Interleukin-11 PEGylation reaction intermediate composition

Abstract

Compositions are presented that include a recombinant IL-11 PEGylated at equimolar to low molar excess of PEG to achieve a highly pure monoconjugate preparation, which provides improved half-life in serum while having desirable therapeutic activity and presenting fewer side-effects. Most preferably, the IL-11 is an N-terminally truncated human or humanized IL-11 and has a 20 Kd or 40 Kd branched PEG moiety, Y- or comb-shaped in particular, coupled to the N-terminal amino group. Such compounds are characterized by substantially increased stability in serum and sustained biological activity while exhibiting significantly reduced plasma expansion.

Claims

1. An interleukin 11 (IL-11) conjugation reaction intermediate of an IL-11 PEG conjugation process, comprising: an IL-11 PEG conjugate comprising an IL-11 polypeptide chain coupled to a bound PEG moiety, wherein the IL-11 PEG process selectively couples an amine-reactive PEG moiety to the N-terminus of the IL-11 polypeptide chain, and wherein the amine reactive PEG moiety has an average molecular weight of between 10-50 Kd and has at least two branches; and residual PEG moiety derived from the amine-reactive PEG moiety that did not couple to the IL-11 polypeptide chain during coupling, wherein a total PEG moiety represents a summation of the bound PEG moiety and the residual PEG moiety, and where the total PEG moiety of the IL-11 conjugation reaction intermediate is equimolar to 2-fold molar excess relative to IL-11 polypeptide chain provided to the IL-11 PEG conjugation process; wherein the amine reactive PEG moiety and the IL-11 polypeptide chain are selected such that the IL-11 conjugation reaction intermediate comprises a monosubstituted IL-11 PEG conjugate at a purity of greater than 93%; and wherein the IL-11 polypeptide chain is a human IL-11 or truncated human IL-11 polypeptide chain.

2. The interleukin 11 (IL-11) conjugation reaction intermediate of claim 1 wherein the IL-11 polypeptide chain is a human IL-11 polypeptide chain.

3. The interleukin 11 (IL-11) conjugation reaction intermediate of claim 1 wherein the IL-11 polypeptide chain is shortened by deletion of an N-terminal proline.

4. The interleukin 11 (IL-11) conjugation reaction intermediate of claim 1 wherein the IL-11 polypeptide chain has a sequence according to SEQ ID NO:1.

5. The interleukin 11 (IL-11) conjugation reaction intermediate of claim 1 wherein the amine-reactive PEG moiety has an average molecular weight of 20 Kd or 40 Kd.

6. The interleukin 11 (IL-11) conjugation reaction intermediate of claim 1 wherein the amine-reactive PEG moiety has a Y shape.

7. The interleukin 11 (IL-11) conjugation reaction intermediate of claim 1 wherein the bound PEG moiety is covalently bound to the N-terminal amino acid of the IL-11 polypeptide via an amine bond.

Description

BRIEF DESCRIPTION OF THE DRAWING

(1) FIG. 1 depicts the primary sequence of IL-11 without N-terminal proline.

(2) FIG. 2 are images of SDS-PAGE gels with molecular weight markers and various PEGylated forms of IL-11 as indicated.

(3) FIG. 3 is a graph depicting plasma concentrations of various IL-11 compositions after single i.v. administration.

(4) FIG. 4 is a graph depicting platelet increment after single i.v. administration of various IL-11 compositions.

(5) FIG. 5 depicts chromatograms of peptide maps for tryptic digestion of unconjugated IL-11, I40NY and I40KY in comparison.

(6) FIG. 6 is a graph depicting platelet increment after s.c. administration of various IL-11 compositions (Daily injection for IL-11 in consecutive 14 days; and weekly injection for PEGylated counterparts).

(7) FIG. 7 is a graph depicting hematocrit reduction after s.c. administration of various IL-11 compositions (Daily injection for IL-11 in consecutive 14 days; and weekly injection for PEGylated counterparts).

(8) FIG. 8 is a graph suggesting a correlation between maximal platelet induction and maximal reduction of hematocrit.

(9) FIG. 9 are graphs depicting cell proliferation activity of PEGylated compounds in 7TD1 assays, in comparison to unconjugated IL-11.

(10) FIG. 10 is an image of a non-reducing SDS-PAGE gel with silver stain illustrating the purity of I40NY at various loading quantities.

(11) FIG. 11 is a HPLC chromatogram depicting the product purity of mono-PEGylated component for I40NY.

(12) FIG. 12 is a pharmacokinetic profile depicting the kinetics of the plasma concentration of I40NY after single s.c. administration, in comparison to single s.c. administration of unconjugated IL-11.

(13) FIG. 13 is an overlay of circular dichroism spectra of IL-11 and I40NY.

(14) FIG. 14 is an ellipticity plot for IL-11 and I40NY as a function of temperature.

(15) FIG. 15 is a pharmacodynamics profile depicting the platelet production of contemplated compounds in an animal model of myelosuppressive rats.

(16) FIG. 16 is a graph depicting the hematocrit reduction of contemplated compounds in an animal model of myelosuppressive rats.

DETAILED DESCRIPTION

(17) The inventors have discovered that the type of PEG compound, the location of covalent attachment, and the primary sequence of IL-11 are determinants to the stability and activity of so modified IL-11. In particularly preferred and unexpected aspects, the inventors discovered that IL-11 has substantially improved stability when truncated at the N-terminus by one amino acid that is then PEGylated. Furthermore, the inventors also discovered that the particular type and molecular weight of the PEG moiety is an additional determinant of stability, activity, and toxicity as is further described in more detail below.

(18) Contemplated Compounds

(19) To investigate the influence of type, molecular weight, and attachment position of PEG to IL-11, the inventors prepared various PEGylated IL-11 molecules from recombinant human IL-11 having a primary sequence as shown in FIG. 1 (identical with the native human IL-11 sequence, but lacking the N-terminal proline). It is generally preferred that the IL-11 protein is a N-terminal truncated or modified human IL-11. For example, especially preferred truncated forms include IL-11 molecules that lack at least one or two or three (or more) N-terminal amino acids. Alternatively, the IL-11 may also be modified to have a N-terminal amino acid that is different from the human unmodified counterpart. For example, a modified IL-11 may lack the first N-terminal amino acid and may have a second amino acid that is other than the second amino acid found in unmodified human IL-11 (e.g., lacking P and having G replaced by V). Most typically, N-terminal amino acids will be stabilizing amino acids and therefore especially include M, G, A, S, T, V, or P, and in further contemplated aspects, destabilizing amino acids (e.g., F, Q, N, R, etc.) may be replaced by stabilizing amino acids. Deletions of one or more amino acids from the N-terminal end will typically be limited to the first ten, or the first five, or the first three amino acids. On the other hand, in less preferred aspects, deletions of one, two, three, four, five, or more amino acids may also be implemented at the C-terminus of the IL-11 moiety. As a general guidance, deletions will generally be limited to those that do not or only moderately adversely affect biological activity and/or stability (e.g., loss of activity and/or stability is less than 20%, and more typically less than 10%). Alternatively, or additionally, contemplated IL-11 molecules also include fusion proteins with IL-11 an exemplary fusion proteins include those described in US 2010/0143973, which is incorporated by reference herein. Most typically, the IL-11 is a recombinant protein and may be expressed in a suitable expression system, and most preferably in a prokaryotic system (e.g., E. coli) or yeast system (e.g., Pichia pasteuris). Of course, it should also be recognized that particularly preferred forms of IL-11 are mature forms (i.e., without leader sequence)

(20) Moreover, it should be appreciated that suitable IL-11 molecules need not be human IL-11 but may be of any other (typically mammalian) origin. Therefore, suitable IL-11 sources (recombinant or native) include primate, murine, porcine, equine, etc. These sequences may then be at least partially humanized to reduce immunogenicity and/or increase stability and/or activity in human. Similarly, synthetic consensus sequences are also contemplated herein.

(21) PEGylation of contemplated IL-11 molecules may be performed in numerous manners and includes covalent as well as non-covalent methods. However, it is generally preferred that the PEGylation uses covalent binding to the IL-11. There are numerous manners known in the art to covalently attach a PEG group to a protein and suitable methods include those that react the N-terminal amino group or the C-terminal carboxylic acid group with a suitable reactive group on the PEG moiety (e.g., aldehyde, maleimide, acid chloride, etc.), or sulfhydryl reactive groups (e.g., maleimide, pyridyl disulfide, vinyl sulfone, etc.) that allow for disulfide bonding to cysteine groups, or amino reactive reagents that react with an -amino group of a lysine amino acid (e.g., NHS-esters, NHS-carbonates, triazine, groups, etc). Therefore, it is also contemplated that one or more amino acids may be added to the N- and/or C-terminus to introduce a reactive group suitable for attachment of a PEGylation group. For example, serine or threonine may be added to allow for enzymatic attachment using a N-actylgalactosamine or PEG sialic derivative, or a lysine for covalent attachment to the -amino group, or a phenyl alanine or threonine group for attachment to a hydroxyl group.

(22) With respect to suitable PEG molecules for use herein, it is generally contemplated that various molecular weights for PEG are appropriate, and contemplated molecular weights are between 2 Kd and 200 Kd (average or nominal molecular weight). However, particularly preferred molecular weights (average or nominal molecular weight) include those between 10-50 Kd per linear chain in a PEGylation moiety. Moreover, it is generally preferred that the PEG moiety will have a single linear, or Y-shaped PEG moiety, and even more preferably that such PEG moiety will have a molecular weight of between 20-40 Kd. Alternatively, suitable PEG moieties may also include dendrimeric PEG constructs, as well as PEG moieties with more than two linear chains. Where the PEG moieties have more than one linear PEG chain, it is generally preferred that the chains have an average molecular weight that is substantially the same (average molecular weight difference less than 15%).

(23) In further preferred aspects, the PEG moiety is covalently attached to the IL-11 via the N-terminal amino group of IL-11 and/or (optionally) to an -amino group of an internal lysine or the ring nitrogen of histidine. Due to the N-terminal covalent bond, it is preferred that the molar ratio of IL-11 to PEG moiety is 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 appreciated that moderate levels of PEGylation may be present at internal amino acid residues (e.g. between 10%-20%, or between 1%-10% of all IL-11 may carry an extra PEGylated internal amino acid). For example, a second PEG moiety may be attached to an -amino group of an internal lysine or histidine. As is further shown in more detail below, a particularly preferred form of PEGylated IL-11 is I40NY, comprising human IL-11 (lacking N-terminal proline), to which is attached on the N-terminus a Y-shaped PEG moiety with an average molecular weight of 40 Kd.

(24) In still further alternative aspects, it should be appreciated that the PEGylation may be mixed with respect to the attachment position of the PEG moiety and/or kind of attachment. Therefore, IL-11 may be subjected to random non-covalent PEGylation and site specific PEGylation at an N-terminal amino acid, or subjected to different site specific PEGylations at the N-terminal amino acid and an internal amino acid. For example, and most preferably, IL-11 (or any modified form thereof) may be PEGylated at the N-terminal amino acid and optionally at an internal amino acid via a nitrogen atom (e.g., from lysine or histidine) in addition to the N-terminal modification.

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

(26) TABLE-US-00001 TABLE 1 Manufacturer/ Cat. No. Structure/Molecular size NOF/ SUNBRIGHT ME-200AL 20 KD Jenkem/ Y-PLAD-40K 40 KD NOF/ SUNBRIGHT ME-120TS 12 KD NOF/ SUNBRIGHT GL2-400TS 40 KD NOF/ SUNBRIGHT GL2-200AL3 20 KD NOF/ SUNBRIGHT ME-050TS 5 KD NOF/ SUNBRIGHT GL4-400AL3 40 KD

(27) After PEGylation of the truncated IL-11, the so obtained compounds were purified as also addressed in more detail below, and the various PEGylated IL-11 molecules had the following designations as shown in Table 2:

(28) TABLE-US-00002 TABLE 2 Compound PEG Structure code size/KDa of PEG Conjugation site (bonding) I20NL Single 20 linear N-terminal (amine bond) I40NY Single 40 Y-shaped N-terminal (amine bond) I12KL Single 12 linear N-terminal (amide bond) I40KY Single 40 Y-shaped N-terminal (amide bond) I20NY Single 20 Y-shaped N-terminal (amine bond) I20NL2 20 1~3 linear N-terminal/lysine or histidine (amine bond) I20NY2 20 1~3 Y-shaped N-terminal/lysine or histidine (amine bond) I05KL4 5 4~5 Linear N-terminal/lysine/histidine (amide bond) I40NX Single 40 4-arm N-terminal (amine bond)

(29) Most notably, the inventors have discovered that the type of PEG moiety and the site of attachment (and to some degree the sequence of the IL-11) had unexpected and substantial influence on biological activity and stability in vivo. As is more evident from the experimental data below, especially preferred PEGylations are at the N-terminal amino acid using a single Y-shaped PEG moiety, particularly where the IL-11 was truncated.

(30) Contemplated Compositions

(31) Based on the inventors' discovery of extended biological activity of contemplated compounds, it is generally contemplated that the compounds according to the inventive subject matter may be formulated for treatment of various diseases associated with a lack of IL-11 or characterized by a therapeutic response to treatment with IL-11. Therefore, and among other contemplated uses, the inventors especially contemplate that pharmaceutical compositions comprising contemplated compounds may be effective for the treatment or prevention of (a) chemotherapy-induced thrombocytopenia, (b) nuclear accident/radiation induced bone and gastrointestinal (GI) damage; (c) chemotherapy induced bone and GI damage; (d) burn induced thrombocytopenia and GI damage; (e) other causes of thrombocytopenia; (f) other causes of GI damage, including inflammatory bowel diseases like Crohn's Disease and ulcerative colitis, as well as pseudomembraneous colitis, (g) free radical-induced lung damage, and/or (h) cardiovascular diseases, wherein contemplated pharmaceutical compositions comprise a therapeutically effective amount of contemplated compounds (or pharmaceutically acceptable salt, hydrate, or prodrug thereof), and a pharmaceutically acceptable carrier. For example, in one aspect of the inventive subject matter, contemplated compositions are formulated for treatment of chemotherapy-induced thrombocytopenia or GI damage or radiation induced bone and gastrointestinal (GI) damage. Alternatively, or additionally, it should also be appreciated that contemplated compositions may be formulated to induce acute phase proteins, and/or to modulate antigen-antibody responses.

(32) It is particularly preferred that contemplated compounds are included in a composition that is 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 appreciated that pharmaceutical compositions according to the inventive subject matter may be administered to humans and other (typically mammalian) animals using various routes, including parenterally, orally, intraperitoneally, and topically.

(33) For example, suitable pharmaceutical compositions for injection preferably comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, emulsions, or suspensions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions prior to use. Examples of suitable aqueous and nonaqueous 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 may be ensured by inclusion of antibacterial and/or antifungal agents (e.g., paraben, phenol sorbic acid, chlorobutanol, etc.), as well as by filtration across sub-micron membranes (e.g., 0.45 M or 0.22 M pore size), autoclaving or pasteurizing, and radiation (e.g., gamma or e-beam). Where appropriate, osmotically active agents may be included (e.g., sugars, sodium chloride, etc.). While not limiting to the inventive subject matter, contemplated formulations for injection are typically in a pH range of 3-9, more typically 6-8, and most typically 7.4+/0.3. Of course, it should also be recognized that all liquid formulations may be preserved in various manners to facilitate long-term storage/stockpiling. For example, contemplated manners of stabilization include water/solvent removal using lyophilization, spray-drying, crystallization, adsorption on (preferably biocompatible or pharmaceutically acceptable) solid phases, etc.

(34) The compositions according to the inventive subject matter may be administered using various routes, including orally, parenterally, by inhalation, topically, rectally, nasally, or via an implanted reservoir, wherein the term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrathecal, intrahepatic, intralesional, and intracranial administration (typically injection or infusion). Preferably, the compositions are administered via injection, typically intravenously, and more preferably subcutaneously. Contemplated pharmaceutical compositions may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, the lower intestinal tract, or areas exposed during surgical intervention. There are numerous topical formulations known in the art, and all of such formulations are deemed suitable for use herein.

(35) With respect to the amount of contemplated compounds in the composition, it should be recognized that the particular quantity will typically depend on the specific formulation and desired purpose. Therefore, it should be recognized that the amount of contemplated compounds will vary significantly. However, it is generally preferred that the compounds are present in a minimum amount effective to deliver a therapeutic effect in vitro and/or in vivo.

(36) Thus, in most preferred embodiments, contemplated compounds will be present in an amount of between about 0.1 g/ml to about 100 mg/ml, more typically in an amount of between about 10 g/ml to about 10 mg/ml, and most typically between about 5 g/ml to about 100 g/ml. With respect to a dosage unit, it is generally contemplated that contemplated compounds are administered at a dosage effective to achieve a desired therapeutic effect, typically 10-100 g/kg, and more preferably 30-70 g/kg. However, alternate dosage units may be between 0.1-10 g/kg, or 50-80 g/kg, or 80-120 g/kg, or 120-200 g/kg, or even higher. Viewed from a different perspective, it should be appreciated that a single-use unit of contemplated formulations may include between about 0.3 mg to 3.0 mg of PEGylated IL-11, or between about 3 mg to 7 mg of PEGylated IL-11, or between about 7 mg to 10 mg of PEGylated IL-11 (most typically with a specific activity of 7-910.sup.6 U/mg). Unless the context dictates the contrary, all ranges set forth herein should be interpreted as being inclusive of their endpoints, and open-ended ranges should be interpreted to include commercially practical values. Similarly, all lists of values should be considered as inclusive of intermediate values unless the context indicates the contrary.

(37) In addition, 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 be separately made available (in a different type of formulation or the same), or be sold as a kit. For example, suitable additional pharmaceutically active agents include various steroids (e.g., corticosteroids), agents that stimulate platelet production in bone marrow (e.g., Li.sub.2CO.sub.3, folic acid, etc.), antibodies, analgesics, and anti-inflammatory agents.

(38) Contemplated Uses

(39) Contemplated compounds may be particularly useful as therapeutic agents for single or combination use in the treatment of (a) nuclear accident/radiation induced bone and gastrointestinal (GI) damage; (b) chemotherapy induced bone and GI damage; (c) burn induced thrombocytopenia and GI damage; (d) other causes of thrombocytopenia; (e) other causes of GI damage, including inflammatory bowel diseases like Crohn's Disease and ulcerative colitis, as well as pseudomembraneous colitis, (f) free radical-induced lung damage, and (g) cardiovascular diseases.

(40) Consequently, the inventors also contemplate use of the compounds presented herein for the manufacture of a drug for treatment of (a) nuclear accident/radiation induced bone and GI damage; (b) chemotherapy induced bone and GI damage; (c) burn induced thrombocytopenia and GI damage; (d) other causes of thrombocytopenia; (e) other causes of GI damage, including inflammatory bowel diseases like Crohn's Disease and ulcerative colitis, as well as pseudomembraneous colitis, (f) free radical-induced lung damage, and (g) cardiovascular diseases.

(41) Viewed from another perspective, the inventors also contemplate methods of treatment of (a) nuclear accident/radiation induced bone and GI damage; (b) chemotherapy induced bone and GI damage; (c) burn induced thrombocytopenia and GI damage; (d) other causes of thrombocytopenia; (e) other causes of GI damage, including inflammatory bowel diseases like Crohn's Disease and ulcerative colitis, as well as pseudomembraneous colitis, (f) free radical-induced lung damage, and (g) cardiovascular diseases in a human in need thereof in which contemplated compounds are administered at a therapeutically effective dosage.

Experiments and Experimental Data

(42) Materials:

(43) Purified bulk of recombinant human IL-11, derived from yeast was provided by Hangzhou Jiuyuan Gene Engineering Company (Lot#20121005/1006/1007/1008). 7TD1 murine hybridoma cell line, was acquired from DSMZ (No. ACC 23). Paraplatin injection (generic name: carboplatin) 10 mg/mL (Lot: 5A03935) was manufactured by Bristol-Myers Squibb Company. Trypsin of sequencing grade, modified from bovine pancreas (Cat. No. 11418025001) was purchased from Roche diagnostics. Mouse IL-11 receptor alpha was acquired from MyBioSource, Inc. (Cat. No. MBS553276). CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (MTS) (Cat. No. G5430) was purchased from Promega for 7TD1 cell assay. The DuoSet ELISA development kit for Human IL-11 was purchased from R&D Systems Inc. (Cat. No. DY218). The Purification resin-MacroCap SP (Product code 17-5440-01) was acquired from GE Healthcare Life Sciences. The Precise Tris-Glycine 8-16% polyacrylamide gels were purchased from Thermo Scientific. Trifluoroacetic acid (Cat. No. 302031) and acetonitril (Cat. No. 34967) for HPLC use were purchased from Sigma-Aldrich.

(44) Monofunctional PEG of various forms with Cat. No. 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. Molecular structures of the PEG reagents were shown above in Table 1.

(45) Preparation of I12KL/I40KY/I05KL4:

(46) 5 mg/mL protein was introduced with mixture of 1 to 2-fold molar ratio of respective PEG reagent (NOF/SUNBRIGHT ME-120TS for I12KL; SUNBRIGHT GL2-400TS for I40KY) and 50 mM NaHCO.sub.3 at pH about 8. I05KL4 (PEG reagent: NOF/SUNBRIGHT ME-120TS) was prepared with the same manner except the molar ratio of PEG to protein was added at 12-fold. The reaction mixture was incubated at room temperature for 2 hours, followed by quenching with 2 mM glycine. PEGylated product was isolated using chromatographic purification procedures as followed. The PEG molecule was linked to protein by amide bonding.

(47) Preparation of I20NL/I40NY/I20NY/I20NL2/I20NY2/I40NX:

(48) 5 mg/mL protein was introduced with mixture of 1 to 2-fold molar ratio of respective PEG reagent (NOF/SUNBRIGHT ME-200AL for I20NL and I20NL2; Jenkem/Y-PLAD-40 for I40NY; NOF/SUNBRIGHT GL2-200AL3 for I20NY and I20NY2; NOF/SUNBRIGHT GL4-400AL3 for I40NX), 10 mM sodium cyanoborohydride and 50 mM NaH.sub.2PO.sub.4. For conjugating onto two sites, the molar ratio of PEG was added at 3.5 to 5.5-fold. The pH was adjusted to about 4.5-5.0. The reaction mixture was incubated at room temperature for 24 hours, followed by quenching with 2 mM glycine. The PEG molecule was linked to protein by a more stable amine bonding. PEGylated product was isolated using chromatographic purification as followed.

(49) Chromatographic Purification:

(50) The pH of protein solution was adjusted to 4-5 with 1M acetic acid, followed by centrifugation or filtration to remove particulates. Four volumes of water was introduced. For conjugate containing PEG over 20 KDa: The protein solution was loaded onto a MacroCap SP column (16 cm) that was equilibrated with buffer A containing 20 mM sodium acetate pH 5. The protein was eluted with gradient- or step-elution of buffer B, containing 20 mM sodium acetate pH 5 and 1M NaCl. For conjugate containing PEG below 20 KDa: The protein solution was loaded onto a MacroCap SP column (16 cm) that was equilibrated with buffer A containing 20 mM sodium phosphate pH 7. The protein was eluted with gradient- or step-elution of buffer B, containing 20 mM sodium phosphate pH 7 and 1M NaCl. A typical final product as analyzed in a SDS PAGE gel can be seen in FIG. 2. Here, the left lane was loaded with molecular weight markers, and various PEGylated forms of IL-11 were loaded into the remaining lanes. Note that I40NY ran at an apparent molecular weight of over 100 Kd, bigger than the estimated one of 60 Kd, which is likely due to the Y-shaped of its PEG moiety. In further particularly preferred aspects, purification of contemplated compounds is performed as a one-step purification process, which provides added advantages in a downstream scale-ups.

(51) Purity Check by RP-HPLC:

(52) The content of each PEGamer was analyzed by reverse-phase (RP) chromatography employing the UPLC coupled with diode-array detector-UltiMate 3000 Rapid Separation LC Systems from Thermo Scientific. The chromatographic procedure was carried out using: Column: Acquity C18, 1.7 m, 2.1150 mm, 300 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; Inject 20 g and run gradient as in Table 3 below

(53) TABLE-US-00003 TABLE 3 Time (min) A % B % 0 100 0 2 100 0 9.9 80 20 9.95 0 100 11.3 0 100 11.31 100 0 17.5 100 0

(54) Determination of Protein Content:

(55) The protein content was determined by the UV/Vis microplate and cuvette spectrophotometerMultiskan GO from Thermo Scientific. Extinction coefficient in units of M.sup.1 cm.sup.1, at 280 nm measured in water is 17,990. Alternatively protein concentration is directly determined by ultraviolet spectroscopy at wavelength 280 nm, using the absorbency value of 0.944 for a 0.1% (1 mg/ml) solution. Protein quantitation using absorbance at 280 nm measures the absorbance of aromatic amino acids such as tryptophan and tyrosine, leaving PEG moiety undetected. As a result, protein concentration by weight stated herein does not contain PEG molecule.

(56) Pharmacokinetics (PK) Study in Healthy Rats:

(57) The in vivo manipulation was carried out in 3 male Sprague-Dawley rats following single dose administration of contemplated compounds by intravenous or subcutaneous route at a dosing level of 100-150 g/kg. Blood samples were collected in numerous time points in heparin tubes, followed by plasma separation and storage at 20 C. The concentration of immunoreactive IL-11 in plasma samples were determined by the DuoSet ELISA kit for Human IL-11 (R&D Systems Inc. Cat. No. DY218). The parameters of pharmacokinetics were yielded by the WinNonlin 5.3 software using non-compartment model.

(58) Pharmacodynamics (PD) Study in Healthy Rats:

(59) The pharmacodynamics assessment was carried out in 4 male Sprague-Dawley rats using intravenous or subcutaneous administration of respective contemplated compounds at dosing strength of 100-150 g/kg. Blood samples were collected in numerous time points in heparin tubes, followed by plasma separation and storage at 20 C. The blood cell count was carried out on a Cell-DYN 3500 hematology analyzer.

(60) Pharmacodynamics (PD) Study in Myelosuppressive Rats:

(61) The pharmacodynamics assessment was carried out in 4 male Sprague-Dawley rats, using intravenous administration of carboplatin at 40 mg/kg on Day 0 to induce myelosuppression. Contemplated compounds were subcutaneously injected on Day 1 at 150 g/kg. Blood samples were collected in numerous time points in heparin tubes, followed by plasma separation and storage at 20 C. The blood cell count was carried out on a Cell-DYN 3500 hematology analyzer.

(62) Tryptic Mapping:

(63) The reaction solution was prepared in 50 mM Tris pH 8.3 buffer containing 2 mg/mL protein and 1/50 (W/W) trypsin. Incubated at room temperature for 6 hours, followed by adding equal volume of 0.2% TFA (trifluoroacetic acid) solution. Any particulate matters were removed by centrifuge prior injection onto HPLC. The chromatographic procedure was carried out using: Column: Zorbax 300 SB-C8, 2.1150 mm, 5 m, 300 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; Inject 10 g and run gradient as in Table 4 below.

(64) TABLE-US-00004 TABLE 4 Time (min) A % B % 0 100 0 3 100 0 8 95 5 45 55 45 45.1 0 100 52 0 100 52.1 100 0 65 100 0
The identification of proteolytic peptide was carried out with HPLC coupled with MS spectrometry (Thermo LCQ Advantage).

(65) In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term about. Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed 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 the specific examples are reported as precisely 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.

(66) While it is generally known that PEG can impart the plasma stability of its conjugates, it is not predictable what attachment type, chain length, and molecular structure will produce a specific result with respect to therapeutic effect and/or pharmacologic parameter. In a first example, the truncated IL-11 conjugates of various PEG size were investigated with regard to plasma stability in normal rats. After intravenous administration, the observed plasma half-life of un-PEGylated IL-11 was found to be very short, less than 10 minutes as compared to those (3.5-13.7 hr) of its PEGylated counterparts. Among the latter, higher molecular-weight PEG imparted greater plasma stability in the following order: I40KY (13.7 hr)I40NY (8.5 hr)>I20NL (3.8 hr)I12KL (3.5 hr). FIG. 3 illustrates the plasma concentration of the various forms of PEGylated IL-11 after single intravenous administration. Each sample was dosed at 100 g/kg in rats. It was concluded that in this example larger or longer PEG chain resulted in longer serum half-life.

(67) In a second example, pharmacodynamics assessment was carried out with healthy Sprague-Dawley rats following intravenous route measuring platelet increment after single intravenous administration at 100 g/kg. As can be seen, from FIG. 4, 40-KD conjugates (I40NY and I40KY) induced more platelet increment (60-75%) than I20NL (50%), where Y represented the Y-shape PEG and L the linear shape PEG. The results also suggested multiple conjugation (I05KL4, 5-KD PEG conjugated on four sites) to be less effective than a single long PEG chain on the N-terminus, as multiples with shorter PEG conjugates had only limited efficacy at about 25% of platelet increment. It was concluded in this example that longer PEG chain on single site resulted in a higher efficacy in the respect of platelet induction. This appears to be opposite to the effect of PEGylation on recombinant human growth hormone.

(68) In a third example, conjugation sites were investigated by tryptic mapping, coupled with LC/MS identification of proteolytic peptides. FIG. 5 depicts tryptic maps of IL-11 (unconjugated), I40NY, and I40KY, and Table 5 below provides the tryptic peptides.

(69) TABLE-US-00005 TABLE5 Peptide Mass Position # PeptideSequence 773.9 1-8 T1 GPPPGPPR 669.7 9-14 T2 VSPDPR 1217.4 15-25 T3 AELDSTVLLTR 774.9 26-32 T4 SLLADTR 798.9 33-39 T5 QLAAQLR 261.3 40-41 T6 DK 3319.8 42-74 T7 FPADGDHNLDSLPTLAMSAG ALGALQLPGVLTR 287.4 75-76 T8 LR 950.1 77-84 T9 ADLLSYLR 839.0 85-90 T10 HVQWLR 174.2 91-91 T11 R 618.7 92-98 T12 AGGSSLK 1327.5 99-110 T13 TLEPELGTLQAR 402.5 111-113 T14 LDR 400.5 114-116 T15 LLR 174.2 117-117 T16 R 860.1 118-124 T17 LQLLMSR 2600.0 125-150 T18 LALPQPPPDPPAPPLAPPSSAWGGIR 1914.2 151-168 T19 AAHAILGGLHLTLDWAVR 655.9 169-174 T20 GLLLLK 275.3 175-176 T21 TR 131.2 177-177 T22 L

(70) Here it can be seen that the peak corresponding to the T1 peptide was noticeably reduced in the tryptic maps of I40NY and I40KY. This indicates that both conjugates with PEG were linked onto T1 peptides, where the N-terminal amine was the only site for the chemical conjugation. As a result, I40NY and I40KY were both N-terminally linked, however only differed in chemical bonding with amine bond for I40NY and amide bond for I40KY. Notably, both I40NY and I40KY exhibited similar effects in serum half-life and platelet induction via intravenous administration.

(71) Since an amine bond is more stable than an amide bond, and yield of mono-PEGylated product was more homogeneous for selective PEGylation using reductive amination, in the next study, various N-terminal conjugates were investigated in terms of efficacy in platelet production, and were evaluated by their respective associated side-effect via subcutaneous administration in healthy rats. FIG. 6 depicts results of a pharmacodynamics study of six PEGylated IL-11 conjugates in rats with subcutaneous administration at 150 g/kg. IL-11 was administered daily for continuous 14 days while PEGylated IL-11 were injected once weekly. PEG shape can influence the function of the conjugate. Particularly, the non-linear shape PEG molecule imparts better plasma stability and more potency than its linear counterpart. As displayed in FIG. 6, I20NY induced more platelet increment (58-70%) than I20NL (46-55%), where Y represented the Y-shape PEG and L the linear shape PEG. These results suggested that a Y-shaped PEG had a greater effectiveness than the linear form of the same molecular weight. However, I40NY (Y-shape) and I40NX (4-arm comb-shape) were comparable in platelet production as both increased up to about 65-70%, suggesting the influence of PEG shape became saturated when the PEG size was about or over 40 KD. Notably, double PEGylation of the same PEG length reduced the in vivo efficacy, as I20NL2 (linear PEG onto two sites) and I20NY2 (Y-shaped PEG onto two sites) had lower platelet production than their single PEGylated counterparts. It was therefore concluded that I40NY, I40NX and I20NY exerted higher efficacy among various N-terminal conjugated IL-11s. Moreover, it was noted that the effect of second administration was somehow down-regulated for smaller PEG conjugates such as 20-KD PEG regardless of number of conjugation site. As a result, I40NY and I40NX were unexpectedly effective compounds with desirable biological properties and relatively moderate adverse effects (especially plasma expansion). Moreover, the biological data further suggest that the so modified IL-11 compounds can be administered less frequently, and most preferably twice weekly, once weekly, or even less. Such schedule is particularly relevant where contemplated compounds are employed in the treatment of thrombocytopenia in a large population (e.g., exposed to radiologic exposure).

(72) The inventors also studied the side-effect associated with IL-11 conjugates in healthy rats. Hematocrit status is usually used as a marker for assessment of side-effect in clinical use of IL-11, because patients might experience dilutional anemia due to plasma expansion. In the animal study, IL-11 was administered subcutaneously for continuous 14 days at 150 g/kg while PEGylated IL-11 were injected once weekly at the same dose. As shown in FIG. 7, all drugs resulted in decreased hematocrit but I40NY was found to have less reduction while maintained higher activity than the rest of PEGylated conjugates. The mitigated dilutional anemia of dosing with I40NY is more prominent when compared in the same chart with other individual animal experiments using conjugated and unconjugated IL-11s. A correlation between platelet production and side-effect as manifested by reduction of hematocrit was established in FIG. 8, suggesting a trend of intensifying side-effect along with the increasing and dose-dependent efficacy when various modified and unmodified IL-11s were plotted in the chart. I40NYs, at different doses, were distinctly located on the right upper side of the trend, indicating less plasma expansion than some other compounds and unmodified IL-11 on the basis of comparable efficacy. In terms of product characterization, the inventors characterized physicochemical and pharmacological properties of a preferred compound, I40NY.

(73) Cell-Based Assay for IL-11 Conjugates:

(74) The biological activities of conjugated IL-11 were tested in a cell proliferation assay, using 7TD1 cell-line (DSMZ, Germany) In brief, 7TD1 cells at 4,000 cells per well were grown at 37 C. in a humidified atmosphere of 5% CO.sub.2 in response to different IL-11 concentrations for two days in the presence of 2 g/mL murine IL-11 receptor (MyBioSource, USA, MBS553276) (Biochem. J., 318:489-495). After addition of MTS, the EC50 of dose response curve was determined by plotting the absorbance of 490 nm on the y-axis against IL-11 concentrations on the y-axis, by fitting sigmoid dose-response curves with GraphPad software Prism 6. Before animal studies, the biological activities of newly synthesized conjugates were tested in a cell proliferation assay, using 7TD1 cell line. Not all PEGylation preparations gave similar products, and the actual product formation depends on the amino acid residues of the IL-11 being conjugated, and on the size and the shape of the PEG molecule used. 7TD1 cells grew in response to different conjugate concentrations. After addition of developers, whose chemical signal has a linear relationship with the cell number, absorbance at 490 nm was read in an ELISA plate-reader. The results were shown in FIG. 9.

(75) Due to the steric hindrance of the PEG moiety, all the conjugates expectedly showed a reduction in bioactivity in the cell-based assay as compared to that of the un-PEGylated IL-11 with the following order of potency: IL-11 (100%)>I20NL, I20NY (both about 16%)>I40NY, I40NX (both 11%)>I40KY (6%)>I20NY2 (3%). Notably, the steric interference is a dominant factor in determining biological activity of PEGylated conjugates and can be seen as a drastic decrease in bioactivity of the conjugate whose overall PEG moiety is bigger than 20 KDa. From some cell-based studies, there was a report showing that a small carbohydrate attachment at the non-core region of the IL-11 molecule, such as N-terminal sequence, enhanced the biological activity when compared with those conjugates at other attachment sites (J. Biol. Chem. Vol 286, No. 10, pp 8085-8093), which was consistent with less reduction of bioactivity by PEG molecule at the N-terminal sequence of IL-11. Although the in vitro bioactivity of I40NY retained only about 11% of native IL-11, in vivo efficacy was beneficially affected and could not be predicted from the in vitro bioactivity data. Table 6 below schematically illustrates bioactivity ratios of various compounds relative to unmodified IL-11.

(76) TABLE-US-00006 TABLE 6 Bioactivity ratio to Identification unmodified IL-11 IL-11 1 I20NL 0.13 I20NY 0.16 I20NY2 0.03 I40NY 0.11 I40NX 0.11 I40KY 0.06

(77) Chemical modification of proteins with PEG is an established technology and has been applied to biopharmaceutical industry to enhance the solubility and physical-chemical stability of proteins. While this chemical reaction is easy to carry out, it often results in a complex mixtures of different PEGylated forms, containing PEGamer and positional isomers. Multiple chromatographic purifications steps are employed to isolate product with high recovery. To develop a commercially viable process in terms of cost and yield, many factors such as protein concentration, quality of PEG, protein/PEG ratio, reaction temperature, and buffer pH, as well as purification process, are required to be optimized.

(78) I40NY was constructed by conjugation with a Y-shaped polyethylene glycol chain on amines forming stable amine bond, with relatively high selectivity to the N-terminal amine driven by conjugation chemistry (reductive amination of aldehyde coupling group in PEG moiety). I40KY on the other hand was conjugated using a functionalized NHS reagent at pH 8 on accessible amines forming the corresponding amide bonds. More specifically, I40NY is the mono-PEGylated IL-11 produced with site-specific reaction under acidic conditions because functionalized aldehyde is largely selective for the N-terminal -amine, whose pKa is lower compared to other nucleophiles. PEG to protein ratio, reaction concentration, pH and kinetics were investigated in the conjugation reaction. Reactions were taken place for 24 hours at room temperature (22-27 C.) in small scale at about 0.05-0.5 mL in volume in the presence of 10 mM sodium cyanoborohydride. Yield of each reaction under investigation was determined by RP-UPLC. Using different pH for selected reactions, optimum conjugation yield was at pH 4.5-5.5. In addition, the inventors noted that the concentration of reactants played an important factor in product yield and discovered that conjugation with IL-11 at concentrations larger than 5 mg/mL were optimal. Likewise, the PEG to protein ratio and conjugation kinetics of the reaction with 5 mg/mL protein at room temperature in the presence of reducing agent were investigated and suggested an optimum molar ratio of 2 for PEG to protein, and reaction extension to 16 hours sufficient for mono-PEGylation.

(79) Purification of PEGylated protein usually employs ion-exchange chromatography in large-scale preparation. However a satisfactory resolution to separate the mono-PEGylated from the oligo-PEGylated is not achievable when conventional ion-exchanger is loaded with reaction product at a loading capacity as little as 1 mg/mL resin. The low capacity of this resin often limits its application for larger scale production. To isolate N-terminally mono-PEGylated IL-11 at high purity, various cation-exchange resins were tested. Notably, high porosity resins (e.g., MacroCap SP from GE Healthcare Life Sciences) provided high capacity with retained resolution, and offered high purity and yield of mono-PEGylated targets at high load conditions. The purification process was demonstrated with a batch size of 400 mg IL-11, prepared in 5 mg/mL in sodium phosphate pH 4.5-5 buffer containing 2 molar ratio of aldehyde-activated 40-KD Y-shaped PEG reagent 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 m membrane, the resulting crude was loaded onto a MacroCap SP column (2.6 (diameter)10 (height) cm at a loading capacity of about 7.5 mg/mL resin). After charging, the column was washed with 20 mM sodium acetate pH 5 buffer over 10 column volume, followed by additional wash with 20 mM sodium acetate pH 5 buffer containing 0.1 M NaCl over 20 column volume. The product was then eluted with 20 mM sodium acetate pH 5 buffer containing 0.3 M NaCl. The overall yield of isolating I40NY was 26.6%. The product purity of I40NY was examined by SDS-PAGE and reverse-phase HPLC, and FIG. 10 shows purity of I40NY on a silver-stained SDS-PAGE gel with quantities of I40NY as indicated above the lanes. The purity of mono-PEGylated IL-11 was larger than 93% as determined by C18-HPLC as the chromatogram displayed in FIG. 11.

(80) To determine the pharmacokinetic parameters of I40NY via subcutaneous route, 3 male Sprague-Dawley rats were injected with 0.15 mg/kg of PEGylated IL-11 via single subcutaneous administration. FIG. 12 displayed the plasma concentration of immunoreactive IL-11 in rats after single subcutaneous administration. Plasma concentration of conjugated IL-11 achieved a maximal level at about 12 hr and remained effective over 50 hr after administration. On the contrary, the recombinant human IL-11 reached a maximal concentration at about 2 hr, and was cleared from the circulation blood stream rapidly as the elimination half-life in plasma was about 1.3 hr. The pharmacokinetic parameters of I40NY via subcutaneous route were summarized in Table 7 below.

(81) TABLE-US-00007 TABLE 7 unit I40NY IL-11 T.sub.1/2, terminal half-life hr 18.6 1.1 T.sub.max, time to maximal concentration hr 12 2 C.sub.max, maximal plasma concentration ng/mL 142 147 AUC.sub.all, area under curve Hr*ng/mL 3947 700 AUC.sub.inf, area under curve to infinity Hr*ng/mL 4421 701 Vz, relative volume of distribution mL/kg 909 347 Cl, relative clearance mL/hr/Kg 33.9 214 MRT, mean residence time hr 27.2 3.7

(82) The secondary structure of I40NY was investigated using circular dichroism. In the chromatogram of a circular dichroism analyzed in the far-UV region, the inventors demonstrated that I40NY maintained the same secondary structure as its unconjugated counterpart, as can be seen from both spectra superimposed in FIG. 13. Moreover the thermal stability of I40NY was demonstrated by circular dichroism by measuring the change of their secondary structures (mean residue ellipticity) in response to thermal stress. FIG. 14 indicated the structural change being less for I40NY in response to temperature increment.

(83) The effectiveness of I40NY in myelosuppressive rats was also demonstrated in carboplatin-treated rats. Male Sprague-Dawley rats were injected with 40 mg/kg of carboplatin via intravenous administration to induce damaged function of bone marrow leading to thrombocytopenia. Medical intervention using daily injection (consecutive 7 days) of IL-11 or single dose of I40NY at the same 0.15 mg/kg dosage was subcutaneously administered immediately after 24 hours of carboplatin treatment. The platelet level was displayed in FIG. 15. Without treatment, subjects experienced about two days of severe thrombocytopenia (less than of normal platelet count), suggesting the untreated having a high risk of life threatening internal bleeding. The efficacy of IL-11 treatment was marginal as the nadir of daily dosing was very close to the threshold of severe thrombocytopenia. Single dose of I40NY, on the contrary, not only prevented the occurrence of severe thrombocytopenia but also accelerated the recovery of platelet levels, as the platelet count returning to the initial number was 1.3 days earlier than the other two groups.

(84) Meanwhile, the side effects as manifested on the reduction of hematocrit was also investigated in the myelosuppressive model. In FIG. 16, treating with IL-11s caused hematocrit reduction in a rapid manner compared to the untreated group. However single dose of I40NY alleviated the nadir, suggesting less intensified side-effect than daily dosing with IL-11. Thus, it should be appreciated that I40NY has proven effective in preventing severe thrombocytopenia induced by chemotherapy, while ameliorating the syndrome of plasma expansion.

(85) Further comparative data between I40NY and another form of a PEGylated IL-11 (as described in U.S. Pat. No. 8,133,480, data not shown) reveal that contemplated compounds, and especially I20NY and I40NY had significantly enhanced in vivo effectiveness and reduced syndrome of side-effect as compared to the other form of PEGylated IL-11 as described in the '480 patent.

(86) It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms comprises and comprising should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refers to at least one of something 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 plus N, or B plus N, etc.