Non-antigenic amine derived polymers and polymer conjugates

Details Non-antigenic amine derived polymers and polymer conjugates Non-antigenic amine derived polymers and polymer conjugates

1998-11-03

2001-01-23

Harrison, Robert H. (Department: 1619)

Drug, bio-affecting and body treating compositions

Nonspecific immunoeffector, per se ; or nonspecific...

C424S078180, C424S279100, C526S352000, C526S243000, C526S936000, C530S402000, C530S815000, C546S001000, C546S157000

Reexamination Certificate

active

06177087

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to polymers which are useful in extending the in vivo circulating life of biologically active materials. The invention also relates to conjugates made with the polymers.
2. Description of Prior Art
Some of the initial concepts of coupling peptides or polypeptide to poly(ethylene glycol) PEG and similar water-soluble poly(alkylene oxides) are disclosed in U.S. Pat. No. 4,179,337, the disclosure of which is incorporated herein by reference. Polypeptide modified with these polymers exhibit reduced immunogenicity or antigenicity and circulate in the bloodstream longer than unmodified versions.
To conjugate poly(alkylene oxides), one of the hydroxyl end-groups is converted into a reactive functional group. This process is frequently referred to as “activation” and the product is called an “activated poly(alqylene oxide)”. Other substantially non-anitigenic polymers are similarly “activated” or fimctionalized.
The activated polymers are reacted with a therapeutic agent having nucleophilic functional group(s) that serve as attachment site(s). One nucleophilic functional group commonly used as an attachment site is the epsilon-amino group of lysines. Free carboxylic acid groups, suitably activated carbonyl groups, oxidized carbohydrate moieties, hydroxyl and mercapto groups have also been used as attachment sites.
Over the years, one shortcoming observed with polymer conjugation is that the conjugate has a different pI than the unmodified protein, enzyme, etc. For example, pegylation, i.e. attachment of the polymer, of lysine amino groups result in a decrease in the isoelectric point and changes the pH optimum, i.e the pH at which maximum bioactivity is observed. In some cases, it would be beneficial to restore the original pI or even alter the pI value of the polymer conjugate in order to optimize bioactivity at physiologic pH.
The present invention provides solutions to this problem.
SUMMARY OF THE INVENTION
In one aspect of the invention, there are provided amine-based polymer intermediates having the formula:
R—B  (I)
wherein (R) is a water-soluble, substantially non-antigenic polymer residue and (B) is preferably a secondary amine or a tertiary amine. Suitable secondary amines are of the formula:
—NH(CH
2
)
n
X  (II)
wherein:
(n) is an integer from 1-12, inclusive; and
X is selected from the group consisting of H; OH; CO
2
R
1
, where R
1
is H or a C
1-6
alkyl; halogen, atyl moieties and heteroaromatic moieties.
Suitable tertiary amines can be selected from:
wherein
(m) is an integer from 0-12, inclusive;
(X) is selected from the group consisting of H, OH, CO
2
R
1
, where R
1
is H or a C
1-6
alkyl, halogen, aryl moieties and heteroaromatic moieties;
(Y) is N or CH; and
(Z) is a C
1-6
alkyl.
The polymer is preferably a poly(alkylene oxide) residue such as a poly(ethylene glycol) having a molecular weight between about 200 and about 100,000. In addition, the polymer is preferably capped with a C
1-4
alkyl group on the end opposite (i.e. distal to) the B moiety.
In another aspect of the invention, there are provided substantially non-antigenic polymers of the formula:
 R—B′—W—L  (VII)
wherein (R) is as defined above; (B′) is NY, where Y is H i.e., a secondary amine or a C
1-6
alkyl or cycloalky i.e., a tertiary amine, (W) is a spacer group, such as a C
1-6
alkyl and (L) is a functional group capable of bonding with nucleophdies.
Other aspects of the invention include polymer conjugates containing biologically-active materials, described as nucleophiles herein as well as methods of conjugate preparation. In these aspects of the invention, the conjugates are of the formula:
[R—B′—W—L
1
]
z
-(nucleophile)
wherein:
(R), (B′) and (W) are as described above;
(L
1
) represents a covalent linkage between (R—B′—W) and a nucleophile; and
(z) represents the number of polymers attached to the nucleophile.
The biologically active materials include proteins, peptides, enzymes, medicinal chemicals or organic moieties whether synthesized or isolated from nature. The conjugation methods include contacting a nucleophile, i.e. a biologically active material, capable of undergoing a substitution reaction with a polymer described. The reaction is carried out under conditions sufficient to effect attachment while maintaining at least a portion of the biological activity.
The present invention also includes methods of treating various maladies and conditions. In this aspect, a mammal in need of treatment is administered an effective amount of a conjugate containing a biologically-active material such as a protein, enzyme or organic moiety attached to a polymer of the present invention.
Some of the chief advantages of the present invention are that the amine-derived polymers impart a pI andlor pH modulating effect to the conjugate. Thus, the isoelectric point of bio-active materials included in the conjugate can be adjusted to a desired point. This is to be contrasted with the effect observed with conventional activated polymers where shifts in isoelectric points can be observed, often to the detriment of optimal activity.
In addition, in those aspects of the invention where anionic groups are included as part of the aminebearing moiety, the in vivo circulating life of the conjugates is further prolonged due to reduced renal filtration.


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