Chemically modified polypeptides

Details Chemically modified polypeptides Chemically modified polypeptides

1999-02-03

2003-06-24

Borin, Michael (Department: 1631)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C530S345000, C516S012000, C424S280100

Reexamination Certificate

active

06583267

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a chemically modified polypeptide in which at least one of the hydroxyl groups in the polypeptide molecule is modified with a polyalkylene glycol derivative; a method for producing the modified polypeptide; a method of treating a patient having reduced granulocytes or thrombocytes using the modified polypeptide; a composition for the treatment comprising the modified polypeptide; and use of the modified polypeptide.
BACKGROUND ART
A chemically modified polypeptide in which at least one of amino groups, carboxyl groups, mercapto groups or guanidino groups in the polypeptide molecule is modified with a polyalkylene glycol derivative (WO 95/023165) and modification of free thiol groups of cysteine residues in the polypeptide molecule are known (EP 0668353). However, when at least one of such amino groups, carboxyl groups, mercapto groups, guanidino groups or free thiol groups of cysteine residues in the polypeptide molecule is modified with a polyalkylene glycol derivative, the activity of the polypeptide may be markedly or completely lost.
For example, the activity of interleukin-15 completely disappears when its amino group(s) are modified with polyethylene glycol [
J. Biol. Chem.,
272:2312 (1997)].
Nothing is known about a chemically modified polypeptide in which at least one of the hydroxyl groups in the polypeptide molecule is modified with a polyalkylene glycol derivative.
Great attention has been directed toward the development of
(1) a method for the analysis of influences of hydroxyl groups upon the activity of a polypeptide, in a case where the hydroxyl group concerned is in the active site of the polypeptide,
(2) a novel chemical modification method which can avoid a probable case where the biological activity of a polypeptide is considerably spoiled when the polypeptide is treated by a conventional chemical modification method, and a chemically modified polypeptide obtained by the method, and
(3) a novel method which can improve resistance of polypeptide against protease, freezing-thawing and denaturing agents.
DISCLOSURE OF THE INVENTION
The present invention relates to a chemically modified polypeptide in which at least one of the hydroxyl groups in the polypeptide molecule is modified with a polyalkylene glycol derivative; a method for producing the modifying polypeptide; a method of treating a patient having reduced granulocytes or thrombocytes using the modifying polypeptide; use of the modifying polypeptide; a pharmaceutical preparation comprising the modifying polypeptide; and a composition for the treatment comprising the modifying polypeptide.
With regard to the polypeptide which can be used in the present invention, any polypeptide can be used so long as it contains a hydroxyl group and has a physiological activity or a pharmacological activity. Examples include those having an activity, such as asparaginase, glutaminase, uricase, superoxide dismutase, lactoferin, streptokinase, plasmin, adenosine deaminase, interleukin-1 to 13, interleukin-15, interferon-&agr;, interferon-&bgr;, interferon-&ggr;, human granulocyte colony-stimulating factor (hereinafter referred to as “hG-CSF”), and the like.
Examples of a polypeptide having an hG-CSF activity include a polypeptide comprising the amino acid sequence represented by SEQ ID NO:1, a polypeptide comprising a partial amino acid sequence of the sequence, a polypeptide comprising an amino acid sequence in which some parts of amino acids of the sequence are substituted by different amino acids [
Nature,
319:415 (1986), Japanese Published Unexamined Patent Application No. 267292/88, Japanese Published Unexamined Patent Application No. 299/88, WO 87/01132] and the like. Specific examples of the polypeptide comprising an amino acid sequence in which some parts of amino acids of the amino acid sequence represented by SEQ ID NO:1 are substituted by different amino acids (hG-CSF derivatives) are shown in Table 1.
TABLE 1
Position from
the N-terminal
amino acid
(hG-CSF of SEQ
Substituted amino acid in various hG-CSF derivatives
ID NO:1)
a)
b)
c)
d)
e)
f)
g)
h)
i)
j)
k)
l)
 1st (Thr)
*
Val
Cys
Tyr
Arg
*
Asn
Ile
Ser
*
Ala
*
 3rd (Leu)
Glu
Ile
Ile
Ile
Thr
Thr
Glu
Thr
Thr
*
Thr
*
 4th (Gly)
Lys
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Tyr
*
 5th (Pro)
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
*
Arg
*
17th (Cys)
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
*Unsubstituted amino acid
Examples of the polyalkylene glycol derivative include polyethylene glycol derivatives, polypropylene glycol derivatives, polyethylene glycol-polypropylene glycol copolymer derivatives, and the like.
The chemically modified polypeptide of the present invention can be produced using a chemical modifying agent comprising the above polyalkylene glycol derivative, and compounds represented by the following formula (I) can be exemplified as preferred chemical modifying agents.
The compounds include polyalkylene glycol derivatives represented by
R
1
—(M)
n
—X—R
2
  (I)
{wherein R
1
represents an alkyl group or an alkanoyl group; M represents
—OCH
2
CH
2
—, —OCH
2
CH
2
CH
2
—
or
—(OCH
2
CH
2
)
r
—(OCH
2
CH
2
CH
2
)
s
—
(wherein r and s are the same or different, and each represents an optionally changeable positive integer); n is an optionally changeable positive integer; X represents a bond, O, NH or S; and R
2
represents
<wherein R
3
represents OH, halogen or
—X
a
—(M
a
)
na
—R
1a
(wherein X
a
, M
a
, R
1a
and na each has the same meanings as the above X, M, R
1
and n, respectively); and Y represents halogen or
—Z—(CH
2
)
p
—(O)
m
—W
[wherein Z represents O, S or NH; and W represents a carboxyl group or a reactive derivative thereof, or
(wherein R
4
represents an alkyl group, and Hal represents halogen); p is an integer of 0 to 6; and m is 0 or 1]>,
—(CO)
m
—(CH
2
)
t
—W
(wherein t is an integer of 0 to 6; and m and W have the same meanings as defined above),
(wherein Hal
a
, pa and R
4a
each has the same meanings as the above Hal, p and R
4
, respectively),
(wherein R
3
and W have the same meanings as defined above),
(wherein R
3
, t and W have the same meanings as defined above),
(wherein W has the same meaning as defined above),
(wherein R
5
represents a residue in which an amino group and a carboxyl group are removed from an amino acid; and W has the same meaning as defined above)}.
With regard to the above compound represented by formula (I), examples of the alkyl group represented by R
1
, R
4
or the like include straight or branched alkyl groups having 1 to 18 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, isohexyl, heptyl, octyl, isooctyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, and the like; examples of the alkanoyl group represented by R
1
include straight or branched alkanoyl groups having 1 to 18 carbon atoms, such as formyl, acetyl, propionyl, butyryl, valeryl, pivaloyl, pentanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, and the like; examples of the halogen represented by R3, Y, Hal or the like include chlorine, bromine and iodine atoms; examples of the reactive derivative of a carboxyl group represented by W or the like include acid halides, such as an acid chloride, an acid bromide, and the like, active esters, such as a p-nitrophenyl ester, an N-oxysuccinimide ester, and the like, and mixed acid anhydrides with monoethyl carbonate, monoisobutyl carbonate and the like; and examples of the amino acid represented by R
5
include glycine, L-alanine, L-valine, L-leucine, L-serine, D-alanine, D-valine, D-leucine, D-serine, &bgr;-alanine, and the like. The positive integer represented by n, r or s is 1 to 20,000, preferably 50 to 5,000 for n, and 1 to 5,000 for r and s.
The polyalkylene glycol derivatives have a molecular weight of 500 to 1,000,000, preferably 3,000 to 1,000,000.
A plurality of hydroxyl groups may be present in the polypeptide molecule, and chemical modification of at least one of these groups may be suff

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