WO2008066729A2

WO2008066729A2 - Boronic acid containing compositions

Info

Publication number: WO2008066729A2
Application number: PCT/US2007/024173
Authority: WO
Inventors: Michael A. Tyo
Original assignee: Dara Biosciences, Inc.
Priority date: 2006-11-22
Filing date: 2007-11-19
Publication date: 2008-06-05
Also published as: WO2008066729A3

Abstract

The invention provides clinical grade compositions comprising peptide boronic acids, and methods of manufacturing the same to avoid degradation and maintain stability and potency.

Description

BQRONIC ACID CONTAINING COMPOSITIONS

Related Applications

This application claims priority under 35 U. S. C. §119(e) to the provisional patent application entitled "Boronic Acid Containing Compositions" filed November 22, 2006, and assigned serial number 60/860,686, the entire contents of which are incorporated herein by reference.

Field of the Invention The invention relates to peptide boronic acid compositions and formulations and methods of manufacture.

Background of the Invention

Certain peptide boronic acids are post-prolyl cleaving enzyme inhibitors. Post-prolyl cleaving enzymes have been implicated in a number of conditions including cancer and diabetes. Peptide boronic acids are currently in clinical trials for the treatment of these and other indications. Pharmaceutical compositions comprising these compounds that maintain potency and stability during storage are necessary for wide-spread clinical applications.

Summary of the Invention

The invention in its broadest sense relates to formulations, including clinical formulations for amino boronic acid compounds and methods related to such formulations, including analysis methods and methods of use. The invention is premised in part on the discovery of an optimum formulation for such compounds, as well as optimal processes for the synthesis of such compounds including methods for detecting and/or excluding impurities and/or degradants.

In one aspect, the invention provides a method for determining an impurity in a material comprising Formula I (as shown herein) comprising injecting into an HPLC column a sample solution containing the material and containing a known amount of a known first reference compound having a known chemical structure of Compound G and/or a known amount of a known second reference compound having a known chemical structure of Compound H, obtaining an HPLC chromatogram, and determining the presence and/or the amount of Compound G and/or Compound H in the material. In another aspect, the invention provides a method for determining an impurity in a material consisting essentially of Formula I comprising injecting into an HPLC column a sample solution containing the material and containing a known amount of a known first reference compound having a known chemical structure of Compound G, and/or a known amount of a known second reference compound having a known chemical structure of Compound H, obtaining an HPLC chromatogram, and determining the presence and/or the amount of Compound G and/or Compound H in the material.

In some embodiments, the reference may contain both Compounds G and H and the method may not require a determination of the separate levels of such compounds, particularly if an HPLC column is used to detect the Compounds. In some embodiments, the individual amounts of Compounds G and H are measured using capillary electrophoresis. In a variant of this method, the reference compound has a known chemical structure of Compound F.

Several embodiments equally apply to the foregoing aspects of the invention. In one embodiment, the method further comprises documenting in a written form the chemical identity and the amount as an impurity of Compound G and/or Compound H.

In one embodiment, the reference sample contains a known amount of a known third reference compound having a known chemical structure of Compound F, and the method further comprises determining the presence and/or the amount of Compound F in the material. In a related embodiment, the method further comprises documenting in a written form the chemical identity and the amount as an impurity of Compound F.

In one embodiment, the reference sample contains a known amount of a known third reference compound having a known chemical structure of Compound I, and the method further comprises determining the presence and/or the amount of Compound I in the material. In a related embodiment, the method further comprises documenting in a written form the chemical identity and the amount as an impurity of Compound I.

In one embodiment, the HPLC column is a reverse phase column and the column is eluted using a mobile phase comprising sodium dodecyl sulfate, trifluoroacetic acid, water, and acetonitrile. In various embodiments, the compound of Formula I is talabostat mesylate. In one aspect, the invention provides a method for determining an impurity in a material comprising Formula I comprising injecting a solution in which the material is dissolved into an HPLC column and obtaining an HPLC chromatogram, determining the amount in the material of a compound known to have the structure of Compound G or Compound H, and documenting in a written form the chemical identity and amount of Compound G or Compound H in the material.

In another aspect, the invention provides a method for determining an impurity in a material consisting essentially of Formula I comprising injecting a solution in which the material is dissolved into an HPLC column and obtaining an HPLC chromatogram, determining the amount in the material of a compound known to have the structure of Compound G or Compound H, and documenting in a written form the chemical identity and amount of Compound G or Compound H in the material.

In some embodiments, a determination of the separate levels of Compounds G and H is not required and thus the HPLC column method is suitable. In other embodiments, the individual amounts of Compounds G and H are measured using capillary electrophoresis. In a variant of this method, the amount of Compound F is determined rather than that of Compound G and/or H.

In one embodiment, the amount of Compound G or Compound H in the material is determined by (i) identifying a peak on the chromatogram that corresponds to a peak on a control chromatogram, (ii) identifying a peak on the chromatogram that corresponds to a relative retention time of Compound G or Compound H, and/or (iii) identifying a peak on the chromatogram that corresponds to a known amount of a spike of a compound known to have the structure of Compound G or Compound H. In another embodiment, the method further comprises determining the amount in the material of a compound known to have the structure of Compound F, and documenting in a written form the chemical identity and amount of Compound F as an impurity in the material. In still another embodiment, the method further comprises determining the amount in the material of a compound known to have the structure of Compound I, and documenting in a written form the chemical identity and amount of Compound I as an impurity in the material.

In one embodiment, the HPLC column is a reverse phase column and the column is eluted using a mobile phase comprising sodium dodecyl sulfate, trifluoroacetic acid, water, and acetonitrile. In another embodiment, the compound of Formula I is talabostat mesylate. In another aspect, the invention provides a method for evaluating the purity of a composition comprising Formula I comprising injecting a reference solution containing impurities comprising Compound F, Compound G, Compound H and/or Compound I into an HPLC column under a set of conditions to obtain a first HPLC chromatogram, wherein the amount and identity of the impurities present in the reference solution was previously determined, injecting a sample solution comprising Formula I into the HPLC column under said set of conditions to obtain a second HPLC chromatogram, and determining the amount of the impurities in the sample solution. In one embodiment, the method further comprises reporting in writing that the amounts of the compounds in the Formula I composition are for Compound F: less than or equal to 2.0%, less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1%, for Compound G: less than or equal to 2.0%, less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1%, for Compound H: less than or equal to 2.0%, less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1%, and for Compound I: less than or equal to 2.0%, less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1%.

In one embodiment, the HPLC column is a reverse phase column and the column is eluted using a mobile phase comprising sodium dodecyl sulfate, trifluoroacetic acid, water, and acetonitrile. In another embodiment, the compound of Formula I is talabostat mesylate.

In one aspect, the invention provides a method for manufacturing a material comprising a compound of Formula I comprising obtaining a composition comprising the compound of Formula I, purifying the composition by chromatography, recrystallization or a combination thereof, conducting HPLC on a sample of purified first composition using a known amount of impurities Compound F, Compound G, Compound H and Compound I as a standard, and determining the presence or absence of the impurities in the sample.

In another aspect, the invention provides a method for manufacturing a material consisting essentially of a compound of Formula I comprising obtaining a composition containing the compound of Formula I, purifying the composition by chromatography, recrystallization or a combination thereof, conducting HPLC on a sample of purified first composition using a known amount of impurities Compound F, Compound G, Compound H and Compound I as a standard, and determining the presence or absence of the impurities in the sample.

In one embodiment, the purifying is carried out until the impurities are less than or equal to 2.0%, 1.0%, 0.5%, 0.1%, 0.4%, 0.3%, 0.2%, 0.15%, 0.1%, 0.05%, even are absent from the purified first composition as determined by HPLC with a detection limit of 0.01- 0.03% and a quantitation limit of 0.03%. In another embodiment, the method further comprises then packaging the purified first composition. In another embodiment, the method further comprises providing indicia on or with the packaged purified first composition indicating a level of the impurities in the packaged first purified composition. In one embodiment, the compound of Formula I is talabostat mesylate.

In one aspect, the invention provides a package containing a composition comprising Formula I and indicia on or contained within the package indicating a level of one or more impurities comprising Compound F, Compound G, Compound H and Compound I, wherein the one or more impurities are identified by chemical name, by chemical structure or by chemical formula.

In one embodiment, the indicia indicates a content for each of these compounds as follows: Compound F: less than or equal to 2.0%, less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1%, for Compound G: less than or equal to 2.0%, less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1%, for Compound H: less than or equal to 2.0%, less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1%, and for Compound I: less than or equal to 2.0%, less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1%. In one embodiment, the indicia indicates a level of impurity of Compounds F, G, H and I of less than or equal to 2.0%. In one embodiment, the compound of Formula I is talabostat mesylate. In one embodiment, the compound of Formula I is greater than 95% linear while in another embodiment it is greater than 98% linear.

In one aspect, the invention provides a method for determining whether a condition protects against degradation of Formula I comprising applying the condition to a composition comprising Formula I, and determining whether one or more of the following impurities selected from Compounds A, B, C, and D form as a result of the applying of the conditions.

In one embodiment, the condition is combining Formula I with a pharmaceutically acceptable carrier. In other embodiments, the conditions are (i) adjusting the pH of the composition, (ii) exposing the composition to light, (iii) exposing the composition to a chemical, (iv) heating the composition, (v) processing the composition into a pharmaceutical dosage form, and/or (vi) storing the composition.

In one embodiment, the impurities comprise Compound A and Compound B, and the indicia indicates levels of impurities as follows: for Compound A: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%, and for Compound B: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

In one embodiment, the impurities comprise Compound C, and the indicia indicates a level of impurity for Compound C of less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

In one embodiment, the impurities comprise Compound D, and the indicia indicates a level of impurity for Compound D of less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%. In one embodiment, the compound of Formula I is talabostat mesylate.

In another aspect, the invention provides a pharmaceutical comprising particles consisting essentially of a compound of Formula I, lactose, microcrystalline cellulose, and an acidic salt.

In one embodiment, the acidic salt is sodium phosphate. In one embodiment, the compound of Formula I is present at between 0.1% and 0.2% w/w relative to the total weight of the lactose and the microcrystalline cellulose. In another embodiment, the compound of Formula I is present at between 0.1% and 0.2% w/w relative to the weight of the lactose.

In one embodiment, the compound of Formula I is talabostat mesylate. In one embodiment, the compound of Formula I is talabostat mesylate and the pharmaceutical has a level of impurity of less than or equal to 3%, equal to or less than 2%, less than or equal to 1.0%, less than or equal to 0.5%, or less than or equal to 0.1% for one or more of Compound A, B, C and/or D. In one embodiment, the total level of impurity of Compounds A-D is less than or equal to 2.0%. In one embodiment, the compound of Formula I is 15-40% cyclic. In another aspect, the invention provides a pharmaceutical preparation comprising agglomerated filler particles, the filler particles coated over their surface with a compound of Formula I, the agglomerated filler particles also coated over their surface with an acidic salt, the agglomerated particles free of compounds that are electron withdrawing and oxidative respecting boronic acid of the compound of Formula I, wherein the compound of Formula I is present between 0.1% and 0.2% w/w relative to the weight of the filler particles in the agglomerated filler particles.

In one embodiment, the preparation further comprises a binder mixed with the agglomerated filler particles, and the mixture is compressed as a tablet. In one embodiment, the filler particles comprise lactose and the agglomerated particles are free of a binder. In another embodiment, the agglomerated particles are formed in the absence of hydroxypropylmethylcellulose and cross-linked N-vinyl-2-pyrrolidone.

In one embodiment, the compound of Formula I is talabostat mesylate. In one embodiment, the compound of Formula I is 15-40% cyclic.

In yet another aspect, the invention provides a pharmaceutical tablet comprising particles consisting essentially of a compound of Formula I, lactose, microcrystalline cellulose, and an acidic salt, the particles compressed in a mixture with one or more of a binder, a lubricant and a disintegrant. In one embodiment, the mixture is a mixture of the particles with a binder and a disintegrant. In another embodiment, the mixture is a mixture of the particles with a binder, a lubricant and a disintegrant. In still another embodiment, the mixture is a mixture of the particles with pre-gelatinized starch.

In one embodiment, the binder is pre-gelatinized starch. In one embodiment, the disintegrant is cross-linked N-vinyl-2-pyrrolidone. In one embodiment, the lubricant is stearic acid.

In one embodiment, the compound of Formula I is present at between 0.1% and 0.2% w/w relative to the weight of lactose, microcrystalline cellulose, and acidic salt. In one embodiment, the compound of Formula I is present at between 0.1% and 0.2% w/w relative to the weight of lactose.

In one embodiment, the compound of Formula I is present at between 50 and 800 micrograms. In one embodiment, the compound of Formula I is talabostat mesylate. In one embodiment, the compound of Formula I is 15-40% cyclic.

In one embodiment, the compound of Formula I is talabostat mesylate and the tablet has a level of impurity of less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1% for one or more of Compound A, B, C and/or D. In one embodiment, the level of impurity of Compounds A-D combined is less than or equal to 10.0%.

In one aspect, the invention provides a pharmaceutical preparation comprising a tablet of the compound of Formula I, wherein the tablet after storage at 2-8 degrees centigrade for 3 months has a total amount of Formula I degradation products that does not exceed 10% of the compound of Formula I in the preparation.

In one embodiment, the amount of Formula I degradation products does not exceed 1.5% of the compound of Formula I in the preparation. In another embodiment, the amount of Formula I degradation products does not exceed 1.0% of the compound of Formula I in the preparation. In still another embodiment, the amount of Formula I degradation products does not exceed 0.5% of the compound of Formula I in the preparation. And in yet another embodiment, the amount of Formula I degradation products does not exceed 0.25% of the compound of Formula I in the preparation. In one embodiment, the amount of each Formula I degradation product does not exceed 0.5% of the compound of Formula I in the preparation, does not exceed 0.25% of the compound of Formula I in the preparation, does not exceed 0.15% of the compound of Formula I in the preparation, or even does not exceed 0.10% of the compound of Formula I in the preparation. In one embodiment, the compound of Formula I in the pharmaceutical preparation is protected from oxidation. In a related embodiment, the compound of Formula I in the pharmaceutical preparation is protected from oxidation by coating the compound of Formula I onto particles that are not oxidative with respect to the compound of Formula I. In one embodiment, the compound of Formula I is coated onto particles free of (-OCH₂CHOHCH₃) groups. In another embodiment, the compound of Formula I is coated onto particles free of hydroxy propyl methyl cellulose.

In one embodiment, the compound of Formula I is coated onto particles free of vinyl pyrrolidone. In one embodiment, the compound of Formula I is coated onto particles free of crosslinked homopolymer of N-vinyl-2 -pyrrolidone. In one embodiment, the preparation further comprises a pH modifying salt. In a related embodiment, the pH modifying salt is sodium phosphate monobasic, monohydrate and the preparation further comprises a pH modifying acid that is phosphoric acid.

In one embodiment, the compound of Formula I in the pharmaceutical preparation is coated onto particles comprising one or more fillers. In a related embodiment, the one or more fillers comprise lactose monohydrate. In another related embodiment, the one or more fillers comprise lactose monohydrate and microcrystalline cellulose.

In one embodiment, the preparation further comprises a lubricant. In another embodiment, the preparation further comprises a binder.

In one embodiment, the compound of Formula I is 15-40% cyclic. In another aspect, the invention provides a method of manufacturing a pharmaceutical tablet comprising providing a solution of a compound of Formula I, wherein the pH of the solution is between 1.5 and 2.9 and wherein the compound of Formula I is at a concentration of about 4000 - 6000 mg/liter, spraying the solution onto particles of a filler material to form agglomerated particles coated with and containing the compound of Formula I, drying the agglomerating particles, and mixing the agglomerated particles with one or more of a binder, a lubricant and a disintegrant, and compressing the mixture into a tablet.

In one embodiment, the filler material is non-oxidative respecting the boronic acid of the compound of Formula I in the presence of both an acidic salt and trace amounts of water at 2-8 degrees centigrade.

In one embodiment, the filler material comprises lactose. In one embodiment, the filler material comprises microcrystalline cellulose. In another embodiment, the filler material comprises lactose and microcrystalline cellulose. In one embodiment, the agglomerated particles are mixed with a binder and a disintegrant. In one embodiment, the agglomerated particles are mixed with a binder, a lubricant and a disintegrant. In one embodiment, the agglomerated particles are mixed with a binder that is pre-gelatinized starch. In one embodiment, the agglomerated particles are mixed with a disintegrant that is cross-linked N-vinyl-2-pyrrolidone. In one embodiment, the agglomerated particles are mixed with a lubricant that is stearic acid. In one embodiment, the binder is pre-gelatinized starch, the disintegrant is cross-linked N-vinyl-2-pyrrolidone, and the lubricant is stearic acid.

In one embodiment, the compound of Formula I is present in the tablet at between 0.1% and 0.2% w/w relative to lactose and microcrystalline cellulose. In one embodiment, the compound of Formula I is present at between 0.1% and 0.2% w/w relative to lactose.

In one embodiment, the compound of Formula I is present in the tablet at between 180 and 220 micrograms. In one embodiment, the compound of Formula I is present in the tablet at between 280 and 320 micrograms. In one embodiment, the pH of the solution is about 2.0 to 2.5. In one embodiment, the pH of the solution is about 2.2. In one embodiment, the compound of Formula I is talabostat mesylate.

In one embodiment, the compound of Formula I is talabostat mesylate and the method further comprises testing for one or more of Compounds A, B, C and/or D. In one embodiment, the tablet is tested for impurities after storage for 3 months at 2-8 degrees centigrade. In one embodiment, the method further comprises packaging the tablet and providing indicia on or with the packaged tablet indicating a level of the one or more impurities in the packaged tablet.

The invention further provides a pharmaceutical tablet made by any of the foregoing processes. In another aspect, the invention provides a kit comprising a package containing a pharmaceutical tablet comprising particles comprising a compound of Formula I, lactose, microcrystalline cellulose, and an acidic salt, the particles compressed in a mixture of the particles with one or more of a binder, a lubricant and a disintegrant, and indicia on or contained within the package indicating the chemical identity of the compound of Formula I.

In another aspect, the invention provides a kit comprising a package containing a pharmaceutical tablet comprising particles consisting essentially of a compound of Formula I, lactose, microcrystalline cellulose, and an acidic salt, the particles compressed in a mixture of the particles with one or more of a binder, a lubricant and a disintegrant, and indicia on or contained within the package indicating the chemical identity of the compound of Formula I.

In one embodiment, the compound of Formula I is talabostat mesylate.

In another aspect, the invention provides a kit comprising a package containing a pharmaceutical tablet comprising particles consisting essentially of a compound of Formula I, lactose, microcrystalline cellulose, and an acidic salt, the particles compressed in a mixture of the particles with one or more of a binder, a lubricant and a disintegrant, and indicia on or contained within the package indicating the chemical identity of the compound of Formula I, wherein the compound of Formula I is talabostat mesylate and wherein the tablet has a level of impurity of less than or equal to 3.0%, less than or equal to 2%, less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1% for one or more of Compounds A, B, C and D.

In one embodiment, the indicia also indicate the chemical identity of the one or more impurities and the amount of the one or more impurities. In one embodiment, the level of impurity of Compounds A-D combined is less than or equal to 10.0%.

In one embodiment, the compound of Formula I is 15-40% cyclic. In another aspect, the invention provides a package containing a tablet comprising

Formula I, and indicia on or contained within the package indicating a level of impurity for one or more impurities comprising Compound A, Compound B, Compound C, and Compound D, wherein the one or more impurities are identified by chemical name, by chemical structure or by chemical formula. In one embodiment, the impurities comprise Compound A and Compound B, and the indicia indicates levels of impurities as follows: for Compound A: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%, and for Compound B: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

In one embodiment, the impurities comprise Compound D, and the indicia indicates a level of impurity for Compound D of less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

In one embodiment, the indicia indicates that the level of impurity combined for Compounds A-D is less than or equal to 10.0%.

In another aspect, the invention provides a method for evaluating the purity of a Formula I composition comprising injecting a reference solution containing impurities comprising Compound A and Compound B into an HPLC column under a set of conditions to obtain a first HPLC chromatogram, wherein the amount and identity of the compounds present in the reference solution is known, injecting a sample solution comprising Formula I into the HPLC column under said set of conditions to obtain a second HPLC chromatogram, and determining the amount of the compounds in the sample solution.

In one embodiment, the method further comprises reporting in writing that the amounts of the compounds in the Formula I composition are for Compound A: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%, and for Compound B: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

In one embodiment, the impurities further comprise a compound of Compound C, and optionally the method further comprises reporting in writing that the amount of the compound of Compound C in the Formula I composition is less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

In one embodiment, the impurities further comprise a compound of Compound D, and optionally the method further comprises reporting in writing that the amount of the compound of Compound D in the Formula I composition is less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%. In one embodiment, the HPLC column is a reverse phase column and the column is eluted using a mobile phase comprising sodium dodecyl sulfate, phosphoric acid, sodium hydroxide, water, and acetonitrile.

In another aspect, the invention provides a method for determining an impurity in a material comprising Formula I comprising injecting a solution comprising the material into an HPLC column and obtaining an HPLC chromatogram, and determining the amount in the material of impurities known to have the structure of Compound A or Compound B, and documenting in a written form the chemical identity of the impurities and the amount of the impurities in the material. In another aspect, the invention provides a method for determining an impurity in a material consisting essentially of Formula I comprising injecting a solution comprising the material into an HPLC column and obtaining an HPLC chromatogram, and determining the amount in the material of impurities known to have the structure of Compound A or Compound B, and documenting in a written form the chemical identity of the impurities and the amount of the impurities in the material.

In one embodiment, the amounts of the compounds in the material are documented in writing to be as follows: for the compound of Compound A: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%, and for the compound of Compound B: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

In one embodiment, the impurities further comprise a compound of Compound C, and optionally the amount of the compound of Compound C in the material is documented in writing to be: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

In one embodiment, the impurities further comprise a compound of Compound D, and optionally the amount of the compound of Compound D in the material is documented in writing to be: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%. In one embodiment, the HPLC column is a reverse phase column and the column is eluted using a mobile phase comprising sodium dodecyl sulfate, phosphoric acid, sodium hydroxide, water, and acetonitrile. In another embodiments, the amount in the material of each compound is determined by (i) identifying a peak on the chromatogram that corresponds to a peak on a control chromatogram of compounds known to have the structure of Compound A and/or Compound B, (ii) identifying a peak on the chromatogram that corresponds to a relative retention time of a compound known to have the structure of Compound A and/or

Compound B, and/or (iii) identifying a peak on the chromatogram that corresponds to a known amount of a spike of the compound known to have the structure of Compound A and/or Compound B.

In another aspect, the invention provides a method for manufacturing a material comprising a compound of Formula I, comprising obtaining a first composition containing the compound of Formula I, purifying the composition by chromatography, recrystallization or a combination thereof, conducting HPLC on a sample of purified first composition using a known amount of one or more of impurities Compound A, Compound B, Compound C and Compound D as a standard, and determining the presence or absence of the impurities in the sample.

In another aspect, the invention provides a method for manufacturing a material consisting essentially of a compound of Formula I comprising obtaining a first composition containing the compound of Formula I, purifying the composition by chromatography, recrystallization or a combination thereof, conducting HPLC on a sample of purified first composition using a known amount of one or more of impurities Compound A, Compound B, Compound C and Compound D as a standard, and determining the presence or absence of the impurities in the sample.

In one embodiment, the purifying is carried out until the impurities are less than 2.0%, 1.5%, 1.0%, 0.9%, 0.8%, 0,7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.15%, 0.1%, 0.05%, or are absent from the purified first composition as determined by HPLC with a detection limit of 0.01 to 0.03% and a quantitation limit of 0.03%.

In another embodiment, the method further comprises then packaging the purified first composition.

In another embodiment, the method further comprises providing indicia on or with the packaged purified first composition indicating a level of the impurities in the packaged purified first composition. The foregoing aspects and embodiments of the invention share some common embodiments, and these are recited below once for the sake of convenience and brevity. Compound A has a chemical structure as follows:

C CO₂H NH

NH₂ Compound B has a chemical structure as follows:

Compound C has a chemical structure as follows:

Compound D has a chemical structure as follows:

Compound E has a chemical structure as follows:

Compound F has a chemical structure as follows:

Compound G has a chemical structure as follows:

Compound H has a chemical structure as follows:

Compound I has a chemical structure as follows:

These and other aspects and embodiments will be described in greater detail herein.

Each of the limitations of the invention can encompass various embodiments of the invention. It is therefore anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and/or the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways.

The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including", "comprising", or "having", "containing", "involving", and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

Brief Description of the Figures FIG. 1 is a schematic showing the chemical synthesis pathway for the API of the invention.

FIG. 2 shows a number of impurities that may arise in the manufacture of the API. FIG. 3 shows a number of impurities that may arise in the API and/or the tablet formulation comprising compounds of Formula I. FIG. 4A and FIG. 4B show alternative flow charts relating to the tablet manufacture.

FIG. 5 is a schematic showing the chemical synthesis pathway for the 2-hydroxy and aldehyde degradants. FIG. 6A is a schematic showing the chemical synthesis pathway for the butyric acid degradant.

FIG. 6B is a schematic showing the chemical synthesis pathway for the 2-keto degradant. FIG. 6C is a schematic showing the degradation pathway from talabostat to the butyric acid and 2-keto degradants.

It is to be understood that the Figures are not required for enablement of the invention.

Detailed Description of the Invention

Active pharmaceutical ingredient (API)

The active pharmaceutical ingredient (API) of the invention is referred to interchangeably as PT-100, talabostat (USAN), and [(2R)-I-I [(2S)-2-amino-3 -methyl- 1- oxobutyl]-2-pyrrolidinyl]boronic acid. Talabostat has a CAS registration number of 149682- 77-9. The API has the following structure:

In most clinical formulations, talabostat is provided as a salt form. Preferably, the salt form is made by combining talabostat as a free base with methane sulphonate salt. The salt form is referred to herein as talabostat mesylate. Talabostat mesylate has a CAS registration number of 150080-09-4. Talabostat has a molecular formula Of C₉H₁₉BN₂O₃ ^• CH₃SO₃H. The molecular weight of talabostat as a free base is 214.1 g/mol and as the methane sulphonate salt is 310.2 g/mol. It takes the form of a white to off-white solid (e.g., powder). The structure of talabostat mesylate (Formula I) is as follows^":

H₂SO₃H Using hot state microscopy and differential scanning calorimetry (DSC), talabostat has a melt range of about 150°C-200°C, or about 160°C-190°C, or about 170°C-180°C. The compound is freely soluble in water and ethanol (i.e., > 100 mg/ml), slightly soluble in isopropyl alchohol (e.g., 30-70 mg/ml or 40-50 mg/ml), and practically insoluble in acetonitrile, acetone and ethyl acetate (i.e., < 0.1 mg/ml) at 22+/-3⁰C. A 2 mg/ml solution of talabostat in water has a pH of about 3.5-4.1, preferably about 3.8.

The API is a single enantiomer having an R, S configuration as shown below:

Chiral purity derives from the use of-(tert-butoxycarbonyl)-L-valine as a starting material and NTO 189 as an intermediate in the synthesis reaction.

Talabostat can exist as both linear and cyclic forms. The equilibrium between the linear and cyclic forms is dependent upon pH and matrix. The structure of linear (free base) talabostat is as follows:

The structure of cyclic (free base) talabostat is as follows:

H

The API when synthesized according to the protocol of FIG. 1 and described below may be more than 98% linear, more than 98.5% linear, more than 99%, or more than 99.5% linear. API Method of Manufacture

The API is synthesized as shown in FIG. 1. The starting materials for making the API are N-(ter-Butoxycarbonyl)-L-valine (i.e., BOC-L-valine), (lS,2S,3R,5S)-(+)-pinanediol, and pyrrolidone. The pinanediol controls the chiral purity of the API since it defines the chiral center at the carbon-boron bond. The BOC-L-valine similarly defines the chiral center at the carbon-nitrogen bond on the valine substituent. The remaining compounds used in the synthesis method are reagents, solvents, or other miscellaneous compounds.

Once synthesized, the API is confirmed using a variety of analyses including HPLC, differential scanning calorimetry (DSC), head-space gas chromatography (GC-HS), capillary electrophoresis (CE), X-ray powder diffraction (XPRD), Raman, NMR (¹H), and mass spectroscopy.

Clinical (Tablet) Formulation of API

In addition to the API, the invention also provides compositions and methods relating to the formulation of the API into a clinical grade product.

Fillers and Filler Particles

The invention envisions pharmaceutical preparations comprising filler particles, and more particularly agglomerated filler particles. The particles comprise the compound of Formula I, fillers and pH modifying agents. In preferred embodiments, the particles do not comprise oxidizing constituents (ingredients) as these can lead to the degradation of talabostat. Thus the particles may comprise the compound of Formula I, fillers and pH modifying agents in the absence of oxidizing agents. Some embodiments provides particles that consist essentially of the compound of Formula I, fillers and pH modifying agents. In the context of these particles, "consist essentially of or "consisting essentially of means that the particles do not contain components that materially alter the compound of Formula I. The compound of Formula I is materially altered, for example, when it significantly degrades, for example via oxidative and hydrolytic cleavage, or when it contains a significant level of impurities. Degradation is usually accompanied by a loss of activity. Thus, degradation can be measured by, for example, measuring activity (e.g., in an in vitro assay of DP-IV inhibition, as described in U.S. Patent Nos. 5462928 and 5965532, and U.S. Patent Application Publication Nos. US20040077601A1, US20060063719A1, and US20060094693A1) . Degradation and/or impurities can be measured by analyzing the sample directly by, for example, HPLC, as described herein. In this latter instance, degradation or impurity is observed by the presence of peaks that do not correspond to talabostat. The level of degradation or impurity is measured by comparing the areas under the curve for the talabostat peak and the additional peaks. Significant degradation or significant impurity, in the context of the filler particles, means that the talabostat peak represents less than 90% of the total area under the curve. In some embodiments, significant degradation or significant impurity, in the context of filler particles, means that a level of degradation of talabostat or the level of impurity is more than that measured in particles consisting of lactose monohydrate, microcrystalline cellulose, and phosphoric acid/sodium phosphate salt (about pH 2.2). Since talabostat is known to degrade by oxidative and hydrolytic cleavage, a particle consisting essentially of talabostat, fillers and pH modifying agents will lack compounds that cause its oxidative and hydrolytic cleavage in excess of that which occurs in the presence of lactose monohydrate, microcrystalline cellulose, and phosphoric acid/sodium phosphate salt (about pH 2.2). It should be understood that the level of tolerated degradation of talabostat (and thus the definition of significant degradation) and the level of tolerated impurities will depend upon the composition being analyzed. In the context of the API composition, significant degradation or significant impurity means that the talabostat peak represents less than 98% of the total area under the curve. In the context of the tablet composition, significant degradation or significant impurity means that the talabostat peak represents less than 90% of the total area under the curve.

There may be one or more fillers used in these particles. Examples of suitable fillers include lactose, microcrystalline cellulose, sucrose, glucose, mannitol, sorbitol, and dibasic calcium phosphate. These are commercially available from a number of sources including but not limited to Pharmatose, Avicel, Emcompress, and the like. In one preferred embodiment, the fillers are lactose monohydrate (e.g., Pharmatose 100M) and microcrystalline cellulose (e.g., Avicel PHlOl). The ratio of these fillers (by weight) can vary but in a preferred embodiment is about 9:1 (lactose monohydrate to microcrystalline cellulose). It is to be understood that virtually all ingredients used in the clinical grade product should be clinical grade themselves. Clinical grade materials may have the National Formulary (NF), U.S. Pharmacopia (USP) or other designation, indicating that they meet the associated specifications. The particles can also be defined physically. These physical attributes are generally the result of the process used to synthesize the particles. Briefly, the synthesis process involves spraying a solution containing the Formula I compound and preferably the acidic salt (and possibly the pH modifying acid) onto a mixture of filler particles, including for example soluble lactose monohydrate filler particles and insoluble microcrystalline cellulose filler particles. When the solution contacts the soluble filler particles, the surface of such particles dissolves to an extent that allows agglomeration of filler particles to each other. As a result, the filler particles both before and after agglomeration (i.e., the agglomerated particles) are coated over their surface with the compound of Formula I and optionally the acidic salt. "Coated over their surface" as used herein means that the compound of Formula I, and optionally the acidic salt, is present on or slightly imbedded within the surface of the soluble and insoluble particles. The resultant agglomerated particles therefore will contain the compound of Formula I, and optionally the acidic salt, on their surface as well as internally by virtue of the coating of the soluble and insoluble filler particles. Based on the method of manufacture, however, the compound of Formula I, and optionally the acidic salt, can be present on any region on the surface rather than any discrete portion thereof (i.e., the compound is expected to be generally equally distributed on the surface of the particle). The ratio of the compound of Formula I to the filler particles is as described herein.

In some important embodiments, the particles including the agglomerated particles are free of compounds that would lead to the degradation of the compound of Formula I. Such compounds have an electron withdrawing and/or oxidative effect on the boronic acid of the compound of Formula I, thereby leading to its degradation and the appearance of some of the impurities mentioned herein. In preferred embodiments, the filler particles are free of hydroxypropylmethylcellulose.

pH Modifying Agents

There may be one or more pH modifying agents present in or used in the synthesis of the filler particles. The pH modifying agents may be acid and base pairs but are not so limited. Preferably, the pH modifying agents are able to buffer a solution at a pH of about 1- 4, more preferably 1.5-2.9, and even more preferably about 1.9-2.5, and most preferably a pH of about 2.2 (e.g., +/- 10% or +/- 5%). In some embodiments, the pH may be 2.1 , 2.2 or 2.3. If an acid-base pair is used, the acid is used as need for pH adjustment. As a result, the salt is referred to herein as an acidic salt. In one embodiment, the pH modifying salt is sodium phosphate (e.g., monobasic, monohydrate) and the pH modifying acid (if present) is phosphoric acid. (e.g., 85%). Other salt-acid pairings are also envisioned by the invention. The ratio of filler to pH modifying agent may vary. In a preferred embodiment, the ratio of microcrystalline cellulose to sodium phosphate (monobasic, monohydrate) is about 158.

Formula I Compound

The amount of free base of compound of Formula I present in such particles may vary but is generally between about 0.05% and 0.4%, and more preferably between about 0.1% and 0.2%, expressed as a percentage of weight relative to the weight of the filler(s). In one embodiment, the free base is present in such particles in an amount of 0.11 % w/w of lactose monohydrate and microcrystalline cellulose combined. The amount of the compound of Formula I or its free base can also be expressed relative to the weight of a single filler such as, according to some embodiments, lactose monohydrate. In this regard, in one embodiment, the amount of the compound of Formula I present in the particles may be between 0.05% and 0.4%, including between about 0.1 % and 0.2%, and more preferably about 0.12% w/w. It is to be understood that these ratios are guidelines and that the invention embraces various modes of expressing the amount of Formula I compound (or other ingredients) in the particles.

In the tablet formulations provided herein, preferably the compound of Formula I (salt form) represents about 0.15% w/w relative to the entire tablet weight, whereas the free base represents about 0.1% w/w.

Thus, in one aspect, the invention provides a pharmaceutical that comprises the above- noted particles. The particles may comprise talabostat mesylate even though the preceding discussion provided ratios based on free base weights. One of ordinary skill in the art will be able to determine ratios based on total weight of talabostat mesylate given the weights provided herein.

In some important embodiments, the compound of Formula I is protected from oxidation. One way of accomplishing this is through the use of particles that are not oxidative relative to the compound of Formula I. That is, the constituents of the particles will not react in an oxidative manner with the compound of Formula I, including the boronic acid moiety of such compounds. As an example, the compound of Formula I is coated on or slightly embedded within particles free of (-OCH₂CHOHCH₃) groups, particles free of hydroxypropylmethyl cellulose, and/or particles free of vinyl pyrrolidone or crosslinked homopolymer of N-vinyl-2-pyrrolidone.

Other ingredients may be mixed with the particles to arrive at a final pharmaceutical preparation. These include binders such as but not limited to pregelatinized starch (e.g., Starch 1500), sucrose, cellulose, acacia, Tragacanth, and polyvinyl pyrrolidine, disintegrants such as but not limited to crospovidone (i.e., cross-linked N-vinyl-2-pyrrolidone), sodium starch glycolate, sodium carboxymethyl cellulose, sodium alginate, and agar, and lubricants such as but not limited to stearic acid, magnesium stearate, talc, and silica. Preferably, the pharmaceutical preparation is prepared into a tablet form. Thus, in one aspect, the invention provides a pharmaceutical tablet that comprises particles consisting essentially of a compound of Formula I, lactose (e.g., lactose monohydrate), microcrystalline cellulose, and an acidic salt. These particles are mixed with one or more of a binder, a lubricant and a disintegrant, and then compressed into a tablet form. It is to be understood that the particles may be mixed with just a binder, just a disintegrant, just a lubricant, a binder and a disintegrant, a binder and a lubricant, a disintegrant and a lubricant, or a binder, a disintegrant and a lubricant. The compound of Formula I may be present in amounts by weight relative to the other particles constituents of lactose, monocrystalline cellulose and/or acidic salt, as stated herein.

The tablets may comprise one or more lubricants. A suitable lubricant is stearic acid. Other lubricants include magnesium stearate, talc, and silica. The lubricant may be present in a w/w ratio relative to total filler ranging from about 55:1 - 75:1, more preferably about 65:1 - 70: 1 , and most preferably about 67: 1. The lubricant amount may also be expressed as a w/w ratio relative to lactose ranging from about 50:1 - 65:1, more preferably about 55:1 - 60: 1 , and most preferably about 58:1. The tablets may comprise one or more disintegrants. A suitable disintegrant is crospovidone. Other disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, sodium alginate, and agar. The disintegrant may be present in a w/w ratio relative to total filler ranging from about 30:1 - 45:1, more preferably about 35:1 - 40:1, and most preferably about 38:1. The disintegrant amount may also be expressed as a w/w ratio relative to lactose ranging from about 25:1 - 50:1, more preferably about 37:1 - 43:1, and most preferably about 40: 1.

The tablets may comprise one or more binders. A suitable binder is pregelatinized starch. Other binders include sucrose, cellulose, acacia, Tragacanth, and polyvinyl pyrrolidone. The binder may be present in a w/w ratio relative to total filler ranging from about 85:1 - 115:1, more preferably about 95 : 1 - 105 : 1 , and most preferably about 100: 1. The binder amount may also be expressed as a w/w ratio relative to lactose ranging from about 75:1 - 95:1, more preferably about 80:1 - 90:1, and most preferably about 85:1. The resultant tablets may have any dosage strength of the compound of Formula I including about 50 μg, about 100 μg, about 150 μg, about 200 μg, about 250 μg, about 300 μg, about 350 μg, about 400 μg, or more. In some preferred embodiments, the tablets are of about 200 μg or about 300 μg dosage strengths. The invention contemplates that the amount of talabostat in these tablets will vary due to manufacturing processes. Thus, the amount of talabostat may vary by at least +/- 20%, at least +/- 10%, at least +/-5%, at least +/- 1 % of the desired amount.

The tablets may be shaped in any way that is suitable in the pharmaceutical arts include round, oval, oblong, capsule- or caplet-shaped, and the like. Additionally, the tablets may be biconvex. The compound of Formula I may be present in a mixture of cyclic and linear forms in the tablet. The percentage of cyclic form (relative to total Formula I compound) may be up to 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, or more. That is, the percentage of cyclic form may range from 5% to 40%, from 5% to 35%, from 10% to 30%, from 15% to 25%, or from 20% to 25%, including 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38% and 39% cyclic. The manufacturing processes described herein can result in lineaπcyclic ranges of about 15-40% cyclic (relative to total compound).

Tablet Man ufacture

The invention further provides a method for manufacturing the afore-mentioned pharmaceutical tablets. FIG. 4 shows alternative flow charts that illustrate manufacturing processes for the pharmaceutical tablets. The processes involve the steps of (a) preparation of a granulation media that comprises the compound of Formula I in solution (e.g., water) with the pH modifying agents, (b) preparation of the granulation powder blend (i.e., filler material) comprising the filler particles, (c) "spraying" of the granulation media onto the granulation powder blend by a wet granulation process thereby producing the agglomerated filler particles referred to herein, (d) optionally wet sieving the agglomerated filler particles, (e) drying the agglomerated filler particles by a hot air process (e.g., a fluidized bed dryer), (f) performing a granule size exclusion by a dry sieving process, (g) combining the sized granule preparation with one or more binders, disintegrants, and lubricants, (h) compressing the final mixture into the pharmaceutical tablet form. The compound of Formula I is present in the granulation media at a concentration of between about 4000 mg/liter and 7000 mg/liter, about 4250 mg/liter and 6400 mg/liter, or about 5000 mg/liter and 5500 mg/liter, or about 5200 mg/liter and 5400 mg/liter, or about 5300 mg/liter (including but not limited to 5311, 5312, 5313, 5314, 5315, 5316, 5317, 5318, 5319, 5320, 5321 and 5322 mg/liter), provided that the correct absolute amount of the Formula I compound is ultimately sprayed onto the filler particles. The spraying/wet granulation technique is commonly used when the active ingredient (i.e., the compound of Formula I) is present in very small quantities, as is the case here, to ensure a more uniform mixing of active ingredient with the inactive ingredients.

One or more of these steps may be accomplished in a single batch or in multiple sub- batches. For example, the wet granulation, dry granulation and granule sizing processes can be run in one or more batches (i.e., sub batch). Wet sieving can be achieved using for example a No. 12 mesh sieve. Dry sieving can be achieved using for example a No. 20 mesh screen or a Quadro Mill with Screen. Blending of the dry granules with excipients such as the binder(s), disintegrant(s) and/or lubricant(s) can be accomplished simultaneously or consecutively. The tablets are compressed to any level of hardness that is acceptable in the pharmaceutical arts. For example, the level of hardness can be in the range of 5-12 kilopascals (kp).

The pH of the granulation media is generally between about 1-4, more preferably between about 1.5 and 2.9, and even more preferably about 2.0-2.4, and most preferably about 2.2. These pHs can be achieved using sodium phosphate (monobasic, monohydrate) as the acidic salt and phosphoric acid as its accompanying acid. Phosphoric acid is used as required to adjust pH accordingly.

The filler particles may comprise lactose (e.g., lactose monohydrate) and/or microcrystalline cellulose. The filler is preferably non-oxidative with respect to the boronic acid moiety of the compound of Formula I in the presence of both an acid salt and trace amounts of water at 2-8 degrees centigrade in order to ensure stability during storage.

The agglomerated filler particles can be blended with a binder and a disintegrant, a binder and a lubricant, a disintegrant and a lubricant, or a binder, disintegrant and lubricant. Tablet Testing

Batches can be tested prior to compression. A batch may be tested for physical appearance, blend uniformity, bulk/tapped density, and water content. Testing can also occur following compression into tablets. These latter tests include content uniformity, size, hardness, and friability.

The invention also contemplates certain standards or specifications for the release of pharmaceutical tablets. These include measures of appearance, identification, potency, impurity content, chiral purity (e.g., as assessed by CE), dissolution, water content, weight variation, friability, hardness, disintegration, blend uniformity, and the like. Tablets suitable for release may meet one or more of the following parameters: a white to off white appearance, a retention time within 5% of the retention time of a standard, a potency that is 95% - 110% (including 95%- 105%) of the expected amount, individual impurity level of less than or equal to 3% or total impurity level that is less than or equal to 10%, dissolution factor (Q) of 65% at 45 minutes, and blend uniformity of 85-115% for individual tablets and/or 90- 110% as a batch average. The characteristics of an exemplary tablet batch are described in the Examples.

API and Tablet Impurities As used herein, impurities are undesired compounds present in the API or the tablet formulation. Impurities include but are not limited to degradation products of talabostat. They do not include compounds that are intended to be present (and in most cases, are deliberately added) to the sample, including salts, pH modifying agents, fillers, lubricants, disintegrants, etc. Impurities in the reaction mixtures and final product can be organic impurities, inorganic impurities, or residual solvents. Organic impurities can result from the manufacturing process or the storage protocol. These can include starting materials, byproducts, intermediates, degradation products, reagents, ligands and catalysts. Inorganic impurities can also result from the manufacturing process or the storage protocol. These can include reagents, ligands, catalysts, heavy metals or other residual materials, inorganic salts, and other materials such as filter aids. Solvents are organic or inorganic liquids used in the preparation of solutions or suspensions during API synthesis. Process-related impurities are shown in FIG. 2. Impurity 1 is a side product of the Stage 6 reaction deriving from phenylboronic acid, and can measured by HPLC. Impurity 2 is an undesired enantiomer of the Stage 4 intermediate, and can measured by HPLC. Impurity 3 is a pinanediol boronate ester of talabostat, and can measured by HPLC. Impurities 4-6 are undesired talabostat enantiomer and diastereomers, and can measured by CE or HPLC. Impurity 7 is phenylboronic acid. Impurity 8 is an undesired enantiomer of the Stage 5 intermediate, and can measured by HPLC. Impurity 9 is BOC-protected talabostat, and can measured by HPLC. Impurity 10 is talabostat-like structure without the boronic acid functional group, and can measured by HPLC. It is to be understood that these impurities may exist as free or salt forms, although for the sake of simplicity they are shown as free forms herein. It is expected that upon analysis such as an HPLC analysis, the free base forms will be analyzed as the salts will dissociate therefrom. Accordingly, peaks on for example an HPLC will correspond to a free base form rather than a salt form of talabostat or one of its impurities. Impurities include degradation products (i.e., degradants) of talabostat. Degradation products may result from the oxidative and hydrolytic cleavage of the carbon-boron bond of talabostat. Impurities that are degradation products are shown in FIG. 3. Impurities 11 and 12 have been observed in talabostat final product. Impurities 13, 14, 16, 17 and 18 are hypothetical as they have not yet been observed in aged or forced degradation samples. Some of these are commercially available (e.g., impurities 13 and 14) while others must be synthesized in order to be used as or in reference samples (e.g., impurities 11 and 12). Impurities 11 and 12 can be synthesized according the methods provided in Example 5. The presence of degradants can be determined using for example HPLC.

Oxidative cleavage of the carbon-boron bond in talabostat results in the 2-hydroxy compound (impurity 19). Impurity 19 and impurity 20 (i.e., the aldehyde compound) undergo pH-dependent interconversion, and thereby exist in an equilibrium. As a result, the presence of one of these degradants may be an indication of the presence of the other. The oxidation product of this latter degradant is the 2-keto compound (impurity 11, (ιS)-l-(2-amino-3- methylbutyryl)-pyrrolidin-2-one). Impurity 11 further degrades, via oxidation, causing ring opening and formation of the butyric acid degradant (impurity 12, (S)-4-(2-amino-3- methylbutyrylamino)-butyric acid) and/or the formation of 2-pyrrolidinone and valine (impurities 13 and 14). Impurity 19 in some instances is the main degradant seen in talabostat API samples. Forced degradation conditions have also been used to test the integrity of the API. These conditions may involve elevated temperature (e.g., 14O⁰C) and/or elevated humidity (e.g., 75% relative humidity (RH)), photolytic, acidic, basic and oxidizing conditions, possibly for extended periods of time. Such conditions can result in the formation of some impurities including undesired talabostat diastereomers. These conditions also can result in the formation of additional peaks with relative retention times of 1.06 and 1.16 (i.e., RRT 1.06 and 1.16). When impurity 23 (i.e., the 8-membered ring imine) is placed in aqueous solution, HPLC peaks at RRT 1.06 and 1.16 are observed. Thus the RRT 1.06 and 1.16 peaks may be degradants of the 8-membered ring imine. These peaks can also be derived from impurities 19 and 20.

In-process and after-process controls are used to ensure that the desired API is synthesized, as well as to determine the level of impurities present. The method of analysis will depend upon the purpose of the analysis. For example, high pressure liquid chromatography (HPLC) is used at the end of each stage in FIG. 1 to determine if the reaction has gone to completion as well as the purity of final product. Other analysis methods can be also be used including but not limited to gas chromatography. Following Stage 6, the API may be subjected to a number of other analyses, each aimed at determining a particular parameter. For example, chiral purity is determined using capillary electrophoresis (CE) with suitable products having < 0.5% (of total area) of an undesired enantiomer. It is to be understood that total impurity content is the sum total of impurities in a talabostat sample (whether it is an API or a tablet sample) whether the identity of each is known or unknown. It can be measured in a number of ways, as will be apparent to those of ordinary skill. As an example, it can be determined by summing the areas (under the curve) of each non-talabostat peak in an HPLC retention time graph and dividing that area by the total areas under the curve for the graph (thereby resulting in a percentage under the curve measurement).

With respect to identification and purity of the API, suitable products will give rise to talabostat peaks that are > 98% (of area) with no single impurity > 1.0% (of area) and with total impurity content of < 2% (of area). The main peak is defined by retention time and the identity of the peak is determined by comparing it to a reference standard of talabostat. API identification can also be accomplished using infrared spectroscopy and ¹H-NMR by comparison with a reference standard spectrum. When stored for extended periods of time and/or under particular conditions, API samples having a stability characterized by < 4% (of total area in a HPLC graph) total impurity content and < 2% (of total area in a HPLC graph) for any single impurity are preferred. The invention provides methods for manufacturing a material comprising a compound of Formula I. In one aspect, the method is a method of manufacturing a material consisting essentially of a compound of Formula I. The method comprises (a) obtaining a composition containing the compound of Formula I, (b) purifying the composition by chromatography, recrystallization or a combination thereof, (c) conducting HPLC on a sample of purified composition using a known amount of impurities as a standard, and (d) determining the presence or absence of the impurities in the sample.

The impurities can be any of those listed herein or known at the time of analysis. Impurities may be present in the API and/or the tablet formulation. Some notable impurities are shown below. Compound A is the butyric acid degradant (impurity 12 in FIG. 3) having an HPLC retention time (relative to talabostat) (i.e., an RRT) of about 0.89 as measured using the early eluting HPLC method described in Example 6 with the API having a relative retention time of 1.00. Compound A can be formed by deliberately (or otherwise) oxidizing the API. It has a structure as follows:

Compound B is the 2-hydroxy pyrrolidine degradant (impurity 19 in FIG. 3) having an HPLC RRT of about 1.05 as measured using the early eluting HPLC method described in Example 6. Compound B is observed in deliberately oxidized batches of the API. Compound B is also a main degradant in the talabostat tablets. It has a structure as follows:

Compound C is the 2-hydroxy pyrrolidine aldehyde degradant (impurity 20 in FIG. 3) having an HPLC RRT of about 1.15 as measured using the early eluting HPLC method described in Example 6. Compound C is observed in deliberately oxidized batches of the API. Compound C is also a main degradant in the talabostat tablets. It has a structure as follows:

Compounds B and C may be observed following tablet manufacture even if they are not present in the API, possibly as a result of the compression stage.

Compound D is the 2-keto degradant (impurity 11 in FIG. 3) having an HPLC RRT of about 0.90 as measured using the early eluting HPLC method described in Example 6. Compound D can be formed by deliberately (or otherwise) oxidizing the API. It has a structure as follows:

Compound E is an undesired talabostat enantiomer (impurity 4 in FIG. 2) having an HPLC RRT of about 1.31 as measured using the early eluting HPLC method described in Example 6. The undesired talabostat enantiomer can be separated from the desired enantiomer and from the undesired diastereomers using capillary electrophoresis as described in Example 9. The undesired enantiomer has a migration time in the capillary electrophoresis method of about 22.8 minutes. It has a structure as follows:

Compound F is phenylboronic acid (impurity 7 in FIG. 2). It is one of the main impurities in the talabostat tablets described herein. It has an HPLC RRT of about 0.30 using the early eluting HPLC method described in Example 6 and a structure as follows:

Compound G is one of the talabostat diastereomer (impurity 5). This compound can be seen using the early eluting HPLC method described in Example 6. Using this method, it elutes with a RRT of 1.31. However it co-elutes with the other talabostat diastereomer (i.e., Compound H) using this technique. In instances where it is only desired to determine the presence of either or both diastereomers without regard to distinguishing between them, then the HPLC method is sufficient. It is possible to resolve these diastereomers from each other using capillary electrophoresis. Compound G has a capillary electrophoresis migration time of 23.3 minutes using the method described in Example 9, and a structure as follows:

Compound H is the other talabostat diastereomer (impurity 6). This compound can be seen using the early eluting HPLC method described in Example 6. Using this method, it elutes with a RRT of 1.31. However it co-elutes with the other talabostat diastereomer (i.e., Compound G) using this technique. In instances where it is only desired to determine the presence of either or both diastereomers without regard to distinguishing between them, then the HPLC method is sufficient. It is possible to resolve these diastereomers from each other using capillary electrophoresis. It has a capillary electrophoresis migration time of about 22.4 minutes using the method described in Example 9, and a structure as follows:

NH₂

Compound I is pinane protected version of talabostat (impurity 3). It has an HPLC RRT of about 4.16 using the method described in Example 6 (late eluting method), and a structure as follows:

Other impurities include but are not limited to pyrrolidinone, and L-valine.

"Purifying the composition" according to this aspect means that the percent by weight of compound of Formula I is increased and the percent by weight of impurities is decreased as a result of the physical manipulation. Chromatography can be any form of chromatography available and suitable to this purpose. It includes but is not limited to HPLC. Recrystallization can be used to increase the relative percentage of R,S stereoisomer over other isomeric forms present in the composition.

Following the purification step, the composition is analyzed for the presence of impurities. This can be done by comparing the impurity content of the composition to one or more reference standards that contain a known amount of one or more impurities that are being assayed. This can also be done by adding a known amount of one or more impurities to the composition and measuring the excess amount of impurities in the sample (i.e., the amounts in excess of the added amounts).

It is envisioned that the purification and analysis steps may be repeated one or more times until a desired impurity level is achieved. The impurity level may be assessed on a single impurity or a total impurity basis. For example, the purification and/or analysis steps may be performed until the impurity level is less than or equal to 2.0%, 1.0%, 0.5%, 0.1%, 0.4%, 0.3%, 0.2%, 0.15%, 0.1%, 0.05%, or even absent from the purified composition. These levels may be determined using HPLC. Generally, HPLC has a detection limit of 0.01-0.03% and a quantitation limit of 0.03%. The level of impurity may be recorded per purification and analysis cycle, or it may be recorded once the final desired level is reached. The levels of impurity (either single or total) may also be indicated on any packaging that houses the purified composition.

In another aspect, the invention provides a method for determining an impurity in a material consisting essentially of Formula I. The method comprises dissolving the material in a solution, injecting that solution into an HPLC column and obtaining an HPLC chromatogram. The amount and potentially identity of the impurity is then determined through analysis of the chromatogram. The impurity may be any of those described herein or otherwise known at the time of analysis including but not limited to Compound A, B, C, D, E, F, G, H and I. The method may also be used to determine the presence and/or amount of more than one impurity.

The amount of impurity in the material can be determined by (i) identifying a peak on the test (or material) chromatogram that corresponds to a peak on a control (or impurity reference) chromatogram, (ii) identifying a peak on the test (or material) chromatogram that corresponds to a relative retention time of the impurity, and/or (iii) identifying a peak on the test (or material) chromatogram that corresponds to a known amount of a spike of the impurity. In another aspect, the invention provides a method for determining the presence and/or level of one or more impurities in a material consisting essentially of Formula I (as shown above). The method comprises injecting into an HPLC column a sample containing the material to be tested and containing a known amount of one or more known (now or at the time of testing) impurities as reference compounds. For example, the known reference compound may be any one of Compounds A, B, C, D, E, F, G, H or I, or it may be another known impurity. Any number and combination of known reference compounds may be added to the material (e.g., Compounds G and H may be combined), as the invention is not limited in this regard. The method further comprises obtaining an HPLC chromatogram for the sample with the reference compound (i.e., the test chromatogram), and determining the presence and/or the amount of the impurity in the material based on that chromatogram. The presence and/or amount of the impurity may be determined in any of a number of ways. For example, these may be determined by comparing the sample chromatogram to a chromatogram of the isolated reference compound (i.e., the reference chromatogram) when present in a comparable diluent (to form a reference sample). Preferably, the isolated reference compound is present in the reference sample at the same concentration as in the "spiked" test sample. In this way, the amounts of impurity can be compared potentially by direct overlay of the chromatograms. In an alternative method, the reference chromatogram can be a chromatogram generated prior to analysis of the test sample. Various reference chromatograms can be generated based on amount, type and number of impurities, and stored for future comparison with test chromatograms.

In still another aspect, the invention provides a method for evaluating the purity of a Formula I composition. The method comprises injecting a reference solution containing one or more impurities, including but not limited to Compound A, B, C, D, E, F, G, H or I into an HPLC column under a set of conditions to obtain a first HPLC (control or impurity reference) chromatogram. The amount and identity of the impurities in the reference solution generally are known (e.g., are previously determined). The method further involves injecting a sample solution comprising Formula I into the HPLC column under said set of conditions to obtain a second HPLC (test or sample) chromatogram. A comparison of the two chromatograms is then used to determine the amount of the compounds in the sample solution.

As stated herein, the amount of the compounds present in the sample solution may then be recorded. Such recording may involve reporting the impurity amounts to one or more parties, including the FDA. The amounts of impurities in the sample solution reporting in this way include but are not limited to less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2.0%, less than or equal to 1.0%, less than or equal to 0.5%, less than or equal to 0.15%, less than or equal to 0.1%, less than or equal to 0.05%, or even lower amounts. The percentage signs refer to % of the area under the curve represented by the impurity relative to the compound of Formula I.

The pharmaceutical tablet may also contain impurities. Such impurities may be carried over from the API (i.e., talabostat, the compound of Formula I) or may derive from the excipients or the pharmaceutical formulation synthesis process itself. Excipient HPLC peaks are usually also present in placebo controls. In one exemplary formulation, excipient peaks were observed having an HPLC RT ranging from 1-4 (with the exception of 1.72), 7.5-9 (with the exception of 8.6), and 17-18. There may be one or more impurities in the pharmaceutical, including any of those described herein or any known prior to the synthesis of the pharmaceutical. Impurities may be detected using for example HPLC. The level of impurity may be expressed as a level of total impurity or a level of impurity for one or more single impurities. An acceptable level of impurity will also depend on whether the composition is the API or the tablet. The level of any single impurity in the API, as described above, is preferably less than 1%, less than 0.5%, 0.1%, or even undetectable, and the total level of all impurities combined is preferably less than or equal to 2%, 1.5%, 1.0%, 0.5%, or 0.1 % or even undetectable.

The level of any single impurity in the tablet may be less than or equal to 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or even undetectable (preferably using a detection system with sufficient sensitivity). The total level of all impurities may be less than or equal to 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or even undetectable (preferably using a detection system with sufficient sensitivity).

The identity of certain peaks on the HPLC can be determined using commercially available reagents (e.g., 2-pyrrolidinone and L-valine are both available from commercial sources), or by synthesizing the expected impurity de novo. For example, the 2-hydroxy and aldehyde degradants are made according to a method described in Example 4. The 2-keto degradant and the butyric acid degradant can be made as described in Example 5.

In yet another aspect, the invention provides a pharmaceutical preparation comprising a tablet of the compound of Formula I as defined by its level of impurity following exposure to one or more conditions. As an example, in one embodiment, the tablet is stored at 2-8⁰C for a period of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months) and which has a total amount of impurities (including Formula I degradation products) that does not exceed 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the compound of Formula I in the preparation. The percentage refers to the area under the curve of all impurity peaks relative to the peak of the Formula I compound. Formula I degradation products are impurities resulting from the degradation of the Formula I compound. These include but are not limited to Compound A, B, C, D, E, F, G, H and I.

In even more stringent embodiments, the total amount of Formula I degradation products does not exceed 2.5%, 2.0%, 1.5%, 1.0%, 0.5%, or 0.25% of the amount of Formula I compound (as determined for example by area under an HPLC curve). The amount of single Formula I degradation products in some instances may not exceed 1%, 0.5%, 0.25%, 0.1%, 0.05%, or may be undetectable (using a detection system with sufficient sensitivity).

In one aspect the invention provides compositions comprising an impurity from those recited herein in an isolated form. As used herein, an isolated form of an impurity is a form that lacks detectable levels of other impurities provided herein. The invention similarly contemplates combining such compositions in order to arrive at a composition that comprises two or more impurities. In this latter instance, at least one of the two impurities is provided herein. The other impurities may be ones identified at a later date. As will be apparent from the methods of the invention, such compositions can be used as reference standards for comparison of for example clinical materials prior to release or for regulatory purposes.

API and Tablet Storage and Stability

In view of the degradation profiles described herein, it is preferred that talabostat is stored in a dry, dark and cool environment. For example, a dry environment can be achieved by use of a desiccant in the container, such as a silica gel desiccant. The container may be made of high density polyethylene (HDPE). Talabostat may be placed first in a polyethylene bag which is placed in an HDPE container having a lid. The container can then be placed at - 18⁰C to -2O⁰C. Storage at these conditions has provided stability of the API without detectable degradation, indicating that at least the API can be stored at these conditions indefinitely.

The invention therefore provides in another aspect, a method for determining if a particular condition protects against or promotes degradation of a compound of Formula I. The condition may be one that is used for short term and/or long term storage, for transportation, and the like, although storage itself may also be a condition. The method involves applying the condition to a composition, including a pharmaceutical composition, comprising a compound of Formula I and determining whether one or more impurities are formed, or increased in amount, as a result of the condition. The presence and/or amount of impurity may be determined using any of the techniques described herein including but not limited to HPLC and CE. The impurity may be one of those described herein or one that is known at the time of the analysis. Examples of impurities that may be detected include but are not limited to Compound A, B, C, D, E, F, G, H and I, or any combination thereof, or any other impurity or combination of impurities from those described herein or known at the time of analysis.

The composition may be solid or liquid in form, although prior to analysis it will usually be preferable to dissolve the composition in a liquid carrier for ease of analysis. Notwithstanding this, the conditions to be applied to the composition include contact (and dissolution into) a liquid carrier such as but not limited to a pharmaceutically acceptable carrier. Other conditions include exposure to humidity, increased temperature (e.g., heating), decreased temperature (e.g., freezing), visible light or other forms of radiation (including X- ray and UV radiation), low or high pH environment, chemicals including preservatives, or a condition that may be applied in the course of processing the composition into a pharmaceutical dosage form. The condition may also be simply storing the composition for a period of time, optionally under optimal conditions.

The level of impurity may vary depending upon the severity of the condition. The level of impurity may also be recorded for example for the purpose of reporting stability and storage information to a third party such as but not limited to the FDA. The impurity identity and/or level may also or alternatively be indicated on a package that houses the composition. In this way, an end user of the pharmaceutical is able to determine whether the pharmaceutical has been stored improperly based on the presence and/or amount of one or more impurities. The level of impurity may also be set as a tolerated level of impurity (i.e., a level after which the pharmaceutical may be discarded or may be considered not suitable for clinical use). The level of impurity therefore may be less than or equal to 5%, 4%, 3%, 2%, 1% 0.5%, 0.25%, 0.15%, 0.1%, 0.05%, or even lower (e.g., undetectable using a method with sufficient sensitivity). The percentage represents a percent of area under the curve on a graph such as but not limited to an HPLC chromatogram as compared to the area of the under the curve for the compound of Formula I. The impurity may be one or more of those discussed herein or may be another known at the time of analysis. Such impurities therefore include but are not limited to Compound A (impurity 12), Compound B (impurity 19), Compound C (impurity 20), Compound D (impurity 11), Compound E (impurity 4), Compound F (impurity 7), Compound G (impurity 5), Compound H (impurity 6), and/or Compound I (impurity 3). Tablets can also be tested for stability for example after exposure to one or more conditions and/or after a period of storage (e.g., 1, 2, 3, 4, 5, 6 months or more) under normal or stressed conditions. Whether in bulk or in packages, the tablets are preferably stored between 2-8⁰C and protected from humidity.

Tablets manufactured according to the methods provided herein have been tested for stability following storage for 12-24 months at 2-8⁰C or for 6-12 months at accelerated conditions (e.g., 25⁰C and 60% relative humidity). Tablets stored for these times and under these conditions can be tested according to any number of criteria as shown herein including but not limited to appearance, potency, impurity/degradant content, dissolution, water content, and hardness.

API and Tablet Kits

The invention further contemplates packaged material, including kits that comprise the material containing the Formula I compound. For example, tablets can be stored in housing such as bags (e.g., double polyethylene bags) or containers (e.g., HDPE bottles). Placing the tablets in such housing is referred to herein as "packaging". Humidity can be reduced or avoided by including a desiccant such as a silica gel desiccant in the housing.

These packages or kits optionally comprise instructions and/or indicia. Such indicia may be on the package itself or contained within the package. These instructions and/or indicia may provide a range of information including identity of the drug substance by name, chemical formula and/or chemical structure, dosage strength, instructions for use including indication to be treated and/or recommended administration schedule, identity and/or amount of individual impurities, and/or amount of total impurities. The impurities may be identified in the indicia by chemical formula, chemical formula, RRT, or chemical or trade name.

The indicia may indicate that the one or more impurities are present in an amount that is less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2.0%, less than or equal to 1.0%, less than or equal to 0.5%, less than or equal to 0.15%, less than or equal to 0.1%, less than or equal to 0.05%, or lower for each impurity. Total impurity level may also be indicated. For example, the indicia may indicate that the total impurity amount is than or equal to 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or lower. The indicia may also indicate the percentage of the Formula I compound that is present in either a linear or cyclic form in the material. For example, the indicia may state that the cyclic form represents about 0.01%, 0.05%, 0.1%, 0.25%, 0.5%, 0.75%, 1% , 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% or more of the total amount of Formula I compound present in the material. The impurities include those described herein or known at the time of packaging.

These include but are not limited to Compound A, B, C, D, E, F, G, H and I.

Thus, in one aspect, the invention provides a kit comprising a package containing a pharmaceutical tablet comprising particles consisting essentially of a compound of Formula I, lactose (e.g., lactose monohydrate), microcrystalline cellulose, and an acidic salt (e.g., sodium phosphate). The particles may be blended with one or more of a binder, a lubricant and a disintegrant and then compressed. On or within the package there may be indicia indicating the chemical identity of the compound of Formula I.

HPLC and CE specifications Various aspects of the invention involve HPLC analysis. In some embodiments, the

HPLC column is a reverse phase column. In some embodiments, the HPLC column is eluted using a mobile phase comprising sodium dodecyl sulfate (SDS), phosphoric acid, sodium hydroxide, water, trifluoroacetic acid (TFA), and/or acetonitrile. The exact composition and percentages of mobile phase constituents may vary depending on whether the API or the tablet (or some intermediate formulation therebetween) is being analyzed. For example, in the case of API analysis, the mobile phase may consist of 25 mM SDS/acetonitrile (65/35 v/v) and 0.05% TFA. Exemplary HPLC and capillary electrophoresis separation and analysis methods are provided in the Examples.

Recording impurity identity and amount in written form

In these and various other methods embraced by the invention, it is further contemplated that the amount and/or chemical identity of the impurity present in the test sample is recorded. Such recordation can take any form including a hand-written form, a typewritten form, an electronic form, an online submission, a graphic form, a verbal form recorded on a tape or other recording medium, and the like. The invention is not to be limited in this regard.

Uses

The compositions of the invention are used for a number of indications including hematopoietic stimulation, immune response induction via induction of IL-I (e.g., as adjuvants to be combined with antigens or antibodies), and treatment of proliferative disorders (e.g., cancer). Subjects to be administered the compositions of the invention are those in need of hematopoietic and/or immune stimulation, those having an infectious disease, and those having a proliferative disorder such as cancer.

Thus, the invention embraces methods for treating cancer by administering any of the foregoing clinical formulations and compositions to a subject in need thereof in an effective amount to inhibit the cancer. In certain embodiments, the cancer may be non-small cell lung cancer, pancreatic cancer, melanoma (including metastatic melanoma), chronic lymphocytic leukemia, Non-Hodgkin's lymphoma, and breast cancer. In some embodiments, the method further comprises administering an anti-cancer chemotherapeutic agent to the subject prior to, at the same time as, and/or after the clinical formulation or composition of the invention. Examples of suitable chemotherapeutic agents can be found in U.S. application publication US 2005/02702703, and these are incorporated by reference herein. Examples include but are not limited to docetaxel (TAXOTERE), cisplatin, gemcitabine, pemetrexed (ALIMTA), erlotinib (TARCEVA), gefitinib (IRESSA), temozolomide (TEMODAR), carboplatin, cyclophosphamide or doxorubicin.

In other aspects, the invention embraces methods for stimulating immune responses comprising administering any of the foregoing clinical formulations and compositions to a subject in need thereof in an effective amount to stimulate an immune response. In some embodiments, the method further comprises administering an antigen or an antibody to the subject prior to, at the same time as, and/or after the clinical formulation or composition of the invention. Examples of suitable antibodies can be found in U.S. application publication US 2005/0084490, and these are incorporated by reference herein. Examples include but are not limited to rituximab (RITUXAN), bevacizumab (AVASTIN), cetuximab (ERBITUX), trastuzumab (HERCEPTIN), tositumomab (BEXXAR), or alemtuzumab (CAMPATH), mitumomab (BEC2), CeaVac, centuximab (IMC-C225), epratuzumab (LYMPHOCIDE), MDX-210, gemtuzumab ozogamicin (MYLOTARG), edrecolomab (PANOREX), pemtumomab (THERAGYN), Zamyl, and ibritumomab tituxetan (ZEVALIN). The invention also covers antibody fragments thereof. Subjects in need of immune stimulation may be those having or at risk of having an infectious disease such as a bacterial infection (e.g., anthrax infection) or a viral infection (e.g., SARS infection). Other infections are provided in U.S. application publication US 2005/0084490, and these are incorporated by reference herein.

The following Examples are provided to illustrate specific instances of the practice of the present invention and are not intended to limit the scope of the invention. As will be apparent to one of ordinary skill in the art, the present invention will find application in a variety of compositions and methods.

Examples Example 1: Synthesis of the API

The process shown in FIG. 1 has been used to synthesize small batches on the order of 350 g as well as large batches on the order of 6-20 kg. The process does not require isolation of the Stage 3 material and rather can proceed directly to Stage 4 without such isolation as shown. Stage 4 uses an in situ source of hydrochloride gas as generated by the reaction of acetyl chloride and isopropyl alcohol. Stage 6 used ethanol to increase crystallization control. If not otherwise defined in FIG. 1, pH adjustment is made using aqueous hydrochloric acid or sodium hydrogen carbonate.

Stage 1 fPreparation ofNTOlSoi To a solution of di-tert-butyl dicarbonate (3wt, 66.0kg ± 1%, 0.98eq) in tert-butyl methyl ether (3,7wt, 81.4kg ±5%) at 25-35°C is added a solution of pyrrolidine (l.Owt, 22.0kg, leq.) in tert-bvXy\ methyl ether (3.7wt, 81.4kg ±5%) over at least 45 minutes, maintaining the temperature in the range 25-35⁰C (gas evolution). This is followed by a tert- butyl methyl ether line rise (0.4wt, 8.8kg ±5%). The reaction mixture is stirred at 25-35°C until the reaction is complete.¹ The reaction mixture is then cooled to 5-10°C, quenched with water (l.Ovol, 22.0L ±5%) and the pH of the aqueous phase adjusted to pH 3.0-3.5 by adding

¹ Stage 1 In-process Testing: TLC (reaction completion). Preliminary pass criteria: <5% pyrrolidine and <5% di-tert-butyl dicarbonate. Expected reaction time is 1 to 4 hours. 0.5M aqueous hydrochloric acid solution (0.8-1.28vol, 17.6-28.2L), maintaining the temperature at 5-10°C. The reaction mixture is then heated to 20-25⁰C, and the pH of the aqueous phase re-checked and re-adjusted, if necessary, to pH 3.0-3.5. The aqueous phase is separated and the organic phase is washed with water (2vol, 44.0L ±5%). The pH of the aqueous phase is checked to ensure it is in the range 6.5-7.5 and if not, the organic phase is washed with additional water (2vol, 44.0L ±5%) and the aqueous phase is separated. The organic phase is stirred with sodium sulfate (1.5wt, 33.0kg ±5%) for 45-60 minutes at 20-25°C.² The reaction mixture is then filtered, the filter-cake is washed with tert-butyl methyl ether (2 x 1.5wt, 2 x 33.0kg ±5%), and the filtrates are combined and concentrated under vacuum at 35-45⁰C to 3vol (66L).³ The yield of the Stage 1 product (NTOl 86) is determined⁴ (42.2-52.8kg, 80-100%th, 192-240%w/w). tert-Butyl methyl ether (l.lwt, 24.2kg ±5%) is then charged and the Stage 1 product is ready to proceed directly to Stage 2.

Stage 2 (BIl (Preparation of NT0187-Part Bl) The Stage 1 product (NTO 186) in tert-butyl methyl ether (corrected for tert-butyl methyl ether [1 wt, 20kg, l.Oeq]) is charged to a vessel and the volume adjusted to 2.5vol with tert-butyl methyl ether. Tetrahydrofuran (3.5wt, 70kg ±5%) is charged and the solution cooled to -70 to -80⁰C. sec-Butyl-lithium (1.3M solution in cyclohexane) (5.2vol, 104L ±1 %, 1.15eq) is charged slowly while maintaining the internal temperature within the range -65 to -8O⁰C, over approximately 1 to 3 hours. tørt-Butyl methyl ether (0.4wt, 8.0kg ±5%) is then charged as a line rinse. Following completion of the addition, the temperature is established in the range -65 to -80⁰C and stirring continued for approximately 2 hours. Trimethyl borate (0.79wt, 15.8kg ±1%) is charged to the reaction mixture, maintaining the reaction temperature in the range -60 to -8O⁰C, followed by tetrahydrofuran (0.4wt, 8.0kg ±5%) as a line rinse. The resultant is warmed to 20-25⁰C over 14-16 hours and then quenched with aqueous hydrochloric acid solution (IM, approximately 6.5-8.0vol, 130— 160L) to a pH of 3.0 to 3.5. The resulting biphasic mixture is stirred at 20-25⁰C for 60-90 minutes.

² Stage 1 In-process testing: Karl Fischer analysis for water content. If water content is >2%, repeat the sodium sulfate addition step. Repeat until analytical result is <2%.

³ Stage 1 In-process Testing: Karl Fischer analysis for water content and ¹H NMR for /erf-butyl content. If water content is >2% or tert-butanol content is >10%w/w, charge ter t-buty\ methyl ether (3 wt, 66kg ±5%), concentrate to 3vol under vacuum at 35-45°C and reanalyze.

⁴ Stage 1 In-process Testing: ¹H NMR to determine yield of product in solution. The phases are separated, the organic phase is retained, and the aqueous phase extracted with tert-butyl methyl ether (2x 2.2wt, 2x 44kg ±5%).⁵ The combined organic phase is washed with water (3vol, 6OL ±5%), and the pH of the aqueous layer checked; if the pH is not in the range of 6.5-7.5, further water washes (3vol, 6OL ±5%) may be carried out. The combined water washes are extracted with tert-butyl methyl ether (2x 2.2wt, 2x 22kg ±5%).⁶ The phases are separated and the organic phases are combined, sodium sulfate (2.0wt, 40kg ±5%) is charged, and the mixture is stirred at 20-25°C for 60-90 minutes. The resulting slurry is filtered and the filter-cake is washed with tert-butyl methyl ether (1.5wt, 30kg ±5%). The combined organic phase is concentrated under vacuum at <25°C to approximately 2.3vol. The mixture is cooled to 5-10°C, and checked for precipitation; once precipitation⁷ has occurred, heptanes (4.1wt, 82kg ±5%) are added, maintaining the temperature at 5-1O⁰C. The mixture is stirred at -10⁰C for 60-90 minutes, and the precipitate collected by filtration with vacuum. The filter-cake is washed with heptanes (2x 2.0wt, 2x 40kg ±5%) and the solid is dried under vacuum⁸ to afford the Stage 2 product (NTO 187) as an off-white solid (12.6-20.0kg, 50-80%th, 63-100%w/w).

Stage 2 (BD (Preparation of NTO 187 Part B21 Repeat steps outlined in 4.1.5.4.

Stage 3/4 (Preparation of NTOl 89)

To a suspension of the Stage 2 product (l.Owt, 29.8kg, l.Oeq) in tert-buty\ methyl ether (7.4wt, 220.3kg ±5%) at 20-25⁰C is added (+)-pinanediol (0.82wt, 24.44kg ±1%, 1.04eq), and the mixture stirred for up to 45 minutes to ensure complete dissolution. Sodium sulfate (1.Owt, 29.8kg ±1%, 2.5eq) is charged, and the reaction mixture stirred until reaction complete.⁹ If specification (Stage 2 content: <3% area) is not met after 4 hours, additional (+)-pinanediol (0.079wt) and sodium sulfate (0.09wt) are added and the mixture is stirred for 1 hour and rechecked for reaction completion. Steps are repeated

⁵ Stage 2 In-process Testing: Loss of product to the aqueous layer is checked by TLC; further /erf-butyl methyl ether extractions may be carried out, if required

⁶ Stage 2 In-process Testing: Loss of product to the aqueous layer is checked by TLC; further /er/-butyl methyl ether extractions may be carried out, if required.

⁷ If precipitation has not started, the solution should be aged for a further 4-5 hours. If still no precipitate is present at the end of the aging period, continue to concentrate at <25°C, until solvent collection has ceased.

⁸ Stage 2 In-process Testing: residual tert-buty\ methyl ether by ¹H NMR; pass criterion: <10%w/w.

⁹ Stage 3 In-process Testing: reaction completion by HPLC. until reaction completion is confirmed. The reaction mixture is filtered and the filter-cake washed with tert-buty\ methyl ether (1.5wt, 44.7kg ±5%). The combined filtrates are concentrated under vacuum at <40°C to approximately 3vol (89L), isopropyl alcohol (3.9wt, 116.2kg ±5%) is charged, and the mixture re-concentrated under vacuum at <40°C to approximately 3vol (89L). The tert-buty\ methyl ether content of the solution is determined.¹⁰ Isopropyl alcohol (2.7wt, 80.5kg ±5%) is then charged to the product- containing solution.

Isopropyl alcohol (3.81wt, 113.5kg ±5%) is charged to a separate flask and cooled to 5 to 10°C. Acetyl chloride (1.46wt, 43.5kg ±5%) is charged over at least 30 minutes, keeping the temperature at 5 to 2O⁰C (exothermic and generation of hydrogen chloride gas). Isopropyl alcohol (0.63wt, 18.8kg ±5%) is charged as a line rinse and the temperature of the mixture adjusted to 15 to 2O⁰C and stirred for an additional 30 to 45 minutes.

The isopropyl alcohol/product containing solution is heated to 40 to 45⁰C and the isopropyl alcohol/acetyl chloride mixture charged over at least 30 minutes, keeping the temperature at 15 to 45⁰C. Isopropyl alcohol (0.63wt, 18.8kg ±5%) is charged as a line rinse, and reaction stirred at 40 to 45°C for 30 to 60 minutes. The reaction is checked to ensure precipitation has occurred, and then cooled to 0 to 5⁰C over at least 4 hours. The resulting slurry is stirred at 0 to 5⁰C for 15 to 16 hours." The resulting white precipitate is collected by filtration and the filter-cake washed with cold (0 to 5⁰C) isopropyl alcohol (1.26wt, 37.1kg ±5%). The isopropyl alcohol content¹² and the Diastereomeric Excess¹³ of the crude product are determined.

The crude Stage 4 product, corrected for isopropyl alcohol content (lwt, 16.3kg) is suspended in isopropyl alcohol (15.7wt, 255.9kg ±5%) and heated to reflux (80 to 85°C). Reflux conditions are maintained for 10 to 15 minutes and the mixture is cooled with stirring to 0 to 5⁰C over approximately 8 hours (precipitation expected at approximately 55 to 60⁰C). The contents are stirred at 0 to 5⁰C for a minimum of 8 hours, and the precipitate is collected by vacuum filtration. The collected solids are washed with cold (0 to 5⁰C) isopropyl alcohol (1.26wt, 20.54kg ±5%). The product is dried under vacuum at up to

¹⁰ Stage 3 In-process Testing: ¹H NMR IPC to determine level of tert-butyl methyl ether in isopropyl alcohol solution. If >5%w/w, the addition/concentration cycle is repeated.

¹¹ Stage 4 In-process Testing: reaction completion by TLC, <5% Stage 3 product present. If specification is not met, a further charge of acetyl chloride in isopropyl alcohol may be added.

¹² Stage 4 In-process Testing: Isopropyl alcohol determination by ¹H NMR.

¹³ Stage 4 In-process Testing: Diastereomeric Excess of crude product by HPLC. 45⁰C¹⁴ to afford the Stage 4 product as a white solid (8.04-12.80kg, 20-32%th, 27-43%w/w).

Stage 5 (Preparation of NTO 1901 A suspension of the Stage 4 product (NT0189)(1.0wt, 8.00kg, l.Oeq), N-tert- butoxy-carbonyl-L-valine (BOC-L-valine) (0.84wt, 6.72kg ±1%, l.leq) and 1- hydroxybenzotriazole (HOBt)(0.5wt, 4.0kg ±1%, 1.05eq) in dichloromethane (18.5wt, 148.0kg ±5%) at 15 to 25⁰C is treated with triethylamine (0.7wt, 5.6g ±5%, 2.0eq, exothermic), keeping the temperature in the range of 15 to 25°C. l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (0.74wt, 5.92kg 1%, l.leq) is charged and the resultant stirred vigorously at 15 to 25°C until the reaction is complete, (approximately 9 to 12 hours).¹⁵ The reaction mixture is then transferred to an evaporator and concentrated under vacuum at up to 4O⁰C to afford a yellow gum. The residue is diluted with tert-butyl methyl ether (1.5wt, 12.0kg ±5%) and concentrated under vacuum at up to 4O⁰C. The resulting gum is analyzed for dichloromethane, content.¹ tert- Butyl methyl ether (1.5wt, 12.0kg ±5%) is charged to the residue and treated with cold (<10°C) 0.2M aqueous hydrochloric acid (l.Ovol, 8.0L ±5%); this biphasic solution is transferred to a vessel at <10°C, followed by a tert-buty\ methyl ether (3.7wt, 29.6kg ±5%) line rinse. The biphasic mixture is warmed to 20 to 25°C and checked for full dissolution; the pH of the aqueous layer is checked to confirm that it is within the range of 3 to 5. The aqueous phase is separated and water (2.0vol, 16.0L ±5%) charged to the organic phase. The mixture is stirred for 15 to 20 minutes at 20 to 25⁰C, and the pH adjusted to 3 to 4 (pH strips) by adding chilled (<10°C) aqueous hydrochloric acid (approximately 1 vol, 8.0L, ±5%). The aqueous phase is separated. Half-saturated aqueous sodium hydrogen carbonate solution (5.0vol, 40.0L ±5%, gas evolution) is added to the organic phase. The mixture is stirred for 15 to 20 minutes at 20 to 25⁰C and the layers are allowed to separate. Water (5.0vol, 40.0L ±5%) is added to the organic phase; the resulting mixture is stirred for 15 to 20 minutes and the layers allowed to separate. The pH of the aqueous phase is checked and if not in the range 6 to 8, a further water wash (5.0vol, 40.0L ±5%) is carried out, and the aqueous layer rechecked. Sodium sulfate (lwt, 8.0kg ±5%) is charged to the organic layer,

¹⁴ Stage 4 In-process Testing: Residual isopropyl alcohol content by GC headspace.

¹⁵ Stage 5 In-process Testing: Reaction completion: TLC & HPLC. Pass criterion: >95% Stage 5 product.

¹⁶ Stage 5 In-process Testing: Dichloromethane content by ¹H NMR. If the levels are >2%, further tert-butyl methyl ether is charged and the solution re-concentrated and reanalyzed. and the slurry stirred for 30 to 45 minutes at 20 to 25⁰C. The mixture is checked for water content , the solid filtered, and the filter-cake washed with tert-butyl methyl ether (2x 1.5 wt, 2x 12kg ±5%). The combined filtrates and washes are concentrated under vacuum to approximately 3.6vol (29L) at 35 to 45°C, and isopropyl alcohol (3.9wt, 31.2kg ±5%) is charged. The mixture is again concentrated under vacuum to approximately 3.6vol at 35 to 45⁰C, and the level of tert-butyl methyl ether determined. The mixture is then heated to 45 to 55°C, maintained at this temperature for 10 to 15 minutes and complete dissolution confirmed. Hot (45 to 55°C) water (12.7wt, 101.6kg ±5%) is charged over not less than 30 minutes, keeping the temperature in the range 45 to 55⁰C. The resulting slurry is cooled to 0 to 5⁰C over at least 2 hours and held at this temperature for a further 60 to 90 minutes. The solid is filtered and the filter-cake washed with cold (0 to 5⁰C) water (2x 2vol, 2x 16L ±5%). The solid is dried under vacuum at 45 to 5O⁰C¹⁹ to afford the Stage 5 product NT0190 as a white solid (8.80-11.28kg, 70-90%th,l 10-141%w/w).

Stage 6 (Preparation of talabostat mesylate)

To a solution of the Stage 5 output (NT0190, lwt, 9.58kg, l.Oeq) and phenylboronic acid (0.34wt, 3.26kg ±1 %, 1.25eq) tert-butyl methyl ether (5.9wt, 56.5kg ±5%) is added and the mixture stirred at 20 to 25°C until dissolution is complete. The solution is clarified through an in-line filter, followed by a tert-butyl methyl ether (1.5wt, 14.4kg ±5%) line rinse.

Methanesulfonic acid (0.32wt, 3,07kg ±1%, 1.52eq) is diluted with tert-butyl methyl ether (0.7wt, 6.71kg ±5%) and added to the phenylboronic acid mixture through an in-line filter, followed by a line rinse of tert-butyl methyl ether (0.7wt, 6.71kg ±5%). Water (0.1 vol, 1.0L ±5%) is added and the reaction mixture is stirred at 20 to 25⁰C until conversion is complete.²⁰ The suspension is heated to reflux (55 to 65°) followed by the addition of absolute ethanol (1.6wt, 15.3kg ±5%), maintaining reflux and stirring for an additional 30 to 40 minutes. The suspension is cooled to 15 to 25°C over a minimum of 2 hours. tert-Butyl methyl ether (5.9wt, 56.5kg ±5%) is added and stirring continued for an additional 1 to 2 hours at 15 to 25⁰C. The suspension is filtered under vacuum and the

¹⁷ Stage 5 In-process Testing: Water content by Karl Fischer (KF). Pass criterion: <1.0%w/w.

¹⁸ Stage 5 In-process Testing: tert-bυty\ methyl ether content determination by ¹H NMR. If >5%w/w, additional isopropyl alcohol charges (3.9wt, 31.2kg ±5%) and concentrations may be carried out.

¹⁹ Stage 5 In-process Testing: Water content by KF. Pass criterion: <0.5%w/w.

²⁰ Stage 6 In-process testing: HPLC (reaction completion). Pass criteria: <0.5% area Stage 5, <3.0% area de- BOC Stage 5 (impurity 3), expected: 20 to 28 hours. filter-cake washed with tert-buty\ methyl ether (3x 3.7wt, 3x 35.4kg ±5%).²¹ The collected solids are dried under vacuum at 45 to 5O⁰C²² to constant weight to afford talabostat mesylate as a white solid (4.98-6.61kg, 75-100%th, 52-69%w/w).

Example 2: Talabostat (API) characteristics

The final API made in accordance with Example 1 was tested for the presence of impurities and none were detected at levels exceeding 0.5% individual or 0.9% (total) (using assays such as CE and HPLC having limits of detection (LOD) of 0.01% and 0.03%). The most commonly detected intermediates were impurities 3, 5, 6 and 7. Using hot state microscopy and differential scanning calorimetry (DSC), talabostat was found to have a melt onset of about 158⁰C which was completed by about 192⁰C. The compound was found to be freely soluble in water and ethanol (i.e., > 100 mg/ml), slightly soluble in isopropyl alchohol (i.e., about 45 mg/ml), and practically insoluble in acetonitrile, acetone and ethyl acetate (i.e., < 0.1 mg/ml) at 22+/-3⁰C. A 2 mg/ml solution of talabostat in water had a pH of about 3.8.

Storage of the API at accelerated degradation conditions (e.g., 25+/-2⁰C and 60+/-5% relative humidity) led to some degradation (i.e., an increase from an impurity area under the curve percentage of 0.06% to 0.21% after 6 months under these conditions). Storage of talabostat at -18 to -2O⁰C for extended times (e.g., up to three years) did not lead to the formation of the RRT 1.06 and 1.16 impurities.

Example 3 : Talabostat tablet characteristics

An exemplary batch of tablets was prepared according to the methods provided herein. This batch had the following characteristics: off-white appearance, retention time within 5% of the standard, 97-98% potency as compared to the asserted level, content uniformity average of 99.9% to 102.5%, no impurities detected, average dissolution of 100%, 5.6% water content, average weight of 199.1mg (for a 200 mg tablet), 0.09% friability test weight loss, an average hardness of 6.1 kp, and a disintegration of 13-19 seconds.

Example 4: Preparation of the 2-hydroxy and aldehyde degradants

²¹ Stage 6 In-process Testing: HPLC (Purity). Pass criterion: >98% area, with no single impurity >1.0% area (confirms reaction completion and confirms removal of phenylboronic acid and pinanediol phenylboronate).

²² Stage 6 In-process Testing: GC for /er/-butyl methyl ether and ethanol content. Pass criteria: <0.3%w/w tert-buty\ methyl ether and <ethanol. Preparation of (2SV2-benzyloxycarbonylamino-3-methylbutyric acid pentafluorophenyl ester

CBz-L-valine (1Og, 0.040 mol) was combined with pentafluorophenol (0.042 mol) in dichloromethane in the presence of the coupling agent l-(3-dimethylaminopropyl)-3-ethyl- carbodiimide hydrochloride (EDC1»HC1). The crude product was purified by column chromatography on silica eluting with heptanes: ethyl acetate (9:1) to yield the desired product (15.86 g, 95% yield). ¹H NMR (CDCl₃) δ: 1.0 (d, 3H), 1.1 (d, 3H), 2.4 (m, IH), 4.7 (m, IH),

5.2 (s, 2H), 5.3 (d, IH), 7.4 (m, 5H).

Preparation of [l-(4,4-DimethoxybutylcarbamoylV2-methyl-propyl]-carbamic acid benzyl ester (11 in FIG. 5).

Compound 10 (15.86 g, 0.038 mol) was combined with 4-aminobutyraldehyde dimethylacetal (0.025) in THF to yield a crude product, which was purified by column chromatography on silica eluting with heptanes:ethyl acetate (1 : 1) to yield the desired product (8.33 g, 60% yield) as a white solid. ¹H NMR (CDCl₃) δ: 1.5 (m, 2H), 1.5 (m, 2H), 1.85 (d,

3H), 1.9 (d, 3H), 2.1 (m, IH), 3.2 (m, 2H), 3.25 (s, 3H), 3.85 (t, IH), 4.3 (t, IH), 5.0 (dd, 2H),

5.3 (br d, IH), 6.15 (br t, IH), 7.4 (m, 5H). ¹³C NMR (CDCl₃) δ: 18.0, 19.9, 24.8, 24.8, 31.5, 39.6, 53.4, 53.5, 61.0, 67.4, 104.7, 128.4, 128.6, 129.0, 136.7, 156.9, 171.6. LCMS: m/z = 303 da (M - 2x OMe), m/z 335 da (M - OMe), 367 da (MH⁺), m/z 389 da (M + Na).

Preparation of iV-CBz-protected 2-hydroxypyrrolidine/aldehyde degradants (12 in FIG. 5).

Compound 11 (500 mg) was stirred in 1.25M HCl/MeOH for 24 hours at room temperature. NMR analysis showed the presence of the 2-methoxy pyrrolidine product. Water was then added, and the solution was stirred for an additional 24 hours at room temperature. NMR analysis showed the presence of both the 2-methoxy pyrrolidine product, the iV-CBz-protected 2-hydroxypyrrolidine degradant (12a), and the iV-CBz-protected aldehyde degradant (12b). Finally, the solution was heated at 50 ⁰C for 4 hours, to give a mixture of 12a and 12b (444 mg). NMR and LCMS data for the N-CBz-protected 2- hydroxypyrrolidine degradant as a mixture of stereoisomers are given. H NMR (CDCl₃) δ: 0.8-1.2 (m, 6H), 1.7-2.3 (m, 2H), 1.7-2.3 (m, 2H), 1.9-20 (m, IH), 3.6-3.8 (m, 2H), 4.1-4.4 (t, IH), 5.0-5.3 (t, 2H), 5.6-5.8 (m, IH), 7.3-7.5 (m, 5H). ¹³C NMR (CDCl₃) δ: 18-20, 21-24, 31- 32, 32-35, 46-47, 58-59, 67.3, 68.0, 81.4, 82.5, 128-129, 136.2, 136.8, 156.8, 158.0, 171-173. LCMS: m/z = 303 da (M - HOH), m/z = 321 da (MH⁺), m/z 343 da (M + Na). Preparation of the 2-hydroxypyrrolidine/aldehyde degradants 03 in FIG. 5).

A sample (-70 mg) of compound 12, containing a mixture of both 12a and 12b, was hydrogenated over 10% Pd/C wet catalyst in MeOH/H₂O) for 16 hours to yield a mixture of the 2-hydroxypyrrolidine degradant (13a) and the aldehyde degradant (13b).

Example 5: Preparation of the 2-keto degradant and butyric acid degradant. I. De novo synthesis a. Butyric acid degradant Preparation of 4-aminobutyric acid benzyl ester toluene-4-sulfonate.

.OBn

H₂N^'

. TsOH O

Toluene-4-sulfonic acid monohydrate (209.3 g, 1.1 mol) was added to a mixture of 4- aminobutyric acid (75.0 g, 0.73 mol), benzyl alcohol (375 mL) and toluene (1500 mL). The reaction was allowed to reflux for 5 hours under Dean-Stark conditions (ca 33 mL water collected vs. ca 26 mL theory). The solution was concentrated at reduced pressure and crystallization occurred upon standing. The solid was collected by filtration and was washed with toluene (3 x 175 mL). The solid was dried on the filter for 1.25 hours and was then transferred to a vacuum oven at 33 ^°C for 19 h to afford the title compound as a white solid (175.9 g, 66% yield, corrected for 7.4% w/w residual benzyl alcohol). The mother liquors were placed in the refrigerator and a second crop of solid was crystallized from the mother liquors. The solid was collected by filtration and was washed with toluene (2 x 100 mL). The solid was dried on the filter for 3.25 hours and was then transferred to a vacuum oven at 37 ^"C for 18 h to afford the title compound as a white solid (43.6 g, 16% yield, corrected for 3.2% w/w residual benzyl alcohol and 22.5% w/w tolueve-4-sulfonic acid. The overall yield of the reaction was 82%, corrected for residual benzyl alcohol and toluene-4-sulfonic acid.

Preparation of f2S)-2-fert-butoxycarbonylamino-3-methylbutyric acid pentafluorophenyl ester.

l-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (196.8 g, 1.02 mol) was added over 12 minutes to a stirred solution of BOC-Z-valine (174.2 g, 0.82 mol) and pentafluorophenol (PfpOH) (158.5 g, 0.86 mol) in dichloromethane (1740 mL) at room temperature. A line rinse of dichloromethane (350 mL) was added. After 18 hours of stirring at room temperature the reaction was quenched by the addition of water (700 mL). The layers were separated, and the organic layer was washed with 0.5 M hydrochloric acid (700 mL) and dried (Na₂SO₄, 82.5 g). The solid was removed by filtration, washed with dichloromethane (350 mL) and the combined filtrates were concentrated at reduced pressure to afford the crude (303.2 g).

Dry flash chromatography on silica using heptanes and then a mixture of ethyl acetate :heptanes (1 : 19) as eluant afforded the title compound as a clear oil, which crystallized on standing (235.2 g, 77% yield, corrected for 8.8%w/w entrained heptanes).

Preparation of (S)-4-(2-fert-butoxycarbonylamino-3-methylbutyrylamino^')butyric acid benzyl ester.

TV.iV-Diisopropylethylamine (630 mL, 3.62 mol) was added over 12 minutes to a stirred solution of 4-aminobutyric acid benzyl ester toluene-4-sulfonate (219.5 g, 0.60 mol) in DMF (440 mL) at 0-10 ^"C. A solution of f2$-2-tert-butoxycarbonylamino-3-methylbutyric acid pentafluorophenyl ester (216.6 g, 0.57 mol) in THF (1760 mL) was added over 32 minutes at 0-10 ^"C before the reaction was allowed to warm to room temperature. After 14.25 hours, the starting material was consumed, as evidenced by TLC. The reaction was then concentrated at reduced pressure. The residue was partitioned between 0.5 MHCI (2.2 L) and ethyl acetate (4.4 L) and the organic layer was further washed with saturated aqueous sodium hydrogen carbonate (880 mL). The organic layer was dried (Na₂SO₄, 124.3 g), and the solid was removed by filtration and washed with ethyl acetate (440 mL). The filtrate was concentrated at reduced pressure to afford the crude product (382.6 g). Dry flash chromatography on silica using a mixture of ethyl acetate :heptanes (1 :4, then 1 :1) as eluant afforded the title compound as a while solid (186.1 g, 84% yield).

Preparation of (£)-4-(2-amino-3-methylbutyrylamino)butyric acid trifluoroacetic acid salt.

Trifluoroacetic acid (23OmL, 2.99 mol) was added to a solution of (5)-4-(2-tert- butoxycarbonylamino-3-methylbutyrylamino)butyric acid benzyl ester (150.4g, 0.38 mol) in dichloromethane (1500 mL). Gentle bubbling was observed immediately. ¹H NMR analysis after 22.5 hours still showed the presence of 10.4 mol % starting material. The reaction mixture was concentrated at reduced pressure. Portions of dichloromethane (3 x 1500 mL) were added to the residue and re-concentrated to afford the deprotected intermediate as a clear oil (242.9 g, 0.3 mol % starting material), which was then dissolved in water (500 mL plus 100 mL line rinse) containing 10% Pd/C (17.4 g). The slurry was purged with nitrogen prior to being placed under an atmosphere of hydrogen at room temperature and stirred for a total of 43.0 h. Analysis by ¹H NMR showed complete reaction. The slurry was filtered through celite (67 g). The celite pad was washed with water (300 mL and 150 mL) and the aqueous solution was washed with dichloromethane (3 x 300 mL) to remove un-reacted intermediate from the solution. The aqueous was freeze-dried to afford the title product as a white solid (73.5g, 61% yield, corrected for 0.7%w/w water). The isolated material has been stored at below -18⁰C in a glass jar bagged in a polyethylene bag containing desiccant.

b. 2-keto degradant

Preparation of (S>[2 methyl- l-(^"2-oxopyrrolidine-l-carbonyl)proplv]carbamic acid fert-butyl ester.

Lithium hexamethyldisilazide (390 mL of 1.0 M in THF, 0.39 mol) was added over 21 minutes to a stirred solution of 2-pyrrolidinone (37.1 g, 0.44 mol) in THF (1370 mL) at 0-5 °C. The milky suspension was allowed to warm to 15-25 ⁰C for 10 minutes, and then was cooled. A solution of BOC-Z-valine-OPfp ester (137.0 g, 0.36 mol), prepared according to the procedure described in Example 2, in THF (1100 mL) was added over 33 minutes at 0-5 °C, followed by a line rinse of THF (270 mL), and the reaction mixture was allowed to warm to room temperature. TLC analysis (ethyl acetate-heptanes, 1 :2, ninhydrin) after 1 hour showed complete consumption of the starting material and the reaction mixture was concentrated at reduced pressure to give crude 247.6 g. Dry flash chromatography on silica using a mixture of ethyl acetate-heptanes (1 :3) afforded the title compound as white solid (145.3 g, 72% yield, corrected for 0.7% w/w entrained heptanes).

Preparation of (S^*)-l-(2-amino-3-methylbutyryl)pyrrolidin-2-one trifluoroacetic acid salt.

(S)-[2 Methyl- l-(2-oxopyrrolidine-l-carbonyl)proply]carbamic acid tert-butyl ester (127.6 g, 0.45 mol) was dissolved in ethyl acetate (160 mL) and heptanes (480 mL) at 50- 70°C. The pink solution was allowed to cool to 25-30 °C over 1 hour to effect crystallization, and the mixture was further cooled to 0-5⁰C and aged for 0.5h, upon which solids formed. The solid was collected by filtration, washed with heptanes (130 mL) and air-dried on the filter for 20 minutes to afford 92.2 g. The recrystallized solid (88.2 g, 0.31 mol) was dissolved in dichloromethane (440 mL) at 15-25 ⁰C, and trifluoroacetic acid (190 mL, 2.47 mol) was added to the solution. Gentle bubbling was observed immediately. TLC after 1.25 hours showed complete consumption of the starting material and the reaction mixture was concentrated at reduced pressure. Dichloromethane (440 mL) was added to the residue and re-concentrated to afford the crude product (181.1 g). TBME (440 mL) was added to the crude product, causing immediate crystallization. The mixture was allowed to stir at 15-25 ⁰C for 1 hour before the solid was collected by filtration. The solid was washed with TBME (2 x 180 mL) and left to dry at the filter under nitrogen for 2 hours. The solid was then transferred to a Bϋchi for further drying at 41 °C under vacuum for 2 hours to afford the title compound (90.9 g, 71% yield). The isolated 2-keto degradant has been stored at below -18⁰C in a glass jar bagged in a polyethylene bag containing desiccant.

//. From the degradation of PT-100

The 2-keto degradant and butyric acid degradant were synthesized by the forced degradation of PT-100. Exposure of PT-100 to elevated temperature (e.g., 140 °C, or 140 ⁰C and 75% room humidity), photolytic conditions, acidic conditions, basic conditions, or oxidizing conditions (e.g., H₂O₂) produced the 2-keto degradant, which can further degrade into the butyric acid degradant, valine, and/or 2-pyrrolidinone. The products were analyzed via HPLC. The degradation pathway is shown in FIG. 6.

Example 6: Determination of PT-100 and impurities/degradants using early and late eluting HPL C separations

An HPLC method for the determination of PT-100 and related impurities/degradants was developed. An early eluting method is described which tends to detect degradants. A late eluting method is also described which tends to detect process-related impurities. Acetonitrile (HPLC grade), trifluoroacetic acid (spectrometric grade), deionized water, sodium dodecyl sulfate (high purity grade) were used for the mobile phase. An assay standard (AWS 400) (for %w/w assays only) and system suitability sample (AWS744) were also used.

The following procedure was used to prepare the mobile phase solution. A first solution containing sodium dodecyl sulfate (SDS) (14.4 g), 2000 mL of deionized water, and trifluoroacetic acid (1 mL) was prepared. A second solution containing acetonitrile (2000 mL), trifluoroacetic acid (1 mL) was then prepared. The first solution and the second solution were then combined in the desired proportion and placed in an ultra-sonic bath for 5 minutes. The solution was filtered through a 0.45 μm nylon membrane filter before use.

The sample solutions were prepared by dissolving a 100 mg sample in 100 mL of the mobile phase solution. The sample solutions were prepared and were immediately injected into the HPLC column. Compounds having retention times earlier than 2.5 minutes were eluted using HPLC Method I, while compounds having retention times later than 2.5 minutes were eluted using HPLC Method II. The retention times of the compounds are shown in Table 1 and Table 2.

HPLC Method I:

Column: YMC-Pack Pro C 18, 150 mm x 4.6 mm, 5 μm

Injection Volume: 20 μL

Detection: UV at 210 nm Mobile phase: Isocratic elution

25mM sodium dodecyl sulphate/acetonitrile 65/35 v/v + 0.05% TFA

Flow rate: 1 mL/min

Temperature: 30 °C

Run time: 30 minutes Wash vial: Water/ Acetonitrile 1/1 v/v

HPLC Method II: Column: YMC-Pack Pro C 18, 150 mm x 4.6 mm, 5 μm

Injection Volume: 20 μL Detection: UV at 210 nm Mobile phase: Isocratic elution

25mM sodium dodecyl sulphate/acetonitrile 40/60 v/v + 0.05% TFA

Flow rate: 1.5 mL/min Temperature: 30 °C Run time: 35 minutes Wash vial: Water/ Acetonitrile 1/1 v/v

Table 1: Compounds having retention times earlier than 2.5 minutes.

Table 2: Compounds having retention times later than 2.5 minutes.

Example 7: Determination of peaks having retention times earlier than 2.5 minutes in talabostat tablets

A HPLC method for the determination of Talabostat Mesylate related substances for the peaks having retention times earlier than 2.5 minutes in Talabostat Mesylate tablets (tablet strength: 0.1 mg, 0.2 mg, and 0.3 mg/tablet) was developed. Acetonitrile (HPLC grade), trifluoroacetic acid (HPLC grade or equivalent ), water (HPLC grade), sodium dodecyl sulphate (OmniPur or equivalent) were used for the mobile phase. 1-Hydroxybenzotriazole hydrate (HOBT) was used as a reference standard. The following procedure was used to prepare the mobile phase solution. A 20 mM solution containing sodium dodecyl sulphate SDS (5.8 g) and 1000 mL of water. The solution was then combined with the appropriate amount of acetonitrile and trifluoroacetic acid. The solution was filtered through a 0.45 μm nylon membrane filter and degassed before use. A 20 mM sodium dodecyl sulphate:acetonitrile:TFA (950:50:1) solution was prepared as a diluent for the tablets.

To prepare the tablet samples, a number of tablets (see Table 3) was combined with 10.0 mL of diluent. The mixture was sonicated for 1 minute (at 25 ⁰C or below, to avoid degradation) and mechanically shaken for ten minutes, to dissolve the tablets. The mixture was allowed to stand until most of the excipients sank to the bottom of the container. A portion of the clear solution was then filtered through a 0.45 pm Nylon syringe filter into and transferred to an HPLC vial, discarding the first couple of drops. The concentration of Talabostat Mesylate (free base) in the sample was approximately 300 μg/mL.

For stability analysis where placebo controls are identified in the protocol, a placebo control sample was prepared in a similar manner as described above.

The samples were then analyzed using HPLC Method III.

Table 3. Number of Tablets used for Sample Preparation.

HPLC Method III:

Column: YMC-Pack Pro C 18, 150 mm x 4.6 mm, 5 μm

Injection Volume: 50 μL Detection: UV at 210 nm Mobile phase A: 20 mM sodium dodecyl sulphate: acetonitrile: TFA 950:50:1 Mobile phase B: 20 mM sodium dodecyl sulphate:acetonitrile:TFA 600:400:1 Flow rate: 1 mL/min (2 mL/min for column cleaning) Temperature: 40 °C Run time: 40 minutes

Example 8: Determination of peaks having retention times later than 2.5 minutes in talabostat tablets A HPLC method for the determination of Talabostat Mesylate related substances for the peaks having retention times later than 2.5 minutes in Talabostat Mesylate tablets (strength: 0.1 mg, 0.2 mg, and 0.3 mg/tablet) was developed. Acetonitrile (HPLC grade), water (HPLC grade), sodium dodecyl sulphate (OmniPur or equivalent), phosphoric acid (85%, HPLC grade), were used for the mobile phase.

The following procedure was used to prepare the mobile phase solution. A buffer solution was prepared by dissolving sodium dodecyl sulfate (5.8 g) in 1000 mL of deionized water and 85% phosphoric acid (1 mL). The pH was adjusted to 2.50 ± 0.05 with IO N sodium hydroxide solution. The buffer solution was then combined with the appropriate amount of acetonitrile and to form the mobile phase. A solution containing SDS buffer: acetonitrile :phosphoric acid (65:35:0.2) was prepared as a diluent for the tablets. The solutions were filtered through a 0.45 μm nylon membrane filter and degassed before use.

To prepare the tablet samples, a number of tablets (Table 3) was combined with 10.0 mL of diluent. The mixture was sonicated for 1 minute (at 25 °C or below, to avoid degradation) and mechanically shaken for ten minutes, to dissolve the tablets. The mixture was allowed to stand until most of the excipients sank to the bottom of the container. A portion of the clear solution was then filtered through a 0.45 pm Nylon syringe filter into and transferred to an HPLC vial, discarding the first 3-5 drops. The concentration of Talabostat Mesylate (free base) in the sample was approximately 300 μg/mL. The sample solution was analyzed within one day of preparation.

HPLC Method IV:

Column: YMC-Pack Pro C 18, 150 mm x 4.6 mm, 5 μm

Injection Volume: 50 μL Detection: UV at 210 nm

Mobile phase : 65:35 sodium dodecyl sulphate buffeπacetonitrile

Flow rate: 1 mL/min

Temperature: 40 °C

Run time: 30 minutes

Example 9: Determination of chiral purity of PT-100 using capillary electrophoresis

A capillary electrophoresis (CE) method for the determination of the chiral purity of PT-100 was developed. Sodium dihydrogen orthophosphate (AR grade), beta-cyclodextrin (sulfated sodium salt) (Sigma catalogue number 389153 AR grade), orthophosphoric acid (AR grade), and deionized water were used to prepare a run buffer solution. A system suitability sample (AWS824) was also used.

The following procedure was used to prepare the run buffer solution. A phosphate buffer solution was prepared by combining sodium dihydrogen orthophosphate (7.8 g) with 500 raL of deionized water and adjusting the pH of the solution to 3.0 with orthophosphoric acid. In another container, p-cyclodextrin sulfate (sodium salt) (800 mg) was dissolved in 2OmL of the phosphate buffer solution, to form the run buffer solution. The run buffer solution was filtered through a 0.45 μm nylon membrane filter prior to use.

The sample solutions were prepared by dissolving a 20 mg sample in 50 mL of the run buffer solution, and were immediately injected into the CE column. The samples were analyzed using CE Method I, and the chiral purity of the samples are shown in Table 4.

CE Method I

Capillary: Capital Analytical EKT (or equivalent) zero flow neutral capillary 54 cm x 50μm (ID) (43 cm to detector).

Injection time: 10 seconds @ 2.0 psi Detection: UV © 210 nm Run buffer: aqueous 20 mM beta cyclodextrin (sulphated sodium salt) in NaH₂PO₄

(10OmM; pH 3)

Sample diluent: NaH₂PO₄ (10OmM; pH 3) Separation voltage: +15kV (normal polarity) Capillary Temp: 15°C Sample Temp: Ambient Run time: 30 min

Table 4: Chiral Purity of PT-100 Determined by Capillary Electrophoresis

Equivalents

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

All references, patents and patent publications that are recited in this application are incorporated in their entirety herein by reference unless otherwise stated.

What is claimed is:

Claims

1. A method for determining an impurity in a material comprising Formula I, comprising: injecting into an HPLC column a sample solution containing the material and containing a known amount of a known first reference compound having a known chemical structure of Compound G,

and/or a known amount of a known second reference compound having a known chemical structure of Compound H,

Q^ .OH

AA OH

^VO NH₂ obtaining an HPLC chromatogram, and determining the presence and/or the amount of Compound G and/or Compound H in the material.

2. A method for determining an impurity in a material consisting essentially of Formula I, comprising: injecting into an HPLC column a sample solution containing the material and containing a known amount of a known first reference compound having a known chemical structure of Compound G,

obtaining an HPLC chromatogram, and determining the presence and/or the amount of Compound G and/or Compound H in the material.

3. The method of claim 1 or 2, further comprising documenting in a written form the chemical identity and the amount as an impurity of Compound G and/or Compound H.

4. The method of claim 1 or 2, wherein the reference sample contains a known amount of a known third reference compound having a known chemical structure of Compound F,

and further comprising determining the presence and/or the amount of Compound F in the material.

5. The method of claim 4, further comprising documenting in a written form the chemical identity and the amount as an impurity of Compound F.

6. The method of claim 1 or 2, wherein the reference sample contains a known amount of a known third reference compound having a known chemical structure of Compound I,

and further comprising determining the presence and/or the amount of Compound I in the material.

7. The method of claim 6, further comprising documenting in a written form the chemical identity and the amount as an impurity of Compound I.

8. The method of claim 1 or 2, wherein the HPLC column is a reverse phase column and the column is eluted using a mobile phase comprising sodium dodecyl sulfate, trifluoroacetic acid, water, and acetonitrile.

9. The method of claim 1 -7 or 8, wherein the compound of Formula I is talabostat mesylate.

10. A method for determining an impurity in a material comprising Formula I, comprising: injecting a solution in which the material is dissolved into an HPLC column and obtaining an HPLC chromatogram; determining the amount in the material of a compound known to have the structure of Compound G or Compound H, and documenting in a written form the chemical identity and amount of Compound G or Compound H in the material.

11. A method for determining an impurity in a material consisting essentially of Formula I, comprising: injecting a solution in which the material is dissolved into an HPLC column and obtaining an HPLC chromatogram; determining the amount in the material of a compound known to have the structure of Compound G or Compound H, and documenting in a written form the chemical identity and amount of Compound G or Compound H in the material.

12. The method of claim 10 or 11, wherein the amount of Compound G or Compound H in the material is determined by (i) identifying a peak on the chromatogram that corresponds to a peak on a control chromatogram, (ii) identifying a peak on the chromatogram that corresponds to a relative retention time of Compound G or Compound H, and/or (iii) identifying a peak on the chromatogram that corresponds to a known amount of a spike of a compound known to have the structure of Compound G or Compound H.

13. The method of claim 10 or 11 , further comprising, determining the amount in the material of a compound known to have the structure of Compound F, and documenting in a written form the chemical identity and amount of Compound F as an impurity in the material.

14. The method of claim 10 or 11, further comprising, determining the amount in the material of a compound known to have the structure of Compound I, and documenting in a written form the chemical identity and amount of Compound I as an impurity in the material.

15. The method of claim 10 or 11 , wherein the HPLC column is a reverse phase column and the column is eluted using a mobile phase comprising sodium dodecyl sulfate, trifluoroacetic acid, water, and acetonitrile.

16. The method of claim 10-14 or 15, wherein the compound of Formula I is talabostat mesylate.

17. A method for evaluating the purity of a composition comprising Formula I, comprising, injecting a reference solution containing impurities comprising Compound F, Compound G, Compound H and/or Compound I into an HPLC column under a set of conditions to obtain a first HPLC chromatogram, wherein the amount and identity of the impurities present in the reference solution was previously determined; injecting a sample solution comprising Formula I into the HPLC column under said set of conditions to obtain a second HPLC chromatogram; and determining the amount of the impurities in the sample solution, wherein Compounds F-I are: Compound F:

Compound G:

Compound H:

Compound I:

18. The method of claim 17, further comprising reporting in writing that the amounts of the compounds in the Formula I composition are: for Compound F: less than or equal to 2.0%, less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1%, for Compound G: less than or equal to 2.0%, less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1%, for Compound H: less than or equal to 2.0%, less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1%, and for Compound I: less than or equal to 2.0%, less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1%.

19. The method of claim 18, wherein the HPLC column is a reverse phase column and the column is eluted using a mobile phase comprising sodium dodecyl sulfate, trifluoroacetic acid, water, and acetonitrile.

20. The method of claim 17, 18 or 19, wherein the compound of Formula I is talabostat mesylate.

21. A method for manufacturing a material comprising a compound of Formula I, comprising obtaining a composition comprising the compound of Formula I, purifying the composition by chromatography, recrystallization or a combination thereof, conducting HPLC on a sample of purified first composition using a known amount of impurities Compound F, Compound G, Compound H and Compound I as a standard, and determining the presence or absence of the impurities in the sample.

22. A method for manufacturing a material consisting essentially of a compound of Formula I, comprising obtaining a composition containing the compound of Formula I, purifying the composition by chromatography, recrystallization or a combination thereof, conducting HPLC on a sample of purified first composition using a known amount of impurities Compound F, Compound G, Compound H and Compound I as a standard, and determining the presence or absence of the impurities in the sample.

23. The method of claim 21 or 22, wherein the purifying is carried out until the impurities are less than or equal to 2.0%, 1.0%, 0.5%, 0.1%, 0.4%, 0.3%, 0.2%, 0.15%, 0.1%, 0.05%, even are absent from the purified first composition as determined by HPLC with a detection limit of 0.01-0.03% and a quantitation limit of 0.03%.

24. The method of claim 23, further comprising then packaging the purified first composition.

25. The method of claim 24, further comprising providing indicia on or with the packaged purified first composition indicating a level of the impurities in the packaged first purified composition.

26. The method of claim 21-24 or 25, wherein the compound of Formula I is talabostat mesylate.

27. A package containing a composition comprising Formula I and indicia on or contained within the package indicating a level of one or more impurities comprising Compound F, Compound G, Compound H and Compound I, wherein the one or more impurities are identified by chemical name, by chemical structure or by chemical formula wherein Compound F, Compound G, Compound H and Compound I are:

Compound F:

Compound G:

Compound H:

Compound I:

28. The package of claim 27, wherein the indicia indicates specifically that: for Compound F: less than or equal to 2.0%, less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1%, for Compound G: less than or equal to 2.0%, less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1%, for Compound H: less than or equal to 2.0%, less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1%, and for Compound I: less than or equal to 2.0%, less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1%.

29. The package of claim 27 or 28, wherein the compound of Formula I is talabostat mesylate.

30. The package of claim 27, 28 or 29, wherein the compound of Formula I is greater than 95% linear.

31. The package of claim 27, 28 or 29, wherein the compound of Formula I is greater than 98% linear.

32. The package of claim 27, wherein the indicia indicates a level of impurity of Compounds F, G, H and I of less than or equal to 2.0%.

33. A method for determining whether a condition protects against degradation of Formula I, comprising applying the condition to a composition comprising Formula I, and determining whether one or more of the following impurities form as a result of the applying of the conditions: Compound A:

Compound B:

Compound C:

Compound D:

34. The method of claim 33, wherein the condition is combining Formula I with a pharmaceutically acceptable carrier.

35. The method of claim 33, wherein the conditions are (i) adjusting the pH of the composition, (ii) exposing the composition to light, (iii) exposing the composition to a chemical, (iv) heating the composition, (v) processing the composition into a pharmaceutical dosage form, and/or (vi) storing the composition.

36. The method of claim 33, wherein the impurities comprise Compound A and Compound B, and wherein the indicia indicates specifically that: for Compound A: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%, and for Compound B: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

37. The method of claim 34, wherein the impurities comprise Compound C, and wherein the indicia indicates specifically that: for Compound C: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

38. The method of claim 33, wherein the impurities comprise Compound D, and wherein the indicia indicates specifically that: for Compound D: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

39. The method of claim 37, wherein the impurities comprise Compound D, and wherein the indicia indicates specifically that: for Compound D: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

40. The method of claim 33-36 or 37, wherein the compound of Formula I is talabostat mesylate.

41. A pharmaceutical comprising, particles consisting essentially of a compound of Formula I, lactose, microcrystalline cellulose, and an acidic salt.

42. The pharmaceutical of claim 41, wherein the acidic salt is sodium phosphate.

43. The pharmaceutical of claim 41 , wherein the compound of Formula I is present at between 0.1% and 0.2% w/w relative to the total weight of the lactose and the microcrystalline cellulose.

44. The pharmaceutical of claim 43, wherein the compound of Formula I is present at between 0.1% and 0.2% w/w relative to the weight of the lactose.

45. The pharmaceutical of claim 41 -43 or 44, wherein the compound of Formula I is talabostat mesylate.

46. The pharmaceutical of claim 41 -43 or 44, wherein the compound of Formula I is talabostat mesylate and wherein the pharmaceutical has a level of impurity of less than or equal to 3%, equal to or less than 2%, less than or equal to 1.0%, less than or equal to 0.5%, or less than or equal to 0.1% for one or more of the following impurities:

Compound A:

Compound B:

Compound C:

Compound D:

47. The pharmaceutical of claim 46, wherein the level of impurity of Compounds A-D is less than or equal to 2.0%.

48. The pharmaceutical of claim 41 , wherein the compound of Formula I is 15-40% cyclic.

49. A pharmaceutical preparation comprising agglomerated filler particles, the filler particles coated over their surface with a compound of Formula I, the agglomerated filler particles also coated over their surface with an acidic salt, the agglomerated particles free of compounds that are electron withdrawing and oxidative respecting boronic acid of the compound of Formula I, and wherein the compound of Formula I is present between 0.1% and 0.2% w/w relative to the weight of the filler particles in the agglomerated filler particles.

50. The pharmaceutical preparation of claim 49, further comprising a binder mixed with the agglomerated filler particles, wherein the mixture is compressed as a tablet.

51. The pharmaceutical preparation of claim 50, wherein the filler particles comprise lactose and wherein the agglomerated particles are free of a binder.

52. The pharmaceutical preparation of claim 50, wherein the agglomerated particles are formed in the absence of hydroxypropylmethylcellulose and cross-linked N-vinyl-2- pyrrolidone.

53. The pharmaceutical preparation of claim 49-51 or 52, wherein the compound of Formula I is talabostat mesylate.

54. The pharmaceutical preparation of claim 49, wherein the compound of Formula I is 15-40% cyclic.

55. A pharmaceutical tablet comprising, particles consisting essentially of a compound of Formula I, lactose, microcrystalline cellulose, and an acidic salt, the particles compressed in a mixture with one or more of a binder, a lubricant and a disintegrant.

56. The pharmaceutical tablet of claim 55, wherein the mixture is a mixture of the particles with a binder and a disintegrant.

57. The pharmaceutical tablet of claim 55, wherein the mixture is a mixture of the particles with a binder, a lubricant and a disintegrant.

58. The pharmaceutical tablet of claim 55, wherein the mixture is a mixture of the particles with pre-gelatinized starch.

59. The pharmaceutical tablet of claim 56, wherein the binder is pre-gelatinized starch.

60. The pharmaceutical tablet of claim 59, wherein the disintegrant is cross-linked N- vinyl-2-pyrrolidone.

61. The pharmaceutical tablet of claim 57, wherein the binder is pre-gelatinized starch.

62. The pharmaceutical tablet of claim 61, wherein the disintegrant is cross-linked N- vinyl-2-pyrrolidone.

63. The pharmaceutical tablet of claim 55 or 62, wherein the lubricant is stearic acid.

64. The pharmaceutical tablet of claim 55, wherein the compound of Formula I is present at between 0.1% and 0.2% w/w relative to the weight of lactose, microcrystalline cellulose, and acidic salt.

65. The pharmaceutical tablet of claim 55 or 64, wherein the compound of Formula I is present at between 50 and 800 micrograms.

66. The pharmaceutical tablet of claim 55, wherein the compound of Formula I is present at between 0.1% and 0.2% w/w relative to the weight of lactose.

67. The pharmaceutical tablet of claim 66, wherein the compound of Formula I is talabostat mesylate.

68. The pharmaceutical tablet of claim 55, wherein the compound of Formula I is talabostat mesylate and wherein the tablet has a level of impurity of less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1% for one or more of the following impurities: Compound A:

Compound B: Compound C:

Compound D:

69. The pharmaceutical tablet of claim 68, wherein the level of impurity of Compounds A-D combined is less than or equal to 10.0%.

70. The pharmaceutical tablet of claim 55, wherein the compound of Formula I is 15-40% cyclic.

71. A pharmaceutical preparation comprising a tablet of the compound of Formula I, wherein the tablet after storage at 2-8 degrees centigrade for 3 months has a total amount of Formula I degradation products that does not exceed 10% of the compound of Formula I in the preparation.

72. The pharmaceutical preparation of claim 71 , wherein the amount of Formula I degradation products does not exceed 1.5% of the compound of Formula I in the preparation.

73. The pharmaceutical preparation of claim 71 , wherein the amount of Formula I degradation products does not exceed 1.0% of the compound of Formula I in the preparation.

74. The pharmaceutical preparation of claim 71 , wherein the amount of Formula I degradation products does not exceed 0.5% of the compound of Formula I in the preparation.

75. The pharmaceutical preparation of claim 71 , wherein the amount of Formula I degradation products does not exceed 0.25% of the compound of Formula I in the preparation.

76. The pharmaceutical preparation of claim 71 -74, or 75, wherein the amount of each Formula I degradation product does not exceed 0.5% of the compound of Formula I in the preparation, does not exceed 0.25% of the compound of Formula I in the preparation, does not exceed 0.15% of the compound of Formula I in the preparation, or even does not exceed 0.10% of the compound of Formula I in the preparation.

77. The pharmaceutical preparation of claim 71-74 or 75, wherein the compound of Formula I in the pharmaceutical preparation is protected from oxidation.

78. The pharmaceutical preparation of claim 77, wherein the compound of Formula I in the pharmaceutical preparation is protected from oxidation by coating the compound of

Formula I onto particles that are not oxidative with respect to the compound of Formula I.

79. The pharmaceutical preparation of claim 78, wherein the compound of Formula I is coated onto particles free of (-OCH₂CHOHCH₃) groups.

80. The pharmaceutical preparation of claim 79, wherein the compound of Formula I is coated onto particles free of hydroxy propyl methyl cellulose.

81. The pharmaceutical preparation of claim 78, wherein the compound of Formula I is coated onto particles free of vinyl pyrrolidone.

82. The pharmaceutical preparation of claim 79, wherein the compound of Formula I is coated onto particles free of vinyl pyrrolidone.

83. The pharmaceutical preparation of claim 80, wherein the compound of Formula I is coated onto particles free of vinyl pyrrolidone.

84. The pharmaceutical preparation of claim 79, wherein the compound of Formula I is coated onto particles free of crosslinked homopolymer of N-vinyl-2-pyrrolidone.

85. The pharmaceutical preparation of claim 80, wherein the compound of Formula I is coated onto particles free of crosslinked homopolymer of N-vinyl-2-pyrrolidone.

86. The pharmaceutical preparation of claim 71 -74 or 75, further comprising a pH modifying salt.

87. The pharmaceutical preparation of claim 86, wherein the pH modifying salt is sodium phosphate monobasic, monohydrate and further comprising a pH modifying acid that is phosphoric acid.

88. The pharmaceutical preparation of claim 79, further comprising a pH modifying salt.

89. The pharmaceutical preparation of claim 88, wherein the pH modifying salt is sodium phosphate monobasic, monohydrate and further comprising a pH modifying acid that is phosphoric acid.

90. The pharmaceutical preparation of claim 78, wherein the compound of Formula I in the pharmaceutical preparation is coated onto particles comprising one or more fillers.

91. The pharmaceutical preparation of claim 90, wherein the one or more fillers comprise lactose monohydrate.

92. The pharmaceutical preparation of claim 90, wherein the one or more fillers comprise lactose monohydrate and microcrystalline cellulose.

93. The pharmaceutical preparation of claim 79, wherein the compound of Formula I in the pharmaceutical preparation is coated onto particles comprising one or more fillers.

94. The pharmaceutical preparation of claim 93, wherein the one or more fillers comprise lactose monohydrate.

95. The pharmaceutical preparation of claim 93, wherein the one or more fillers comprise lactose monohydrate and microcrystalline cellulose.

96. The pharmaceutical preparation of claim 88, wherein the compound of Formula I in the pharmaceutical preparation is coated onto particles comprising one or more fillers.

97. The pharmaceutical preparation of claim 96, wherein the one or more fillers comprise lactose monohydrate.

98. The pharmaceutical preparation of claim 96, wherein the one or more fillers comprise lactose monohydrate and microcrystalline cellulose.

99. The pharmaceutical preparation of claim 96, further comprising a lubricant.

100. The pharmaceutical preparation of claim 99, further comprising a binder.

101. The pharmaceutical preparation of claim 71 , wherein the compound of Formula I is 15-40% cyclic.

102. A method of manufacturing a pharmaceutical tablet comprising, providing a solution of a compound of Formula I, wherein the pH of the solution is between 1.5 and 2.9 and wherein the compound of Formula I is at a concentration of about 4000 - 6000 mg/liter, spraying the solution onto particles of a filler material to form agglomerated particles coated with and containing the compound of Formula I, drying the agglomerating particles, and mixing the agglomerated particles with one or more of a binder, a lubricant and a disintegrant, and compressing the mixture into a tablet.

103. The method of claim 102, wherein the filler material is non-oxidative respecting the boronic acid of the compound of Formula I in the presence of both an acidic salt and trace amounts of water at 2-8 degrees centigrade.

104. The method of claim 102, wherein the filler material comprises lactose.

105. The method of claim 102, wherein the filler material comprises microcrystalline cellulose.

106. The method of claim 102, wherein the filler material comprises lactose and microcrystalline cellulose.

107. The method of claim 102, wherein the filler material consists of lactose and microcrystalline cellulose.

108. The method of claim 102, wherein the agglomerated particles are mixed with a binder and a disintegrant.

109. The method of claim 102, wherein the agglomerated particles are mixed with a binder, a lubricant and a disintegrant.

110. The method of claim 102, wherein the agglomerated particles are mixed with a binder that is pre-gelatinized starch.

11 1. The method of claim 108, wherein the agglomerated particles are mixed with a binder that is pre-gelatinized starch.

1 12. The method of claim 109, wherein the agglomerated particles are mixed with a binder that is pre-gelatinized starch.

113. The method of claim 102, wherein the agglomerated particles are mixed with a disintegrant that is cross-linked N-vinyl-2-pyrrolidone.

114. The method of claim 108, wherein the agglomerated particles are mixed with a disintegrant that is cross-linked N-vinyl-2-pyrrolidone.

115. The method of claim 109, wherein the agglomerated particles are mixed with a disintegrant that is cross-linked N-vinyl-2-pyrrolidone.

116. The method of claim 102, wherein the agglomerated particles are mixed with a lubricant that is stearic acid.

117. The method of claim 108, wherein the agglomerated particles are mixed with a lubricant that is stearic acid.

118. The method of claim 109, wherein the agglomerated particles are mixed with a lubricant that is stearic acid.

119. The method of claim 102 or 109, wherein the binder is pre-gelatinized starch, the disintegrant is cross-linked N-vinyl-2-pyrrolidone, and the lubricant is stearic acid.

120. The method of claim 106, wherein the binder is pre-gelatinized starch, the disintegrant is cross-linked N-vinyl-2-pyrrolidone, and the lubricant is stearic acid.

121. The method of claim 102, wherein the compound of Formula I is talabostat mesylate.

122. The method of claim 119, wherein the compound of Formula I is talabostat mesylate.

123. The method of claim 120, wherein the compound of Formula I is talabostat mesylate.

124. The method of claim 106, wherein the compound of Formula I is present in the tablet at between 0.1% and 0.2% w/w relative to lactose and microcrystalline cellulose.

125. The method of claim 106, wherein the compound of Formula I is present in the tablet at between 180 and 220 micrograms.

126. The method of claim 106, wherein the compound of Formula I is present in the tablet at between 280 and 320 micrograms.

127. The method of claim 104, wherein the compound of Formula I is present at between 0.1% and 0.2% w/w relative to lactose.

128. The method of claim 102, wherein the compound of Formula I is talabostat mesylate and the method further comprises testing for one or more of the following impurities:

Compound A:

Compound B:

Compound C:

Compound D:

129. The method of claim 128, wherein the tablet is tested for impurities after storage for 3 months at 2-8 degrees centigrade.

130. The method of claim 128, further comprising packaging the tablet and providing indicia on or with the packaged tablet indicating a level of the one or more impurities in the packaged tablet.

131. The method of claim 102, wherein the pH of the solution is about 2.0 to 2.5.

132. The method of claim 102, wherein the pH of the solution is about 2.2.

133. A pharmaceutical tablet made by the process of claim 102-131 or 132.

134. A kit comprising, a package containing a pharmaceutical tablet comprising particles comprising a compound of Formula I, lactose, microcrystalline cellulose, and an acidic salt, the particles compressed in a mixture of the particles with one or more of a binder, a lubricant and a disintegrant, and indicia on or contained within the package indicating the chemical identity of the compound of Formula I.

135. A kit comprising, a package containing a pharmaceutical tablet comprising particles consisting essentially of a compound of Formula I, lactose, microcrystalline cellulose, and an acidic salt, the particles compressed in a mixture of the particles with one or more of a binder, a lubricant and a disintegrant, and indicia on or contained within the package indicating the chemical identity of the compound of Formula I.

136. The kit of claim 134 or 135, wherein the compound of Formula I is talabostat mesylate.

137. A kit comprising, a package containing a pharmaceutical tablet comprising particles consisting essentially of a compound of Formula I, lactose, microcrystalline cellulose, and an acidic salt, the particles compressed in a mixture of the particles with one or more of a binder, a lubricant and a disintegrant, and indicia on or contained within the package indicating the chemical identity of the compound of Formula I, wherein the compound of Formula I is talabostat mesylate and wherein the tablet has a level of impurity of less than or equal to 3.0%, less than or equal to 2%, less than or equal to 1.0%, less than or equal to 0.5%, or even less than or equal to 0.1% for one or more of the following impurities:

Compound A:

Compound B:

Compound C:

NH

NH₂

Compound D:

138. The kit of claim 137, wherein the indicia also indicate the chemical identity of the one or more impurities and the amount of the one or more impurities.

139. The kit of claim 137, wherein the level of impurity of Compounds A-D combined is less than or equal to 10.0%.

140. The kit of claim 135, wherein the compound of Formula I is 15-40% cyclic.

141. A package containing a tablet comprising Formula I, and indicia on or contained within the package indicating a level of impurity for one or more impurities comprising Compound A, Compound B, Compound C, and Compound D, wherein the one or more impurities are identified by chemical name, by chemical structure or by chemical formula wherein Compound A, Compound B, Compound C and Compound D are:

Compound A:

Compound B:

Compound C:

Compound D:

142. The package of claim 141, wherein the impurities comprise Compound A and Compound B, and wherein the indicia indicates specifically that: for Compound A: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%, and for Compound B: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

143. The package of claim 142, wherein the impurities comprise Compound C, and wherein the indicia indicates specifically that: for Compound C: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

144. The package of claim 142, wherein the impurities comprise Compound D, and wherein the indicia indicates specifically that: for Compound D: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

145. The package of claim 143, wherein the impurities comprise Compound D, and wherein the indicia indicates specifically that: for Compound D: less than or equal to 3%, less than or equal to 2%, less than or equal to 1 %, less than or equal to 0.5%, or even less than or equal to 0.15%.

146. The package of claim 141-144 or 145, wherein the indicia indicates that the level of impurity combined for Compounds A-D is less than or equal to 10.0%.

147. A method for evaluating the purity of a Formula I composition, comprising, injecting a reference solution containing impurities comprising Compound A and Compound B into an HPLC column under a set of conditions to obtain a first HPLC chromatogram, wherein the amount and identity of the compounds present in the reference solution is known; injecting a sample solution comprising Formula I into the HPLC column under said set of conditions to obtain a second HPLC chromatogram; and determining the amount of the compounds in the sample solution, wherein Compound A and Compound B are:

Compound A:

Compound B:

148. The method of claim 147, further comprising reporting in writing that the amounts of the compounds in the Formula I composition are: for Compound A: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%, and for Compound B: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

149. The method of claim 147, wherein the impurities further comprise a compound of Compound C, wherein the compound of Compound C is:

150. The method of claim 147, wherein the impurities further comprise a compound of Compound C, wherein the compound of Compound C is:

NH -O

NH₂ , and further comprising reporting in writing that the amount of the compound of Compound C in the Formula I composition is: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

151. The method of claim 147 or 149, wherein the impurities further comprise a compound of Compound D, wherein the compound of Compound D is:

152. The method of claim 147 or 149, wherein the impurities further comprise a compound of Compound D, wherein the compound of Compound D is:

further comprising reporting in writing that the amount of the compound of Compound D in the Formula I composition is: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

153. The method of claim 148, wherein the impurities further comprise a compound of Compound D, wherein the compound of Compound D is:

154. The method of claim 148, wherein the impurities further comprise a compound of Compound D, wherein the compound of Compound D is:

further comprising reporting in writing that the amount of the compound of Compound D in the Formula I composition is: less than or equal to 3%, less than or equal to 2%, less than or equal to 1 %, less than or equal to 0.5%, or even less than or equal to 0.15%.

155. The method of claim 147, wherein the HPLC column is a reverse phase column and the column is eluted using a mobile phase comprising sodium dodecyl sulfate, phosphoric acid, sodium hydroxide, water, and acetonitrile.

156. A method for determining an impurity in a material comprising Formula I, comprising: injecting a solution comprising the material into an HPLC column and obtaining an HPLC chromatogram; and determining the amount in the material of impurities known to have the structure of Compound A or Compound B, and documenting in a written form the chemical identity of the impurities and the amount of the impurities in the material, wherein Compound A and Compound B are: Compound A:

Compound B:

N OH

NH₂

157. A method for determining an impurity in a material consisting essentially of Formula I, comprising: injecting a solution comprising the material into an HPLC column and obtaining an HPLC chromatogram; and determining the amount in the material of impurities known to have the structure of Compound A or Compound B, and documenting in a written form the chemical identity of the impurities and the amount of the impurities in the material, wherein Compound A and Compound B are: Compound A:

Compound B:

158. The method of claim 156 or 157, wherein the amounts of the compounds in the material are documented in writing to be: for the compound of Compound A: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%, and for the compound of Compound B: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%

159. The method of claim 156 or 157, wherein the impurities further comprise a compound of Compound C, wherein the compound of Compound C is:

160. The method of claim 156 or 157, wherein the impurities further comprise a compound of Compound C, wherein the compound of Compound C is:

wherein the amount of the compound of Compound C in the material is documented in writing to be: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

161. The method of claim 156 or 157, wherein the impurities further comprise a compound of Compound D, wherein the compound of Compound D is:

162. The method of claim 156 or 157, wherein the impurities further comprise a compound of Compound D, wherein the compound of Compound D is:

wherein the amount of the compound of Compound D in the material is documented in writing to be: less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.5%, or even less than or equal to 0.15%.

163. The method of claim 159, wherein the impurities further comprise a compound of Compound D, wherein the compound of Compound D is:

164. The method of claim 159, wherein the impurities further comprise a compound of Compound D, wherein the compound of Compound D is:

wherein the amount of the compound of Compound D in the material is documented in writing to be: less than or equal to 3.0%.

165. The method of claim 156 or 157, wherein the HPLC column is a reverse phase column and the column is eluted using a mobile phase comprising sodium dodecyl sulfate, phosphoric acid, sodium hydroxide, water, and acetonitrile.

166. The method of claim 156, 157 or 158, wherein the amount in the material of each compound is determined by (i) identifying a peak on the chromatogram that corresponds to a peak on a control chromatogram of compounds known to have the structure of Compound A and/or Compound B, (ii) identifying a peak on the chromatogram that corresponds to a relative retention time of a compound known to have the structure of Compound A and/or Compound B, and/or (iii) identifying a peak on the chromatogram that corresponds to a known amount of a spike of the compound known to have the structure of Compound A and/or Compound B.

167. A method for manufacturing a material comprising a compound of Formula I, comprising obtaining a first composition containing the compound of Formula I, purifying the composition by chromatography, recrystallization or a combination thereof, conducting HPLC on a sample of purified first composition using a known amount of one or more of impurities Compound A, Compound B, Compound C and Compound D as a standard, and determining the presence or absence of the impurities in the sample.

168. A method for manufacturing a material consisting essentially of a compound of Formula I, comprising obtaining a first composition containing the compound of Formula I, purifying the composition by chromatography, recrystallization or a combination thereof, conducting HPLC on a sample of purified first composition using a known amount of- one or more of impurities Compound A, Compound B, Compound C and Compound D as a standard, and determining the presence or absence of the impurities in the sample.

169. The method of claim 167 or 168, wherein the purifying is carried out until the impurities are less than 2.0%, 1.5%, 1.0%, 0.9%, 0.8%, 0,7%, 0.6%, 0.5%, 0.4%, 0.3%,

0.2%, 0.15%, 0.1%, 0.05%, or are absent from the purified first composition as determined by HPLC with a detection limit of 0.01 to 0.03% and a quantitation limit of 0.03%.

170. The method of claim 167, 168 or 169, further comprising then packaging the purified first composition.

171. The method of claim 170, further comprising providing indicia on or with the packaged purified first composition indicating a level of the impurities in the packaged purified first composition.