl Ketone
	MeOH	Methanol
	MIBK	Methyl Isobutyl Ketone
	N/A	Not Applicable
	PLM	Polarised Light Microscopy
	RT	Room Temperature
	SCXRD	Single Crystal X-Ray Diffraction
	SEM	Scanning Electron Microscope
	TGA	Thermal Gravimetric Analysis
	UV	Ultra Violet
	VT-XRPD	Variable Temperature X-Ray Powder Diffraction
	XRPD	X-Ray Powder Diffraction

Analytical Techniques


	Acronyms	Full Name/Description

	DSC	Differential scanning calorimetry
	mDSC	Modulated differential scanning calorimetry
	NMR	Nuclear magnetic resonance spectroscopy
	TGA	Thermogravimetric analysis
	XRPD	X-ray powder diffraction
	DVS	Dynamic vapor sorption

Methods


	Acronym	Full Name/Description

	CC	Crash cooling
	CP	Crash precipitation
	FC	Fast cooling
	FE	Fast evaporation
	SC	Slow cooling

Miscellaneous


	Acronym	Full Name/Description

	Agg. (agg.)	Aggregates
	API	Active pharmaceutical ingredient
	B/E	Birefringence and extinction
	B	Birefringence
	d	Day(s)
	decomp.	Decomposition
	endo	Endotherm
	exo	Exotherm
	h	Hour(s)
	LIMS	Laboratory information management system
	RT	Room temperature/ambient temperature
	UM	Unknown morphology
	A_w	water activity
	w/	With
	IS	Insufficient amount
	Anh.	Anhydrous
	API	Active pharmaceutical ingredient
	B	Birefringence
	B/E	Birefringence and extinction
	MRZ	Marizomib
	O/N	Overnight
	PLM	Polarized light microscope
	RT	Room temperature/ambient temperature
	Sat'd	Saturated

Solvents


	Acronyms	Full Name/Description

	[EMIm][Cl]	1-Ethyl-3-methylimidazolium chloride
	[EMIm][NTF₂]	1-Ethyl-3-methylimidazolium
		bis(trifluoromethylsulfonyl)imide
	2-Me THF	2-Methyltetrahydrofuran
	ACN	acetonitrile
	CHCl₃	chloroform
	DCM	dichloromethane
	DMF	dimethylformamide
	EtOAc	ethyl acetate
	Formulation	55% propylene glycol +
	System A	5% EtOH + 40% citrate buffer
		(10 mM; pH 5)
	Formulation	14/86 (v/v) H₂O/tBA
	System B	containing 30 mg/ml sucrose,
		and titrated with HCl to pH 3.2 ± 0.2
	IPA	isopropyl alcohol
	MEK	butanone (methyl ethyl ketone)
	MeOAc	methyl acetate
	MIBK	methyl isobutyl ketone
	MTBE	methyl Tertiary Butyl Ether
	NMP	N-Methyl-2-pyrrolidone
	THF	tetrahydrofuran
	t-BuOH	Tert-butanol
	DMSO	Dimethylsulfoxide
	EtOH	Ethanol
	H2O	Water
	H2SO4	Sulfuric acid
	HCl	Hydrochloric acid
	HFIPA




	1,1,1,3,3,3-hexafluoro-2-propanol
	IPrOAc	Isopropyl acetate
	MeOH	Methanol
	NMP	N-methyl-2-pyrrolidone
	1-PrOH	1- propanol
	TFE

	2,2,2-trifluoroethanol

XRPD Terminology

The term “crystalline” is understood to mean a form that exhibits an XRPD pattern with sharp peaks (similar to instrumental peak widths) and weak diffuse scattering (relative to the peaks).
The term “disordered crystalline” is understood to mean a form that exhibits an XRPD pattern with broad peaks (relative to instrumental peak widths) and/or strong diffuse scattering (relative to the peaks). Disordered materials may be microcrystalline; crystalline with large defect density; mixtures of crystalline and x-ray amorphous phases; or a combination of the above. Additional analysis may differentiate among these options.
The term “insufficient signal” is used when insufficient signal above the expected background scattering was observed. This may indicate, for example, that the x-ray beam missed the sample and/or that the sample was of insufficient mass for analysis.
“Particle statistics artifacts” can occur when the particle size distribution contains a small number of large crystals which may in some cases lead to sharp spikes in the XRPD pattern.
“Preferred orientation artifacts” can occur when the particle morphology is prone to non-random orientation in the sample holder which may in some cases lead to changes in relative peak intensities.
“No peaks” can occur when no Bragg peaks are observed in the XRPD pattern. The absence of peaks may in some cases be due to an x-ray amorphous sample and/or insufficient signal.
“Single crystalline phase” can refer to when an XRPD pattern is judged to contain evidence of a single crystalline phase if the Bragg peaks can be indexed with a single unit cell.
“X-ray amorphous” can refer to situations in which diffuse scatter is present, but no evidence for Bragg peaks is found in an XRPD pattern. X-ray amorphous materials may be: nano-crystalline; crystalline with a very large defect density; kinetic amorphous material; thermodynamic amorphous material; or a combination of the above. Additional analysis may differentiate among these options.

Hygroscopicity

The term “low hygroscopicity” refers to samples that exhibit <0.5 weight percent water uptake over a specified relative humidity range.
The term “limited hygroscopicity” refers to samples that exhibit <2.0 weight percent water uptake over a specified relative humidity range.
The term “significant hygroscopicity” refers to samples that exhibit ≥2.0 weight percent water uptake over a specified relative humidity range.
The term “deliquescence” refers to spontaneous liquefaction associated with water sorption from atmospheric moisture.
The term “stoichiometric hydrate” refers to crystalline material with stable stoichiometric water content over an extended relative humidity range. Exemplary stoichiometric hydrates can be, for instance, hemihydrates, monohydrates, sesquihydrates, or dehydrates.
A “variable hydrate” can refer to crystalline material with variable water content over an extended relative humidity range, yet with no phase change.

Experimental Procedures

Examples 3-11 used Sample 4 of marizomib.

X-Ray Powder Diffraction (XRPD)—Samples 1-3

X-Ray Powder Diffraction patterns were collected on a Bruker AXS C2 GADDS diffractometer using Cu Kα radiation (40 kV, 40 mA), automated XYZ stage, laser video microscope for auto-sample positioning and a HiStar 2-dimensional area detector. X-ray optics consists of a single Gobel multilayer mirror coupled with a pinhole collimator of 0.3 mm. A weekly performance check was carried out using a certified standard NIST 1976 Corundum (flat plate).
The beam divergence, i.e. the effective size of the X-ray beam on the sample, was approximately 4 mm. A θ-θ continuous scan mode was employed with a sample-detector distance of 20 cm which gave an effective 2θ range of 3.2°-29.7°. Unless otherwise specified, the sample would be exposed to the X-ray beam for about 120 seconds. The software used for data collection was GADDS for XP/2000 4.1.43 and the data were analyzed and presented using Diffrac Plus EVA v15.0.0.0.
Samples run under ambient conditions were prepared as flat plate specimens using powder as received without grinding. Approximately 1-2 mg of the sample was lightly pressed on a glass slide to obtain a flat surface.
Samples run under non-ambient conditions were mounted on a silicon wafer with heat-conducting compound. The sample was then heated to the appropriate temperature at 20° C./min and subsequently held isothermally for 1 minute before data collection was initiated.
Alternatively, X-Ray Powder Diffraction patterns were collected on a Bruker D8 diffractometer using Cu Kα radiation (40 kV, 40 mA), 0-20 goniometer, and divergence of V4 and receiving slits, a Ge monochromator and a Lynxeye detector. The instrument was performance checked using a certified Corundum standard (NIST 1976). The software used for data collection was Diffrac Plus XRD Commander v2.6.1 and the data were analysed and presented using Diffrac Plus EVA v15.0.0.0.
Samples were run under ambient conditions as flat plate specimens using powder as received. The sample was gently packed into a cavity cut into polished, zero-background (510) silicon wafer. The sample was rotated in its own plane during analysis. The details of the data collection are: Angular range: 2 to 42°2θ; Step size: 0.05°2θ; Collection time: 0.5 s/step.

X-Ray Powder Diffraction (XRPD)—Sample 4

Transmission Mode: XRPD patterns were collected with a PANalytical X'Pert PRO MPD diffractometer using an incident beam of Cu radiation produced using an Optix long, fine-focus source. An elliptically graded multilayer mirror was used to focus Cu Kα X-ray radiation through the specimen and onto the detector. Prior to the analysis, a silicon specimen (NIST SRM 640e) was analyzed to verify the observed position of the Si (111) peak is consistent with the NIST-certified position. A specimen of the sample was sandwiched between 3-μm-thick films and analyzed in transmission geometry. A beam-stop, short antiscatter extension, and antiscatter knife edge, were used to minimize the background generated by air. Soller slits for the incident and diffracted beams were used to minimize broadening from axial divergence. Diffraction patterns were collected using a scanning position-sensitive detector (X'Celerator) located 240 mm from the specimen and Data Collector software v. 2.2b.
Reflection Mode: XRPD patterns were collected with a PANalytical X'Pert PRO MPD diffractometer using an incident beam of Cu Kα radiation produced using a long, fine-focus source and a nickel filter. The diffractometer was configured using the symmetric Bragg-Brentano geometry. Prior to the analysis, a silicon specimen (NIST SRM 640e) was analyzed to verify the observed position of the Si 111 peak is consistent with the NIST-certified position. A specimen of the sample was prepared as a thin, circular layer centered on a silicon zero-background substrate. Antiscatter slits (SS) were used to minimize the background generated by air. Soller slits for the incident and diffracted beams were used to minimize broadening from axial divergence. Diffraction patterns were collected using a scanning position-sensitive detector (X'Celerator) located 240 mm from the sample and Data Collector software v. 2.2b.
A high-resolution XRPD pattern was indexed using proprietary SSCI software, TRIADS™. Indexing and structure refinement are computational studies. Agreement between the allowed peak positions, marked with red bars, and the observed peaks indicates a consistent unit cell determination. Successful indexing of the pattern indicates that the sample is composed primarily of a single crystalline phase. To confirm the tentative indexing solution, the molecular packing motifs within the crystallographic unit cells must be determined. No attempts at molecular packing were performed.

Proton Nuclear Magnetic Resonance (¹H-NMR) Samples 1-3

NMR spectra were collected on a Bruker 400 MHz instrument equipped with an auto-sampler and controlled by a DRX400 console. Automated experiments were acquired using ICON-NMR v4.0.7 running with Topspin v1.3 using the standard Bruker loaded experiments. For non-routine spectroscopy, data were acquired through the use of Topspin alone.
Samples were prepared in DMSO-d6, unless otherwise stated. Off-line analysis was carried out using ACD Spectrus Processor 2014.

Proton Nuclear Magnetic Resonance (¹H-NMR)—Sample 4

Initial characterization solution proton NMIR spectra were acquired with an Agilent DD2-400 spectrometer using deuterated methanol.
Other solution NMR spectra were acquired using a Bruker-Biospin 5 mm gradient broadband probe on a Bruker-Biospin AVANCE II 400 MHz NMR spectrometer. The spectrum was referenced using the tetramethylsilane resonance and set equal to 0.0 ppm. The FID was processed using Bruker TopSpin 2.1.
For the coformer screen and salt screen (Examples 12 and 13), the solution NMIR spectra were acquired with an Agilent DD2-400 spectrometer. Samples were prepared by dissolving approximately 5-10 mg of sample in DMSO-d6 containing TMS. The data acquisition parameters are displayed in each plot of the spectrum in the Data section of this report.

Differential Scanning Calorimetry (DSC)—Samples 1-3

DSC data were collected on a TA Instruments Q2000 equipped with a 50-position auto-sampler. The calibration for thermal capacity was carried out using sapphire and the calibration for energy and temperature was carried out using certified indium. Typically 0.5-3 mg of each sample, in a pin-holed aluminum pan, was heated at 10° C./min from 25° C. to 300° C. A purge of dry nitrogen at 50 ml/min was maintained over the sample.
The instrument control software was Advantage for Q Series v2.8.0.394 and Thermal Advantage v5.5.3 and the data were analyzed using Universal Analysis v4.5A.
Differential scanning calorimetry experiments were performed in duplicate at each of the four heating rates (i.e., 2, 5, 10 and 50° C./min).

Differential Scanning Calorimetry (DSC)—Sample 4

DSC was performed using a TA Instruments Q2000 differential scanning calorimeter. Temperature calibration was performed using NIST-traceable indium metal. The sample was placed into an aluminum Tzero DSC pan, covered with a lid, and the weight was accurately recorded. A weighed aluminum pan configured as the sample pan was placed on the reference side of the cell.
Modular Differential Scanning Calorimetry (mDSC)—Sample 4
mDSC data was obtained on a TA Instruments 2920 differential scanning calorimeter equipped with a refrigerated cooling system (RCS). Temperature calibration was performed using NIST traceable indium metal. The sample was placed into an aluminum DSC pan, covered with a lid, and the weight was accurately recorded. A weighed aluminum pan configured as the sample pan was placed on the reference side of the cell.

Thermo-Gravimetric Analysis (TGA) Samples 1-3

TGA data were collected on a TA Instruments Q500 TGA, equipped with a 16-position auto-sampler. The instrument was temperature calibrated using certified Alumel™ and Nickel. Typically 5-10 mg of each sample was loaded onto a pre-tared aluminum DSC pan and heated at 10° C./min from ambient temperature to 350° C. A nitrogen purge at 60 ml/min was maintained over the sample.
The instrument control software was Advantage for Q Series v2.5.0.256 and Thermal Advantage v5.5.3 and the data were analyzed using Universal Analysis v4.5A.

Thermo-Gravimetric Analysis (TGA)—Sample 4

TG analyses were performed using a TA Instruments Q5000 IR thermogravimetric analyzer. Temperature calibration was performed using nickel and Alumel™. Each sample was placed in an aluminum pan. The sample was hermetically sealed, the lid pierced, then inserted into the TG furnace. The furnace was heated under nitrogen.

Polarized Light Microscopy (PLM) Samples 1-3

Samples were studied on a Leica LM/DM polarized light microscope with a digital video camera for image capture. A small amount of each sample was placed on a glass slide, mounted in immersion oil and covered with a glass slip, the individual particles being separated as well as possible. The sample was viewed with appropriate magnification and partially polarized light, coupled to a λ false-color filter.

Polarized Light Microscopy (PLM)—Sample 4

Light microscopy was performed using a Motic SMZ-168 stereomicroscope. Various objectives typically ranging from 0.8-10× were used with crossed-polarized light to view samples. Samples were either viewed in situ, or in a drop of mineral oil.
For the coformer screen and salt screen (Examples 12 and 13), light microscopy was performed using Fisher Scientific Stereomaster stereomicroscope. Various objectives typically ranging from 0.8-10× were used with crossed-polarized light to view samples.

Hot Stage Microscopy (HSM)

Hot Stage Microscopy was carried out using a Leica LM/DM polarised light microscope combined with a Mettler-Toledo FP82HT hot-stage and a digital video camera for image capture. A small amount of each sample was placed onto a glass slide with individual particles separated as well as possible. The sample was viewed with appropriate magnification and partially polarised light, coupled to a λ false-colour filter, whilst being heated from ambient temperature typically at 10-20° C./min.

Chemical Purity Determination by HPLC

Purity analysis was performed on an Agilent PI 100 series system equipped with a diode array detector and using ChemStation software vB.04.03 using the method set forth in Table-5.

TABLE 5

HPLC Conditions for Purity Determination

	Parameter	Value

	Type of method	Reverse phase with gradient elution
	Sample Preparation	0.5 mg/ml in acetonitrile:water 1:1 +
		0.1% TFA
	Column	Supelco Ascentis Express C18, 100 ×
		4.6 mm, 2.7 μm
	Column
	25
	Temperature (° C.)
	Injection (μl)	5
	Wavelength,	255, 90
	Bandwidth (nm)
	Flow Rate	2
	(ml/min)
	Phase A	0.1% TFA in water
	Phase B	0.085% TFA in acetonitrile

	Time	%	%
Timetable	(min)	Phase A	Phase B

	0	95	5
	6	5	95
	6.2	95	5
	8	95	5

Example 1—Hydrolysis of Marizomib

30 mg of Sample 1 was suspended in the given solvent shown in Table 6 (1 ml) at room temperature. The samples were matured between 50° C. and room temperature heat-cool cycles (8 h per cycle) for 96 h. The solids were filtered, air dried and analyzed by XRPD.

TABLE 6

Preparation and Characterization of Solids from Hydrolysis Experiments

		Obs. post			HPLC
Solvent	Obs. at r.t.	maturation	XRPD	¹H-NMR	Purity

MeOH	Suspension	Suspension	Crystalline -	n/a	n/a
			similar to
			Form I
Water	Suspension	Suspension	Crystalline -	Comparable	74.8%
			similar to	with ref.	(ref. to
			Form I	spectrum	starting
					material:
					98.2%)

After 4 days maturation in water, the chemical purity was down from 98% to 75%, with emphasis on a particular peak on the HPLC chromatogram (RRT=4.75 min) as shown in FIG. 28. However, the solid phase remained the same by XRPD as shown in FIG. 26. Without wishing to be bound by theory, it is proposed that the hydrolysed product crystallizes in the same phase as the starting material (Form I). In other words, XRPD could not be used to distinguish between morphic Form I and the hydrolysis product.

Example 2—Polymorphism Screening

Polymorphism studies on marizomib were carried out in different solvents and conditions to understand the polymorphic behavior of the free form.
Marizomib ( Sample 1, 30 mg) was dissolved or suspended in the given solvent at room temperature. Solvent was added until the material dissolved or up to a maximum of 50 volumes. The suspensions were matured between 50° C. and room temperature heat-cool cycles (8 h per cycle) for 24 h. The solutions were cooled to and held at 5° C. for 48 h. If no solid was obtained then the solutions were allowed to evaporate slowly at room temperature. All the recovered solids were analyzed by XRPD. The results are given in Table 7.

TABLE 7

Polymorphism Screen Results

Obs. at r.t.

Maturation

Cooling at

Evap. at

Solvent	10 vol.	20 vol.	30 vol.	40 vol.	50 vol.	50° C./r.t.	5° C.	r.t.	XRPD

n-Heptane	X	X	X	X	X	X	n/a	n/a	Form I
Ethyl acetate	X	X	X	X	X	✓	✓	Solid	Form I
MIBK	X	X	X	X	X	X	n/a	n/a	Form I
2-Propanol	X	X	X	X	X	X	n/a	n/a	Form I
Acetone	X	X	X	X	X	✓	✓	Solid	Form I
Chloroform	X	✓					✓	Solid	Form I
Dimethyl sulfoxide	X	X	X	X	X	✓	✓	Solid	Form I
tert-Butylmethyl	X	X	X	X	X	X	n/a	n/a	Form I
ether
Anisole	X	X	X	X	X	X	n/a	n/a	Form I
Cumene	X	X	X	X	X	X	n/a	n/a	Form I
MEK	X	X	X	X	X	✓	✓	Solid	Form I
Isopropyl acetate	X	X	X	X	X	X	n/a	n/a	Form I
DMF	X	✓					✓	Solution
								after four
								weeks*
Toluene	X	X	X	X	X	X	n/a	n/a	Form I
Tetrahydrofuran	X	✓					✓	Solid	Form I
Dichloromethane	X	X	X	X	X	X	n/a	n/a	Form I
Acetonitrile	X	X	X	X	X	X	n/a	n/a	Form I
Nitromethane	X	X	X	X	X	X	n/a	n/a	Form I
Ethanol	X	X	X	X	X	X	n/a	n/a	Form I
DMA	X	✓					✓	Solution
								after four
								weeks*

✓ = Soluble X = Insoluble, n/a = not applicable.
Data can be found in Data Section 2.
*No further work was performed on the solution.

Form I was the only form obtained from the screen. Comparison spectra from all of the solvents screened are given in FIGS. 11 and 12, with the exception of DMF and DMA, from which no solid was found to precipitate. Chemical purity profiles of two of the samples isolated from EtOH and EtOAc experiments were recorded to check the material susceptibility to hydrolysis. The results were 97.4% and 96.8% respectively (reference to chemical purity of the starting material, 98.2%) as set forth in FIGS. 29 and 30.

Example 3—Characterization and Approximate Solubility of Sample 4

Prior to any testing, Sample 4 was characterized by XRPD, TGA, DSC, 1HNMR, and DVS. The results are given below in Table-8. The Corresponding figure numbers are given in the right hand column.

TABLE 8

Characterization of Sample 4 Prior to Testing.

	Analysis	Result

	XRPD	crystalline, pattern indexed,
		Form I
	TGA	168° C. (onset, decomp.)
		negligible weight loss prior
		to decomp.
	DSC	166° C. (exo, peak);
		175° C. (endo, peak)
	¹H NMR	consistent with chemical
		structure of MRZ
	DVS	−0.035 wt % (at 5% RH)
		+0.039 wt % (5-75% RH)
		+1.158 wt % (75-95% RH)
		−1.276 wt % (95-5% RH)

Approximate Solubility

Weighed samples of MRZ, Sample 4 were treated with aliquots of the test solvents or solvent mixtures at ambient temperature. Complete dissolution of the test material was determined by visual inspection. Solubility was estimated based on the total solvent volume used to provide complete dissolution. The actual solubility may be greater than the value calculated because of the use of solvent aliquots that were too large or due to a slow rate of dissolution. If complete dissolution was not achieved during the experiment, the solubility is expressed as “less than”. If complete dissolution was achieved by only one aliquot addition, the value is reported as “larger than”. The results are given in Table-9, below. As set forth in Table-9, the ratio of solvent mixtures are given by volume. Solubilities are estimated at ambient temperature and reported to the nearest mg/mL; if complete dissolution was not achieved, the value is reported as “<”; the actual solubility may be greater than the reported value due to the use of solvent aliquots that were too large or due to a slow rate of dissolution. Post-solubility samples were stored at 2-8° C. for 15-16 days followed by 1 day at ambient storage before visual inspection for precipitation. Post-solubility samples were visually inspected after 2-3 days of ambient storage. Formation system A is: 55 propylene glycol+5% ethanol+40% citrate buffer (10 mM; pH 5). Formation system B is: 14/86 (v/v) H₂O/tert-butyl alcohol containing 30 mg/mL sucrose, and titrated with HCl to pH 3.2±0.2.

TABLE 9

Approximate Solubility of Sample 4 Prior to Testing

	Solubility	Precipitates formed
Solvent	(mg/mL)	during storage?

Formulation System A	<1	N/A
Formulation System B	6	no
acetic acid	10	no
IPA	6	no
[EMIm][NTF₂]	1	N/A
[EMIm][Cl]	<2	N/A
ACN	10	no
tert-butanol	3	no
acetone	39	no
anisole	2	no
CHCl₃	2	yes
cyclohexanone	22	no
DMSO	>83	no
DCM	2	no
dioxane	20	no
EtOAc	15	no
Formic acid	15	no
MeOAc	20	no
MIBK	11	no
MEK	33	yes (small amount)
nitromethane	2	no
NMP	5	no
pyridine	36	no
THF	49	no
2-Me THF	32	no
1-propanol	2	no
2,2,2-trifluoroethanol	<1	N/A
50:50 acetone/heptane	12	no
50:50 acetone/iso-octane	12	no
25:75 ACN/MTBE	10	no
20:80 CHCl₃/DCM	3	no
25:75 DMF/anisole	17	no
50:50 EtOAc/cyclohexane	4	no
50:50 EtOAc/isooctane	2	no
20:80 MEK/IPA	10	no
20:80 MEK/MIBK	12	no
50:50 MeOH/Et₂O	21	no
20:80 NMP/IPA (c)	19	no
20:80 NMP/1-propanol	19	no
50:50 THF/hexanes	21	no
20:80 THF/nitromethane	15	no
20:80 2-MeTHF/MeOAc	34	no

Example 4—Polymorph Screen of Marizomib Sample 4 Evaporation Attempts

For slow evaporation (SE), solutions containing marizomib Sample 4 in selected solvents were allowed to evaporate at ambient temperature from vials covered with perforated aluminum foil. For fast evaporation (FE), solutions containing marizomib Sample 4 in selected solvents were allowed to evaporate at ambient temperature from open vials. The results are given below in Table-10. For flash evaporation, solids of disordered marizomib Sample 4 were added dropwise to pre-warmed glass vials, allowing the solvent to flash evaporate immediate upon contact. Resulting solids were collected for analysis. The corresponding figure numbers are given in the right hand column.

TABLE 10

Polymorph Screen of Marizomib Sample 4 Evaporation Attempts

		Observations/	XRPD
Solvent	Condition	Comments	Results

acetone	SE	white solids,	Form I
		dendritic particles
CHCl₃	SE	white solids,	Form I
		needles, agg.
IPA	SE	white solids,	Form I
		needles, B/E
THF	FE	white solids,	Form I
		dendritic particles
MEK	FE	white solids,	Form I
		columns, agg.
ACN	FE	white solids,	Form I
		needles, agg.
DCM	FE	white solids,	Form I
		columns, agg.

Example 5—Polymorph Screen of Marizomib Sample 4 Cooling Attempts

For crash cooling (CC), concentrated solutions of marizomib Sample 4 were prepared at elevated temperature and filtered through pre-warmed 0.2-μm nylon filters into a clean vial pre-chilled in a dry ice/acetone bath. For fast cooling (FC) and slow cooling (SC), solutions containing marizomib Sample 4 in selected solvents at elevated temperatures in sealed vials. Solutions were then filtered into clean vials with 0.2-μm nylon filters and removed from the heating plate and left at ambient conditions (FC) or allowed to cool to specified temperature on the heating plate (SC). The results are given below in Table-11. As set forth below, the ratio of solvent mixtures are given by volume. Temperature and time are approximate.

TABLE 11

Polymorph Screen of Marizomib Sample 4 Cooling Attempts

		Observations/	XRPD
Solvent	Condition	Comments	Results

ACN
	1. CC (65° C.	1. white solids,	Form I
	to −78° C.)	some columns,
		B/E
EtOAc
	1. CC (70° C.	1. clear solution	Form I
	to −78° C.)	2. white solids,
	2. kept at RT	small fibrous
	for 5 min	particles
THF/	1. CC (70° C.	1. clear solution	—
hexanes	to −78° C.)	2. white solids,
(50:50)	2. kept at RT	IS
	for 10 min
MeOAC
	1. CC (65° C.	1. clear solution	Form I
	to −78° C.)	2. white solids,
	2. kept at RT	fine particles,
	for 5 min	UM
MEK/IPA	1. CC (78° C.	1. clear solution	Form I +
(20:80)	to −78° C.)	2. white solids,	additional
	2. kept at RT	fine particles,	peaks
	for 5 min	UM, some B
NMP/	1. CC (78° C.	1. clear solution	—
1-PrOH	to −78° C.)	2. clear solution
(20:80)	2. kept at RT	3. white solids,
	for 5 min	UM small
	3. kept at −10	particles, IS
	to −25° C.
	for 7 d
MIBK
	1. SC	1. white solids,	Form I
	(70° C. to RT)	columns, agg.,
		B/E
cyclohexanone
	1. SC	1. clear solution	Form I
	(70° C. to RT)	2. clear solution
	2. kept at 2-8° C.	3. clear solution
	for 1 d	4. cloudy solution,
	3. kept at −10	white solids,
	to −25° C.	fibrous needles
	for 20 d
	4. cyclohexane
	added, kept
	at 2-8 ° C.
	for 2 d
EtOAc/	1. SC	1. clear solution	Form I
isooctane	(70° C. to RT)	2. clear solution
(50:50)	2. kept at 2-8° C.	3. needles, B/E
	for 1 d
	3. kept at −10
	to −25° C.
	for 6 d
THF/	1. SC	1. white solids,	Form I
nitromethane	(65° C. to RT)	columns, B/E
(20:80)
[EMIm][NTF₂]	1. SC from 77° C.	1. clear solution	Form I
	to RT	2. white solids,
	2. kept at RT	fine particles,
	for 11 d	UM, agg., B
[EMIm][Cl]	1. FC (105° C.	1. orange solution	unique
	to 81° C.) (c)	2. off-white solids,	pattern +
	2. hot H₂O	fine	minor
	added	particles, UM, B	Form I
Formulation
	1. SC from 77° C.	1. white solids,	Form I
System A	to RT	fibrous agg.
Formulation	1. CC (105° C.	1. sample frozen	Form I
System A	to −78° C.)	2. white solids,
	2. kept at 2-8° C.	small particles,
	for 6 d	agg., UM
Formulation
	1. SC (85° C.	1. white fibrous	Form I
System B	to RT)	solids

Example 6—Polymorph Screen of Marizomib Sample 4 Solvent/Antisolvent Addition Attempts

For solvent/antisolvent (SAS) experiments, concentrated solutions of marizomib were prepared in selected solvents and solutions were filtered through 0.2-μm nylon filters, either into antisolvent at ambient conditions, or into clean vials and antisolvents were subsequently added into the solutions. In the case of crash precipitation (CP), concentrated solutions of marizomib were prepared in selected solvents and filtered through 0.2-μm nylon filters and into anti-solvent precooled in an ice/water bath. The results are given below in Table-12. The ratio of solvent mixtures are by volume. Time and temperature are approximate.

TABLE 12

Polymorph Screen of Marizomib Sample 4
Solvent/Antisolvent Addition Attempts

		Observations/	XRPD
Solvent	Condition	Comments	Results

pyridine/	1. CP (solution in	1. no solids	—
Et₂O	pyridine added to
(5:7)	cold Et₂O)
acetone/	1. CP (solution in	1. white solids,	Form I
isooctane	acetone added to	some fibrous
(1:3)	cold isooctane)	particles,
		some B
EtOAc/	1. CP (solution in	1. white solids,	Form I
cyclohexane	EtOAc added to	agg., anh.
(1:2)	cold cyclohexane)	particles
acetone/	1. CP (solution in	1. white solids,	Form I
heptane	acetone added to	agg., anh.
(1:1)	cold heptane)	particles
formic
	1. cold CHCl ₃	1. rose colored	Form I +
acid/CHCl₃	added into sample	solution, clear	additional
(1:1)	solution of	2. no solids	peaks
	6882-21-08	3. sample frozen
	2. kept at 2-8° C.	4. rose colored
	1 day	solution
	3. kept at −10	5. off white
	to −25° C.	solids, agg.,
	overnight	small particles,
	4. kept at 2-8° C.	UM
	5. evaporation at RT,
	purged w/N₂
	after 3 d
MEK/	1. CP (solution in	1. white solids,	Form I
IPE (1:5)	MEK added to	small fibrous
	cold IPE),	particles, UM, B
	manually shaken
THF/	1. CP (solution in	1. white solids,	Form I
hexanes	THF added to	small particles &
(1:5)	cold hexanes),	agg., UM
	manually shaken
ACN/heptane	1. CP (solution in	1. clear	Form I
(3:10)	ACN added to	2. clear solution
	cold heptane),	3. clear solution
	shaken	4. white solids,
	2. shaken at 5° C.	agg., columns,
	on orbital shaker	B/E solution
	for 6 d
	3. shaken at −4
	to 2° C. on
	orbital shaker
	for 3 d
	4. evaporation
	under N₂
acetone/	1. solids	1. clear solution	Form I
Formulation	dissolved in	2. mostly clear
System A (1/2)	acetone	solution, small
	2. solution	particulates
	filtered into	3. white solids,
	Formulation	fine particles,
	System A, shaken	needles, agg.
	3. stored at
	2-8° C. for 1 d
2-Me	1. CP (solution in	1. clear solution	Form I
THF/MTBE	2-Me THF added to	2. clear solution
(1:5)	cold MTBE), shaken	3. clear solution
	2. shaken at 5° C.	4. white solids,
	on orbital shaker	needles, agg.,
	for 6 d	B/E
	3. shaken at −4
	to 2° C. on
	orbital shaker
	for 3 d
	4. evaporation
	under N2
MeOH/	1. CP (solution in	1. clear solution	Form I
Et₂O (1:5)	MeOH added to cold	2. clear solution
	Et₂O), shaken	3. clear solution
	2. shaken at 5° C.	4. white solids,
	on orbital shaker	fibrous needles
	for 6 d
	3. shaken at −4
	to 2° C. on
	orbital shaker
	for 3 d
	4. evaporation
	under N₂
Formulation	1. CP (solution in	1. white solids,	—
System B/	Formulation System	UM, IS
nitromethane	B added to cold
(1:1.5)	nitromethane),
	shaken

Example 7—Polymorph Screen of Marizomib Sample 4 Slurry Attempts

Sufficient amounts of solids of marizomib Sample 4 were added to selected solvents in vials so that excess solids initially persisted. The mixture was then triturated with a stir bar at specified temperatures for an extended period of time. The results are given below in Table-13. The ratio of solvent mixtures are by volume. Time and temperature are approximate.

TABLE 13

Polymorph Screen of Marizomib Sample 4 Slurry Attempts

		Observations/	XRPD
Solvent	Condition	Comments	Results

formic acid	RT, 17 d	all solids dissolved,	—
		solution rose color
acetone/heptane	RT, 7 d	—	Form I
(50:50)
NMP	RT, 7 d	white solids, small	Form I
		particles, UM, B
1-propanol	50° C., 7 d	white solids, pasty,	Form I
		B, UM
anisole	50° C., 7 d	white solids, UM	Form I
nitromethane	50° C., 7 d	white solids, small	Form I
		particles, UM
2-Me THF/MeOAc	RT, 7 d	white solids, some	Form I
(20:80)		columns, B
CHCl₃	RT, 8 d	white solids, fine	Form I
		particles, agg.,
		UM, B
Formulation	RT, 13 d	white solids, agg.,	Form I
System A		UM
Formulation	RT, 7 d	white solids, fine	Form I
System B		particles, UM
acetic acid	RT, 7 d	white solids,	Form I
		agglomerates, B
acetone/H₂O (40:60)	RT, 7 d	white solids, small	Form I
(A_w= 0.92)		particles, UM, B
ACN/H₂O	RT, 7 d	white solids, small	Form I
(50:50)		particles, UM, B
(A_w= 0.90)
IPA/H₂O	RT, 7 d	white solids, small	Form I
(50:50)		particles, UM, B
(A_w= 0.96)
2-BuOH/H₂O	RT, 7 d	white solids, small	Form I
(70:30)		particles, UM, B
(A_w= 1.04)
HFIPA/H₂O	RT, 7 d	white solids, small	Form I
(75:25)		particles, UM, B
(A_w= 1.41)
TFE/H₂O	RT, 7 d	white solids, small	Form I
(50:50)		particles, UM, B
(A_w= 1.08)

Example 8—Polymorph Screen of Marizomib Sample 4 Manual Grinding Attempts

Solids of marizomib Sample 4 were ground by hand at room temperature using a mortar and pestle at five minutes per cycle for two (wet grinding) or four (dry grinding) cycles. In the case of wet grinding, a small amount of select solvent or solvent mixture (10 μL) was added before grinding. The inside of the mortar was scraped between each cycle. The results are given below in Table-14. As set forth below, the ratio of solvent mixtures are given by volume. Times are approximate. Disordered Form I was used as the starting material for the water recrystallization experiment.

TABLE 14

Polymorph Screen of Marizomib Sample
4 Manual Grinding Attempts

		Observations/	XRPD
Solvent	Condition	Comments	Results

—	4 × 5 min	white solids,	disordered
		UM, agg.	Form I
Formulation	2 × 5 min;	white tacky	Form I
System A	2 × 10 μL	solids
		agglomerates, B
Formulation
	2 × 5 min;	white solids,	disordered
System B	2 × 10 μL	fine particles,	Form I
		agg., UM, B
acetone/heptane	2 × 5 min;	white solids,	disordered
(50/50)	2 × 10 μL	fine particles,	Form I
		agg., UM, B
formic acid	2 × 5 min;	white solids,	disordered
	2 × 10 μL	fine particles,	Form I
		agg., UM, B
acetic acid	2 × 5 min;	white solids,	disordered
	2 × 10 μL	fine particles,	Form I
		agg., UM, B
water
	2 × 5 min;	white solids,	disordered
	2 × 10 μL	small particles,	Form I
		agg., UM, B

Example 9—Polymorph Screen of Marizomib Sample 4 Heat Stress Attempts from Disordered Form I

Solids of disordered marizomib Sample 4 were held at designated temperatures for the indicated amount of time. The results are given below in Table-15. Resulting solids were collected for analysis. Temperature and time are approximate.

TABLE 15

Polymorph Screen of Marizomib Sample 4 Heat
Stress Attempts from Disordered Form I

	Observations/
Condition	Comments	XRPD Results

44° C.,	white solids,	Form I w/
overnight	fluffy, agg., UM	some disorder
60° C., 5 h	white solids,	Form I w/
	fluffy, agg., UM	some disorder
80° C., 2 h	white solids,	Form I w/
	fluffy, agg., UM	some disorder

Example 10—Experimental Attempts Targeting Amorphous Marizomib

For lyophilization, a solution of MRZ was prepared in either dioxane or Formulation System B, filtered through 0.2-μm nylon filter, and frozen in a glass flask by rotating it in a cold dry ice/IPA bath. The flask was then purged with N2, submerged in a propylene glycol bath at temperatures below −25° C., and placed under vacuum of 0.028 mm Hg to dry for 2 to 5 days. The resulting solids were collected for analysis.
For fast evaporation (FE), solutions containing MRZ in selected solvents were allowed to evaporate at ambient temperature from open vials. The results are shown in Table-16 below. The ratio of solvent mixtures are by volume. Time and temperature are approximate. The acetone used was anhydrous.

TABLE 16

Experimental Attempts Targeting Amorphous Marizomib

		Observations/	XRPD
Solvent	Condition	Comments	Results

acetone	flash evaporation	white solids,	Form I +
	at 110° C.	some yellowing,	additional
		agg., UM	peaks
acetone	flash evaporation	white solids,	Form I
	at 90° C.	needles, agg.,
		B/E
dioxane	lyophilization	static white	Form I
		powder, UM
dioxane	lyophilization	white powder,	disordered
		static, clumps	Form I
dioxane	lyophilization	white powder,	disordered
		flakey, fluffy	Form I
Formulation	lyophilization	clear, gooey,	disordered
System B		sticky	Form I

Example 11—Further Analysis on Selected Samples from Polymorph Screen

TABLE 17

Analysis of on Selected Samples from Polymorph Screen

Condition	Analysis	Result

flash evaporation	¹H NMR	peaks attributable to MRZ
from acetone		observed + unidentified
(110° C.)		additional peaks with
		substantial integral
		intensities
crash cool from	¹H NMR	spectrum in general
MEK/IPA		consistent with chemical
(78 to −78° C.)		structure of MRZ +
		small additional
		unidentified peaks
lyophilization	mDSC	161° C. (endo, onset)
FC from [EMIm][Cl]	¹H NMR	spectrum not consistent
followed by addition		with chemical structure
of hot H₂O		of MRZ
evaporation from	¹H NMR	peaks attributable to
formic acid/CHCl₃		MRZ observed +
		unidentified additional
		peaks, some peaks with
		substantial integral
		intensities

Example 12—Co-crystal Screen of Marizomib

For crash cooling (CC), a solution of MRZ and coformer was prepared at elevated temperature in a given solvent mixture. The vial was capped and immediately placed in a refrigerator or freezer. For fast cooling (FC), solutions of MRZ and coformer were prepared at elevated temperatures in given solvents or solvent mixtures. The vial was capped and placed on a bench top at room temperature to quickly cool. For slow cooling (SC), a solution of MRZ and coformer was prepared at elevated temperature in a given solvent mixture. The vial was capped and left in a heating block at elevated temperature. The heater then was turned off for the sample to cool down naturally to room temperature. For slow evaporation (SE), solutions of MRZ and coformer were generated at ambient temperature in a given solvent or solvent mixture. The solutions were allowed to evaporate partially or to dryness from a loosely capped vial at ambient conditions. Slurry experiments were carried out by making saturated solutions containing excess solid. The slurries were agitated at ambient or elevated temperatures for a specified amount of time. The solids present were recovered via positive pressure filtration. Solvent assisted grinding experiments were carried out by mixing MRZ and coformer in an agate mortar and pestle. Aliquots of solvent were added and the mixture ground manually for specified amount of time. The solids were scraped from the walls of the mortar and the pestle head. Another aliquot of solvent added and ground for several more minutes. For temperature cycling, solutions or suspensions of MRZ and coformer were made in a given solvent and placed at elevated temperature. The sample was cycled by removing from heat and then reapplying several times. The solids were collected via vacuum filtration upon the last cool from elevated temperature.
As set forth in Table 18 below, “X:Y” refers to marizomib:conformer mole ratio. Temperatures and times are approximate. Solvent ratios are by volume. Approximately 60-90 mg of marizomib was used for each experiment.

TABLE 18

Cocrystal Screen of Marizomib

Coformer			XRPD
(X:Y)	Conditions	Observations	Results

Acetic acid	Slurry, RT	White solids	MRZ
(large excess)		PLM: agglomerates, B
Acetic acid	1) Acetone, stir, RT, 5	1) Slightly turbid solution	MRZ
(1:2)	Days	2) Faint hazy solution
	2) Cool, 2-8° C., 3 days	3) Translucent solids
	3) SE, RT	PLM: agglomerates, prismatics, B
Adenine	EtOH, slurry, RT, 5 days	White solids	MRZ
(1:1)		PLM: Small particles, unknown
		morphology, B
Adenine	1) Add MeOH to	1) White suspension	MRZ +
(1:3)	Adenine	2) White suspension	minor
	2) Add MIBK to MRZ	3) White suspension	adenine
	3) Add adenine	4) White suspension
	suspension to MRZ	5) White solids
	4) Slurry at 60° C. O/N	PLM: small particles, unknown
	5) Slurry, RT, 1 day	morphology
L-alanine	1) Dissolve L-alanine in	1) Clear, colorless solution	—
(1.1)	H₂O	2) Clear, colorless solution
	2) Dissolve MRZ in	3) White gel
	pyridine	4) White suspension insufficient
	3) Add L-alanine to	for XRPD
	MRZ	5) White suspension, insufficient
	4) Slurry, RT, 2 days	for XRPD
	5) Cool to 2-8° C.	6) Clear, tacky gel
	6) SE, RT
L-alanine	1) Add MeOH to L-	1) Solids remain	MRZ
(1:1)	alanine	2) White suspension
	2) Add suspension to	3) White suspension
	MRZ	4) White solids
	3) Slurry at 60° C. O/N	PLM: small particles, unknown
	4) Slurry, RT, 1 da	morphology
L-arginine	1) Dissolved L-arginine	1) Clear, colorless solution	MRZ
(1:1)	in HFIPA	2) Clear, colorless solution
	2) Dissolved MRZ in	3) Turbid solution
	dioxane	4) White solids
	3) Added L-arginine	PLM: small particles, unknown
	solution dropwise to	morphology, B.
	MRZ solution
	4) Stir, RT, ~25 minutes
	SE (FIG. 129 filtrate	Yellow gel	—
	material), RT
L-ascorbic acid	MIBK, FC, 75° C. to RT	White solids	MRZ + L-
(2:1)			ascorbic
			acid
L-ascorbic acid	1) IP A added to MRZ at	1) Scant solids remain	MRZ
(1:1)	50° C.	2) Clear, colorless solution
	2) L-ascorbic acid	3) Clear, colorless solution
	dissolved in MeOH	4) White solids
	3) L-ascorbic acid added	PLM: small particles, unknown
	dropwise to MRZ at	morphology, B.
	50° C.
	4) FC to RT
L-ascorbic acid	ACN, slurry, RT, 7 days	White solids	MRZ + L-
(1.5)		PLM: small particles, unknown	ascorbic
		morphology, B	acid
Benzoic acid	EtOAc, SE, RT	Translucent solids	MRZ
(2:1)		PLM: needles, agglomerates, B/E
		possible singles
Benzoic acid	1) Dissolve benzoic acid	1) Clear, colorless solution	MRZ +
(1:1)	in EtOH	2) Clear, colorless solution	benzoic acid
	2) Dissolve MRZ in	3) Clear, colorless solution
	acetone	4) Clear, colorless solution
	3) Add benzoic acid to	5)Translucent solids
	MRZ	PLM: agglomerates of needles, B
	4) Stir, RT, 2 days
	5) SE at RT
Benzoic acid	MEK:MIBK (20:80),	White solids	MRZ
(1:5)	slurry, RT, 7 days	PLM: small particles, unknown
		morphology, B.
Caffeine	Dioxane, SC, 50° C. to	White solids	MRZ +
(1:1)	RT		caffeine
Caffeine	1) Add THF to caffeine	1) White suspension	Caffeine +
(1:2)	2) Add caffeine	2) White suspension	minor
	suspension to MRZ	3) White solids	MRZ
	3) Slurry, 50° C., 2 days	PLM: small particles,
		agglomerates, unknown
		morphology, B.
Caffeine	DMF:anisole (25:75),	White solids,	Caffeine
(1:3)	slurry, RT, 6 days	PLM: small particles, unknown
		morphology, B
Trans-cinnamic	1) Dissolve trans-	1) Clear, colorless solution	MRZ +
acid (1:1)	cinnamic acid in ACN	2) Clear, colorless solution	trans-
	2) Dissolve MRZ in	3) Clear, colorless solution	Cinnamic
	MEK	4) Clear, colorless solution	acid
	3) Add trans-cinnamic	5) Scant translucent solids
	acid solution to MRZ	PLM: Rods, B/E
	dropwise	6) White solids
	4) Stir, RT, 3 days	PLM: prisms and plates, B
	5) Cool to 2-8° C.
	6) SE, RT
Citric acid	NMP, crash cool, 50° C.	Clear, colorless solution	—
(1:1)	to 2-8° C.
Citric acid	Solvent assisted	White solids	MRZ +
(1:2)	grinding, IPA, (3 × 5		citric acid
	minutes, 3 × 10 μL)
	1) EtOAc, slurry, 50° C.,	1) Scant white solids	MRZ +
	5 days	2) White solids	citric acid
	2) Slurry, RT, 6 days	PLM: small particles, unknown
		morphology, B
Citric acid	MIBK, slurry, RT, 6	White solids,	MRZ +
(1:3)	days	PLM: small particles, unknown	citric acid
		morphology, B
(2-	1) Dissolve (2-	1) Clear, colorless solution	MRZ
hydroxypropyl)-	hydroxy propyl)-β-	2) Clear, colorless solution
β -cyclodextrin	cyclodextrin in MeOH	3) White precipitation
(1:1)	2) Dissolve MRZ in	4) White suspension (gel)
	acetone	5) Clear solution with white gel at
	3) Add conformer	bottom
	solution to MRZ	5) Clear solution with clear gel at
	4) Slurry at RT	bottom
	5) After ~20 minutes,	6) White solids
	place at 50° C.	PLM: needles, spherulites, B
	5) Slurry, 50° C., 2 days
	6) Supernatant solution
	decanted, SE, RT
(2-	1) Dissolve (2-	1) Clear, colorless solution	MRZ
hydroxypropyl)-	hydroxypropyl)-β-	2) Clear, colorless solution
β-cyclodextrin	cyclodextrin in H₂O	3) White suspension
(2:1)	2) Dissolve MRZ in	4) Turbid suspension
	ACN at 50° C.	5) Turbid solution
	3) Add H₂O solution to	6) Translucent solids
	ACN solution at 50° C.	PLM: needles, B/E
	4) Slurry, 50° C., 2 days
	5) Slurry, RT, 6 days
	6) SE, RT
L-cysteine	1) Grind L-cysteine and	1) White solids	MRZ
(1:1)	MRZ ~10 minutes	2) White suspension
	2) Add EtOH/dioxane	3) White suspension
	(50/50)	4) White solids
	3) Sonicated ~2 minutes	PLM: small particles, unknown
	4) Temperature cycle	morphology, B
	between RT and 50° C.
	(4 cycles × 1 hour each
	temperature, held at RT
	overnight)
L-cysteine	1) Add H₂O to L-	1) Slightly turbid solution	Highly
(1:2)	cysteine at 50° C.	2) Clear, colorless solution	disordered
	2) Dissolve MRZ in	3) Immediate white precipitation	crystalline
	ACN at 60° C.	4) Turbid, faintly yellow
	3) L-cysteine solution	suspension
	added to ACN solution	5) Turbid solution
	4) Slurry, 50° C., 2 days	6) Off-white solids
	5) Slurry, RT, 4 days	PLM: Glassy, no apparent B
	6) SE, RT
Cytosine	1) Add 0.5 mL H₂O to	1) White suspension	MRZ +
(1:1)	cytosine, slurry at 50° C.	2) White suspension	unique
	2) Add 0.5 mL ACN to	3) White suspension	peaks +
	MRZ, slurry at 50° C.	4) Faintly yellow solution with	possible
	3) Sonicate 2 minutes	white solids	minor
	4) Add cytosine	5) White solids	cytosine
	suspension to MRZ,	PLM: small particles, unknown
	slurry at 50° C., 2 days	morphology, B
	5) Slurry, RT, 6 days
Cytosine	1) 1.5 mL H₂O added to	1) White suspension	Unique
(1:3)	cytosine	2) Scant solids remain	peaks +
	2) 1 mL dioxane added	3) White suspension	MRZ
	MRZ	4) Light yellow, clear solution	(same as
	3) Cytosine suspension	5) Cream colored solids	observed in
	added to MRZ (dioxane:	PLM: small particles, unknown	FIG. 233)
	water 2:3)	Morphology	(XRPD)
	4) Slurry at 60° C. O/N		Primarily
	5) Stir, RT, 1 day		cytosine
			with
			dioxane
			and trace
			MRZ
			additional
			peaks also
			present
			(NMR)
Fumaric acid	Anh. acetone, SE, RT	Translucent solids	MRZ +
(1:1)		PLM: needles, dendritics, B/E	fumaric
			acid +
			additional
			peak
Fumaric acid	Solvent assisted	White solids	MRZ +
(1:1)	grinding, ACN (2 × 5		fumaric
	minutes, 2 × 20 μL)		acid
Fumaric acid	1) Added TFE to	1) White suspension	MRZ +
(1:3)	fumaric acid at 50° C.	2) Clear, colorless solution	fumaric
	2) Dissolved MRZ in	3) White suspension	acid
	THF at 50° C.	4) White solids
	3) Added fumaric acid to	PLM: small particles, unknown
	MRZ solution	morphology, B
	4) Temp, cycle (50° C. -
	RT) three cycles hold 4
	hours
Gentisic acid	1) Dissolve gentisic acid	1) Clear, colorless solution	MRZ
(1:1)	in t-BuOH at 50° C.	2) White suspension
	2) Add to MRZ at 50° C.	3) White solids
	3) Slurry, 50° C., 2 days	PLM: fibrous and small particles
		with unknown morphology, B
Gentisic acid	ACN, FC, 60° C. to RT	White solids	MRZ
(2:1)		PLM: agglomerates, fines, B.
Gentisic acid	MEK:IPA (20:80),	White solids,	MRZ
(1:5)	slurry, RT, 6 days	PLM: small particles, unknown
		morphology, B
L-glutamine	Anh. acetone, grinding	White solids	MRZ + L-
(1:1)	(2 × 5 minutes, 2 × 20		glutamine
	μL)
L-glutamine	1) Dissolve L-glutamine	1) Clear, colorless solution	MRZ +
(1:3)	in H₂O at 50° C.	2) Clear, colorless solution	minor L-
	2) Dissolve MRZ in 2-	3) Two clear, colorless layers	glutamine
	MeTHF at 50° C.	4) White solids
	3) Add L-glutamine to	PLM: fibrous, B
	MRZ dropwise at 50° C.
	4) FC/FE at RT
L-glutamine	ACN:MTBE (25:75),	White solids,	MRZ + L-
(1:5)	slurry, RT, 6 days	PLM: small particles, unknown	glutamine
		morphology, B
Glutaric acid	1) Dissolve glutaric acid	1) Clear, colorless solution	MRZ +
(1:1)	in IPrOAc	2) Clear, colorless solution	Glutaric
	2) Dissolve MRZ in	3) Clear, colorless solution	acid
	ACN at 50° C.	4) Clear, colorless solution
	3) Add glutaric acid	5) Clear, colorless solution
	solution dropwise	6) Clear, colorless solution
	4) Stir, 50° C., 2 days	7) Translucent solids
	5) FC to RT	PLM: dendrites, needles, B
	6) Cool to 2-8° C.
	7) SE, RT
Glutaric arid	1) Dissolve glutaric acid	1) Clear, colorless solution	MRZ
(1:3)	in EtOH	2) White suspension
	2) Add to MRZ	3) White suspension
	3) Slurry, RT, 6 days	PLM: small particles, unknown
		morphology, B
Glycine	ACN, grinding (2 × 5	White solids	MRZ +
(2:1)	minutes, 2 × 20 μL)		glycine
Glycine	1) Dissolve glycine in	1) Clear, colorless solution	MRZ +
(1:1)	H₂O	2) Clear, colorless solution	glycine
	2) Dissolve MRZ in	3) White precipitate
	acetone	4) White solids
	3) Add glycine to MRZ	PLM: small particles, unknown
	dropwise	morphology, B
	4) Slurry, RT, 2 days
Glycine	EtOAc:cyclohexane	White solids,	MRZ +
(1:5)	(50:50), slurry, RT, 5	PLM: small particles, unknown	glycine
	days	morphology, B
Glycolic acid	1) Melt glycolic acid at	1) Clear melt	MRZ +
(1:1)	85° C.	2) White solids	minor
	2) Add MRZ	3) Clear, colorless solution	glycolic
	3) Dissolve in THF at	4) White solids	acid
	85° C.	PLM: small needles, B.
	4) Evaporate off solvent
	at 85° C.
Glycolic acid	Acetone, SE, RT	White solids	MRZ
(1:2)		PLM: Needles, B.
Hippuric acid	1) MEK, FC, 65° C. to	1) Scant solids	Hippuric
(1:1)	RT	2) White solids	acid +
	2) Cool to 2-8° C.	PLM: equants, B.	MRZ peak
Hippuric acid	1) Add IPA/HFIPA (1:1)	1) White suspension	MRZ +
(1:5)	to Hippuric acid	2) White suspension	hippuric
	2) Add suspension to	3) White suspension	acid
	MRZ	4) White solids
	3) Heat to 50° C.	PLM: small particles, unknown
	4) Slurry, 50° C., 6 days	morphology, B
Hydantoin	1) Add dioxane	1) Scant solids remain	MRZ
(1:1)	2) Add H₂O (2:1	2) White precipitation
	dioxane:H₂O)	3) White solids
	3) Slurry, RT	PLM: small particles, unknown
		morphology, B.
Hydantoin	1) H₂O added to	1) White suspension	MRZ
(1:3)	hydantoin	2) Clear, colorless solution
	2) ACN added to MRZ	3) White suspension
	3) Hydantoin suspension	4) Clear, colorless solution
	added to MRZ	5) White solids
	4) Slurry, 60° C.	PLM: tangled fibers, B.
	overnight
	5) Stir, RT, 1 day
4-	1) Dissolve 4-	1) Clear, colorless solution	MRZ + 4-
hydroxybenzoic	hydroxybenzoic acid in	2) White suspension	hydroxy
acid (1:1)	anh. acetone	3) White suspension	benzoic
	2) Add anh. acetone to	4) White suspension, insufficient	acid
	MRZ	for analysis
	3) Add 4-	5) White solids
	hydroxybenzoic acid	PLM: needles and dendrites, B
	solution to MRZ
	4) Stir, RT
	5) SE, RT
4-	1) 4-hydroxybenzoic	1) Clear, colorless solution	MRZ + 4-
hydroxybenzoic	acid dissolved in EtOH	2) Clear, colorless solution	hydroxy
acid (1:1)	2) MRZ disslolved in	3) Clear, colorless solution	benzoic
	dioxane	4) Clear, colorless solution	acid
	3) EtOH solution added	5) White solids
	to MRZ	PLM: dendrites, needles, B/E.
	4) Stir, RT, 1 day
	5) FE, RT
Imidazole	Pyridine, SC, 45° C. to	Clear, golden yellow solution	—
(1:1)	RT
Imidazole	1) Dissolve imidazole in	1) Clear, colorless solution	—
(1:2)	acetone	2) Clear, colorless solution
	2) Add to MRZ	3) Yellow tacky gel
	3) SE, RT
Imidazole	1) MeOAc, slurry, RT, 2	1) Clear, light yellow solution	—
(1:5)	days	2) Golden oil
	2) SE, RT
L-lysine	1) Dissolve L-lysine in	1) Yellow solution	MRZ
(1:1)	HFIPA	2) Clear, colorless solution
	2) Dissolve MRZ in	3) Beige suspension
	THF	4) White solids
	3) Add L-lysine solution	PLM: small particles, unknown
	dropwise to MRZ	morphology, B.
	4) Stir, RT, ~35 minutes
	SE (FIG. 171 material	Orange gel	—
	filtrate), RT
L-lysine	1) L-lysine dissolved in	1) Clear, colorless solution	—
(1:3)	H₂O	2) Scant solids remain
	2) ACN added to MRZ	3) White suspension
	3) L-lysine solution	4) Orange solution
	added to MRZ	5) Orange solution
	4) Slurry, 60° C. O/N
	5) Stir, RT, 1 day
Maleic acid	Nitromethane, SC, 50° C.	White solids	MRZ
(1:1)	to RT
Maleic acid	1) Added 1-PrOH to	1) Clear, colorless solution	MRZ
(1:3)	maleic acid	2) White suspension
	2) Added to MRZ	3) White solids
	3) Slurry, RT, 6 days	PLM: small particles, unknown
		morphology, B
Maleic acid	Acetone:heptane	White solids	MRZ +
(1:5)	(50:50), slurry, RT, 7	PLM: small agglomerates,	maleic acid
	days	unknown morphology, B.
DL-malic acid	1) Add DCM to DL-	1) White suspension	MR 7 +
(1:1)	malic acid	2) Clear, colorless solution	DL-malic
	2) Dissolve MRZ in	3) White suspension	acid
	THF	4) White solids
	3) Add MRZ solution to	PLM: small particles, unknown
	DL-malic acid	morphology, B.
	suspension
	4) Slurry at RT, 7 days
DL-malic acid	1) Dioxane added to	1) Solids remain	MRZ
(1:3)	DL-malic acid	2) White suspension
	2) Transferred to MRZ	3) Clear, colorless solution
	3) Slurry, 60° C., O/N	4) Clear, colorless solution
	4) Stir, RT, 1 day	5) White wet solids
	5) FE, RT	PLM: small needles, B.
Melamine	1) Add 1 mL DMSO:	1) White suspension	MRZ +
(1:1)	H₂O (1:1) to MRZ. Add	2) White suspension	unique
	melamine.	3) White suspension	peaks
	2) Sonicate ~2 minutes	4) White solids	(XRPD)
	3) Slurry at 50° C., 2	PLM: white solids, unknown	MRZ:
	days	morphology, B.	Melamine
	4) Slurry, RT, 6 days		1:1.6,
			DMSO and
			H₂O
			present,
			numerous
			additional
			peaks
			suggestive
			of some
			level of
			degradation
			(NMR)
			Broad
			endotherm
			at 97.5°
			C. (peak
			max), sharp
			endotherms
			at 107.9° C.
			and 124.1°
			C. (peak
			max) (DSC)
			10.3%
			weight loss
			from 30.2°
			C. to 104.0°
			C. 21.1%
			weight loss
			from 30.2°
			C. to 146.0°
			C. (TGA)
Melamine	1) Add 1 mL DMSO:	1) White suspension	MRZ +
(1:3)	H₂O (1:1) melamine	2) White suspension	melamine +
	2) Added melamine	3) White suspension	unique
	suspension to MRZ	4) White solids	peaks
	3) Slurry at 60° C. O/N	PLM: small particles, unknown
	4) Slurry, RT, 1 day	morphology
Nicotinamide	ACN, FC, 70° C. to RT	White damp solids	MRZ
(1:1)
Nicotinamide	1) Add acetone to	1) White suspension	Nicotin-
(1:3)	nicotinamide	2) White suspension	amide +
	2) Add nicotinamide	3) White solids	MRZ
	suspension to MRZ	PLM: agglomerates, B
	3) Slurry, RT, 6 days
Nicotinamide	MEK:IPA (20:80),	White solids	MRZ +
(1:5)	slurry, RT, 7 days	PLM: small particles, unknown	nicotin-
		morphology, B	amide
Orotic acid	1) Nitromethane added	1) White suspension	MRZ
(2:1)	to orotic acid and 50° C.	2) White suspension
	2) Add to MRZ at 50° C.	3) White solids
	3) Slurry, 50° C., 6 days	PLM: small particles, unknown
		morphology, B
Orotic acid	MEK, grinding (2 × 5	White solids	MRZ +
(1:1)	minutes manual		minor
	grinding)		orotic acid
Orotic acid	MeOAc, slurry, RT, 7	White solids	MRZ +
(1:5)	days	PLM: small particles, unknown	orotic acid
		morphology, B.
Oxalic acid	Anh. acetone, FE, RT	Translucent solids	MRZ +
(1:1)		PLM: dendritics, needles, B.	oxalic acid
Oxalic acid	1) Oxalic acid dissolved	1) Clear, colorless solution	MRZ
(1:2)	in EtOH	2) Clear, colorless solution
	2) MRZ dissolved in	3) Clear, colorless solution
	MEK	4) White solids
	3) Add oxalic acid	PLM: small particles, unknown
	solution to MRZ	morphology, B
	4) Stir, RT, 7 days
Oxalic acid	1) Dissolved MRZ in	1) Clear, colorless solution	MRZ +
(1:5)	anhydrous acetone	2) Clear, colorless solution	oxalic acid
	2) Dissolved oxalic acid	3) Clear, colorless solution
	in EtOH	4) Slightly turbid solution
	3) Added oxalic acid	5) Translucent off-white solids
	solution dropwise to	PLM: needles, prismatics, B/E
	MRZ solution
	4) Stir, RT, 5 days
	5) SE at RT
L-proline	Nitromethane,	White solids	MRZ + L-
(2:1)	temperature cycle		proline
	between 50° C. and RT
	(3 cycles, 1 hour each)
L-proline	1) Dissolved MRZ in	1) Clear, colorless solution	L-proline +
(1:1)	THF	2) Clear, colorless solution	minor
	2) Dissolved L-proline	3) Clear colorless solution	additional
	in MeOH	4) White solids	peaks
	3) Added L-proline	PLM: small particles, unknown	(possibly
	solution dropwise to	morphology, B.	MRZ)
	MRZ
	4) Stir, RT, 4 days
	SE of FIG. 190 material	White solids	MRZ +
	filtrate, RT	PLM: fibrous agglomerates, B.	additional
			peaks
L-proline	1) Added EtOH to L-	1) White suspension	L-proline +
(1:2)	proline at 50° C	2) White suspension	MRZ
	2) Added EtOAc to	3) White suspension
	MRZ at 50° C.	4) White solids
	3) Added EtOH	PLM: small particles, unknown
	suspension to MRZ	morphology, B
	4) Temp cycle (50° C. to
	RT, five cycles, 1 hour
	each)
L-proline	Acetone:iso-octane	White solids,	MRZ + L-
(1:3)	(50:50), slurry, RT, 6	PLM: small particles, unknown	proline +
	days	morphology, B	additional
			peaks
			(possibly
			L-proline
			mono-
			hydrate)
L-proline	Add sat'd L-proline in	White solids	MRZ
(sat'd in IPA at	IPA to MRZ at 50° C.	PLM: needles, B/E
50° C.)	temp cycle (50° C.-RT)
L-proline sat'd	1) Add L-proline to	1) White suspension	L-proline
in H₂O/MeOH	H₂O/MeOH at 50° C.	2) Clear, colorless solution
50/50 v/v	overnight	3) White suspension
	2) Hot filter saturated L-	4) Yellow solution, scant solids
	proline solution	5) Viscous yellow suspension
	3) Add saturated	PLM: unknown morphology, B.
	solution to MRZ at
	50° C.
	4) Slurry, 50° C., 2 days
	5) Slurry, RT, 3 days
L-pyroglutamic	1) Dissolve	1) Clear, colorless solution	MRZ +
acid (1:1)	pyroglutamic acid in	2) Clear, colorless solution	Minor
	EtOH	3) Clear, colorless solution	pyro-
	2) Dissolve MRZ in	4) Clear, colorless solution	glutamic
	acetone	5) Translucent solids	acid
	3) Add pyroglutamic	PLM: dendrites, B/E
	acid to MRZ
	4) Stir, RT, 3 days
	5) SE, RT
L-pyroglutamic	1) L-pyroglutamic acid	1) Clear, colorless solution	MRZ + L-
acid (1:1)	dissolved in MeOH	2) Turbid solution	pyro-
	2) EtOAc added to MRZ	3) Clear, colorless solution	glutamic
	3) MeOH solution added	4) Clear, colorless solution	acid
	to MRZ	5) White solids, PLM: dendrites
	4) Stir, RT, 1 day	and needles, B
	5) FE, RT
L-pyroglutamic	Slurry, ACN, RT, 6 days	White solids	MRZ + L-
acid (1:2)		PLM: small particles, unknown	pyro-
		morphology, B	glutamic
			acid
2-pyrrolidone	1) Dissolve MRZ in	1) Clear, colorless solution	MRZ +
(1:1)	acetone (1 mL) at 50° C.	2) Clear, colorless, solution	extra peaks
	2) Add 25 μL 2-	3) Hazy solution, insufficient for	at 14.1 and
	pyrrolidone at RT to	XRPD	15.5 °2θ
	MRZ solution	4) White solids	(XRPD)
	3) Temperature cycle	PLM: small needles, B.	MRZ:2-
	between 50° C. and RT		pyrrolidone
	(4 cycles × 1 hour at		1:1.2 +
	each temperature, held at		additional
	RT overnight)		minor
	4) SE, RT		peaks, no
			acetone
			detected
			(NMR)
			Sharp
			endotherm
			with an
			onset at
			29.4° C.
			and peak
			max at
			32.8° C.
			Broad
			features
			(endo-
			thermic) at
			91.4° C.
			and 131.3°
			C. (peak
			max.)
			(DSC)
			5.0%
			weight loss
			from 27.1°
			C. to 92.0°
			C. (TGA)
2-pyrrolidone	Manual grinding (2 × 5	White tacky solids	MRZ +
(excess)	minutes, 2 × 10 μL)		extra peaks
			at 14.1 and
			15.5 °2θ
2-pyrrolidone	1) Dissolve MRZ in 500	1) Clear, slightly yellow sol'n	—
(large excess)	μL 2-pyrrolidone at 50° C.	2) Clear, colorless solution
	2) Cool to RT with	3) Clear solution
	stirring
	3) FE, RT
Saccharin	IPA, FC, 70° C. to RT	White damp solids	MRZ
(1:1)
Saccharin	1) Dissolve saccharin in	1) Clear, colorless solution	MRZ +
(1:2)	acetone	2) White suspension	minor
	2) Add to MRZ	3) White solids	saccharin
	3) Slurry, RT, 6 days	PLM: small particles, unknown
		morphology, B
Saccharin	ACN, slurry, RT, 7 days	White solids	MRZ +
(1:5)		PLM: small agglomerates,	Saccharin
		unknown morphology, B
Salicylic acid	EtOAc:toluene (50:50),	White solids	MRZ
(1:1)	FC, 75° C. to RT	PLM: agglomerates, needles,
		B/E. possible singles
Salicylic acid	1) Salicylic acid	1) Clear, colorless solution	MRZ
(1.3)	dissolved in EtOH	2) Clear, colorless solution
	2) MRZ dissolved in	3) Clear, colorless solution
	MIBK	4) White solids,
	3) Salicylic acid solution	PLM: Small particles, unknown
	added to MRZ	morphology, B
	4) Stir, RT
	1) Salicylic acid	1) Clear, colorless solution	MRZ +
	dissolved in EtOH	2) Clear, colorless solution	salicylic
	2) MRZ dissolved in	3) Clear, colorless solution	acid
	MEK	4) Clear, colorless solution
	3) Salicylic acid solution	5) White solids
	added to MRZ	PLM: dendritics and needles, B
	4) Stir, RT, 8 days
	5) SE, RT
Salicylic acid	1) Salicylic acid	1) Clear, colorless solution	MRZ +
(1:5)	dissolved in 1-PrOH,	2) Clear, colorless solution	salicylic
	MRZ dissolved in ACN,	3) Clear, colorless solution	acid
	addition of MRZ	4) White solids
	solution to salicylic acid	PLM: needles, dendrites, B
	solution
	2) Stir, RT, 2 days
	3) Cool to 2-8° C., 4 days
	4) FE, RT
L-Serine	1) Dissolve L-serine in	1) Clear, colorless solution	MRZ
(1:1)	H₂O	2) Clear, colorless solution
	2) Dissolve MRZ in	3) Clear, colorless solution
	THF	4) Slightly turbid solution
	3) Add L-serine to MRZ	5) White solids
	4) Stir, RT, 3 days	PLM: agglomerates, B
	5) SE, RT
L-Serine	1) H₂O added to L-	1) Solids remain	MRZ
(1:3)	serine	2) Solids remain
	2) MeOAc added to	3) Two layers formed
	MRZ	4) Clear, colorless solution
	3) H₂O added to MeOAc	5) Clear, colorless solution
	4) Slurry, 60° C., O/N	6) White solids
	5) Stir, RT, 1 day	PLM: dendrites, needles, B/E
	6) FE, RT
Succinic acid	DCM, slurry, RT, 6 days	White solids	MRZ +
(1:1)		PLM: small particles, unknown	succinic
		morphology, B	acid
Succinic acid	1) Dissolve succinic acid	1) Clear, colorless solution	MRZ
(1.2)	in MeOH	2) Clear, colorless solution
	2) Dissolve MRZ in	3) Clear, colorless solution
	acetone	4) White solids
	3) Add succinic acid	PLM: small particles, unknown
	solution to MRZ	morphology, B
	4) Stir, RT, 5 days
Succinic acid	1) Succinic acid	1) Clear, colorless solution	MRZ +
(1:5)	dissolved in EtOH, MRZ	2) Clear, colorless solution	succinic
	dissolved in THF,	3) Translucent solids	acid
	addition of succinic acid	PLM: needles, rosettes, B/E
	solution to MRZ
	solution
	2) Stir, RT, 2 days
	3) SE at RT
L-(+)-tartaric	1) EtOAc, SC, 50° C. to	1) White suspension, insufficient	MRZ +
acid (2:1)	RT	for analysis	L-(+)-
	2) Cool to 2-8° C.	2) White solids	tartaric
			acid
L-(+)-tartaric	1) Dissolve L-(+)-	1) Clear, colorless solution	MRZ +
acid (1:2)	tartaric acid in EtOH at	2) Clear, colorless solution	minor
	50° C.	3) Clear, colorless solution	L-(+)-
	2) Dissolve MRZ in	4) Clear, colorless solution	tartaric
	dioxane at 50° C.	5) Translucent solids	acid
	3) Add L-(+)-tartaric	PLM: Needles, B/E
	acid solution to MRZ at
	50° C.
	4) Stir at RT for cool
	5) SE at RT
L-(+)-tartaric	THF:nitromethane	White solids	MRZ +
acid (1:5)	(20:80), slurry, RT, 7	PLM: small particles, unknown	L-(+)-
	days	morphology, B	tartaric
			acid
Thymine	1) Add MeOH to	1) White suspension	Thymine
(1:1)	thymine at 50° C.	2) White suspension
	2) Add EtOAc to MRZ	3) White suspension
	at 50° C.	4) White suspension
	3) Add thymine	5) White solids
	suspension	PLM: small particles, unknown
	4) Slurry at 50° C.,	morphology, B
	2 days
	5) Slurry at RT, 6 days
Thymine	1) 1-PrOH added to	1) White suspension	MRZ +
(1:3)	Thymine	2) White suspension	thymine
	2) Added to MRZ	3) White suspension
	3) Slurry at 60° C., O/N	4) White solids
	4) Stir, RT, 1 day	PLM: small particles, unknown
		morphology
Uracil	1) Add H₂O to uracil at	1) White suspension	MRZ +
(1:1)	50° C.	2) White suspension	Uracil
	2) Add acetone to MRZ	3) White suspension
	3) Add uracil suspension	4) White solids
	to MRZ	PLM: small particles, unknown
	4) Slurry, RT	morphology, B.
Uracil	1) Uracil dissolved in	1) Clear, colorless solution	MRZ +
(1:3)	H₂O	2) Clear, colorless solution	uracil
	2) MRZ dissolved in	3) White suspension
	THF	4) White suspension
	3) Uracil solution added	5) White solids
	to MRZ solution	PLM: small particles, unknown
	4) Slurry, 60° C., O/N	morphology
	5) Stir, RT, 1 day
Urea	EtOAc, FC, 50° C. to RT	White solids	MRZ
(2:1)		PLM: small particles, unknown
		morphology, B
Urea	Solvent assisted	White solids	MRZ +
(1:1)	grinding, t-BuOH (3 × 5		urea
	minutes, 3 × 20 μL)
Urea	Dioxane, stir, RT, 7 days	Translucent solids	Urea:
(1:5)		PLM: needles, dendritics	dioxane
			solvate +
			minor
			MRZ
			(XRPD)
			Urea,
			dioxane,
			DMSO,
			water, trace
			MRZ (1H
			NMR)

Example 13—Salt Screen of Marizomib

For crash cooling (CC), a solution of MRZ and coformer was prepared at elevated temperature in a given solvent mixture. The vial was capped and immediately placed in a refrigerator or freezer. For fast cooling (FC), solutions of MRZ and coformer were prepared at elevated temperatures in given solvents or solvent mixtures. The vial was capped and placed on a bench top at room temperature to quickly cool. For slow cooling (SC), a solution of MRZ and coformer was prepared at elevated temperature in a given solvent mixture. The vial was capped and left in a heating block at elevated temperature. The heater then was turned off for the sample to cool down naturally to room temperature. For slow evaporation (SE), solutions of MRZ and coformer were generated at ambient temperature in a given solvent or solvent mixture. The solutions were allowed to evaporate partially or to dryness from a loosely capped vial at ambient conditions. Slurry experiments were carried out by making saturated solutions containing excess solid. The slurries were agitated at ambient or elevated temperatures for a specified amount of time. The solids present were recovered via positive pressure filtration. Solvent assisted grinding experiments were carried out by mixing MRZ and coformer in an agate mortar and pestle. Aliquots of solvent were added and the mixture ground manually for specified amount of time. The solids were scraped from the walls of the mortar and the pestle head. Another aliquot of solvent added and ground for several more minutes. For temperature cycling, solutions or suspensions of MRZ and coformer were made in a given solvent and placed at elevated temperature. The sample was cycled by removing from heat and then reapplying several times. The solids were collected via vacuum filtration upon the last cool from elevated temperature.
As set forth in Table 19 below, “X:Y” refers to marizomib:conformer mole ratio. Temperatures and times are approximate. Solvent ratios are by volume. Approximately 60-90 mg of marizomib was used for each experiment.

TABLE 19

Salt Screen of Marizomib

Counnterion			XRPD
(X:Y)	Conditions	Observations	Results

Benzenesulfonic	Anh. acetone,	Brown solution	—
acid (1:1)	stir, RT,
	2 days
HCl (1:1)	IPA/anh.	White solids	MRZ
	acetone (3:1),	PLM: agglomerates
	stir, RT,	of needles, B/E
	7 days
HCl (1:2)	IPrOAc, stir,	White solids	MRZ
	RT (HCl in	PLM: small particles,
	IPA), 7 days	unknown morph, B.
Methanesulfonic	Anh. acetone,	Dark orange solution,	—
acid (1:1)	stir, RT,	yellow solids,
	1 day	experiment
		discontinued
Phosphoric acid	Dioxane, stir,	Clear, colorless	—
(1:1)	RT, 4 days	solution
Sulfuric acid	2-MeTHF, stir,	Clear, light yellow	—
(1:1)	RT, 2 days	solution
Sulfuric acid	Acetone, stir,	Black solution	—
(large excess)	RT, 2 days
Sulfuric acid	Stir in H₂SO₄,	Dark brown solution	—
(large excess)	RT, 2 days
p-toluenesulfonic	EtOAc, stir,	White solids	MRZ
acid (1:1)	RT, 2 days	PLM: small particles,
		unknown morphology,
		B.
p-toluenesulfonic	1) THF, stir,	1) Clear, colorless	MRZ +
acid (1:2)	RT, 2 days	solution	p-toluene
	2) SE, RT	2) Off-white solids	sulfonic
		PLM: agglomerates,	acid +
		B.	unique peaks
			(7.9, 12.7
			2θ)

EQUIVALENTS

While the present invention has been described in conjunction with the specific embodiments set forth above, many alternatives, modifications and other variations thereof will be apparent to those of ordinary skill in the art. All such alternatives, modifications and variations are intended to fall within the spirit and scope of the present invention.

Claims

1. Morphic Form I of marizomib, characterized by an X-ray powder diffraction pattern including peaks at about 7.2, 14.5, and 36.7°2° using Cu Kα radiation.

2. The morphic Form of claim 1, further including X-ray powder diffraction peaks at about 18.1, 19.6, and 20.8°2θ using Cu Kα radiation.

3. The morphic Form of any of the preceding claims, further including X-ray powder diffraction peaks at about 16.3, 19.8, and 20.5°2θ using Cu Kα radiation.

4. The morphic Form of any of the preceding claims, further including X-ray powder diffraction peaks at about 15.2, 21.5, and 22.3°2θ using Cu Kα radiation.

5. The morphic Form of any of the preceding claims, further including X-ray powder diffraction peaks at about 14.7, 29.2, and 30.0°2° using Cu Kα radiation.

6. The morphic Form of any of the preceding claims, further including X-ray powder diffraction peaks at about 8.2, 14.8, and 27.7°2θ using Cu Kα radiation.

7. The morphic Form of any of the preceding claims, further characterized by an X-ray powder diffraction pattern substantially similar to that set forth in FIG. 1, 2 or 3.

8. The morphic Form of any of the preceding claims, further characterized by a degradation event at about 175° C. measured by thermogravimetric analysis.

9. The morphic Form of any of the preceding claims, further characterized by two exotherms at about 150-180° C. as measured by differential scanning calorimetry at a rate of about 2° C. per minute.

10. The morphic Form of any of the preceding claims, characterized by melting at about 160-175° C. as measured by hot stage microscopy.

11. The morphic Form of any of the preceding claims, wherein the polymorph is at least about 98% pure as measured by HPLC.

12. The morphic Form of claim 11, wherein the polymorph is at least about 99.1% pure as measured by HPLC.

13. A pharmaceutical composition comprising a morphic Form of any of the preceding claims and a pharmaceutically acceptable carrier.

14. A method of treating a disease, comprising administering to a subject in need thereof an effective amount of a morphic Form of any of claims 1-12.

15. The method of claim 14, wherein the disease is cancer.

16. Use of the morphic Form of any of claims 1-12 for the treatment of a disease.

17. Use of the morphic Form of any of claims 1-12 for inhibiting a protease.

18. Use of the morphic Form of any of claims 1-12 in the manufacture of a medicament for the treatment of a disease.

19. A method of preparing a morphic Form of marizomib characterized by an X-ray powder diffraction pattern including peaks at about 7.2, 14.5, and 36.7°2θ using Cu Kα radiation, comprising recrystallizing marizomib from a solvent.

20. The method of claim 19, wherein the solvent is selected from the group consisting of n-heptane, ethyl acetate, methyl-isobutyl ketone, 2-propanol, acetone, chloroform, dimethyl sulfoxide, tert-butyl methyl ether, anisole, cumene, methyl ethyl ketone, isopropyl acetate, dimethylformamide, toluene, tetrahydrofuran, dichloromethane, acetonitrile, nitromethane, ethanol, and dimethylacetamide.