Methods of evaluating small molecule-modified polymers in compositions

Abstract

To provide methods for evaluating polymer compositions comprising polymers modified with a small molecule compound.SOLUTION: In an embodiments, evaluating polymer compositions includes determining the concentration of a small molecule compound within the polymer compositions. The polymer compositions may be used to prepare semi-permeable devices (e.g., hydrogel capsules) that comprise at least one compartment encapsulating a plurality of cells capable of expressing a therapeutic agent, which may be released from the device, e.g., upon implantation in a subject. In an embodiment, certain small molecule compounds bound to the modified polymer are capable of mitigating host foreign body response (FBR) of the polymer compositions and / or semi-permeable device.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] [background] Treatment of chronic and genetic diseases by transplanting cells engineered to produce therapeutic agents that can treat such diseases holds exciting promise for improving the health of patients with such diseases. To fully realize this promise, the transplanted cells must be protected from the patient's immune response so that they can survive, and they must also be capable of producing therapeutic levels of the desired therapeutic agent over a period of weeks, months, or even longer. One approach to delivering such cell therapies is to encapsulate the engineered cells in a semipermeable device (e.g., a hydrogel capsule) so that the device structure isolates the cells from the host's immune system while allowing the entry of nutrients for the cells and the exit of the produced therapeutic agent. In some cases, these semipermeable devices (e.g., hydrogel capsules) can be modified with various substances, such as small molecule compounds. There is a need for improved methods for evaluating these devices, for example, by quantifying the concentration of small molecule compounds bound to these semipermeable devices (e.g., hydrogel capsules), which may affect the long-term stability of these devices and their ability to avoid a host foreign body response (FBR) when implanted in a subject.

[0002] [overview] The present disclosure provides, at least in part, a method for evaluating a polymer composition comprising a polymer modified with a small molecule compound. In some embodiments, the evaluating comprises determining the concentration of the small molecule compound within the polymer composition. The polymer composition can be used to prepare a semipermeable device (e.g., a hydrogel capsule) comprising at least one compartment encapsulating a plurality of cells capable of expressing a therapeutic agent, which can be released from the device upon implantation into a subject, for example. In some embodiments, certain small molecule compounds conjugated to the modified polymer can mitigate the host foreign body response (FBR) of the polymer composition and / or semipermeable device. For example, polymer compositions and semipermeable devices comprising certain small molecule compounds exhibit a lower host FBR compared to the host FBR induced by polymer compositions and semipermeable devices that do not contain the small molecule compound.

[0003] Without wishing to be bound by theory, the concentration of certain small molecule compounds (e.g., antifibrotic compounds) within a polymer composition used to prepare a semipermeable device can significantly affect the ability of the composition and / or device to mitigate host FBR. Therefore, it can be useful to determine the concentration of small molecule compounds included in a polymer composition, for example, to adjust the FBR as needed. However, current methods for quantifying the concentration of certain small molecules are not ideal for use in evaluating the polymer compositions described herein. The method described below is designed to circumvent these obstacles and provide a reliable determination of the concentration of certain small molecule compounds of modified polymers within a polymer composition.

[0004] In one aspect, the disclosure features a method for evaluating a polymer composition including a polymer modified with a small molecule compound, the method including exposing the polymer composition to reaction conditions that allow for release of the small molecule compound from the modified polymer. The method can further include obtaining a value for the concentration of the small molecule compound bound to the modified polymer. In some embodiments, the polymer in the modified polymer is a polysaccharide. In some embodiments, the polysaccharide is an alginate (e.g., an alginate having an average molecular weight of 75 kD to 150 kDa and / or a guluronic acid to mannuronate (G:M) ratio of 1.5 or greater).

[0005] The small molecule compound may be covalently or non-covalently bound to the polymer (e.g., alginate) in the modified polymer. In some embodiments, the small molecule compound is covalently bound to the polymer (e.g., alginate), for example, via a linking group. In some embodiments, the small molecule is an anti-fibrotic compound. In some embodiments, the anti-fibrotic compound has the formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein the variables A, L 1 , M, L 2 , P., L. 3 , and Z, and associated subvariables, are defined herein. In some embodiments, the compound of Formula (I) or a pharmaceutically acceptable salt thereof (e.g., Formula (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (III-c), (III-d), or (III-e)) is a compound described herein, such as one of the compounds shown in Table 1 herein. In certain embodiments, the antifibrotic compound is Compound 100, Compound 101, or Compound 102 shown in Table 1.

[0006] In some embodiments, the reaction conditions that allow release of the small molecule compound from the modified polymer include contacting the polymer with an acidic solution, a basic solution, an enzyme solution, light, microwave irradiation, heating, or a combination thereof. In certain embodiments, the method further includes a separation step, which may be performed before obtaining the concentration of the small molecule compound. Exemplary separation steps include filtration or chromatography (e.g., size exclusion chromatography, ion exchange chromatography, reverse phase chromatography, gel filtration chromatography, or hydrophobic interaction chromatography). In certain embodiments, obtaining the concentration value of the small molecule compound includes determining the area of ​​a chromatogram peak of the small molecule compound. In certain embodiments, obtaining the concentration value of the small molecule compound involves comparison with a standard (e.g., a small molecule compound such as the compound of Formula (I)). The concentration of the small molecule compound bound to the modified polymer may be, for example, in the range of 0.5% to 10% (w / w) modified polymer, for example, about 0.5% to 5%, 1% to 5%, 1% to 4%, or 1 to 3% (w / w) modified polymer.

[0007] In some embodiments, the method further comprises obtaining the concentration of the unconjugated small molecule compound (i.e., "free" small molecule compound) in the polymer composition. The concentration of the free small molecule compound (e.g., free antifibrotic compound) in the polymer composition can be less than about 1% (w / w) of the modified polymer, e.g., less than about 0.9%, 0.8%, 0.7%, 0.6%, or 0.5% (w / w) of the modified polymer.

[0008] In another aspect, the disclosure features a method for evaluating a polymer composition including a polymer (e.g., a modified polymer) modified with a small molecule compound, the method not including exposing the polymer composition to reaction conditions that allow release of the small molecule compound from the modified polymer. In some embodiments, the method further includes obtaining a value for the total small molecule compound conjugated (e.g., covalently attached) to the modified polymer in the polymer composition; and using the obtained value to obtain a value for the concentration of the modified polymer, thereby evaluating the polymer composition. In some embodiments, the modified polymer is a modified polysaccharide. In some embodiments, the polymer in the modified polymer (e.g., the polymer used to prepare the modified polymer) is an alginate (e.g., an alginate having an average molecular weight of 75 kD to 150 kDa and / or a guluronic acid to mannuronate (G:M) ratio of 1.5 or greater). In some embodiments, the small molecule compound is an antifibrotic compound. In some embodiments, the antifibrotic compound is a compound of Formula (I) or a pharmaceutically acceptable salt thereof (e.g., a salt described herein). In some embodiments, the method includes obtaining a refractive index value of the polymer composition, hi some embodiments, obtaining the refractive index value includes obtaining a refractometer reading (nd) at a particular wavelength and / or at a particular temperature.

[0009] In another aspect, the disclosure features modified polymers and polymer compositions that include a concentration of one or more small molecule compounds of the modified polymer of about 0.5%-5%, 1%-5%, 1%-4%, or 1-3% (w / w).

[0010] The details of one or more embodiments of the present disclosure are described herein. Other features, objects, and advantages of the present disclosure will be apparent from the detailed description, drawings, examples, and claims. [Brief explanation of the drawings]

[0011] [Figure 1]1 shows an exemplary HPLC chromatogram (UV) obtained in determining the levels of total antifibrotic compounds in a sample of antifibrotic alginate, as outlined in Example 2. [Figure 2A] Figure 2A shows exemplary HPLC chromatograms obtained in determining the level of unconjugated (e.g., free) antifibrotic compounds in samples of antifibrotic alginate, as outlined in Example 3. Figure 2A shows a UV chromatogram corresponding to a standard solution. [Figure 2B] FIG. 2B shows the UV chromatogram corresponding to the free antifibrotic compound in solution.

[0012] [Detailed explanation] The present disclosure features methods for evaluating polymer compositions comprising at least one polymer modified with a small molecule compound (e.g., an anti-fibrotic compound). The polymer compositions can be used to encapsulate a plurality of cells (e.g., living cells) that can express a therapeutic agent when the device is implanted in a subject (e.g., a human). In some embodiments, the polymer compositions and devices can mitigate host FBR. In some embodiments, the polymer compositions and devices further comprise a means for improving the viability and / or productivity of the living cells contained therein.

[0013] In some embodiments, a polymer composition is evaluated by quantifying the concentration of small molecule compounds bound (covalently or non-covalently) to the polymer in the polymer composition. The concentration of the bound small molecule compounds can be evaluated, for example, by obtaining a value for the total concentration of small molecule compounds (bound and unbound) in a first sample of the polymer composition (i.e., "total small molecules"), obtaining a value for the concentration of unbound small molecule compounds (i.e., "free small molecules") in a second sample of the polymer composition, and subtracting the concentration of the free small molecules from the total small molecule concentration. In another embodiment, a polymer composition is evaluated by obtaining a value for the refractive index of the polymer composition. The polymer composition may or may not be subjected to conditions for hydrolyzing the small molecule compounds from the modified polymer. Methods for determining the concentration of small molecule compounds in a polymer composition are described herein.

[0014] Abbreviations and Definitions The following abbreviations are used throughout the detailed description and examples of this disclosure: CBP: cell-associated polypeptide CBP-polymer: a polymer covalently modified with CBP via a linker CM-Alg: Chemically modified alginate CM-LMW-Alg: Chemically modified, low molecular weight alginate CM-HMW-Alg: Chemically modified, high molecular weight alginate CM-MMW-Alg: Chemically modified, medium molecular weight alginate HMW-Alg: high molecular weight alginate MMW-Alg: Medium molecular weight alginate RGD-alginate: alginate covalently modified with a peptide containing the amino acid sequence RGD U-Alg: unmodified alginate U-HMW-Alg: Unmodified high molecular weight alginate U-LMW-Alg: Unmodified low molecular weight alginate U-MMW-Alg: Unmodified medium molecular weight alginate 70:30 CM-Alg:U-Alg: 70:30 mixture of chemically modified and unmodified alginate (V:V), e.g., as described in Example 2

[0015] In order that the present disclosure may be more readily understood, some technical and scientific terms used herein are specifically defined below. Unless specifically defined elsewhere herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0016] As used in this specification, including the appended claims, singular terms such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0017] "About" or "approximately," when used herein to modify a parameter defined as a numerical value (e.g., a physical property of a hydrogel capsule, e.g., diameter, sphericity, number of cells encapsulated therein, number of capsules in a preparation), means that the quoted numerical value is within an acceptable functional range of the defined parameter as determined by one of ordinary skill in the art; this functional range depends in part on how the numerical value is measured or determined, and in part on the limitations of the measurement system, such as the acceptable error range of the measurement system. For example, "about" can mean a range of 20% above and below the quoted numerical value. In some embodiments, the term "about" means that the modified parameter can vary by 15%, 10%, or 5% above or below the numerical value stated for that parameter. Alternatively, particularly with respect to certain characteristics of the devices described herein (e.g., the density of small molecule compounds in a polymer composition), the term "about" can mean within an order of magnitude (e.g., within 5-fold, 4-fold, 3-fold, 2-fold, or 1-fold) above or below the quoted value.

[0018] As used herein, "acquire" or "acquiring" refers to achieving the acquisition of a value (e.g., a numerical value or image) or a physical entity (e.g., a sample) by "directly acquiring" or "indirectly acquiring" the value or physical entity. "Directly acquiring" means performing a process (e.g., performing an analytical method or protocol) to obtain the value or physical entity. "Indirectly acquiring" refers to receiving a value or physical entity from another party or provider (e.g., a third-party laboratory that directly acquired the physical entity or value). Directly acquiring a value or physical entity includes performing a process that involves a physical change in a physical substance or the use of an instrument or device. An example of directly acquiring a value is obtaining a sample from a human subject. Directly acquiring a value includes performing a process using an instrument or device, such as obtaining fluorescence microscopy data using a fluorescence microscope or obtaining a refractive index using a refractometer.

[0019] "Administer," "administering," or "administration," as used herein, refers to implanting, absorbing, ingesting, injecting, or otherwise introducing into a subject an entity described herein (e.g., a device, or a preparation of a device), or providing such an entity to a subject for administration.

[0020] "Antifibrotic," as used herein, refers to a compound or substance that reduces foreign body reactions (FBR). For example, implantation of a device (e.g., a hydrogel capsule) containing an antifibrotic compound into a tissue induces a reduced amount of FBR in biological tissue compared to the FBR induced by implantation of a non-antifibrotic reference device (i.e., a device lacking any antifibrotic compound but having substantially the same composition (e.g., same CBP-polymer, same cell type) and structure (e.g., size, shape, number of compartments)). In embodiments, the degree of FBR can be measured using assays known in the art, for example, those described in WO 2017 / 075630, or assays / methods described in Vegas, A., et al., Nature Biotechnol (supra) (e.g., subcutaneous cathepsin measurement of implanted capsules, Masson's Trichrome (MT), hematoxylin or eosin staining of tissue sections, quantification of collagen density, macrophage (CD68 or F4 / 80) cell staining and confocal microscopy, myofibroblast (alpha-smooth muscle actin) staining and confocal microscopy), and the like. The immune response in tissues containing the implanted device (e.g., hydrogel capsules) may include, for example, protein adsorption, macrophages, multinucleated foreign body giant cells, fibroblasts, and angiogenesis, using one or more of the following: quantification of RNA sequences of known inflammatory factors and immune cell markers, such as actin (SMA), or general cell adhesion, or 79 RNA sequences of known inflammatory factors and immune cell markers, or FACS analysis for macrophages and neutrophils in devices (e.g., capsules) retrieved after 14 days in the peritoneal cavity of suitable subjects, e.g., immunocompetent mice. In embodiments, FBR is assessed by measuring the levels of one or more biomarkers of immune response, e.g., cathepsin, TNF-α, IL-13, IL-6, G-CSF, GM-CSF, IL-4, CCL2, or CCL4, in tissues containing the semipermeable device.In some embodiments, the FBR induced by a semipermeable device of the invention (e.g., a hydrogel capsule comprising an anti-fibrotic compound disposed on its exterior surface) is at least about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% lower than the FBR induced by a reference device without an FBR (e.g., a semipermeable device substantially identical to the test or claimed device but lacking a means for mitigating FBR (e.g., a hydrogel capsule that does not include an anti-fibrotic compound but is otherwise substantially identical to the claimed capsule)). In some embodiments, the FBR (e.g., biomarker level) is measured after about 30 minutes, about 1 hour, about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, or about more.

[0021] "Cell," as used herein, refers to a recombinant or non-recombinant cell. In certain embodiments, the cell is an immortalized cell or a recombinant cell derived from an immortalized cell. In certain embodiments, the cell is a viable cell, e.g., alive as measured by any technique described herein or known in the art.

[0022] As used herein, "cell-binding peptide (CBP)" refers to a linear or cyclic peptide comprising an amino acid sequence derived from the cell-binding domain of a ligand of a cell adhesion molecule (CAM) (e.g., a CAM that mediates cell-matrix or cell-cell junctions). In certain embodiments, a CBP can mimic at least one activity of a ligand of a cell adhesion molecule (CAM) or other cell surface molecule that mediates cell-matrix or cell-cell junctions or other receptor-mediated signaling. A CBP can be less than 50, 40, 30, 25, 20, 15, or 10 amino acids in length. In certain embodiments, a CBP is 3-12 amino acids in length, 4-10 amino acids in length, or 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. The amino acid sequence of a CBP can be identical to a naturally occurring binding domain sequence or a conservatively substituted variant thereof. In certain embodiments, a CAM ligand is a mammalian protein. In some embodiments, the CBP is an RGD peptide, meaning that the peptide comprises the amino acid sequence RGD and, optionally, one or more additional amino acids located at one or both of the N-terminus and C-terminus. In some embodiments, the CBP is a cyclic peptide comprising RGD, such as one of the cyclic RGD peptides described in Vilaca, H. et al., Tetrahedron 70(35):5420-5427 (2014). In some embodiments, the CBP is a linear peptide comprising RGD and is less than 6 amino acids in length. In some embodiments, the CBP is a linear peptide consisting essentially of RGD or RGDSP.

[0023] "CBP-polymer," as used herein, refers to a polymer comprising at least one CBP covalently attached to the polymer via a linker. In some embodiments, the polymer is not a peptide or polypeptide. In some embodiments, the polymer in a CBP-polymer does not contain any amino acids. In some embodiments, the polymer in a CBP-polymer is a synthetic or naturally occurring polysaccharide, such as an alginate, e.g., sodium alginate. In some embodiments, the number of polysaccharide moieties to which CBP is covalently attached is less than any of the following values: 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1%.

[0024] As used throughout this specification and claims, "consists essentially of," and variations such as "consist essentially of" or "consisting essentially of," indicate the inclusion of any recited element or group of elements, and the optional inclusion of other elements of similar or different nature to the recited elements, that do not substantially alter the basic or novel properties of the specified molecule, composition, device, or method. As a non-limiting example, a peptide or polypeptide consisting essentially of a recited amino acid sequence may also contain one or more amino acids that include a substitution in the recited amino acid sequence of one or more amino acid residues that does not substantially affect the relevant biological activity of the cell-binding peptide or therapeutic protein, respectively. As another non-limiting example, a peptide or polypeptide consisting essentially of a recited amino acid sequence may include one or more covalently attached moieties (e.g., radioactive or fluorescent labels) that do not substantially alter the relevant biological activity of the cell-binding peptide (e.g., the ability to increase the viability or productivity of encapsulated cells as described herein).

[0025] "Derived from," as used herein with respect to cells, refers to cells obtained from a tissue, cell line, or cell that is then optionally cultured, passaged, differentiated, induced, etc., to produce derived cells. For example, mesenchymal stem cells can be derived from mesenchymal tissue and then differentiated into various cell types.

[0026] "Device," as used herein, refers to any implantable object (e.g., particle, hydrogel capsule, implant, medical device) that contains cells (e.g., living cells) that can express a therapeutic agent after implantation of the device and has a structure that supports the viability of the cells by allowing cellular nutrients to enter the device. In some embodiments, the device is a semipermeable device, allowing the release of metabolic byproducts produced by the living cells and / or the therapeutic agent from the device.

[0027] As used herein, "effective amount" refers to an amount of a device, device composition, or component of a device or device composition, e.g., a plurality of hydrogel capsules containing cells (e.g., recombinant cells), or an agent (e.g., a therapeutic agent) produced by cells, sufficient to elicit a biological response, e.g., to treat a disease, disorder, or condition. In some embodiments, the term "effective amount" refers to the amount of a component of a device, e.g., the number of cells in the device, the concentration of an antifibrotic compound disposed on the surface and / or in the barrier compartment of the device, or the concentration of a polypeptide (e.g., CBP) in the cell-containing compartment. As will be recognized by those skilled in the art, an effective amount can vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the therapeutic agent, composition, or device (e.g., capsule, particle), the condition being treated, the mode of administration, and the age and health of the subject. An effective amount encompasses both therapeutic and prophylactic treatments. As an example, an effective amount of a small molecule compound (e.g., an antifibrotic compound) conjugated to a modified polymer in a semipermeable device can modulate host FBR. An effective amount of a device, composition, or component (eg, a small molecule compound) can be determined by any technique known in the art or described herein.

[0028] As used herein, a "recombinant cell" is a cell with a non-naturally occurring modification, typically comprising a nucleic acid sequence (e.g., DNA or RNA) or polypeptide that is not present (or is present at different levels) in other similar cells that have not been modified under similar conditions (an exogenous nucleic acid sequence). In embodiments, a recombinant cell comprises an exogenous nucleic acid (e.g., a vector or modified chromosomal sequence). In embodiments, a recombinant cell comprises an exogenous polypeptide or an exogenous nucleic acid sequence, e.g., a sequence, e.g., DNA or RNA, that is not present in a similar cell that has not been modified. In embodiments, the exogenous nucleic acid sequence is chromosomal, e.g., the exogenous nucleic acid sequence is an exogenous sequence placed within an endogenous chromosomal sequence. In embodiments, the exogenous nucleic acid sequence is chromosomal or extrachromosomal, e.g., a non-integrating vector. In embodiments, the exogenous nucleic acid sequence comprises a chromosomal or extrachromosomal nucleic acid sequence that comprises a sequence that encodes a polypeptide or a sequence that is expressed as a polypeptide. In certain embodiments, a recombinant cell comprises a therapeutic agent that is present at a level or distribution that differs from that found in a similar cell that has not been modified. In embodiments, the recombinant cell comprises RPE engineered to produce RNA or a therapeutic polypeptide. In embodiments, the recombinant cell may comprise an exogenous nucleic acid sequence, including a chromosomal or extrachromosomal exogenous nucleic acid sequence comprising a sequence to be expressed as RNA, such as a sequence comprising mRNA or regulatory RNA. In embodiments, the recombinant cell comprises an exogenous nucleic acid sequence, including a chromosomal or extrachromosomal nucleic acid sequence comprising a sequence encoding a polypeptide or a sequence to be expressed as a polypeptide. In some embodiments, the polypeptide is encoded with a codon-optimized sequence to achieve higher expression of the polypeptide compared to the naturally occurring coding sequence.The codon-optimized sequence can be generated using commercially available algorithms, such as GeneOptimizer (ThermoFisher Scientific), OptimumGene™ (GenScript, Piscataway, NJ USA), GeneGPS® (ATUM, Newark, CA USA), or the Java Codon Adaptation Tool (JCat, www.jcat.de; Grote, A. et al., Nucleic Acids Research, Vol. 33, Issue suppl_2, pp. W526-W531 (2005)). In some embodiments, the recombinant cell (e.g., RPE cell) comprises an exogenous nucleic acid sequence that controls the conformation or expression of an endogenous sequence. In some embodiments, the recombinant cell (e.g., RPE cell) is cultured from a population of stably transfected cells or from a monoclonal cell line.

[0029] As used herein, unless otherwise specified, "Factor VII protein" or "FVII protein" refers to a polypeptide comprising the amino acid sequence of a native Factor VII protein or a variant thereof, having FVII biological activity, e.g., promoting blood clotting, as determined by art-recognized assays. Native FVII exists as a single-chain zymogen, a zymogen-like two-chain polypeptide, and a fully activated two-chain form (FVIIa). In some embodiments, reference to FVII includes its single-chain and two-chain forms, including zymogen-like and FVIIa. FVII proteins that can be produced by the devices described herein, e.g., devices comprising recombinant RPE cells, include wild-type primate (e.g., human), porcine, canine, and murine proteins, as well as variants of such wild-type proteins, including fragments, mutants, and variants having one or more amino acid substitutions and / or deletions. In some embodiments, the mutant FVII proteins are capable of activation to a fully activated two-chain form (Factor VIIa) having at least 50%, 75%, 90% or more (including >100%) of the activity of wild-type Factor VIIa. Variants of FVII and FVIIa are known, such as marzeptacog alfa (activated) (MarzAA) and the variants described in EP 1373493, U.S. Pat. No. 7771996, U.S. Pat. No. 9476037, and U.S. Patent Application Publication No. 20080058255.

[0030] FVII bioactivity can be quantified by art-recognized assays, unless otherwise specified. For example, FVII bioactivity in a sample of biological fluid, such as plasma, can be quantified by (i) measuring the amount of factor Xa produced in a system containing lipid membranes and tissue factor (TF) embedded in factor X (Persson et al., J. Biol. Chem. 272:19919-19924, 1997); (ii) measuring factor X hydrolysis in an aqueous system; (iii) measuring its physical binding to TF using an instrument based on surface plasmon resonance (Persson, FEBS Letts. 413:359-363, 1997); or (iv) measuring the hydrolysis of synthetic substrates; and / or (v) measuring thrombin generation in a TF-independent in vitro system. In an embodiment, FVII activity is assessed by a commercially available chromogenic assay (BIOPHEN FVII, HYPHEN BioMed Neuville sur Oise, France), in which a biological sample containing FVII is mixed with thromboplastin calcium, factor X, and SXa-11 (a chromogenic substrate specific for factor Xa).

[0031] As used herein, "factor VIII protein" or "FVIII protein," unless otherwise specified, refers to a polypeptide comprising the amino acid sequence of a native factor VIII polypeptide or a variant thereof, having FVIII biological activity, e.g., clotting activity, as determined by art-recognized assays. FVIII proteins that can be expressed by the devices described herein, e.g., devices comprising recombinant RPE cells, include wild-type primate (e.g., human), porcine, canine, and murine proteins, as well as variants of such wild-type proteins, including fragments, mutants, and variants having one or more amino acid substitutions and / or deletions, B-domain deleted (BDD) variants, single-chain variants, and fusions of any of the above wild-type or variants with half-life-extending polypeptides. In embodiments, cells are engineered to encode a precursor factor VIII polypeptide (e.g., having a signal sequence) having a complete or partial deletion of the B domain. In embodiments, cells are engineered to encode single-chain factor VIII polypeptides, and the mutant FVIII proteins preferably have at least 50%, 75%, 90% or more (including >100%) of the clotting activity of the corresponding wild-type factor VIII. Assays for measuring the clotting activity of FVIII proteins include one-stage or two-stage clotting assays (Rizza et al., 1982, Coagulation assay of FVIII:C and FIXa in Bloom ed. The Hemophelias. NY Churchill Livingston 1992) or chromogenic substrate FVIII:C assays (Rosen, S. 1984, Scand J Haematol 33:139-145, supplement).

[0032] Several FVIII-BDD mutants are known, and are described, for example, in the following U.S. Patents: 4,868,112 (e.g., column 2, line 2 to column 19, line 21 and Table 2); 5,112,950 (e.g., column 2, lines 55-68, Figure 2, and Example 1); 5,171,844 (e.g., column 4, line 122 to column 5, line 36); 5,543,502 (e.g., column 2, lines 17-46); 5,595,886; 5,610,278; 5,789,203 (e.g., column 2, lines 26-51 and Examples 5-8); and 5,972,885 (e.g., column 1, line 25 to column 2, line 40). ); and mutants having complete or partial B domain deletions as disclosed in any of the following: No. 6,048,720 (e.g., column 6, lines 1-22 and Example 1); No. 6,060,447; No. 6,228,620; No. 6,316,226 (e.g., column 4, lines 4 to column 5, line 28 and Examples 1-5); No. 6,346,513; No. 6,458,563 (e.g., column 4, lines 25-53) and No. 7,041,635 (e.g., column 2, lines 1 to 3, line 19, column 3, lines 40 to 4, line 67, column 7, lines 43 to 8, line 26, and column 11, lines 5 to 13, line 39).

[0033] In some embodiments, the FVIII-BDD protein produced by the device described herein (e.g., expressed by recombinant cells contained in the device) has one or more of the following deletions of amino acids in the B domain: (i) most of the B domain, excluding the amino-terminal B domain sequence essential for intracellular processing of the primary translation product into two polypeptide chains (WO 91 / 09122); (ii) amino acids 747-1638 (Hoeben RC, et al. J. Biol. Chem. 265(13):7318-7323 (1990)); amino acids 771-1666 or amino acids 868-1562 (Meulien P., et al. Protein Eng. 2(4):301-6 (1988)); amino acids 982-1562 or 760-1639 (Toole et al. al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986); amino acids 797-1562 (Eaton et al., Biochemistry 25:8343-8347 (1986)); 741-1646 (Kaufman, WO 87 / 04187)), 747-1560 (Sarver et al., DNA 6:553-564 (1987)); amino acids 741-1648 (Pasek, WO 88 / 00831)), amino acids 816-1598 or 741-1689 (Lagner, Behring Inst. Mitt. (1988) No. 82:16-25, EP295597); deletions involving one or more residues in the Furin protease recognition sequence, for example, LKRHQR (SEQ ID NO: 65) at amino acids 1643 to 1648, including any of the specific deletions described in U.S. Pat. No. 9,956,269, column 10, line 65 to column 11, line 36.

[0034] In other embodiments, the FVIII-BDD protein retains any of the following B-domain amino acids or amino acid sequences: (i) one or more N-linked glycosylation sites in the B-domain, e.g., residues 757, 784, 828, 900, 963, or optionally 943, the first 226 amino acids, or the first 163 amino acids (Miao, HZ, et al., Blood 103(a):3412-3419 (2004); Kasuda, A., et al., J. Thromb. Haemost. 6:1352-1359 (2008); and Pipe, SW, et al., J. Thromb. Haemost. 9:2235-2242 (2011)).

[0035] In some embodiments, the FVIII-BDD protein is a single-chain mutant generated by substitution of one or more amino acids in the Furin protease recognition sequence (LKRHQR at amino acids 1643-1648 (SEQ ID NO: 65)) that prevents proteolytic cleavage at this site, including any of the substitutions at positions R1645 and / or R1648 described in U.S. Pat. Nos. 10,023,628, 9,394,353, and 9,670,267.

[0036] In some embodiments, any of the above-described FVIII-BDD proteins may further comprise one or more of the following mutations: an F309S substitution to improve expression of the FVIII-BDD protein (Miao, HZ, et al., Blood 103(a):3412-3419 (2004)); an albumin fusion (WO 2011 / 020866); and an Fc fusion (WO 04 / 101740).

[0037] Unless otherwise specified, all FVIII-BDD amino acid positions referred to herein refer to positions in full-length human FVIII.

[0038] As used herein, unless otherwise specified, "factor IX protein" or "FIX protein" refers to a polypeptide comprising the amino acid sequence of a native factor IX protein or a variant thereof, having FIX biological activity, e.g., clotting activity, as determined by art-recognized assays. FIX is produced as an inactive zymogen, which is converted to its active form by removal of the activation peptide factor XIa to produce heavy and light chains held together by one or more disulfide bonds. FIX proteins that can be produced by the devices described herein (e.g., semipermeable devices containing recombinant RPE cells) include wild-type primate (e.g., human), porcine, canine, and murine proteins, as well as variants of such wild-type proteins, including fragments, mutants, and variants with one or more amino acid substitutions and / or deletions and fusions of any of the above wild-type or variant proteins with half-life-extending polypeptides. In embodiments, cells are engineered to encode a full-length wild-type human factor IX polypeptide (e.g., with a signal sequence) or a functional variant thereof. The mutant FIX protein preferably has at least 50%, 75%, 90% or more (including >100%) of the clotting activity of wild-type factor VIX. Assays for measuring the clotting activity of FIX proteins include the Biophen Factor IX Assay (Hyphen BioMed) and one-stage clotting assays (activated partial thromboplastin time (aPTT) (e.g., as described in EP 2032 607), thrombin generation time assay (TGA), and rotational thromboelastometry (e.g., as described in WO 2012 / 006624).

[0039] Several functional FIX mutants are known and are described in the following international patent publications: WO 02 / 040544, page 4, lines 9-30 and page 15, lines 6-31; WO 03 / 020764 A2, Tables 2 and 3, pages 14-24, and page 12, lines 1-27; WO 2007 / 149406, page 4, line 1 to page 19, line 11; WO 2007 / 149406, page 19, line 12 to page 20, line 9; WO 08 / 118507, page 5, line 14 to page 6, line 5; WO 09 / 051717, page 9, line 11 to page 20, line 2; WO 09 / 137254, page 2, paragraph

[0006] to page 5, paragraph

[0011] and page 16, paragraph

[0044] to page 24, paragraph

[0057] ; WO 09 / 130198 A2, page 4, line 26 to page 12, line 6; WO 09 / 140015, page 11, paragraph

[0043] to page 13, paragraph

[0053] ; WO 2012 / 006624; or WO 2015 / 086406, which may be expressed by recombinant cells encapsulated in the device described herein, including any of the functional FIX variants described therein.

[0040] In certain embodiments, the FIX polypeptide comprises a wild-type or mutant sequence fused to a heterologous polypeptide or non-polypeptide moiety that extends the half-life of the FIX protein. Exemplary half-life extending moieties include Fc, albumin, PAS sequence, transferrin, CTP (the 28-amino acid C-terminal peptide (CTP) of human chorionic gonadotropin (hCG) with its four O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES), albumin-binding polypeptides, albumin-binding small molecules, or any combination thereof. An exemplary FIX polypeptide is the rFIXFc protein described in WO 2012 / 006624, which is a FIXFc single chain (FIXF c-sc) and an Fc single chain (Fc-sc) linked together via two disulfide bonds in the hinge region of Fc.

[0041] FIX variants also include gain-of-function and loss-of-function variants. An example of a gain-of-function variant is the "Padua" variant of human FIX, which has an L (leucine) at position 338 of the mature protein instead of an R (arginine) (corresponding to amino acid position 384 of SEQ ID NO: 2), and has higher catalytic and coagulation activity compared to wild-type human FIX (Chang et al., J. Biol. Chem., 273:12089-94 (1998)). An example of a loss-of-function variant is an alanine substituted for a lysine at the fifth amino acid position from the start of the mature protein, which results in a protein with reduced binding to collagen IV (e.g., loss of function).

[0042] As used herein, unless otherwise specified, "interleukin-2 protein" or "IL-2 protein" refers to a polypeptide comprising the amino acid sequence of a native IL-2 protein or a variant thereof that has IL-2 biological activity, e.g., activates IL-2 receptor signaling in Treg cells, as determined by art-recognized assays. IL-2 proteins that can be produced by the devices described herein, e.g., devices comprising recombinant RPE cells, include wild-type primate (e.g., human), porcine, canine, and murine proteins, as well as variants of such wild-type proteins. Variant IL-2 proteins preferably have at least 50%, 75%, 90%, or more (including >100%) of the biological activity of the corresponding wild-type IL-2. A biological activity assay for IL-2 proteins is described in U.S. Pat. No. 10,035,836 and includes, for example, measuring the level of phosphorylated STAT5 protein in Treg cells compared to CD4+CD25- / low T cells or NK cells. Mutant IL-2 proteins that can be produced by a device of the present disclosure (e.g., a device comprising recombinant RPE cells) include proteins with one or more of the following amino acid substitutions: N88R, N88I, N88G, D20H, Q126L, Q126F, and C125S or C125A. "Medium molecular weight alginate" or "MMW-Alg," as used herein, refers to an alginate having an approximate molecular weight of 75 kDa to 150 kDa.

[0043] "Polymer composition," as used herein, refers to a composition (e.g., solution, mixture) that includes one or more polymers. As a class, "polymers" include homopolymers, heteropolymers, copolymers, block polymers, and block copolymers, and can be both natural and synthetic. Homopolymers contain one type of building block (i.e., monomer), while copolymers contain multiple types of monomers.

[0044] As used herein, "polypeptide" refers to a polymer comprising amino acid residues joined via peptide bonds and having at least 2, and in some embodiments at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, or 200 amino acid residues.

[0045] As used herein, "prevention," "prevent," and "preventing" refer to treatments that involve the administration or application of a therapy, e.g., administering an encapsulated cell composition (e.g., as described herein) prior to the onset of a disease, disorder, or condition to prevent physical manifestations of the disease, disorder, or condition. In some embodiments, "prevention," "prevent," and "preventing" require that no sign or symptom of the disease, disorder, or condition has yet occurred or been observed. In some embodiments, treatment includes prevention, and in other embodiments, it does not.

[0046] "Reference device," as used herein with respect to a claimed device (e.g., a hydrogel capsule), means a device (e.g., a hydrogel capsule) that (i) lacks a particular feature, e.g., a barrier compartment containing a FBR mitigation measure (e.g., a small molecule compound, e.g., an antifibrotic compound); (ii) encapsulates in the cell-containing compartment approximately the same amount of cells of the same cell type as in the claimed device; and (iii) has a substantially similar polymer composition and structure as in the claimed device, except for the lack of the particular feature (e.g., an antifibrotic compound). In certain embodiments, the number of viable cells in the cell-containing compartment of the reference device is within 80-120%, or within 90-110%, of the number of viable cells in the cell-containing compartment of the claimed device. In certain embodiments, the cells in the reference device and the cells in the claimed device are obtained from the same cell culture. In some embodiments, substantially similar polymer composition means that all polymers (e.g., polymer components of any anti-fibrous polymer) in the reference device and in the claimed device are of the same chemical class and molecular weight class, if applicable (e.g., alginates with high G content and the same molecular weight range).

[0047] "Refractive index," as used herein, refers to a dimensionless value that represents the relative ratio of the speed of light in a medium (e.g., a sample) to the speed of light in a vacuum. The refractive index can be used to describe the phase velocity of light through a medium (e.g., how much the light path is bent when it enters a medium (e.g., a sample)). The refractive index of a sample can be measured with a refractometer and is dependent on temperature and wavelength. Exemplary refractive indices include water (nD=1.333 at 20°C) and ethanol (nD=1.36 at 20°C).

[0048] "Sequence identity" or "percent identical," as used herein to refer to two nucleotide sequences or two amino acid sequences, means that when the two sequences are compared and aligned for maximum correspondence over a comparison window or designated region, the two sequences are the same within a specified region or have the same nucleotides or amino acids at a specified percentage of nucleotide or amino acid positions within the specified region. Sequence identity can be determined using standard techniques known in the art, including, but not limited to, any of the algorithms described in U.S. Patent Application Publication No. 2017 / 02334455. In embodiments, the specified percentage of identical nucleotide or amino acid positions is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0049] As used herein, "subject" refers to a human or a non-human animal. In embodiments, the subject is, for example, a human (i.e., male or female) of any age group, a pediatric subject (e.g., infant, child, adolescent), or an adult subject (e.g., young adult, middle-aged adult, or geriatric). In embodiments, the subject is a non-human animal, for example, a mammal (e.g., a mouse, a dog, a primate (e.g., a cynomolgus monkey or a rhesus monkey). In embodiments, the subject is a commercially relevant mammal (e.g., a cow, a pig, a horse, a sheep, a goat, a cat, or a dog)) or a bird (e.g., a commercially relevant bird such as a chicken, a duck, a goose, or a turkey). In certain embodiments, the animal is a mammal. The animal can be male or female and at any stage of development. The non-human animal can be a genetically modified animal.

[0050] As used herein, "treatment," "treat," and "treating" refer to one or more of the reduction, reversal, alleviation, or delay in the onset of a disease, disorder, or condition, or the inhibition of the progression of one or more symptoms, signs, or underlying causes of a disease, disorder, or condition. In embodiments, treatment includes the reduction, reversal, alleviation, or delay in the onset of a disease, disorder, or condition, or the inhibition of the progression of a symptom of a disease, disorder, or condition. In embodiments, treatment includes the reduction, reversal, alleviation, or delay in the onset of a disease, disorder, or condition, or the inhibition of the progression of a sign of a disease, disorder, or condition. In embodiments, treatment includes the reduction, reversal, alleviation, reduction, or delay in the onset of an underlying cause of a disease, disorder, or condition. In some embodiments, "treatment," "treat," and "treating" require that a sign or symptom of a disease, disorder, or condition has occurred or has been observed. In other embodiments, treatment may be administered even when signs or symptoms of a disease or condition are absent, for example, as a prophylactic treatment. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, e.g., to delay or prevent recurrence. In some embodiments, treatment includes prevention, while in other embodiments it does not.

[0051] Selected Chemical Definitions Definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are defined as defined in Handbook of Chemistry and Physics, 75 thThe general principles of organic chemistry, as well as the moieties and reactivities of specific functional groups, are identified in accordance with the Periodic Table of the Elements, CAS version, inside cover of the Ed., and specific functional groups are defined conventionally as they appear therein. Also, general principles of organic chemistry, as well as specific functional group moieties and reactivities, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5 th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987.

[0052] Abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulas set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0053] When a range of values ​​is listed, it is intended that each value and subrange within the range be included. For example, "C1-C6 alkyl" is intended to include C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6 alkyl.

[0054] As used herein, "alkyl" refers to an alkyl group having 1 to 24 carbon atoms ("C1-C 24In some embodiments, an alkyl group refers to a radical of a linear or branched saturated hydrocarbon group having 1 to 12 carbon atoms ("C1-C 12 alkyl), 1 to 10 carbon atoms (C1 to C 12 In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C-C alkyl"). In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C-C alkyl"). Examples of C1-C6 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8), and the like. Each instance of an alkyl group can independently be optionally substituted, i.e., unsubstituted (an "unsubstituted alkyl") or substituted with one or more substituents (e.g., for illustration purposes, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent) (a "substituted alkyl").

[0055] As used herein, "alkenyl" refers to a radical of a straight-chain or branched hydrocarbon group having 2 to 24 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds ("C2-C 24 In some embodiments, an alkenyl group refers to an alkyl group having 2 to 10 carbon atoms ("C-C"). 10C2-C4 alkenyl"), 2 to 8 carbon atoms ("C2-C8 alkenyl"), 2 to 6 carbon atoms ("C2-C6 alkenyl"), 2 to 5 carbon atoms ("C2-C5 alkenyl"), 2 to 4 carbon atoms ("C2-C4 alkenyl"), 2 to 3 carbon atoms ("C2-C3 alkenyl"), or 2 carbon atoms ("C2 alkenyl"). The one or more carbon-carbon double bonds can be intermediate (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C2-C4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-C6 alkenyl groups include the aforementioned C 2~4 Alkenyl groups include pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Each instance of an alkenyl group can independently be optionally substituted, i.e., unsubstituted (an "unsubstituted alkenyl") or substituted with one or more substituents (e.g., illustratively 1 to 5 substituents, 1 to 3 substituents, or 1 substituent) (a "substituted alkenyl").

[0056] As used herein, the term "alkynyl" refers to a radical of a straight-chain or branched hydrocarbon group having 2 to 24 carbon atoms and one or more carbon-carbon triple bonds ("C2-C 24 In some embodiments, an alkynyl group refers to an alkynyl group having 2 to 10 carbon atoms ("C-C"). 10C-C alkynyl groups include 2 to 8 carbon atoms ("C-C alkynyl"), 2 to 6 carbon atoms ("C-C alkynyl"), 2 to 5 carbon atoms ("C-C alkynyl"), 2 to 4 carbon atoms ("C-C alkynyl"), 2 to 3 carbon atoms ("C-C alkynyl"), or 2 carbon atoms ("C alkynyl"). The one or more carbon-carbon triple bonds can be intermediate (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C-C alkynyl groups include ethynyl (C), 1-propynyl (C), 2-propynyl (C), 1-butynyl (C), 2-butynyl (C), and the like. Each instance of an alkynyl group can independently be optionally substituted, i.e., unsubstituted (an "unsubstituted alkynyl") or substituted with one or more substituents (e.g., illustratively 1 to 5 substituents, 1 to 3 substituents, or 1 substituent) (a "substituted alkynyl").

[0057] As used herein, the term "heteroalkyl" refers to an acyclic, stable, straight or branched chain, or combination thereof, containing at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. The heteroatoms O, N, P, S, and Si may be located at any position of the heteroalkyl group. Exemplary heteroalkyl groups include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, and -O-CH2-CH3. Up to two or three heteroatoms may be consecutive (e.g., -CH2-NH-OCH3 and -CH2-O-Si(CH3)3). When "heteroalkyl" is referenced followed by a specific heteroalkyl group (e.g., -CHO, -NR C R D etc.), the terms heteroalkyl and -CH2O or -NR C R D It will be understood that the heteroalkyl groups are not redundant or mutually exclusive. Rather, specific heteroalkyl groups are recited for added clarity. Thus, the term "heteroalkyl" includes -CHO, -NR C R D Nothing herein should be construed as excluding specific heteroalkyl groups such as: Each instance of a heteroalkyl group independently can be optionally substituted, i.e., unsubstituted (an "unsubstituted heteroalkyl") or substituted with, for example, one or more substituents, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (a "substituted heteroalkyl").

[0058] The terms "alkylene," "alkenylene," "alkynylene," or "heteroalkylene," by themselves or as part of another substituent, mean, unless otherwise stated, a divalent radical derived from an alkyl, alkenyl, alkynyl, or heteroalkyl, respectively. An alkylene, alkenylene, alkynylene, or heteroalkylene group may be described, for example, as a C-C-membered alkylene, a C-C-membered alkenylene, a C-C-membered alkynylene, or a C-C-membered heteroalkylene, where the term "membered" refers to a non-hydrogen atom in the moiety. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Additionally, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'- can represent both -C(O)2R'- and -R'C(O)2-.

[0059] As used herein, "aryl" refers to an aromatic ring system having 6 to 14 ring carbon atoms and zero heteroatoms ("C6-C 14 "aryl" refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in the cyclic array). In some embodiments, an aryl group has 6 ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C 10 Aryl"; e.g., naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms ("C 14 Aryl"; for example, anthracyl). The aryl group is, for example, C6-C 10A membered aryl may be described as a substituted or unsubstituted aryl, where the term "membered" refers to a non-hydrogen ring atom in the moiety. Aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each instance of an aryl group can independently be optionally substituted, i.e., unsubstituted (an "unsubstituted aryl") or substituted with one or more substituents (a "substituted aryl").

[0060] As used herein, "heteroaryl" refers to the radical of a 5- to 10-membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π-electrons shared in the cyclic array) having ring carbon atoms and 1 to 4 ring heteroatoms in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5- to 10-membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be at a carbon or nitrogen atom, as valence allows. Heteroaryl bicyclic ring systems can contain one or more heteroatoms in one or both rings. "Heteroaryl" also includes ring systems in which a heteroaryl ring, as defined above, is fused to one or more aryl groups, with the point of attachment being at either the aryl or heteroaryl ring; in such instances, the number of ring members refers to the number of ring members in the fused (aryl / heteroaryl) ring system. In bicyclic heteroaryl groups in which one ring does not contain heteroatoms (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be at either ring, i.e., the point of attachment can be at either the ring with a heteroatom (e.g., 2-indolyl) or the ring without a heteroatom (e.g., 5-indolyl). Heteroaryl groups can be described, for example, as 6- to 10-membered heteroaryl, where the term "member" refers to a non-hydrogen ring atom in the moiety.

[0061] In some embodiments, a heteroaryl group is a 5- to 10-membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5- to 10-membered heteroaryl"). In some embodiments, a heteroaryl group is a 5- to 8-membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5- to 8-membered heteroaryl"). In some embodiments, a heteroaryl group is a 5- to 6-membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5- to 6-membered heteroaryl"). In some embodiments, a 5- to 6-membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, a 5- to 6-membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heteroaryl has one ring heteroatom selected from nitrogen, oxygen, and sulfur. Each instance of a heteroaryl group can independently be optionally substituted, i.e., unsubstituted (an "unsubstituted heteroaryl") or substituted with one or more substituents (a "substituted heteroaryl").

[0062] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Other exemplary heteroaryl groups include heme and heme derivatives.

[0063] As used herein, the terms "arylene" and "heteroarylene," alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively.

[0064] As used herein, "cycloalkyl" refers to a group having 3 to 10 ring carbon atoms ("C3-C 10 "Cycloalkyl" refers to the radical of a non-aromatic cyclic hydrocarbon group having 3 to 8 ring carbon atoms ("C-C cycloalkyl"), 3 to 6 ring carbon atoms ("C-C cycloalkyl"), or 5 to 10 ring carbon atoms ("C-C 10 Cycloalkyl groups may be described, for example, as C4-C7 membered cycloalkyl, where the term "member" refers to a non-hydrogen ring atom in the moiety. Exemplary C3-C6 cycloalkyl groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-C8 cycloalkyl groups include, but are not limited to, the aforementioned C3-C6 cycloalkyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), cubanyl (C8), bicyclo[1.1.1]pentanyl (C5), bicyclo[2.2.2]octanyl (C8), bicyclo[2.1.1]hexanyl (C6), bicyclo[3.1.1]heptanyl (C7), and the like. 10 The cycloalkyl group includes, but is not limited to, the above-mentioned C3 to C8 cycloalkyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C 10), spiro[4.5]decanyl (C 10 As illustrated in the examples above, in certain embodiments, a cycloalkyl group is monocyclic ("monocyclic cycloalkyl") or contains fused, bridged, or spiro ring systems, such as bicyclic ring systems ("bicyclic cycloalkyl"), and can be saturated or partially unsaturated. "Cycloalkyl" also includes ring systems in which a cycloalkyl ring, as defined above, is fused to one or more aryl groups, and the point of attachment is at the cycloalkyl ring; in such instances, the number of carbons continues to refer to the number of carbons in the cycloalkyl ring system. Each instance of a cycloalkyl group can independently be optionally substituted, i.e., unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl").

[0065] As used herein, "heterocyclyl" refers to the radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("3- to 10-membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment can be at a carbon or nitrogen atom, as valence allows. Heterocyclyl groups can be monocyclic ("monocyclic heterocyclyl") or fused, bridged, or spiro ring systems, such as bicyclic ring systems ("bicyclic heterocyclyl"), and can be saturated or partially unsaturated. Heterocyclyl bicyclic ring systems can contain one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which a heterocyclyl ring, as defined above, is fused to one or more cycloalkyl groups, with the point of attachment being at either the cycloalkyl or heterocyclyl ring, or in which a heterocyclyl ring, as defined above, is fused to one or more aryl or heteroaryl groups, with the point of attachment being at the heterocyclyl ring; in such instances, the number of ring members continues to indicate the number of ring members in the heterocyclyl ring system. A heterocyclyl group may be described, for example, as a 3- to 7-membered heterocyclyl, where the term "member" refers to the non-hydrogen ring atoms in the moiety, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. Each instance of heterocyclyl may independently be optionally substituted, i.e., unsubstituted ("unsubstituted heterocyclyl") or substituted with one or more substituents ("substituted heterocyclyl"). In certain embodiments, the heterocyclyl group is an unsubstituted 3- to 10-membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-10 membered heterocyclyl.

[0066] In some embodiments, a heterocyclyl group is a 5- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (a "5- to 10-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 8-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (a "5- to 8-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 6-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (a "5- to 6-membered heterocyclyl"). In some embodiments, a 5- to 6-membered heterocyclyl has 1 to 3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heterocyclyl has 1 to 2 ring heteroatoms selected from nitrogen, oxygen, and sulfur, In some embodiments, the 5- to 6-membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0067] Exemplary 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azirdinyl, oxiranyl, and thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl, piperazinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, triazinanyl or thiomorpholinyl-1,1-dioxide. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azocanyl, oxecanyl, and thiocanyl. Exemplary 5-membered heterocyclyl groups (also referred to herein as 5,6-bicyclic heterocycles) fused to a C6 aryl ring include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like.Exemplary 6-membered heterocyclyl groups (also referred to herein as 6,6-bicyclic heterocycles) fused to an aryl ring include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0068] As used herein, "amino" refers to the radical -NR 70 R 71 where R 70 and R 71 are each independently hydrogen, C1-C8 alkyl, C3-C 10 Cycloalkyl, C4-C 10 Heterocyclyl, C6-C 10 Aryl and C5-C 10 Heteroaryl. In some embodiments, amino refers to NH2.

[0069] As used herein, "cyano" refers to the radical --CN.

[0070] As used herein, "halo" or "halogen," independently or as part of another substituent, means, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom.

[0071] As used herein, "hydroxy" refers to the radical --OH.

[0072] As defined herein, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups are optionally substituted (e.g., a "substituted" or "unsubstituted" alkyl, a "substituted" or "unsubstituted" alkenyl, a "substituted" or "unsubstituted" alkynyl, a "substituted" or "unsubstituted" heteroalkyl, a "substituted" or "unsubstituted" cycloalkyl, a "substituted" or "unsubstituted" heterocyclyl, a "substituted" or "unsubstituted" aryl, or a "substituted" or "unsubstituted" heteroaryl group). In general, the term "substituted," whether preceded by the term "optionally," means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, for example, one that results in a stable compound (e.g., a compound that does not undergo spontaneous transformation, such as by rearrangement, cyclization, elimination, or other reaction). Unless otherwise specified, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituents may be the same or different at each position. The term "substituted" is intended to include substitution with all permissible substituents of organic compounds, including any of the substituents described herein, that result in the formation of stable compounds. The present disclosure contemplates all such combinations that result in stable compounds. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituents described herein that satisfy the valence of the heteroatom and result in the formation of a stable moiety.

[0073] Two or more substituents may optionally be linked to form an aryl, heteroaryl, cycloalkyl, or heterocyclyl group. Such so-called ring-forming substituents are typically, but not necessarily, found attached to a ring base structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of a ring base structure form a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of a ring base structure form a spirocyclic structure. In yet another embodiment, the ring-forming substituents are attached to non-adjacent members of the base structure.

[0074] The compounds of formula (I) described herein may contain one or more asymmetric centers and therefore may exist in various isomeric forms, e.g., enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of individual enantiomers, diastereomers, or geometric isomers, or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high-pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present disclosure further includes the compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0075] As used herein, a pure enantiomer is substantially free (i.e., in enantiomeric excess) of the other enantiomer or stereoisomer of the compound. In other words, the "S" form of the compound is substantially free from the "R" form of the compound, and the "R" form is therefore in enantiomeric excess. The terms "enantiomerically pure" or "pure enantiomer" indicate that a compound contains more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, more than 99.5%, or more than 99.9% by weight of an enantiomer. In certain embodiments, the weight is based on the total weight of all enantiomers or stereoisomers of the compound.

[0076] The compounds of formula (I) described herein may also contain one or more isotopic substitutions. For example, H is: 1 H, 2 H (D or deuterium), and 3 H can be in any isotopic form, including T or tritium; C can be 12 C. 13 C, and 14 C can be any isotopic form, including 16 O and 18 It can be in any isotopic form, including O, etc.

[0077] The term "pharmaceutically acceptable salts" is intended to include salts of active compounds prepared with relatively non-toxic acids or bases, depending on the specific substituents found in the compounds described herein. When the compounds of Formula (I) used in providing the devices of the present disclosure contain a relatively acidic functional group, a base addition salt can be obtained by contacting such a neutral form of the compound with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts. When the compounds used in the present disclosure contain a relatively basic functional group, an acid addition salt can be obtained by contacting such a neutral form of the compound with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphate, dihydrogenphosphate, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, as well as salts derived from organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc. Also included are salts of amino acids such as arginate, and salts of organic acids such as glucuronic acid or galacturonic acid (see, e.g., Berge et al., Journal of Pharmaceutical Science 66:1-19 (1977)). The compounds used in certain specific devices of the present disclosure (e.g., particles, hydrogel capsules) contain both basic and acidic functionalities that allow the compounds to be converted into base or acid addition salts. These salts can be prepared by methods known to those of ordinary skill in the art. Other pharmaceutically acceptable carriers known to those of ordinary skill in the art are suitable for use in the present disclosure.

[0078] Certain compounds of formula (I) can exist in unsolvated forms as well as solvated forms, including hydrated forms. Generally, solvated forms are equivalent to unsolvated forms and are included within the scope of the present disclosure. Certain compounds of formula (I) can exist in multiple crystalline or amorphous forms. Generally, all physical forms are equivalent for the uses envisioned by the present disclosure and are intended to be within the scope of the present disclosure.

[0079] The term "solvate" refers to a form of a compound associated with a solvent, usually by solvolysis. This physical association may involve hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein may be prepared, for example, in crystalline form, and may be solvated. Suitable solvates include pharmaceutically acceptable solvates, and further include both stoichiometric and non-stoichiometric solvates.

[0080] The term "hydrate" refers to a compound associated with water. Typically, a hydrate of a compound contains a defined ratio of water molecules to the number of compound molecules in the hydrate. Thus, a hydrate of a compound can be represented, for example, by the general formula R x HO, where R is the compound and x is a number greater than 0.

[0081] The term "tautomer," as used herein, refers to interconvertible forms of a compound structure that vary in the displacement of hydrogen atoms and electrons. Thus, two structures can be in equilibrium through the movement of π electrons and atoms (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Tautomers can be relevant for achieving optimal chemical reactivity and biological activity of a compound of interest.

[0082] As used herein, the symbols [ka] refers to the binding to the surface of an entity, e.g., a polymer (e.g., a hydrogel-forming polymer such as alginate) or an implantable device, e.g., a particle, hydrogel capsule. [ka] The bond represented by may refer to a direct bond to an entity, e.g., a polymer or implantable element, or may refer to a linkage to an entity via a linking group. As used herein, "linking group" refers to a moiety for linking a compound of formula (I) to an entity (e.g., a polymer or implantable element (e.g., a device) described herein) and may include any linking chemistry known in the art. A list of exemplary linking groups can be found in Bioconjugate Techniques (3), which is incorporated herein by reference in its entirety. rd ed. Greg T. Hermanson, Waltham, MA: Elsevier, Inc., 2013). In some embodiments, the linking group is selected from alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, —C(O)—, —OC(O)—, —N(R C )-, -N(R C )C(O)-, -C(O)N(R C )-, -N(R C )N(R D )-, -NCN-, -C(=N(R C )(R D ))O-, -S-, -S(O) x -, -OS(O) x -, -N(R C )S(O) x -, -S(O) x N(R C )-, -P(R F ) y -, -Si(OR A )2-, -Si(R G )(OR A )-, -B(OR A )- or metal, where R A, R C , R D , R F , R G Each of x, y, and x is independently as described herein. In some embodiments, the linking group comprises an amine, a ketone, an ester, an amide, an alkyl, an alkenyl, an alkynyl, or a thiol. In some embodiments, the linking group is a crosslinker. In some embodiments, the linking group is -C(O)(C1-C6-alkylene)-, where alkylene is R 1 is replaced by R 1 are as described herein. In some embodiments, the linking group is -C(O)(C1-C6-alkylene)-, where the alkylene is substituted with 1-2 alkyl groups (e.g., 1-2 methyl groups). In some embodiments, the linking group is -C(O)C(CH3)2-. In some embodiments, the linking group is -C(O)(methylene)-, where the alkylene is substituted with 1-2 alkyl groups (e.g., 1-2 methyl groups). In some embodiments, the linking group is -C(O)CH(CH3)-. In some embodiments, the linking group is -C(O)C(CH3)-.

[0083] Polymer composition characteristics The present disclosure features methods for evaluating polymer compositions comprising at least one polymer modified with a small molecule compound. For example, the methods described herein can be used to determine the total concentration of the small molecule compound bound to the modified polymer. The polymer compositions can be used to encapsulate cells (e.g., living cells) expressing a therapeutic agent to form a semipermeable device that can release the therapeutic agent upon implantation of the device into a subject (e.g., a human or other mammalian subject). The methods described herein can provide a means for reliably determining the concentration of the small molecule compound within the polymer composition, which can be useful, for example, for measuring the stability of small molecule compound modifications over time.

[0084] The small molecule compounds attached to the modified polymer in the polymer composition can include naturally occurring or non-naturally occurring small molecules and variants thereof. In some embodiments, the small molecule compounds include alcohols, amines, carboxylic acids, esters, thiols, aryls, heteroaryls, ethers, alkenes, alkynes, azides, sulfonic acids, polyethylene glycol, or other moieties. The small molecule compounds can be linked to the modified polymer in any manner (e.g., via an amide, ester, thioester, disulfide, or ether linkage). In addition, the small molecule compounds for use with the present disclosure can be modified in some manner (e.g., by chemical or enzymatic modification (e.g., glycosylation, phosphorylation)).

[0085] In some embodiments, small molecule compounds have an average molecular weight of 100 Da, 200 Da, 250 Da, 300 Da, 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1 kDa, 1.5 kDa, 2 kDa, 2.5 kDa, 5 kDa, or more. In some embodiments, small molecule compounds have an average molecular weight of 5 kDa, 2.5 kDa, 2 kDa, 1.5 kDa, 1 kDa, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 250 Da, 200 Da, 150 Da, 100 Da, or less. In some embodiments, small molecule compounds have an average molecular weight of more than 100 Da. In some embodiments, the small molecule compound has an average molecular weight of more than 250 Da. In some embodiments, the small molecule compound has an average molecular weight of less than 500 Da. In some embodiments, the small molecule compound has an average molecular weight of less than 1 kDa. In some embodiments, the small molecule compound has an average molecular weight of 100 Da to 5 kDa, e.g., 100 Da to 2.5 kDa, 100 Da to 2 kDa, 100 Da to 1 kDa, 200 Da to 5 kDa, 200 Da to 2.5 kDa, 200 Da to 2 kDa, 200 Da to 1 kDa, 250 Da to 5 kDa, 250 Da to 2.5 kDa, 250 Da to 2 kDa, 250 Da to 1 kDa, 500 Da to 5 kDa, 500 Da to 2.5 kDa, 500 Da to 2 kDa, or 500 Da to 1 kDa.

[0086] In some embodiments, the small molecule compound is an anti-fibrotic compound. In certain embodiments, the anti-fibrotic compound has Formula (I): [ka] or a salt thereof (e.g., a pharmaceutically acceptable salt), wherein: A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R C )-, -N(R C )C(O)-, -C(O)N(RC )-, -N(R C )C(O)(C1-C6-alkylene)-, -N(R C )C(O)(C1-C6-alkenylene)-, -N(R C )N(R D )-, -NCN-, -C(=N(R C )(R D ))O-, -S-, -S(O) x -, -OS(O) x -, -N(R C )S(O) x -, -S(O) x N(R C )-, -P(R F ) y -, -Si(OR A )2-, -Si(R G )(OR A )-, -B(OR A )-, or a metal, each of which is optionally bonded to a linking group (e.g., a linking group described herein) and one or more R 1 is optionally replaced by; L 1 and L 3 is independently a bond, alkyl, or heteroalkyl, wherein each of the alkyl and heteroalkyl is selected from one or more R 2 is optionally replaced by; L 2 is a bond; M is absent or each is one or more R 3 alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl optionally substituted with; P is absent or each contains one or more R 4 cycloalkyl, heterocyclyl, or heteroaryl optionally substituted with Z is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, -OR A , -C(O)R A , -C(O)OR A , -C(O)N(R C )(R D ), -N(RC )C(O)R A , cycloalkyl, heterocyclyl, aryl, or heteroaryl, where each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is selected from one or more R 5 is optionally replaced by; R A , R B , R C , R D , R E , R F , and R G is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, azido, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is selected from the group consisting of one or more R 6 is optionally replaced by; or R C and R D together with the nitrogen atom to which they are attached, form one or more R 6 forming an optionally substituted ring (e.g., a 5- to 7-membered ring) with R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 each independently represents alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )C(O)R B1 , -C(O)N(R C1 ), SR E1 , S(O) x R E1 , -OS(O) x R E1 , -N(RC1 )S(O) x R E1 , -S(O) x N(R C1 )(R D1 ), -P(R F1 ) y , cycloalkyl, heterocyclyl, aryl, heteroaryl, where each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is selected from one or more R 7 is optionally replaced by; R A1 , R B1 , R C1 , R D1 , R E1 , and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is selected from the group consisting of one or more R 7 is optionally replaced by; Each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl; x is 1 or 2; y is 2, 3, or 4.

[0087] In some embodiments, the compound of Formula (I) has the formula (Ia): [ka] or a pharmaceutically acceptable salt thereof, wherein A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R C )-, -N(R C)C(O)-, -C(O)N(R C )-, -N(R C )N(R D )-, N(R C )C(O)(C1-C6-alkylene)-, -N(R C )C(O)(C1-C6-alkenylene)-, -NCN-, -C(=N(R C )(R D ))O-, -S-, -S(O) x -, -OS(O) x -, -N(R C )S(O) x -, -S(O) x N(R C )-, -P(R F ) y -, -Si(OR A )2-, -Si(R G )(OR A )-, -B(OR A )-, or a metal, each of which is optionally bonded to a linking group (e.g., a linking group described herein) and one or more R 1 is optionally replaced by; L 1 and L 3 is independently a bond, alkyl, or heteroalkyl, wherein each of the alkyl and heteroalkyl is selected from one or more R 2 is optionally replaced by; L 2 is a bond; M is absent or each is one or more R 3 alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl optionally substituted with; P is one or more R 4 is heteroaryl optionally substituted with Each Z is one or more R 5 alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl optionally substituted with R A , R B , RC , R D , R E , R F , and R G is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, azido, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is selected from the group consisting of one or more R 6 is optionally replaced by; or R C and R D together with the nitrogen atom to which they are attached, form one or more R 6 forming an optionally substituted ring (e.g., a 5- to 7-membered ring) with R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 each independently represents alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )C(O)R B1 , -C(O)N(R C1 ), SR E1 , S(O) x R E1 , -OS(O) x R E1 , -N(R C1 )S(O) x R E1 , -S(O) x N(R C1 )(R D1 ), -P(R F1 ) y, cycloalkyl, heterocyclyl, aryl, heteroaryl, where each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is selected from one or more R 7 is optionally replaced by; R A1 , R B1 , R C1 , R D1 , R E1 , and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is selected from the group consisting of one or more R 7 is optionally replaced by; Each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl; x is 1 or 2; y is 2, 3, or 4.

[0088] In some embodiments, for Formula (I) and (Ia), A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, —O—, —C(O)O—, —C(O)—, —OC(O)—, —N(R C )C(O)-, -N(R C )C(O)(C1-C6-alkylene)-, -N(R C )C(O)(C1-C6-alkenylene)-, or -N(R C In some embodiments, A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, —O—, —C(O)O—, —C(O)—, —OC(O)—, or —N(R CIn some embodiments, A is alkyl, alkenyl, alkynyl, heteroalkyl, —O—, —C(O)O—, —C(O)—, —OC(O—, or —N(R C In some embodiments, A is alkyl, —O—, —C(O)O—, —C(O)—, —OC(O), or —N(R C In some embodiments, A is —N(R C )C(O)-, -N(R C )C(O)(C1-C6-alkylene)-, or -N(R C )C(O)(C1-C6-alkenylene)-. In some embodiments, A is -N(R C In some embodiments, A is —N(R C )- and R C and R D is independently hydrogen or alkyl. In some embodiments, A is -NH-. In some embodiments, A is -N(R C )C(O)(C1-C6-alkylene)-, where alkylene is R 1 In some embodiments, A is substituted with -N(R C )C(O)(C1-C6-alkylene)-, and R 1 is alkyl (e.g., methyl). In some embodiments, A is -NHC(O)CH(CH3)2-. In some embodiments, A is -N(R C )C(O)(methylene)-, and R 1 is alkyl (e.g., methyl). In some embodiments, A is -NHC(O)CH(CH3)-. In some embodiments, A is -NHC(O)C(CH3)-.

[0089] In some embodiments, for Formulas (I) and (Ia), L 1 is a bond, alkyl, or heteroalkyl. In some embodiments, L 1 is a bond or alkyl. In some embodiments, L 1is a bond. In some embodiments, L 1 is alkyl. In some embodiments, L 1 is C1-C6 alkyl. In some embodiments, L 1 is -CH2-, -CH(CH3)-, -CH2CH2CH2, or -CH2CH2-. In some embodiments, L 1 is —CH2— or —CH2CH2—.

[0090] In some embodiments, for Formulas (I) and (Ia), L 3 is a bond, alkyl, or heteroalkyl. In some embodiments, L 3 is a bond. In some embodiments, L 3 is alkyl. In some embodiments, L 3 is C1~C 12 In some embodiments, L 3 is C1-C6 alkyl. In some embodiments, L 3 is -CH-. In some embodiments, L 3 is heteroalkyl. In some embodiments, L 3 is one or more R 2 C1-C optionally substituted with (e.g., oxo) 12 In some embodiments, L is heteroalkyl. 3 is one or more R 2 (e.g., oxo). In some embodiments, L 3 is —C(O)OCH—, —CH(OCHCH)—, —CH(OCHCH)—, CHCHO—, or —CHO—. In some embodiments, L 3 is -CH2O-.

[0091] In some embodiments, for Formula (I) and (Ia), M is absent, alkyl, heteroalkyl, aryl, or heteroaryl. In some embodiments, M is heteroalkyl, aryl, or heteroaryl. In some embodiments, M is absent. In some embodiments, M is alkyl (e.g., C1-C6 alkyl). In some embodiments, M is -CH2-. In some embodiments, M is heteroalkyl (e.g., C1-C6 heteroalkyl). In some embodiments, M is (-OCH2CH2-)z, where z is an integer selected from 1 to 10. In some embodiments, z is an integer selected from 1 to 5. In some embodiments, M is -OCH2CH2-, (-OCH2CH2-)2, (-OCH2CH2-)3, (-OCH2CH2-)4, or (-OCH2CH2-)5. In some embodiments, M is -OCH2CH2-, (-OCH2CH2-)2, (-OCH2CH2-)3, or (-OCH2CH2-)4. In some embodiments, M is (-OCH2CH2-)3. In some embodiments, M is aryl. In some embodiments, M is phenyl. In some embodiments, M is unsubstituted phenyl. In some embodiments, M is [ka] In some embodiments, M is R 7 (For example, one R 7 In some embodiments, M is phenyl substituted with [ka] In some embodiments, R 7 is CF3.

[0092] In some embodiments, for Formulas (I) and (Ia), P is absent, heterocyclyl, or heteroaryl. In some embodiments, P is absent. In some embodiments, for Formulas (I) and (Ia), P is tricyclic, bicyclic, or monocyclic heteroaryl. In some embodiments, P is monocyclic heteroaryl. In some embodiments, P is nitrogen-containing heteroaryl. In some embodiments, P is monocyclic, nitrogen-containing heteroaryl. In some embodiments, P is 5-membered heteroaryl. In some embodiments, P is 5-membered nitrogen-containing heteroaryl. In some embodiments, P is tetrazolyl, imidazolyl, pyrazolyl, or triazolyl, pyrrolyl, oxazolyl, or thiazolyl. In some embodiments, P is tetrazolyl, imidazolyl, pyrazolyl, or triazolyl, or pyrrolyl. In some embodiments, P is imidazolyl. In some embodiments, P is [ka] In some embodiments, P is triazolyl. In some embodiments, P is 1,2,3-triazolyl. In some embodiments, P is [ka] is.

[0093] In some embodiments, P is heterocyclyl. In some embodiments, P is 5-membered heterocyclyl or 6-membered heterocyclyl. In some embodiments, P is imidazolidinonyl. In some embodiments, P is [ka] In some embodiments, P is thiomorpholinyl-1,1-dioxidyl.

[0094] In some embodiments, P is [ka] is.

[0095] In some embodiments, for Formula (I) or (Ia), Z is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. In some embodiments, Z is heterocyclyl. In some embodiments, Z is monocyclic or bicyclic heterocyclyl. In some embodiments, Z is an oxygen-containing heterocyclyl. In some embodiments, Z is a 4-membered heterocyclyl, a 5-membered heterocyclyl, or a 6-membered heterocyclyl. In some embodiments, Z is a 6-membered oxygen-containing heterocyclyl. In some embodiments, Z is tetrahydropyranyl. In some embodiments, Z is [ka] In some embodiments, Z is a 4-membered oxygen-containing heterocyclyl. In some embodiments, Z is [ka] is.

[0096] In some embodiments, Z is a bicyclic oxygen-containing heterocyclyl. In some embodiments, Z is phthalic anhydridyl. In some embodiments, Z is a sulfur-containing heterocyclyl. In some embodiments, Z is a 6-membered sulfur-containing heterocyclyl. In some embodiments, Z is a 6-membered heterocyclyl containing a nitrogen atom and a sulfur atom. In some embodiments, Z is thiomorpholinyl-1,1-dioxidyl. In some embodiments, Z is [ka] In some embodiments, Z is a nitrogen-containing heterocyclyl. In some embodiments, Z is a 6-membered nitrogen-containing heterocyclyl. In some embodiments, Z is [ka] is.

[0097] In some embodiments, Z is a bicyclic heterocyclyl. In some embodiments, Z is a bicyclic heterocyclyl. In some embodiments, Z is a bicyclic heterocyclyl. 5 In some embodiments, Z is 2-oxa-7-azaspiro[3.5]nonanyl. In some embodiments, Z is [ka] In some embodiments, Z is 1-oxa-3,8-diazaspiro[4.5]decan-2-one. In some embodiments, Z is [ka] is.

[0098] In some embodiments, for Formula (I) and (Ia), Z is aryl. In some embodiments, Z is monocyclic aryl. In some embodiments, Z is phenyl. In some embodiments, Z is monosubstituted phenyl (e.g., one R 5 In some embodiments, Z is a monosubstituted phenyl, wherein one R 5 is a nitrogen-containing group. In some embodiments, Z is a monosubstituted phenyl, wherein one R 5 is NH. In some embodiments, Z is a monosubstituted phenyl, wherein one R 5 is an oxygen-containing group. In some embodiments, Z is a monosubstituted phenyl, wherein one R 5 is an oxygen-containing heteroalkyl. In some embodiments, Z is a monosubstituted phenyl, wherein one R 5 is OCH. In some embodiments, Z is a monosubstituted phenyl, wherein one R 5 In some embodiments, Z is a monosubstituted phenyl, wherein one R 5 In some embodiments, Z is a monosubstituted phenyl, wherein one R 5 is in para position.

[0099] In some embodiments, for formula (I) and (Ia), Z is alkyl. In some embodiments, Z is C1-C 12 In some embodiments, Z is C1-C 10 In some embodiments, Z is C1-C8 alkyl. In some embodiments, Z is selected from 1 to 5 R 5 In some embodiments, Z is C1-C8 alkyl substituted with one R 5 In some embodiments, Z is C1-C8 alkyl substituted with one R 5 C1-C8 alkyl substituted with, where R 5is alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , or -N(R C1 )(R D1 In some embodiments, Z is one R 5 C1-C8 alkyl substituted with, where R 5 -OR A1 or -C(O)OR A1 In some embodiments, Z is one R 5 C1-C8 alkyl substituted with, where R 5 -OR A1 Or -C(O)OH. In some embodiments, Z is -CH3.

[0100] In some embodiments, for formula (I) and (Ia), Z is heteroalkyl. In some embodiments, Z is C1-C 12 In some embodiments, Z is a C1-C 10 In some embodiments, Z is a C1-C8 heteroalkyl. In some embodiments, Z is a C1-C6 heteroalkyl. In some embodiments, Z is selected from the group consisting of one or more R 5 In some embodiments, Z is a nitrogen-containing heteroalkyl optionally substituted with 1 to 5 R 5 In some embodiments, Z is N-methyl-2-(methylsulfonyl)ethane-1-aminyl.

[0101] In some embodiments, Z is -OR A or -C(O)OR A In some embodiments, Z is -OR A (e.g., —OH or —OCH). In some embodiments, Z is OCH. In some embodiments, Z is —C(O)ORA (e.g., —C(O)OH).

[0102] In some embodiments, Z is hydrogen.

[0103] In some embodiments, L 2 is a bond, and P and L 3 is independently absent. 2 is a bond, P is heteroaryl, and L 3 is a bond and Z is hydrogen. In some embodiments, P is heteroaryl and L 3 is heteroalkyl and Z is alkyl.

[0104] In some embodiments, the compound of Formula (I) has the formula (Ib): [ka] or a pharmaceutically acceptable salt thereof, wherein ring M 1 Each contains 1 to 5 R 3 cycloalkyl, heterocyclyl, aryl, or heteroaryl optionally substituted with 1 is 1 to 5 R 5 cycloalkyl, heterocyclyl, aryl, or heteroaryl, optionally substituted with R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclyl, aryl, or heteroaryl, or R 2a and R 2b or R 2c and R 2d together form an oxo group; X is absent, N(R 10 )(R 11 ), O, or S; R Cis hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, where each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is selected from 1 to 6 R 6 optionally substituted with R 3 , R 5 , and R 6 each independently represents alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )C(O)R B1 , -C(O)N(R C1 ), SR E1 , cycloalkyl, heterocyclyl, aryl, or heteroaryl; R 10 and R 11 each independently represents hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, —C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -C(O)N(R C1 ), cycloalkyl, heterocyclyl, aryl, or heteroaryl; R A1 , R B1 , R C1 , R D1 , and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is selected from 1 to 6 R 7 and each R 7is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl; each m and n is independently 1, 2, 3, 4, 5, or 6; [ka] refers to a bond to a linking group or polymer described herein. In some embodiments, each R 3 and R 5 For each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally and independently substituted with halogen, oxo, cyano, cycloalkyl, or heterocyclyl.

[0105] In some embodiments, the compound of Formula (Ib) has the formula (Ibi): [ka] or a pharmaceutically acceptable salt thereof, wherein ring M 2 is one or more R 3 aryl or heteroaryl optionally substituted with; ring Z 2 is cycloalkyl, heterocyclyl, aryl, or heteroaryl; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b or R 2c and R 2d taken together form an oxo group; X is absent, O, or S; and each R 3 and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1wherein each of the alkyl and heteroalkyl is optionally substituted with halogen; or two R 5 Together, we form Ring Z 2 form a 5- to 6-membered ring fused to each R A1 and R B1 are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4, 5, or 6; [ka] refers to a bond to a linking group or polymer as described herein.

[0106] In some embodiments, the compound of formula (Ibi) has the formula (Ib-ii): [ka] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is cycloalkyl, heterocyclyl, aryl or heteroaryl; R 2c and R 2d each is independently hydrogen, alkyl, or heteroalkyl, or R 2c and R 2d together form an oxo group; each R 3 and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 wherein each of the alkyl and heteroalkyl is optionally substituted with halogen; and each R A1 and R B1 are independently hydrogen, alkyl, or heteroalkyl; p and q are each independently 0, 1, 2, 3, 4, 5, or 6; [ka] refers to a bond to a linking group or polymer as described herein.

[0107] In some embodiments, the compound of Formula (I) has the formula (Ic): [ka] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is cycloalkyl, heterocyclyl, aryl or heteroaryl; R 2c and R 2d each is independently hydrogen, alkyl, or heteroalkyl, or R 2c and R 2d together form an oxo group; each R 3 and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 wherein each of the alkyl and heteroalkyl is optionally substituted with halogen; and each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; m is 1, 2, 3, 4, 5, or 6; p and q are each independently 0, 1, 2, 3, 4, 5, or 6; [ka] refers to a bond to a linking group or polymer as described herein.

[0108] In some embodiments, the compound of Formula (I) has the formula (Id): [ka] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is cycloalkyl, heterocyclyl, aryl, or heteroaryl; X is absent, O, or S; R 2a, R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b or R 2c and R 2d together form an oxo group; each R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 wherein each of the alkyl and heteroalkyl is optionally substituted with halogen; and each R A1 and R B1 are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4, 5, or 6; [ka] refers to a bond to a linking group or polymer as described herein.

[0109] In some embodiments, the compound of Formula (I) has the formula (Ie): [ka] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is cycloalkyl, heterocyclyl, aryl, or heteroaryl; X is absent, O, or S; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b or R 2c and R 2d together form an oxo group; each R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1, -C(O)OR A1 , or -C(O)R B1 and each R A1 and R B1 are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4, 5, or 6; [ka] refers to a bond to a linking group or polymer as described herein.

[0110] In some embodiments, the compound of Formula (I) has the formula (If): [ka] or a pharmaceutically acceptable salt thereof, wherein M is one or more R 3 ring P is alkyl optionally substituted with one or more R 4 is heteroaryl optionally substituted with 3 is one or more R 2 each Z is alkyl or heteroalkyl optionally substituted with one or more R 5 alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl optionally substituted with; R 2a and R 2b each is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b together form an oxo group; R 2 , R 3 , R 4 , and R 5 each independently represents alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 and each R A1 and R B1is independently hydrogen, alkyl, or heteroalkyl; n is independently 1, 2, 3, 4, 5, or 6; [ka] refers to a bond to a linking group or polymer as described herein.

[0111] In some embodiments, the compound of formula (I) has formula (II): [ka] or a pharmaceutically acceptable salt thereof, wherein M is a bond, alkyl, or aryl, wherein alkyl and aryl are selected from the group consisting of one or more R 3 and optionally substituted with; L 3 is one or more R 2 and Z is hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, or -OR A where alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl are each independently selected from one or more R 5 optionally substituted with R A is hydrogen; R 2a and R 2b each is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b together form an oxo group; R 2 , R 3 , and R 5 each independently represents alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 and each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; n is independently 1, 2, 3, 4, 5, or 6; [ka] refers to a bond to a linking group or polymer as described herein.

[0112] In some embodiments, the compound of formula (II) has the formula (II-a): [ka] or a pharmaceutically acceptable salt thereof, wherein L 3 are each one or more R 2 Z is hydrogen, alkyl, heteroalkyl, or -OR A where alkyl and heteroalkyl are each independently one or more R 5 optionally substituted with R 2a and R 2b each is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b together form an oxo group; R 2 , R 3 , and R 5 each independently represents alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 and;R A is hydrogen; each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; n is independently 1, 2, 3, 4, 5, or 6; [ka] refers to a bond to a linking group or polymer as described herein.

[0113] In some embodiments, the compound of formula (I) has formula (III): [ka] or a pharmaceutically acceptable salt thereof, wherein Z 1 Each contains 1 to 5 R 5 alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl optionally substituted with; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclyl, aryl, or heteroaryl; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R C is hydrogen, alkyl, alkenyl, alkynyl, or heteroalkyl, where each alkyl, alkenyl, alkynyl, or heteroalkyl is selected from 1 to 6 R 6 optionally substituted with R 3 , R 5 , and R 6 each independently represents alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 and each R A1 and R B1 are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; q is an integer from 0 to 25; [ka] refers to a bond to a linking group or polymer as described herein.

[0114] In some embodiments, the compound of Formula (III) has the formula (III-a): [ka] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 Each contains 1 to 5 R 5 cycloalkyl, heterocyclyl, aryl, or heteroaryl optionally substituted with R 2a , R 2b , R 2c , and R 2d each independently is hydrogen, alkyl, heteroalkyl, halo; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R 3 and R 5 each independently represents alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 and each R A1 and R B1 are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; o and p are each independently 0, 1, 2, 3, 4, or 5; q is an integer from 0 to 25; [ka] refers to a bond to a linking group or polymer as described herein.

[0115] In some embodiments, the compound of formula (III-a) has formula (III-b): [ka] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 Each contains 1 to 5 R 5 cycloalkyl, heterocyclyl, aryl, or heteroaryl optionally substituted with R 2a , R 2b , R 2c, and R 2d each independently is hydrogen, alkyl, heteroalkyl, halo; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R 3 and R 5 each independently represents alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 and each R A1 and R B1 are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; o and p are each independently 0, 1, 2, 3, 4, or 5; q is an integer from 0 to 25; [ka] refers to a bond to a linking group or polymer as described herein.

[0116] In some embodiments, the compound of formula (III-a) has formula (III-c): [ka] or a pharmaceutically acceptable salt thereof, wherein X is C(R')(R"), N(R'), or S(O) x each of R' and R" is independently hydrogen, alkyl, halogen, or cycloalkyl; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, heteroalkyl, or halo; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R 3 and R 5 each independently represents alkyl, heteroalkyl, halogen, oxo, -ORA1 , -C(O)OR A1 , or -C(O)R B1 and each R A1 and R B1 are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4, or 5; q is an integer from 0 to 25; x is 0, 1, or 2; [ka] refers to a bond to a linking group or polymer as described herein.

[0117] In some embodiments, the compound of formula (III-c) has formula (III-d): [ka] or a pharmaceutically acceptable salt thereof, wherein X is C(R')(R"), N(R'), or S(O) x each of R' and R" is independently hydrogen, alkyl, halogen, or cycloalkyl; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, heteroalkyl, or halo; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R 3 and R 5 each independently represents alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 and each R A1 and R B1 are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4, or 5; q is an integer from 0 to 25; x is 0, 1, or 2; [ka] refers to a bond to a linking group or polymer as described herein.

[0118] In some embodiments, the compound is a compound of formula (I). 2 is a bond, and P and L 3 is independent and absent.

[0119] In some embodiments, the compound is a compound of formula (Ia). In some embodiments of formula (II-a), L 2 is a bond, P is heteroaryl, and L 3 is a bond and Z is hydrogen. In some embodiments, P is heteroaryl and L 3 is heteroalkyl and Z is alkyl. In some embodiments, L 2 is a bond, and P and L 3 is independently absent. 2 is a bond, P is heteroaryl, and L 3 is a bond and Z is hydrogen. In some embodiments, P is heteroaryl and L 3 is heteroalkyl and Z is alkyl.

[0120] In some embodiments, the compound is a compound of formula (Ib). In some embodiments, P is absent and L 1 is -NHCH2, and L 2 is a bond, M is aryl (e.g., phenyl), and L 3 is —CHO and Z is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, such as thiomorpholinyl-1,1-dioxide). In some embodiments, the compound of Formula (Ib) is compound 116.

[0121] In some embodiments of Formula (Ib), P is absent and L 1 is -NHCH2, and L 2 is a bond, M is absent, and L 3 is a bond and Z is heterocyclyl (eg, an oxygen-containing heterocyclyl such as tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl). In some embodiments, the compound of Formula (Ib) is compound 105.

[0122] In some embodiments, the compound is of formula (Ibi). In some embodiments of formula (Ibi), R 2a and R 2b is independently hydrogen or CH3, and R 2c and R 2d are independently hydrogen, m is 1 or 2, n is 1, X is O, p is 0, and M 2 is one or more R 3 and R is phenyl optionally substituted with 3 is -CF3 and Z 2 is heterocyclyl (e.g., an oxygen-containing heterocyclyl, such as tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl). In some embodiments, the compound of Formula (Ibi) is Compound 100, Compound 106, Compound 107, Compound 108, Compound 109, or Compound 111.

[0123] In some embodiments, the compound is of formula (Ib-ii). In some embodiments of formula (Ib-ii), R 2a , R 2b , R 2c , and R 2d is independently hydrogen, q is 0, p is 0, m is 1, and Z 2 is heterocyclyl (eg, an oxygen-containing heterocyclyl, such as tetrahydropyranyl). In some embodiments, the compound of Formula (Ib-ii) is compound 100.

[0124] In some embodiments, the compound is of formula (Ic). In some embodiments of formula (Ic), R 2c and R 2d are independently hydrogen, m is 1, p is 1, q is 0, and R 5 is —CH 3 and Z is heterocyclyl (e.g., nitrogen-containing heterocyclyl, e.g., piperazinyl). In some embodiments, the compound of Formula (Ic) is compound 113.

[0125] In some embodiments, the compound is of formula (Id). In some embodiments of formula (Id), R 2a , R 2b , R 2c , and R 2d is independently hydrogen, m is 1, n is 3, X is O, p is 0, and Z is heterocyclyl (e.g., an oxygen-containing heterocyclyl such as tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl). In some embodiments, the compound of Formula (Id) is compound 110 or compound 114.

[0126] In some embodiments, the compound is of formula (If). In some embodiments of formula (If), R 2a and R 2b are independently hydrogen, n is 1, M is —CH—, P is a nitrogen-containing heteroaryl (e.g., imidazolyl), and L 3 is —C(O)OCH 2 — and Z is CH 3 . In some embodiments, the compound of Formula (If) is compound 115.

[0127] In some embodiments, the compound is a compound of formula (II-a). In some embodiments of formula (II-a), R 2a and R 2b are independently hydrogen, n is 1, q is 0, and L 3is —CH 2 (OCH 2 CH 2 ) 2 and Z is —OCH 3 . In some embodiments, the compound of Formula (II-a) is compound 112.

[0128] In some embodiments of Formula (II-a), R 2a and R 2b are independently hydrogen, n is 1, and L 3 is a bond or —CH 2 , and Z is hydrogen or —OH. In some embodiments, the compound of Formula (II-a) is Compound 103 or Compound 104.

[0129] In some embodiments, the compound is a compound of formula (III). In some embodiments of formula (III), R 2a , R 2b , R 2c , and R 2d are independently hydrogen, m is 1, n is 2, q is 3, p is 0, and R C is hydrogen, and Z 1 is R 5 (e.g., —N(CH)(CHCH)S(O)CH). In some embodiments, the compound of Formula (III) is compound 120.

[0130] In some embodiments, the compound is of formula (III-b). In some embodiments of formula (III-b), R 2a , R 2b , R 2c , and R 2d are independently hydrogen, m is 0, n is 2, q is 3, p is 0, and Z 2 is one R 5 (e.g., —NH 2 ). In some embodiments, the compound of Formula (III-b) is Compound 102.

[0131] In some embodiments, the compound is of formula (III-b). In some embodiments of formula (III-b), R 2a , R 2b , R 2c , and R 2d are independently hydrogen, m is 1, n is 2, q is 3, p is 0, and R C is hydrogen, and Z 2 is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, e.g., a nitrogen-containing spiroheterocyclyl, e.g., 2-oxa-7-azaspiro[3.5]nonanyl). In some embodiments, the compound of Formula (III-b) is compound 121.

[0132] In some embodiments, the compound is of formula (III-d). In some embodiments of formula (III-d), R 2a , R 2b , R 2c , and R 2d are independently hydrogen, m is 1, n is 2, q is 1, 2, 3, or 4, p is 0, and X is S(O). In some embodiments of Formula (III-d), R 2a and R 2b is independently hydrogen, m is 1, n is 2, q is 1, 2, 3, or 4, p is 0, and X is S(O). In some embodiments, the compound of Formula (III-d) is Compound 101, Compound 117, Compound 118, or Compound 119.

[0133] In some embodiments, the compound is a compound of Formula (Ib), (Id), or (Ie). In some embodiments, the compound is a compound of Formula (Ib), (Id), or (II). In some embodiments, the compound is a compound of Formula (Ib), (Id), or (If). In some embodiments, the compound is a compound of Formula (Ib), (Id), or (III).

[0134] In some embodiments, the compound of Formula (I) is not a compound disclosed in WO 2012 / 112982, WO 2012 / 167223, WO 2014 / 153126, WO 2016 / 019391, or WO 2017 / 075630. In certain embodiments, the compound of Formula (I) is a compound disclosed in any one of WO 2018 / 067615 and WO 2019 / 169333, each of which is incorporated herein by reference in its entirety.

[0135] In some embodiments, the anti-fibrotic compound (a compound of Formula (I)) comprises a compound shown in Table 1 or a pharmaceutically acceptable salt thereof.

[0136] [Table 1]

[0137] [Table 2]

[0138] [Table 3]

[0139] [Table 4]

[0140] In some embodiments, the small molecule compound attached to the modified polymer is a compound of Formula (I) (e.g., Formula (Ia), (Ib), (Ic), (Id), (Ie), (If), (II), (II-a), (III), (III-a), (III-b), (III-c), or (III-d)), or a pharmaceutically acceptable salt thereof; [ka] or a pharmaceutically acceptable salt thereof.

[0141] In some embodiments, the modified polymers described herein are [ka] or a pharmaceutically acceptable salt of either compound.

[0142] Each small molecule compound (e.g., an anti-fibrotic compound) can be attached to a polymer in the polymer composition via a linking group. The linking group can include an alkyl, carbonyl, amide, ester, thioester, disulfide bond, alkene, or other moiety. In some embodiments, the linking group is —C(O)(C1-C6-alkylene)—, where alkylene is R 1 is substituted with R 1 is as described herein. In some embodiments, the linking group is -C(O)(C1-C6-alkylene)-, where the alkylene is substituted with 1 to 2 alkyl groups (e.g., 1 to 2 methyl groups). In some embodiments, the linking group is -C(O)C(CH3)2-. In some embodiments, the linking group is -C(O)(methylene)-, where the alkylene is substituted with 1 to 2 alkyl groups (e.g., 1 to 2 methyl groups). In some embodiments, the linking group is -C(O)CH(CH3)-. In some embodiments, the linking group is -C(O)C(CH3)-.

[0143] In some embodiments, the polymer composition comprises a single polymer modified with a small molecule compound. In other embodiments, the polymer composition comprises multiple polymers modified with small molecule compounds, for example, at least two modified polymers, at least three modified polymers, or at least four modified polymers. The polymer composition may comprise multiple polymers modified with the same small molecule compound or multiple polymers modified with different small molecule compounds. The polymer composition may comprise a mixture of modified and unmodified polymers.

[0144] In one embodiment, the polymer composition comprises a first modified polymer, e.g., a first modified polymer modified with a compound of formula (I) (e.g., a compound described in Table 1) or a pharmaceutically acceptable salt thereof. In another embodiment, the polymer composition comprises a second modified polymer, e.g., a compound of formula (I) (e.g., a compound described in Table 1) or a pharmaceutically acceptable salt thereof.

[0145] Each of the modified and unmodified polymers in the polymer composition may be linear, branched, or crosslinked, and may be a polymer selected for its molecular weight range, degree of polymerization, viscosity, or melt flow rate. Exemplary polymers within the polymer composition include naturally occurring polymers and non-naturally occurring polymers (e.g., synthetic polymers). For example, the polymer composition may include polystyrene, polyethylene, polypropylene, polyacetylene, poly(vinyl chloride) (PVC), polyvinyl alcohol (PVA), polyolefin copolymers, poly(urethane), polyacrylates and polymethacrylates, polyacrylamides and polymethacrylamides, poly(methyl methacrylate), poly(2-hydroxyethyl methacrylate), polyesters, polysiloxanes, polydimethylsiloxanes (PDMS), polyethers, poly(orthoesters), poly(carbonates), poly(hydroxyalkanoates), polyfluorocarbons, PEEK®, Teflon® (polytetrafluoroethylene, PTFE), PEEK, silicones, epoxy resins, Kevlar®, Dacron® ) (condensation polymers derived from ethylene glycol and terephthalic acid), polyethylene glycol, nylon, polyalkenes, phenolic resins, natural and synthetic elastomers, adhesives and sealants, biopolymers such as polyolefins, polysulfones, polyacrylonitriles, polysaccharides and natural latex, collagen, cellulose polymers (e.g., alkylcelluloses), polyethylene glycol and 2-hydroxyethyl methacrylate (HEMA), polysaccharides, poly(glycolic acid), poly(L-lactic acid) (PLLA), poly(lactic-glycolic acid) (PLGA), polydioxanone (PDA), or racemic poly(lactic acid), polycarbonates, (e.g., polyamides (e.g., nylon)), fluoropolymers, carbon fibers, agarose, alginates, chitosan, and blends or copolymers thereof. Branched polymers can include one or more of the following types: star polymers, comb polymers, brush polymers, dendronized polymers, ladders, and dendrimers.The polymer can be a thermoresponsive polymer, for example, a gel (e.g., that becomes solid or liquid upon exposure to heat or a certain temperature) or a photocrosslinkable polymer. The amount of modified polymer in the polymer composition (e.g., weight % of the polymer composition, actual weight of the polymer) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more (e.g., w / w); or can be less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less.

[0146] In some embodiments, the polymer composition may comprise a polymer selected from alginate, chitosan, hyaluronate, polyethylene glycol (PEG), polyacrylamide, gelatin, poly(L-lactic acid) (PLLA), poly(lactic-co-glycolic acid) (PLGA), carboxymethylcellulose, and carboxymethylchitosan. In some embodiments, the polymer composition does not comprise a polyamide polymer. In addition to the polymers described herein, the polymer composition may comprise one or more unmodified naturally occurring or synthetic polymers from any of the above-listed polymer classes to provide structural integrity to the semipermeable device and / or to provide a scaffold to support the encapsulated cells.

[0147] In some embodiments, the modified polymer is alginate. Alginate is a polysaccharide composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G). In some embodiments, the alginate is a high guluronic acid (G) alginate, comprising greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more guluronic acid (G). In some embodiments, the G:M ratio is at least 1.3, 1.5, or greater than 1.5. In some embodiments, the alginate is a high mannuronic acid (M) alginate, comprising greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more mannuronic acid (M). In some embodiments, the M:G ratio is about 1. In some embodiments, the M:G ratio is less than 1. In certain embodiments, the polymer composition comprises a modified alginate and an unmodified alginate.

[0148] The polymers can be modified with small molecule compounds using any of a variety of methods known in the art, for example, Bioconjugate Techniques (3 rd ed., Greg T. Hermanson, Waltham, MA: Elsevier, Inc., 2013. For example, if the polymer to be modified is alginate and the small molecule compound contains a terminal amine, the small molecule can be covalently attached to the carboxylate group of the alginate monomer using an approach similar to that described in Example 1 herein.

[0149] In some embodiments, the polymer composition further comprises a polymer modified with a polypeptide, such as a cell-binding peptide (CBP). Exemplary CBPs include sequences such as RGD, RGDSP, GRGD, GRGDSP, DGEA, PHSRN, YIGSR, or CAM ligand proteins. In certain embodiments, the modified polymer comprises a mixture of two, three, or more CBPs (e.g., RGD+DGEA, RGD+PHSRN, RGD+DGEA+PHSRN).

[0150] The modified polymers described herein can be used to prepare semipermeable devices that encapsulate cells. In some embodiments, the semipermeable devices can include a single type of modified polymer or multiple types of modified polymers. These semipermeable devices include at least one cell-containing compartment containing multiple cells (e.g., living cells). In some embodiments, the devices include two, three, four, or more cell-containing compartments. The cells can be a variety of different cell types (e.g., human cells), such as epithelial cells, endothelial cells, fibroblasts, mesenchymal stem cells, keratinocytes, and pancreatic islet cells. Exemplary cell types include those listed in International Publication No. WO 2017 / 075631, which is incorporated herein by reference in its entirety. In some embodiments, the cells are recombinant cells, e.g., recombinant cells engineered to express a therapeutic agent (e.g., a protein, e.g., an antibody, enzyme, blood clotting factor, hormone, or growth factor).

[0151] In certain embodiments, the cells contained in the disclosed devices include RPE cells or MSC cells (e.g., recombinant RPE cells or recombinant MSC cells). In some embodiments, the semi-permeable device does not include pancreatic islet cells. In certain embodiments, the cells contained in the disclosed devices have one or more of the following characteristics: (i) are incapable of producing insulin (e.g., insulin A chain, insulin B chain, or proinsulin) in amounts effective to treat diabetes or another disease or condition that can be treated with insulin; (ii) are incapable of producing insulin in response to glucose; or (iii) are not derived from induced pluripotent stem cells that have been recombined or differentiated into insulin-producing pancreatic beta cells.

[0152] In embodiments, the plurality of cells is in the form of a cell suspension before being encapsulated in a semipermeable device described herein, e.g., a hydrogel capsule. The cells in the suspension may be in the form of single cells (e.g., from a monolayer cell culture) or may be provided in another form, for example, disposed on a microcarrier (e.g., a bead or matrix) or as a three-dimensional aggregate of cells (e.g., a cell cluster or spheroid). The cell suspension may include a plurality of cell clusters (e.g., as a spheroid) or microcarriers.

[0153] In some embodiments, the plurality of cells is engineered to produce a therapeutic agent. In certain embodiments, the therapeutic agent is for the prevention or treatment of a disease, disorder, or condition (e.g., those described in WO 2017 / 075631). The therapeutic agent can be any biological substance, such as a nucleic acid (e.g., a nucleotide, DNA, or RNA), a polypeptide, a lipid, a sugar (e.g., a monosaccharide, disaccharide, oligosaccharide, or polysaccharide), or a small molecule. Exemplary therapeutic agents include those listed in WO 2017 / 075631.

[0154] In some embodiments, the therapeutic agent is a protein, e.g., a hormone, enzyme, cytokine (e.g., a pro-inflammatory cytokine or an anti-inflammatory cytokine), growth factor, clotting factor, or lipoprotein. A peptide or polypeptide (e.g., a protein, e.g., a hormone, growth factor, clotting factor, or coagulation factor, antibody molecule, enzyme, cytokine, cytokine receptor, or chimeric protein comprising a cytokine or cytokine receptor) produced by a recombinant cell can have a native amino acid sequence or can include a variant of a native sequence. A variant can be a naturally occurring or non-naturally occurring amino acid substitution, mutation, deletion, or addition to a reference sequence, e.g., a native sequence. A native amino acid sequence can be a polymorphic variant. A native amino acid sequence can be a human or non-human amino acid sequence. In some embodiments, a native amino acid sequence or a naturally occurring variant thereof is a human sequence. Additionally, proteins for use with the present disclosure can be modified in some way, for example, by chemical or enzymatic modification (e.g., glycosylation, phosphorylation). In some embodiments, the protein has an average molecular weight of 5 kD, 10 kD, 25 kD, 50 kD, 100 kD, 150 kD, 200 kD, 250 kD, 500 kD, or more.

[0155] In some embodiments, the protein is a clotting factor or coagulation factor, e.g., a blood clotting factor or blood coagulation factor, hi some embodiments, the protein is a protein involved in coagulation, the process by which blood is converted from a liquid to a solid or gel. Exemplary clotting and coagulation factors include factor I (e.g., fibrinogen), factor II (e.g., prothrombin), factor III (e.g., tissue factor), factor V (e.g., proaccelerin, labile factor), factor VI, factor VII (e.g., stable factor, proconvertin), factor VIII (e.g., antihemophilic factor A), factor VIIIC, factor IX (e.g., antihemophilic factor B), factor X (e.g., Stuart-Prower factor), factor XI (e.g., plasma thromboplastin precursor), factor XII (e.g., Hageman factor), factor XIII (e.g., fibrin-stabilizing factor), von Willebrand factor (vWF), prekallikrein, heparin cofactor II, high molecular weight kininogen (e.g., Fitzgerald factor), antithrombin III, and fibronectin. In some embodiments, the protein is an anticoagulant such as protein C.

[0156] In some embodiments, the protein is a replacement therapy or replacement protein, hi some embodiments, the replacement therapy or replacement protein is a clotting factor or coagulation factor, such as factor VIII (e.g., comprising the naturally occurring human factor VIII amino acid sequence or a variant thereof) or factor IX (e.g., comprising the naturally occurring human factor IX amino acid sequence or a variant thereof).

[0157] In some embodiments, the encapsulated cells described herein are engineered to express a human factor VIII protein (e.g., recombinant factor VIII). In some embodiments, the recombinant factor VIII is B-domain deleted recombinant factor VIII (FVIII-BDD). In some embodiments, the cells are engineered to express factor IX (e.g., human factor IX (FIX) protein).

[0158] In some embodiments, the encapsulated cells are derived from a human RPE cell line and comprise an exogenous nucleic acid sequence comprising a promoter sequence operably linked to a coding sequence for a polypeptide, in embodiments, the coding sequence is a codon-optimized FVIII-BDD coding sequence or a codon-optimized FIX-padua coding sequence.

[0159] In certain embodiments, the concentration of a small molecule compound (e.g., a compound of Formula (I)) in a modified polymer (e.g., an antifibrotic alginate) is defined as w / w%, e.g., the % weight of the small molecule in a solution (e.g., saline) divided by the weight of the modified polymer, as determined, for example, by an assay described herein. In certain embodiments, the concentration of a small molecule compound (e.g., a compound of Formula (I), e.g., Compound 101) is about 1.0 w / w% to about 3.0 w / w%, about 1.3 w / w% to about 2.5 w / w%, or about 1.5 w / w% to about 2.2 w / w%.

[0160] In some embodiments, the concentration of the small molecule compound (e.g., an antifibrotic compound, e.g., a compound of Formula (I)) present in the modified polymer correlates with a particular parameter (e.g., retention time on a chromatogram or area under the curve on a chromatogram). In certain embodiments, the small molecule compound may be conjugated to the modified polymer or may be present free in the sample, e.g., after hydrolysis. In certain embodiments, the concentration of the small molecule compound (e.g., a small molecule compound conjugated to a polymer (e.g., alginate)) is 0.1% to 10% (w / w). For example, the concentration of the small molecule compound (e.g., a small molecule compound conjugated to a polymer (e.g., alginate)) is 1% to 10%, 2% to 8%, 3% to 6%, 2% to 4%, 4% to 6%, or 6% to 8% (w / w). In certain embodiments, the concentration of the small molecule compound (e.g., a small molecule compound conjugated to a polymer (e.g., alginate)) is 4% to 6% (w / w). In certain embodiments, the concentration of the small molecule compound (eg, a small molecule compound conjugated to a polymer, such as alginate) is 2% to 4% (w / w).

[0161] In some embodiments, the semi-permeable device is not any capsule, device, implant, or other object disclosed in any of WO 2012 / 112982, WO 2012 / 167223, WO 2014 / 153126, WO 2016 / 019391, WO 2016 / 187225, U.S. Patent Application Publication Nos. 2012-0213708, 2016-0030359, and 2016-0030360.

[0162] Methods for evaluating modified polymers The present disclosure features methods for evaluating polymers modified with a small molecule compound (e.g., an antifibrotic compound) within a polymer composition. The evaluation can include determining the concentration of the small molecule compound (e.g., an antifibrotic compound) in the modified polymer, identifying the small molecule compound in the modified polymer, or querying the polymer composition for a particular impurity. The methods described herein include exposing a sample of the modified polymer to reaction conditions that allow for the release of the small molecule compound from the modified polymer. Upon release of the small molecule compound (e.g., an antifibrotic compound) from the modified polymer, the concentration of the small molecule compound (e.g., an antifibrotic compound) bound to the modified polymer can be determined, for example, by chromatographic analysis.

[0163] Any reaction conditions can be used to liberate a small molecule compound (e.g., an antifibrotic compound) from the modified polymer. For example, the polymer composition can be subjected to an acidic solution, a basic solution, an enzyme solution, heating, light, microwave irradiation, hydrogenation, or a combination thereof. In some embodiments, to release a small molecule compound (e.g., an antifibrotic compound) from the modified polymer, the polymer composition is exposed to an acidic solution (e.g., a solution containing HCl, HBr, HF, H2SO4, HNO3, HClO4, CF3COOH, CH3COOH, or CF3SO3H). In some embodiments, the acidic solution has a pH of less than 7, e.g., less than 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, or less. The polymer composition can be heated, for example, at a temperature of about 25°C, 40°C, 60°C, 80°C, 100°C, greater than 120°C, or higher. In some embodiments, the polymer composition can be subjected to, for example, simultaneously, an acidic solution and heat to release the small molecule compound (e.g., an anti-fibrotic compound) from the modified polymer. In some embodiments, the polymer composition is stirred while being heated, for example, using a stir plate.

[0164] In some embodiments, the polymer composition is exposed to microwave irradiation for, for example, at least 1 second, 2 seconds, 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, or 55 seconds, 1 minute, 2.5 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 1 hour. In some embodiments, the polymer composition is subjected to microwave irradiation for 1 minute to 1 hour, for example, 1 minute to 45 minutes, 1 minute to 30 minutes, 1 minute to 15 minutes, 5 minutes to 1 hour, 5 minutes to 45 minutes, 5 minutes to 30 minutes, 5 minutes to 15 minutes, 10 minutes to 1 hour, 10 minutes to 45 minutes, 10 minutes to 30 minutes, 15 minutes to 45 minutes, and 15 minutes to 30 minutes. In some embodiments, the polymer composition is exposed to microwave irradiation for 5 to 45 minutes. In some embodiments, the polymer composition is stirred while microwave irradiation is applied, for example, in a microwave reactor (e.g., an Anton-Paar Monowave microwave reactor), at a speed of, for example, 100 revolutions per minute (rpm), 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, or more. In some embodiments, the polymer composition is exposed to the acidic solution and microwave irradiation, for example, simultaneously.

[0165] The methods described herein further include obtaining a concentration value of a small molecule compound (e.g., an antifibrotic compound) bound to the modified polymer in the polymer composition. To this end, in some embodiments, the hydrolyzed polymer composition can be subjected to a separation process. Any known separation process (e.g., filtration, electrophoresis, or chromatography) can be used to separate the unconjugated small molecule compound (i.e., the "free" small molecule compound) from the polymer. Exemplary chromatographic methods include size exclusion chromatography, ion exchange chromatography, gel filtration chromatography, reversed-phase chromatography, hydrophobic interaction chromatography, or thin-layer chromatography. In some embodiments, filtration is used to separate the unconjugated small molecule compound from the polymer in the polymer composition. After filtration, in some embodiments, the concentration of the free small molecule compound is subjected to reversed-phase chromatography to determine the concentration of the small molecule compound bound to the modified polymer.

[0166] To determine the concentration of a small molecule compound in a polymer composition, the methods described herein can further include comparing the concentration of the small molecule compound in the sample to a known standard. Exemplary procedures are described herein, for example, in Examples 2-5 below.

[0167] The methods detailed herein may also be used to assess the impurity profile of a polymer composition. In certain embodiments, the method further comprises obtaining the concentration of free small molecule compounds (i.e., unconjugated small molecule compounds, e.g., compounds not previously attached to a modified polymer) in the polymer composition.

[0168] The present disclosure features methods for evaluating modified polymers that include small molecule compounds within the polymer composition, without subjecting the polymer composition to degradative conditions (e.g., hydrolysis conditions). For example, evaluating can include performing a non-degradative analytical method (e.g., refractive index measurement or spectroscopic measurement) on a sample of the polymer composition. In some embodiments, the non-degradative method of evaluation can include determining the total concentration of the modified polymer in the sample, determining the concentration of the small molecule compound bound to the modified polymer in the sample, identifying a physical parameter (e.g., chemical formula, molecular weight, or atomic composition) of the small molecule compound bound to the modified polymer in the sample, or probing the polymer composition for a particular impurity. In some embodiments, evaluating the polymer composition (e.g., with a non-degradative method) can include, for example, obtaining a refractive index value for the polymer composition in a solid or liquid sample.

[0169] For example, to use refractive index to evaluate a polymer composition described herein, the polymer composition can be dissolved in an aqueous medium (e.g., saline) or other buffer solution (e.g., HEPES, sodium phosphate, Tris, or PBS). The refractive index of the polymer composition can be compared to a control sample that contains only an aqueous medium, for example, without the polymer composition. In some embodiments, all samples are analyzed at the same wavelength of light and temperature.

[0170] In some embodiments, the relative refractive index increment (i.e., "dn / dc value") of each sample is determined. The relative refractive index increment relates to the amount by which the refractive index of a polymer composition changes with respect to another parameter, such as the amount of small molecule compound (e.g., conjugated or unconjugated small molecule compound) in the composition. In some embodiments, the relative refractive index increment is expressed in any dimension (e.g., g / mol or (w / w%)). For example, in the case of a polymer composition comprising alginate modified with a small molecule compound (e.g., a compound of Formula (I), e.g., Compound 101), the relative refractive index increment can be determined for each of the small molecule compound and a representative component of alginate (e.g., guluronic acid moiety). An exemplary method is provided in Example 5 herein.

[0171] The refractive index (nd) of the polymer composition can be, for example, 1.3300 to 1.3400 at a particular temperature or temperature range (e.g., about 2 to 8°C or about 22 to 28°C). For example, the refractive index of the polymer compositions described herein can be 1.3310 to 1.3400, such as 1.3320 to 1.3400, 1.3330 to 1.3400, 1.3340 to 1.3400, 1.3360 to 1.3400, 1.3370 to 1.3400, and 1.3380 to 1.3400. In some embodiments, the refractive index of the polymer composition is 1.3350 to 1.3400.

[0172] Enumeration of Embodiments 1. A method for evaluating a polymer composition comprising a polymer, the polymer having formula (Ib): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, Ring M 1 is aryl or heteroaryl, and each aryl and heteroaryl is selected from one or more R 3 optionally substituted with; Ring Z 1is a cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each cycloalkyl, heterocyclyl, aryl, or heteroaryl is selected from 1 to 5 R 5 optionally substituted with; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b , or R 2c and R 2d taken together form an oxo group; X is absent, O, or S; Each R 3 and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 wherein each alkyl and heteroalkyl is optionally substituted with one or more halogen, oxo, cyano, cycloalkyl, or heterocyclyl; or two R 5 Together, we form Ring Z 1 forming a 5-6 membered ring fused to; R c is hydrogen or alkyl; Each R A1 and R B1 are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; [ka] refers to a linking group or bond to the polymer, or a pharmaceutically acceptable salt thereof, The method comprises: (a) exposing the polymer composition to reaction conditions that allow for release of the compound of formula (Ib) from the modified polymer; and (b) obtaining a value for the concentration of the compound of formula (Ib) bound to the modified polymer; Including, thereby evaluating the polymer composition or semi-permeable device. method.

[0173] 2. A method for evaluating a polymer composition comprising a polymer, the polymer having formula (III-a): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, Ring Z 2 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 1 to 5 R 5 optionally substituted with; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, heteroalkyl, halo; or R 2a and R 2b , or R 2c and R 2d together form an oxo group; R 3 and R 5 each independently represents alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 and; Each R A1 and R B1 are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; o and p are each independently 0, 1, 2, 3, 4, or 5; q is an integer from 0 to 25; [ka] refers to a linking group or bond to the polymer, or a pharmaceutically acceptable salt thereof, The method comprises: (a) exposing the polymer composition to reaction conditions that allow for release of the compound of formula (III-a) from the modified polymer; and (b) obtaining the concentration value of the compound of formula (III-a); Including, thereby evaluating the polymer composition or semi-permeable device. method.

[0174] 3. The method of embodiment 1 or 2, wherein the polymer in the modified polymer is a polysaccharide.

[0175] 4. The method of embodiment 3, wherein the polysaccharide is alginate.

[0176] 5. The method of embodiment 4, wherein the alginate has an average molecular weight of 75 kDa to 150 kDa.

[0177] 6. The method of embodiment 4 or 5, wherein the alginate has a guluronic acid to mannuronate (G:M) ratio of 1.5 or greater.

[0178] 7. The method of any one of embodiments 1 to 6, wherein the compound of formula (Ib) or formula (III-a) is covalently attached to the polymer (e.g., alginate) in the modified polymer.

[0179] 8. The method of any one of embodiments 1 to 6, wherein the compound of formula (Ib) or formula (III-a) is non-covalently bound to the polymer (e.g., alginate) in the modified polymer.

[0180] 9. The compound of formula (Ib) or formula (III-a) may have a linking group (e.g., —C(O)(C1-C6-alkylene)-, where alkylene is R 1 is substituted with R 1 7. The method of any one of embodiments 1 to 6, wherein the modified polymer (e.g., modified alginate) is covalently attached via a modified polymer (e.g., modified alginate) via a carboxylate bond (wherein carboxylate is as defined herein).

[0181] 10. The method of any one of embodiments 1 to 9, wherein the polymer composition comprises a single type of modified polymer (e.g., a modified alginate).

[0182] 11. The method of any one of embodiments 1-9, wherein the polymer composition comprises a plurality of modified polymers (eg, at least two modified polymers, at least three polymers).

[0183] 12. The method of any one of embodiments 1-11, wherein said compound of formula (Ib) or formula (III-a) is a compound listed in Table 1.

[0184] 13. The method of any one of embodiments 1 to 12, wherein the compound of formula (Ib) or formula (III-a) is selected from compound 100, compound 101, compound 110, compound 113, and compound 114 listed in Table 1.

[0185] 14. The method of any one of embodiments 1-13, wherein said compound of formula (Ib) or formula (III-a) is compound 100 listed in Table 1.

[0186] 15. The method of any one of embodiments 1-13, wherein said compound of formula (Ib) or formula (III-a) is compound 101 listed in Table 1.

[0187] 16. The method of any one of embodiments 1-13, wherein said compound of formula (Ib) or formula (III-a) is compound 110 listed in Table 1.

[0188] 17. The method of any one of embodiments 1-13, wherein said compound of formula (Ib) or formula (III-a) is compound 113 listed in Table 1.

[0189] 18. The method of any one of embodiments 1-13, wherein said compound of formula (Ib) or formula (III-a) is compound 114 listed in Table 1.

[0190] 19. The method of any one of the preceding embodiments, wherein the reaction conditions in step (a) comprise contacting the polymer composition with an acidic solution (e.g., an HCl solution, e.g., a 1N to 8N HCl solution).

[0191] 20. The method of any one of the preceding embodiments, wherein the reaction conditions in step (a) include heating the polymer composition (e.g., at a temperature greater than about 25°C, 40°C, 60°C, 80°C, 100°C, 120°C, or higher).

[0192] 21. The method of any one of the preceding embodiments, wherein the reaction conditions in step (a) comprise exposing the polymer composition to microwave radiation.

[0193] 22. The method according to any one of embodiments 1 to 21, further comprising a separation step.

[0194] 23. The method of embodiment 22, wherein the separating step is performed between step (a) and step (b).

[0195] 24. The method of embodiment 22 or 23, wherein the separation step comprises chromatography (e.g., size exclusion chromatography, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, or hydrophobic interaction chromatography).

[0196] 25. The method of any one of embodiments 1 to 24, wherein obtaining the concentration value in step (b) comprises determining the area of ​​a chromatogram peak of the compound of formula (Ib) or formula (III-a).

[0197] 26. The method of embodiment 25, wherein obtaining the concentration value in step (b) further comprises comparing the area of ​​the chromatogram peak with a standard (e.g., a compound of formula (Ib) or formula (III-a)).

[0198] 27. The method of any one of embodiments 1-26, further comprising obtaining a value for the concentration of the unconjugated compound of Formula (Ib) (i.e., the "free" compound of Formula (Ib)) or the unconjugated compound of Formula (III-a) (i.e., the "free" compound of Formula (III-a)) in the polymer composition.

[0199] 28. Obtaining a value for the concentration of the unconjugated compound of formula (Ib) (i.e., the "free" compound of formula (Ib)) or the unconjugated compound of formula (III-a) (i.e., the "free" compound of formula (III-a)) in the polymer composition, (a') separating the polymer composition into a polymer-bound fraction and a non-polymer-bound fraction. 28. The method of embodiment 27, comprising:

[0200] 29. The method of embodiment 27 or 28, further comprising (b') retaining said non-polymer-bound fraction.

[0201] 30. (c') obtaining a concentration value of the unconjugated compound of Formula (Ib) (i.e., the "free" compound of Formula (Ib)) or the unconjugated compound of Formula (III-a) (i.e., the "free" compound of Formula (III-a)) in the non-polymer-bound fraction. 30. The method of any one of embodiments 27 to 29, further comprising:

[0202] 31. The method of any one of embodiments 27-30, wherein said separating in step (a') comprises filtration, for example through a molecular weight cut-off filter.

[0203] 32. The method of any one of embodiments 27 to 31, wherein step (c') comprises a separation step.

[0204] 33. The method of embodiment 32, wherein the separating step comprises chromatography (e.g., size exclusion chromatography, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, or hydrophobic interaction chromatography).

[0205] 34. The method of any one of embodiments 27 to 33, wherein obtaining the concentration value in step (c') further comprises determining the area of ​​a chromatogram peak of a sample of the unconjugated compound of Formula (Ib) (i.e., the "free" compound of Formula (Ib)) or the unconjugated compound of Formula (III-a) (i.e., the "free" compound of Formula (III-a)) in the non-polymer-bound fraction.

[0206] 35. The method of embodiment 34, wherein obtaining the concentration value in step (c') further comprises comparing the area of ​​the chromatogram peak with a standard (e.g., a compound of formula (Ib) or formula (III-a)).

[0207] 36. The method of any one of embodiments 1 to 35, wherein evaluating the polymer composition comprises determining the concentration of the compound of formula (Ib) or formula (III-a) bound to the modified polymer in the polymer composition.

[0208] 37. Determining the concentration of the compound of formula (Ib) or formula (III-a) bound to the modified polymer in the polymer composition comprises: (a'') obtaining a value of the total concentration of the compound of formula (Ib) or formula (III-a) in the polymer composition; (b'') obtaining a value for the concentration of the unconjugated compound of Formula (Ib) (i.e., the "free" compound of Formula (Ib)) or the unconjugated compound of Formula (III-a) (i.e., the "free" compound of Formula (III-a)) in the polymer composition; and / or (c'') subtracting the concentration of the free compound of Formula (Ib) or the free compound of Formula (III-a) (e.g., as determined in step (b'')) from the total concentration of the compound of Formula (Ib) or Formula (III-a) in the polymer composition. Including, thereby determining the concentration of the compound of formula (Ib) or formula (III-a) conjugated (e.g., covalently attached) to the modified polymer in the polymer composition; 37. The method of embodiment 36.

[0209] 38. The method of embodiment 37, comprising (a'').

[0210] 39. The method of embodiment 37 or 38, comprising (b'').

[0211] 40. The method of any one of embodiments 37-39, comprising (c'').

[0212] 41. A method for determining the concentration of a polymer composition comprising an alginate, wherein the alginate has the formula (Ib): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, Ring M 1 is aryl or heteroaryl, and each aryl and heteroaryl is selected from one or more R 3 optionally substituted with; Ring Z 1is a cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each cycloalkyl, heterocyclyl, aryl, or heteroaryl is selected from 1 to 5 R 5 optionally substituted with; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b , or R 2c and R 2d taken together form an oxo group; X is absent, O, or S; Each R 3 and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 wherein each alkyl and heteroalkyl is optionally substituted with one or more halogen, oxo, cyano, cycloalkyl, or heterocyclyl; or two R 5 Together, we form Ring Z 1 forming a 5-6 membered ring fused to; R c is hydrogen or alkyl; Each R A1 and R B1 are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; [ka] refers to a linking group or bond to the polymer, or a pharmaceutically acceptable salt thereof, or Formula (III-a): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, Ring Z 2 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 1 to 5 R 5 optionally substituted with; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, heteroalkyl, halo; or R 2a and R 2b , or R 2c and R 2d together form an oxo group; R 3 and R 5 each independently represents alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 and; Each R A1 and R B1 are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; o and p are each independently 0, 1, 2, 3, 4, or 5; q is an integer from 0 to 25; [ka] refers to a linking group or bond to the polymer, or a pharmaceutically acceptable salt thereof, The method comprises: (a) obtaining a value for the total concentration of the compound of formula (Ib) or formula (III-a) in the polymer composition; A method comprising:

[0213] 42. (b) obtaining a value for the concentration of the unconjugated compound of formula (Ib) (i.e., the "free" compound of formula (Ib)) or the unconjugated compound of formula (III-a) (i.e., the "free" compound of formula (III-a)) in the polymer composition; 42. The method of embodiment 41, further comprising:

[0214] 43. (c) subtracting the concentration of the free compound of formula (Ib) or the free compound of formula (III-a) (e.g., as determined in step (b)) from the total concentration of the compound of formula (Ib) or formula (III-a) in the polymer composition. 43. The method of embodiment 41 or 42, further comprising:

[0215] 44. The method of any one of embodiments 41-43, wherein step (a) is performed before step (b).

[0216] 45. The method of any one of embodiments 41-43, wherein step (b) is performed before step (a).

[0217] 46. ​​Step (a) is (a) exposing the polymer composition to reaction conditions that allow for release of the compound of Formula (Ib) or Formula (III-a) from the modified polymer; and (b) obtaining a concentration value of the compound of formula (Ib) or the compound of formula (III-a); 46. ​​The method of any one of embodiments 41 to 45, comprising:

[0218] 47. The method of any one of embodiments 41-46, wherein the compound is a compound of formula (Ib).

[0219] 48. The method of embodiment 47, wherein the compound is Compound 100, Compound 110, Compound 113, or Compound 114, or a pharmaceutically acceptable salt thereof.

[0220] 49. The method of any one of embodiments 41-46, wherein the compound is a compound of formula (III-a).

[0221] 50. The method of embodiment 49, wherein the compound is compound 101 or a pharmaceutically acceptable salt thereof.

[0222] 51. The method of any one of embodiments 1 to 50, wherein the concentration of the compound of formula (Ib) or the compound of formula (III-a) bound to the polymer in the modified polymer (e.g., modified alginate) is about 0.5% to about 10% (w / w) of the modified polymer (e.g., modified alginate), for example, about 0.5% to 5%, 1% to 5%, 1% to 4%, or 1 to 3% (w / w) of the modified polymer (e.g., modified alginate).

[0223] 52. The method of any one of embodiments 1 to 51, wherein the concentration of the free compound of formula (Ib) or the free compound of formula (III-a) in the polymer composition is less than about 1% (w / w) of the modified polymer (e.g., modified alginate), for example, less than about 0.9%, 0.8%, 0.7%, 0.6%, or 0.5% (w / w) of the modified polymer (e.g., modified alginate).

[0224] 53. A method for evaluating a polymer composition comprising a polymer, the polymer having formula (Ib): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, Ring M 1 is aryl or heteroaryl, and each aryl and heteroaryl is selected from one or more R 3 optionally substituted with; Ring Z 1 is a cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each cycloalkyl, heterocyclyl, aryl, or heteroaryl is selected from 1 to 5 R 5optionally substituted with; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b , or R 2c and R 2d taken together form an oxo group; X is absent, O, or S; Each R 3 and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 wherein each alkyl and heteroalkyl is optionally substituted with one or more halogen, oxo, cyano, cycloalkyl, or heterocyclyl; or two R 5 Together, we form Ring Z 1 forming a 5-6 membered ring fused to; R c is hydrogen or alkyl; Each R A1 and R B1 are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; [ka] refers to a linking group or bond to the polymer, or a pharmaceutically acceptable salt thereof, or Formula (III-a): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, Ring Z 2 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 1 to 5 R 5 optionally substituted with; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, heteroalkyl, halo; or R 2a and R 2b , or R 2c and R 2d together form an oxo group; R 3 and R 5 each independently represents alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 and; Each R A1 and R B1 are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; o and p are each independently 0, 1, 2, 3, 4, or 5; q is an integer from 0 to 25; [ka] refers to a linking group or bond to the polymer, or a pharmaceutically acceptable salt thereof, The method comprises: (a) obtaining a refractive index value of said polymer composition; A method comprising:

[0225] 54. (b) Obtaining the value of the total unconjugated compounds of formula (Ib) (i.e., "free" compounds of formula (Ib)) or the total unconjugated compounds of formula (III-a) (i.e., "free" compounds of formula (III-a)) in the polymer composition. 54. The method of embodiment 53, further comprising:

[0226] 55. (c) Obtaining the concentration value of the polymer modified with the compound of formula (Ib) or formula (III-a). 55. The method of embodiment 53 or 54, further comprising:

[0227] 56. The method of embodiment 55, wherein said obtaining includes using the values ​​obtained in each of steps (a) and (b).

[0228] 57. The method of any one of embodiments 53-56, wherein the polymer is alginate.

[0229] 58. The method of embodiment 57, wherein the alginate has an average molecular weight of 75 kDa to 150 kDa.

[0230] 59. The method of embodiment 57 or 58, wherein the alginate has a guluronic acid to mannuronate (G:M) ratio of 1.5 or greater.

[0231] 60. The method of any one of embodiments 53-59, wherein the compound of formula (Ib) or formula (III-a) is covalently attached to the polymer (e.g., alginate) in the modified polymer.

[0232] 61. The method of any one of embodiments 53-59, wherein the compound of formula (Ib) or formula (III-a) is non-covalently bound to the polymer (e.g., alginate) in the modified polymer.

[0233] 62. The compound of formula (Ib) or formula (III-a) may have a linking group (e.g., —C(O)(C1-C6-alkylene)-, where alkylene is R 1 is substituted with R 1 60. The method of any one of embodiments 53-59, wherein the modified polymer (e.g., modified alginate) is covalently attached via a carboxyl group (C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C44, C45, C46, ​​C47, C48, C49, C51, C52, C53, C54, C65, C76, C77, C78, ​​C79, C80, C81, C82, C83, C84, C85, C96

[0234] 63. The method of any one of embodiments 53-62, wherein the polymer composition comprises a single type of modified polymer (e.g., modified alginate).

[0235] 64. The method of any one of embodiments 53-62, wherein the polymer composition comprises a plurality of modified polymers (eg, at least two modified polymers, at least three polymers).

[0236] 65. The method of any one of embodiments 53-64, wherein said compound of formula (Ib) or formula (III-a) is a compound listed in Table 1.

[0237] 66. The method of any one of embodiments 53-65, wherein the compound of formula (Ib) or formula (III-a) is selected from compound 100, compound 101, compound 110, compound 113, and compound 114 listed in Table 1.

[0238] 67. The method of any one of embodiments 53-66, wherein said compound of formula (Ib) or formula (III-a) is compound 100 listed in Table 1.

[0239] 68. The method of any one of embodiments 53-66, wherein said compound of formula (Ib) or formula (III-a) is compound 101 listed in Table 1.

[0240] 69. The method of any one of embodiments 53-66, wherein said compound of formula (Ib) or formula (III-a) is compound 110 listed in Table 1.

[0241] 70. The method of any one of embodiments 53-66, wherein said compound of formula (Ib) or formula (III-a) is compound 113 listed in Table 1.

[0242] 71. The method of any one of embodiments 53-66, wherein said compound of formula (Ib) or formula (III-a) is compound 114 listed in Table 1.

[0243] 72. Obtaining the value of all compounds of formula (Ib) or formula (III-a) conjugated (e.g., covalently attached) to the modified polymer composition of step (b) includes: (b') the polymer composition, (i) releasing the compound of formula (Ib) or formula (III-a) from the modified polymer; subjecting the mixture to reaction conditions that allow for and (b'') obtaining the concentration value of the compound of formula (Ib) or the compound of formula (III-a); 72. The method of any one of embodiments 53 to 71, comprising:

[0244] 73. The method of any one of embodiments 53-71, further comprising obtaining dn / dc values ​​for components of the polymer composition (eg, components of the modified polymer).

[0245] 74. The method of embodiment 73, further comprising using the dn / dc value in step (c) of the method to obtain a value for the concentration of the polymer modified with, for example, the compound of formula (Ib) or formula (III-a).

[0246] 75. A method for determining the concentration of a compound of formula (Ib) or formula (III-a) bound to alginate in a polymer composition, comprising: (a) obtaining a refractive index value of said polymer composition; (b) obtaining the value of all compounds of Formula (Ib) or Formula (III-a) conjugated (e.g., covalently attached) to the modified polymer in the polymer composition; and / or (c) using the values ​​obtained in each of steps (a) and (b) to obtain a concentration value of the compound of formula (Ib) or formula (III-a) conjugated to the modified polymer; Including, Thereby, the concentration of the compound of formula (Ib) or formula (III-a) bound to the polymer in the polymer composition is determined. method.

[0247] 76. The method of embodiment 75, comprising (a).

[0248] 77. The method of embodiment 75 or 76, comprising (b).

[0249] 78. The method of any one of embodiments 75-77, including (c).

[0250] 79. The method of any one of embodiments 75-78, wherein the compound is a compound of formula (Ib).

[0251] 80. The method of embodiment 79, wherein the compound is Compound 100, Compound 110, Compound 113, or Compound 114, or a pharmaceutically acceptable salt thereof.

[0252] 81. The method of any one of embodiments 75-78, wherein the compound is a compound of formula (III-a).

[0253] 82. The method of embodiment 81, wherein the compound is compound 101 or a pharmaceutically acceptable salt thereof.

[0254] 83. A polymer composition comprising a polymer modified with a compound of formula (Ib) or a compound of formula (III-a), wherein the concentration of the compound of formula (Ib) or the compound of formula (III-a) is 0.5% to 5% (w / w) of modified alginate, for example, as determined by the method of any one of embodiments 1 to 82.

[0255] 84. A polymer composition comprising an alginate modified with a compound of formula (Ib) or a compound of formula (III-a), wherein the concentration of the compound of formula (Ib) or the compound of formula (III-a) is 0.5% to 5% (w / w) of the modified alginate, for example, as determined by the method described in any one of embodiments 1 to 82.

[0256] 85. A polymer composition comprising an alginate modified with a compound selected from Compound 100, Compound 101, Compound 110, Compound 112, Compound 113, and / or Compound 114 shown in Table 1, wherein the concentration of the compound is 0.5% to 5% (w / w) of the modified alginate, for example, as determined by the method described in any one of embodiments 1 to 82.

[0257] [Example] In order that the present disclosure may be more fully understood, the following examples are set forth. The examples described in this application are provided to illustrate the devices (e.g., capsules, particles, chemical modifications, compositions, and methods) provided herein and should not be construed as limiting the scope thereof in any way.

[0258] Example 1. Preparation of exemplary modified polymers. Using methods known in the art, the polymeric material can be chemically modified with a small molecule compound (e.g., an anti-fibrotic compound) or a pharmaceutically acceptable salt thereof prior to formation of the semipermeable device (e.g., a hydrogel capsule).

[0259] For example, in the case of alginate, the alginate carboxylic acid is activated for coupling to one or more amine-functionalized compounds to obtain an antifibrotic compound, e.g., alginate modified with a compound of Formula (I). The alginate polymer is dissolved in water (30 mL / gram polymer) and treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.5 equivalents) and N-methylmorpholine (1 equivalent). To this mixture is added a solution of the compound of interest (e.g., compound 101 shown in Table 4) in acetonitrile (0.3 M). The compound of interest can be prepared using methods known in the art (e.g., as described in WO 2018 / 067615, the entire contents of which are incorporated herein by reference).

[0260] The amount of compound and coupling reagent added depends on the desired concentration of compound bound to alginate. To prepare the CM-LMW-Alg-101-Medium polymer solution, dissolved unmodified low-molecular-weight alginate (approximate molecular weight <75 kDa, G:M ratio ≥1.5) was treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (5.1 mmol / g alginate), N-methylmorpholine (10.2 mmol / g alginate), and Compound 101 (5.4 mmol / g alginate). To prepare the CM-LMW-Alg-101-High polymer solution, dissolved unmodified low molecular weight alginate (approximate molecular weight <75 kDa, G:M ratio ≥1.5) was treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (5.1 mmol / g alginate) and N-methylmorpholine (10.2 mmol / g alginate) and Compound 101 (5.4 mmol / g alginate).

[0261] The reaction is warmed to 55°C for 16 hours, then cooled to room temperature and gently concentrated by rotary evaporation. The residue is then dissolved in water. The mixture is filtered through a bed of cyano-modified silica gel (Silicycle), and the filter cake is washed with water. The resulting solution is then extensively dialyzed (10,000 MWCO membrane). The alginate solution is concentrated by lyophilization to obtain the desired chemically modified alginate as a solid, or by any suitable technique to produce a chemically modified alginate solution with a viscosity of 25 cP to 35 cP.

[0262] In one embodiment, the concentration of alginate-bound small molecule compounds is determined using the protocols described in Examples 2-5 below, or as described in Example 27 below.

[0263] Example 2. Determination of total antifibrotic compounds in samples modified with antifibrotic compounds. To determine the total amount of antifibrotic compound (including both free and conjugated antifibrotic compounds) in a polymer composition, the polymer composition can be subjected to acid hydrolysis to cleave the conjugated antifibrotic compound from the modified polymer. The concentration of the resulting unconjugated antifibrotic compound can then be determined using, for example, high-pressure liquid chromatography (HPLC). Here, the total antifibrotic compound is quantified in a sample of the modified alginate, as described below.

[0264] Acid hydrolysis of modified polymers. 50 ± 5 mg of lyophilized solid antifibrotic compound-modified alginate or a saline solution (1000 ± 50 mg) of antifibrotic compound-modified alginate was weighed into a reaction vial, ensuring that the solid remained at the bottom of the vial. The vial was then charged with 10 mL of 2N HCl along with a stir bar. The sealed vial was heated to 120 °C, stirred at 400 rpm for 120 min, and then cooled to ambient temperature. The entire solution was then transferred to a 25 mL volumetric flask, and the empty reaction vial was thoroughly rinsed three times with 5 mL of LCMS-grade water (using the same pipette to ensure complete sample transfer) and transferred to the volumetric flask. The volumetric flask was then filled to volume with LCMS-grade water, transferred to a 50 mL centrifuge tube, and centrifuged at 3000 revolutions per minute (rpm) for 10 min. The supernatant was then analyzed by HPLC analysis and / or stored at 2-8°C until use.

[0265] Alternatively, hydrolysis of alginate modified with an antifibrotic compound (alginate conjugated with Compound 101) was carried out by microwave irradiation. Briefly, 1000±50 mg of alginate conjugated with an antifibrotic compound in saline solution was weighed into a microwave vial. Then, 10 mL of 6N HCl solution was added to the vial along with a stir bar, and the vial was capped and placed in a microwave autosampler. Each sample was treated according to the following conditions:

[0266] [Table 5]

[0267] Each vial was removed, the contents transferred to a volumetric flask, and 2 mL of LCMS-grade water was added (x2). The flask was capped and inverted to mix, then the contents were transferred to a 50 mL centrifuge tube. The sample was centrifuged at 5000 rpm for approximately 10 minutes. 1 mL of the supernatant was then transferred to an LC vial and stored overnight at 2-8°C. The next day, the sample was heated to 60°C and thoroughly dried by placing a needle directing a stream of nitrogen directly over the sample (ensuring that the needle did not touch the sample). After drying, the LC vial was charged with 0.25 mL of a 0.1 μmol / mL internal standard mixture (see preparation below), the solution was vortexed thoroughly, and then transferred to another LC vial containing a 0.25 mL insert. The sample was stored at 2-8°C until use.

[0268] HPLC conditions. HPLC was performed on an Agilent 1260 LC with DAD and SQ MS using an XBridge C18, 2.5 μm, 4.6 × 50 mm (Waters 186006037) column and equipped with an API-ES MS detector. The mobile aqueous phase used was 0.1% ammonia, and the mobile organic phase was 0.1% ammonia in acetonitrile at a flow rate of 1.0 mL / min. The column was at 30°C. The UV detector was set to a wavelength of 220 nm.

[0269] Samples were injected in 10 μL volumes according to a set sequence. First, two blank samples of water were injected in sequence, followed by five repeats of a 0.01 mg / mL free amine standard. Samples were then run in sequence, with injections of the standard free antifibrotic compound solution interleaved every 10 samples. The HPLC sequence was completed by injection of a 1.0 mg / mL antifibrotic compound standard, followed by a column wash with 50 / 50 water-acetonitrile. An exemplary HPLC chromatogram obtained from an acid-hydrolyzed sample for determining total antifibrotic compound concentration is shown in Figure 1.

[0270] System suitability criteria. Data were checked against the following suitability criteria: no significant interference in UV and TIC (optional); relative standard deviation (RSD) of retention time in the first five injections less than 2%; RSD of area in the first five injections less than 10%; RSD of retention time in the first five injections and all bracket injections less than 2%; RSD of area in the first five injections and all bracket injections less than 10%; and, optionally, an anti-fibrotic compound peak with m / z 392.1±0.5.

[0271] Data Analysis and Calculations. The total antifibrotic compound concentration (mg / mL) in the samples was calculated using the following formula: [Total Antifibrotic Compound Concentration (mg / mL)] = [Area (Hydrolyzed Sample)] / [Area (1.0 mg / mL Standard)] × [Concentration (1.0 mg / mL Standard)].

[0272] The concentration (%) of total antifibrotic compounds in the modified alginate was calculated using the following formula: Concentration (%) of total antifibrotic compounds in the modified alginate = [Concentration (mg / mL) of total antifibrotic compounds in the sample] × 25 mL / [Weight (mg) of modified alginate × 100].

[0273] Example 3. Determination of free antifibrotic compounds in alginates modified with antifibrotic compounds. To determine the amount of unconjugated antifibrotic compound (i.e., "free" antifibrotic compound) in a sample of total modified polymer (e.g., antifibrotic alginate), the free antifibrotic compound can be separated from the modified polymer and quantified. Here, the following procedure was used to separate and quantitate the free antifibrotic compound from a sample of modified alginate.

[0274] Sample preparation. A lyophilized solid sample of antifibrotic alginate (50 ± 5 mg) was weighed into a scintillation vial, followed by the addition of 5.0 mL of saline. The mixture was then shaken and vortexed for 10 minutes to completely dissolve the solution, and then transferred to a tube fitted with a molecular weight cut-off (MWCO) filter. The MWCO tube was then centrifuged at 5,000 rpm for 60 minutes. After centrifugation, the supernatant above the MWCO filter, containing the antifibrotic alginate, was removed from the tube and discarded. A sample from the bottom of the MWCO tube, containing the free antifibrotic compound, was then transferred to a 5 mL volumetric flask, made to volume with water or saline, and thoroughly mixed by inversion. The solution was then stored in the scintillation vial at 2-8°C.

[0275] A solution of antifibrous alginate in saline solution was also analyzed using the same method outlined above by placing 1000±50 mg of antifibrous alginate in saline into a MWCO tube containing 4 mL of saline, and centrifuging the tube for 90 minutes.

[0276] Preparation of free amine standard solution. 50 ± 5 mg of antifibrotic compound (standard) was weighed into a scintillation vial, followed by approximately 10 mL of LCMS-grade water, which was shaken and vortexed to completely dissolve the solids. The solution was then transferred to a 50 mL volumetric flask, and the vial was rinsed twice with LCMS-grade water, which was then transferred to the volumetric flask. The volumetric flask was then brought to volume and mixed thoroughly to obtain a 1 mg / mL free antifibrotic compound standard solution. A 0.01 mg / mL free antifibrotic compound standard solution was also prepared by transferring 100 μL of the 1 mg / mL standard solution to a 10 mL volumetric flask, bringing the flask to volume with LCMS-grade water, and mixing thoroughly. Both standard solutions were stored at 2–8°C.

[0277] HPLC conditions. HPLC was performed as outlined in Example 2.

[0278] Samples were injected in 10 μL volumes according to a set sequence. First, two blank samples of water were injected in sequence, followed by five repeats of a 0.01 mg / mL free antifibrotic compound standard. Samples were then run in sequence, with injections of the standard free antifibrotic compound solution sandwiched between every 10 samples. The HPLC sequence was completed by injection of a 1.0 mg / mL free antifibrotic compound standard, followed by a column wash with 50 / 50 water-acetonitrile. Exemplary HPLC chromatograms can be found in Figures 2A-2B, including a standard sample (Figure 2A) and an exemplary sample containing a free antifibrotic compound (Figure 2B).

[0279] System suitability criteria. Data were checked against the following suitability criteria: no significant interference in UV and TIC (optional); relative standard deviation (RSD) of retention time in the first five injections less than 2%; RSD of area in the first five injections less than 10%; RSD of retention time in the first five injections and all bracket injections less than 2%; RSD of area in the first five injections and all bracket injections less than 10%; and, optionally, an anti-fibrotic compound peak with m / z 392.1±0.5.

[0280] Data Analysis and Calculations. The free antifibrotic compound peak was confirmed to have an m / z of 392.1±0.5 and a retention time consistent with that found in the standard free antifibrotic compound solution.

[0281] The concentration (mg / mL) of the standard free antifibrotic compound solution was calculated using the following formula: Standard free antifibrotic compound concentration (mg / mL) = [weight of free antifibrotic compound (mg)] / 50 mL / dilution factor, where the dilution factor is 1 for a 1.0 mg / mL free antifibrotic compound standard and 100 for a 0.01 mg / mL free amine standard.

[0282] The concentration of free antifibrotic compound in the sample (mg / mL) was calculated from the HPLC data using the following formula: Concentration of free antifibrotic compound in sample (mg / mL) = Area (free antifibrotic compound sample) / [Area (free antifibrotic compound standard)] × [Concentration of free antifibrotic compound standard (mg / mL)].

[0283] The concentration (%) of free antifibrotic compound in the modified alginate was then calculated using the following formula: Concentration (%) of free antifibrotic compound in the modified alginate = {[Concentration (mg / mL) of free antifibrotic compound in the sample] × 5 mL} / [(mass of modified alginate) × 100].

[0284] Example 4. Determination of conjugated antifibrotic compounds in polymers modified with antifibrotic compounds. If the amounts of free antifibrotic compound and total antifibrotic compound (free antifibrotic compound + conjugated antifibrotic compound) in a sample of modified polymer are known, the amount of conjugated antifibrotic compound in the modified polymer can be easily calculated. If the amount of free antifibrotic compound is less than 2%, the amount of conjugated antifibrotic compound can be reported as equal to the value of total antifibrotic compound. Alternatively, the concentration of conjugated antifibrotic compound can be calculated by subtracting the amount of free antifibrotic compound (e.g., the amount calculated in Example 3) from the amount of total antifibrotic compound (e.g., the amount calculated in Example 2). This calculation can be expressed as follows: Conjugated Antifibrotic Compound (w / w%) = Total Antifibrotic Compound (w / w%) - Free Antifibrotic Compound (w / w%).

[0285] Conversion of the amount of conjugated antifibrotic compound in saline solution to an equivalent solid sample. The amount of conjugated antifibrotic compound in a saline solution of modified polymer (e.g., antifibrotic alginate) can be converted to the corresponding amount of conjugated antifibrotic compound in the equivalent form as a lyophilized solid using a simple conversion. This conversion requires determining the concentration of the modified polymer (e.g., antifibrotic alginate) in the saline solution, which can be obtained according to the protocol outlined in Example 5. The amount of conjugated antifibrotic compound in the equivalent solid sample can then be derived by simply dividing the amount of conjugated antifibrotic compound obtained for the saline solution by the concentration of the modified polymer in the saline solution (multiplying by 100). This calculation is represented by the following formula: conjugated antifibrotic compound (w / w%) in solid sample = [conjugated antifibrotic compound (w / w%) in saline] / ([concentration of modified alginate (w / w%) in saline] × 100).

[0286] For example, the antifibrotic alginate in a saline solution was calculated to have a total antifibrotic compound of 1.97 w / w% and a free antifibrotic compound of less than 0.01 w / w%. Because the amount of free antifibrotic compound was less than 2%, the amount of conjugated antifibrotic compound was determined to be 1.97 w / w% (equivalent to the total antifibrotic compound). The concentration of antifibrotic alginate in the saline sample was determined to be 4.91 w / w% (according to the protocol in Example 5). The calculated amount of conjugated antifibrotic compound in the corresponding solid sample was then calculated to be 40.12 w / w% ([1.97 w / w%] / [{4.91 w / w% x 100}] = 40.12 w / w%).

[0287] Example 5. Determining the concentration of modified polymer in solution using refractive index. Refractive index (RI) was used to determine the concentration of modified polymers (e.g., antifibrotic alginate) in solution without removing the polymer modifier (e.g., antifibrotic compound) from the polymer (i.e., without performing acid hydrolysis). A refractometer can be used to determine the concentration of modified polymers in saline solution. Here, the concentration of antifibrotic alginate was determined using a refractometer according to the protocol outlined below.

[0288] Standard Preparation. Approximately 1200 mg of antifibrous alginate in lyophilized solid form was weighed into a scintillation vial and the actual weight (Ws) was recorded. Sterile saline solution was then added in the amount necessary to achieve a final concentration of 6.0%, and the actual final weight (Wt) was recorded. This mixture was then gently mixed on a tube rotator at 20 rpm for at least 2 hours and then stored overnight at 2-8°C to ensure full dissolution. The next day, the sample was warmed to room temperature and gently mixed on a tube rotator at 20 rpm for at least 4 hours. The resulting 6.0% stock solution should be homogenous and free of any solid residue.

[0289] Dilutions: A series of dilutions was performed to prepare approximately 5 g solutions at target concentrations of 5.5%, 5.0%, 4.5%, and 4.0%. For each concentration, an appropriate amount of the 6.0% stock solution prepared above was transferred to a vial, and the actual weight (Wx) was recorded. Approximately 5,000 mg of sterile saline solution was then added, and the actual total weight (Wt) was recorded. Each vial was then gently mixed on a tube rotator at 20 rpm for at least 1 hour and stored at 2-8°C. The actual weights, concentrations, and refractive indices obtained for the triazole-modified alginates are shown in Table 2 below.

[0290] [Table 6]

[0291] Refractometer Measurements. At least five drops of sample (ambient temperature) were added to a clean, dry prism of a LAXCO RBD-5001 refractometer so that the sample completely covered the base of the prism and any air bubbles on the prism were broken or removed. Measurements were taken at a temperature of 20°C. A blank measurement was taken by rinsing the prism twice with saline solution and measuring the second saline rinse; this blank measurement had an nD of 1.3343 to 1.3347. All measurements were taken in triplicate.

[0292] Exemplary Test Sequence. A series of sample measurements were performed using the following protocol: a saline solution was measured, followed by a standard solution. Sample 1 was then measured in triplicate, followed by each subsequent sample in triplicate. Finally, the standard was measured again, followed by a saline solution. The prism was rinsed between all measurements.

[0293] Standard calibration curve. The refractive index (nd) measured for each of the standard solutions prepared above (6.0%, 5.5%, 5.0%, 4.5%, and 4.0% standard solutions) was plotted against the calculated concentrations (shown in Table 2), and a linear regression was performed to obtain the slope and Y-intercept.

[0294] Suitability criteria. The refractive index reading for 0.9% saline should be within 1.3343-1.3347, and the calculated concentration should be within 90%-110% of the theoretical concentration.

[0295] Extrapolation of the standard curve. The concentration of the samples was determined by one of two methods. In the first method, the concentration of each antifibrotic alginate was determined by extrapolation from the standard curve. For example, the concentration of the sample was calculated as follows: concentration = m × refractive index + b, where m is the slope of the line fitted to the curve and b is the Y-intercept. The coefficient of determination was 0.95 or greater.

[0296] Determination of dn / dc: In the second method, the concentration of antifibrous alginate in solution was determined by comparing the specific refractive index increment (i.e., dn / dc value). This value was calculated for both Compound 101 and the glucuronic acid moiety by first determining the actual concentration (μmol / g) or (w / w%) of each component (Compound 101 or glucuronic acid) using the actual Ws and Wt, and then plotting it against the corrected refractive index value. The dn / dc value is meant to be a constant for each component at a specific wavelength and temperature. For example, standard concentrations were determined as follows: Concentration of standard (μmol / g) = Ws (mg) / MW (g / mol) / Wt (mg) × 1,000,000; or Concentration of standard (w / w%) = Ws (mg) / Wt (mg) × 100.

[0297] The corrected refractive index value was then calculated as follows: RI standard - RI saline = dn / dC x concentration of standard, where differential RI (dn / dC) is the slope of the line fitted to the curve.

[0298] A summary of exemplary dn / dc values ​​is summarized in Table 3.

[0299] [Table 7]

[0300] Sample Analysis: To calculate the total concentration of anti-fibrous alginate in the solution in this method, the refractive index value of the sample was measured (RI, sample) and used in the following formula to determine the anti-fibrous alginate concentration:

[0301] Concentration of unmodified alginate (w / w% or μmol / g) = [((RI, sample) - (RI, saline) - (dn / dc, compound 101)) x (concentration, compound 101)] / (dn / dc, GA)

[0302] For example, the concentrations of anti-fibrotic alginate as determined by RI are shown below.

[0303] [Table 8]

[0304] Example 6: Preparation of hydrogel capsules Capsules encapsulating RPE cells as single cells. Immediately prior to encapsulation, single ARPE-19 cells engineered to express a therapeutic protein were centrifuged at 1,400 rpm for 1 minute and washed with calcium-free Krebs-Henseleit (KH) buffer (4.7 mM KCl, 25 mM HEPES, 1.2 mM KH2PO4, 1.2 mM MgSO4 x 7H2O, 135 mM NaCl, pH 7.4, approximately 290 mOsm). After washing, the cells were centrifuged again, and all of the supernatant was aspirated. In some experiments, the cell pellet was then resuspended in a 70:30 CM-LMW-Alg:U-HMW-Alg solution (control capsules) or one of the modified alginate solutions described in Table 4 of Example 1 at the desired density of suspended single cells per ml of alginate solution.

[0305] Prior to fabrication of the one- and two-compartment hydrogel capsules, the buffer and alginate solutions were sterilized by filtration through a 0.2 μm filter using an aseptic process.

[0306] To fabricate single-compartment hydrogel capsules approximately 1.5 mm in diameter that encapsulate cells, the desired number of cells was suspended in the desired alginate solution (e.g., the 70:30 CM-LMW-Alg:U-HMW-Alg solution used in the control capsules or one of the experimental alginate solutions described in Example 1). The resulting cell suspension was loaded into a syringe and capped with an 18-gauge blunt-tip needle (SAI Infusion Technologies). The syringe was placed in a syringe pump oriented vertically above a dish containing the crosslinking solution. A high-voltage generator was connected to the needle and grounded to a biosafety cabinet. The syringe pump and generator were turned on, and the alginate solution was extruded through the needle at a flow rate of 0.16 mL / min or 10 mL / h, adjusting the voltage between 5 and 9 kV until a drop rate of 12 drops per 10 seconds was achieved.

[0307] To prepare devices configured as two-compartment hydrogel millicapsules with a diameter of approximately 1.5 mm, the electrostatic droplet generator was set up as follows: an ES series 0-100 kV, 20-watt high-voltage generator (EQ series, Matsuda, NC, USA) was connected to the upper and lower ends of a concentric needle (22 G inner lumen, 18 G outer lumen, Rame-Hart Instrument Co., Succasunna, NJ, USA). The inner lumen was attached to a first BD disposable 5 ml syringe with a BD Luer-Lok™ tip (BD (catalog no. 309646), Franklin Lakes, NJ, USA), and the syringe was connected to a vertically oriented syringe pump (Pump 11 Pico Plus, Harvard Apparatus, Holliston, MA, USA). The outer lumen was connected to a second 5 ml Luer-lock syringe via a Luer coupling, which was connected to a second vertically oriented syringe pump (Pump 11 Pico Plus). To encapsulate cells in only the first (inner) compartment, a first alginate solution containing cells (as a single-cell suspension) (a 70:30 CM-Alg-101:UM-Alg solution (as a control) or one of the experimental alginate solutions described in Example 1) was placed in the first syringe, and a second cell-free alginate solution containing an antifibrotic compound (e.g., CM-LMW-Alg-101 and U-HMW-Alg) was placed in the second syringe. In the case of the control two-compartment hydrogel capsules in the following examples, the second (outer) compartment was formed using a 70:30 CM-LMW-Alg-101:U-HMW-Alg solution. Two syringe pumps moved the first and second alginate solutions from the syringes through both lumens of the concentric needle, and a single droplet containing both alginate solutions was extruded from the needle into a glass dish containing the cross-linking solution. Each Pico Plus syringe pump was set to a diameter of 12.06 mm, and the flow rate of each pump was adjusted to achieve a 1:1 flow ratio for the two alginate solutions. Thus, with the total flow rate set at 10 ml / h, the flow rate of each alginate solution was approximately 5 ml / h.

[0308] For both two-compartment and one-compartment millicapsule fabrication, after extrusion of the desired volume of alginate solution, the alginate droplets were crosslinked for 5 min in a crosslinking solution containing 25 mM HEPES, 20 mM BaCl2, 0.2 M mannitol, and poloxamer 188. Capsules that fell to the bottom of the crosslinking vessel were collected by pipetting into a conical tube. After the capsules settled in the tube, the crosslinking buffer was removed and the capsules were washed. Cell-free capsules were washed four times with HEPES buffer (15.428 g NaCl, 0.70 g KCl, 0.488 g MgCl2·6H2O, 50 ml HEPES (1 M) buffer in 2 L deionized water (Gibco, Life Technologies, California, USA)) and stored at 4 °C until use. The capsules encapsulating the cells were washed four times with HEPES buffer, twice with 0.9% saline, and twice with culture medium, and stored in an incubator at 37°C.

[0309] Equivalents and Scope This application references various issued patents, published patent applications, journal articles, books, manuals, and other publications, all of which are incorporated herein by reference in their entirety. In the event of a conflict between any of the incorporated documents and this specification, the present specification shall control. In addition, any specific embodiments of the present disclosure that fall within the prior art may be explicitly excluded from any one or more claims. Because such embodiments are deemed known to those of ordinary skill in the art, they may be excluded even if not expressly excluded herein. Any specific embodiments of the present disclosure may be excluded from any claim for any reason, whether related to the existence of prior art or not.

[0310] Those skilled in the art will recognize or be able to ascertain, without undue routine experimentation, equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above specification, drawings, or examples, but rather is intended to be as set forth in the appended claims. Those skilled in the art will recognize that various changes and modifications to the description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

1. A method for evaluating a polymer composition comprising a polymer, the polymer having formula (I-b): 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, During the ceremony, Ring M １ is aryl or heteroaryl, and each aryl and heteroaryl is selected from one or more R ３ optionally substituted with; Ring Z １ is a cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each cycloalkyl, heterocyclyl, aryl, or heteroaryl is selected from 1 to 5 R ５ optionally substituted with; R ２ａ , R ２ｂ , R ２ｃ , and R ２ｄ Each of is independently hydrogen, alkyl, or heteroalkyl, or R ２ａ and R ２ｂ , or R ２ｃ and R ２ｄ taken together form an oxo group; X is absent, O, or S; Each R ３ and R ５ are independently alkyl, heteroalkyl, halogen, oxo, -OR Ａ１ , -C(O)OR Ａ１ , or -C(O)R Ｂ１ wherein each alkyl and heteroalkyl is optionally substituted with one or more halogen, oxo, cyano, cycloalkyl, or heterocyclyl; or two R ５ Together, we form Ring Z １ forming a 5-6 membered ring fused to R ｃ is hydrogen or alkyl; Each R Ａ１ and R Ｂ１ is independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; 【Chemistry 2】 refers to a linking group or bond to the polymer, or a pharmaceutically acceptable salt thereof, The method comprises: (a) exposing the polymer composition to reaction conditions that allow for release of the compound of formula (Ib) from the modified polymer; and (b) obtaining a concentration value of the compound of formula (Ib) bound to the modified polymer; Including, thereby evaluating the polymer composition or semi-permeable device. method.

2. A method for evaluating a polymer composition comprising a polymer, the polymer having formula (III-a): 【Transformation 3】 or a pharmaceutically acceptable salt thereof, During the ceremony, Ring Z ２ is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 1 to 5 R ５ optionally substituted with; R ２ａ , R ２ｂ , R ２ｃ , and R ２ｄ each is independently hydrogen, alkyl, heteroalkyl, halo; or R ２ａ and R ２ｂ , or R ２ｃ and R ２ｄ taken together form an oxo group; R ３ and R ５ each independently represents alkyl, heteroalkyl, halogen, oxo, -OR Ａ１ , -C(O)OR Ａ１ , or -C(O)R Ｂ１ and Each R Ａ１ and R Ｂ１ is independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; o and p are each independently 0, 1, 2, 3, 4, or 5; q is an integer from 0 to 25; 【Chemistry 4】 refers to a linking group or bond to the polymer, or a pharmaceutically acceptable salt thereof, The method comprises: (a) exposing the polymer composition to reaction conditions that allow for the release of the compound of formula (III-a) from the modified polymer; and (b) obtaining the concentration value of the compound of formula (III-a); Including, thereby evaluating the polymer composition or semi-permeable device. method.

3. The method of claim 1 or 2, wherein the polymer in the modified polymer is a polysaccharide.

4. The method of claim 3 , wherein the polysaccharide is alginate.

5. The method of claim 4, wherein the alginate has an average molecular weight of 75 kDa to 150 kDa.

6. 5. The method of claim 4, wherein the alginate has a guluronic acid to mannuronate (G:M) ratio of 1.5 or greater.

7. 2. The method of claim 1, wherein the compound of formula (Ib) or formula (III-a) is covalently attached to the polymer (eg, alginate) in the modified polymer.

8. 2. The method of claim 1, wherein the compound of formula (Ib) or formula (III-a) is non-covalently bound to the polymer (eg, alginate) in the modified polymer.

9. The compounds of formula (I-b) or formula (III-a) may contain a linking group (e.g., —C(O)(C １ ~C ６ -alkylene)-, where alkylene is R １ and R １ The method of claim 1, wherein the modified polymer (e.g., modified alginate) is covalently attached to the modified polymer via a carboxyl group (C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C44, C45, C46, ​​C47, C48, C49, C50, C51, C52, C53, C64, C65, C76, C77, C78, ​​C79, C80, C81, C82, C83, C84, C85, C96, C9

10. The method of claim 1 , wherein the polymer composition comprises a single type of modified polymer (e.g., a modified alginate).

11. The method of claim 1 , wherein the polymer composition comprises a plurality of modified polymers (e.g., at least two modified polymers, at least three polymers).

12. The method of claim 1, wherein the compound of formula (Ib) or formula (III-a) is a compound listed in Table 1.

13. 2. The method of claim 1, wherein the compound of formula (I-b) or formula (III-a) is selected from Compound 100, Compound 101, Compound 110, Compound 113, and Compound 114 listed in Table 1.

14. The method of claim 1, wherein the reaction conditions in step (a) include contacting the polymer composition with an acidic solution (e.g., an HCl solution, e.g., a 1N to 8N HCl solution).

15. 10. The method of claim 1, wherein the reaction conditions in step (a) include heating the polymer composition (e.g., at a temperature greater than about 25°C, 40°C, 60°C, 80°C, 100°C, 120°C, or higher).

16. 10. The method of claim 1, wherein the reaction conditions in step (a) comprise exposing the polymer composition to microwave radiation.

17. The method of claim 1 further comprising a separation step.

18. 18. The method of claim 17, wherein the separation step occurs between step (a) and step (b).

19. 19. The method of claim 17 or 18, wherein the separation step comprises chromatography (e.g., size exclusion chromatography, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, or hydrophobic interaction chromatography).

20. 2. The method of claim 1, wherein obtaining the concentration value in step (b) comprises determining the area of ​​a chromatogram peak of the compound of formula (I-b) or formula (III-a).

21. 21. The method of claim 20, wherein obtaining the concentration value in step (b) further comprises comparing the area of ​​the chromatogram peak with a standard (e.g., a compound of formula (I-b) or formula (III-a)).

22. 10. The method of claim 1, further comprising obtaining a value for the concentration of the unconjugated compound of formula (I-b) (i.e., the "free" compound of formula (I-b)) or the unconjugated compound of formula (III-a) (i.e., the "free" compound of formula (III-a)) in the polymer composition.

23. Obtaining a value for the concentration of the unconjugated compound of formula (I-b) (i.e., the "free" compound of formula (I-b)) or the unconjugated compound of formula (III-a) (i.e., the "free" compound of formula (III-a)) in the polymer composition comprises: (a') separating the polymer composition into a polymer-bound fraction and a non-polymer-bound fraction; (b') retaining said non-polymer-bound fraction; and (c') obtaining a value for the concentration of the unconjugated compound of formula (I-b) (i.e., the "free" compound of formula (I-b)) or the unconjugated compound of formula (III-a) (i.e., the "free" compound of formula (III-a)) in the non-polymer-bound fraction.

23. The method of claim 22, comprising:

24. 24. The method of claim 23, wherein the separating in step (a') comprises filtration, for example through a molecular weight cut-off filter.

25. 25. The method of claim 23 or 24, wherein step (c') comprises a separation step.

26. 26. The method of claim 25, wherein the separation step comprises chromatography (e.g., size exclusion chromatography, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, or hydrophobic interaction chromatography).

27. 24. The method of claim 23, wherein obtaining the concentration value in step (c') further comprises determining the area of ​​a chromatogram peak of a sample of the unconjugated compound of formula (I-b) (i.e., the "free" compound of formula (I-b)) or the unconjugated compound of formula (III-a) (i.e., the "free" compound of formula (III-a)) in the non-polymer-bound fraction.

28. 28. The method of claim 27, wherein obtaining the concentration value in step (c') further comprises comparing the area of ​​the chromatogram peak with a standard (e.g., a compound of formula (I-b) or formula (III-a)).

29. 2. The method of claim 1, wherein evaluating the polymer composition comprises determining the concentration of the compound of formula (I-b) or formula (III-a) bound to the modified polymer in the polymer composition.

30. Determining the concentration of the compound of formula (I-b) or formula (III-a) bound to the modified polymer in the polymer composition includes: (a'') obtaining a value for the total concentration of the compound of formula (I-b) or formula (III-a) in the polymer composition; (b'') obtaining a value for the concentration of the unconjugated compound of formula (I-b) (i.e., the "free" compound of formula (I-b)) or the unconjugated compound of formula (III-a) (i.e., the "free" compound of formula (III-a)) in the polymer composition; and / or (c'') subtracting the concentration of the free compound of formula (I-b) or the free compound of formula (III-a) (e.g., as determined in step (b'')) from the total concentration of the compound of formula (I-b) or formula (III-a) in the polymer composition. Including, thereby determining the concentration of the compound of formula (I-b) or formula (III-a) conjugated (e.g., covalently attached) to the modified polymer in the polymer composition; 30. The method of claim 29.

31. 1. A method for determining the concentration of a polymer composition comprising an alginate, wherein the alginate has the formula (I-b): 【Transformation 5】 or a pharmaceutically acceptable salt thereof, During the ceremony, Ring M １ is aryl or heteroaryl, and each aryl and heteroaryl is selected from one or more R ３ optionally substituted with; Ring Z １ is a cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each cycloalkyl, heterocyclyl, aryl, or heteroaryl is selected from 1 to 5 R ５ optionally substituted with; R ２ａ , R ２ｂ , R ２ｃ , and R ２ｄ Each of is independently hydrogen, alkyl, or heteroalkyl, or R ２ａ and R ２ｂ , or R ２ｃ and R ２ｄ taken together form an oxo group; X is absent, O, or S; Each R ３ and R ５ are independently alkyl, heteroalkyl, halogen, oxo, -OR Ａ１ , -C(O)OR Ａ１ , or -C(O)R Ｂ１ wherein each alkyl and heteroalkyl is optionally substituted with one or more halogen, oxo, cyano, cycloalkyl, or heterocyclyl; or two R ５ Together, we form Ring Z １ forming a 5-6 membered ring fused to R ｃ is hydrogen or alkyl; Each R Ａ１ and R Ｂ１ are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; 【Transformation 6】 refers to a linking group or bond to the polymer, or a pharmaceutically acceptable salt thereof, or Formula (III-a): 【Transformation 7】 or a pharmaceutically acceptable salt thereof, During the ceremony, Ring Z ２ is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 1 to 5 R ５ optionally substituted with; R ２ａ , R ２ｂ , R ２ｃ , and R ２ｄ each is independently hydrogen, alkyl, heteroalkyl, halo; or R ２ａ and R ２ｂ , or R ２ｃ and R ２ｄ taken together form an oxo group; R ３ and R ５ each independently represents alkyl, heteroalkyl, halogen, oxo, -OR Ａ１ , -C(O)OR Ａ１ , or -C(O)R Ｂ１ and Each R Ａ１ and R Ｂ１ are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; o and p are each independently 0, 1, 2, 3, 4, or 5; q is an integer from 0 to 25; 【Transformation 8】 refers to a linking group or bond to the polymer, or a pharmaceutically acceptable salt thereof, The method comprises: (a) obtaining a value for the total concentration of the compound of formula (I-b) or formula (III-a) in the polymer composition; (b) obtaining a value for the concentration of the unconjugated compound of formula (I-b) (i.e., the "free" compound of formula (I-b)) or the unconjugated compound of formula (III-a) (i.e., the "free" compound of formula (III-a)) in the polymer composition; and / or (c) subtracting the concentration of the free compound of formula (I-b) or the free compound of formula (III-a) (e.g., as determined in step (b)) from the total concentration of the compound of formula (I-b) or formula (III-a) in the polymer composition. Including, thereby determining the concentration of the compound of formula (I-b) or formula (III-a) conjugated (e.g., covalently attached) to the modified polymer in the polymer composition; method.

32. 32. The method of claim 31 , wherein step (a) is performed before step (b).

33. 32. The method of claim 31 , wherein step (b) is performed before step (a).

34. Step (a) is (a) exposing the polymer composition to reaction conditions that allow for the release of the compound of Formula (I-b) or Formula (III-a) from the modified polymer; and (b) obtaining a concentration value of the compound of formula (I-b) or the compound of formula (III-a); 32. The method of claim 31 , comprising:

35. The method of claim 31, wherein the compound is a compound of formula (Ib).

36. 36. The method of claim 35, wherein the compound is Compound 100, Compound 110, Compound 113, or Compound 114, or a pharmaceutically acceptable salt thereof.

37. 32. The method of claim 31, wherein the compound is a compound of formula (III-a).

38. 38. The method of claim 37, wherein the compound is compound 101 or a pharmaceutically acceptable salt thereof.

39. 32. The method of claim 31, wherein the concentration of the compound of formula (I-b) or the compound of formula (III-a) bound to a polymer in the modified polymer (e.g., modified alginate) is about 0.5% to about 10% (w / w) of the modified polymer (e.g., modified alginate), such as about 0.5% to 5%, 1% to 5%, 1% to 4%, or 1 to 3% (w / w) of the modified polymer (e.g., modified alginate).

40. 32. The method of claim 31, wherein the concentration of the free compound of formula (I-b) or the free compound of formula (III-a) in the polymer composition is less than about 1% (w / w) of the modified polymer (e.g., modified alginate), such as less than about 0.9%, 0.8%, 0.7%, 0.6%, or 0.5% (w / w) of the modified polymer (e.g., modified alginate).

41. A method for evaluating a polymer composition comprising a polymer, the polymer having formula (I-b): 【Chemistry 9】 or a pharmaceutically acceptable salt thereof, During the ceremony, Ring M １ is aryl or heteroaryl, and each aryl and heteroaryl is selected from one or more R ３ optionally substituted with; Ring Z １ is a cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each cycloalkyl, heterocyclyl, aryl, or heteroaryl is selected from 1 to 5 R ５ optionally substituted with; R ２ａ , R ２ｂ , R ２ｃ , and R ２ｄ Each of is independently hydrogen, alkyl, or heteroalkyl, or R ２ａ and R ２ｂ , or R ２ｃ and R ２ｄ taken together form an oxo group; X is absent, O, or S; Each R ３ and R ５ are independently alkyl, heteroalkyl, halogen, oxo, -OR Ａ１ , -C(O)OR Ａ１ , or -C(O)R Ｂ１ wherein each alkyl and heteroalkyl is optionally substituted with one or more halogen, oxo, cyano, cycloalkyl, or heterocyclyl; or two R ５ Together, we form Ring Z １ forming a 5-6 membered ring fused to R ｃ is hydrogen or alkyl; Each R Ａ１ and R Ｂ１ are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; 【Chemistry 10】 refers to a linking group or bond to the polymer, or a pharmaceutically acceptable salt thereof, or Formula (III-a): 【Chemistry 11】 or a pharmaceutically acceptable salt thereof, During the ceremony, Ring Z ２ is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 1 to 5 R ５ optionally substituted with; R ２ａ , R ２ｂ , R ２ｃ , and R ２ｄ each is independently hydrogen, alkyl, heteroalkyl, halo; or R ２ａ and R ２ｂ , or R ２ｃ and R ２ｄ taken together form an oxo group; R ３ and R ５ each independently represents alkyl, heteroalkyl, halogen, oxo, -OR Ａ１ , -C(O)OR Ａ１ , or -C(O)R Ｂ１ and Each R Ａ１ and R Ｂ１ are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; o and p are each independently 0, 1, 2, 3, 4, or 5; q is an integer from 0 to 25; 【Chemistry 12】 refers to a linking group or bond to the polymer, or a pharmaceutically acceptable salt thereof, The method comprises: (a) obtaining a refractive index value for said polymer composition; (b) obtaining a value for the total unconjugated compounds of formula (I-b) (i.e., "free" compounds of formula (I-b)) or the total unconjugated compounds of formula (III-a) (i.e., "free" compounds of formula (III-a)) in the polymer composition; and / or (c) using the values ​​obtained in each of steps (a) and (b) to obtain a value for the concentration of the polymer modified with the compound of formula (I-b) or formula (III-a). Including, thereby evaluating the polymer composition. method.

42. 42. The method of claim 41, wherein the alginate has an average molecular weight of 75 kDa to 150 kDa.

43. 42. The method of claim 41, wherein the alginate has a guluronic acid to mannuronate (G:M) ratio of 1.5 or greater.

44. 42. The method of claim 41, wherein the compound of formula (Ib) or formula (III-a) is covalently attached to the polymer (e.g., alginate) in the modified polymer.

45. 42. The method of claim 41, wherein the compound of formula (Ib) or formula (III-a) is non-covalently bound to the polymer (e.g., alginate) in the modified polymer.

46. The compounds of formula (I-b) or formula (III-a) may contain a linking group (e.g., —C(O)(C １ ~C ６ -alkylene)-, where alkylene is R １ and R １ 42. The method of claim 41, wherein the modified polymer (e.g., modified alginate) is covalently attached to the modified polymer via a carboxyl group (C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C

47. 42. The method of claim 41, wherein the polymer composition comprises a single type of modified polymer (e.g., a modified alginate).

48. 42. The method of claim 41, wherein the polymer composition comprises a plurality of modified polymers (e.g., at least two modified polymers, at least three polymers).

49. 42. The method of claim 41, wherein the compound of formula (Ib) or formula (III-a) is a compound listed in Table 1.

50. 42. The method of claim 41, wherein the compound of formula (I-b) or formula (III-a) is selected from compound 100, compound 101, compound 110, compound 113, and compound 114 listed in Table 1.

51. Obtaining the value of all compounds of formula (I-b) or formula (III-a) conjugated (e.g., covalently attached) to the modified polymer composition of step (b) comprises: (b') the polymer composition, (i) releasing the compound of formula (Ib) or formula (III-a) from the modified polymer; exposing the reaction mixture to reaction conditions that allow (b'') obtaining the concentration value of the compound of formula (I-b) or the compound of formula (III-a); 42. The method of claim 41 , comprising:

52. 42. The method of claim 41, further comprising obtaining dn / dc values ​​for components of the polymer composition (e.g., components of the modified polymer).

53. 53. The method of claim 52, further comprising using the dn / dc value in step (c) of the method to obtain a value for the concentration of the polymer modified with a compound of Formula (I-b) or Formula (III-a).

54. 1. A method for determining the concentration of alginate-bound compounds of formula (I-b) or formula (III-a) in a polymer composition, comprising: (a) obtaining a refractive index value for said polymer composition; (b) obtaining the value of all compounds of formula (I-b) or formula (III-a) conjugated (e.g., covalently attached) to the modified polymer in said polymer composition; (c) using the values ​​obtained in each of steps (a) and (b) to obtain a value for the concentration of the compound of formula (I-b) or formula (III-a) conjugated to the modified polymer; Including, Thereby, the concentration of the compound of formula (I-b) or formula (III-a) bound to the polymer in the polymer composition is determined. method.

55. 55. The method of claim 54, wherein the compound is a compound of formula (Ib).

56. 56. The method of claim 55, wherein the compound is Compound 100, Compound 110, Compound 113, or Compound 114, or a pharmaceutically acceptable salt thereof.

57. 55. The method of claim 54, wherein the compound is a compound of formula (III-a).

58. 58. The method of claim 57, wherein the compound is compound 101 or a pharmaceutically acceptable salt thereof.

59. 59. A polymer composition comprising a polymer modified with a compound of formula (I-b) or a compound of formula (III-a), wherein the concentration of the compound of formula (I-b) or the compound of formula (III-a) is 0.5% to 5% (w / w) of modified alginate, for example as determined by the method of any one of claims 1 to 58.

60. 59. A polymer composition comprising an alginate modified with a compound of formula (I-b) or a compound of formula (III-a), wherein the concentration of the compound of formula (I-b) or the compound of formula (III-a) is 0.5% to 5% (w / w) of the modified alginate, for example as determined by the method of any one of claims 1 to 58.

61. 100. A polymer composition comprising an alginate modified with a compound selected from compound 100, compound 101, compound 110, compound 112, compound 113, and / or compound 114 shown in Table 1, wherein the concentration of the compound is 0.5% to 5% (w / w) of the modified alginate, for example, as determined by the method of any one of claims 1 to 58.

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