WO2010077260A1 - Hémoglobines pegylées stabilisées à la valine-1 (alpha), leurs procédés de préparation et leur utilisation - Google Patents

Hémoglobines pegylées stabilisées à la valine-1 (alpha), leurs procédés de préparation et leur utilisation Download PDF

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WO2010077260A1
WO2010077260A1 PCT/US2009/006079 US2009006079W WO2010077260A1 WO 2010077260 A1 WO2010077260 A1 WO 2010077260A1 US 2009006079 W US2009006079 W US 2009006079W WO 2010077260 A1 WO2010077260 A1 WO 2010077260A1
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pegylated
propyl
vai
alkyl
hemoglobin
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PCT/US2009/006079
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Seetharama A. Acharya
Tao Hu
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Albert Einstein College Of Medicine Of Yeshiva University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/41—Porphyrin- or corrin-ring-containing peptides
    • A61K38/42—Haemoglobins; Myoglobins

Definitions

  • the present invention generally relates to hemoglobin-based blood substitutes.
  • the invention is directed to PEGylated hemoglobins (Hbs) modified at Valine- 1 (alpha) (VaI- l( ⁇ )) to increase tetramer stability of hemoglobin, to methods of making these modified hemoglobins, and to their use as blood substitutes.
  • Hbs PEGylated hemoglobins
  • Hb hemoglobin
  • HBOCs hemoglobin based oxygen carriers
  • Hb undergoes dissociation from its native tetrameric state ( ⁇ 2 ⁇ 2 ) to a dimeric state ( ⁇ dimer) [6].
  • ⁇ dimer a dimeric state
  • the in vivo rapid dissociation of acellular Hb to ⁇ dimers leads to nephrotoxicity [7].
  • PEGylation induces extensive tetramer-dimer dissociation and leads to significant structural and functional perturbation of Hb [8].
  • (Propyl-PEG5K) 6 -Hb is essentially in ⁇ dimers [8].
  • (Propyl-PEG5K) 6 -Hb is a heterogeneous mixture and not an ideal model protein to investigate the tetramer-dimer dissociation of the PEGylated Hb.
  • (Propyl- PEG5K) 6 -Hb is a hexaPEGylated Hb where four polyethylene glycol (PEG) chains are conjugated at Val-l( ⁇ ) and Val-l( ⁇ ) and the other two at the Lysine (Lys) residues [9].
  • PEGylation at VaI- l( ⁇ ) and VaI- l( ⁇ ) may play a stronger influence than at the Lys residues on the tetramer stability of Hb.
  • VaI- l( ⁇ ) and VaI- l( ⁇ ) are located topologically at the ⁇ end and the ⁇ end of the central cavity of HbA, respectively [10]. They are involved in a number of well-known functions, such as the transition from R state to T state and the cooperative release or uptake of oxygen [2]. Variants of Hb with amino acid substitutions (e.g. glutamic acid [H]) or chemical modification (e.g., carbamylation [12], carboxymethylation [13] and reductive hydroxyethylation [14]) at the amino terminus have been used to investigate the structure and the function of Hb.
  • amino acid substitutions e.g. glutamic acid [H]
  • chemical modification e.g., carbamylation [12], carboxymethylation [13] and reductive hydroxyethylation [14]
  • the tetramer-dimer equilibrium of Hb is influenced by external factors (e.g., ion strength and pH) or by allosteric effectors (e.g., Cl ' and organic phosphate) [18-19].
  • the PEG chain provides a unique balance of hydrophobicity/hydrophilicity and the propensity to occupy a large volume in an aqueous environment [20]. This is partially due to the chain flexibility as well as an extensive degree of its hydration [20]. Conjugation of the PEG chains to Hb may perturb the ⁇ l ⁇ 2 interface and alter the tetramer stability of Hb by weakening the hydrogen bond and the hydrophobic interactions of Hb. Accordingly, there is a need for a PEGylated Hb having increased tetramer stability. The present invention addresses that need.
  • the invention provides a PEGylated hemoglobin (Hb) having a chemical moiety conjugated to Valine- 1 (alpha) of Hb to prevent PEGylation of the Hb at Valine- 1 (alpha).
  • Hb PEGylated hemoglobin
  • the invention also provides a PEGylated hemoglobin (Hb) comprising polyethylene glycol (PEG) conjugated to Valine- 1 (beta) of Hb but not to Valine- 1 (alpha) of
  • the invention further provides a method of preparing a PEGylated hemoglobin (Hb), the method comprising a) conjugating a chemical moiety to Valine-
  • the present invention is directed to pharmaceutical compositions and blood substitutes comprising the PEGylated hemoglobins disclosed herein and to methods of treating a subject comprising administering to the subject any of the PEGylated hemoglobins disclosed herein.
  • FIG. 1A-1B Purification of the PEGylated Hbs by ion exchange chromatography.
  • the PEGylated Hb in the absence (A) and in the presence (B) of IHP were purified by a Q Sepharose HP column (2.6 x 65 cm 2 ).
  • the column was equilibrated with 50 mM Tris-Ac buffer (pH 8.5) and eluted with 8 column volumes of 50 mM Tris-Ac buffer using a pH gradient of 8.5-7.0 at a flow rate of 2 ml/min.
  • FIG. 2 Characterization of the PEGylated Hbs. RP-HPLC analysis of HbA
  • the left inset shows the isoelectric focusing of HbA (lane 1), [Propyl-Val- l( ⁇ )] 2 -Hb (lane 2), Sample B (lane 3) and Sample A (lane 4).
  • the + and - signs indicate the anode and the cathode during electrofocusing, respectively.
  • FIG. 3A-3F Characterization of the sites of PEGylation.
  • MALDI-TOF/TOF spectra are shown for tryptic digestion of ⁇ -globin (A), ⁇ -globin (B), PEGylated ⁇ -globin from Fig. 2C (C) and PEGylated ⁇ -globin from Fig. 2D (D), respectively.
  • MS fragmentation is shown for the peptide at m/z 6178.3 Da in Fig. 3C (E) and the peptide at m/z 5957.3 Da in Fig. 3D (F).
  • FIG. 4 Size exclusion chromatography analysis of the PEGylated Hbs. HbA
  • FIG. 5A-5D Preparation and characterization of the propylated Hb at VaI- l( ⁇ ).
  • the propylated Hb in the presence of IHP was purified by cation exchange chromatography on a Mono S column (0.5 x 5 cm 2 ) (A).
  • the column was equilibrated with 50 mM Tris-Ac buffer (pH 6.0) and eluted with 40 column volumes of 50 mM Tris-Ac buffer at a pH gradient of 6.0-7.0 at a flow rate of 0.8 ml/min.
  • Globin chain analysis of the propylated Hb was carried out by RP-HPLC (B).
  • FIG. 6. S2o ; w of the modified Hbs a function of the protein concentration.
  • HbA Sedimentation velocity measurements of HbA (a), [Propyl-Val-l( ⁇ )] 2 -Hb (b), [Propyl- PEG5K-Val-l( ⁇ )] 2 -Hb (c), [Propyl-PEG5K-Val-l( ⁇ )] 2 -Hb (d) and [Propyl-PEG5K-Val- l( ⁇ )] 2 - ⁇ -Hb (e) were conducted in a Beckman XL-I analytical ultracentrifuge in PBS buffer at pH 7.4, 25 0 C and 55,000 rpm. Boundary movement was followed at 405 nm using the centrifuge's absorption optics.
  • FIG. 7A-7B Structural studies of the modified Hb.
  • the circular dichroism spectra (A) of HbA (a), [Propyl-Val-l( ⁇ )] 2 -Hb (b), [Propyl-PEG5K-Val-l( ⁇ )] 2 -Hb (c) and [Propyl-PEG5K-Val-l( ⁇ )] 2 -Hb (d) were recorded at 25 0 C with a 0.2-cm light path cuvette (310 ⁇ l).
  • the molar ellipticity ( ⁇ ) is expressed in deg.cm 2 /dmol on a heme basis.
  • Intrinsic fluorescence emission spectra (B) of HbA (a), [Propyl-Val-l( ⁇ )] 2 -Hb (b), [Propyl-PEG5K- Val-l( ⁇ )] 2 -Hb (c) and [Propyl-PEG5K-Val-l( ⁇ )] 2 -Hb (d) were recorded at room temperature.
  • the excitation wavelength was 280 nm.
  • FIG. 8 The pH dependent oxygen affinity and the Hill coefficients of the modified Hbs.
  • Oxygen affinity (P 50 ) of HbA (a), [Propyl-Val-l( ⁇ )] 2 -Hb (b), [Propyl-PEG5K- Val-l( ⁇ )] 2 -Hb (c) and [Propyl-PEG5K-Val-l( ⁇ )] 2 -Hb (d) was measured using Hem-O-Scan (Aminco) in 100 mM sodium phosphate buffer as a function of pH at 37 0 C. Each point presents an average of two duplicate results.
  • FIG. 9 Size exclusion chromatography analysis of the PEGylated Hbs. HbA
  • the invention provides a PEGylated hemoglobin (Hb) having a chemical moiety conjugated to Valine- 1 (alpha) of Hb to prevent PEGylation of the Hb at Valine- 1 (alpha).
  • the chemical moiety can be, for example, an alkyl, aralkyl, aryl or heteroaryl group, and is preferably an alkyl.
  • the alkyl is Cj-C 8 alkyl (i.e., Ci, C 2 , C 3 , C 4 , C 5 , C 6 , C 7 or C 8 alkyl) or Ci-C 6 alkyl, more preferably C 3 -C 8 alkyl or C 2 -C 4 alkyl, and most preferably C 3 alkyl (propyl).
  • the alkyl, aralkyl, aryl or heteroaryl can be unsubstituted or optionally substituted with OH, NH 2 , halogen or Ci-C 6 alkyl.
  • the chemical moiety can be specifically bound to Valine- 1 (alpha).
  • the PEGylated hemoglobin comprises PEGylation at Valine- 1 (beta).
  • conjugation of the chemical moiety to Valine- 1 (alpha) improves the tetramer stability of the PEGylated hemoglobin compared to a PEGylated hemoglobin that does not have the chemical moiety conjugated to Valine- 1 (alpha).
  • PEG polyethylene glycol
  • PEGylation means conjugating with PEG
  • the invention also provides a PEGylated hemoglobin (Hb) comprising polyethylene glycol (PEG) conjugated to Valine- 1 (beta) of Hb but not to Valine- 1 (alpha) of Hb.
  • the PEGylated hemoglobin (Hb) further comprises PEG conjugated to Lysine residues of Hb.
  • the PEGylated hemoglobin (Hb) can have a chemical moiety such as, for example, an alkyl, aralkyl, aryl or heteroaryl group, conjugated to Valine- 1 (alpha) of Hb.
  • the chemical moiety is an alkyl.
  • the alkyl is Ci-C 8 alkyl, more preferably C 3 -C 8 alkyl or C 2 -C 4 alkyl, and most preferably C 3 alkyl (propyl).
  • the alkyl, aralkyl, aryl or heteroaryl can be unsubstituted or optionally substituted with OH, NH 2 , halogen or Ci-C 6 alkyl.
  • the PEGylated hemoglobin (Hb) without a PEG conjugated to Valine- 1 (alpha) has improved tetramer stability compared to a PEGylated hemoglobin that is PEGylated at Valine- 1 (alpha).
  • the PEGylated Hb can be PEGylated with different numbers of PEG chains.
  • the hemoglobin is PEGylated with 2 to 8 PEG chains. More preferably, the hemoglobin is PEGylated with 2, 4 or most preferably 6 PEGs. Preferably, each globin chain of the hemoglobin is PEGylated.
  • the PEGylated hemoglobin can comprise a PEG with a molecular weight of, for example, 200-40,000 daltons.
  • PEGs of various molecular weights, conjugated to various groups can be obtained commercially, for example from Nektar Therapeutics, Huntsville, Alabama.
  • the PEG has a molecular weight of 2,000-20,000 daltons, 2,000- 10,000 daltons, or 3,000-8,000 daltons, more preferably 4,000-6,000 daltons, and most preferably about 5,000 daltons, for example 2000, 3000, 5000, 7500 or 10,000 daltons.
  • the PEGylated hemoglobin is not PEGylated at Cysteine-93(beta).
  • the PEG can be attached to Hb via a linker and/or an extension arm.
  • an "extension arm” refers to a carbon chain - thiol group that is attached to the amino group of Hb during a thiolation process using, for example, iminothiolane or dithio sulfo succinimidyl propinate (DTSSP).
  • the linker or extension arm can comprise, for example, an alkyl, aryl and/or heteroaryl group.
  • the alkyl group can be a Ci-C 6 alkyl such as a propyl group
  • the aryl group can be a phenyl group.
  • the linker or extension arm can, for example, comprise a ⁇ -mercapto butyrimidyl chain or a ⁇ -mercapto propylamide chain or a ⁇ -malimido caproic amide.
  • the PEG is provided as thio PEG.
  • Types of polyethylene glycols (PEGs) include, but are not limited to, methoxy
  • the maleimide PEG can be, for example, a maleimide PEG comprising an alkyl linker or a maleimide phenyl PEG.
  • Preferred PEGylated hemoglobins include (Propyl-PEG5 K) 6 - [Propyl- VaI- l(alpha)] 2 -Hb, (SP-PEG5K) 6 -[Propyl-Val-l(alpha)] 2 -Hb where SP is succinimidophenyl, (TCP-PEG5K) 6 -[Propyl-Val-l(alpha)] 2 -Hb where TCP is thiocarbamoylphenyl, and (Propionyl-PEG5K) 6 -[Propyl-Val-l(alpha)] 2 -Hb.
  • the number of PEG chain in these PEGylated Hbs can vary; for example, the number of PEG chains in the PEGylated Hb could be two or four.
  • the invention further provides a method of preparing a PEGylated hemoglobin (Hb), the method comprising a) conjugating a chemical moiety to Valine- 1 (alpha) of the Hb to prevent PEGylation of the Hb at Valine- 1 (alpha), and b) conjugating PEG chains to the Hb to generate a PEGylated Hb.
  • the chemical moiety can be, for example, an alkyl, aralkyl, aryl or heteroaryl group, and is preferably an alkyl.
  • the alkyl is Ci-C 8 alkyl, more preferably C 3 -C 8 alkyl or C 2 -C 4 alkyl, and most preferably C 3 alkyl (propyl).
  • the alkyl, aralkyl, aryl or heteroaryl can be unsubstituted or optionally substituted with OH, NH 2 , halogen or Cj-C 6 alkyl.
  • the chemical moiety can be specifically bound to Valine- 1 (alpha).
  • the PEGylated hemoglobin comprises PEGylation at Valine- 1 (beta).
  • conjugation of the chemical moiety to Valine- 1 (alpha) improves the tetramer stability of the PEGylated hemoglobin compared to a PEGylated hemoglobin that does not have the chemical moiety conjugated to Valine- 1 (alpha).
  • the invention provides a PEGylated Hb prepared by any of the methods disclosed herein.
  • the invention also provides for the use of any of the PEGylated Hbs described herein for the preparation of a pharmaceutical composition or blood substitute. [0036] The invention also provides a composition comprising any of the PEGylated
  • Hbs disclosed herein, or prepared by any of the methods disclosed herein, and a pharmaceutically acceptable carrier include, but are not limited to, additive solution-3 (AS-3), saline, phosphate buffered saline, Ringer's solution, lactated Ringer's solution, Locke-Ringer's solution, Krebs Ringer's solution, Hartmann's balanced saline solution, and/or heparinized sodium citrate acid dextrose solution.
  • the pharmaceutical compositions also may comprise known plasma substitutes and plasma expanders.
  • the pharmaceutical compositions of the present invention may be used as blood substitutes, and may be administered by conventional means including but not limited to transfusion and injection.
  • the invention provides a method of treating a subject which comprises administering to the subject any of the PEGylated hemoglobins or blood substitutes disclosed herein or any PEGylated hemoglobin or blood substitute prepared by any of the methods disclosed herein.
  • the subject Prior to treatment, the subject may have a reduced red blood cell count or a reduced blood volume. The reduced blood volume may be due to a wound or to surgery.
  • the subject may have a disease characterized by vaso-occlusion or impaired blood flow. Such diseases include, but are not limited to, sickle cell disease, myocardial infarction and/or shock.
  • the treatment does not produce hypertension in the subject.
  • the treatment does not affect arteriolar diameter or venular diameter in the subject.
  • the PEGylated hemoglobin does not cause constriction of arterioles.
  • the treatment does not increase vascular resistance in the subject.
  • the treatment does not affect the subject's heart rate.
  • the PEGylated hemoglobin described herein can be prepared using hemoglobin of human, canine, chicken, sheep, murine, or feline origin.
  • the invention may be used in the treatment of human subjects or in veterinary medicine.
  • the present study focused on investigating the influence of PEGylation on the structure (including the tetramer stability) and function of Hb. This influence may come from the sites of PEGylation (e.g., Val-l( ⁇ )) and the PEG chains. Accordingly, two PEGylated Hbs site-specifically modified at VaI- l( ⁇ ) and at VaI- l( ⁇ ) and one propylated Hb site- specifically modified at VaI- l( ⁇ ) were prepared and characterized. Size exclusion chromatography (SEC) and equilibrium and velocity analytical ultracentrifugation were used to evaluate the tetramer stability of the modified Hbs. Structural and functional properties of the three modified Hbs were also measured.
  • SEC Size exclusion chromatography
  • equilibrium and velocity analytical ultracentrifugation were used to evaluate the tetramer stability of the modified Hbs. Structural and functional properties of the three modified Hbs were also measured.
  • Hb hemoglobin
  • PEG polyethylene glycol
  • PEGylated conjugated with PEG
  • PEGylation conjugation with PEG
  • PBS phosphate buffered saline
  • DPG 2,3 diphospho glycerate
  • IHP inositol hexaphosphate
  • TFA trifluoro acetic acid
  • K d the dissociation constant
  • HBOC hemoglobin based oxygen carrier
  • IEF isoelectric focusing
  • P 50 partial oxygen pressure at half saturation
  • S sedimentation coefficient
  • SEC size exclusion chromatography
  • RP-HPLC reverse phase high performance liquid chromatography
  • CD circular dichroism
  • SAS solvent accessible surface
  • ⁇ H + the number of protons per heme released upon full oxygenation
  • n Hill coefficient.
  • HbA Human adult hemoglobin
  • IHP inositol hexaphosphate
  • HbA (0.25 mM) or IHP bound HbA (0.25 mM) were reacted with 1.5 mM PEG5K propionaldehyde (Sunbio Inc., Korea) and 7.5 mM NaCNBH 3 (Sigma Chemical Co., MO) in 50 mM BisTris-Ac buffer (pH 6.5) overnight at 4 0 C.
  • the reaction mixture was dialyzed against 50 mM Tris-Ac buffer (pH 8.5) and loaded on a Q Sepharose High Performance (HP) column (2.6 x 65 cm 2 ) equilibrated with 50 mM Tris-Ac buffer (pH 8.5).
  • the column was equilibrated by 50 mM Tris-Ac buffer (pH 6.0) and eluted by 40 column volumes of 50 mM Tris-Ac buffer using a pH gradient of 6.0-7.0 at a flow rate of 0.8 ml/min. The desired fractions were pooled and concentrated.
  • HbA Reductive Carboxymethylation of Hb.
  • HbA was carboxymethylated using glyoxylate. Briefly, 0.5 mM HbA was incubated with 2 mM glyoxylate and 25 mM sodium cyanoborohydride at 4 0 C in 50 mM Bis-Tris buffer, pH 6.5 for 4 h. The excess unreacted materials were removed by dialyzing against PBS at 4 0 C overnight with 13,000 Mw cutoff dialyzing membrane. The product was further dialysed against Tris-acetate buffer, pH 8.0 at 4 0 C for 4 h, then purified and then applied to a Q-Sepherose high performance column (2.6 * 65 cm, 350 mL of column volume, Pharmacia).
  • the column was equilibrated with 50 mM Tris acetate at pH 8.0 (buffer A) using an AKTA Purifier 10 System (Amersham Pharmacia Biotech.), and eluted at 4 0 C with two linear gradient of 0-90% buffer B (50 mM Tris acetate at pH 6.8) in 6 column volumes and then 90-100% buffer B in 8 column volumes.
  • the column was eluted at a flow rate of 2 ml/min.
  • the effluent was monitored at 540 and 630 nm. The peaks were collected and dialyzed against PBS overnight, and then concentrated and stored in -8O 0 C freezer. [0046] Analytical methods.
  • Size exclusion chromatography (SEC) of the Hb samples was carried out at room temperature on two Superose 12 columns (Amersham-Pharmacia Biotech, 1 x 31 cm 2 ) connected in series. The columns were equilibrated and eluted by PBS buffer (pH 7.4) at a flow rate of 0.5 ml/min.
  • Reverse Phase (RP)-HPLC analysis of the globin chains was carried out on a Vydac C4 column (0.46 x 25 cm 2 ) using a linear gradient of 35- 50% acetonitrile containing 0.1% trifluoro acetic acid (TFA) in 100 min at a flow rate of 1.0 ml/min.
  • the g(s*) distributions were determined using DCDT+ v2.0.4 (http://www.jphilo.mailway.com) using values of v of 0.74 mL/g for HbA and [Propyl- VaI-I ( ⁇ )] 2 -Hb [16] and 0.806 mL/g for the PEGylated Hbs [17].
  • the g(s*) was normalized to standard conditions (S 2O1 W and £ ) 20 ,w) by correcting for buffer density and viscosity.
  • Sedimentation equilibrium experiments were conducted at 10,000 and 15,000 rpm using six channel centerpieces. Three protein concentrations 0.4 and 0.8) were analyzed for each sample. The identity of scans taken at 22 h and 24 h confirmed that the samples had reached equilibrium. The data was globally analyzed using HeteroAnalysis vl.1.33 (J.L. Cole & J. W. Lary, Analytical Ultracentrifugation Facility, Biotechnology Services Center, University of Connecticut, Storrs, CT 06269). The best-fit values and joint confidence intervals are reported.
  • Tetramer-dimer dissociation constant measured by SEC was measured essentially as described by Manning et al. [24]. Briefly, Hb samples were diluted to a series of Hb concentrations and subjected to two Superose 12 columns (1 x 31 cm 2 ) in series. PBS buffer at pH 7.4, 6.5 or 8.5 was used as indicated in the text. NaOH or HAc was used to adjust the pH of the PBS buffer. The columns were equilibrated and eluted at a flow rate of 0.5 ml/min.
  • ⁇ -fumaryl Hb was set as the tetramers for HbA and [Propyl-Val-l( ⁇ )] 2 -Hb.
  • [Propyl-PEG5K-Val-l( ⁇ )] 2 - ⁇ -Hb was set as the tetramers for the two PEGylated Hbs.
  • Circular dichroism spectroscopy Circular dichroism spectroscopy.
  • Circular dichroism (CD) spectra of the Hb samples were recorded on a JASCO-720 spectropolarimeter (Tokyo, Japan) at 25 0 C with a 0.2-cm light path cuvette (310 ⁇ l).
  • CD spectra from 250 nm to 200 nm
  • the Hb concentration was 1.3 ⁇ M as tetramer.
  • the Hb concentration was 26.0 ⁇ M as tetramer. All the Hb samples were in PBS buffer, pH 7.4.
  • the molar ellipticity ( ⁇ ) is expressed in deg.cm 2 /dmol on a heme basis.
  • Front-face fluorescence measurements Intrinsic front-face fluorescence measurements were performed using Shimadzu RF-5301 spectrofluorimeter at room temperature. The emission spectra were recorded from 300 nm to 400 nm using an excitation wavelength of 280 nm. Excitation and emission slit widths were both 5 nm. All the samples used were at Hb concentration of 6.0 mg/ml in PBS, pH 7.4. Cuvette with 1 cm path-length was used.
  • Oxygen affinity measurements Oxygen equilibrium curves of the Hb samples
  • the PEG model was PEG5K molecule consisting of 1 13 consecutive ethylene oxide moieties and was generated using Accelrys Insight II software. The model was refined in water by simulated annealing to derive a globular structure. The simulation was carried out using DISCOVER program in Accelrys suite of programs. The consistent valence force field potential was used. All the four N-terminals are covalently connected to propyl end of the PEG chain with single bond. The calculations of surface accessible area (SAS) were carried out using the algorithm of Lee & Richards [27] and the program was developed at Scripps [28]. The Insight Graphics was mainly used for computer modeling. The loss of SAS due to association was then calculated.
  • SAS surface accessible area
  • VaI- l( ⁇ ) and VaI- l( ⁇ ) show an unusually higher reactivity to aldehyde group than Lys residues, due to their lower pK a (6.95 for VaI-I ( ⁇ ) and 7.05 for Val-l( ⁇ )) [29].
  • IHP an allosteric effector can reversibly bind VaI- l( ⁇ ) and efficiently inhibit the modification at VaI- l( ⁇ ).
  • site-specific PEGylation of VaI-I ( ⁇ ) can be achieved at low pH in the presence of IHP.
  • the PEGylated Hb was purified by anion exchange chromatography on a Q Sepharose HP column (Fig.
  • Peak b corresponds to the elution position of Hb.
  • Peak b presumably corresponds to the PEGylated Hb at VaI-I ( ⁇ ).
  • Peaks c and a presumably correspond to the PEGylated Hb at VaI- l( ⁇ ) and Hb conjugated with multiple PEG chains.
  • Peak b (Sample A) and Peak c (Sample B) were pooled for characterization.
  • Sample B is a PEGylated Hb site-specifically modified at VaI- l( ⁇ ) and referred as [Propyl-PEG5K- Val-l( ⁇ )] 2 -Hb.
  • Fig. 3E As compared to the tryptic peptides of ⁇ globin (Fig.
  • Sample A is a PEGylated Hb site-specifically modified at VaI-I ( ⁇ ) and referred as [Propyl-PEG5K-Val-l( ⁇ )] 2 -Hb.
  • PEGylated Hbs exhibited higher hydrodynamic volume than HbA (Fig. 4).
  • the PEGylated ⁇ -fumaryl Hb site-specifically modified at VaI- l( ⁇ ) [Propyl-PEG5K-Val-l( ⁇ )] 2 - ⁇ -Hb was eluted earlier than that of [Propyl-PEG5K-Val-l( ⁇ )] 2 -Hb and close to that of [Propyl- PEG5K-Val-l( ⁇ )] 2 -Hb.
  • PEGylation at VaI- l( ⁇ ) presumably led to extensive tetramer- dimer dissociation of Hb.
  • HbA and [Propyl-PEG5K-Val-l( ⁇ )] 2 -Hb showed 12-fold and 46-fold increase in the K d , respectively.
  • the Kd of [Propyl-PEG5K-Val-l( ⁇ )] 2 -Hb could not be measured due to its very high K d at pH 6.5.
  • the K ⁇ of [Propyl-PEG5K-Val-l( ⁇ )] 2 -Hb is less sensitive to pH than that of [Propyl-PEG5K-Val-l( ⁇ )] 2 -Hb and more sensitive than that of HbA.
  • Peaks c and a correspond to unmodified Hb and Hb conjugated with multiple propyl moieties, respectively.
  • Peak b is pooled and subjected to RP-HPLC analysis.
  • the peak of ⁇ globin for Peak b is not shifted and the peak of ⁇ globin is slightly right-shifted as compared with that of HbA.
  • MALDI-TOF showed that this shifted peak has a M w of 15170.0 Da, corresponding to ⁇ globin (15127.3 Da) conjugated with one propyl unit (43.0 Da) (Table 1).
  • IEF analysis showed that the propylated Hb migrated as a single band during IEF, reflecting its homogeneity (the left inset, Fig. 2).
  • SDS-PAGE analysis showed that it migrated as a single band, comparable to that of HbA (the right inset, Fig. 2).
  • Hbs have been carried out by the CD spectroscopy and the intrinsic front-face fluorescence spectroscopy.
  • the region around 285 run is considered to be indicative of the transition from the R state to the T state [31].
  • the ellipticity at 285 nm was increased by propylation at
  • PEGylation of Hb may alter the quaternary structure of Hb.
  • the Soret band of Hb is informative on the interactions of the heme prosthetic group with the surrounding aromatic residues [32].
  • the ellipticity of HbA was not altered by propylation at VaI- l( ⁇ ). However, it was increased by PEGylation at VaI- l( ⁇ ) with the maximal ellipticity at 421 nm blue-shifted to 419 nm.
  • the PEG chains at VaI- l( ⁇ ) was increased by PEGylation at VaI- l( ⁇ ) with the maximal ellipticity at 421 nm blue-shifted to 419 nm.
  • VaI- l( ⁇ ) significantly altered the heme environment of Hb.
  • the ellipticity of Hb was slightly increased by PEGylation at VaI- l( ⁇ ).
  • Intrinsic front-face fluorescence Intrinsic front-face fluorescence. Intrinsic front-face fluorescence of Hb could be used to monitor the quaternary conformational changes. This sensitivity of the signal was assigned mostly to Trp-37( ⁇ ). When excited at 280 nm, the fluorescence intensity of
  • HbA slightly increased upon propylation at VaI- l( ⁇ ) and increased further by conjugation of the PEG chains with a peak position at 320 nm (Fig. 7B).
  • the fluorescence sensitivity to the structural changes relies significantly on the conditions for quenching by Forster-type energy transfer to the hemes [33].
  • Trp-37( ⁇ ) is close to the neighboring ⁇ subunit heme, which is liable to become perturbed by changes at the ⁇ l ⁇ 2 interface [33].
  • the increase in the fluorescence of Trp-37( ⁇ ) partially results from the tetramer-dimer dissociation of Hb.
  • Hbs The oxygen affinity of the three modified Hbs was investigated in PBS (pH 7.4) at 37 0 C. As shown in Table 5, the oxygen affinity of HbA increased upon propylation at VaI- l( ⁇ ) and did not increase further by conjugation of the PEG5K chains. The Hill coefficient (n) of HbA was slightly decreased upon propylation at VaI- l( ⁇ ) and significantly decreased upon PEGylation at VaI- l( ⁇ ). This indicated that the PEG chains at VaI- l( ⁇ ) can decrease the n of HbA, presumably due to the tetramer-dimer dissociation of Hb upon PEGylation.
  • Chloride and L35 can bind at the ⁇ end of the central cavity and are quite opposite to DPG and IHP that bind at the ⁇ end.
  • Propylation at VaI- l( ⁇ ) did not alter the sensitivity of HbA to DPG and IHP but increase the sensitivity to NaCl and L35 (Table 5).
  • PEGylation at VaI- l( ⁇ ) did not alter the sensitivity of HbA to DPG and IHP but decrease the sensitivity to NaCl and L35.
  • the PEG chains at VaI- l( ⁇ ) can partially inhibit the binding of chloride and L35 at the ⁇ end of Hb.
  • PEGylation at Val-l( ⁇ ) did not alter the sensitivity to NaCl and L35 but essentially diminished the sensitivity to DPG and IHP.
  • Bohr effect of the modified Hbs (the decrease in oxygen affinity as function of a decrease in pH) is expressed as the number of Bohr protons released upon oxygenation.
  • Fig. 8 shows the oxygen affinity of the modified Hbs as a function of pH.
  • the Bohr effect estimated from the slopes ( ⁇ H + ) between pH 6.5 and 8.0 is 0.38 proton/heme for HbA.
  • Propylation at VaI- l( ⁇ ) leads to an increase in the H + release (0.52 proton/heme) and enhances the Bohr effect of HbA.
  • PEGylation at VaI- l( ⁇ ) result in a decrease in the H + release (0.25 proton/heme).
  • the PEG chains conjugated at VaI- l( ⁇ ) can significantly lower the Bohr effect of HbA.
  • PEGylation at VaI- l( ⁇ ) led to a lower H + release (0.17 proton/heme) than PEGylation at VaI- l( ⁇ ).
  • the Bohr effect of the PEGylated Hb is dependent on the sites of PEGylation.
  • the n of HbA and [Propyl-Val-l( ⁇ )] 2 -Hb slightly changed as a function of pH.
  • the n of the two PEGylated Hbs significantly increased as pH increased from 6.5 to 8.0.
  • VaI- l( ⁇ ) is given in Table 7. It may be noted that the product also has a high oxygen affinity just as hemospan, which is generated by extension arm facilitated PEGylation and is in phase III clinical trial. The stability of this hexaPEGylated tetramer is higher than that of hemospan.
  • Hb VaI- l( ⁇ ) and VaI-I ( ⁇ ) are PEGylated quantitatively, and by using these carboxymethylated and propylated Hbs, PEGylation at these sites are avoided, and accordingly can generate a PEG-Hb adduct with a native like tetramer stability, if the PEGylation at the respective sites is directly responsible for the enhanced tetramer dissociation.
  • Hb site specifically carboxymethylation at of VaI-I ( ⁇ ) or VaI- l( ⁇ ) has been used for reductive PEGylation to avoid PEGylation at these two sites.
  • HexaPEGylation of [Cm-VaI- l( ⁇ )] 2 -Hb and [Cm-VaI- l( ⁇ )] 2 -Hb exhibit an elution pattern on FPLC comparable to that of hexaPEGylated Hb.
  • Elution position of all these three hexaPEGylated uncrosslinked Hbs is later than that of hexaPEGylated intramolecularly crosslinked Hb.
  • the PEGylated uncrosslinked carboxymethylated Hbs exists essentially as PEGylated dimers under the physiological conditions. Avoiding the PEGylation on the ⁇ -amino groups of Hb during reductive hexaPEGylation of the molecule by pre-derivatization of these sites with carboxymethylation (Cm) does not afford the native Hb like tetramer stability to reductively hexaPEGylated tetramers.
  • the PEGylated globin chains isolated by RP-HPLC were analyzed by MALDI-TOF.
  • the number of the PEG chains was calculated from the difference between their mass and the unPEGylated globin.
  • the molecular volume of the modified Hbs was calculated from the molecular radius measured by dynamic light scattering at Hb concentration of 1 mg/ml.
  • HbA PEGylatedHb 3 PEGylatedHb" Propylated Hb c pH6.5 4.18 ⁇ 0.01 30.8 2.69 ⁇ 0.01 ND f 2.80 ⁇ 0.01 1134.0 4.64 ⁇ 0.01 3.12 pH7.4 4.58 ⁇ 0.01 2.5 2.98 ⁇ 0.01 724.0 3.32 ⁇ 0.01 24.5 4.86 ⁇ 0.01 0.30 pH8.5 4.67 ⁇ 0.01 1.5 3.02 ⁇ 0.01 309.1 3.58 ⁇ 0.01 1.8 4.86 ⁇ 0.01 0.31 a [Propyl-PEG5K-Val-l( ⁇ )] 2 -Hb b [Propyl-PEG5K-Val-l( ⁇ )] 2 -Hb '[Propyl- VaI
  • Oxygen equilibrium curves of the samples were measured at 37 ° C in PBS (pH 7.4) at a Hb tetramer concentration of 0.5 mM.
  • the Hill coefficient is given in the parenthesis.
  • P 50 is the partial oxygen pressure at 50% saturation and is expressed in mmHg on the left of the Hill coefficient.
  • Oxygen equilibrium curves of the samples were measured at 37 ° C in PBS (pH 7.4) at Hb tetramer concentration of 0.5 mM.
  • n is the Hill coefficient.
  • P 50 is the partial oxygen pressure at 50% saturation and is expressed in mmHg on the left of the Hill coefficient.
  • the dissociation constant (Kd) was measured in PBS buffer, pH 7.4.
  • the present study was aimed at two primary objectives.
  • the first one focused on providing a biochemical insight into the influence of PEGylation (the sites of PEGylation and the length of the PEG chain) on the tetramer-dimer dissociation of Hb.
  • the result shows that this influence comes from the PEG chains conjugated at VaI- l( ⁇ ).
  • the second objective was the design of a new strategy to increase the tetramer stability of the PEGylated Hb. The result indicate that this objective can be achieved by avoiding PEGylation at Val-l( ⁇ ).
  • the tetramer stability of Hb depends on the polar interactions (e.g., hydrogen bonds) and the hydrophobic interactions [18].
  • the pH dependent dissociation of Hb is ascribed to the breaking or to the formation of hydrogen bonds upon protonation/deprotonation of one of the residues involved in their proximity [18].
  • the PEG chains at VaI- l( ⁇ ) enhance the breaking of the hydrogen bonds (Table 3).
  • the quaternary structure of oxyHb generates a central cavity that can accommodate at least 80 water molecules.
  • the PEG chains perturb the hydration layer of Hb and the organization of the water molecules around Hb. This presumably attenuates the number and the strength of the hydrogen bond.
  • the PEG chains at VaI- l( ⁇ ) also lead to the greater hydration of Hb, force the exposure of a more extensive hydrophobic surface to solvent and decrease the hydrophobic interactions of Hb.
  • the ⁇ end of the central cavity is not extensive in liganded Hb and very close to the tetramer-dimer allosteric interface.
  • the ⁇ end of the central cavity is involved in more extensive interactions than does the ⁇ end [15].
  • the breaking of hydrogen bonds at the ⁇ end makes more contribution on the tetramer-dimer dissociation of Hb than that at the ⁇ end, even though PEGylation at VaI- l( ⁇ ) has a lower propensity than that at VaI- l( ⁇ ) to weaken the hydrogen bonds.
  • the substitution of Ala and acetyl Ser for VaI- l( ⁇ ) results in an insignificant influence on the K d of Hb [15].
  • the tetramer-dimer dissociation of the PEGylated Hb is dependent on the site of PEGylation.
  • Association of Hb dimers to tetramers is driven primarily by the interactions between residues at the ⁇ l ⁇ 2 interface [16].
  • the ⁇ l ⁇ 2 interface is highly polar and dynamic, undergoing large structural changes as transition of the R to T state.
  • Variants with substitutions at the ⁇ l ⁇ 2 interface e.g., Hb Rothschild ( ⁇ 37 Trp ⁇ Arg) [37-38] and Hb Kansas ( ⁇ lO2 Asn— »Thr) [39-40] may destabilize the ⁇ l ⁇ 2 interface and lead to the extensive dissociation of Hb tetramer.
  • VaI- l( ⁇ ) is very close to the ⁇ l ⁇ 2 interface
  • the PEG chains at VaI- l( ⁇ ) may alter the polarity, hydrophobicity and spatial orientation of residues at the ⁇ l ⁇ 2 interface. This alteration may destabilize the ⁇ l ⁇ 2 interface and perturb the heme environment and quaternary structure of Hb.
  • the molecular models show that the PEG5K chains conjugated at VaI- l( ⁇ ) and at VaI- l( ⁇ ) form loosely organized domains on the Hb surface (Fig. 10).
  • the interdomain distances for the two PEG chains at the ⁇ and ⁇ end are 66.9 A and 16.0 A, respectively.
  • the proximity of the two PEG domains at the ⁇ end facilitated the interaction with each other (Fig. 10B).
  • This structural aspect is absent in the diPEGylated Hb at VaI- l( ⁇ ) (Fig. 10A). This difference in the interactions of the two PEG chains at the two ends, apparently, impact the local hydration layer of Hb and may impact the tetramer stability of Hb.
  • the differential orientation of the PEG domains also leads to the loss of the solvent accessible surface (SAS) area at a different level (Table 6).
  • SAS solvent accessible surface
  • the loss in the SAS area of the diPEGylated Hb at VaI- l( ⁇ ) is higher than that at VaI- l( ⁇ ).
  • the proximity of the two PEG domains at the ⁇ -end may result in their significant loss of the SAS area of the PEG chains. However, such a contribution is absent in the distal PEG domains at the ⁇ end.
  • the interdomain interactions of the PEG chains conjugated at the ⁇ end may act as a pseudo crosslinker outside the central cavity to strengthen the tetramer stability of Hb.
  • the anticipated superior structural and functional properties of this product are (i) the lower Kd of new hexaPEGylated molecule (6.3 ⁇ M vs. 25 ⁇ M); (ii) expected low autoxidation rate in view of free Cys-93( ⁇ ); (iii) expected similar levels of tissue oxygenation for comparable oxygen affinity and (iv) more efficient vasodilation through nitrite reductase activity and transport of NO in view of free Cys-93( ⁇ ).

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Abstract

L'invention porte sur des hémoglobines PEGylées (Hbs) modifiées à la valine-1(alpha) de façon à augmenter la stabilité des tétramères de l'hémoglobine, sur des procédés de production de ces hémoglobines modifiées et sur leur utilisation en tant que substituts sanguins.
PCT/US2009/006079 2008-12-08 2009-11-12 Hémoglobines pegylées stabilisées à la valine-1 (alpha), leurs procédés de préparation et leur utilisation WO2010077260A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11529421B2 (en) 2017-12-20 2022-12-20 University Of Essex Enterprises Limited Modified globin proteins

Non-Patent Citations (1)

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Title
HU T. ET AL: "Influence of the chemistry of conjugation of polyethylene glycol to hemoglobin on the oxygen binding and solution properties of the PEG-Hemoglobin conjugate", BIOCHEMICAL JOUMAL IMMEDIATE PUBLICATION, 19 August 2005 (2005-08-19), pages 1 - 33, Retrieved from the Internet <URL:http://www.biochemj.org/bj/imps_x/pdf/BJ20050663.pdf> [retrieved on 20100105] *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11529421B2 (en) 2017-12-20 2022-12-20 University Of Essex Enterprises Limited Modified globin proteins

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