111111
`
`1111111111111111111111111111111111111111111111111111111111111111111111111111
`US 20030104996Al
`
`(19) United States
`(12) Patent Application Publication
`Li et al.
`
`(10) Pub. No.: US 2003/0104996 Al
`Jun. 5, 2003
`( 43) Pub. Date:
`
`(54) L-METHIONINE AS A STABILIZER FOR
`NESP/EPO IN HSA-FREE FORMULATIONS
`
`(22) Filed:
`
`Aug. 30, 2001
`
`(76)
`
`Inventors: Tiansheng Li, Thousand Oaks, CA
`(US); Byeong Chang, Thousand Oaks,
`CA (US); Christopher Sloey, Sherman
`Oaks, CA (US)
`
`Correspondence Address:
`U.S. Patent Operations/ CAC
`Dept. 4300, M/S 27-4-A
`AMGEN INC.
`One Amgen Center Drive
`Thousand Oaks, CA 91320-1799 (US)
`
`(21)
`
`Appl. No.:
`
`09/945,517
`
`Publication Classification
`
`(51)
`Int. CI? ..................................................... A61K 38/22
`(52) U.S. Cl. ................................................................ 514/12
`
`(57)
`
`ABSTRACT
`
`The present invention relates to single use and multi-dose
`pharmaceutical formulations comprising a biologically
`active agent and methionine, wherein said formulations
`demonstrate improved stability, and wherein said formula(cid:173)
`tions do not contain human serum albumin.
`
`
`
`
`MAIA Exhibit 1018
`MAIA V. BRACCO
`IPR PETITION
`
`

`

`Patent Application Publication
`
`Jun. 5, 2003 Sheet 1 of 8
`
`US 2003/0104996 A1
`
`Figure 1
`
`r::
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`
`................................... ·······
`+ 1 0 mM methionine
`
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`No methionine
`
`.... ··········
`
`30
`
`31
`
`32
`
`33
`
`34
`
`35
`
`Retention Time (minutes)
`
`
`
`
`

`

`Patent Application Publication
`
`Jun. 5, 2003 Sheet 2 of 8
`
`US 2003/0104996 Al
`
`Figure 2
`
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`
`20
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`10
`15
`0
`Methionine Concentration (mM)
`
`25
`
`
`
`
`

`

`Patent Application Publication
`
`Jun. 5, 2003 Sheet 3 of 8
`
`US 2003/0104996 Al
`
`Figure 3
`
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`

`

`Patent Application Publication
`
`Jun. 5, 2003 Sheet 4 of 8
`
`US 2003/0104996 Al
`
`Figure 4
`
`- • - 1 00 JlQ/m L
`-o- 500 JlQ/m L
`
`0.2
`
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`
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`G)
`
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`
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`20
`0
`10
`5
`15
`Methionine Concentration (mM)
`
`
`
`
`

`

`Patent Application Publication
`
`Jun. 5, 2003 Sheet 5 of 8
`
`US 2003/0104996 Al
`
`Figure 5
`
`0.6----------------------------------------~
`
`•
`
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`-o- 500 J..LQ/m L
`
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`
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`
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`
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`
`0
`
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`
`5
`10
`15
`20
`Methionine Concentration (mM}
`
`
`
`
`

`

`Patent Application Publication
`
`Jun. 5, 2003 Sheet 6 of 8
`
`US 2003/0104996 Al
`
`Figure 6
`
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`
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`I I
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`\ ________ _
`\. _____ )
`
`EPO control
`
`93
`
`94
`
`96
`95
`Retention Time (minutes)
`
`97
`
`98
`
`
`
`
`

`

`Patcllt J\.pplicatioll Publicatioll
`
`.Tun. S, 2003 Sheet 7 of 8
`
`Figure 7
`
`Vs 2003!0104996 A1
`
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`
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`
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`•
`
`
`
`
`

`

`Patent Application Publication
`
`Jun. 5, 2003 Sheet 8 of 8
`
`US 2003/0104996 Al
`
`Figure 8
`
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`
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`
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`
`OxidizedTryptophan 51 ___ _____Jt
`
`Reduced peak area due
`to oxidized histidine
`
`Retention Time
`
`
`
`
`

`

`US 2003/0104996 Al
`
`Jun.5,2003
`
`1
`
`L-METHIONINE AS A STABILIZER FOR
`NESP/EPO IN HSA-FREE FORMULATIONS
`
`BACKGROUND OF THE INVENTION
`
`[0001] Due to recent advances in genetic and cell engi(cid:173)
`neering technologies, proteins known to exhibit various
`pharmacological actions in vivo are capable of being pro(cid:173)
`duced in large amounts for pharmaceutical applications.
`Such proteins include erythropoietin (EPO), granulocyte
`colony-stimulating factor (G-CSF), interferons (alpha, beta,
`gamma, consensus), tumor necrosis factor binding proteins
`(TNFbp ), interleukin-1 receptor antagonist (IL-1ra), brain(cid:173)
`derived neurotrophic factor (BDNF), keratinocyte growth
`factor (KGF), stem cell factor (SCF), megakaryocyte growth
`differentiation factor (MGDF), osteoprotegerin (OPG), glial
`cell line derived neurotrophic factor (GDNF), obesity pro(cid:173)
`tein (OB protein), and novel erythropoiesis stimulating
`protein (NESP).
`
`[0002] EPO is a glycoprotein hormone necessary for the
`maturation of erythroid progenitor cells into erythrocytes. It
`is produced in the kidney and is essential in regulating levels
`of red blood cells in the circulation. Conditions marked by
`low levels of tissue oxygen signal increased production of
`EPO, which in turn stimulates erythropoiesis. A loss of
`kidney function as is seen in chronic renal failure (CRF), for
`example, typically results in decreased production of EPO
`and a concomitant reduction in red blood cells. Human
`urinary EPO was purified by Miyake et al., J. Bioi. Chern.,
`252:5558 (1977) from patients with aplastic anemia. How(cid:173)
`ever, the amount of purified EPO protein obtained from this
`source was insufficient for therapeutic applications. The
`identification and cloning of the gene encoding human EPO
`and expression of recombinant protein was disclosed in U.S.
`Pat. No. 4,703,008 to Lin, the disclosure of which is
`incorporated herein by reference. A method for purification
`of recombinant human erythropoietin from cell medium is
`disclosed in U.S. Pat. No. 4,667,016 to Lai et. al., which is
`incorporated herein by reference. The production of biologi(cid:173)
`cally active EPO from mammalian host cells has made
`available, for the first time, quantities of EPO suitable for
`therapeutic applications. In addition, knowledge of the gene
`sequence and the increased availability of purified protein
`has led to a better understanding of the mode of action of this
`protein.
`
`[0003] Both human urinary derived EPO (Miyake et al.
`supra) and recombinant human EPO expressed in mamma(cid:173)
`lian cells contain three N -linked and one 0-linked oligosac(cid:173)
`charide chains which together comprise about 40% of the
`total molecular weight of the glycoprotein. N-linked glyco(cid:173)
`sylation occurs at asparagine residues located at positions
`24, 38 and 83 while 0-linked glycosylation occurs at a
`serine residue located at position 126 (see Lai et al., J. Bioi.
`Chern., 261:3116 (1986); Broudy et al., Arch. Biochem.
`Biophys, 265:329 (1988)). The oligosaccharide chains have
`been shown to be modified with terminal sialic acid residues
`with N-linked chains typically having up to four sialic acids
`per chain and 0-linked chains having up to two sialic acids.
`An EPO polypeptide may therefore accommodate up to a
`total of 14 sialic acids.
`
`[0004] Various studies have shown that alterations of EPO
`carbohydrate chains can affect biological activity. In one
`study, however, the removal of N-linked or 0-linked oli-
`
`gosaccharide chains singly or together by mutagenesis of
`asparagine or serine residues that are glycosylation sites
`sharply reduces in vitro activity of the altered EPO that is
`produced in mammalian cells; Dube et. al., J. Bioi. Chern.,
`263:17516 (1988). However, DeLorme et al., Biochemistry,
`31:9871-9876 (1992) reported that removal of N-linked
`glycosylation sites in EPO reduced in vivo but not in vitro
`biological activity.
`[0005] The relationship between the sialic acid content of
`EPO and in vivo biological activity was disclosed by deter(cid:173)
`mining the in vivo activity of isolated EPO isoforms. It was
`found that a stepwise increase in sialic acid content per EPO
`molecule gave a corresponding stepwise increase in in vivo
`biological activity as measured by the ability of equimolar
`concentrations of isolated EPO isoforms to raise the hema(cid:173)
`tocrit of normal mice; Egrie et al., Glycoconjugate J., 10:263
`(1993). Those EPO isoforms having higher sialic acid con(cid:173)
`tent also exhibited a longer serum half-life but decreased
`affinity for the EPO receptor, suggesting that serum half-life
`is an important determinant of in vivo biological activity.
`
`[0006]
`In the U.S., EPO has been used in the treatment of
`chronic renal failure maintained on dialysis as well as
`pre-dialysis, and in the treatment anemia secondary to
`chemotherapy treatment in cancer and in anemia associated
`with zidovudine treatment of HIV infection. Worldwide,
`EPO has been used to treat anemia associated with prema(cid:173)
`turity, sickle cell anemia, rheumatoid arthritis, and bone
`marrow transplantation; Markham et al., Drugs, 49:232-254
`(1995).
`
`[0007] NESP is a hyperglycosylated erythropoietin analog
`having five changes in the amino acid sequence of rHuEPO
`which provide for two additional carbohydrate chains. More
`specifically, NESP contains two additional N-linked carbo(cid:173)
`hydrate 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 refer(cid:173)
`ence in its entirety). NESP is biochemically distinct from
`EPO, having a longer serum half-life and higher in vivo
`biological activity; Egrie et al., ASH 97, Blood, 90:56a
`(1997). NESP has been shown to have -3 fold increase in
`serum half-life in mice, rats, dogs and man; Id. In mice, the
`longer serum 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.
`
`[0008] A pharmacokinetic study demonstrated that, con(cid:173)
`sistent with the animal studies, NESP 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 Nephrology, 8:268A (1997). A less frequent
`dosing schedule would be more convenient to both physi(cid:173)
`cians 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.
`
`[0009] Although commercially available EPO and NESP
`formulations are generally well tolerated and stable, con(cid:173)
`sideration should be given to the fact that, under extreme
`conditions, such proteins may be unstable and undergo
`
`
`
`
`

`

`US 2003/0104996 Al
`
`Jun.5,2003
`
`2
`
`various undesirable physiochemical degradations during
`manufacturing, handling, and storage conditions. Such deg(cid:173)
`radations include aggregation, inactivation, and oxidation of
`methionine residues, and such degradations may be accel(cid:173)
`erated by external factors such as heat and light, or in
`formulations that are free of human blood products such as
`albumin, or in multi-dose formulations which contain pre(cid:173)
`servatives such as benzyl alcohol.
`
`[0010] Methods of inhibiting oxidation in methionine(cid:173)
`containing polypeptides have been described; Takruri et al.,
`U.S. Pat. No. 5,272,135 (Dec. 21, 1993). Specifically, Tak(cid:173)
`ruri describes methods of inhibiting the oxidation of
`methionine residue(s) in liquid or semi-liquid preparations,
`said preparations comprising polypeptides having amino
`acid sequences comprising at least one methionine residue.
`The prevention of methionine oxidation is said to be accom(cid:173)
`plished by the addition of free L-methionine to the prepa(cid:173)
`rations in an amount sufficient to inhibit oxidation of the
`methionine residue(s) in the polypeptide. The oxidation of
`the methionine residues is said to be associated with the
`plastic containers, e.g., polypropylene or low density poly(cid:173)
`ethylene (LDPE), which are readily permeable to oxygen,
`and within which the preparations are stored. The polypep(cid:173)
`tides contemplated for use in Takruri are growth factors, and
`the preparations tested are ophthalmic aqueous-based prepa(cid:173)
`rations of epidermal growth factor (EGF). Preparations
`containing EPO or NESP, or any other glycosylated protein
`are not discussed, nor are formulations which are HSA-free,
`multi-dose, or HSA-free multi-dose discussed.
`
`SUMMARY OF THE INVENTION
`
`[0011] The present invention provides pharmaceutical for(cid:173)
`mulations of EPO and/or NESP wherein the incorporation of
`methionine and other stabilizing agents into said formula(cid:173)
`tions provide for a more stable formulation, even in extreme
`conditions wherein critical degradations induced by light,
`heat, impurities in additives, leacheates in the prefilled
`syringes, the manufacturing process, storage, transportation,
`and handling may otherwise occur.
`
`[0012]
`Importantly, the formulations also demonstrate
`improved stability in HSA-free formulations and HSA-free
`multi-dose formulations containing preservatives, wherein
`the critical degradations may be more pronounced.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0013] FIG. 1 is a graph depicting the effect of free
`methionine on the aggregation of NESP during exposure to
`light. NESP in phosphate buffered saline was exposed to
`ultraviolet light for 4 hours at room temperature.
`
`[0014] FIG. 2 is a graph depicting the effect of free
`methionine on the aggregation of NESP in the presence of
`1% benzyl alcohol during storage at 2-8° C. Samples con(cid:173)
`taining 500 ,ug/mL of NESP were stored for 13 months.
`
`[0015] FIG. 3 is a graph depicting the effect of various
`additives and treatment on the oxidation of methionine 54
`residue in NESP during incubation for 90 days at 37° C. %
`oxidation was determined by tryptic mapping followed by
`Reversed-phase HPLC and mass spectrometry.
`
`[0016] FIG. 4 is a graph depicting the effect of free
`methionine on the oxidation of NESP in a preserved formu-
`
`lation containing 1% benzyl alcohol. 0-20 mM free methion(cid:173)
`ine was tested and samples were incubated at 4 o C. for 56
`days.
`[0017] FIG. 5 is a graph depicting the effect of free
`methionine on the oxidation of NESP in a preserved formu(cid:173)
`lation containing 1% benzyl alcohol. 0-20 mM free methion(cid:173)
`ine was tested and samples were incubated at 29° C. for 56
`days.
`
`[0018] FIG. 6 compares the tryptic maps of EPO in
`solutions at pH 7.0±benzyl alcohol and ±free L-methionine.
`
`[0019] FIG. 7 is a graph comparing NESP methionine
`oxidation rates with and without purging (10 minutes) with
`nitrogen. % methionine oxidation is plotted versus benzal(cid:173)
`dehyde concentration. 0.1 mg/ml NESP was tested.
`[0020] FIG. 8 compares the tryptic maps of over-oxidized
`NESP samples. Met-54 was fully oxidized for all samples
`shown in the figure. Numbers depicted on the figure repre(cid:173)
`sent the concentration of methionine added to the samples.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`[0021]
`"Excipient" is defined herein as a non-therapeutic
`agent added to a pharmaceutical composition to provide a
`desired effect, e.g. stabilization, isotonicity.
`
`[0022]
`"Polypeptide" is defined herein as natural, syn(cid:173)
`thetic, and recombinant proteins or peptides having more
`than about 10 amino acids, and having a desired biological
`activity.
`[0023] As used herein, biologically active agents refers to
`recombinant or naturally occurring polypeptides, whether
`human or animal, useful for prophylactic, therapeutic or
`diagnostic application. The biologically active agent can be
`natural, synthetic, semi-synthetic or derivatives thereof.
`Contemplated active agents include peptides, small mol(cid:173)
`ecules, carbohydrates, nucleic acids, lipids, proteins, and
`analogs thereof. One skilled in the art will readily be able to
`adapt a desired biologically active agent to the compositions
`of present invention.
`
`[0024] Proteins contemplated for use would include but
`are not limited to interferon consensus (see, U.S. Pat. Nos.
`5,372,808, 5,541,293 4,897,471, and 4,695,623 hereby
`incorporated by reference including drawings), granulocyte(cid:173)
`colony stimulating factors (see, U.S. Pat. Nos. 4,810,643,
`4,999,291, 5,581,476, 5,582,823, and PCT Publication No.
`94/17185, hereby incorporated by reference including draw(cid:173)
`ings), interleukins (see, U.S. Pat. No. 5,075,222, hereby
`incorporated by reference including drawings), erythropoi(cid:173)
`etins (see, U.S. Pat. Nos. 4,703,008, 5,441,868, 5,618,698
`5,547,933, and 5,621,080 hereby incorporated by reference
`including drawings), stem cell factor (PCT Publication Nos.
`91/05795, 92/17505 and 95/17206, hereby incorporated by
`reference including drawings), osteoprotegerin (PCT Publi(cid:173)
`cation No. 97/23614, hereby incorporated by reference
`including drawings), novel erythropoiesis stimulating pro(cid:173)
`tein (NESP) (PCT Publication No. 94/09257, hereby incor(cid:173)
`porated by reference including drawings), leptin (OB pro(cid:173)
`tein) (see PCT publication Nos. 96/40912, 96/05309,
`97/00128, 97/01010 and 97/06816 hereby incorporated by
`reference including figures), megakaryocyte growth differ(cid:173)
`entiation factor (see, PCT Publication No. 95/26746 hereby
`
`
`
`
`

`

`US 2003/0104996 Al
`
`Jun.5,2003
`
`3
`
`incorporated by reference including figures),tumor necrosis
`factor-binding protein (TNF-bp ), interleukin-1 receptor
`antagonist (IL-lra), brain derived neurotrophic factor
`(BDNF), glial derived neurotrophic factor (GDNF), kerati(cid:173)
`nocyte growth factor (KGF) and thrombopoietin. The term
`proteins, as used herein, includes peptides, polypeptides,
`consensus molecules, analogs, derivatives or combinations
`thereof.
`
`[0025]
`In general, EPO useful in the present invention has
`the sequence of human erythropoietin, or closely related
`analogues thereof. The EPO may be produced by mamma(cid:173)
`lian cells outside the body, or it may be isolated from natural
`sources. Preferably, the EPO is recombinant human EPO
`(rHuEPO) produced as described in U.S. Pat. No. 4,703,008
`to Lin, the disclosure of which is incorporated herein by
`reference. The amino acid sequence of EPO is that depicted
`herein in SEQ ID NO: 1. The preferred host cells are Chinese
`Hamster Ovary (CHO) cells as described in Example 10 of
`the Lin patent. Other host cells known in the art, e.g. baby
`hamster kidney cells, may also be used to produce EPO
`useful in the present invention. While the procedures of
`Example 10 in the Lin patent are the preferred method for
`producing rEPO, modifications and changes could be made
`to that process as known in the art.
`
`[0026] NESP of the present invention is a hyperglycosy(cid:173)
`lated 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 NESP are provided in co-owned PCT Appli(cid:173)
`cation 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 Ovary (CHO) host cells and the
`expressed polypeptide was analyzed for the presence of
`additional carbohydrate chains. In a preferred embodiment,
`NESP will have two additional N-linked carbohydrate
`chains at residues 30 and 88. The numbering of the amino
`acid sequence is that of human erythropoietin (EPO). The
`amino acid sequence of NESP is that depicted herein in SEQ
`ID N0:2. It is understood that NESP will have the normal
`complement of N-linked and 0-linked glycosylation sites in
`addition to the new sites.
`
`[0027] The EPO and NESP of the present invention may
`also include conservative amino acid changes at one or more
`residues in SEQ ID NOs: 1 and 2. These changes do not
`result in addition of a carbohydrate chain and will have little
`effect on the biological activity of the analog. These are set
`forth in Table 1, below. See generally, Creighton, Proteins,
`passim (W. H. Freeman and Company, N.Y., 1984); Ford et
`al., Protein Expression and Purification 2:95-107 (1991),
`which are herein incorporated by reference.
`
`TABLE 1
`
`Conservative Amino Acid Substitutions
`
`Basic:
`
`Acidic:
`
`arginine
`lysine
`histidine
`glutamic acid
`aspartic acid
`
`TABLE 1-continued
`
`Conservative Amino Acid Substitutions
`
`Polar:
`
`Hydrophobic:
`
`Aromatic:
`
`Small:
`
`glutamine
`asparagine
`leucine
`isoleucine
`valine
`phenylalanine
`tryptophan
`tyrosine
`glycine
`alanine
`serine
`threonine
`methionine
`
`[0028] Therapeutic uses of the compositions of the present
`invention depend on the biologically active agent used. One
`skilled in the art will readily be able to adapt a desired
`biologically active agent to the present invention for its
`intended therapeutic uses. Therapeutic uses for such agents
`are set forth in greater detail in the following publications
`hereby
`incorporated by reference
`including drawings.
`Therapeutic uses include but are not limited to uses for
`proteins like consensus interferon (see, U.S. Pat. Nos. 5,372,
`808, 5,541,293, hereby incorporated by reference including
`drawings), interleukins (see, U.S. Pat. No. 5,075,222, hereby
`incorporated by reference including drawings), erythropoi(cid:173)
`etins (see, U.S. Pat. Nos. 4,703,008, 5,441,868, 5,618,698
`5,547,933, 5,621,080, 5,756,349, and 5,955,422, hereby
`incorporated by reference including drawings), granulocyte(cid:173)
`colony stimulating factors (see, U.S. Pat. Nos. 4,999,291,
`5,581,476, 5,582,823, 4,810,643 and PCT Publication No.
`94/17185, hereby incorporated by reference including draw(cid:173)
`ings), megakaryocyte growth differentiation factor (see,
`PCT Publication No. 95/26746), stem cell factor (PCT
`Publication Nos. 91/05795, 92/17505 and 95/17206, hereby
`incorporated by reference including drawings), OB protein
`(see PCT publication Nos. 96/40912, 96/05309, 97/00128,
`97/01010 and 97/06816 hereby incorporated by reference
`including figures), and novel erythropoiesis stimulating pro(cid:173)
`tein (PCT Publication No. 94/09257, hereby incorporated by
`reference including drawings). In addition, the present com(cid:173)
`positions may also be used for manufacture of one or more
`medicaments for treatment or amelioration of the conditions
`the biologically active agent is intended to treat.
`
`[0029] As relates specifically to NESP, the present inven(cid:173)
`tion provides for a method of raising and maintaining
`hematocrit in a mammal comprising administering a thera(cid:173)
`peutically effective amount of NESP in a pharmaceutical
`composition of the present invention, wherein the NESP is
`administered less frequently than an equivalent molar
`amount of rHuEPO to obtain a comparable target hemat(cid:173)
`ocrit. The dosing frequency of the present invention in order
`to reach a patient's optimal hematocrit range is less than
`three times per week. Dosing frequencies may be two times
`per week, one time per week, or less than one time per week,
`such as one time every other week, once per month or once
`every two months. The dosing frequency required to main(cid:173)
`tain a patient's target hematocrit is less than three times per
`week. Dosing frequencies may be two times per week, one
`
`
`
`
`

`

`US 2003/0104996 Al
`
`Jun.5,2003
`
`4
`
`time per week, or less than one time per week, such as one
`time every two weeks, once per month or once every two
`months.
`
`[0030] The invention may be employed with any condi(cid:173)
`tion resulting in a decrease in red blood cell levels, such as
`anemia associated with a decline or loss of kidney function,
`(chronic renal failure) myelosuppressive therapy, cancer,
`viral infection, chronic disease and excessive loss of blood
`during surgical procedures.
`
`[0031]
`It is envisioned that the formulations of the present
`invention will additionally contain a buffering agent, e.g.,
`alkali salts (sodium or potassium phosphate or their hydro(cid:173)
`gen or dihydrogen salts), sodium citrate/citric acid, sodium
`acetate/acetic acid, and any other pharmaceutically accept(cid:173)
`able ph buffering agent known in the art, to maintain the pH
`of the solution within a desired range. Mixtures of these
`buffering agents may also be used. The amount of buffering
`agent useful in the composition depends 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 of the preferred formulations will be
`in the range of 5.0 to 7.0, and pH-adjusting agents such as
`hydrochloric acid, citric acid, sodium hydroxide, or a salt
`thereof, may also be included in order to obtain the desired
`pH.
`
`[0032] The formulations will also contain sorbitan mono-
`9-octadecenoate
`poly( oxy-1,2-ethanediyl)
`derivatives,
`including but not limited to, polysorbate 80 or polysorbate
`20. Other derivatives are well known in the art. The amount
`of polysorbate 20 or 80 to be used will be in the range of
`0.001% to 0.1% (w/v). The preferred amount is 0.005%
`(w/v) in the single use and multi-dose formulations.
`
`[0033]
`In order to provide EPO and/or NESP pharmaceu(cid:173)
`tical formulations having superior stability, free L-methion(cid:173)
`ine will be included in the formulations. The amount of free
`L-methionine included will be in the range of 0.05 mM to 50
`mM. In HSA-containing formulations, the preferred amount
`in the single use formulations is 0.05 mM to 5 mM, and the
`preferred amount in the multi-dose formulations is 1 mM to
`10 mM. In HSA-free formulations, the preferred amount in
`the single use formulations is 0.05 mM to 5 mM, and the
`preferred amount in the multi-dose formulations is 1 mM to
`10mM.
`
`[0034] Preservatives contemplated for use in the multi(cid:173)
`dose formulations of the present invention include benzyl
`alcohol, benzalkonium chloride, chlorobutanol, cresol, phe(cid:173)
`nol, and parabens. The amount of preservative included will
`be in the range of 0% to 2% (w/v) and the preferred amount
`in the formulations is 1% (w/v).
`
`[0035] The formulations of the present invention may
`further include an isotonicity adjusting agent to render the
`solution isotonic and more compatible for injection. Typical
`tonicity agents are well known in the art and include but are
`not limited to sodium chloride, mannitol, glycine, and sor(cid:173)
`bitol. The preferred agent is sodium chloride within a
`concentration range of 0 mM to 200 mM.
`
`[0036]
`It is also envisioned that other anti-oxidants may be
`included in the formulations of the present invention. Anti(cid:173)
`oxidants contemplated for use in the preparation of the
`formulations include amino acids such as glycine and lysine,
`
`chelating agents such as EDTA and DTPA, and free-radical
`scavengers such as sorbitol and mannitol.
`
`[0037] Preferred NESP formulations contemplated for use
`in the present invention will contain 10 mM to 30 mM
`phosphate buffer, 100 mM to 200 mM NaCl, 0.001% to
`0.1 %(w/v) polysorbate 80, and 0.5 mM to 50 mM L-me(cid:173)
`thionine, pH 5.0-7.0; and more preferably, 20 mm phosphate
`buffer, 140 mM NaCl, 0.005%(w/v) polysorbate 80, and 1
`mM L-methionine, pH 6.2.
`
`[0038] Preferred EPO formulations contemplated for use
`in the present invention will contain 0.01 mM to 5 mM
`phosphate buffer, 0.01 mM to 150 mM NaCl, 5 mM to 50
`mM L-arginine or L-histidine or salt thereof, 0.001% to
`0.1% (w/v) polysorbate 80, and 0.5 mM to 50 mM L-me(cid:173)
`thionine, pH 5.0-7.0; and more preferably, 2 mM phosphate
`buffer, 110 mM NaCl, 43.1 mM L-arginine HCl, 0.006%
`(w/v) polysorbate 80, and 0.5, 1, 2, 3 or 5 mM L-methionine,
`pH 6.0; or 2 mM phosphate buffer, 142 mM NaCl, 9.54 mM
`L-histidine HCl, 0.006% (w/v) polysorbate 80, and 0.5, 1, 2,
`3 or 5 mM L-methionine, pH 6.0.
`
`[0039] Also contemplated for use in inhibiting oxidation
`of methionine is nitrogen overlay. Nitrogen overlay can be
`introduced to the headspace of a vial or prefilled syringe by
`purging nitrogen during the filling process.
`
`[0040] The following examples are offered to more fully
`illustrate the invention, but are not to be construed as
`limiting the scope thereof.
`
`EXAMPLE 1
`
`[0041] This example describes the preparation ofEPO and
`NESP HSA containing and HSA-free single use and multi(cid:173)
`dose formulations. The EPO and NESP protein preparations
`were prepared as described in the Materials and Methods
`section below.
`
`[0042] NESP and/or EPO HSA-containing formulations
`were then prepared by adding 0.1-1% albumin, the appro(cid:173)
`priate buffering agents (e.g., sodium phosphate), and a
`tonicity modifier (e.g., sodium chloride) to the protein
`preparation to obtain formulations having the desired con(cid:173)
`centrations of protein and excipients. NESP and/or EPO
`HSA-free formulations were prepared by replacing the albu(cid:173)
`min with other recombinant proteins or pharmaceutically
`acceptable surfactants (e.g. polysorbate 20 or 80). Multi(cid:173)
`dose formulations were prepared by introducing preserva(cid:173)
`tive(s) (e.g. benzyl alcohol) to the HSA-containing or HSA(cid:173)
`free formulations.
`
`EXAMPLE 2
`[0043] This example describes experiments wherein the
`effect of free L-methionine on the aggregation (introduced
`by light) of NESP was evaluated. Although the underlying
`mechanism is not clear, under extreme conditions, light
`introduces significant aggregation to the NESP formula(cid:173)
`tions. NESP single use, HSA-free formulations prepared as
`described in Example 1 were used in the experiment.
`
`[0044] The glass vials containing the protein were placed
`into a UV light box and were incubated overnight (16 hours)
`with continuous UV light exposure. The samples were
`analyzed with SEC-HPLC. As depicted in FIG. 1, addition
`of 10 mM free methionine significantly decreased the rate of
`aggregation.
`
`
`
`
`

`

`US 2003/0104996 Al
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`Jun.5,2003
`
`5
`
`EXAMPLE 3
`
`[0045] This example describes experiments wherein the
`effect of free L-methionine on the aggregation of NESP in
`the presence of benzyl alcohol was evaluated. Although the
`underlying mechanism is not clear, benzyl alcohol intro(cid:173)
`duces very minor aggregation to the NESP formulations
`even during storage at 2-8° C. NESP multi-dose, HSA-free
`formulations prepared as described in Example 1 were used
`in the experiment.
`
`[0046] Multi-dose formulations containing 1% benzyl
`alcohol were incubated for 13 months at 2-8° C. and
`analyzed with SEC-HPLC method. As depicted in FIG. 2,
`addition of 1 mM-20 mM free methionine significantly
`decreased the rate of aggregation.
`
`EXAMPLE 4
`
`[0047] This example describes experiments wherein vari(cid:173)
`ous additives and treatments were tested for their ability to
`inhibit methionine oxidation in the NESP HSA-free single
`use formulations. NESP HSA-free single use formulations
`prepared as described in Example 1 were used in the
`experiments.
`
`[0048] First, the protective effect of various anti-oxidants
`on NESP was examined by hydrogen peroxide spiking
`experiment (described in the Materials and Methods section
`below). Free amino acids L-lysine, glycine and L-methion(cid:173)
`ine were tested and the % oxidation was determined by
`tryptic mapping as described in the Materials and Methods
`section below. It was demonstrated convincingly that free
`L-methionine prevents the oxidation of the Met-54 residue
`of NESP in the presence of excess hydrogen peroxide (see
`Table 1).
`
`TABLE 1
`
`Anti-Oxidant
`
`NESP Met-54 Oxidation (%)
`
`Glycine
`Lysine
`Methionine
`Glycine + Lysine
`Glycine + Methionine
`Lysine + Glycine + Methionine
`
`100
`100
`37.3
`100
`35.3
`32.9
`
`[0049] Next, the protective effect of various add