Reaction intermediates for producing polyethylene glycol (PEG)-interleukin 11 (IL-11) conjugates and methods for purifying PEG-IL-11 conjugates

Abstract

To provide a reactive intermediate for generating polyethylene glycol (PEG)-interleukin 11 (IL-11) conjugate, which reduces adverse effect while stabilizing IL-11, and a method for purifying a PEG-IL-11 conjugate.SOLUTION: The present invention discloses a reactive intermediate for generating a polyethylene glycol (PEG)-interleukin 11 (IL-11) conjugate, which achieves improved half-life in serum while having desirable therapeutic activity and presenting less side-effects, and a method for purifying a PEG-IL-11 conjugate. Most preferably, the IL-11 in the PEG-IL-11 conjugate is an N-terminally truncated human or humanized IL-11 and has a 20Kd or 40Kd branched PEG moiety, which is Y- or comb-shaped and, in particular, coupled to an N-terminal amino group.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] This application claims priority to the inventors' co-pending U.S. Provisional Patent Application No. 62 / 127,748, filed March 3, 2015, which is incorporated herein by reference.

[0002] Technical Field The field of the invention is pharmaceutical compositions and methods, particularly relating to pegylated interleukin-11 (IL-11). [Background technology]

[0003] The background description includes information that may be helpful in understanding the present invention. No admission is made that any of the information provided herein is prior art or related to the present invention, or that any of the publications specifically or implicitly referenced are prior art. All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In the event that a definition or use of a term in an incorporated reference contradicts or is contrary to the definition of that word provided herein, the definition of that word provided herein applies and the definition of that word in the reference does not apply.

[0004] Current treatment regimens involve platelet transfusions, which are often in short supply and carry the risk of viral contamination, making chemotherapy-induced thrombocytopenia an unmet medical need. Recombinant human IL-11 can be administered to patients to stimulate platelet production. However, IL-11 administration requires daily administration, resulting in limited clinical efficacy and plasma volume expansion.

[0005] IL-11 is a cytokine that acts as a major signaling factor in hematopoiesis, particularly in stimulating megakaryocyte maturation. IL-11 action is typically mediated by the IL-11 receptor and glycoprotein gp130, followed by gp130 phosphorylation / activation. Clinical uses of IL-11 include the treatment of chemotherapy-related side effects, where it is thought to enhance megakaryopoiesis and increase platelet counts. Recombinant human IL-11 is commercially available as NEUMEGA® (Oprelvekin, Wyeth-Ayerst) and is approved for the prevention of severe thrombocytopenia and reduction of the need for platelet transfusions after myelosuppressive chemotherapy in adult patients with non-myeloid malignancies at high risk for severe thrombocytopenia. NEUMEGA® is typically supplied in single-use vials containing 5 mg of IL-11 as a lyophilized powder that is reconstituted with 1 mL of sterile water for injection (administered at a dose of 25–50 μg / kg / day). The most frequent adverse events associated with NEUMEGA™ are life-threatening atrial arrhythmias, syncope, dyspnea, congestive heart failure, and plasma expansion leading to pulmonary edema.

[0006] IL-11 is relatively rapidly removed from the circulation, thus requiring frequent injections. For example, Neumega™ administered subcutaneously to healthy men has a terminal half-life of approximately 6.9 hours (Neumega™ package insert). Poor pharmacokinetics, including rapid renal excretion and proteolysis, and associated side effects often result in poor clinical outcomes. Furthermore, daily injections also require hospitalization to manage adverse events, which not only increases medical costs but also impairs patients' quality of life. As a result, platelet transfusions remain the gold standard for treating chemotherapy-induced thrombocytopenia (CIT).

[0007] Several attempts have been made in the art to increase serum stability while maintaining the beneficial therapeutic potential of such compositions. For example, U.S. Patent Application Publication No. 2010 / 0098658 (Patent Document 1) reports an IL-11 analog (mIL-11) associated with a polymer (PEG) that exhibits enhanced resistance to acid degradation and increased serum half-life. In another attempt to stabilize IL-11, cysteine ​​mutants of IL-11 were prepared, and selected muteins were further modified with PEG to increase serum stability, as described in U.S. Patent No. 8,133,480 (Patent Document 2). While these modifications have improved the serum stability or half-life of IL-11 at least to some extent, they still suffer from one or more drawbacks, including low efficacy in myelosuppressed animals, complex manufacturing procedures, repeated administration, and formulation into injectable solutions.

[0008] Because IL-11 lacks a cysteine ​​residue, U.S. Patent No. 8,133,480 (Patent Document 2) describes the insertion of a cysteine ​​residue into the C-terminal amino acid sequence, providing a functional group that allows for conjugation of thiol-reactive polyethylene glycol chains. While biological activity is preserved, the introduction of cysteine ​​may result in intermolecular dimerization, and insect cell production yields may be lower than those from bacterial production. Furthermore, the serum half-life of such modified IL-11 is less desirable, approximately 5.6 hours for 40 kD PEG when administered intravenously to male Sprague-Dawley rats. Furthermore, animal studies using cyclophosphamide-treated rats showed minimal efficacy with a the-other-day dosing scheme. Another PEG conjugation technique using 20 kD PEG via an amine or amide bond to an N-terminally truncated sequence of IL-11 was described in US 2010 / 0098658. Although N-terminal truncation does not reduce its biological activity, its serum half-life after subcutaneous administration to male Sprague-Dawley rats was approximately 8.5 hours, again below the desired stability. Furthermore, its efficacy in animal disease models was unclear.

[0009] A 20 kD linear or branched PEG conjugated to the amine groups of IL-11 has been reported (Takagi et al. 2007, "Enhanced pharmacological activity of recombinant human interleukin-11 (rhIL11) by chemical modification with polyethylene glycol." J Control Release, 119(3):271-278 (Non-Patent Document 1)), and such non-specific conjugation often resulted in multiple PEGylation by reaction with lysine, histidine, and tyrosine residues and the N-terminal amine.

[0010] Other reports have demonstrated that certain carbohydrate modifications on the "non-core" regions of IL-11, such as the N-terminus and loops, enhanced cell-stimulating activity, suggesting that these regions are likely designed to limit the biological activity of IL-11 (Yanaka et al. 2011, "Non-core region modulates interleukin-11 signaling activity: generation of agonist and antagonist variants," J. Biol. Chem., 286:8085-8093). However, no desirable modifications have been reported that have higher stability and activity than unmodified IL-11. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent Application Publication No. 2010 / 0098658 [Patent Document 2] U.S. Patent No. 8,133,480 [Non-patent literature]

[0012] [Non-Patent Document 1] Takagi et al. 2007, “Enhanced pharmacological activity of recombinant human interleukin-11(rhIL11) by chemical modification with polyethylene glycol.”J Control Release,119(3):271-278 [Non-patent document 2] Yanaka et al.2011,“Non-core region modulates interleukin-11 signaling activity:generation of agonist and antagonist variants.”J. Biol. Chem.,286:8085-8093 Summary of the Invention [Problem to be solved by the invention]

[0013] Therefore, although several methods for stabilizing IL-11 are known in the art, all or almost all have one or more drawbacks, such as limited effectiveness and the need for repeated administration.More importantly, even in modified forms, the adverse effects of IL-11 (e.g., plasma volume expansion) are not alleviated.Therefore, there remains a need for improved compositions and methods that stabilize IL-11 while simultaneously alleviating its adverse effects. [Means for solving the problem]

[0014] Summary of the Invention The subject matter of the invention relates to compounds, compositions, and methods for improving the stability and half-life of IL-11 in serum while maintaining biological activity and reducing side effects. In a particularly preferred embodiment, the inventors have discovered that the amino acid position, the method of attachment, and the type of PEG are important for producing stable and biologically active PEGylated IL-11, and that particularly preferred PEGylated IL-11 has the same sequence as native human IL-11 but lacks the first amino acid, proline, at the N-terminus. Furthermore, such IL-11 is preferably covalently modified at the N-terminus, at a possible second site of a lysine residue within the polypeptide chain. Most typically, the average molar ratio of IL-11 to the PEG compound attached to IL-11 is 1:1.

[0015] In one aspect of the present subject matter, the inventors contemplate a modified interleukin-11 (IL-11) compound comprising an IL-11 polypeptide chain covalently attached to a PEG moiety, wherein the PEG moiety has an average molecular weight of 10-50 Kd and has different first and second PEG moieties, wherein the PEG moiety is covalently attached to the N-terminal amino acid, and wherein the IL-11 polypeptide chain is a human or humanized polypeptide chain.

[0016] Most commonly, the IL-11 polypeptide chain is a human IL-11 polypeptide chain and / or may be truncated by deletion of an N-terminal proline. For example, a particularly suitable IL-11 polypeptide chain may have the sequence of SEQ ID NO: 1. With respect to the PEG moiety, it is generally preferred that the moiety have an average molecular weight of 20 Kd or 40 Kd and / or that the PEG moiety have a Y-shape. While not limiting the subject matter, it is preferred that the molar ratio of polypeptide chain to PEG moiety is about 1:1 (e.g., 0.9:1 to 1:0.9, or 0.8:1 to 1:0.8). In addition, it is contemplated that a second PEG moiety may be covalently attached to the modified IL-11 via an amino group on the IL-11 polypeptide chain. Furthermore, it is generally preferred that the PEG moiety be covalently attached to the N-terminal amino acid via an amine bond (although an amide bond is also specifically contemplated).

[0017] From another perspective, the inventors also contemplate pharmaceutical compositions comprising a therapeutically effective amount of the subject IL-11 compound (e.g., as described above) in combination with a pharmaceutically acceptable carrier. If desired, the compositions can be formulated for injection and may include an IL-11 compound, where the IL-11 compound is present in an amount providing a dosage unit of 10-100 μg / kg for a pediatric or adult patient. Furthermore, it is contemplated that the compositions can be lyophilized or in liquid form for injection or infusion. Most preferably, the pharmaceutical composition may further comprise a second pharmaceutically active compound, either separately or in admixture with the IL-11 compound. Accordingly, kits containing the contemplated pharmaceutical compositions together with other ingredients (e.g., a second pharmaceutically active compound, such as a steroid, an agent that stimulates platelet production in the bone marrow, an antibody, an analgesic, or an anti-inflammatory agent, or a solvent for reconstitution) are expressly contemplated herein.

[0018] Therefore, the inventors also contemplate the use of the IL-11 compounds of the present invention in the manufacture of pharmaceutical compositions. While not limited to the present invention, specific contemplated treatments include: (a) nuclear accident / radiation-induced bone and gastrointestinal disorders, (b) chemotherapy-induced bone and gastrointestinal disorders, (c) burn-induced thrombocytopenia and gastrointestinal disorders, (d) chemotherapy-induced thrombocytopenia, (e) trauma-, cancer-, or infection-induced gastrointestinal disorders or inflammatory bowel disease, (f) free radical-induced lung disorders, and (g) cardiovascular diseases. As mentioned above, it is generally contemplated to formulate the pharmaceutical composition for injection and / or lyophilize the pharmaceutical composition.

[0019] Thus, in a further aspect of the inventive subject matter, the inventors also contemplate a method for increasing the serum half-life of an interleukin-11 (IL-11) compound. A preferred method includes covalently linking an IL-11 polypeptide chain to a PEG moiety, wherein the PEG moiety has an average molecular weight of 10-50 Kd and has different first and second PEG moieties, the PEG moiety is covalently linked to the N-terminal amino acid, and the IL-11 polypeptide chain is a human or humanized polypeptide chain. Most commonly, the IL-11 polypeptide chain is a human IL-11 polypeptide chain and / or the IL-11 polypeptide chain is truncated by deletion of an N-terminal proline (e.g., having the sequence of SEQ ID NO: 1).

[0020] In further contemplated methods, the PEG moiety can have an average molecular weight of 20 Kd or 40 Kd and / or have a Y-shape. If desired, the molar ratio of polypeptide chain to PEG moiety is about 1:1, and it is further contemplated that the method can further comprise covalently attaching a second PEG moiety through an amino group in the IL-11 polypeptide chain. As before, it is contemplated that the PEG moiety is covalently attached to the N-terminal amino acid through an amine bond.

[0021] In further contemplated methods, the inventors contemplate methods for treating conditions responsive to administration of IL-11. Such methods typically involve administering a therapeutically effective amount of a contemplated pharmaceutical composition to a patient in need thereof. For example, suitable conditions can be selected from the group consisting of (a) nuclear accident / radiation-induced bone and gastrointestinal disorders, (b) chemotherapy-induced bone and gastrointestinal disorders, (c) burn-induced thrombocytopenia and gastrointestinal disorders, (d) chemotherapy-induced thrombocytopenia, (e) trauma-, cancer-, or infection-induced gastrointestinal disorders or inflammatory bowel diseases, (f) free radical-induced lung disorders, and (g) cardiovascular diseases. Exemplary preferred pharmaceutical compositions for these methods can include IL-11 I40NY or I20NY, and it is further contemplated that IL-11 is administered (e.g., subcutaneously) at a dose of 10-100 μg / kg.

[0022] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings, in which like numerals represent like components. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows the primary sequence of IL-11 without the N-terminal proline. [Figure 2] FIG. 2 is an image of an SDS-PAGE gel with molecular weight markers and various PEGylated forms of IL-11 as indicated. [Figure 3] FIG. 3 is a graph depicting plasma concentrations of various IL-11 compositions after a single intravenous administration. [Figure 4] FIG. 4 is a graph depicting platelet increases following a single intravenous administration of various IL-11 compositions. [Figure 5] FIG. 5 shows chromatograms comparing peptide maps for tryptic digests of unconjugated IL-11, I40NY and I40KY. [Figure 6] FIG. 6 is a graph depicting platelet expansion after subcutaneous administration of various IL-11 compositions (daily injections for 14 consecutive days for IL-11 and weekly injections for the pegylated counterpart). [Figure 7] FIG. 7 is a graph depicting hematocrit reduction following subcutaneous administration of various IL-11 compositions (daily injections for 14 consecutive days for IL-11 and weekly injections for the pegylated counterpart). [Figure 8] FIG. 8 is a graph suggesting a correlation between maximum platelet induction and maximum decrease in hematocrit. [Figure 9] FIG. 9 is a graph depicting the cell proliferation activity of PEGylated compounds in the 7TD1 assay compared to unconjugated IL-11. [Figure 10] FIG. 10 is an image of a silver-stained non-reducing SDS-PAGE gel showing the purity of I40NY at various loading amounts. [Figure 11]FIG. 11 is an HPLC chromatogram showing the product purity of the monoPEGylated components of I40NY. [Figure 12] FIG. 12 is a pharmacokinetic profile showing the kinetics of plasma concentrations of I40NY after a single subcutaneous dose compared to a single subcutaneous dose of unconjugated IL-11. [Figure 13] FIG. 13 is an overlay of circular dichroism spectra of IL-11 and I40NY. [Figure 14] FIG. 14 is an ellipticity plot for IL-11 and I40NY as a function of temperature. [Figure 15] FIG. 15 is a pharmacodynamic profile showing platelet production of a putative compound in a myelosuppressed rat animal model. [Figure 16] FIG. 16 is a graph showing hematocrit reduction of putative compounds in a myelosuppressed rat animal model. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present inventors have found that the type of PEG compound, the location of covalent attachment, and the primary sequence of IL-11 are determinants of the stability and activity of such modified IL-11. In a particularly preferred and unexpected embodiment, the present inventors have found that when N-terminally truncated by one amino acid and then PEGylated, IL-11 has substantially improved stability. Furthermore, the present inventors have also found that the specific type and molecular weight of the PEG moiety are further determinants of stability, activity, and toxicity, as further described in more detail below.

[0025] Possible compounds To investigate the effects of PEG type, molecular weight, and attachment position on IL-11, we prepared various PEGylated IL-11 molecules from recombinant human IL-11 having the primary sequence shown in Figure 1 (identical to the native human IL-11 sequence but lacking the N-terminal proline). It is generally preferred that the IL-11 protein be an N-terminally truncated or modified human IL-11. For example, particularly preferred truncated forms include IL-11 molecules lacking at least one, two, or three (or more) N-terminal amino acids. Alternatively, IL-11 may also be modified to have an N-terminal amino acid different from that of its human unmodified counterpart. For example, modified IL-11 may lack the first N-terminal amino acid and may have a second amino acid other than that found in unmodified human IL-11 (e.g., P deleted and G replaced with V). Most commonly, the N-terminal amino acid is a stabilizing amino acid and thus includes, inter alia, M, G, A, S, T, V, or P; in further contemplated embodiments, a destabilizing amino acid (e.g., F, Q, N, R, etc.) may be replaced by a stabilizing amino acid. Deletion of one or more amino acids from the N-terminus is usually limited to the first 10, or the first 5, or the first 3 amino acids, while in less preferred embodiments, deletion of one, two, three, four, five, or more amino acids may also occur at the C-terminus of the IL-11 component. As a general guideline, deletions are generally limited to those that do not adversely affect, or have only a minimal effect on, biological activity and / or stability (e.g., less than a 20%, more usually less than a 10% loss of activity and / or stability). Alternatively, or in addition, contemplated IL-11 molecules also include fusion proteins with IL-11, exemplary fusion proteins include those described in US2010 / 0143973, which is incorporated herein by reference. Most typically, IL-11 is a recombinant protein, which may be expressed in a suitable expression system, most preferably in a prokaryotic system (e.g., E. coli) or a yeast system (e.g., Pichia pasteuris).Of course, it should also be understood that a particularly preferred form of IL-11 is the mature form (ie, without the leader sequence).

[0026] Furthermore, it should be understood that a suitable IL-11 molecule need not be human IL-11, but may be of any other (generally mammalian) origin. Thus, suitable sources of IL-11 (recombinant or natural) include primate, murine, porcine, equine, etc. These sequences may therefore be at least partially humanized to reduce immunogenicity in humans and / or increase stability and / or activity. Similarly, synthetic consensus sequences are also contemplated herein.

[0027] The PEGylation of the contemplated IL-11 molecule can be carried out in many ways, including covalent and non-covalent methods. However, it is generally preferred that the PEGylation utilize a covalent bond to IL-11. Many methods are known in the art for covalently linking a PEG group to a protein. Suitable methods include reacting the N-terminal amino group or C-terminal carboxylic acid group with a suitable reactive group on the PEG moiety (e.g., aldehyde, maleimide, acid chloride, etc.) or a sulfhydryl-reactive group (e.g., maleimide, pyridyl disulfide, vinyl sulfone, etc.), thereby enabling disulfide bonding to an amino-reactive reagent that reacts with a cysteine ​​group or the ε-amino group of a lysine amino acid (e.g., NHS-ester, NHS-carbonate, triazine group, etc.). Therefore, it is also contemplated that one or more amino acids can be added to the N- and / or C-terminus to introduce a suitable reactive group for attaching a PEGylated group. For example, serine or threonine can be added to allow for enzymatic conjugation using N-acetylgalactosamine or PEG sialic derivatives, or lysine can be added for covalent attachment to the ε-amino group, or phenylalanine or threonine groups for attachment to the hydroxyl group.

[0028] With respect to PEG molecules suitable for use herein, a variety of molecular weights for PEG are generally considered suitable, with contemplated molecular weights ranging from 2 Kd to 200 Kd (average or nominal molecular weight). However, particularly preferred molecular weights (average or nominal molecular weights) include those of 10 to 50 Kd per linear chain in the PEGylated moiety. Furthermore, it is generally preferred that the PEG moiety have a single linear or Y-shaped PEG moiety, with such PEG moieties having molecular weights of 20 to 40 Kd even more preferred. Alternatively, suitable PEG moieties may also include dendritic PEG structures, and the PEG moiety may have more than two linear chains. When the PEG moiety has more than one linear PEG chain, it is generally preferred that the chains have substantially the same average molecular weight (average molecular weights differing by less than 15%).

[0029] In a further preferred embodiment, the PEG moiety is covalently attached to IL-11 via its N-terminal amino group and / or (optionally) to the epsilon-amino group of an internal lysine or the ring nitrogen of a histidine. For N-terminal covalent attachment, the molar ratio of IL-11 to the PEG moiety is preferably about 1:1 (e.g., 0.9:1 to 1:0.9, or 0.8:1 to 1:0.8, etc.). In addition, it should be understood that moderate levels of PEGylation may be present at internal amino acid residues (e.g., 10% to 20%, or 1% to 10% of the total IL-11 may have additional PEGylated internal amino acids). For example, a second PEG moiety may be attached to the epsilon-amino group of an internal lysine or histidine. As further described in more detail below, a particularly preferred form of PEGylated IL-11 is I40NY, which comprises human IL-11 (lacking an N-terminal proline) and to which a Y-shaped PEG moiety having an average molecular weight of 40 Kd is attached at the N-terminus.

[0030] In yet another embodiment, it should be understood that PEGylation can be mixed with respect to the attachment position and / or type of attachment of the PEG moiety. Thus, IL-11 can be subjected to random non-covalent PEGylation and site-specific PEGylation at the N-terminal amino acid, or can be subjected to different site-specific PEGylation at the N-terminal amino acid and at an internal amino acid. For example, and most preferably, IL-11 (or any modified form thereof) can be PEGylated at the N-terminal amino acid, and optionally, in addition to the N-terminal modification, can be PEGylated at an internal amino acid via a nitrogen atom (e.g., from lysine or histidine).

[0031] For example, and using truncated IL-11 as shown in FIG. 1, following the experimental protocol provided by the manufacturer and as described in more detail below, we performed PEGylation using PEG reagents as shown in Table 1 (where n and m are independently integers between 80 and 1000, depending on the molecular weight of the compound).

[0032] [Table 1]

[0033] After PEGylation of truncated IL-11, the compounds so obtained were purified as further detailed below, and the various PEGylated IL-11 molecules were given the following designations as shown in Table 2.

[0034] [Table 2]

[0035] Most notably, the inventors have found that the type and attachment site of the PEG moiety (and to some extent the sequence of IL-11) have unexpected and substantial effects on biological activity and in vivo stability. As will become more apparent from the experimental data below, a particularly preferred PEGylation is at the N-terminal amino acid using a single Y-shaped PEG moiety, especially when IL-11 is truncated.

[0036] Contemplated Composition : Based on the inventors' discovery of the expanded biological activity of the contemplated compounds, it is generally contemplated that the subject compounds of the present invention can be formulated for the treatment of a variety of diseases associated with a deficiency of IL-11 or characterized by a therapeutic response to treatment with IL-11. Accordingly, and among other contemplated uses, the inventors particularly believe that pharmaceutical compositions comprising the contemplated compounds may be effective for the treatment or prevention of (a) chemotherapy-induced thrombocytopenia, (b) nuclear accident / radiation-induced bone and gastrointestinal (GI) disorders, (c) chemotherapy-induced bone and GI disorders, (d) burn-induced thrombocytopenia and GI disorders, (e) other causes of thrombocytopenia, (f) inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, and other causes of GI disorders, including pseudomembranous colitis, (g) free radical-induced lung disorders, and / or (h) cardiovascular disease, in which case the contemplated pharmaceutical compositions comprise a therapeutically effective amount of the contemplated compound (or a pharmaceutically acceptable salt, hydrate, or prodrug thereof) and a pharmaceutically acceptable carrier. For example, in one embodiment of the inventive subject matter, contemplated compositions are formulated for the treatment of chemotherapy-induced thrombocytopenia or GI disorders or radiation-induced bone and gastrointestinal (GI) disorders. It should also be understood that contemplated compositions can alternatively, or additionally, be formulated to induce acute phase proteins and / or modulate antigen-antibody responses.

[0037] It is particularly preferred that the contemplated compounds are included in compositions formulated with one or more non-toxic pharmaceutically acceptable carriers.Suitable pharmaceutical compositions are preferably formulated for injection or infusion, or for oral administration in solid or liquid form.Thus, it should be understood that the pharmaceutical compositions of the present subject matter can be administered to humans and other (usually mammalian) animals using a variety of routes, including parenteral, oral, intraperitoneal, and topical.

[0038] For example, pharmaceutical compositions suitable for injection preferably include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, emulsions, or suspensions, as well as sterile powders to be reconstituted into sterile injectable solutions or dispersions before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, Ringer's solution, and isotonic sodium chloride solution, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, oils, and injectable organic esters (e.g., ethyl oleate). Contemplated compositions may also contain various inactive ingredients, including preservatives, wetting agents, emulsifying agents, and / or dispersing agents. Sterility can be ensured by the inclusion of antibacterial and / or antifungal agents (e.g., parabens, phenol sorbic acid, chlorobutanol, etc.), filtration through submicron membranes (e.g., 0.45 μM or 0.22 μM pore size), autoclaving or pasteurization, and irradiation (e.g., gamma or E-beam). Where appropriate, osmotically active agents can be included (e.g., sugars, sodium chloride, etc.). While not limiting to the subject matter of the present invention, contemplated injectable formulations generally have a pH range of 3-9, more commonly 6-8, and most commonly 7.4 + / - 0.3. Of course, it should be understood that all liquid formulations can be preserved in a variety of ways to facilitate long-term storage / stockpiling. For example, contemplated stabilization methods include water / solvent removal using lyophilization, spray drying, crystallization, adsorption onto a (preferably biocompatible or pharmaceutically acceptable) solid phase, etc.

[0039] Compositions according to the present subject matter can be administered using a variety of routes, including orally, parenterally, by inhalation, topically, rectally, nasally, or via an implanted reservoir, where the term "parenteral," as used herein, includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrathecal, intrahepatic, intralesional, and intracranial administration (generally by injection or infusion). Preferably, the compositions are administered by injection, typically intravenously, and more preferably subcutaneously. Contemplated pharmaceutical compositions can also be applied topically, particularly when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, skin, lower intestinal tract, or areas exposed during surgical intervention. Many topical formulations are known in the art, and all such formulations are considered suitable for use herein.

[0040] With regard to the amount of compound that is intended in the composition, it should be recognized that the specific amount typically depends on specific formulation and desired purpose.Therefore, it should be understood that the amount of compound that is intended can vary significantly.However, it is generally preferred that compound is present in the minimum amount that is effective for delivering therapeutic effect in vitro and / or in vivo.

[0041] Thus, in the most preferred embodiments, a contemplated compound is present in an amount of about 0.1 μg / ml to about 100 mg / ml, more typically about 10 μg / ml to about 10 mg / ml, and most typically about 5 μg / ml to about 100 μg / ml. With respect to dosage units, it is generally contemplated that a contemplated compound will be administered at a dosage effective to achieve the desired therapeutic effect, typically 10-100 μg / kg, more preferably 30-70 μg / kg. However, alternate-day dosage units can be 0.1-10 μg / kg, or 50-80 μg / kg, or 80-120 μg / kg, or 120-200 μg / kg, or even higher. From a different perspective, the single-use unit of the envisioned formulation may be about 0.3 mg to 3.0 mg of PEGylated IL-11, or about 3 mg to 7 mg of PEGylated IL-11, or about 7 mg to 10 mg of PEGylated IL-11 (most commonly 7 to 9 × 10 mg). 6 It should be understood that values ​​may include any range (having a specific activity in U / mg). Unless the context dictates otherwise, all ranges set forth herein should be construed to be inclusive of their endpoints, and open-ended ranges should be construed to include commercially feasible values. Similarly, all lists of values ​​should be deemed to include intermediate values ​​unless the context dictates otherwise. It should be.

[0042] Additionally, it should be noted that contemplated formulations may include one or more additional pharmaceutically active agents, which may be present in the same formulation or may be available separately (in different or the same formulation) or sold as a kit. For example, suitable additional pharmaceutically active agents include various steroids (e.g., corticosteroids), agents that stimulate platelet production in the bone marrow (e.g., Li2CO3, folic acid, etc.), antibodies, analgesics, and anti-inflammatory agents.

[0043] Intended Use : The contemplated compounds may be particularly useful as therapeutic agents for use alone or in combination in the treatment of (a) nuclear accident / radiation-induced bone and gastrointestinal (GI) disorders, (b) chemotherapy-induced bone and GI disorders, (c) burn-induced thrombocytopenia and GI disorders, (d) other causes of thrombocytopenia, (e) inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, and other causes of GI disorders including pseudomembranous colitis, (f) free radical-induced lung disorders, and (g) cardiovascular diseases.

[0044] As a result, the inventors also contemplate the use of the compounds provided herein for the manufacture of a medicament for the treatment of (a) nuclear accident / radiation-induced bone and GI disorders, (b) chemotherapy-induced bone and GI disorders, (c) burn-induced thrombocytopenia and GI disorders, (d) other causes of thrombocytopenia, (e) inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, and other causes of GI disorders, including pseudomembranous colitis, (f) free radical-induced lung disorders, and (g) cardiovascular diseases.

[0045] From another perspective, the inventors also contemplate methods for treating (a) nuclear accident / radiation-induced bone and GI disorders, (b) chemotherapy-induced bone and GI disorders, (c) burn-induced thrombocytopenia and GI disorders, (d) other causes of thrombocytopenia, (e) inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, and other causes of GI disorders including pseudomembranous colitis, (f) free radical-induced lung disorders, and (g) cardiovascular diseases in a human in need thereof, comprising administering a therapeutically effective amount of a contemplated compound.

[0046] Experiments and Experimental Data : material : Purified bulk recombinant human IL-11 from yeast was provided by Hangzhou Jiuyuan Gene Engineering Company (Lot# 20121005 / 1006 / 1007 / 1008). 7TD1 mouse hybridoma cell line was obtained from DSMZ (No. ACC23). Paraplatin® injection (generic name: carboplatin) 10 mg / mL (Lot: 5A03935) was from Bristol-Myers Squibb Company. Sequencing-grade trypsin modified from bovine pancreas (Cat. No. 11418025001) was purchased from Roche diagnostics. Mouse IL-11 receptor alpha was obtained from MyBioSource, Inc. (Cat. No. MBS553276). CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay (MTS) (catalog number G5430) was purchased from Promega for the 7TD1 cell assay. The DuoSet ELISA development kit for human IL-11 was purchased from R&D Systems Inc. (catalog number DY218). Purification resin MacroCap SP (product code 17-5440-01) was obtained from GE Healthcare Life Sciences. Precise Tris-Glycine 8-16% polyacrylamide gel was purchased from Thermo Scientific. HPLC-grade trifluoroacetic acid (catalog number 302031) and acetonitrile (catalog number 34967) were purchased from Sigma-Aldrich.

[0047] Various forms of monofunctional PEGs with catalog numbers SUNBRIGHT® ME-120TS, ME-200AL, GL2-400TS, ME-050TS, GL4-400AL3, GL2-200AL3 were purchased from NOF Corporation, and Y-PALD-40K was purchased from Jenkem Technology USA. The molecular structures of the PEG reagents are listed above in Table 1.

[0048] Preparation of I12KL / I40KY / I05KL4 : 5 mg / mL of protein was introduced into a 1-2x molar mixture of each PEG reagent (NOF / SUNBRIGHT ME-120TS for I12KL and SUNBRIGHT GL2-400TS for I40KY) and 50 mM NaHCO3 at pH 8. I05KL4 (PEG reagent: NOF / SUNBRIGHT ME-120TS) was prepared in the same manner, except that a 12x molar ratio of PEG to protein was added. The reaction mixture was incubated at room temperature for 2 hours and then quenched with 2 mM glycine. The PEGylated product was isolated using a chromatographic purification procedure as follows: PEG molecules were attached to the protein via amide bonds.

[0049] Preparation of I20NL / I40NY / I20NY / I20NL2 / I20NY2 / I40NX : 5 mg / mL of protein was introduced into the reaction mixture with a 1-2x molar mixture of each PEG reagent (NOF / SUNBRIGHT ME-200AL for I20NL and I20NL2, Jenkem / Y-PLAD-40 for I40NY, NOF / SUNBRIGHT GL2-200AL3 for I20NY and I20NY2, and NOF / SUNBRIGHT GL4-400AL3 for I40NX), 10 mM sodium cyanoborohydride, and 50 mM NaH2PO4. To conjugate onto two sites, PEG was added at a 3.5-5.5x molar ratio. The pH was adjusted to approximately 4.5-5.0. The reaction mixture was incubated at room temperature for 24 hours and subsequently quenched with 2 mM glycine. PEG molecules were attached to the protein via the more stable amine bond. The PEGylated products were isolated using chromatographic purification as follows:

[0050] Chromatographic purification : The pH of the protein solution was adjusted to 4-5 with 1 M acetic acid, followed by centrifugation or filtration to remove particulate matter. Four volumes of water were added. For conjugates containing PEG greater than 20 kDa, the protein solution was loaded onto a MacroCap SP column (1 x 6 cm) equilibrated with Buffer A containing 20 mM sodium acetate, pH 5. The protein was eluted by gradient or step elution with Buffer B containing 20 mM sodium acetate, pH 5, and 1 M NaCl. For conjugates containing PEG less than 20 kDa, the protein solution was loaded onto a MacroCap SP column (1 x 6 cm) equilibrated with Buffer A containing 20 mM sodium phosphate, pH 7. The protein was eluted by gradient or step elution with Buffer B containing 20 mM sodium phosphate, pH 7, and 1 M NaCl. A typical final product analyzed by SDS-PAGE gel can be seen in Figure 2. Here, molecular weight markers were loaded in the left lane, and various PEGylated forms of IL-11 were loaded in the remaining lanes. It should be noted that I40NY eluted with an apparent molecular weight of over 100 Kd, which is greater than the estimated 60 Kd, likely due to the Y-shaped nature of its PEG moiety. In a further particularly preferred embodiment, purification of the contemplated compound is carried out as a one-step purification process, which provides added advantages in downstream scale-up.

[0051] Purity check by RP-HPLC : The content of each PEGamer was analyzed by reversed-phase (RP) chromatography using UPLC coupled with a diode array detector—Thermo Scientific UltiMate 3000 Rapid Separation LC Systems. The chromatography procedure was performed using the following: Column: Acquity C18, 1.7 μm, 2.1 × 150 mm, 300 Å pore size, equipped with a guard cartridge; Mobile Phase A: 0.1% (v / v) TFA in 50% (v / v) acetonitrile; Mobile Phase B: 0.1% (v / v) TFA in 95% (v / v) acetonitrile; Flow Rate: 0.4 ml / min; Column Temperature: 65°C; Detection: 214 nm; 20 μg was injected, and the gradient was as shown in Table 3 below.

[0052] [Table 3]

[0053] Protein content measurement : Protein content was measured by UV / Vis microplate and cuvette spectrophotometer - Multiskan GO from Thermo Scientific. Units M at 280 nm measured in water. -1 cm - The extinction coefficient of 1 is 17,990. Alternatively, protein concentration is measured directly by ultraviolet spectroscopy at a wavelength of 280 nm using an absorbance value of 0.944 for a 0.1% (1 mg / ml) solution. Protein quantification using absorbance at 280 nm measures the absorbance of aromatic amino acids such as tryptophan and tyrosine, and PEG components remain undetectable. As a result, the protein concentrations reported herein by weight do not include PEG molecules.

[0054] Pharmacokinetic (PK) studies in healthy rats : In vivo manipulations were performed in three male Sprague-Dawley rats after a single intravenous or subcutaneous administration of the intended compound at a dose level of 100–150 μg / kg. Blood samples were collected at multiple time points into heparinized tubes, followed by plasma separation and storage at -20°C. The concentration of immunoreactive IL-11 in the plasma samples was measured using a DuoSet ELISA kit for human IL-11 (R&D Systems Inc., catalog number DY218). Pharmacokinetic parameters were obtained using WinNonlin 5.3 software using a noncompartmental model.

[0055] Pharmacodynamic (PD) studies in healthy rats : Pharmacodynamic evaluation was performed in four male Sprague-Dawley rats using intravenous or subcutaneous administration of each of the proposed compounds at dosage strengths of 100-150 μg / kg. Blood samples were collected at multiple time points into heparinized tubes, followed by plasma separation and storage at -20°C. Blood cell counts were performed on a Cell-DYN 3500 hematology analyzer.

[0056] Pharmacodynamic (PD) studies in myelosuppressed rats : Pharmacodynamic evaluation was performed in four male Sprague-Dawley rats by inducing myelosuppression using intravenous administration of 40 mg / kg carboplatin on day 0. The proposed compound was injected subcutaneously at 150 μg / kg on day 1. Blood samples were collected at multiple time points into heparinized tubes, followed by plasma separation and storage at -20°C. Blood cell counts were performed on a Cell-DYN3500 hematology analyzer.

[0057] Trypsin mapping : A reaction solution was prepared in 50 mM Tris pH 8.3 buffer containing 2 mg / mL protein and 1 / 50 (w / w) trypsin. After incubation at room temperature for 6 hours, an equal volume of 0.2% TFA (trifluoroacetic acid) solution was added. Particulate matter was removed by centrifugation and then injected into the HPLC. Chromatography was performed using a Zorbax 300SB-C8 column, 2.1 x 150 mm, 5 μm, 300 Å pore size. Mobile phase A: 0.1% (v / v) TFA. Mobile phase B: 0.1% (v / v) TFA in 95% (v / v) acetonitrile. Flow rate: 0.2 ml / min. Detection: 214 nm. 10 μg was injected, and the gradient was as shown in Table 4 below.

[0058] [Table 4]

[0059] Identification of proteolytic peptides was performed by HPLC coupled with MS spectroscopy (Thermo LCQ Advantage).

[0060] In some embodiments, numbers expressing properties such as amounts and concentrations of ingredients, reaction conditions, and the like, used to describe and claim certain embodiments of the invention should be understood to be modified in some instances by the use of the word "about." Accordingly, in some embodiments, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties targeted by particular embodiments. In some embodiments, the numerical parameters should be analyzed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values ​​set forth in certain embodiments are reported as precise as practicable. The numerical values ​​presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0061] Although it is generally known that PEG can confer plasma stability to its conjugates, it is impossible to predict which bond type, chain length, and molecular structure will result in specific results regarding therapeutic efficacy and / or pharmacological parameters. In the first example, truncated IL-11 conjugates with various PEG sizes were investigated for plasma stability in normal rats. After intravenous administration, the observed plasma half-life of non-PEGylated IL-11 was found to be very short, less than 10 minutes, compared to the plasma half-life of its PEGylated counterpart (3.5-13.7 hours). Among the latter, higher molecular weight PEGs conferred greater plasma stability in the following order: I40KY (13.7 hours) - I40NY (8.5 hours) > I20NL (3.8 hours) - I12KL (3.5 hours). Figure 3 shows the plasma concentrations of various forms of PEGylated IL-11 after a single intravenous administration. Each sample was administered at 100 μg / kg in rats. In this example, it was concluded that the larger or longer the PEG chain, the longer the serum half-life.

[0062] In a second example, pharmacodynamic evaluation was performed in healthy Sprague-Dawley rats via the intravenous route to measure platelet induction after a single intravenous dose of 100 μg / kg. As shown in Figure 4, the 40-KD conjugates (I40NY and I40KY) induced a greater platelet increase (60-75%) than I20NL (50%), where Y represents Y-shaped PEG and L represents linear PEG. The results also suggest that multiple conjugations (I05KL4, a 5-KD PEG conjugated at four sites) were less effective than a single long PEG chain at the N-terminus, as multiple short PEG conjugates had only limited efficacy, with platelet induction of approximately 25%. In this example, it was concluded that the longer the PEG chain at one site, the greater the efficacy in terms of platelet induction. This appears to be the opposite of the effect of PEGylation on recombinant human growth hormone.

[0063] In a third example, the conjugation sites were investigated by tryptic mapping in conjunction with LC / MS identification of proteolytic peptides. Figure 5 shows the tryptic maps of IL-11 (unconjugated), I40NY, and I40KY, and Table 5 below provides the tryptic peptides.

[0064] [Table 5]

[0065] Here, it can be seen that the peak corresponding to the T1 peptide was significantly reduced in the tryptic maps of I40NY and I40KY. This indicates that both conjugates were PEG-linked to the T1 peptide, where the N-terminal amine is the only site for chemical conjugation. Consequently, I40NY and I40KY were linked to both N-termini, but the only difference was that I40NY was linked via an amine bond, whereas I40KY was linked via an amide bond. Notably, both I40NY and I40KY showed similar serum half-life and platelet induction following intravenous administration.

[0066] Amine bonds are more stable than amide bonds, and the yield of mono-PEGylated products is more uniform with selective PEGylation using reductive amination. In the next study, various N-terminal conjugates were investigated for their efficacy in platelet production and their associated side effects were evaluated by subcutaneous administration in healthy rats. Figure 6 shows the results of a pharmacodynamic study of six PEGylated IL-11 conjugates administered subcutaneously at 150 μg / kg in rats. IL-11 was administered daily for 14 consecutive days, while PEGylated IL-11 was injected once weekly. PEG shape can affect the function of the conjugate. In particular, nonlinear PEG molecules confer better plasma stability and higher efficacy than their linear counterparts. As shown in Figure 6, I20NY induced a greater platelet increase (58-70%) than I20NL (46-55%), where Y represents Y-shaped PEG and L represents linear PEG. These results suggested that Y-shaped PEGs had higher efficacy than linear forms of the same molecular weight. However, I40NY (Y-shaped) and I40NX (four-comb) were comparable in platelet production, both increasing by approximately 65–70%, suggesting that the effect of PEG shape was saturated when PEG size was approximately 40 kD or larger. Notably, I20NL2 (two-site linear PEG) and I20NY2 (two-site Y-shaped PEG) had lower platelet production than their single-PEGylated counterparts, so double PEGylation of the same PEG length reduced in vivo efficacy. Therefore, it was concluded that I40NY, I40NX, and I20NY exerted higher efficacy among various N-terminally conjugated IL-1Is. Furthermore, it should be noted that the effect of the second administration was somewhat downregulated for smaller PEG conjugates, such as 20-kD PEG, regardless of the number of conjugation sites. As a result, I40NY and I40NX were unexpectedly effective compounds with desirable biological properties and relatively few adverse effects, particularly plasma volume expansion. Furthermore, the biological data further suggest that such modified IL-11 compounds can be administered less frequently, most preferably twice a week, once a week, or less frequently.Such a schedule is particularly relevant when the contemplated compound is used in the treatment of thrombocytopenia in larger populations (eg, those exposed to radiation exposure).

[0067] We also studied the side effects associated with IL-11 conjugates in healthy rats. Because patients may experience dilutional anemia due to plasma volume expansion, hematocrit status is typically used as a marker for evaluating side effects in clinical IL-11 use. In animal studies, IL-11 was administered subcutaneously at 150 μg / kg for 14 consecutive days, while PEGylated IL-11 was injected once a week at the same dose. As shown in Figure 7, all drugs resulted in a decrease in hematocrit, but I40NY was found to cause less of a decrease while maintaining higher activity than the remaining PEGylated conjugates. The alleviation of dilutional anemia with I40NY administration is more pronounced when compared on the same chart with other individual animal studies using conjugated and unconjugated IL-11. The correlation between platelet production and side effects, as indicated by a decrease in hematocrit, is demonstrated in Figure 8, which suggests a trend toward increasing side effects with increasing dose-dependent efficacy when various modified and unmodified IL-11 are plotted on a chart. I40NY at various doses clearly falls in the upper right corner, which means that it increases plasma volume less than certain other compounds and unmodified IL-11 on a comparable efficacy basis. In terms of product characterization, the inventors characterized the physicochemical and pharmacological properties of the preferred compound, I40NY.

[0068] Cell-based assay of IL-11 conjugates : The biological activity of conjugated IL-11 was tested in a cell proliferation assay using the 7TD1 cell line (DSMZ, Germany). Briefly, 4,000 7TD1 cells per well were grown at 37°C in a humidified atmosphere with 5% CO2 for 2 days in the presence of 2 μg / mL mouse IL-11 receptor (MyBioSource, USA, MBS553276) against various IL-11 concentrations (Biochem. J., 318:489-495). After adding MTS, the absorbance at 490 nm on the y-axis was plotted against the IL-11 concentration on the y-axis, and the EC50 of the dose-response curve was determined by fitting a sigmoidal dose-response curve using GraphPad software Prism 6. Prior to animal testing, the biological activity of the newly synthesized conjugates was tested in a cell proliferation assay using the 7TD1 cell line. Not all PEGylated preparations yield similar products; actual product formation depends on the amino acid residue of IL-11 conjugated and the size and shape of the PEG molecule used. 7TD1 cells proliferated in response to different conjugate concentrations. After adding a developer, whose chemical signal has a linear relationship with cell number, the absorbance at 490 nm was read using an ELISA plate reader. The results are shown in Figure 9.

[0069] Due to steric hindrance of the PEG moiety, all conjugates showed predicted decreased bioactivity in cell-based assays compared with non-PEGylated IL-11, with the following order of potency: IL-11 (100%) > I20NL, I20NY (both approximately 16%) > I40NY, I40NX (both 11%) > I40KY (6%) > I20NY2 (3%). Steric hindrance is a dominant factor in determining the biological activity of PEGylated conjugates, resulting in a significant decrease in bioactivity for conjugates with an overall PEG content greater than 20 kDa. Some cell-based studies have shown that small hydrocarbon linkages in non-core regions of the IL-11 molecule, such as the N-terminal sequence, enhanced bioactivity compared with conjugates at other linkage sites ( J. Biol. Chem. Vol. 286, No. 10, pp. 8085–8093 ). This is consistent with the minimal decrease in bioactivity due to PEG molecules at the N-terminal sequence of IL-11. Although the in vitro bioactivity of I40NY retains only about 11% of that of native IL-II, the in vivo efficacy is favorably affected and was not predicted from the in vitro bioactivity data. Table 6 below summarizes the bioactivity ratios of various compounds relative to unmodified IL-11.

[0070] [Table 6]

[0071] Chemical modification of proteins with PEG is an established technique and has been applied in the biopharmaceutical industry to enhance protein solubility and physicochemical stability. While this chemical reaction is easy to perform, it often results in a complex mixture of different PEGylated forms, including PEGamers and positional isomers. Multiple chromatographic purification steps are used to isolate the product with high yields. To develop a commercially viable process in terms of cost and yield, it is necessary to optimize many factors, including protein concentration, PEG quality, protein / PEG ratio, reaction temperature, and buffer pH, as well as the purification process.

[0072] I40NY was constructed by conjugation of a Y-shaped polyethylene glycol chain on the amine moiety, which forms a stable amine bond with relatively high selectivity to the N-terminal amine, driven by conjugation chemistry (reductive amination of the aldehyde coupling group in the PEG moiety). I40KY, on the other hand, was conjugated using an NHS reagent functionalized at pH 8 on accessible amines to form the corresponding amide bond. More specifically, since the functionalized aldehyde is highly selective for the N-terminal α-amine, whose pKa is lower than that of other nucleophiles, I40NY is produced by a site-specific reaction under acidic conditions. The PEG-to-protein ratio, reaction concentration, pH, and kinetics were investigated in the conjugation reactions. The reactions were carried out in a small scale (approximately 0.05–0.5 mL) at room temperature (22–27 °C) for 24 h in the presence of 10 mM sodium cyanoborohydride. The yield of each investigated reaction was measured by RP-UPLC. Using different pH values ​​for selected reactions, the optimal conjugation yield was found to be at pH 4.5-5.5. Additionally, we found that reactant concentration plays an important role in product yield, with conjugation with IL-11 at concentrations greater than 5 mg / mL being optimal. Similarly, we investigated the PEG-to-protein ratio and conjugation kinetics of reactions with 5 mg / mL protein at room temperature in the presence of a reducing agent, suggesting that an optimal PEG-to-protein molar ratio of 2 was achieved by extending the reaction for up to 16 hours, sufficient for monoPEGylation.

[0073] Purification of PEGylated proteins typically involves the use of ion exchange chromatography in large-scale preparations. However, satisfactory resolution for separating mono- and oligo-PEGylated products is not achieved when loading reaction products onto conventional ion exchangers at loading capacities as low as 1 mg per mL of resin. This low capacity of resins often limits their application for larger-scale production. To isolate N-terminally mono-PEGylated IL-11 with high purity, various cation exchange resins were tested. In particular, high-porosity resins (e.g., MacroCap SP from GE Healthcare Life Sciences) offered high capacity while retaining resolution, providing high purity and yield of the mono-PEGylated target under high loading conditions. The purification process was demonstrated with a batch size of 400 mg of IL-11 prepared at 5 mg / mL in sodium phosphate pH 4.5-5 buffer containing a 40-kD Y-shaped PEG reagent activated with a 2 molar ratio of aldehyde in the presence of 10 mM sodium cyanoborohydride. The reaction solution was quenched by adding 2 M glycine, followed by dilution with 4× volume of deionized water. After filtration through a 0.2 μm membrane, the resulting crude material was loaded onto a MacroCap SP column (2.6 (diameter) × 10 (height) cm with a loading capacity of approximately 7.5 mg / mL resin). After loading, the column was washed with over 10 column volumes of 20 mM sodium acetate pH 5 buffer, followed by an additional wash with over 20 column volumes of 20 mM sodium acetate pH 5 buffer containing 0.1 M NaCl. The product was then eluted with 20 mM sodium acetate pH 5 buffer containing 0.3 M NaCl. The overall yield of isolated I40NY was 26.6%. Product purity of I40NY was examined by SDS-PAGE and reverse-phase HPLC, and Figure 10 shows the purity of I40NY on a silver-stained SDS-PAGE gel with the amount of I40NY as indicated on the lanes. The purity of monoPEGylated IL-11 was greater than 93% as measured by C18-HPLC as the chromatogram displayed in FIG.

[0074] To measure the pharmacokinetic parameters of I40NY administered subcutaneously, three male Sprague-Dawley rats were injected with a single subcutaneous dose of 0.15 mg / kg PEGylated IL-11. Figure 12 shows the plasma concentrations of immunoreactive IL-11 in rats after a single subcutaneous administration. The plasma concentration of conjugated IL-11 reached a maximum level at approximately 12 hours and remained effective for 50 hours after administration. In contrast, recombinant human IL-11 reached a maximum concentration at approximately 2 hours and was rapidly removed from the circulation due to its plasma elimination half-life of approximately 1.3 hours. The pharmacokinetic parameters of I40NY administered subcutaneously are summarized in Table 7 below.

[0075] [Table 7]

[0076] The secondary structure of I40NY was investigated using circular dichroism. In circular dichroism chromatograms analyzed in the far-UV region, we demonstrated that I40NY maintains the same secondary structure as its unbound counterpart, as can be seen from the overlaid spectra in Figure 13. Furthermore, the thermal stability of I40NY was demonstrated by circular dichroism by measuring the changes in their secondary structure (mean residue ellipticity) in response to thermal stress. Figure 14 showed less structural change for I40NY in response to increasing temperature.

[0077] The efficacy of I40NY in myelosuppressed rats was also demonstrated in rats treated with carboplatin. Male Sprague-Dawley rats were injected intravenously with 40 mg / kg carboplatin to induce bone marrow dysfunction and lead to thrombocytopenia. Medical intervention using daily injections of IL-11 (for 7 consecutive days) or a single dose of I40NY at the same 0.15 mg / kg dose was administered subcutaneously immediately after 24 hours of carboplatin treatment. Platelet levels are shown in Figure 15. Without treatment, subjects experienced approximately 2 days of severe thrombocytopenia (less than one-third of normal platelet count), which, if untreated, indicates a high risk of life-threatening internal bleeding. Because the worst-case scenario with daily administration is very close to the threshold for severe thrombocytopenia, the efficacy of IL-11 treatment is low. In contrast, a single dose of I40NY not only prevented the occurrence of severe thrombocytopenia, but also accelerated the recovery of platelet levels, with platelet counts returning to their starting counts 1.3 days earlier than in the other two groups.

[0078] Meanwhile, the side effects of hematocrit reduction were also investigated in a myelosuppression model. As shown in Figure 16, IL-11 treatment rapidly reduced hematocrit compared with the untreated group. However, a single dose of I40NY alleviated the worst symptoms, suggesting that its side effects are weaker than those of daily IL-11 administration. Therefore, I40NY has been proven effective in preventing chemotherapy-induced severe thrombocytopenia, while also alleviating the syndrome of plasma volume expansion.

[0079] Further comparative data between I40NY and other forms of pegylated IL-11 (as described in US8133480, data not shown) reveal that the contemplated compounds, particularly I20NY and I40NY, have significantly improved in vivo efficacy and reduced side effect symptoms compared to other forms of pegylated IL-11 described in the '480 patent.

[0080] It will be apparent to those skilled in the art that many further modifications besides those already described are possible without departing from the inventive concepts herein. Accordingly, the inventive subject matter is not limited except as by the appended claims. Moreover, in interpreting both the specification and the claims, all words should be interpreted in the broadest possible manner consistent with the context. In particular, the words "comprises" and "comprising" should be interpreted to refer non-exclusively to elements, components, or steps, indicating that the referenced element, component, or step may be present, utilized, or combined with other elements, components, or steps not explicitly mentioned. When a claim of the specification refers to at least one selected from the group consisting of A, B, C... and N, the text should be interpreted as requiring only one element from the group, not A+N, or B+N, etc.

Claims

1. 1. A reaction intermediate composition for producing a polyethylene glycol (PEG)-interleukin-11 (IL-11) conjugate, comprising: a first amount of IL-11 consisting of unreacted IL-11 polypeptide; a reactive PEG comprising a second amount of a branched PEG attached to a reactive group selected to selectively form an amide bond with the N-terminal amine of the IL-11 polypeptide upon reductive amination; and an IL-11 conjugate comprising a third amount of an IL-11 polypeptide and a fourth amount of PEG, the IL-11 conjugate comprising a branched PEG moiety covalently attached to the N-terminus of the IL-11 polypeptide chain; wherein the sum of the second amount and the fourth amount represents the total amount of branched PEG in the reaction intermediate composition, the sum of the first amount and the second amount represents the total amount of IL-11 in the reaction intermediate composition, and the ratio of the total amount of branched PEG to the total amount of IL-11 is 1:1 to 1:

2. wherein the reactive PEG and the PEG component each have an average molecular weight of 10 Kd to 50 Kd and have a Y-shape; the IL-11 polypeptide is a human or humanized polypeptide chain; the reaction intermediate composition is present at a pH of 4.5 to 5.0; A reaction intermediate composition, wherein ion exchange chromatography of the reaction intermediate composition using SP ion exchange media yields a mono-PEGylated IL-11 conjugate with a purity of greater than 93%.

2. 2. The reaction intermediate composition of claim 1, wherein the IL-11 polypeptide is truncated by deletion of an N-terminal proline.

3. The reaction intermediate composition according to claim 1, wherein the PEG-IL-11 conjugate is I40NY.

4. 1. A method for purifying a PEG-IL-11 conjugate, comprising: A step of obtaining the reaction intermediate composition of claim 1; adjusting the pH of the reaction intermediate composition of claim 1 to between pH 4.5 and pH 5 to produce a pH-adjusted reaction intermediate composition; applying the pH-adjusted reaction intermediate composition to a cation exchange medium; eluting the bound PEG-IL-11 conjugate from the cation exchange medium with an elution buffer having a pH of 7; wherein the reaction intermediate composition comprises a reactive PEG and a branched PEG component, each having an average molecular weight of 10 Kd to 50 Kd and having a Y-shape; the reaction intermediate composition comprises an IL-11 polypeptide that is a human or humanized polypeptide chain; The method wherein the reaction intermediate composition is subjected to ion exchange chromatography of the reaction intermediate composition using SP ion exchange media to obtain a mono-PEGylated IL-11 conjugate with a purity of greater than 93%.

5. 5. The method of claim 4, wherein the elution buffer comprises 1 M NaCl.

6. 5. The method of claim 4, wherein the reaction intermediate composition comprises an IL-11 polypeptide that is truncated by deletion of an N-terminal proline.

7. The method of claim 4, wherein the PEG-IL-11 conjugate is I40NY.

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