US20060154865A1 - Conjugates of insulin-like growth factor-1 and poly(ethylene glycol) - Google Patents
Conjugates of insulin-like growth factor-1 and poly(ethylene glycol) Download PDFInfo
- Publication number
- US20060154865A1 US20060154865A1 US11/313,101 US31310105A US2006154865A1 US 20060154865 A1 US20060154865 A1 US 20060154865A1 US 31310105 A US31310105 A US 31310105A US 2006154865 A1 US2006154865 A1 US 2006154865A1
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- US
- United States
- Prior art keywords
- igf
- variant
- amino acid
- poly
- ethylene glycol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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Definitions
- This invention relates to conjugates of insulin-like growth factor-I(IGF-I) with poly(ethylene glycol) (PEG), pharmaceutical compositions containing such conjugates, and methods for the production and methods of use of such conjugates.
- PEG poly(ethylene glycol)
- AD Alzheimer's disease
- NFT neurofibrillary tangle
- amyloid plaque core One of the major components of the amyloid plaque core is the pathologically deposited small amyloid-beta-peptide (A ⁇ ), which is cleaved by secretases from amyloid precursor protein (APP).
- a ⁇ a self-aggregating peptide of 39-43 residues (MW ⁇ 4 kDa), is synthesized as part of the larger APP (110- 120 kDa).
- APP is a type I integral membrane glycoprotein with a large N-terminal extracellular domain, a single transmembrane domain and a short cytoplasmic tail.
- the A ⁇ region spans portions of the extracellular and transmembrane domains of APP.
- the most common hypothesis for the participation of APP in neuronal cell death in AD is the amyloid hypothesis. This hypothesis postulates that plaque amyloid depositions or partially aggregated soluble A ⁇ trigger a neurotoxic cascade, thereby causing neurodegeneration similar to AD pathology.
- IGF-I Insulin-like growth factor I
- IGF-I Insulin-like growth factor I
- somatomedin C a circulating hormone structurally related to insulin.
- Use, activity and production are mentioned in, e.g., EP 0 123 228; EP 0 128 733; U.S. Pat. Nos. 5,861,373; 5,714,460; EP 0 597 033; WO 02/32449; WO 93/02695.
- IGF-I function The regulation of IGF-I function is quite complex. In the circulation, only 0.2 % of IGF-I exists in the free form whereas the majority is bound to IGF-binding proteins (IGFBP's), which have very high affinities to IGF's and modulate IGF-I function. The factor can be locally liberated by mechanisms releasing IGF-I such as proteolysis of IGFBPs by proteases.
- IGFBP's IGF-binding proteins
- IGF-I plays a paracrine role in the developing and mature brain, and in vitro studies indicate that IGF-I is a potent non-selective trophic agent for several types of neurons in the CNS.
- IGF-I Reduction of brain and serum levels of free IGF-I has been related to the pathogenesis of sporadic and familial forms of AD. Furthermore, IGF-I protects neurons against A ⁇ -induced neurotoxicity. Peripherally administered IGF-I is capable of reducing brain A ⁇ levels in rats and mice and that in a transgenic AD mouse model prolonged IGF-I treatment significantly reduced brain amyloid plaque load. These data strongly support the idea that IGF-I is able to reduce brain A ⁇ levels and plaque-associated brain dementia by clearing A ⁇ from the brain.
- PEG poly(ethylene glycol)
- a common method for the PEGylation of proteins is the use of poly(ethylene glycol) activated with amino-reactive reagents like N-hydroxysuccinimide (NHS). With such reagents poly(ethylene glycol) is attached to the proteins at free primary amino groups such as the N-terminal ⁇ -amino group and the ⁇ -amino groups of lysine residues.
- WO 94/12219 and WO 95/32003 claim polyethylene glycol conjugates comprising PEG and IGF or a cysteine-mutated IGF, where the PEG is attached to said mutein at a free cysteine in the N-terminal region of the mutein.
- WO 2004/60300 describes N-terminally PEGylated IGF-I.
- the invention comprises an IGF-I variant having an amino acid alteration at at least one of amino acid positions 27, 37, 65 and 68 of the wild-type IGF-I amino acid sequence so that one or more of amino acids 37, 65, 68 is/are lysine(K) and amino acid 27 is a polar amino acid but not lysine.
- Amino acid 27 can be, for example, arginine.
- IGF-I variants are useful as intermediates (IGF-I intermediates) for the production of lysine-PEGylated IGF-I.
- a lysine-PEGylated IGF-I variant (amino-reactive PEGylated IGF-I variant), preferably a 20 kDa to 100 kDa lysine-PEGylated IGF-I variant, and especially preferably a lysine-monoPEGylated IGF-I variant, has superior properties in regard to therapeutic applicability.
- a further embodiment of the invention is a composition containing, inter alia, both a lysine-PEGylated IGF-I variant according to the invention and an IGF-I variant which is N-terminally PEGylated.
- the molecular ratio can be, for example, within the range of 9:1 to 1:9, such as a composition wherein the molar ratio is at least 1:1 ( at least one part lysine-PEGylated IGF-I variant per one part of N-terminally PEGylated IGF-I variant), for example at least 6:4 ( at least six parts lysine-PEGylated IGF-I variant per four parts of N-terminally PEGylated IGF-I variant).
- the lysine-PEGylated IGF-I variant and the N-terminally PEGylated IGF-I variant may be monoPEGylated.
- the variant may be identical in both the lysine-PEGylated IGF-I variant and the N-terminally PEGylated IGF-I variant.
- the variant may be selected from the group consisting of RRKK, RRKR, RRRK, RKRR.
- PEG may typically have an average molecular weight of 30 to 45 kDa, especially 30 kDa or 40 kDa.
- Lysine-PEGylated IGF-I variant and N-terminally PEGylated IGF-I variant show comparable affinities in binding of IGF binding proteins (e.g. BP4 and BP5), but different activities in regard to IGF-IR phosphorylation.
- the present invention provides a conjugate containing an IGF-I variant and poly(ethylene glycol) group(s), where the IGF-I variant has amino acid alteration(s) at at least one of amino acid positions 27, 37, 65 and 68 of the wild-type IGF-I amino acid sequence so that one or more of amino acids 37, 65, 68 is/are lysine(K), amino acid 27 is a polar amino acid but not lysine and said PEG is conjugated to said IGF-I variant via primary amino group(s), typically via primary amino group(s) of lysine(s).
- the poly(ethylene glycol) group(s) may have an overall molecular weight of at least 20 kDa, such as from about 20 to 100 kDa or such as from 20 to 80 kDa.
- the poly(ethylene glycol) group(s) may be conjugated to the IGF-I variant via the primary amino group(s) of lysine at one or more amino acid positions 37, 65, 68 (amino-reactive PEGylation) and are optionally PEGylated at the N-terminal amino acid.
- the conjugate may be mono- or diPEGylated at lysine residue(s) at one or more amino acid position(s) 37, 65, 68 and optionally PEGylated at the N-terminal amino acid.
- the conjugate may be monoPEGylated at K65, K68, or K37 or diPEGylated at K65 and K68 and optionally PEGylated at the N-terminal amino acid.
- Preferably not more than 20% of the PEGylated IGF-I variant is additionally N-terminal PEGylated.
- IGF-I variants are designated herein as follows: K65 means that amino acid 65 is lysine, R27 means that amino acid 27 is arginine etc.
- An IGF-I variant bearing the amino acids R27, K37, K65, K68 is designated RKKK.
- IGF-I wildtype is designated KRKK.
- IGF-I variants and variants in the conjugates may be, for example, RRKK, RRKR, RRRK, RKRR.
- the (PEGylated) IGF-I variant according to the invention may be a variant in which up to three (preferably all three) amino acids at the N-terminus are truncated.
- the respective wild type mutant is named Des(1-3)-IGF-I and lacks the amino acid residues gly, pro and glu from the N-terminal.
- the poly(ethylene glycol) group(s) may be either linear or branched.
- the invention further comprises methods for the preparation of a conjugate according to the invention using the IGF-I intermediates.
- the method comprises the preparation of a conjugate comprising an IGF-I variant and one or two poly(ethylene glycol) group(s), where the poly(ethylene glycol) group(s) may have an overall molecular weight of at least 20 kDa, or from about 20 to 100 kDa, or from 20 to 80 kDa, by reacting the IGF-I intermediate with activated (polyethylene) glycol under conditions such that the (polyethylene) glycol is chemically bound to the IGF-I intermediate via primary lysine amino group(s) of IGF-I variant.
- the invention further comprises pharmaceutical compositions containing a conjugate according to the invention, which may also include a pharmaceutically acceptable carrier.
- the invention further comprises methods for the production of pharmaceutical compositions containing a conjugate according to the invention.
- the invention further comprises methods for the treatment of AD, comprising the administration of a pharmaceutically effective amount of amino-reactive PEGylated IGF-I variant to a patient in need of such treatment, in, for example, one to two applications per week.
- FIG. 1 IEC-HPLC of PEG-IGF. Pure positional isomers were separated from the PEGylation mixture using a preparative strong-cation exchange column (TOSOH-BIOSEP, SP-5PW). Five different peak fractions (numbered 0-4) were isolated and processed for further analysis.
- TOSOH-BIOSEP preparative strong-cation exchange column
- FIG. 2 SDS-PAGE analysis of monoPEG-IGF-I peaks.
- the five purified peak fractions (numbered 0-4) were electrophoresed by 4-12% Tris-glycine SDS-PAGE under non-reduced (A) and reduced (B) conditions and gels were stained for protein with Coomassie blue.
- MW molecular weight marker.
- FIG. 3 Peptide analysis of monoPEGylated IGF-I peaks 1, 2 and 3.
- the purified monoPEGylated peaks 1, 2 and 3 were digested with Asp-N and the peptide fragments separated by HPLC.
- Peptide sequences of the PEGylated fragments were obtained by Edman N-terminal peptide degradation as described.
- HPLC analysis yielded 6 different peptide fragments for rhIGF-I at retention times between 30 and 45 minutes and a major fragment 7 (and a minor fragment 7′) for the PEGylated peaks at ⁇ 70 minutes retention time.
- Arrows indicate the major relative changes in peptide fragments in the different peaks as compared to rhIGF-I.
- B. Peptide sequence of the 6 fragments obtained from Asp-N cleavage of rhIGF-I.
- the lysines (K) are marked in bold and occur in fragments 3 and 4.
- Fragment 5 illustrates the N-terminal peptide.
- C. Peptide sequence of the PEGylated peptide fragments 7 after Edman degradation. Cystein and PEGylated lysine residues deliver breaks in the peptide sequence.
- FIG. 4 Hierarchical clustering of AD of the different global expression profile from IGF-I and the monopegylated isomers. The incubation time of the IGF-I and the pegylated variants was 24 h; usind the mouse cell line NIH-3T3. The conditions for the data analysis are described in the text.
- A Clusters of downregulated genes.
- B Clusters of upregulated genes.
- FIG. 5 IGF-IR phosphorylation by rhIGF-I and monoPEG-IGF-I.
- NIH-3T3 cells stably transfected with human IGF-IR were serum-starved over night and incubated with increasing doses (0.01-10 [g/ml) of rhIGF-I or monoPEG-IGF-I isomers (peak 1, 2, 3, peak mix, Des(1-3) peak 3) for 30 minutes. Subsequently, cells were processed for Western or in-cell analysis as described in Methods. Dose response curves with rhIGF-I (A), peak 1 (B), peak 2 (C), peak 3 (D), peak mix (E) and Des(1-3) peak 3 (F), respectively. The phosphorylation signal was normalized for IGF-IR levels in the individual wells. Data points show means ⁇ SEM of 6 measurements from 3 independent cultures.
- FIG. 6 Quantitative analysis of IGF-IR phosphorylation in NIH-3T3 cells. Dose response curves obtained form the experiments in Example 7 were fitted with one-site binding kinetics to obtain specific binding (B Max ), halfmaximal binding concentrations (EC 50 ) and Hill coefficients (n H ). Data represent means ⁇ SEM of 6 measurements from 3 independent cultures.
- FIG. 10 Time course of in vivo Abeta lowering in AAP and PS2APP mice by peak 3.
- Mixed populations of two months old APP and PS2APP mice were treated for 2, 6, 24, 48 or 72 h with peak 3 (50 ⁇ g/kg i.p.).
- APP, C99, Abeta and actin levels were evaluated and ratios calculated as described.
- A, Abeta/APP and B, C99/Abeta ratios at 6, 24 and 48 h after injection (*, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001 vs. untreated control).
- PEGylated IGF-I variant or “amino-reactive PEGylation” as used herein means an IGF-I variant that is covalently bonded to one or two poly(ethylene glycol) groups by amino-reactive coupling to one or two lysines of the IGF-I variant molecule.
- the PEG group(s) is/are attached at the sites of the IGF-I variant molecule that are the primary ⁇ -amino groups of the lysine side chains. It is further possible that PEGylation occurs in addition on the N-terminal ⁇ -amino group.
- PEGylated IGF-I variant can consist of a mixture of IGF-I variants, PEGylated at K65, K68 and/or K37 with or without N-terminal PEGylation, whereby the sites of PEGylation can be different in different molecules or can be substantially homogeneous in regard to the amount of poly(ethylene glycol) side chains per molecule and/or the site of PEGylation in the molecule.
- the IGF-I variants are mono- and/or diPEGylated and especially purified from N-terminal PEGylated IGF-I variants.
- Amino-reactive PEGylation designates a method of randomly attaching poly(ethylene glycol) chains to primary lysine amino group(s) of the IGF-I variant by the use of reactive (activated) poly(ethylene glycol), preferably by the use of N-hydroxysuccinimidyl esters of, preferably, methoxypoly(ethylene glycol).
- the coupling reaction attaches poly(ethylene glycol) to reactive primary ⁇ -amino groups of lysine residues and optionally the a-amino group of the N-terminal amino acid of IGF-I.
- Such amino group conjugation of PEG to proteins is well known in the art. For example, review of such methods is given by Veronese, F.
- the conjugation of PEG to primary amino groups of proteins can be performed by using activated PEGs which perform an alkylation of said primary amino groups.
- activated alkylating PEGs for example PEG aldehyde, PEG-tresyl chloride or PEG epoxide can be used.
- Further useful reagents are acylating PEGs such as hydroxysuccinimidyl esters of carboxylated PEGs or PEGs in which the terminal hydroxy group is activated by chloroformates or carbonylimidazole.
- Further useful PEG reagents are PEGs with amino acid arms.
- Such reagents can contain the so-called branched PEGs, whereby at least two identical or different PEG molecules are linked together by a peptidic spacer (preferably lysine) and, for example, bound to IGF-I variant as activated carboxylate of the lysine spacer.
- a peptidic spacer preferably lysine
- IGF-I variant as activated carboxylate of the lysine spacer.
- Mono-N-terminal coupling is also described by Kinstler, O., et al., Adv. Drug Deliv. Rev. 54 (2002) 477-485.
- PEG or poly(ethylene glycol) as used herein means a water soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art.
- the term “PEG” is used broadly to encompass any polyethylene glycol molecule, wherein the number of ethylene glycol units is at least 460, preferably 460 to 2300 and especially preferably 460 to 1840 (230 PEG units refers to an molecular weight of about 10 kDa). The upper number of PEG units is only limited by solubility of the conjugate. Generally, PEGs which are larger than PEGs containing 2300 units are not used.
- a PEG used in the invention terminates on one end with hydroxy or methoxy (methoxy PEG, mPEG) and is on the other end covalently attached to a linker moiety via an ether oxygen bond.
- the polymer is either linear or branched. Branched PEGs are e.g. described in Veronese, F. M., et al., Journal of Bioactive and Compatible Polymers 12 (1997) 196-207.
- Useful PEG reagents are e.g. available form Nektar Therapeutics.
- any molecular mass for a PEG can be used as practically desired, e.g., from about 20 kDaltons (Da) to 100 kDa (n is 460 to 2300).
- the number of repeating units “n” in the PEG is approximated for the molecular mass described in Daltons. For example, if two PEG molecules are attached to a linker, where each PEG molecule has the same molecular mass of 10 kDa (each n is about 230), then the total molecular mass of PEG on the linker is about 20 kDa.
- the molecular masses of the PEG attached to the linker can also be different, e.g., of two molecules on a linker one PEG molecule can be 5 kDa and one PEG molecule can be 15 kDa.
- Molecular mass means always average molecular mass.
- the invention provides PEGylated forms of IGF-I variant with improved properties.
- PEGylated IGF-I variant conjugates contain one or two PEG groups, which may be linear or branched and randomly attached thereto, whereby the overall molecular weight of all PEG groups in the conjugate is generally about 20 to 80 kDa. Small deviations from this range of molecular weight are possible as long as the PEGylated IGF-I variant does show activity in lowering Abeta peptide levels in the brain. Also PEGylation of IGF-I variants with PEG having molecular weights of more than 80 kDa results in higher bioavailability.
- the range of 20 to 100 kDa for the molecular weight of PEG has been found by the inventors to be optimal range for a conjugate of PEG and IGF-I variant useful for an efficient treatment of a patient suffering from AD.
- molecular weight means the mean molecular weight of the PEG.
- a monoPEGylated IGF-I selected from the group consisting of RRKK, RRKR, RRRK and RKRR, wherein the branched PEG group has a molecular weight of 30-45, preferably 40-45 kDa (about 920 PEG units) .
- the calculated average molecular weight for such a monoPEG-IGF-I is about 51.6 kDa.
- PEG or PEG group means a residue containing poly(ethylene glycol) as an essential part.
- a PEG can contain further chemical groups which are necessary for binding reactions; which results from the chemical synthesis of the molecule; or which is a spacer for optimal distance of the parts of the molecule from one another.
- PEG can consist of one or more PEG side-chains which are linked together.
- PEG groups with more than one PEG chain are called multiarmed or branched PEGs.
- Branched PEGs can be prepared, for example, by the addition of polyethylene oxide to various polyols, including glycerol, pentaerythriol, and sorbitol.
- a four-armed branched PEG can be prepared from pentaerythriol and ethylene oxide.
- Branched PEGs usually have 2 to 8 arms and are described in, for example, EP-A 0 473 084 and U.S. Pat. No. 5,932,462.
- PEGs with two PEG side-chains (PEG2) linked via the primary amino groups of a lysine (Monfardini, C., et al., Bioconjugate Chem. 6 (1995) 62-69).
- Substantially homogeneous as used herein means that the only PEGylated IGF-I variant molecules produced, contained or used are those having one or two PEG group(s) attached.
- the preparation may contain small amounts of unreacted (i.e., lacking PEG group) protein.
- As ascertained by peptide mapping and N-terminal sequencing, one example below provides for the preparation which is at least 90% PEG-IGF-I variant conjugate and at most 5% unreacted protein. Isolation and purification of such homogeneous preparations of PEGylated IGF-I variant can be performed by usual purification methods, preferably size exclusion chromatography.
- “MonoPEGylated” as used herein means that IGF-I variant is PEGylated at only one lysine per IGF-I variant molecule, whereby only one PEG group is attached covalently at this site. Such a monoPEGylated IGF-I can be in addition PEGylated at the N-terminus to a certain extent.
- the pure monoPEGylated IGF-I variant (without N-terminal PEGylation) is at least 80% of the preparation, preferably 90%, and most preferably, monoPEGylated IGF-I variant is 92%, or more, of the preparation, the remainder being e.g.
- the monoPEGylated IGF-I variant preparations according to the invention are therefore homogeneous enough to display the advantages of a homogeneous preparation, e.g., in a pharmaceutical application. The same applies to the diPEGylated species.
- Activated PEGs or activated PEG reagents are well-known in the state of the art.
- electrophilically activated PEGs such as alkoxybutyric acid succinimidyl esters of poly(ethylene glycol) (“lower alkoxy-PEG-SBA”) or alkoxypropionic acid succinimidyl esters of poly(ethylene glycol) (“lower alkoxy-PEG-SPA”) or N-hydroxysuccinimide activated PEGs.
- Any conventional method of reacting an activated ester with an amine to form an amide can be utilized.
- the exemplified succinimidyl ester is a leaving group causing the amide formation.
- succinimidyl esters to produce conjugates with proteins is disclosed in U.S. Pat. No. 5,672,662.
- the reaction conditions used have an influence on the relative amount of differently PEGylated IGF-I variants.
- the reaction conditions e.g., ratio of reagents, pH, temperature, protein concentration, time of reaction etc.
- the relative amounts of the different PEGylated species can be varied.
- the reaction is performed in a buffered aqueous solution pH 8-10, containing 5-15% (v/v) ethanol and 0.5-4% (v/v) ethyleneglycol.
- the protein:PEG ratio may be 1:0.5 to 1:2 if monoPEGylated variants are desired and 1:2.2 to 1:5 if diPEGylated variants are desired.
- Reaction temperature and reaction time can be varied according to the knowledge of a skilled artisan, whereby high temperature and long reaction time results in increased PEGylation. If monoPEGylated variants are desired, it is therefore typical to work at 4° C. and for up to 30 minutes.
- a reaction buffer which consists of 50 mM sodium borate, 10% ethanol and 1% di(ethylene glycol) (DEG) at a pH of about 9.0, a protein:PEG ratio of about 1:1.5, and a reaction temperature of from 4° C., a mixture of mono-, di-, and trace amounts of the tri-PEGylated species are produced.
- a reaction buffer which consists of 50 mM sodium borate, 10% ethanol and 1% di(ethylene glycol) (DEG) at a pH of about 9.0
- DEG di(ethylene glycol)
- reaction temperature of from 4° C.
- the protein:PEG ratio is about 1:3, primarily the di- and oligo-PEGylated species is produced.
- IGF-I variant conjugates according to the invention may be prepared by covalently reacting a primary lysine amino group of an IGF-I variant with a bifunctional reagent to form an intermediate with an amide linkage and covalently reacting the intermediate containing amide linkage with an activated poly(ethylene glycol) derivative to form an IGF-I variant conjugate.
- the bifunctional reagent is preferably N-succinimidyl-S-acetylthiopropionate or N-succinimidyl-S-acetylthioacetate
- the activated poly(ethylene glycol) derivative is preferably selected from the group consisting of iodo-acetyl-methoxy-PEG, methoxy-PEG-vinylsulfone, and methoxy-PEG-maleimide.
- mPEG2-NHS N-Hydroxysuccinimidyl activated branched PEG ester
- the IGF-I variant conjugates may be prepared by amino-reactive covalent linking of thiol groups to IGF-I variant (“activation”) and coupling the resulting activated IGF-I variant with a poly(ethylene glycol) (PEG) derivative.
- the first step comprises covalent linking of thiol groups via lysine NH 2 -groups of IGF-I variant.
- This activation of IGF-I variant is performed with bifunctional reagents which carry a protected thiol group and an additional reactive group, such as active esters (e.g., a succinimidylester), anhydrides, esters of sulphonic acids, halogenides of carboxylic acids and sulphonic acids, respectively.
- the reaction is carried out, for example, in an aqueous buffer solution, pH 6.5-8.0, e.g., in 10 mM potassium phosphate, 300 mM NaCl, pH 7.3.
- the bifunctional reagent may be added in DMSO. After completion of the reaction, preferably after 30 minutes, the reaction is stopped by addition of lysine. Excess bifunctional reagent may be separated by methods known in the art, e.g., by dialysis or column filtration.
- the average number of thiol groups added to IGF-I variant can be determined by photometric methods described in, for example, Grasetti, D. R,. and Murray, J. F., in Arch. Biochem. Biophys. 119 (1967) 41-49.
- the above reaction is followed by covalent coupling of an activated poly(ethylene glycol) (PEG) derivative.
- PEG poly(ethylene glycol)
- Activated PEG derivatives are known in the art and are described in, for example, Morpurgo, M., et al., J. Bioconjugate Chem. 7 (1996) 363-368 for PEG-vinylsulfone.
- Linear chain and branched chain PEG species are suitable for the preparation of the compounds of formula I.
- reactive PEG reagents are iodo-acetyl-methoxy-PEG and methoxy-PEG-vinylsulfone.
- the use of these iodo-activated substances is known in the art and is described e.g. by Hermanson, G. T., in Bioconjugate Techniques, Academic Press, San Diego (1996) pp. 147-148.
- compositions contain an effective amount of the substance according to the invention, for example from about 0.1 to 100 mg/ml, together with a suitable amount of a carrier.
- the compositions may be administered parenterally.
- the PEGylated IGF-I according to the invention is administered preferably via intraperitoneal, subcutaneous, intravenous or intranasal application.
- compositions may be used for administration for injection or infusion, preferably via intraperitoneal, subcutaneous, intravenous or intranasal application and contain an effective amount of the PEGylated IGF-I variant together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
- Such compositions include diluents of various buffer contents (e.g. arginine, acetate, phosphate), pH and ionic strength, additives such as detergents and solubilizing agents (e.g. TweenTM 80/polysorbate, pluronicTM F68), antioxidants (e.g.
- compositions may influence the physical state stability rate of release and clearance of the monoPEGylated IGF-I according to the invention.
- patients are treated with dosages in the range between 0.001 to 3 mg, preferably 0.01 to 3 mg of PEGylated IGF-I variant per kg per day over a certain period of time, lasting from one day to about 30 days or even longer.
- Drug is applied as a single daily subcutaneous or i.v. or i.p. (intraperitoneal) bolus injection or infusion of a pharmaceutical formulation containing 0.1 to 100 mg PEGylated IGF-I per ml.
- This treatment can be combined with any standard (e.g. chemotherapeutic) treatment, by applying PEGylated IGF-I before, during or after the standard treatment. This results in an improved outcome compared to standard treatment alone.
- the PEGylated IGF-I used is preferably monoPEGylated IGF-I, preferably as a composition of a lysine-PEGylated IGF-I variant according to the invention and an IGF-I variant which is N-terminally PEGylated wherein the molar ratio is at least 1:1.
- SEQ ID NO: 1 amino acid sequence of human IGF-I (amino acids 1-70 IGF-I; amino acids 71-105 propeptide according to SwissProt P01343).
- rhIGF-I human insulin-like growth factor
- PeproTech Rocky Hill, N.J., USA
- Des(1-3)-IGF-I was obtained from GroPep (Adelaide, Australia).
- the polyethylene glycol (PEG) reagent was delivered from Nektar Ltd. (San Carlos, Calif., USA). All other chemicals and solvents for this investigation were of the highest purity available.
- IGF-I variants can be produced recombinantly according to the state of the art by e.g. using site directed mutagenesis in combination with expression methods preferably in E. coli as e.g. described in U.S. Pat. Nos. 6,509,443 or 6,403,764.
- IGF-I isomers with PEGylation at a single site were produced.
- MonoPEG-IGF-I was prepared by the conjugation of lysine ⁇ -amino groups at the surface or on the N-terminus of the IGF-I molecule with an activated branched PEG moiety of 40 kDa molecular weight.
- PEGylation reaction mixture contained rhIGF-I and 40 kDa PEG-NHS reagent at 1:1.5 molar ratio in 50 mM sodium borate buffer (10% etanol and 1% DEG), pH 9.0. The reaction was performed for 30 min at 4° C.
- a purification scheme with a preparative strong-cation exchange column (TOSOH-BIOSEP, SP-5PW, 30 mm i.d. and 75 mm length) was used to prepare pure monoPEG-IGF-I isoforms.
- the buffer system consisted of 7.5 mM sodium acetate, 10% ethanol and 1% diethylene glycol, adjusted to pH 4.5 (buffer A) and 20 mM potassium phosphate, 10% ethanol and 1% diethylene glycol, adjusted to pH 6.5 (buffer B).
- the column was pre-equilibrated with 25% buffer B. After loading the PEG-IGF-I samples, the column was washed with 35% buffer B, followed by an ascending linear gradient to 65% buffer B to separate the isomers. For the elution of the nonPEGylated IGF-I the system was switched to modified buffer B with a pH. 8.0. The flow rate was 8 ml/min and the detection was performed at 218 nm. The resulting protein samples were collected manually and stored aliqouted at ⁇ 20° C. to be analyzed by a variety of protein chemical and biological assays (see below).
- the purified monoPEG-IGF isomers were cleaved with Asp-N to identify the four possible PEGylation sites at the N-terminus, at K27, K65 or K68.
- the cleavage buffer consisted of 100 mM Tris/HCl pH 8.0 with 0.04 ⁇ g/ ⁇ L Asp-N (Roche Diagnostics GmbH, DE). 20 ⁇ g monoPEG-IGF were incubated in cleavage buffer for 16 h at 37° C. with 1 ⁇ g Asp-N. After 16 h the reaction mixture was reduced by addition of TCEP (10 mM) for 1.5 h at 37° C.
- reaction solution was quenched by addition of 1/20 volume of 10% TFA to finalize the cleavage reaction.
- the obtained peptide mixtures were either directly analyzed by online-HPLC ESI mass spectrometry, HPLC or stored at ⁇ 80° C.
- ESI-LC-MS analysis the peptide mixtures were separated on an Agilent 1100 HPLC system equipped with a Phenomenex Jupiter C18 reversed phase column (1 ⁇ 250 mm, 5 ⁇ m, 300 ⁇ ) with a flow rate of 40 ⁇ l/min. The WV signal was also recorded at 220 nm. A Q-ToF II or LCT mass spectrometer (Micromass) was directly coupled to the HPLC system. ESI-ToF spectra were recorded with 1 scan/s in the mass range from 200-2000 m/z. UV and TIC spectra were evalulated and each peptide could be assigned to a single peak in the chromatogram.
- peptide mixtures were separated on an Agilent 1100 HPLC system equipped with a Phenomenex Jupiter C18 reversed phase column (1 ⁇ 250 mm, 5 ⁇ m, 300 ⁇ ) with a flow rate of 40 ⁇ l/min.
- the UV signal was also recorded at 220 nm.
- PEG peptides were shifted in retention time towards higher acetonitrile concentrations and were manually collected and submitted to N-terminal Edman degradation.
- N-terminal Edman degradation sequencing was conducted on an Applied Biosystems Procise system according to N-terminal Edman degradation sequencing was conducted on an Applied Biosystems Proteinsequencer Procise 492 with the procise system control software according to the manufacturers instructions.
- 20 ⁇ l of fractions collected from HPLC runs were directly applied to BioBrene PlusTM conditioned micro TFA filters. Filters were dried under argon covered with cartridge seals and introduced into the proteinsequencer. Automatic standard programs were used to conduct the sequential degradation of the polypeptide.
- Analysis of the HPLC chromatograms from each degradation cycle with Applied Biosystems data evaluation software 610A revealed the positions each amino acid. Cysteins as well as modified amino acids like pegylated lysine appeared as a gap in the chromatogram.
- IGF-IR stably transfected NIH-3T3 cells were serum-starved for 2 h and incubated in the absence of serum over 24 h with 0.1 or 1 ⁇ g/ml rhIGF and 1 ⁇ g/ml of the respective monoPEG-IGF-I isomers or the mixture of all isomers obtained without separation. Subsequently, the cultured cells were harvested and the total cellular RNA was extracted with RNA-BeeTM. From each sample 10 ⁇ g RNA were reversely transcribed, labelled and processed by using commercial kits according to the supplier's instructions (Invitrogen, Basel, Switzerland; Ambion, Huntingdon, UK).
- the mRNA levels value of the treated cells had to be at least five fold higher or five fold lower as compared to the untreated cells.
- the standard deviation must be significantly smaller than the absolute change in average difference and the calculated confidence level of a gene was set greater than 97% (p ⁇ 0.03).
- NIH-3T3 cells stably transfected with human IGF-IR were used for these experiments between passages 2 and 4.
- Cells were cultivated either in uncoated 24- or in 96-well plates and grown until 70% confluency. Subsequently, cells were serum-starved over night and then incubated with rhIGF-I or the respective PEG-IGF-I peaks (or peak mix) for 30 minutes. Cells were then either lysed in Laemmli buffer for Western or fixed with 4% paraformaldehyde for 30 minutes for in-cell analysis of IGF-IR phosphorylation. For Western analysis, protein extracts were separated by 10% Bis-Tris SDS-PAGE and blotted onto nitrocellulose membranes.
- the PS2APP mouse model has been shown to develop an amyloidosis with measurable Abeta levels at 2 months and onset of plaque deposition at 8 months of age (Richards, J. G., et al., J.
- Injections were performed i.p. under slight isoflurane anesthesia. At different times points after injection (2 h, 6 h, 24 h, 48 h or 72 h) animals were sacrificed under isoflurane anesthesia. Mice were decapitated and brains were removed for isolation of the telencephalon (cortex including hippocampus). Cortical protein extracts were prepared in hypotonic lysis buffer containing 4 mM Tris pH 7.4 and 320 mM Sucrose (both Fluka) with protease and phosphatase inhibitor cocktails (both Sigma). Sample buffer was Laemmli containing 8 M urea (Fluka).
- Proteins were separated by 4-12% Bis-Tris SDS-PAGE and blotted onto nitrocellulose membranes. Blots were co-incubated with mouse-anti-amyloid precursor protein (APP) (WO-2 clone, 1:5000; The Genetics Company) detecting APP, the C99 fragment and A ⁇ and goat-anti-Actin (C-11, 1:5000; Santa Cruz) primary antibodies and labeled with anti-mouse-Alexa680 (1:10000; Molecular Probes) and anti-goat-IRDye800 secondary antibodies (1:10000; Jackson). Fluorescent detection of protein bands was performed with the Odyssey imaging system (Licor Biosciences). From digital images, pixel intensity of protein bands was analyzed using GraphPad Prism software. Data were expressed as means ⁇ SEM.
- APP mouse-anti-amyloid precursor protein
- IGF-I contains 4 amino groups as potential PEGylation sites and 4 possible monoPEGylated IGF-I (monoPEG-IGF-I) isomers were expected. Further derivates were expected to be oligoPEGylated depending on the reaction conditions.
- a strong-cation high pressure liquid chromatography method (IEC-HPLC) was developed for the separation of PEG-IGF-I or -Des(1-3)-IGF-I isomers based on their local charge differences.
- a preparative elution profile of 5 mg PEG-IGF-I is shown in FIG. 1 . The result of this method was a separation into 5 major peaks, 3 peaks with baseline separation and 2 with partial separation.
- peaks 1-3 were designated as 3 different monoPEG-IGF-I isomers.
- the expected molecular weight for monoPEG-IGF-I (51.6 kDa) and the apparent size of ⁇ 70 kDa; however, the observed higher apparent molecular weight can be explained by the larger hydrodynamic volume of PEG due to water binding considerably slowing the electrophoretic motility of PEG-IGF-I and increasing the apparent molecular weight (Foser, S., et al., Pharmacogenomic J. 3 (2003) 319).
- FIG. 11B shows the peptide sequences of the respective fragments as obtained by Asp-N cleavage of rhlGF-I.
- Edman N-terminal peptide degradation we analyzed the peptide sequences of the major fragments 7 obtained with the PEGylated peaks 1, 2 and 3. Thereby, cysteins and PEGylated amino acids delivered breaks in the peptide sequence.
- peak 1 could be clearly mapped as rhIGF-I being PEGylated at K27 ( FIG. 3C ).
- the major fragment 7 (>90%) was mapped as K65 being PEGylated since K68 was confirmed as unmodified lysine (K) by Edman degradation.
- the fraction of peak 3 delivered a PEGylation at K68 (gap in the sequence) with K65 resulting in a signal in the Edman degradation HPLC chromatogram ( FIG. 3C ).
- the minor peak 7′ could not be sequenced by Edman degradation indicating that the N-terminus was not accessible to the reaction, most probably by PEGylation.
- peak 1 consists of the K27 PEGylated isomer
- peak 2 is IGF-I PEGylated at K65
- peak 3 is K68 PEGylated with a significant amount of N-terminally PEGylated IGF-I.
- DNA micorarrays we determined the transcriptional response of NIH-3T3 cells stably transfected with human IGF-IR over a 24h treatment with rhIGF-I or the PEG-IGF-I isomers. Therefore, we stimulated the cells with 0.1 and 1 ⁇ g/ml of IGF-I or with 1 ⁇ g/ml of monoPEG-IGF-I peaks or the peak mixture obtained from the PEGylation reaction.
- the mRNA abundance was expressed as average difference (AD) between perfect match oligonucleotide probes and a corresponding probe with mismatch in the center position.
- AD average difference
- This analysis yielded to a total of 162 genes, 86 upregulated and 76 downregulated by all IGF-I variants.
- a general correlation profile for transcriptional activity of different monoPEG-IGF-I isomers is illustrated in form of a hierarchical cluster of the up- and downregulated genes in FIG. 4 . An inspection of the induction levels of individual gene of 0.1 ⁇ g/ml and 1.0 ⁇ g/ml IGF-I shows that the selected clusters are very similar.
- Peak 3 generates a similar expression profile as unpegylated IGF-I at the same concentration and it is more potent than the PEG-IGF mixture and the other peaks.
- peak 2 triggers a similar transcriptional response like the PEG-IGF mixture. Consistent with the biological activity Peak 1 shows the weakest transcriptional response, indicating that pegylation interferes with receptor interaction.
- NIH-3T3 cells stably expressing the human IGF-IR were used. After serum starvation over night, cells were treated with increasing doses of rhIGF-I or the respective PEG-IGF-I isomer (0.003-10 ⁇ g/ml).
- Western analysis of phosphorylated IGF-IR was performed according to described above and the obtained dose response curves were fitted with a one-site binding kinetics including the Hill coefficient (nH); quantitative data of the association curves are shown in the table in FIG. 6 .
- the dose response of rhIGF-I FIG.
- the peak mixture demonstrated a similar IGF-IR activation pattern with slightly lower affinity with an EC 50 of 28.8 nM and regular nH of 1.28 ( FIG. 5E , 7).
- PEG-Des(1-3)-IGF-I peak 3 had the highest affinity of all PEG isomers with an EC 50 of 10.8 nM and regular nH of 1.08 ( FIG. 5F , 7).
- the data indicate that all peaks with exception of peak 1 specifically activated the human IGF-IR with a 2-5 fold loss of affinity as compared to rhlGF-I.
- Double-transgenic PS2APP mice were used to analyze the short-term effects of rhIGF-I on brain Abeta load.
- FIG. 7A shows Western Blots of brain extracts from 2 months old mice. Whereas APP, C99 and control protein (albumin) levels appear unchanged by rhIGF-I, Abeta levels were reduced 2 h after rhIGF-I injection. Quantitative analysis was performed and ratios of the respective pixel intensities were calculated to obtain information about potential effects of rhIGF-I on Abeta production.
- peak 3 was the only compound active at low doses (15-50 ⁇ g/kg) in increasing the C99/Abeta ratio representative for an increased Abeta clearance. Also in this evaluation, peak 1 was inactive and peak 2 only exerted significant effects at 500 ⁇ g/kg. Taken together, these data suggest that peak 3 is the most active monoPEG-IGF-I isomer in this in vivo experimental paradigm.
- FIG. 11 show results in solely double-transgenic PS2APP mouse model.
- IGFBP4 BP4
- IGFBP5 BP5
- IGFBP4 (SwissProt 22692) was identified and cloned by Shimasaki, S., Mol. Endocrinol. 4 (1990) 1451-1458.
- IGFBP5 (SwissProt 24593) was identified and cloned by Kiefer, M. C., Biochem. Biophys. Res. Commun. 176 (1991) 219-225. Both binding proteins were produced recombinantly in E. coli.
- Pegylated IGF-1 samples (analytes) were injected at 5 concentrations between 1.23 nM and 100 nM for 5 minutes (association phase) and washed with HBS-P for five minutes at a flow rate of 50 ⁇ l/min.
- the chip surface was regenerated by one injection of 100 mM HCl for 1 min.
- Negative control data e.g. buffer curves
- IGF-IR overexpressing cells were stimulated by the polypeptides of the invention and IGF-IR phosphorylation status was analyzed subsequently by ELISA.
- 96-Well streptavidin coated polystyrene plates (Nunc) were coated with 100 ⁇ l monoclonal antibody against human IGF-1R ⁇ (0.5 mg/ml) diluted 1:350 in PBST with 3% BSA. After incubation for 1 hour at room temperature on a plate shaker, the coating solution was removed and plates were washed thrice with 200 ⁇ l PBST per well.
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US20090253628A1 (en) * | 2008-04-03 | 2009-10-08 | Bettina Holtmann | Use of PEGylated IGF-I variants for the treatment of neuromuscular disorders |
US20110183903A1 (en) * | 2008-04-03 | 2011-07-28 | Bettina Holtmann | USE OF PEGylated IGF-I VARIANTS FOR THE TREATMENT OF NEUROMUSCULAR DISORDERS |
WO2011041376A1 (fr) * | 2009-09-30 | 2011-04-07 | Prolong Pharmaceuticals | Facteur modifié de stimulation des colonies de granulocytes (g-csf) |
CN102740896A (zh) * | 2010-02-11 | 2012-10-17 | 霍夫曼-拉罗奇有限公司 | Igf-i聚乙二醇缀合物 |
WO2011098400A1 (fr) * | 2010-02-11 | 2011-08-18 | F. Hoffmann-La Roche Ag | Conjugués de l'igf-i et du poly(éthylène glycol) |
KR101485646B1 (ko) | 2010-02-11 | 2015-01-22 | 에프. 호프만-라 로슈 아게 | Igf―ⅰ 폴리(에틸렌 글리콜) 컨쥬게이트 |
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WO2014097116A1 (fr) | 2012-12-18 | 2014-06-26 | Novartis Ag | Polypeptides stabilisés de facteur de croissance de type insuline |
EP3626735A1 (fr) | 2012-12-18 | 2020-03-25 | Novartis AG | Polypeptides stabilisés de facteur de croissance de type insuline |
US11266626B2 (en) * | 2015-09-09 | 2022-03-08 | The Trustees Of Columbia University In The City Of New York | Reduction of ER-MAM-localized APP-C99 and methods of treating alzheimer's disease |
WO2017147298A1 (fr) | 2016-02-23 | 2017-08-31 | The Regents Of The University Of Colorado, A Body Corporate | Procédés à base de peptides pour traiter une lésion neurologique |
WO2020150313A1 (fr) | 2019-01-18 | 2020-07-23 | The Regents Of The University Of Colorado, A Body Corporate | Peptides antimicrobiens alpha-hélicoïdaux amphipathiques pour le traitement d'infections par des agents pathogènes à gram-négatif |
WO2022198020A1 (fr) * | 2021-03-19 | 2022-09-22 | Quest Diagnostics Investments Llc | Identification et quantification de variants du facteur de croissance i analogue à l'insuline par spectrométrie de masse |
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