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(11) Patent Number: KE 168 Kenya Ihdustial Prep, NM..
(45) Date of grant: 13/01/2003
A 61K 47/48
(21) Application Number: 2000/ 000310
(22) Filing Date: 30/06/2000
(30) Priority data:
60/142/254 20/07/1999 US; 60/150,225 23/08/1999 US; 60/151,548 31/08/1999 US and 60/166,151 17/11/1999 US
(73) Owner: F. HOFFMAN-LA ROCHE AG of , 124 GRENZACHERSTRASSE CH-4070 BASLE, Kenya
Pascal Sebastian Bail=
(74) Agent/Address of correspondence: JANE WANYAGA & CO. P.o.Box 535 Nairobi
The present invention refers to conjugates of erythropoietin with (ethlyleneglycol) comprising an erythropoietin glycoprotein having the in vivo biological activity of causing bone marrow cells to increase the production of retculocytes and the red blood cells and selected from the group consisting of human erythropoietin and analogs thereof which have sequence of human erythropoietin modified by the addition from 1-6 glycosylation sites or the rearrangement of at least one glycosylation site ; said glycoprotein being covalently linked to "n" poly ( ethylene glycol ) groups of formula- -00-(CH2) X OCH2C142)M -OR WITH THE CARBONAYLOF EACH POLY(ETHYLENEGLYCOL )group forming an amide bond with one of said amino groups ; wherein R is a lower alkyl ; Xis 2 or 3; M is about 450 to about 900 ; n is from 1-3 and n&m are chosen so that the molecular weight of the conjugate minus the erythropoeitin glycoprotein is from 20 kiodaltons to 100 kilodaltons.
CHEMICALLY MODIFIED NOVEL ERYTHROPOIETIN STIMULATING
PROTEIN COMPOSITIONS AND METHODS
BACKGROUND OF THE INVENTION
Novel erythropoietin stimulating protein (NESP) is a hyperglycosylated erythropoietin analog having five changes in the amino acid sequence of rHuEPO
which provide for two additional carbohydrate chains.
10 More specifically, NESE, contains two additional N-linked carbohydrate chains at amino acid residues 30 and 88 (numbering corresponding to the sequence of human
EPO) (see PCT Application No. US94/02957, herein
incorporated by reference in its entirety). NESP is
15 biochemically distinct from EPO, having a longer serum half-life and higher in vivo biological activity; Egrie
at al., ASH 97, Blood, 22:56a (1997). NESS has been shown to have -3 fold increase in serum half-life in mice, rats, dogs and man; Id. In mice, the longer serum
20 half-life and higher in vivo activity allow for less frequent dosing (once weekly or once every other week)• compared to rHuEPO to obtain the same biological response; Id.
A pharmacokinetic study demonstrated that,
25 consistent with the animal studies, NESS has a
significantly longer serum half-life than rHuEPO in chronic renal failure patients, suggesting that a less frequent dosing schedule may also be employed in humans; MacDougall, et al., J American Society of Brephrology,
30 11:268A (1997). A less frequent dosing schedule would be more convenient to both physicians and patients, and would be particularly helpful to those patients involved in self-administration. Other advantages to less
frequent dosing may include less drug being introduced into patients, a reduction in the nature or severity of the few side-effects seen with rHuEPO administration, and increased compliance.
5 Although the extended hilf-life of NESP offers
the advantage of less frequent dosing relative to EPO, there are still potential indications, such as chemotherapy, which may require an even longer therapeutic half-life than NESP currently demonstrates.
10 A common approach often used to extend the
half-lives of proteins in vivo is the chemical conjugation of a water soluble polymer, such as polyethylene glycol (PEG), to the protein of interest. Generally, polyethylene glycol molecules are connected
15 to the protein via a reactive group found on the protein. Amino groups, such as those on lysine residues or at the N-terminus, are convenient for such 'attachment.
A variety of approaches have been used to
20 attach the polyethylene glycol molecules to the protein (PEGylation). For example, Royer (U.S. Patent
No. 4,002,531) states that reductive alkylation was used for attachment of polyethylene glycol molecules to an enzyme. Davis et al. (U.S. Patent No. 4,179,337)
25 disclose PEG:protein conjugates involving, for example, enzymes and insulin. Shaw (U.S. Patent No. 4,904,584) disclose the modification of the number of lysine residues in proteins for the attachment of polyethylene glycol molecules via reactive amine groups. Hakimi et
30 al. (U.S. Patent No. 5,834,594) disclose substantially non-immunogenic water soluble PEG:protein conjugates, involving for example, the proteins IL-2, interferon alpha, and IL-lra. The methods of Hakimi et al. involve the utilization of unique linkers to connect the various
free amino groups in the protein to PEG. Kinstler et
al. (U.S. Patent Nos. 5,824,784 and 5,985,265) teach methods allowing for selectively N-terminally chemically modified proteins and analogs thereof,
5 including G-CSF and consensus interferon. Importantly, these modified proteins have advantages as relates to protein stability, as well as providing for processing advantages.
PEGylation approaches such as those described
10 above are traditionally applied to non-glycosylated proteins derived from bacterial expression systems in order to render improvements in solubility and in vivo circulating half-lives (such properties are typically conferred to glycosylated proteins (glycoproteins)
15 through the carbohydrate moieties added in the course of eukaryotic expression). The effects of PEGylation on the in vivo half-lives of non-glycosylated proteins is generally thought to derive from the physicochemical and dynamic properties of PEG conferring a larger
20 hydrodynamic volume and total mass to the conjugate, thus reducing the rate of renal clearance. Additional benefits typically include increased solubility and decreased immunogenicity for the conjugate. However,• not all proteins respond equally to PEGylation and there
25 is no guarantee of improved performance.
The present invention is based upon the surprising finding that a highly glycosylated protein, e.g., NESP, can be PEGylated to provide a pharmaceutical composition with an even more dramatic sustained
30 duration profile than NESP, allowing for a once every 4¬6 week dosing for raising hematocrit and treating anemia, and thus providing tremendous therapeutic advantage.
plymARy. OF THE INVENTION
The present invention relates to a
5 substantially homogenous preparation of chemically modified NESP (or analog thereof) and related methods.
The present invention further relates to a substantially homogenous preparation of N-terminally chemically modified NESP (or analog thereof).
10 The present invention further relates to a
preparation of chemically modified NESS represented as a mixed population of either monosubstituted positional isoforms or polysubstituted forms.
15 DRIER DESCRIPTION OF THE FIGURES
Figure 1 depicts the design strategy for NESS PEGylation: (A) PEG polymer size is varied from 5kD, 20kD and 30kD; (B) PEG polymer conformation can be
20 either linear or branched with total molecular weights of 10kD, 20kD or 4.0kD PEG; and (C) preparations of PEG;NESP with different degrees of substitution can be isolated to include: mono-PEG, di-PEG or, in some cases, tri-PEG NESP.
Figure 2 depicts the various reaction chemistries for PEGylation of NESP: (A) reductive alkylation of NESP with PEG-aldehyde; (B) acylation of NESP with N-succinimidyl ester of PEG; and
30 (C) PEGylation of the NESP polysaccharide side chains by limited periodate oxidation of the carbohydrate with the resultant aldehyde reacted with PEG-hydrazide to form a hydrazone linkage followed by subsequent reduction with sodium cyanoborohydride to stabilize the linkage.
Figure 3 is a graph depicting in vivo activity data of various 5kD poly-PEG:NESP conjugates vs. unmodified NESP (11). Samples and -1-
5 are mixtures of 5kD poly-PEG:NESP with progressively lower degrees of substitution. % iron uptake is plotted vs. ng/mL administered.
Figure 4 is a graph depicting prolongation of
10 elevated hemoglobin (HGB) levels in response to treatment with various PEG:NESP conjugates relative to unmodified NESP. Single bolus injection of 100 pg/kg NESP (4), 20kD linear mono-PEG:NESP conjugate derived from NHS-ester activated methoxy-PEG (E), 20kD linear
15 (-80% mono-PEG:NESP and 20% di-PEG:NESP) conjugate derived by reductive alkylation from aldehyde activated PEG (V), and a saline control (•). HGB (g/dL) is plotted vs. # days post-treatment.
20 Figure 5 is a graph depicting prolongation of
elevated reticulocyte levels in response to treatment with various PEG:NESP conjugates relative to unmodified NESP. Single bolus injections of 100 pg/kg NESP (0), 20kD linear mono-PEG:NESP (•), 5kD linear mono-PEG:NESP
25 (V) and 5kD linear di-PEG:NESP conjugates (4) derived by reductive alkylation from aldehyde activated methoxy¬PEG, a 20kD branched mono-PEG:NESP (1). conjugate from NHS-ester activated PEG, and a saline control (A). Absolute reticulocyte count is plotted vs. # days post-
Figure 6 is a graph depicting prolongation of elevated hemoglobin levels in response to treatment with various PEG:NESP conjugates relative to unmodified NESP.
Single bolus injections of 100 gg/kg NESP (0), 20kD linear mono-PEG:NESP (0), 5kD linear mono-PEG:NESP (V) and 5kD linear di-PEG:11E5P conjugates (4) derived by reductive alkylation from aldehyde activated methoxy-PEG
5 and a 20kD branched mono-PEG:NESP conjugate (II) from NHS-ester activated PEG. HGB (g/dL) is plotted vs. 4 days post-treatment.
Figure 7 depicts a Q Sepharose HP column
10 chromatogram of the 5kD poly-PEG:NESP conjugate. The column was a HiTrap Q Sepharose HP column which utilized a 50mM Had to 200mM Had linear gradient to elute the product.
15 Figure 8 depicts a Q Sepharose HP column
chromatogram of the 20kD mono-PEG:NESP conjugate. The column was a HiTrap Q Sepharose HP column which utilized a 50MM NaCI to 200mM Neel linear gradient to elute the product.
Figure 9 depicts a Q Sepharose HP column chromatogram of the 30kD mono-PEG:NESP conjugate. The column was a HiTrap Q Sepharose HP column which utilized a 50mM NaC1 to 200mM NaC1 linear gradient to elute the
Figure 10 is a graph depicting reticulocyte response of anemic mice after single bolus injections of 3 gg/kg 30kD mono-PEG:NESP conjugate (V), 3 gg/kg 20kD
30 mono-PEG:NESP conjugate (M), and 3 Kg/kg 5kD poly¬PEG:NESP conjugate mixture AU. Absolute reticulocyte count is plotted vs. # days post-treatment.
Figure 11 is a graph depicting reticulocyte response of anemic mice after single bolus injections of 10 pg/kg 30kD mono-PEG:NESP conjugate (r), 10 pg/kg 20kD
mono-PEG:NESP conjugate (■), and 10 pg/kg 5kD poly-
5 PBG:NESP conjugate mixture (0). Absolute reticulocyte count is plotted vs. # days post-treatment.
Figure 12 is a graph depicting reticulocyte response of anemic mice after single bolus injections of
10 30 pg/kg 30kD mono-PEG:NESP conjugate (r), 30 pg/kg 20kD mono-PEG:NESP conjugate (U), and 30 pg/kg 5kD poly-PEG:NESP.conjugate mixture (S) vs. 30 pg/kg unmodified NESP (0). Absolute reticulocyte count is plotted vs. # days post-treatment.
Figure 13 is a graph depicting hemoglobin response of anemic mice after single bolus injections of 3 pg/kg 30kD mono-PEG:NESP conjugate (r), 3 Ng/kg 20kD mono-PEG:NESP conjugate (■), and 3 pg/kg 5kD poly-
20 PEG:NESP conjugate mixture (•. HGB (g/dL) is plotted vs. # days post-treatment.
Figure 14 is a graph depicting hemoglobin response of anemic mice after single bolus injections of
25 10 pg/kg 30kD mono-PEG:NESP conjugate (r), 10 pg/kg 20kD mono-PEG:NESP conjugate (M), and 10 pg/kg 5kD poly-PEG:NESP conjugate mixture (•). HGB (g/dL) is plotted vs. # days post-treatment.
30 Figure 15 is a graph depicting hemoglobin
response of anemic mice after single bolus injections of 30 pg/kg 30kD mono-PEG:NESP conjugate (r), 30 gg/kg 20kD
mono-PEG:NESP conjugate (■), and 30 µg/kg 5kD poly¬PEG:NESP conjugate mixture (•) vs. 30 gg/kg unmodified NESP (0). 0G9 (g/dL) is plotted vs. it days post¬treatment.
Figure 16 is a graph depicting reticulocyte response of normal mice after single bolus injections of 3 pg/kg 30kD mono-PEG:NESP conjugate (r), 3 pg/kg 20kD mono-PEG:NESP conjugate (M), and 3 gg/kg 5k0 poly-
10 PEG:NESP conjugate mixture M. Absolute reticulocyte count is plotted vs. I days post-treatment.
Figure 17 is a graph depicting reticulocyte response of normal mice after single bolus injections of
15 10 gg/kg 30k0 mono-PEG:NESE, conjugate (I), 10 gg/kg 20kD mono-PEG:NESP conjugate (■), and 10 pg/kg 5kD poly¬PEG:NESP conjugate mixture (S). Absolute reticulocyte count is plotted vs. it days post-treatment.
20 Figure 18 is a graph depicting reticulocyte
response of normal mice after single bolus injections of 30 gg/kg 30kD mono-PEG:NESP conjugate (Y), 30 gg/kg 20kD
mono-PEG:NESP conjugate (U), and 30 pg/kg 5kD poly¬PEG:NESP conjugate mixture (•) vs. 30 µg/kg unmodified
25 NESP (0). Absolute reticulocyte count is plotted vs. ft days post-treatment.
Figure 19 is a graph depicting hemoglobin response of normal mice after single bolus injections of
30 3 gg/kg 30kD mono-PEG:NESP conjugate (f), 3 pg/kg 20kD
mono-PEG:NESP conjugate (U), and 3 pg/kg 5kD poly-
PEG:NESP conjugate mixture (11). HGB (g/dL) is plotted vs. # days post-treatment.
Figure 20 is a graph depicting hemoglobin
5 response of normal mice after single bolus injections of 10 µg/kg 30kD mono-PEG:NESP conjugate on, 10 pg/kg 20kD
mono-PEG:NESP conjugate (M), and 10 µg/kg 5kD poly¬PEG:NESP conjugate mixture (4). HGB (g/clL) is plotted vs. it days post-treatment.
Figure 21 is a graph depicting hemoglobin response of normal mice after single bolus injections of 30 gg/kg 30kD mono-PEG:2,1E8P conjugate (V), 30 pg/kg 20kD mono-PEG:NESP conjugate (I), and 30 µg/kg 5kD poly-
15 PEG:NESP conjugate mixture (G) vs. 30 pg/kg unmodified
NESP (0). KGB (g/dL) is plotted vs. # days post-
Figure 22 depicts size exclusion HPLC
20 chromatograms of the 5kD poly-PEG:NESP (--), the 20kD mono-PEG:NESP (- - 1 and 30kD mono-PEG:NESP (---). The SEC column was. a Tosohaas TSK 3000 SWX1 (5 micron - 7.8 mm X 30 cm) which utilized 100mM NaHP00 10% ethanol, 150MM Nadi, pH 6.9, to elute the products.
DETAILED DESCRIPTION 07 3n INVENTION
To discover if the in vivo therapeutic half-life of a glycoprotein such as NESP would benefit from
30 PEGylation, a variety of different PEG:NESP conjugates were synthesized and tested in vivo for prolonged erythropoiesis.
In order to both optimize the potential effects of PEGylation and to identify the preferred sites and chemistries of PEG attachment, a design strategy was employed wherein polymer length,
5 conformation, and both the degree and sites of attachment were varied (see Figure 1).
Methods for preparing the PEGylated NESP of the present invention generally comprise the steps of (a) reacting NESP with polyethylene glycol (such as a
10 reactive ester or aldehyde derivative of PEG) under conditions whereby NESP becomes attached to one or more PEG groups, and (b) obtaining the reaction product(s). Because the specific sites of NESP modification might significantly alter the intrinsic activity of the
15 conjugate, three different PEGylation chemistries were explored (see Figure 2). The first approach utilizes reductive alkylation to conjugate a PEG-aldehyde (0-(3- Oxopropy1)-0'-methylpolyethylene glycol) to a primary amine of NESP. Under appropriate conditions, this
20 approach has been demonstrated to yield PEG conjugates predominately modified through the a-amine at the protein N-terminus. Because the PEG is linked through a
secondary amine by reductive alkylation there is the potential to preserve the charge at the protein N-
The second chemistry applied to PEGylation of
NESS was the acylation of the primary amines of NESS using the ENS-ester of methoxy-PEG (0-((N-Succinimidyloxycarbony1)-methy1]-0'-methylpolyethylene
30 glycol). In contrast to the previous chemistry, acylation with mathoxy-PEG-NHS results in an amide linkage which will eliminate the charge from the original primary amine.
The final attachment chemistry evaluated utilized a mild oxidation of NESP under conditions selected to target the pendant diol of the penultimate glycosyl unit sialic acid for oxidation to an aldehyde.
5 The resultant glycoaldehyde was then reacted with a methoxy-PEG-hydrazide (0-(Hydrazinocarbonylmethyl)-0'- methylpolyethylene glycol) to form a semi-stable hydrazone between the PEG and NESP. The hydrazone was subsequently reduced by sodium cyanoborohydride to
10 produce a stable PEG:NESP conjugate.
The present methods each provide for a
substantially homogenous mixture of polymer:protein conjugate. "Substantially homogenous" as used herein means that only polymer:protein conjugate molecules are
15 observed. As ascertained by peptide mapping and N-terminal sequencing, one example below provides for a preparation which is at least 90% polymer:protein conjugate, and at most 10% unreacted protein. Preferably, the PEGylated material is at least 95% of
20 the preparation (as in the working example below) and most preferably, the PEGylated material is 99% of the preparation or more. The polymer:protein conjugate has biological activity and the present 'substantially homogenous. PEGylated NESP preparations provided herein
25 are those which are homogenous enough -to display the advantages of a homogenous preparation, e.g., ease in clinical application in predictability of lot to lot pharmacokinetics.
One may also choose to prepare a mixture of
30 polymer:protein conjugate molecules, and the advantage provided herein is that one may select the proportion of mono-polymer:protein conjugate to include in the mixture. Thus, if desired, one may prepare a mixture of various protein with various numbers of polymer moieties
attached (i.e., di-, tri-, tetra-, etc.) and combine said conjugates with the mono-polymer:protein conjugate prepared using the present methods, and have a mixture with a predetermined proportion of mono-polymer:protein
5 conjugate. .
Initial experiments designed to evaluate and optimize PEG:protein reaction stoichiometries revealed that PEGylation by reductive alkylation using PEG-aldehyde was surprisingly somewhat inefficient,
10 requiring substantially higher molar ratios of PEG to protein than typically observed with non-glycosylated proteins. Similarly, acylation with PEG-ESOS esters was also slower and less efficient than expected. It was thus evident that the PEGylation of non-glycosylated
15 proteins was not necessarily predictive of the PEGylation of glycosylated proteins and that further optimization of reaction conditions was necessary.
The polymer molecules contemplated for use in the PEGylation approaches described herein may be
20 selected from among water soluble polymers or a mixture thereof. The water soluble polymer may be selected from the group consisting of, for example, polyethylene glycol, monomethoxy-polyethylene glycol, dextran, poly¬(N-vinyl pyrrolidone), propylene glycol homopolymers,'a
25 polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol), dextran, HPMA, Fleximer., and polyvinyl alcohol. The polymer selected should be water soluble so that the protein to which it is attached does not precipitate in an aqueous
30 environment, such as a physiological environment. For the acylation reactions, the polymer(s) selected should have a single reactive ester group. For the present reductive alkylation, the polymer(s) selected should have a single reactive aldehyde group. A preferred
reactive PEG-aldehyde is polyethylene glycol propionaldehyde, which is water stable, or mono C1-C10 alkoxy or aryloxy derivatives thereof (see, U.S. Patent 5,252,714). The polymer may be branched or unbranched.
5 Preferably, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable.
A particularly preferred water-soluble polymer for use herein is polyethylene glycol, abbreviated PEG.
10 As used herein, polyethylene glycol is meant to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono-(Cl-C10) alkoxy¬or aryloxy-polyethylene glycol.
The proportion of polyethylene glycol
15 molecules to protein molecules will vary, as will their concentrations in the reaction mixture. In general, the
optimum ratio (in terms of efficiency of reaction in that there is no excess unreacted protein or polymer) will be determined by the molecular weight of the
20 polyethylene glycol selected and on the number of available reactive groups (typically ..or 3 amino groups) available. As relates to molecular weight, the higher the molecular weight of the polymer, the fewer number of polymer molecules which may be attached to the protein.
25 Similarly, branching of the polymer should be taken into account when optimizing these parameters. Generally, the higher the molecular weight (or the more branches) the higher the polymer:protein ratio. In the present invention, several different linear PEG polymer lengths
30 were evaluated (5kD, 20kD and 30kD). Similarly,
conjugates of two-armed branched PEG polymers (10kD, 20kD and 40kD) were also tested. From each preparation, samples of mono-substituted and di-substituted PEG:NES?
were isolated to investigate the effects of secondary sites of PEGylation.
In general, for the PEGylation reactions contemplated herein, the preferred average molecular
5 weight is about 2kDa to about 100kDa (the term "about"
indicating ± lkDa). More preferably, the average
molecular weight is about SkDa to about 40kDa. The ratio of water-soluble polymer to NESP will generally range from 1:1 for monoPEG-, 2:1 for diPEG etc, and the
10 mass ratios for PEG:protein would run -1:7 for 5kD mono-PEG to -1:1.3 for 30kD monoPEG.
The method of obtaining the PEGylated NESP preparation may be by purification of the PEGylated material from a population of non-PEGylated NESP
15 molecules. For example, presented below is an example where mono- and/or di-PEGylated NESP is separated using ion exchange size chromatography. Size exclusion chromatography is used as an analytical tool to characterize the purified products.
20 The present invention also provides a method
for selectively obtaining N-terminally chemically modified NESP. The method comprises reductive
alkylation which exploits.differential reactivity of different types of primary amino groups (lysine versus
25 the N-terminal) available for derivatization in a
particular protein. Under the appropriate reaction conditions, substantially selective derivatization of
the protein at the N-terminus with a carbonyl group containing polymer is achieved. The reaction is
30 performed at pH which allows one to take advantage of the pKa differences between the e-amino groups of the
lysine residues and that of the a-amino group of the N-
terminal residue of the protein. By such selective derivatization attachment of a water soluble polymer to
a protein is controlled: the conjugation with the polymer takes place predominantly at the N-terminus of
the protein and no significant modification of other reactive groups, such as the lysine side chain amino
5 groups, occurs. The preparation will preferably be greater than 80% mono-polymer:protein conjugate, and more preferably' greater than' 95% mono-polymer:protein conjugate.
NESP of the present invention is a
10 hyperglycosylated EPO analog comprising two additional
glycosylation sites with an additional carbohydrate chain attached to each site. NESP was constructed using site-directed mutagenesis and expressed in mammalian host cells. Details of the production of NEST, are
15 provided in co-owned PCT Application No. US94/02957. New N-linked glycosylation sites for rHuEPO were introduced by alterations in the DNA sequence to encode the amino acids Asn-X-Ser/Thr in the polypeptide chain. DNA encoding NESP was transfected into Chinese Hamster
20 Ovary (CHO) host cells and the expressed polypeptide was
analyzed for the presence of additional_ carbohydrate
chains. In a preferred embodiment, NESE. will have two
additional N-linked carbohydrate chains at residues 30
and 88. The numbering of the amino acid sequence is
25 that of human erythropoietin (EPO). The amino acid sequence of NESS is that depicted in SEQ ID NO: 1. It is understood that NESP will have the normal complement of N-linked and 0-linked glycosylation sites in addition to the new sites.
30 The NESP of the present invention may also
include conservative amino acid changes at one or more residues in SEQ ID NO: 1. These changes do not result in addition of a carbohydrate chain and will have little effect on the biological activity of the analog.
In general, comprehended by the present invention are pharmaceutical compositions comprising effective amounts of protein or derivative products of the invention together with pharmaceutically acceptable
5 diluents, stabilizers, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content
(e.g., Tris-HC1, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents
10 (e.g., Polysorbate 20, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), .
preservatives (e.g., Thimerosol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); see, e.g.,
Remington's Pharmaceutical Sciences, 18th Ed. (1990,
15 Mack Publishing Co., Easton, PA 18042) pages 1435:1712 which are herein incorporated by reference. An effective amount of active ingredient is a
therapeutically, prophylactically, or diagnostically effective amount, which can be readily determined by a
20 person skilled in the art by taking into consideration
such factors as body weight, age, and therapeutic or prophylactic goal.
The PEG:NESP compositions of the present invention may also include a buffering agent to maintain
25 the pH of the solution within a desired range. Preferred agents include sodium acetate, sodium phosphate, and sodium citrate. Mixtures of these buffering agents may also be used. The amount of buffering agent useful in the composition depends
30 largely on the particular buffer used and the pH of the solution. For example, acetate is a more efficient buffer at pH 5 than pH 6 so less acetate may be used in a solution at pH 5 than at pH 6. The preferred pH range
for the compositions of the present invention is pH 3.0 - 7.5.
The compositions of the present invention may further include an isotonicity adjusting agent to render
5 the solution isotonic and more compatible for injection. The moat preferred agent is sodium chloride within a concentration range of 0 - 150m06.
As used herein, and when contemplating
PEG:NESP conjugates, the term '"therapeutically effective
10 amount" refers to an amount which gives an increase in
hematocrit that provides benefit to a patient. The amount will vary from one individual to another and will depend upon a number of factors, including the overall physical condition of the patient and the underlying
15 cause of anemia. For example, a therapeutically effective amount of rHuEPO for a patient suffering from chronic renal failure is 50 to 150 units/kg three times per week. The amount of rHuEPO used for therapy gives an acceptable rate of hematocrit increase and maintains
20 the hematocrit at a beneficial level (usually at least about 30% and typically in a range of 30% to 36%). A therapeutically effective amount of the present compositions may be readily ascertained by one skilled in the art using publicly available materials and
The invention provides for administering
PEG:NESP conjugates less frequently than NESP and/or EPO. The dosing frequency will vary depending upon the condition being treated, but in general will be about
30 one time per 4-6 weeks. It is understood that the dosing frequencies actually used may vary somewhat from the frequencies disclosed herein due to variations in responses by different individuals to the PEG:NESP
conjugates; the term "about° is intended to reflect such variations.
The present invention may thus be used to stimulate red blood cell production and correct
5 depressed red cell levels. Most commonly, red cell levels are decreased due to anemia. Among the conditions treatable by the present invention include anemia associated with a decline or loss of kidney function (chronic renal failure), anemia associated with
10 myelosuppressive thereby, such as chemotherapeutic or anti-viral drugs (such as AZT), anemia associated with the progression of non-myeloid cancers, and anemia associated with viral infections (such as HIV). Also treatable are conditions which may lead to anemia in an
15 otherwise healthy individual, such as an anticipated loss of blood during surgery. In general, any condition treatable with rHuEPO and/or NESP may also be treated with the PEG.NESP conjugates of the invention.
The invention also provides for administration
20 of a therapeutically effective amount of iron in order
to maintain increased erythropoiesis during therapy. The amount to be given may be readily determined by one skilled in the art based upon therapy with rHuEPO.
PEG:NESP conjugates prepared in accordance
25 with the present invention is preferably administered by injection intraperitoneally, subcutaneously, or
intramuscularly. However, it would be clear to one skilled in the art that other routes of delivery could also be effectively utilized using the compositions of
30 the present invention.
The following examples are offered to more fully illustrate the invention, but are not to be
construed as limiting the scope thereof. Example 1
describes the preparation and testing of PEG:NESP conjugates prepared by coupling either 5kD or 20kD methoxy-PEG-hydrazides to NESP through aldehydes generated in the NESS carbohydrate chains by sodium
5 periodate oxidation. Example 2 describes the preparation and testing of PEG:NESP conjugates prepared utilizing 20kD PEG polymers as NHS-PEG esters and PEG-aldehydes to produce PEG-NESP conjugates by acylation and reductive alkylation respectively. Example 3
10 demonstrates the effects on activity of the degree of
substitution and variations of the polymer size and conformation for various PEG:NESP conjugates. Example 4
describes the efficacy of three PEG:NESP conjugates: 20kD mono-PEG:NESP; the 5kD poly-PEG:NESP mixture; and
15 30kD mono-PEG:NESP, as examined at three different doses
relative to a NESP control, in an anemic mouse model.
In Example 5,. three different PEG-NESP conjugates were
evaluated in a normal mouse bioassay to compare and contrast their erythropoietic potential and duration.
PEG:NESP conjugates were produced by coupling either 5kD or 20kD methoxy-PEG-hydrazides to NESS
25 through aldehydes generated in the NESP carbohydrate chains by sodium periodate oxidation. The degree of modification was controlled by varying the sodium periodate concentration during oxidation.
The conjugates were prepared by first
30 oxidizing NESS (2-4 mg/ml in 50th sodium acetate) with
either ltM or 10MM sodium meta-periodate (Sigma) for thirty minutes at room temperature in 100MM sodium acetate, pH 5.6. The periodate is then removed by buffer exchange into 100mM sodium acetate, pH 5.4.
Methoxy-PEG-hydrazide (Shearwater Polymers) is then added at 5-100 fold molar excess polymer:protein, with 100-fold excess preferred. The intermediate hydrazone linkage was further reduced by addition of 15mM sodium
5 cyanoborohydride (Sigma) and allowed to react overnight at 4°C. The resultant conjugates were then fractionated by size exclusion FPLC using a Superdex 75, 26 ram x 60 cm column (Pharmacia) eluted with 20mM sodium phosphate, 150mM NaC1, pH 7.2. The resultant preparations ranged
10 in size from -40kO to -200kD, as estimated by SOS-PAGE.
Samples of PEG:NESP were tested for receptor binding in an in vitro EIA format. The in vitro assay is
a displacement assay wherein the PEG:NESP conjugates compete for binding of the EPO receptor with an EPO-HRP
15 conjugate used as a reporter. The in vitro assay results suggest that the PEG:NESP conjugates had a lower apparent affinity for the NESP receptor.
Bioactivity of various PEG:NESE, conjugates was
then evaluated in vivo by monitoring iron uptake in
20 rodents after a single subcutaneous dose of conjugate. In the assay, mice are preconditioned in a hyperbaric chamber to suppress expression of endogenous erythropoietin, then dosed with a single, subcutaneous bolus injection of NES? or a PEG:NESP conjugate. After
25 five days, the mice receive an intravenous injection of Fe" isotope as a tracer to monitor iron uptake in the red blood cells. Two days after the administration of Fe", the animals are sacrificed and analyzed for iron uptake as a function of dose.
30 Initially, several pools of 5kO poly-PEG:NESP
conjugates with varying degrees of PEGylation were tested for iron uptake as a function of conjugate dose. The in vivo assay results are depicted in Figure 3, and demonstrated that the PEG:NESP conjugates prepared by
coupling PEG-hydrazide to oxidized NEST perform comparably to NESP alone in the iron uptake bioassay.
This example describes the preparation and
testing of PEG:NESP conjugates prepared utilizing NHS-PEG esters and PEG-aldehydes produced from 20kD PEG polymers. Reaction stoichiometries and buffer
10 conditions were optimized for each chemistry to produce
20kD mono-PEG:NESP conjugates in good yield. A 20kD mono-PEG:NESP derived by acylation of NESP with the 2010 methoxy-PEG-NHS ester was prepared, as well as a mixture (-80%/20%) of 20kD mono/di-PEG:NESP derived by reductive
15 alkylation of NESP with 20kD methoxy-PEG-aldehyde.
The reaction with methoxy-PEG-aldehyde
(Shearwater Polymers) can be carried out from pH 4-6 with the optimum being at pH 5.2. The concentration of NESP in the reaction mixture was 4 mg/ma in 50mM sodium
20 acetate. The molar excess of PEG aldehyde used was 5-20 fold, and sodium cyanoborohydride was added to a final. 15mM concentration. The reaction was stirred for 1 hour
at ambient temperature and then for 18 hours at 5°C.
Upon completion of the reaction, the mixture.was diluted
25 to a conductivity of less than 5 mS/cm, the pH raised to 7.0, and the mixture loaded onto a Q Sepharose HP column (Pharmacia). The products were elute& from the column utilizing a linear gradient from 50mM NaCl to 200mM NaCl buffered in 10mM sis-Tris-Propane, pH 7.0. This
30 purification allows for separation of species based on the number of PEG molecules attached to NESP.
The reaction with PEG activated NHS ester, methoxy-SPA-PEG (Shearwater Polymers), was carried out at pH 8.0 at a NESP concentration from 2-4 mg/ml in 50mM
Bicine buffer. A buffered solution of NESP was added to 10-20 molar equivalents of PEG. The reaction was stirred for 1 hour at ambient temperature. Upon completion of the reaction, the mixture was diluted to a
5 conductivity of less than 5 mS/cm, the pH raised to 7.0,
and the sample loaded onto a QHP column (Pharmacia).
The products were eluted with a linear gradient from 50MM NaCl to 200mM Had buffered in 10mM Bis-Tris¬Propane, pH 7.0.
10 The two isolated PEG:NESP conjugates, a 20kD
mono-PEG:NESP (NHS) and a mixture (-80%/20%) of 20kD mono/di-PEG:NESP (aldehyde) were then tested in a murine in vivo bioassay. The murine bioassay measures reticulocytes, a red blood cell precursor, and
15 hemoglobin as monitors of erythropoiesis in response to a single dose of NESP or PEG:NESP in normal mice. Specifically, the bioassay measures the intensity and duration of an increased hemoglobin and reticulocyte response resulting from subcutaneous bolus injections of
20 100 gg/kg in female BDF 1 mice. The assay results are depicted in Figure 4, and the results of the study indicated a significant increase and prolongation of the hemoglobin response from the PEG:NESP conjugates relative to an equivalent dose of NESP alone.
This example demonstrates the effects on activity of the degree of substitution and variations of
30 the polymer size and conformation for PEG:NESP conjugates.
Using both methoxy-PEG-aldehyde and methoxy¬PEG-NHS based chemistries, a variety of PEG:NESP
conjugates were synthesized from 5kD, 20kD and 30kD
linear polymers as well as 10kD, 20kD and 40kD branched polymers. From these reactions, preparations of mono-substituted and di-substituted PEG.NESP were isolated chromatographically and tested for prolonged
5 erythropoiesis in the mouse bioassay.
The reaction with methoxy-PEG-aldehyde (Shearwater Polymers) was run with a NESP concentration of 4 mg/ml and a 25-fold molar excess of PEG in 20mM NaOAc, pH 5.0, with sodium cyanoborohydride added to a
10 final concentration of 20mM. The reaction was stirred overnight at 4°C, diluted 4-fold with 20mM Tris, pH 7.2, and the pH adjusted to pH 7.4 with NaOH. The diluted reaction mixture was then loaded onto a 5 ml HiTrap Q Sepharose HP column (Pharmacia). The PEGylated NESP
15 isoforms were resolved by elution with a 0-150mM Had gradient in 20mM Tris, pH 7.2.
The reaction with methoxy-PEG-NHS ester (Shearwater Polymers) was run with a NESP concentration of 4 mg/ma and a 5-7 fold molar excess of PEG in 50mM
20 Bicine buffer, pH 8. The reaction was stirred overnight
at 4°C, then diluted 4-fold with 20mM Tris, pH 7.2 and
the pH adjusted to pH 7.4 with NaOH. The diluted
reaction mixture was then loaded onto a 5 ml HiTrap Q
Sepharose HP column (Pharmacia). The PHGylated NESP
25 isoforme were resolved by elution with a 0-150mM NaC1
gradient in 20mM Tris, pH 7.2 (see Figures 5-7).
These process schemes were employed for each of the 5kD, 20kD and 30kD linear polymers as well as the 10kD, 20kD and 40kD branched PEG-NHS esters. The
30 various conjugates are listed in Table 1 below:
Chemistry Degree of
5kD linear 20kD linear 20kD linear 30kD linear 30kD linear mPEG-NHS ester mPEG-NHS ester mPEG-NHS ester mPEG-NHS ester mPEG-NHS ester mono/di-PEG mono-PEG di-PEG mono-PEG di-PEG
5kD linear 5kD linear 20kD linear 30kD linear 30kD linear mPEG-aldehyde mPEG-aldehyde mPEG-aldehyde mPEG-aldehyde mPEG-aldehyde mono-PEG di-PEG mono-PEG mono-PEG di-PEG
40kD branched branched mPEG-NHS
40kD branched hmnamduTECi-aldehyde
5kD linear mPEG-hydrazide
mPEG-hydrazide high (>7 PEGs)
low (1-5 PEGs)
20kD linear mPEG-hydrazide
mPEG-hydrazide high (>7 PEGs)
medium (-4-7 PEGs)
low (1-5 PEGs)
Each purified isoform was then tested in a
5 murine in vivo bioassay for prolonged erythropoietic
activity as measured by changes in reticulocyte and
hemoglobin determinations after single, subcutaneous
bolus injections of 100 Rg/kg in normal, female EDF 1 mice. Each mono-substituted PEG:NESP conjugate from the
linear and branched polymer series showed significant and comparable prolongation of the erythropoietic effect
5 (see Figures 8 and 9). The di-substituted PEG:NESP
conjugates from the 20kD and 30kD PEG polymers were considerably less active, but unexpectedly, the 5kD di-substituted PEG:NESP conjugate demonstrated an
equivalent activity to the mono-substituted counterpart.
10 All.of the mono-substituted, branched PEG:NESP conjugates demonstrated prolonged activity comparable to the analogous mono-substituted linear PEG:NESP
These examples thus demonstrate the enhanced
15 duration of erythropoietic stimulation by a variety of PEG:NESP conjugates using single-dose, bolus injections in normal mouse models.
This example describes the efficacy of three'
PEG:NESP conjugates: 20kD mono-PEG:NESP; the 5kD poly¬PEG:NESP mixture; 'and 30kD mono-PEG:NESP, as examined at three different doses relative to a NESP control, in an
25 anemic mouse model.
To induce an anemic condition, mice were pretreated with cis-platinin at 1 mg/kg/day for 3 days, followed by a 7 day rest period. After 3 ten day cycles, the mice were dosed with single, bolus
30 injections of 30 Rg/kg, 10 µg/kg or 3 µg/kg of the 20kD mono-PEG:NESP, 30kD mono-PEG:NESP or the 5kD poly-PEG:NESP conjugates and compared to a NESP alone control
at 30 µg/kg. Reticulocyte and hemoglobin levels were
monitored as a function of time and in response to the single dose of each drug (see Figures 10-15).
These data demonstrate the unexpected
advantages of an -3 fold dose reduction and significant
5 increases in erythropoietic half-life for the PEG:NESP conjugates relative to NESP alone, in that the results demonstrate a clear dose dependence for both the
magnitude and duration of either the reticulocyte or hemoglobin response to the PEG:NESP conjugates. In some
10 cases the 30kD mono-PEG:NESP conjugate appears to modestly outperform the 5kD poly-PEG:NESP conjugate, which modestly outperforms the 20kD mono-PEG:NESP conjugate, suggesting that the 30kD mono-PEG:NESP conjugate might be a preferred configuration.
In this example, three different PEG-NESP conjugates were evaluated in a normal mouse bioassay to
20 compare and contrast their erythropoietic potential and duration. The three compounds tested were: 30kD mono-
PEG:NESP derived by acylation with the 30kD PEG-NHS ester, the 20kD mono-PEG:NESP derived, by reductive alkylation with the 20kD PEG-aldehyde and the 5kD poly-
25 PEG:NESP mixture derived by reductive alkylation with the 5kD PEG-aldehyde. Each PEG:NESP conjugate was tested as a single bolus, subcutaneous dose at 30 pg/kg,
10 pg/kg or 3 µg/kg. Unmodified NESP was used as a control at 30 gg/kg in a single, bolus injection. The
30 erythropoietic response and duration were monitored as a function of reticulocyte counts or hemoglobin concentration (see Figures 16-21) as a function of time. These data show that all three PEG:NESP forms are
capable of inducing a strong erythropoietic response
with significant dose reduction. Moreover, these
PEG:NESP conjugates demonstrate a prolonged efficacy relative to the unmodified NESP.
5 Materials and Methods
The present NESP may be prepared according to the above incorporated-by-reference PCT Application No. US94/02957.
10 The conjugates prepared herein were also
characterized using size exclusion chromatography (SEC)
as an analytical tool. The SEC column was a Tosohaas TSK 3000 SWx1 (5 micron - 7.8 mm X 30 cm) which utilized 100mM NaHPO, 10% ethanol, 150mM NaC1, pH 6.9, to elute
15 the products. A representative chromatograph is depicted in Figure 22.
While the present invention has been described in terms of certain preferred embodiments, it is
20 understood that, variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations which .come within the scope of the invention as claimed.
1. A substantially homogenous preparation of chemically modified NESS, optionally in a
5 pharmaceutically acceptable diluent, carrier or adjuvant.
2. A preparation of claim 1 where said NESP is chemically modified with a chemical selected from the
10 group consisting of dextran, poly(n-vinyl pyurrolidone), polyethylene glycols, propropylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols and polyvinyl alcohols.
15 3. A preparation of claim 2 where said NESP
or analog thereof is chemically modified with polyethylene glycol.
4. A preparation of claim 3 said polyethylene 20 glycol has a molecular weight of between about 2kD and 100kD.
5. A preparation of claim 4 wherein said polyethylene glycol has a molecular weight of between 25 about 5kD and 30kD.
6. A preparation of claim 1 wherein said preparation is comprised of a mixed population of mono-PEGylated NESP and poly-PEGylated NESP.
7. A preparation of claim 1 wherein said preparation is comprised of at least 95% N-terminally mono-PEGylated NESS and at most 5% unPEGylated NESS.
8. A preparation of claim 1 wherein said NESP has the sequence identified in SEQ. ID No. 1.
9. A pharmaceutical composition comprising:
5 (a) a substantially homogenous preparation of
mono-PEGylated NESP, said mono-PEGylated NESP consisting
of a polyethylene glycol moiety connected to a NESP moiety solely at the N-terminus thereof;
(b) fewer than 5% non-pegylated NESP
10 molecules; and
(c) a pharmaceutically acceptable diluent, adjuvant or carrier.
10. A pharmaceutical composition comprising:
15 (a) a substantially homogenous preparation of
mono-PEGylated NESP, said mono-PEGylated NESP consisting
of a polyethylene glycol moiety connected to a NESP
moiety through aldehydes generated in said NESP carbohydrate chains;
20 (b) fewer than 5% non-pegylated NESP
(c) a pharmaceutically acceptable diluent, adjuvant or carrier.
25 11. A pharmaceutical composition comprising:
(a) a substantially homogenous preparation of mono-PEGylated NESS, said mono-PEGylated NESP consisting
of a polyethylene glycol moiety connected to a NESP moiety using methoxy-PEG-NHS chemistry;
30 (b) fewer than 5% non-pegylated NESP
(c) a pharmaceutically acceptable diluent, adjuvant or carrier.
12. A pharmaceutical composition comprising:
(a) a substantially homogenous preparation of PEGylated NESP, said PEGylated NESP comprising a mixed population of mono-PEGylated NESP and poly-PEGylated
(b) fewer than 5% non-pegylated NESP molecules; and
(c) a pharmaceutically acceptable diluent, adjuvant or carrier.
13. A method of treating a hematopoietic disorder comprising administering a therapeutically effective dose of a preparation of Claim 1.
A: PEG Size
--,vWwvv,WrVvV NEAP NESP
5kD PEG . 20kD PEG
B: PEG Conformation
10kD, 20k0 and 40kD PEG
C: Degree of Substitution
rnono-PEG di-PEG tri-PEG
14 100 1000
calm! Sample hinted
12 r U11111111 12
0 2 4 6 8 10 12 14 18 18 20 22 24 26 28 30 32
Days after NESP treatment
1000 5kD polyP EG-N ESP _ 120
= 100 = 80 = 60 = 40
Absorbance (280 nm)
400 200 L
-200 0 _ r I I I I I
50 100 150
Elution Volume (ml)
2500 2000 1500 1000 500
-5 0 5 10 15 20 25 30
-10 10 20 30 40 50 60
20 18 16 14 12 .10
-10 0 10 20 30 40 50 60
,...: 1500 1
k 1000 -
o 5 10 15 20 25
20 - 19 - 18 - 17 : 16 -15 -
13 1 I i 1 L I 1 1 1 I 1 o
4 6 8 10 12 14 16 18
<110> AmGEN WT.
< 120> CHEMICALLY MODIFIED NOVEL ERYTHAOPOIETIN STIMULATING PROTEIN COMPOSITIONS AND METHODS
< 140> [Not Yet Assigned]
<170> PatentIn Ver. 2.1
<213> Homo sapiens
Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu
1 5 10 15
Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Asn Glu Thr
20 25 30
Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Vol Asn Phe
35 40 45
Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp
50 55 60
Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu
65 70 75
Leu Val Asn Ser Ser Gln Val Asn Glu Thr Leu Gln Leu His Val Asp
85 50 95
Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leo Leu Arg Ala Leu
100 105 110
Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Se> Ala Ala
115 120 125
Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Lou Phe Arg Val
130 135 140
Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly Asp