Substituted phenyl boronic acid-containing polymers and methods of use

Abstract

To provide substituted phenyl boronic acid-containing polymers and methods of use.SOLUTION: This disclosure relates to cationic polymers functionalized with substituted phenylboronic acid groups and to methods of using the same to treat metabolic and gastrointestinal disorders. The invention provides polymer compositions for forming a physical barrier in the gastrointestinal (GI) tract of a subject between the intestinal lining and the luminal contents. The polymers of the invention are mucin-interacting agents which form a physical barrier in-situ by interaction with resident mucin in the GI tract. The polymers described herein have improved mucin and mucus complexing activity in comparison to comparable cationic polymers and can effectively condense mucin and mucus at the pH of the duodenum.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 903,328, filed September 20, 2019, the entire disclosure of which is incorporated herein by reference. [Background technology]

[0002] Background of the Invention Approximately 11.3% of the U.S. adult population has type 2 diabetes mellitus (T2DM), and 35% of U.S. adults have prediabetic symptoms. U.S. healthcare costs attributable to diabetes are approaching $200 billion annually. The incidence of T2DM continues to increase in parallel with the obesity epidemic, and most current treatments for T2DM consist of oral medication regimens that can be suboptimal for many patients, in part due to side effects associated with systemic absorption of the medications. Bariatric surgery to bypass or remove the duodenum from the gastrointestinal tract has been shown to improve T2DM. The Diabetes Surgery Summit recommends bariatric surgery for the treatment of T2DM in some obese patients (grade III obesity) and that bariatric surgery should be considered for the treatment of others (see, e.g., Koliaki, C. et al., BMC Endocr Disord. (2017) 17:50 DOI 10.1186 / s12902-017-0202-6).

[0003] Analysis of the typical diabetic patient pathway from first-line medications to insulin and surgery and other highly invasive procedures reveals significant gaps, not least ineffective treatments and clinical inertia. Surgery and other solutions have also not been widely adopted. The inclusion of specialty clinicians in the care pathway contributes to these failures. Thus, effective treatments under the supervision of a primary care physician are likely to reach a much larger segment of the target population and therefore have a greater impact than those requiring a specialist, e.g., an endocrinologist, gastroenterologist, or surgeon.

[0004] US2006 / 0134062 discloses polymers in which certain arylboronic acid moieties are attached to the polymer backbone via long linkers, and the use of such polymers as lipase inhibitors. It is stated that the polymer backbone is not critical for lipase inhibition.

[0005] US 7,943,713 discloses certain polyamine boronic acid derivatives and their use for increasing the wet strength and wet strength of paper. Preferred polymers are characterized by aryl boronic acid moieties attached directly to carbon atoms of the polymer backbone or attached to carbon atoms of the polymer backbone via amide bonds.

[0006] WO2017 / 024237 discloses certain cationic polymers and the use of the polymers to complex mucus to form an occlusive barrier in the duodenum. Seno, M. et al., Materials Science and Engineering C, 62 (2016) 474-479, discloses a specific pH- and sugar-sensitive multilayer film composed of phenylboronic acid-modified poly(allylamine hydrochloride) and poly(vinyl alcohol). Sato, K. et al., Langmuir, 2014, 30, 9247-9250, also discloses a multilayer film composed of phenylboronic acid-modified poly(allylamine hydrochloride) and poly(vinyl alcohol). Thus, there is a need for new medications and non-invasive methods for treating subjects who exhibit T2MD and related metabolic disorders. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] US Patent Application Publication No. 2006 / 0134062 [Patent Document 2] U.S. Patent No. 7,943,713 [Patent Document 3] International Publication No. 2017 / 024237 [Non-patent literature]

[0008] [Non-Patent Document 1] Koliaki, C. et al., BMC Endocr Disord. (2017) 17:50 DOI 10.1186 / s12902-017-0202-6 [Non-patent document 2] Seno, M. et. al, Materials Science and Engineering C, 62 (2016) 474-479 [Non-patent document 3] Sato, K. et al, Langmuir, 2014, 30, 9247-9250 Summary of the Invention [Means for solving the problem]

[0009] Summary of the Invention The present invention provides polymer compositions for forming a physical barrier between the intestinal lining and the luminal contents of the gastrointestinal (GI) tract of a subject. The polymers of the present invention are mucin-interactive agents that form a physical barrier in situ by interacting with resident mucins in the GI tract.

[0010] The present inventors have discovered that the incorporation of pendant substituted phenylboronic acid moieties into certain cationic polymers dramatically improves the mucin complexing activity of the polymers.

[0011] As shown in the examples, the polymers described herein have improved mucin and mucus complexing activity compared to comparable cationic polymers and can effectively concentrate mucin and mucus at duodenal pH. The polymers bind tightly to mucus at duodenal pH, and once bound to mucus, they are resistant to removal by high concentrations of salt (e.g., 1 M NaCl). The resulting polymer-mucus complexes have dramatically different properties compared to free mucus.

[0012] The present disclosure relates to cationic polymers containing pendant substituted phenylboronic acid groups attached directly or indirectly to the polymer backbone via amine or amide bonds, and methods for treating metabolic diseases comprising administering a therapeutically effective amount of such polymers to a subject in need thereof.

[0013] Another aspect of the present invention is a pharmaceutical composition comprising the polymer of the present invention and a carrier or diluent. The pharmaceutical composition may be used for therapy, for example, in the treatment of the disorders described herein. Similarly, the present invention provides the use of the polymer disclosed herein as a medicament, and the use of the polymer disclosed herein in the manufacture of a medicament for treating the disorders described herein. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 illustrates the mucin complexation assay.

[0015] [Figure 2] Figure 2 shows the results of the oral glucose tolerance test for the vehicle control (0.9% saline) and the treatment groups treated with 40 mg / mL of Example 3 and 80 mg / mL of Example 3. Figure 2 shows the blood glucose levels over 120 minutes from each group after glucose administration.

[0016] [Figure 3]FIG. 3 compares the reduction in area under the blood glucose rise curve (iAUC) for the vehicle control (0.9% saline) and treatment groups treated with 40 mg / mL of Example 3 and 80 mg / mL of Example 3, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description of the Invention The present disclosure relates to cationic polymers containing pendant substituted phenylboronic acid groups attached directly or indirectly to the polymer backbone via amine or amide bonds. The cationic polymers are preferably polycations containing amine- or ammonium-containing repeating units, and optionally containing other cationic groups, such as imidazolyl, pyridinyl, and guanidino. The substituted phenylboronic acid polymers preferably contain both cationic repeating units and substituted phenylboronic acid repeating units. The pendant phenylboronic acid moieties are substituted with one or more suitable substituents, including electron-withdrawing and electron-donating groups, as described herein. In some embodiments, the polymer may contain repeating units containing both cationic and substituted phenylboronic acid groups. The cationic polymers may also be copolymers containing any desired neutral or anionic repeating units, as further described herein, provided that the polymer retains a net cationic charge.

[0018] Pharmaceutical compositions comprising these polymers and methods of treatment using these polymers to treat metabolic disorders, such as type 2 diabetes mellitus (T2DM), type 1 diabetes mellitus (T1DM), prediabetes, hyperlipidemia, obesity, overweight, metabolic syndrome, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, and polycystic ovary syndrome (PCOS), are also disclosed.

[0019] Exemplary polymers include repeat units containing substituted phenylboronic acid moieties of formulas (I)-(III). [ka]

[0020] In formulas (I) to (III): R 1 , R 2 , R 3 and R 4 are independently hydrogen or substituted or unsubstituted alkyl; Y 1 is independently in each occurrence a direct bond or -L 1 -A 1 -L 2 -A 2 -And;L 1 is, in each occurrence, -NR 9 -, -NC(O)-, or -C(O)N-; L 2 is absent, -NR, in each occurrence. 9 -, -O- or -S-; Y 2 is independently in each occurrence a direct bond or -L 3 -A 1 -L 2 -A 2 -And;L 3 is, in each occurrence, -C(O)- or absent; L 2 is absent, -NR, in each occurrence. 9 -, -O- or -S-; A 1 and A 2 represents independently in each occurrence an absent or optionally substituted C1 ~C5 alkylene; R 9 , R 10 and R 11 is independently in each occurrence hydrogen or substituted or unsubstituted alkyl (preferably substituted or unsubstituted C1-C6 alkyl); Z, in each occurrence, [ka] and preferably, -B(OH)2 is at the 3- or 4-position of the phenyl ring; X 1 and X 2 is hydrogen, halo, -CN, -NO2, -N + (R 9 )(R 10 )(R 11 ), -CF3, -SO3(R 9 ), -SO2(R 9 ), -CON(R 9 )(R 10 ), -(CH2) m -N(R 9 )(R 10 ), and -OR 9 independently selected from the group consisting of: n is an integer from 1 to 100,000; m is an integer from 0 to 4; However, X 1 and X 2 Not more than one of is hydrogen; X 1 or X 2 Either -(CH2) m -N(R 9 )(R 10 ), it is attached to the carbon atom of the phenyl ring adjacent to the carbon atom of the phenyl ring to which -B(OH)2 is attached, and X 1 and X 2 The other is hydrogen.

[0021] In some embodiments of formulas (I)-(III), X 1 are halo, -CN, -NO2, -N + (R 9 )(R 10 )(R 11 ), -CF3, -SO3(R 9 ), -SO2(R 9 ), or -CON(R 9 )(R 10 ) and X 2 is -(CH2) m -N(R 9 )(R 10 )But-OR 9 nor X;1 is -(CH2) m -N(R 9 )(R 10 ) OR -OR 9 and X 2 Is halo, -CN, -NO2, -N + (R 9 )(R 10 )(R 11 ), -CF3, -SO3H, or -CON(R 9 )(R 10 ) neither.

[0022] In some preferred embodiments, the polymer contains repeat units of formula (I).

[0023] Preferred repeating units of formula (I) include repeating units of formulae (Ia) to (If). [ka]

[0024] In formulas (Ia) to (If), R 1 , R 2 , R 3 , A 1 , A 2 , X 1 , X 2 , m, and n are as described in Formula (I). In some embodiments, -B(OH)2 is at the 3-position of the phenyl ring. In other embodiments, -B(OH)2 is at the 4-position of the phenyl ring.

[0025] In certain instances, the polymer comprises repeat units of formula (Ia), (Ib), (Ic), (Id), (Ie), or (If), and R 1 , R 2 and R 3 are hydrogen atoms.

[0026] In other particular examples, the polymer comprises repeat units of formula (Ia), (Ib), (Ic), (Id), (Ie), or (If), and R 1 and R 2are hydrogen, and R 3 is alkyl, preferably R 3 is methyl.

[0027] On the phenylboronic acid ring, halo, preferably fluoro, -CN, -NO2, -N + (R 9 )(R 10 )(R 11 ), -CF3, -SO3(R 9 ), -SO2(R 9 ), and -CON(R 9 )(R 10 ) is advantageous because these groups are electron-withdrawing in nature and can lower the pKa of the boronic acid.

[0028] On the phenylboronic acid ring adjacent to the boronic acid group, -(CH2) m -N(R 9 )(R 10 ), preferably -(CH2)-N(Me)2, is also advantageous because this group is electron donating in nature and reduces the pKa value due to the interaction between the B and N atoms.

[0029] Without wishing to be bound by any particular theory, it is believed that certain substituted phenylboronic acid polymers have a lower pKa than unsubstituted phenylboronic acid polymers, resulting in greater sensitivity to pH increases between the stomach and duodenum, resulting in increased sensitivity at the desired site of barrier formation. It is believed to enhance the mucin complexing activity of the polymer.

[0030] In formulas (I) to (III), R 1 , R 2 , R 3 , R 4 , Y 1 , Y 2 , m, n and Z are as defined above, and preferably -B(OH)2 is at the 3- or 4-position of the ring, and X 1 and X 2 is at another position on the ring.

[0031] In formulas (I) to (III), R 1 , R 2 , R 3 , R 4 , Y 1 , Y 2 , m, n and Z are as defined above, and preferably -B(OH)2 is at the 3-position of the ring, and X 1 and X 2 is at another position on the ring.

[0032] In formulas (I) to (III), R 1 , R 2 , R 3 , R 4 , Y 1 , Y 2 , m, n and Z are as defined above, and preferably -B(OH)2 is at the 4-position of the ring, and X 1 and X 2 is at another position on the ring.

[0033] In formulas (I) to (III), R 1 , R 2 , R 3 , R 4 , Y 1 , Y 2 , m, n and Z are as defined above, and preferably -B(OH)2 is at the 3- or 4-position of the ring, and X 1 or X 2 Either -(CH2) m -N(R 9 )(R 10 ) and X 2 is bonded to a carbon atom of the phenyl ring adjacent to the carbon atom of the phenyl ring to which -B(OH)2 is bonded, and X 1 and X 2 The other is hydrogen.

[0034] In formulas (I) to (III), R 1 , R 2 , R 3 , R 4 , Y 1 , Y 2, m, n and Z are as defined above, and preferably -B(OH)2 is at the 3-position of the ring, and X 1 or X 2 Either -(CH2) m -N(R 9 )(R 10 ) and X 2 is bonded to a carbon atom of the phenyl ring adjacent to the carbon atom of the phenyl ring to which -B(OH)2 is bonded, and X 1 and X 2 The other is hydrogen.

[0035] In formulas (I) to (III), R 1 , R 2 , R 3 , R 4 , Y 1 , Y 2 , m, n and Z are as defined above, and preferably -B(OH)2 is at the 4-position of the ring, and X 1 or X 2 Either -(CH2) m -N(R 9 )(R 10 ) which is bonded to a carbon atom of the phenyl ring adjacent to the carbon atom of the phenyl ring to which -B(OH)2 is bonded, and X 1 and X 2 The other is hydrogen.

[0036] In formulas (I) to (III), R 1 , R 2 , R 3 , R 4 , Y 1 , Y 2 , m, n and Z are as defined above, and preferably -B(OH)2 is at the 3-position of the ring, and X 1 is at the 5-position of the ring and is halo, preferably fluoro; X 2 is a group containing hydrogen, halo, -CN, -NO2, -N at the 2, 5, or 6 position of the ring. + (R 9 )(R 10 )(R 11 ), -CF3, -SO3(R 9 ), -SO2(R 9), or -CON(R 9 )(R 10 )

[0037] In formulas (I) to (III), R 1 , R 2 , R 3 , R 4 , Y 1 , Y 2 , m, n and Z are as defined above, and preferably -B(OH)2 is at the 4-position of the ring, and X 1 is at the 2-position of the ring and is halo, preferably fluoro; X 2 is a group containing hydrogen, halo, -CN, -NO2, -N at the 3, 5, or 6 position of the ring. + (R 9 )(R 10 )(R 11 ), -CF3, -SO3(R 9 ), -SO2(R 9 ), or -CON(R 9 )(R 10 )

[0038] In formulas (I) to (III), R 1 , R 2 , R 3 , R 4 , Y 1 , Y 2 , m, n and Z are as defined above, and preferably -B(OH)2 is at the 3-position of the ring, and X 1 is at the 5-position of the ring and is halo, preferably fluoro; X 2 is hydrogen.

[0039] In formulas (I) to (III), R 1 , R 2 , R 3 , R 4 , Y 1 , Y 2 , m, n and Z are as defined above, and preferably -B(OH)2 is at the 4-position of the ring, and X 1 is the ring at position 2, is halo, preferably fluoro; X 2 is hydrogen.

[0040] In formulas (I) to (III), R 1 , R 2 , R 3 , R 4 , Y 1 , Y 2 , m, n and Z are as defined above, and preferably -B(OH)2 is at the 4-position of the ring, and X 1 is at the 2-position of the ring and is halo, preferably fluoro; X 2 is halo, preferably fluoro, at the 3, 5, or 6 position of the ring.

[0041] In formulas (I) to (III), R 1 , R 2 , R 3 , R 4 , Y 1 , Y 2 , m, n and Z are as defined above, and preferably -B(OH)2 is at the 4-position of the ring, and X 1 is at the 2-position of the ring and is halo, preferably fluoro; X 2 is at the 3-position of the ring is halo, preferably fluoro.

[0042] In formulas (I) to (III), R 1 , R 2 , R 3 , R 4 , Y 1 , Y 2 , m, n and Z are as defined above, and preferably -B(OH)2 is at the 4-position of the ring, and X 1 is at the 2-position of the ring and is -CF3, and X 2 is hydrogen.

[0043] In formulas (I) to (III), R 1 , R 2 , R 3 , R 4 , Y 1 , Y 2 , m, n and Z are as defined above, and preferably -B(OH)2 is at the 3-position of the ring, and X 1 or X 2 Either -(CH2) m -N(R 9)(R 10 ) which is bonded to a carbon atom of the phenyl ring adjacent to the carbon atom of the phenyl ring to which -B(OH)2 is bonded, and X 1 and X 2 The other is hydrogen.

[0044] In formulas (I) to (III), R 1 , R 2 , R 3 , R 4 , Y 1 , Y 2 , m, n and Z are as defined above, and preferably -B(OH)2 is at the 4-position of the ring, and X 1 or X 2 Either -(CH2) m -N(R 9 )(R 10 ) which is bonded to a carbon atom of the phenyl ring adjacent to the carbon atom of the phenyl ring to which -B(OH)2 is bonded, and X 1 and X 2 The other is hydrogen.

[0045] In formulas (Ia) to (If), R 1 , R 2 , R 3 , A 1 , A 2 , m and n are as described in formula (I), preferably -B(OH)2 is at the 4-position of the ring, and X 1 is at the 2-position of the ring and is halo, preferably fluoro; X 2 is hydrogen.

[0046] In formulas (Ia) to (If), R 1 , R 2 , R 3 , A 1 , A 2 , m and n are as described in formula (I), preferably -B(OH)2 is at the 4-position of the ring, and X 1 is halo, preferably fluoro, at the 2-position of the ring, and X 2 is at the 3-position of the ring is halo, preferably fluoro.

[0047] In formulas (Ia) to (If), R 1 , R 2 , R 3 , A 1 , A 2 , m and n are as described in formula (I), preferably -B(OH)2 is at the 4-position of the ring, and X 1 is -CF3 at the 2-position of the ring, and X 2 is hydrogen.

[0048] In formulas (Ia) to (If), R 1 , R 2 , R 3 , A 1 , A 2 , m and n are as described in formula (I), preferably -B(OH)2 is at the 3-position of the ring, and X 1 At the 4th position of the ring, -(CH2) m -N(R 9 )(R 10 ), preferably —CH—N(Me) and X 2 is hydrogen.

[0049] In formulas (Ia) to (If), R 1 , R 2 , R 3 , A 1 , A 2 , m and n are as described in formula (I), preferably -B(OH)2 is at the 4-position of the ring, and X 1 At the 3rd position of the ring, -(CH2) m -N(R 9 )(R 10 ), preferably —CH—N(Me) and X 2 is hydrogen.

[0050] In other preferred embodiments, the polymer contains repeat units of formula (II). In some examples of polymers containing repeat units of formula (II), Y 2 is a direct bond and Z is [ka] Preferably, -B(OH)2 is at the 3- or 4-position of the ring, and X 1 and X 2 is as above.

[0051] In other preferred embodiments, the polymer contains repeat units of formula (II). In some examples of polymers containing repeat units of formula (II), Y 2 is a direct bond and Z is [ka] Preferably, -B(OH)2 is at the 3- or 4-position of the ring, and X 1 and X 2 is as above.

[0052] In other preferred embodiments, the polymer contains repeat units of formula (III). In some examples of polymers containing repeat units of formula (III), Y 2 is a direct bond and Z is [ka] Preferably, -B(OH)2 is at the 3- or 4-position of the ring, and X 1 and X 2 is as above.

[0053] The polymer may be a homopolymer. In the case of a homopolymer, the polymer contains nitrogen-containing repeating units (e.g., polyamines or polyamides) with pendant boronic acid moieties attached directly or indirectly to the polymer backbone via nitrogen atoms in the repeating units. Thus, the polymer typically contains secondary or tertiary amines, or optionally quaternary ammonium groups, to which boronic acid moieties are attached. The secondary or tertiary amines are protonated at about pH 5-7, providing a cationic polymer.

[0054] Preferably, the polymer is a copolymer containing repeat units of any one of formulas (I) to (III) and one or more other repeat units, which are preferably cationic (e.g., nitrogen-containing repeat units), but may be neutral or anionic, provided that the polymer retains an overall cationic charge.

[0055] Preferred nitrogen-containing repeat units that may be modified to include pendant boronic acid moieties include poly(allylamine) (PAAn), poly(diallylamine) (PDAAn), These include poly(ethyleneimine) (PEI) and poly(methacrylamidopropylamine) (PMAPAn).

[0056] In the polymers disclosed herein, at least about 5% of the repeating chemical units contain a pendant boronic acid group, e.g., a repeating unit of any one of Formulas (I)-(III). In some cases, substantially all of the repeating chemical units in the polymer contain a pendant boronic acid group. Preferably, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, or about 5% to about 15% of the repeating chemical units contain a pendant boronic acid group.

[0057] Suitable nitrogen-containing repeat units for inclusion in polymers are well known in the art and include, for example, polyvinylamine, poly-N-alkylvinylamine, polyacrylamide, polyalkylacrylamide (e.g., polymethacrylamide), poly-N-alkylacrylamide, polyalkyl-N-alkylacrylamide, polyallylamine, poly-N-alkylallylamine, polydiallylamine, poly-N-alkyldiallylamine, polyethyleneimine, polyaminostyrene, polyvinylimidazole, polyvinylpyridine, and the like.

[0058] When the amino nitrogen is protonated, the nitrogen-containing repeat unit is cationic. If desired, the cationic character may be modified using known methods, for example, by converting the amine to a guanidino, biguanide, aromatic, such as imidazolyl and pyridinyl, quaternary ammonium, or by introducing additional amino groups, for example, by alkylating the amine with an alkylamino or alkylammonium group.

[0059] Polyamines are typically highly charged at duodenal pH (approximately pH 5-6), but due to the high density of nearby protonated amine sites, they become slightly deprotonated upon passage from the stomach (pH approximately 2) to the duodenum (pH approximately 5) after ingestion. Even a small amount of neutralization effectively reduces the charge density of the polymer, leading to these polymer chains becoming more coiled, compacted, and poorly hydrated as the pH increases. Without wishing to be bound by theory, this pH responsiveness is believed to contribute to preferential mucus complexation in the duodenum compared to the stomach. Other polymers of the present invention that can respond to increased duodenal pH contain cationic repeat units (e.g., repeat units with protonated amines) with derivative or structural features that result in a lower pKa value than that of standard protonated aliphatic amines. The lower pKa of these protonated polymers makes them more sensitive to the increase in pH that coincides with passage from the stomach to the duodenum. As a result, these types of polymers can be targeted to interact with the loose mucus of the proximal duodenum, and include polyamines substituted with polar groups, such as hydroxyl groups, less than three carbon atoms away from the protonated amine. In some embodiments, the polymers include amines modified to have a lower pKa than the unmodified amine. For example, the cationic polymer may have a pKa value of less than 9.0, more preferably less than 8.0, and most preferably less than or equal to 7.0.

[0060] Boronic acid-containing repeat units suitable for inclusion in the polymers described herein include, but are not limited to, repeat units of formulas (I), (Ia)-(If), (II), and (III): [ka] [ka] [ka] where "m" represents an integer from 1 to 100,000; and X 1 and X 2 is written as above.

[0061] Exemplary polymers containing substituted phenylboronic acid repeat units include the following: [ka]

[0062] Exemplary polymers containing substituted phenylboronic acid repeat units include the following: [ka]

[0063] Suitable repeat units of nitrogen-containing cationic monomers include, but are not limited to, the following: [ka] [ka] [ka] [ka] where "n" represents an integer from 1 to 100,000.

[0064] Optionally, the cationic polymer containing pendant boronic acid groups may further comprise a hydrophobic group, e.g. For example, it may also contain pendant hydrophobic groups. As used herein, a hydrophobic group is a moiety that is more soluble (as an individual chemical) in octanol than in water. For example, an octyl substituent is a hydrophobic group because octane is more soluble in octanol than in water. Suitable hydrophobic groups are, for example, C6 or higher linear, branched, or cyclic hydrocarbons optionally substituted with one or more hydroxy, halo, and / or aryl (e.g., benzyl) groups.

[0065] If desired, additional repeat units that may be included in the polymers described herein include neutral and acid repeat units, such as polyacrylates, polyalkylene glycols, polystyrenes, polyvinyl alcohols, polyvinyl phosphates, polyvinyl sulfates, and the like.

[0066] Specific examples of polymers of the present disclosure include: [ka] There is.

[0067] Additional specific examples of polymers of the present disclosure include: [ka] There is.

[0068] Additional specific examples of polymers of the present disclosure include: [ka] There is.

[0069] Additional specific examples of polymers of the present disclosure include: [ka] There is.

[0070] Additional specific examples of polymers of the present disclosure include: [ka] [ka] There is.

[0071] The copolymers of the present invention may exist in a variety of forms, with suitable forms including block copolymers, graft copolymers, comb copolymers, star copolymers, dendrimers, hyperbranched copolymers, random copolymers, gradient block copolymers, and alternating copolymers.

[0072] The present disclosure also provides cationic polymers containing pendant hydrophobic groups, and Disclosed herein is the use of such polymers to treat metabolic diseases.

[0073] Preferably, the polymers disclosed herein are of a size sufficient to prevent the polymer from being substantially absorbed when orally administered to a subject, such as a human. The molecular weight threshold above which the polymer is not absorbed from the GI tract into the systemic circulation depends on the specific polymer and the conditions in the GI tract, as well as other factors, but it is generally recognized that polymers greater than 1,000 Da are not substantially absorbed from the GI tract into the systemic circulation. Thus, compositions of the present invention that are not substantially absorbed from the GI tract are substantially free of polymer chains less than 1,000 Da, preferably 5,000 Da, or more preferably 10,000 Da, and have an average molecular weight (M) of at least about 10,000 Da, preferably in the range of 20,000 to 250,000 Da, or more. wThe polymers of the invention may contain a distribution of polymer chain lengths and may have a polydispersity index (PDI) in the range of 1.5 to 4.0, but they are substantially free of material less than 1,000 Da, preferably they are free of material less than 5,000 Da, or more preferably they are free of material less than 10,000 Da.

[0074] The polymers of the present invention are soluble and preferably not cross-linked, in some embodiments the polymers may be slightly cross-linked but remain soluble and do not form extensive networks or gels.

[0075] Pharmaceutically acceptable salts of the disclosed polymers are also included in the present invention. For example, polymers having acid functional groups may also exist in anionic form or in combination with a cation in the form of a conjugate base. Suitable cations include alkaline earth metal ions, such as sodium and potassium ions, alkaline earth ions, such as calcium and magnesium ions, and unsubstituted and substituted (primary, secondary, tertiary, and quaternary) ammonium ions. Polymers having basic groups, such as amines, may also be protonated and may be combined with a pharmaceutically acceptable counteranion, such as a halide (Cl). - and Br - ), CH3OSO3 - , HSO4 - , SO4 2- , HCO 3- , CO3 2-, nitrate, hydroxide, persulfate, sulfite, acetate, formate, sulfate, phosphate, lactate, succinate, propionate, oxalate, butyrate, ascorbate, citrate, dihydrogencitrate, tartrate, taurocholate, glycocholate, cholate, hydrogencitrate, maleate, benzoate, folate, amino acid derivatives, nucleotides, lipids, or phospholipids. Similarly, the ammonium group contains a pharmaceutically acceptable counterion. Boronic acid groups can be reacted with anions, such as sodium or potassium hydroxide, alkoxides, or carboxylates, to form salts, such as -B-(OH)3Na. + , -B-(OH)3K + , -B-(OH)2(OCH3)Na + , -B-(OH)2(OCH3)K + , -B-(OH)2(OCOCH3)Na + , -B-(OH)2(OCOCH3)K + etc. can be formed.

[0076] The polymers disclosed herein are typically provided as a mixture of polymer chains with some variation in chain length. This distribution of polymer chain length can be measured using a detector capable of measuring the molar mass of polymers, such as size exclusion chromatography (SEC) and multi-angle laser light scattering (MALLS). This method can also confirm the absence of short, low molecular weight polymer chains. This method can also provide a polydispersity index (PDI), which is typically the ratio M w / M n (M w is the weight fraction average molecular weight, and M n is the number average molecular weight). PDI=M w / M n

[0077] M w , M n and PDI values ​​can be obtained by SEC and MALLS detectors. It is preferred to obtain PDI values ​​greater than 2, or even greater than 3, for synthetic polymeric materials made from standard free radical processes. In contrast, living free radical polymerization processes, such as atom transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer (RAFT), can produce materials with PDIs less than 2, or even less than 1.5.

[0078] The polymers disclosed herein can be prepared using any suitable method, for example, by direct polymerization of one, two or more monomers or by polymer modification.

[0079] Polymerization may be carried out using techniques known in the art of polymer synthesis (see, for example, Shalaby et al., ed., Water-Soluble Polymers, American Chemical Society, (See Washington, DC

[1991] .) Some cationic monomers are They are available as salts and may be polymerized by methods known in the art, for example, via a free radical addition process. In this case, the polymerization mixture includes a free radical initiator. Suitable free radical initiators include azobis(isobutyronitrile), azobis(4-cyanovaleric acid), 2,2'-azobis(2-amidinopropane) dihydrochloride, potassium persulfate, ammonium persulfate, and potassium hydrogen persulfate. Other suitable initiators include ionizing radiation and ultraviolet light. The free radical initiator is preferably present in the reaction mixture in an amount ranging from about 0.01 mole percent to about 5 mole percent relative to the monomers.

[0080] The polymer modification approach uses polyamines, while the copolymer approach uses acrylamide derivatives. In the reaction scheme, "M" represents a group containing a substituted phenylboronic acid moiety.

[0081] Polyamines can serve as mucus-interacting agents and starting materials for chemical modification with boronic acid groups. Exemplary polyamines include polyethyleneimine, hydroxyethylated polyethyleneimine, polyamidoamine (PAMAM) dendrimers, poly(allylamine) (PAAn) and its copolymers, poly(diallylamine) (PDAAn) and its copolymers, poly(vinylamine) and its copolymers, poly(vinyl imidazole) and its copolymers, poly(vinyl pyridine) and its copolymers, poly(vinyl aniline) and its copolymers, and amine-containing acrylamide and methacrylamide copolymers. Preferred polyamines include poly(allylamine) (PAAn), poly(diallylamine) (PDAAn), poly(ethyleneimine) (PEI), and poly(methacrylamidopropylamine) (PMAPAn).

[0082] Polyamine derivatives can be obtained from chemical modification of polyamines via amide-forming chemistry using EDC coupling (Scheme 1). [ka] Scheme 1: Amide-forming reaction of polyamines with carboxylic acids using EDC as a coupling reagent.

[0083] 1% wt / vol of the desired polyamine is prepared in deionized water with the pH adjusted to 5.0. Ethanol or other suitable organic is added to the polymer solution at 50% of the initial polymer solution volume. The M-carboxylic acid to be coupled is placed in water at 25% of the initial polymer solution volume to form a solution or slurry. The EDC coupling agent is dissolved in ethanol or other suitable solvent at 25% of the initial polymer solution volume. The EDC solution is then mixed with the M-carboxylic acid solution or slurry. The combined EDC / M-carboxylic acid solution is added dropwise to the polymer solution via a pipette or pressure-equalizing dropping funnel over approximately 10 minutes. The reaction solution contains approximately 0.5% wt / vol of polymer, approximately 62% vol of water, and approximately 38% vol of ethanol or other suitable organic solvent. The reaction is stirred and the pH is maintained at 5.0. The reaction is stirred at room temperature for approximately 18 hours while the pH is stabilized at 5.0. The polymer is precipitated with an excess (3x volume) of acetone.

[0084] Polyamine derivatives can be obtained from the chemical modification of polyamines via the Michael addition reaction, where polyamines are the nucleophiles and acrylamides are the Michael acceptors (Scheme 2). [ka] Scheme 2: Michael addition reaction of polyamine nucleophiles with acrylamide Michael acceptors.

[0085] 1% wt / vol of the desired polyamine was prepared in deionized water and the pH was adjusted to 8.5. This pH may be increased or decreased depending on the level of modification desired. The desired M-acrylamide was dissolved in ethanol or other suitable solvent at 20% of the initial polymer solution volume. The acrylamide solution was then added to the polymer solution to form a reaction mixture containing approximately 0.83% wt / vol of polymer, approximately 83% vol of water, and approximately 17% vol of ethanol or other suitable solvent. The reaction mixture was heated to 70°C and stirred for 48 hours. The polymer was precipitated with an excess (3x volume) of acetone.

[0086] Polyamine derivatives can be obtained from the chemical modification of polyamines by hydroxyalkylation using epoxide ring-opening chemistry (Scheme 3). [ka] Scheme 3: Reaction of polyamines with epoxides.

[0087] 2% wt / vol of the desired polyamine is prepared in deionized water and the pH adjusted to 6.0. This pH may be increased or decreased depending on the level of modification desired. The desired M-epoxide is dissolved in water / ethanol (25% / 75%) at 100% of the initial polymer solution volume. This solution is then added to the polymer solution to prepare a reaction solution containing approximately 1% wt / vol of polymer, approximately 62% vol of water, and approximately 38% vol of ethanol. The reaction mixture is heated at 60°C for 48 hours. If the epoxide is not in complete solution at 60°C, a small amount of additional ethanol may be added to aid dissolution. The polymer is precipitated with an excess (3 times the volume) of acetone.

[0088] Polyacrylamide derivatives can be obtained, for example, by polymerization of 3-acrylamidopropylamine with the desired M-acrylate or M-acrylamide (Scheme 4). [ka] Scheme 4: Reaction of acrylamide with acrylate.

[0089] The desired amount of the desired M-acrylate or M-acrylamide monomer is placed in a 30 ml glass vial equipped with magnetic stirring and a N2(g) inlet. The desired M-acrylate or M-acrylamide monomer is dissolved in dimethylformamide or another suitable water-miscible organic solvent. The desired amount of cationic, neutral, or anionic comonomer is then added. A small amount of water may be required to fully dissolve the charged comonomer in the binary solvent system. The appropriate amount of AIBN initiator is added. The comonomer solution is purged with N2(g) for at least about 15 minutes. The reaction is then heated at 65°C under N2(g). After heating for several hours, the copolymer solution or suspension is precipitated from acetone. The polymer is isolated by adding some water and adjusting the pH to lower it to facilitate precipitation in acetone. Finally, the product can be dissolved in deionized water, frozen in IPA / dry ice, and lyophilized.

[0090] Accordingly, in some aspects, the present invention provides a method for applying a physical barrier between the intestinal lining and the luminal contents of the gastrointestinal (GI) tract of a subject, the method comprising administering a therapeutically effective amount of a polymer described herein to the GI tract of the subject.

[0091] As used herein, the terms "physical barrier" or "luminal barrier" refer to a polymer-mucus complex that prevents or reduces chyme contact with the mucosal epithelium underlying the polymer-mucous complex in the intestinal tract. The physical barrier is generated when the polymer binds in situ with anionic mucins contained in the mucus lining the intestinal wall. A physical barrier may be substantially complete or partial. A substantially complete physical barrier extends to substantially cover the entire epithelial lining of a target area, e.g., the proximal duodenum. A partial physical barrier extends to cover a portion of a target area, e.g., a portion of the epithelial lining of the duodenum. For example, a partial barrier may cover at least about 1%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, or more of the epithelium at the target site.

[0092] The partial physical barrier may be discontinuous and spatially distributed and may have varying degrees of permeability, for example, a physical barrier may be a semi-permeable complex of a polymer and mucus or mucin on the intestinal luminal surface, preferably in the duodenum.

[0093] In embodiments, the physical barrier or a formulation thereof passes through the subject's natural digestive process. In yet other embodiments, the physical barrier is removable or reversible by ingesting a liquid or solvent.

[0094] The polymers described herein bind tightly to mucus and mucin, forming polymer-mucus complexes that are resistant to dissociation (e.g., by high salt concentrations and low pH). Thus, once formed, the physical barrier typically exists for a "retention period" or "dwell time" before being removed by the natural action of the digestive system. Typical retention periods may range from about 30 minutes to about 7 days, including periods ranging from about 1 hour to about 3 hours, from about 1 hour to about 5 hours, from about 1 hour to about 24 hours, from about 1 to about 3 days, etc.

[0095] The desired residence time may vary depending on the clinical application and may be adjusted based on the amount of polymer administered, as well as the frequency and interval between doses. For example, up to 50% of subjects with T2DM have gastroparesis or delayed gastric emptying, which may require a mucoadhesive lining that remains in place for a longer period of time than prediabetic or nondiabetic obese subjects. Blood glucose levels often spike within the first two hours after a meal, and most often within the first 60 minutes; therefore, in one embodiment, the lining should adhere for at least 60 minutes. In another embodiment, a longer-lasting mucoadhesive lining may be required for prediabetic subjects who may not take medication before every meal and therefore may not be compatible with treatment and may need to change their behavior. In this application, the lining may adhere for a minimum of 6-8 hours, and may be required for up to 24 hours. Residence time is also influenced by the mucus layer to which the polymer has the greatest affinity. For example, a superficial, loosely adherent layer will shed within minutes to hours, while an affinity for a deeper, tightly adherent layer will result in a longer-lasting mucoadhesive coating. Overall, dwell times may be tailored to various clinical and technical considerations in the embodiments outlined in this disclosure.

[0096] The polymers of the present invention are capable of forming an occlusive barrier layer in the proximal intestine, particularly the duodenum. Preferably, the occlusive barrier is formed in the proximal duodenum or duodenal bulb, so that the polymer can be released from the stomach and fully form a barrier layer upon entering the proximal duodenum.

[0097] The polymer is orally administered in any suitable dosage form. A variety of dosage forms suitable for oral administration are well known in the art and include liquid formulations (e.g., solutions, suspensions, slurries, syrups), gels, ointments, powders, tablets, caplets, capsules, and the like.

[0098] In one example, polymer can be administered in liquid form, and is typically sufficiently stable and soluble in the stomach, allowing immediate delivery to the duodenum in an active state, without needing further swelling, solubilization, or equilibration with the surrounding environment.The polymer described herein is typically a polyamine, which undergoes some deprotonation when moving from the highly acidic stomach (pH about 2) to the duodenum (pH about 5), which allows the complexing activity of polymer to be targeted to the duodenum.However, polymer may also form a barrier layer in the stomach.

[0099] In another example, the polymer is administered in a solid form that can hydrate in the stomach. The solid form may be formulated to dissolve slowly, which can protect the polymer from gastric acidity but allow the polymer to enter the proximal duodenum in a fully active state. In another example, the polymer is administered in the form of an enteric-coated tablet, caplet, capsule, or other enteric-coated dosage form to protect the polymer from gastric acidity. In such an example, the enteric coating is formulated to dissolve or degrade as quickly as possible after or during passage through the pyloric valve (when the pH increases from about pH 2), allowing for immediate release of the polymer. Such dosage forms may include a superdisintegrant to facilitate immediate release of the polymer at the desired site in the intestine, for example, the proximal duodenum. Suitable superdisintegrating excipients are well known in the art (e.g., Mohanachandran, PS et al., Superdisintegrants: An Overview, Int. J. Pharma. Sci. Review and Research, (2011) 6:1 pp 105-109). For example, Enteric capsules that can be targeted are described in the literature (e.g., Reix N. et al. Intl J Pharm (2012) 422:1-2 pp. 338-340).

[0100] The polymers of the present invention may dissolve rapidly in the stomach, duodenum or on other mucosal surfaces after oral administration.

[0101] In some embodiments, pharmaceutical formulations of the polymers of the present invention can optionally include a calcium salt, such as calcium chloride or calcium citrate, as it is believed that the formation of a physical barrier can be facilitated in the presence of calcium salts.

[0102] If desired, the polymer may be administered to the gastrointestinal tract of the subject via an endoscope, nasogastric tube, oral gavage tube, or similar device. The polymer may also be sprayed onto the mucosa at the desired site of action; for example, spraying may be performed endoscopically.

[0103] For therapeutic purposes, a "therapeutically effective amount" of the polymer is administered. As used herein, a therapeutically effective amount is an amount sufficient to affect the desired response, including clinical response, under the conditions of administration. A therapeutically effective amount may be, for example, sufficient to improve glucose homeostasis, reduce insulin resistance, cause weight loss, and / or improve other signs and / or symptoms of T1DM, T2DM or other metabolic disorders, such as hyperlipidemia, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, and other conditions, such as obesity and overweight. For example, a therapeutically effective amount may be an amount sufficient to lower blood glucose levels and / or reduce HbA1C.

[0104] The exact amount to be administered will depend on several known factors, including age, weight, sex, the specific condition to be treated and its severity, sensitivity to the drug, and the subject's overall health. A skilled clinician can determine the appropriate amount to administer based on these and other factors. Typically, 1 to 5 tablets / capsules are administered per dose, each sized 0 or 0E or 00 or 00E or 000. Dosages may be administered 1, 2, 3, or 4 times per day. The timing of administration will be based on the underlying indication. Preferably, for the treatment of metabolic conditions, the dose is administered at least 5, 10, 15, 30, or 60 minutes before a meal and within 12 hours before a meal. For other indications, administration immediately before or with a meal may be preferred.

[0105] As used herein, and as is well understood in the art, "treatment" refers to an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results may include, but are not limited to, alleviation or improvement of one or more symptoms or conditions, whether detectable or not, reduction in the severity of the disease or affliction, a stabilized (i.e., non-worsening) state of the disease or affliction, prevention of the spread of the disease or affliction, delay or slowing of the progression of the disease or affliction, improvement or alleviation of the state of the disease or affliction, and remission (whether partial or total). "Treatment" can also mean prolonging survival compared to expected survival if not receiving treatment.

[0106] The present disclosure relates to methods of treating metabolic diseases by administering a therapeutically effective amount of the polymers disclosed herein to a subject in need thereof. Metabolic diseases that can be treated using the methods include, for example, glucose intolerance, T1DM, T2DM, prediabetes, hyperlipidemia, obesity, overweight, obesity, dyslipidemia, hypertension, hyperglycemia, impaired glucose tolerance, insulin resistance, metabolic syndrome, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), and polycystic ovary syndrome (PCOS).

[0107] The present disclosure also relates to methods of treating gastrointestinal disorders by administering a therapeutically effective amount of the polymers disclosed herein to a subject in need thereof. Gastrointestinal disorders that can be treated using the methods include, for example, celiac disease, irritable bowel syndrome, inflammatory bowel disease, colitis, Clostridium difficile, endotoxemia, diarrhea, and constipation.

[0108] The methods of the present invention are also useful in treating leaky gut syndrome and related conditions. Leaky gut syndrome is a technical term describing a condition in which intestinal permeability increases due to alterations / damage to epithelial tight junctions, resulting in impaired epithelial barrier function. This impaired barrier acts as a conduit for intraluminal macromolecules and antigens, allowing them to pass through the intestinal wall and triggering inflammatory immunological responses, which lead to various health conditions. For example, leaky gut syndrome is involved in IBS (irritable bowel syndrome). Certain proteins in food can act as antigens that elicit an immune response. For example, in celiac disease, preventing gluten from contacting the epithelium can reduce the immunological response. Leaky gut is also involved in other immunological conditions, such as inflammatory bowel disease (Crohn's disease, ulcerative colitis). Strengthening the intestinal barrier can reduce endotoxin absorption in the gastrointestinal tract. Some of these endotoxins are the result of normal bacterial metabolism / degradation or bacterial overgrowth. This is particularly relevant for patients with liver dysfunction, for example, in cirrhosis, endotoxin is not metabolized (detoxified) by the liver in patients, resulting in brain dysfunction (called hepatic encephalopathy).Therefore, the method described herein can be used to strengthen the barrier properties of intestine, and can treat or reduce the occurrence of hepatic encephalopathy.Uremia is the impairment of intestinal barrier function. Another condition associated with chronic kidney disease (CKD) is another condition that can be treated or reduced using the methods described herein. Similarly, because clinical evidence demonstrates that intestinal permeability is high in patients with advanced chronic kidney disease (CKD), the methods described herein can be used to treat or reduce the incidence of CKD.

[0109] Therapeutic methods may also provide benefit by reducing clinical biomarkers associated with various disorders, for example, reducing systemic inflammation, oxidative stress, and hyperuricemia.

[0110] The disclosed polymers may be administered to a subject in the form of a pharmaceutical composition that includes a pharmaceutically acceptable carrier, excipient, buffer, or diluent.

[0111] For oral administration, the pharmaceutical compositions of the present invention may be presented in dosage forms such as capsules, tablets, caplets, powders, granules, gels, suspensions, liquids, or other suitable dosage forms. Capsules may be gelatin, soft gel, or solid. Tablet, caplet, and capsule formulations may further contain one or more adjuvants, binders, diluents, disintegrants, excipients, fillers, or lubricants, each of which is known in the art. Examples of such agents include carbohydrates such as lactose or sucrose, anhydrous calcium hydrogen phosphate, corn starch, mannitol, xylitol, cellulose or its derivatives, microcrystalline cellulose, gelatin, stearate, silicon dioxide, talc, sodium starch glycolate, acacia, flavoring agents, preservatives, buffers, disintegrants, and coloring agents.

[0112] Orally administered compositions can contain one or more necessary agents, for example, sweeteners such as fructose, aspartame or saccharin; flavorings such as peppermint, wintergreen oil or cherry; coloring agents; and preservatives, in order to provide pharmaceutically palatable preparations.The pharmaceutical preparation suitable for oral administration and the method for preparing it are well known in the art.See, for example, Remington: The Science and Practice of Pharmacy, twentieth edition, 2000.

[0113] Pharmaceutical preparations that may be used orally include push-fit capsules made of suitable materials, such as gelatin, and soft, sealed capsules made of suitable materials, such as gelatin, and plasticizers, such as glycerol or sorbitol. Push-fit capsules may also contain the active ingredient mixed with a filler, such as lactose, a binder, such as starch, and / or a lubricant, such as talc or magnesium stearate, and, if necessary, a stabilizer. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. Additionally, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. Methods for encapsulating compositions (e.g., in hard gelatin or cyclodextran coatings) are known in the art (Baker, et al., "Controlled Release of Biological Active Agents", John Wiley and Sons, 1986).

[0114] The method of the present invention involves the co-formulation of a polymer composition with probiotics. The probiotic formulation helps to increase beneficial probiotic bacteria in the intestinal tract. Probiotics are known in the art to have a significant effect on reducing blood glucose, HbA1c, insulin levels, and insulin resistance in subjects with diabetes. Suitable probiotics include, but are not limited to, Lactobacillus, Bifidobacteria, S. accharomyces boulardii, and Bacillus coagulans, Akkermansia muciniphila, Bifidobacterium spp., Escherichia spp. Methods for preparing formulations containing probiotics are well known in the art.

[0115] Optionally, the methods of treatment described herein may include co-administration of the polymer composition with one or more additional therapeutic agents. Therapeutic agents for co-administration in subjects with diabetes can include GLP-1 receptor agonists, DPP-4 inhibitors, SGLT-2 inhibitors, glucosidase inhibitors, insulin, metformin, sulfonylureas, and drugs from the thiazolidenedione classes.

[0116] In particular examples, the additional therapeutic agent is one or more drugs indicated for the treatment of diabetes (type 1 and / or type 2), prediabetes, hyperglycemia, impaired glucose tolerance, or insulin resistance. Such drugs include biguanides (e.g., metformin), sulfonylureas (e.g., rimepiride, gliclazide, gilpizide, glimepiride, tolbutamide, glibenclamide (glyburide), gliquidone, and glyclopyramide), meglitinides (e.g., repaglinide and nateglinide), thiazolidinediones (e.g., pioglitazone and rosiglitazone), alpha-glucosidase inhibitors (e.g., acarbose and miglitol), dipeptidyl peptidase 4 (DPP4) inhibitors (e.g., vildagliptin, sitagliptin, saxagliptin, and linagliptin), GLP-1 analogs (e.g., exenatide, lixivistatin, lixivistatin), and the like. senatide, dulaglutide, and liraglutide), sodium-glucose cotransporter 2 (SGLT2) inhibitors (e.g., dapagliflozin, ganagliflozin, and empagliflozin), amylin mimetics (e.g., pramlinitide), D2-dopamine agonists (e.g., bromocriptine), bile acid sequestrants (e.g., cholestyramine, colesevelam, colestilan, and colestimide), and insulins (e.g., human insulin, insulin glulisine, insulin lispro, insulin isophane human, insulin zinc suspension mixed bovine, insulin protamine zinc bovine, insulin isophane porcine, insulin isophane human, etc.).

[0117] Combination therapy can provide several advantages over monotherapy. For example, administering a polymer and an additional therapeutic agent may enhance the efficacy and / or reduce the amount of the additional therapeutic agent required for the desired effect. Thus, undesirable side effects of the additional therapeutic agent may be reduced or eliminated. Furthermore, the polymers of the present invention and the additional therapeutic agent may provide superior therapy compared to each agent as a monotherapy, and the combination therapy may provide additive or synergistic effects.

[0118] The subject to be treated by the method disclosed herein is typically a mammal, preferably a human subject.Suitable subjects include but are not limited to primates, such as humans, monkeys, apes, etc.; cattle, such as cattle, bulls, etc.; sheep, such as sheep; goats, such as goats; pigs, such as pigs, boars, etc.; equines, such as horses, donkeys, zebras, etc.; cats, including wild and domestic cats; canines, including dogs; rabbits, including rabbits, hares, etc.; and rodents, including mice, rats, etc.Preferably, subjects are humans, including but are not limited to fetuses, newborns, infants, juveniles, and adults.

[0119] The terms "a," "an," and "the" as used in this application, including the claims, refer to "one or more." Thus, for example, a reference to "a subject" includes a plurality of subjects unless the context clearly dictates otherwise (e.g., a plurality of subjects).

[0120] Throughout this specification and claims, the terms "comprise," "comprises," and "comprising" are used in a non-exclusive sense unless the context requires otherwise. Similarly, the term "include" and its grammatical variations are intended to be non-limiting such that the recitation of items in a list does not exclude other similar items that may be substituted for or added to the listed items.

[0121] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter described herein belongs.

[0122] The term "about," when referring to a value, means ±20% or ±10. Furthermore, when the term "about" is used in connection with one or more numbers or numerical ranges, it should be understood to refer to all such numbers and to include all numbers within the range, modifying that range by extending the upper and lower limits of the numerical values ​​set forth. Recitation of numerical ranges by endpoints includes all numbers subsumed within that range, e.g., whole integers, including fractions thereof (e.g., recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, etc.), and any range within that range.

[0123] As used herein, the term "alkyl" refers to a monovalent aliphatic hydrocarbon, typically containing 1 to about 6 carbon atoms. The alkyl group may be a straight chain, branched chain, monocyclic or polycyclic moiety, or a combination thereof. Suitable substituents for the alkyl group include aryl, -OH, halogen (-Br, -Cl, -I, and -F), -O(R'), -O-CO-(R'), -CN, -NO, -COOH, -NH, -NH(R'), -N(R'), -COO(R'), -CONH, -CONH(R'), -CON(R'), -S(O)R', -S(O)R', -SH, and -S(R'). Each R' is independently an alkyl group or an aryl group. Substituted alkyl groups may have more than one substituent. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like.

[0124] As used herein, the term "alkylene" refers to an optionally substituted -(CH) x - where x is an integer of from 1 to 5. Preferably, x is from 1 to 3, more preferably x is 1 or 2. Suitable substituents for an alkylene group are the same as those suitable for an alkyl group.

[0125] As used herein, the term "alkoxy" refers to a group of the formula -O-alkyl. Examples of alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), tert-butoxy, and the like.

[0126] As used herein, the term "aryl" refers to a stable, aromatic, monocyclic ring system having 3 to 7 ring atoms, all of which are carbon and may be substituted or unsubstituted. Aryl substituents include -OH, halogen (-Br, -Cl, -I, and -F), -O(R'), -O-CO-(R'), -CN, -NO, -COOH, -NH, -NH(R'), -N(R'), -COO(R'), -CONH, -CONH(R'), -CON(R'), -S(O)R', -S(O)R', -SH, and -S(R'). Each R' is independently an alkyl group.

[0127] As used herein, the term "aryloxy" refers to a group of formula -O-aryl, where the aryl group may be optionally substituted.

[0128] As used herein, the term "electron-withdrawing" refers to an atom or group that attracts electron density from neighboring atoms toward itself, usually by resonance or inductive effects. Suitable electron-withdrawing groups include, but are not limited to, halo, -CN, -NO, -N + (R 9 )(R 10 )(R 11 ), -CF3, -SO3(R 9 ), -SO2(R 9 ), and -CON(R9 )(R 10 ) is available.

[0129] As used herein, the term "electron donating" refers to an atom or group that donates electron density to a neighboring atom, usually by resonance or inductive effects. Suitable electron donating groups include, but are not limited to, alkyl, amino, -(CH) m -N(R 9 )(R 10 ), and -OR 9 There is.

[0130] As used herein, the term "hydroxy" refers to a group of formula --OH.

[0131] As used herein, "halo" or "halogen" refers to F, Cl, Br, or I.

[0132] As used herein, the phrase "optionally substituted" means unsubstituted or substituted. As used herein, the term "substituted" means that a hydrogen atom has been removed and replaced with a substituent. It is understood that substitution at a given atom is limited by valence.

[0133] The following examples are provided to further illustrate embodiments of the present invention, but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used. [Example]

[0134] I. Synthesis: Reagents: Poly(allylamine hydrochloride) was obtained from Nittobo Medical, Japan (PAAn-HCl, catalog number PAA-HCl-3L, 50.3% solution in water) and used as received. Materials were characterized by 1H-NMR, TGA, and size-exclusion chromatography (SEC-MALLS). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was obtained from Chem-Impex International, Wood Dale, IL (EDC-HCl, catalog number 00050, 99.8%) and used as received. Materials were characterized by 1H-NMR, FT-IR, and melting point. 1-Hydroxybenzotriazole hydrate was obtained from Chem-Impex International, Wood Dale, IL (HOBt, catalog number 24755, 99.8% (odb), 21.3% water) and used as received. The material was characterized by 1H-NMR and FT-IR. 4-Carboxyphenylboronic acid was obtained from Chem-Impex International, Wood Dale, IL (CPBA, catalog number 28086, 99.7%) and used as received. The material was characterized by 1H-NMR, FT-IR, and melting point. 3-Trifluoromethyl-4-carboxyphenylboronic acid was obtained from Combi-Blocks Inc., San Diego, CA (CF3-CPBA, catalog number FA-2133, 98%) and used as received. The material was characterized by 1H-NMR and FT-IR. 3-Fluoro-4-carboxyphenylboronic acid was obtained from AOB Chem, Zhuhai, China (F-CPBA, catalog number 10055, 97%). The material was obtained from Combi-Blocks Inc., San Diego, CA (Di-F-CPBA, catalog number FA-3733, 96%) and used as received. The material was characterized by H-NMR, FT-IR, and melting point. 2,3-Difluoro-4-carboxyphenylboronic acid was obtained from Combi-Blocks Inc., San Diego, CA (Di-F-CPBA, catalog number FA-3733, 96%) and used as received. The material was characterized by H-NMR and FT-IR. 3-Dimethylaminomethyl-4-carboxyphenylboronic acid pinacol ester was obtained from Combi-Blocks Inc., San Diego, CA (MeN-CPBA, catalog number PN-4078, 96%) and used as received. The material was characterized by H-NMR and FT-IR.

[0135] Example 1 Synthesis of PAAn modified with 4-carboxyphenylboronic acid: PAAn-HCl 50.3% solution (5.964 g, 32 mmol amine equivalent) was placed in a 250 ml beaker containing a magnetic stir bar and deionized water (90 ml). The resulting clear solution was magnetically stirred and a pH electrode was added. The pH was adjusted to 8.0 by adding 1 N NaOH solution dropwise with stirring. 4-carboxyphenylboronic acid, 99.7% (0.907 g, 5 mmol) was added to the reaction mixture, and the resulting suspension was stirred. After 20 min of stirring, the pH had dropped to 6.8, with some solids remaining suspended in the solution. Additional NaOH solution was added in portions with stirring to completely dissolve the suspension. The pH of the resulting clear solution was 7.5. Solid HOBt hydrate powder (0.046 g, 0.25 mmol) was then added to the clear reaction solution, and after 20 min of stirring, it dissolved. Hydrochloric acid (1N) was added to the reaction mixture to lower the pH to 5.4. The reaction solution remained clear. EDC-HCl (1.152 g, 6 mmol) dissolved in 10 ml of deionized water was slowly pipetted into the reaction mixture. The final volume of the clear reaction mixture was 120 ml, and the pH was 5.6. The reaction mixture was stirred for 18 hours. The pH of the reaction mixture was adjusted to 2.5 with 1 N HCl, and then Pall Minimate TMDialysis was performed against 2.5% NaCl solution using tangential flow filtration in a TFF system. Five diafiltration volumes were removed, and the filtrate was desalted until the conductivity was less than 200 μS / cm, after which the retentate was collected in a lyophilization jar. The solution was frozen in an IPA / dry ice slurry and lyophilized to dryness (3 days). A yield of 1.492 g was obtained as a fluffy white solid. H-NMR spectrum (DO / DCl) confirmed the expected structure.

[0136] Example 2 Synthesis of PAAn modified with 3-trifluoromethyl-4-carboxyphenylboronic acid: PAAn-HCl 50.3% solution (0.5088 g, amine equivalent: 2.74 mmol) was placed in a 30 ml glass vial containing a magnetic stir bar and deionized water (9.0 ml). The resulting clear solution was magnetically stirred and a pH electrode was added. The pH was adjusted to 8.0 by adding 1 N NaOH solution dropwise with stirring. 3-trifluoromethyl-4-carboxyphenylboronic acid, 98% (0.1148 g, 0.491 mmol) was added to the reaction mixture, and the resulting suspension was stirred. After 20 min of stirring, the pH had dropped to 6.8, with some solids remaining suspended in the solution. Additional NaOH solution was added in portions with stirring to completely dissolve the suspension. The pH of the resulting clear solution was 7.5. Solid HOBt hydrate powder (0.0088 g, 0.0514 mmol) was then added to the clear reaction solution, which dissolved after 20 minutes of stirring. Hydrochloric acid (1 N) was added to the reaction mixture to lower the pH to 5.4. The reaction solution remained clear. EDC-HCl (0.1143 g, 0.596 mmol) dissolved in 1.0 ml of deionized water was slowly pipetted into the reaction mixture. The final volume of the clear reaction mixture was 10 ml, and the pH was 5.6. The reaction mixture was stirred for 18 hours. The reaction mixture was precipitated in excess acetone. The precipitated solid was dissolved in water, the pH was adjusted to 2.8 with 1 N HCl, and then the solution was dialyzed using 6K-8k MWCO cellulose membrane dialysis tubing (Speck). The product was purified by dialysis using a centrifuge (Drum Laboratories) for 8 hours, followed by three additional water changes over two days. The solution contained within the dialysis bag was collected in a lyophilization jar. The solution was frozen in an IPA / dry ice slurry and lyophilized to dryness (3 days). The yield was 263 mg as a fluffy white solid. 1H-NMR spectrum (DO / DCl) confirmed the expected structure.

[0137] Example 3 Synthesis of PAAn modified with 3-fluoro-4-carboxyphenylboronic acid: PAAn-HCl 50.3% solution (5.964 g, 32 mmol amine equivalent) was placed in a 250 ml beaker containing a magnetic stir bar and deionized water (90 ml). The resulting clear solution was magnetically stirred and a pH electrode was added. The pH was adjusted to 8.0 by adding 1 N NaOH solution dropwise with stirring. 3-Fluoro-4-carboxyphenylboronic acid, 97% (1.034 g, 5 mmol) was added to the reaction mixture, and the resulting suspension was stirred. After 20 min of stirring, the pH had dropped to 6.8, with some solids remaining suspended in the solution. Additional NaOH solution was added in portions with stirring to completely dissolve the suspension. The pH of the resulting clear solution was 7.5. Solid HOBt hydrate powder (0.046 g, 0.25 mmol) was then added to the clear reaction solution, and after 20 min of stirring, it dissolved. Hydrochloric acid (1N) was added to the reaction mixture to lower the pH to 5.4. The reaction solution remained clear. EDC-HCl (1.152 g, 6 mmol) dissolved in 10 ml of deionized water was slowly pipetted into the reaction mixture. The final volume of the clear reaction mixture was 120 ml, and the pH was 5.6. The reaction mixture was stirred for 18 hours. The pH of the reaction mixture was adjusted to 2.5 with 1 N HCl, and then Pall Minimate TMDialysis was performed against 2.5% NaCl solution using tangential flow filtration in a TFF system. Five diafiltration volumes were removed, and the filtrate was desalted until the conductivity was less than 200 μS / cm, after which the retentate was collected in a lyophilization jar. The solution was frozen in an IPA / dry ice slurry and lyophilized to dryness (3 days). A yield of 1.492 g was obtained as a fluffy white solid. H-NMR spectrum (DO / DCl) confirmed the expected structure.

[0138] Example 4 Synthesis of PAAn modified with 2,3-difluoro-4-carboxyphenylboronic acid: A 50.3% solution of PAAn-HCl (0.5027 g, 2.70 mmol amine equivalent) was placed in a 30 ml glass vial containing a magnetic stir bar and deionized water (9.0 ml). The resulting clear solution was magnetically stirred and a pH electrode was added. The pH was adjusted to 8.0 by adding 1 N NaOH solution dropwise with stirring. 2,3-Difluoro-4-carboxyphenylboronic acid (0.1029 g, 0.510 mmol) was added to the reaction mixture, and the resulting suspension was stirred. After 20 min of stirring, the pH had dropped to 5.6, with some solids remaining suspended in the solution. Additional NaOH solution was added in portions with stirring to completely dissolve the suspension. The pH of the resulting clear solution was 7.5. Solid HOBt powder (0.0082 g, 0.0479 mmol) was then added to the clear reaction solution, which dissolved after 20 minutes of stirring. Hydrochloric acid (1 N) was added to the reaction mixture to lower the pH to 5.5. The reaction solution remained clear. EDC-HCl (0.1018 g, 0.531 mmol) dissolved in 1.0 mL of deionized water was slowly pipetted into the reaction mixture. The final volume of the clear reaction mixture was 10 mL, and the pH was 5.5. The reaction mixture was stirred for 18 hours. The reaction mixture was precipitated with excess acetone. The precipitated solid was dissolved in water, and the pH was adjusted to 2.8 with 1 N HCl. The solution was subjected to dialysis purification for 8 hours using 6K-8K MWCO cellulose membrane dialysis tubing (Spectrum Laboratories) against acidified water (pH = 2-3), followed by three additional water changes over two days. The contents of the dialysis bag were collected in a freeze-drying jar. The solution was frozen in an IPA / dry ice slurry and lyophilized to dryness (3 days). The yield was 235 mg as a white solid. The 1H-NMR spectrum (DO / DCl) confirmed the expected structure.

[0139] Example 5 Synthesis of PAAn modified with 3-dimethylaminomethyl-4-carboxyphenylboronic acid: PAAn-HCl 50.3% solution (0.4973 g, amine equivalent: 2.67 mmol) was placed in a 30 ml glass vial containing a magnetic stir bar and deionized water (9 ml). The resulting clear solution was magnetically stirred and a pH electrode was added. The pH was adjusted to 8.0 by adding 1 N NaOH solution dropwise with stirring. Solid 3-dimethylaminomethyl-4-carboxyphenylboronic acid pinacol ester (0.1565 g, 0.458 mmol) was added to the reaction mixture, and the resulting suspension was stirred. After 20 min of stirring, the pH had dropped to 5.1, with some solids remaining suspended in solution. Additional NaOH solution was added in portions with stirring to completely dissolve the suspension. The pH of the resulting clear solution was 8.0. Solid HOBt powder (0.0088 g, 0.0514 mmol) was then added to the clear reaction solution, which dissolved after 20 minutes of stirring. Hydrochloric acid (1 N) was added to the reaction mixture to lower the pH to 5.5. The reaction solution remained clear. EDC-HCl (0.1048 g, 0.547 mmol) dissolved in 1.0 mL of deionized water was slowly pipetted into the reaction mixture. The final volume of the clear reaction mixture was 10 mL, and the pH was 5.5. The reaction mixture was stirred for 18 hours. The reaction mixture was precipitated twice into excess acetone. The collected precipitate was dissolved in deionized water, and the solution was frozen in an IPA / dry ice slurry and lyophilized to dryness (3 days). A yield of 271 mg was obtained as a fluffy white solid. H-NMR spectrum (DO / DCl) confirmed the expected structure and also indicated that the pinacol ester group had hydrolyzed from the boronic acid. II. Mucin Mixture Observation Assay:

[0140] The scheme of the mucin mixture assay is shown in Figure 1.

[0141] When a water-soluble mucin glycoprotein is mixed with a soluble polymer complexing agent, a variety of results can occur. The solution may remain clear, or it may become cloudy or even opaque. The physical state of the mixture may remain homogeneous and fluid, may contain particles, or may even assume a gel-like appearance. The opacity and physical appearance of the mixture provide a qualitative assessment of the interaction between the complexing agent and the mucin, and more importantly, the physical properties of the complex that is formed.

[0142] The present inventors are seeking polymers that can form a wide range of network structures when mixed with mucin. They have found that when certain polymers are combined with mucin glycoproteins in solution, complexation can be observed by the appearance of a gel-like or opaque mixture with substantial phase separation. Conversely, in the absence of complexation, the solution can remain clear and fluid. In seeking polymers capable of strong mucin complexation, they are interested in classifying such mixtures according to well-defined descriptors of transparency and physical state.

[0143] For example, in some cases, a dispersion may be formed. No particulates are observed in the dispersion. The dispersion may be stable for several days without settling or aggregation. In other cases, a suspension of small particles may be formed. The suspension may settle over time (hours, days), but is generally stable and can be resuspended by mixing. There is prior literature on the use of a turbidity metric derived from absorbance at specific visible light wavelengths and the relationship of this metric to assess the degree of complexation in some related experiments involving complexation involving polymers.

[0144] However, in some cases, mucin complexation with certain polymers results in the gross precipitation of larger particles, sometimes with irregular or complex morphologies. This may indicate extensive network formation. In some cases, these precipitated solids have adhesive properties, adhering to the walls of the vial in which the mixing experiment is performed. In some cases, complex fibrous precipitates or gels are formed. Furthermore, gross precipitation is often accompanied by phase separation (syneresis), in which the polymer / mucin complex deposits as an adhesive film or gel-like mass, and a separate liquid phase (usually clear or slightly hazy) is observed. In such gross precipitations, the degree of complexation may be very high, and it is useful to classify the morphology and appearance of the mucin / polymer complex compared to the related material.

[0145] The purpose of this assay is to determine whether the polymers of the present invention are capable of forming insoluble complexes when mixed with soluble mucin glycoproteins under a set of standard conditions and to observe the general properties of the resulting mixtures.

[0146] The assay results are a clarity descriptor assignment, a physical state descriptor assignment, and optional explanations and comments.

[0147] Isolation of the water-soluble fraction of Sigma (Cat. No. 1778) mucin from porcine stomach type 3 (MPS-3): The protocol is as follows: 1.0 g of MPS-3 was mixed with 40 ml of MilliQ deionized water in a 50 ml conical tube. The tube was attached to a rotating carousel mixer to allow the suspension to stand overnight. The 50 ml conical tube was then centrifuged in a Beckman Avanti centrifuge (5,300 rpm, 60 min). The supernatant was carefully poured into a new 50 ml conical tube, taking care not to disturb the solid pellet at the bottom. The centrifugation of the supernatant was then repeated (Beckman Avanti centrifuge, 5,300 rpm, 60 min). The supernatant was again collected in a weighed 50 ml conical tube. The mucin solution was frozen at -80°C for at least 2 hours. The frozen sample was placed in a freeze dryer for at least 3 days. The lyophilized product was collected, the tubes were weighed, and the % yield was calculated, with an expected recovery of 0.60-0.65 g. The solid was stored in a refrigerator at 2-5°C.

[0148] Preparation of 0.1M MES-Saline Buffer: 21.3 g of (2-(N-morpholino)ethanesulfonic acid monohydrate) (MES, JT Baker Bioreagent, >98%) was added to a 1-liter bottle. 9.0 g of sodium chloride (Fisher Chemical, USP grade) was added to the bottle. A 1-liter graduated cylinder was filled with mQ deionized water (DIW, 18 MΩ). Approximately 750 ml of DIW was added to the bottle, capped, and shaken to dissolve all solids. A magnetic stir bar and pH electrode were added to the bottle, and stirring was initiated. The volume of solution added was noted, and 1 N NaOH solution was added in portions to bring the solution to a pH of 6.0 (or other desired pH). Additional DIW was added as needed to bring the total volume to 1 liter. The bottle was capped, and the buffer was stored at 2–5°C.

[0149] Polymer / Mucin Mixture Turbidity and Observation Assay: A 1.0% w / w solution was made of each test polymer, adjusted to pH 6.0 using MES-saline buffer. A 1.0% w / w solution of aqueous MPS-3 was made, adjusted to pH 6.0 using MES-saline buffer. The mucin solution was slightly hazy. 0.25–0.5 mL of the MPS-3 solution was placed in a 2-dram (4 mL) vial. An equal volume of the test polymer solution was slowly added to the vial. The vial was capped and gently swirled to mix while observing any physical changes. The resulting mixture can be read using one of the following descriptors for its clarity: clear (only slightly hazy, similar to the initial mucin solution); hazy (hazier than the initial mucin solution, difficult to see through it). the text can be read through it); cloudy (not transparent, cannot read through it, but light transmits well); opaque (milk-like, not transparent, does not transmit much light), and not applicable (material shows gross phase separation). The resulting mixture can be read by choosing one of the following descriptors of its physical state: clear; dispersion (no particles visible); suspension (very fine particulates visible), precipitate (large irregular particles, may be sticky), and phase separated (film, gel deposited, clear or hazy supernatant fluid may be visible). The mixture in the capped vial was observed for approximately 1 hour to note any changes.

[0150] Mucin Mixing Observation Assay Results: Following the protocol described above, a series of test compounds were tested for their ability to form insoluble complexes with mucin in MES buffer at pH 5.5, 6.0, and 6.5. Polymer solutions were made at least 1 hour prior to the experiment and gently mixed on a rotisserie. Complete dissolution was confirmed for all test polymers before the experiment began.

[0151] Table 1: [Table 1]

[0152] The results of the mucin mixing assay show that when the parent polymer, PAAn-HCl, is mixed with mucin, hazy dispersions are obtained at all pH values. This result suggests that the polymer / mucin complex does not form an extensive network structure in this case. When the modified PAAn of Example 1 is mixed with mucin, similar hazy dispersions are observed at pH = 5.5 and 6.0. However, at pH = 6.5, a white film is deposited on the container wall, and strong phase separation is observed. At this higher pH value, the polymer of Example 1 can act as a mucin crosslinker, forming an extensive network structure.

[0153] The polymers of Examples 2, 3, and 4 are derivatives of the polymer of Example 1. They contain additional electron-withdrawing substituents on the phenylboronic acid ring. For these polymers, particularly Examples 3 and 4, strong phase separation behavior is observed at all pH values ​​investigated in this experiment. This additional substitution activates the phenylboronic acid, resulting in a stronger interaction with mucin. This stronger interaction results in the formation of a stronger, more robust, and extensive network observed at pH 6.0 and, in some cases, at pH 5.5. III. Surface Plasmon Resonance (SPR) Studies on Mucin-Interacting Polymers

[0154] This example describes an investigation of the interaction of a soluble polymer with a mucin-coated surface. Surface plasmon resonance (SPR) was used to quantify the binding of mucin-interacting polymers in solution flowing over a mucin-coated SPR chip. The binding of these polymers was compared to assess how the degree of substitution affects the polymer upon binding to the mucin surface.

[0155] SPR experiments were performed using a Bio-Rad XPR36 ProteOn TM SPR Instruments and ProteOn TMThis was performed using an LCP sensor chip. Specifically, a set of identical water-soluble poly(allylamine) polymers was observed, differing only in their degree of substitution with the mucosophilic group 3-fluoro-CPBA. The robustness of the surface-deposited materials was then challenged by exposure to 1M NaCl. The results of the study were published in Bio-Rad ProteOn TM Figure 1 shows a sensorgram generated by the XPR36 instrument and associated software. The sensorgram results directly demonstrate quantitative surface attachment of the polymer to the modified surface. A newly purchased LCP sensor chip was used for this experiment.

[0156] Preparation of biotinylated mucin: Biotinylation of MPS-3 water-soluble fraction: Water-soluble fraction MPS-3 was prepared as described in the mucin mixture observation assay. The soluble fraction MPS-3 (42 mg) was dissolved in 30 ml of pH 7.2 PBS buffer and filtered through a 0.45 m PES syringe filter and then a 0.2 m syringe filter to obtain a clear, colorless solution. (+)-Biotin N-hydroxysuccinimide ester (Sigma, #H1759) (25 mg) was dissolved in DMF (2 mL) to obtain a clear solution. The entire 2 mL DMF solution of (+)-biotin N-hydroxysuccinimide ester was added dropwise via pipette to 18 ml of MPS-2 PBS solution. The mixture was covered with aluminum foil and rocked on a shaker at room temperature for 4 hours. The reaction mixture was placed in dialysis tubing (SpectraPor MWCO = 6-8 kDa), and the dialysis bag was placed in a 5-gallon bucket of deionized water with slow stirring. Dialysis was allowed to continue for at least 48 hours. The retentate (pH found to be 6.55) was collected in a lyophilization jar, and the material was frozen in a dry ice / acetone bath and lyophilized. The product was collected as a white fluffy solid (12 mg) and stored in a freezer (-20 °C).

[0157] SPR Operation: Coating of ProteOn chip with biotinylated mucin: A ProteOn LCP chip was inserted into a ProteOn XPR36 instrument. PBS buffer (pH = 6.0) was flowed through all six lanes of the chip at 100 µl / min for 3-5 minutes. A solution of biotinylated mucin (5 µg / ml) in PBS (pH = 6.0) was flowed through all six lanes of the chip at 25 µl / min for 600 seconds. The sensorgram response was evaluated to ensure saturation coverage, indicated by a stable RU reading. PBS buffer (pH = 6.0) was flowed through all six lanes of the chip at 100 µl / min for 3-5 minutes. The SPR sensorgram response was evaluated for stability of the bound mucin layer.

[0158] Testing polymer-mucin interactions and resistance to washout: Six different test polymer solutions (4 μg / ml) were flowed through six lanes of a mucin-modified LCP chip at 100 μl / min for 240 seconds. Sensorgram responses were evaluated to ensure saturation coverage, as indicated by stable RU readings. PBS buffer (pH = 6.0) was flowed through all six lanes of the chip at 100 μl / min for 3-5 minutes. SPR sensorgram responses were evaluated. 1M NaCl PBS buffer (pH = 6.0) was flowed through all six lanes of the chip at 100 μl / min for 18 seconds. PBS buffer (pH = 6.0) was flowed through all six lanes of the chip at 100 μl / min for 3-5 minutes. SPR sensorgram responses to NaCl treatment were evaluated.

[0159] Results of SPR studies: Flowing 5 μg / ml of biotinylated MPS-3 onto a Bio-Rad LCP chip resulted in the formation of a stable mucin layer; a stable baseline was obtained in the running buffer. After establishing the mucin layer, the SPR chip was exposed to a 5 μg / ml solution of biotinylated MPS-3, resulting in a stable accumulation of material on the LCP chip. The sudden increase in signal intensity rolled over and stabilized after 50 seconds, indicating that the surface was saturated with a stable mucin layer.

[0160] The binding of PAAn-fluoro-CPBA polymers to mucin-coated SPR chips was affected by the degree of substitution. After establishing a reproducible and stable mucin layer on a new SPR chip, four different polymer solutions were simultaneously flowed through lanes of the microfluidic chip. The polymer solutions were allowed to flow over the chip for an extended period of time to allow the RU signals to roll over and establish equilibrium for each polymer. All PAAn-fluoro-CPBA derivatives strongly bound to the mucin surface. Surface binding was found to depend on the level of fluoro-CPBA substitution on the polymer. PAAn-fluoro-CPBA (15) showed the strongest surface binding (1470 RU), followed by PAAn-fluoro-CPBA (10) (1286 RU) and PAAn-fluoro-CPBA (6) (1149 RU). Unsubstituted PAAn also showed strong binding to the mucin surface, but to a lesser extent (1002 RU).

[0161] Exposure of the polymer-bound mucin surface to a strong salt solution (1 M NaCl) significantly reduced the adsorbed PAAn surface, whereas the PAAn-(fluoro-CPBA) surface remained stable. After flowing the polymer solution over the surface long enough to reach a near-equilibrium RU signal, buffer was again flowed over the surface, demonstrating the stability of the surface coating as a stable RU signal in each lane. Next, a short burst (18 s) of strong salt solution (1 M NaCl) was passed over the mucin chip, followed by a return to the running buffer.

[0162] Brief exposure to NaCl resulted in the removal of a significant amount of adsorbed PAAn (-26%). In contrast, the RU values ​​of the PAAn-fluoro-CPBA materials showed smaller effects (-2%, -5%, -9%) and appear to be more robust to strong salt treatments with increasing fluoro-CPBA substitution (see Table 2).

[0163] Table 2: [Table 2]

[0164] A set of three PAAn-fluoro-CPBA polymers with varying levels of fluoro-CPBA substitution was evaluated for binding to mucin-modified surfaces by SPR. Maximum surface binding was observed with PAAn-fluoro-CPBA (15), with lesser substituted polymers binding to a lesser extent. Unsubstituted PAAn also bound to the mucin surface, but to a lesser extent.

[0165] SPR surfaces coated with PAAn-fluoro-CPBA polymers were stable to treatment with concentrated salt solutions. This contrasts with the behavior of unsubstituted PAAn, which lost a significant percentage of the bound polymer when treated with salt. This may be due to the exclusive Coulombic binding of PAAn with the mucin surface, which is effectively screened in the presence of salt. In the case of PAAn-fluoro-CPBA materials, the combination of Coulombic and mucophilic interactions of these modified polymers makes the mucin complexes stable against strong salt conditions. IV. Single-Dose Efficacy Studies:

[0166] Animal Model: The Goto-Kakazaki (GK) rat model was selected based on extensive literature evidence regarding its response to surgical and device-induced duodenal emptiness. The GK rat model is one of the most well-validated rodent models for evaluating type 2 diabetes medications. GK rats are a polygenic, non-obese Wistar substrain that develop adult-onset type 2 diabetes early in life. GK rats are an ideal model of type 2 diabetes, exhibiting features such as retinopathy, nephropathy, neuropathy, and cardiovascular complications similar to those seen in humans. Most importantly, this substrain has been extensively studied in weight loss surgery models, demonstrating improved glucose tolerance after weight loss surgery similar to humans. An oral glucose tolerance test (OGTT) was used to evaluate the efficacy of a single dose of polymer therapy in the GK rat model.

[0167] General Procedure: Prior to the experiment, all rats were fasted for 15 hours, with water available. Animals were divided into three test groups, each consisting of 6-7 rats. (Group A) Vehicle control, received 0.9% saline; (Group B) Treatment with Example 3 at a concentration of 40 mg / mL dissolved in 0.9% saline; and (Group C) Treatment with Example 3 at a concentration of 80 mg / mL dissolved in 0.9% saline. All rats received a standard 1.5 mL volume of each solution delivered by oral gavage in the same manner. After 60 minutes, baseline blood glucose levels were measured. Immediately after recording the baseline blood glucose measurement, a 40% glucose solution (2.0 g / kg rat) was administered by oral gavage. Glucose tolerance test samples were then collected from each rat at 30, 60, 90, and 120 minutes after glucose administration (see Figure 2). Time-dependent blood glucose values ​​were analyzed and plotted using GraphPad Prism 8 software. Two-way ANOVA was applied to the raw data to assess multiple comparisons between all groups and time points. The area under the curve was calculated using zero as the baseline for the Y parameter; the percentage reduction was calculated manually, and a t-test was applied to verify the significance between the treatment groups and the vehicle control.

[0168] Results: The 40 mg / mL and 80 mg / mL treatment groups showed statistically significant dose-dependent decreases in area under the elevation curve (iAUC) of 31% and 43%, respectively, when compared to the vehicle control (see Figure 3), thereby demonstrating the efficacy of the polymers of the present disclosure. Significance is shown at times 60, 90, and 120 minutes in each treatment group, with p-values ​​shown in Figures 2 and 3. * p<0.05, ** p<0.01, *** p<0.001, **** Shown as p<0.0001.

[0169] While the present invention has been particularly shown and described with reference to certain preferred embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The present invention provides, for example, the following items. (Item 1) A polymer containing a repeating unit of any one of the repeating units of formulas (I) to (III) [ka] wherein R 1 , R 2 , R 3 and R 4 are independently hydrogen or substituted or unsubstituted alkyl; Y 1 is independently in each occurrence a direct bond or -L 1 -A 1 -L 2 -A 2 -And;L 1 is, in each occurrence, -NR 9 -, -NC(O)-, or -C(O)N-; L 2 is absent, -NR, in each occurrence. 9 -, -O- or -S-; Y 2 is independently in each occurrence a direct bond or -L 3 -A 1 -L 2 -A 2 -And;L 3 is, in each occurrence, -C(O)- or absent; L 2 is absent, -NR, in each occurrence. 9 -, -O- or -S-; A 1 and A 2 is independently in each occurrence absent or optionally substituted C1-C5 alkylene; R9 , R 10 and R 11 is independently in each occurrence hydrogen or substituted or unsubstituted alkyl (preferably substituted or unsubstituted C1-C6 alkyl); Z, in each occurrence, [ka] and preferably said -B(OH)2 is at the 3 or 4 position of said ring; X 1 and X 2 is hydrogen, halo, -CN, -NO2, -N + (R 9 )(R 10 )(R 11 ), -CF3, -SO3(R 9 ), -SO2(R 9 ), -CON(R 9 )(R 10 ), -(CH2) m -N(R 9 )(R 10 ), and -OR 9 independently selected from the group consisting of: n is an integer from 1 to 100,000; m is an integer from 0 to 4; However, X 1 and X 2 Not more than one of is hydrogen; X 1 or X 2 Either -(CH2) m -N(R 9 )(R 10 ), it is attached to a carbon atom of the phenyl ring adjacent to the carbon atom of the phenyl ring to which -B(OH)2 is attached, and X 1 and X 2 The other is hydrogen, polymer. (Item 2) Item 1. The polymer according to item 1, comprising a repeating unit of formula (I): (Item 3) The repeating unit of formula (I) is [ka] and preferably said -B(OH)2 is at the 3 or 4 position of said ring. (Item 4) A polymer comprising repeating units of formulas (Ia) to (If) according to item 3, R 1 , R 2 and R 3 is hydrogen; A 1 and A 2 is independently absent or optionally substituted C1-C5 alkylene. (Item 5) A polymer comprising repeating units of formulas (Ia) to (If) according to item 3, R 1 and R 2 is hydrogen; R 3 is methyl; A 1 and A 2 is independently absent or optionally substituted C1-C5 alkylene. (Item 6) Item 1. The polymer according to item 1, comprising a repeating unit of formula (II): (Item 7) In the repeating unit of formula (II), Y 2 is a direct bond; Z is [ka] and preferably said -B(OH)2 is at the 3 or 4 position of said ring. (Item 8) Item 1. The polymer according to item 1, comprising a repeating unit of formula (III): (Item 9) In the repeating unit of formula (III), Y 2 is a direct bond; Z is [ka] and preferably said -B(OH)2 is at the 3 or 4 position of said ring. (Item 10) [ka] [ka] [ka] wherein "m" represents an integer from 1 to 100,000, and b) [ka] [ka] [ka] [ka] at least one repeating unit selected from the group consisting of: (wherein "n" represents an integer from 1 to 100,000); A polymer comprising: (Item 11) [ka] A polymer selected from the group consisting of: (Item 12) [ka] A polymer selected from the group consisting of: (Item 13) [ka] [ka] [ka] [ka] A polymer selected from the group consisting of: (Item 14) A pharmaceutical composition comprising the polymer of any one of the preceding items and a pharmaceutically acceptable excipient. (Item 15) 15. The pharmaceutical composition according to item 14, wherein the composition is a liquid, tablet, caplet or capsule. (Item 16) 15. The pharmaceutical composition according to item 14, wherein the composition is an enteric coated tablet, caplet or capsule. (Item 17) 17. The pharmaceutical composition of item 16, wherein the enteric coated tablet, caplet, or capsule is for targeted delivery to the duodenum. (Item 18) 18. A method for treating a metabolic disorder in a subject, comprising administering a therapeutically effective amount of the polymer according to any one of items 1 to 13 or the pharmaceutical composition according to any one of items 14 to 17 to a subject in need thereof. (Item 19) 20. The method of item 18, further comprising administering another therapeutic agent to the subject in need thereof. (Item 20) 20. The method of item 18 or item 19, wherein the metabolic disorder is glucose intolerance, T1DM, T2DM, prediabetes, hyperlipidemia, obesity, overweight, dyslipidemia, hypertension, hyperglycemia, impaired glucose tolerance, insulin resistance, metabolic syndrome, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), and polycystic ovary syndrome (PCOS). (Item 21) 20. The method of claim 18 or 19, wherein the metabolic disorder is type 2 diabetes mellitus. (Item 22) 18. A method for treating a gastrointestinal disorder in a subject, comprising administering a therapeutically effective amount of the polymer according to any one of items 1 to 13 or the pharmaceutical composition according to any one of items 14 to 17 to a subject in need thereof.

Claims

[Claim 1] The invention described in the specification.

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