Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2000-04-07
2003-05-20
Russel, Jeffrey E. (Department: 1654)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S345000, C530S409000, C530S410000, C560S158000, C560S182000, C564S159000
Reexamination Certificate
active
06566506
ABSTRACT:
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to branched 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.
Some of the initial concepts of coupling peptides or polypeptides 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. Polypeptides modified with these polymers exhibit reduced immunogenicity/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(alkylene oxide)”. Other substantially non-antigenic polymers are similarly “activated” or functionalized.
The activated polymers are reacted with a therapeutic agent having nucleophilic functional groups that serve as attachment sites. One nucleophilic functional group commonly used as an attachment site is the &egr;-amino groups of lysines. Free carboxylic acid groups, suitably activated carbonyi groups, oxidized carbohydrate moieties and mercapto groups have also been used as attachment sites.
Insulin and hemoglobin were among the first therapeutic agents conjugated. These relatively large polypeptides contain several free &egr;-amino attachment sites. A sufficient number of polymers could be attached to reduce immunogenicity and increase the circulating life without significant loss of biologic activity.
Excessive polymer conjugation and/or conjugation involving a therapeutic moiety's active site where groups associated with bioactivity are found, however, often result in loss of activity and thus therapeutic usefulness. This is often the case with lower molecular weight peptides which have few attachment sites not associated with bioactivity. Many non-peptide therapeutics also lack a sufficient number of attachment sites to obtain the benefit of polymer modification.
One suggestion for overcoming the problems discussed above is to use longer, higher molecular weight polymers. These materials, however, are difficult to prepare and expensive to use. Further, they provide little improvement over more readily available polymers.
Another alternative suggested is to attach two strands of polymer via a triazine ring to amino groups of a protein. See, for example,
Enzyme,
26, 49-53 (1981) and
Proc. Soc. Exper. Biol. Med.,
188, 364-9 (1988). Triazine, however, is a toxic substance which is difficult to reduce to acceptable levels after conjugation. In addition, triazine is a planar group and can only be double-polymer substituted. The planar structure rigidly locks the two polymer chains in place. This limits the benefits of polymer conjugation to about the same as that obtained by increasing polymer chain length. Thus, non-triazine-based activated polymers would offer substantial benefits to the art.
In the above-mentioned cases, however, the biologically active polymer conjugates were formed having substantially hydrolysis-resistant bonds (linkages) between the polymer and the parent biologically-active moiety. Thus, long-lasting conjugates which are of a rather permanent nature rather than prodrugs per se (where the parent molecule is eventually liberated in vivo were prepared.
In addition, over the years, several methods of preparing prodrugs have also been suggested. Prodrugs include chemical derivatives of a biologically-active parent compound which, upon administration, will eventually liberate the active parent compound in vivo. Use of prodrugs allows the artisan to modify the onset and/or duration of action of a biologically-active compound in vivo. Prodrugs are often biologically inert or substantially inactive forms of the parent or active compound. The rate of release of the active drug is influenced by several factors including the rate of hydrolysis of the linker which joins the parent biologically active compound to the prodrug carrier.
Prodrugs based on ester or phosphate linkages have been reported. In most cases, the particular type of ester linkage used to form the prodrug provides T
½
for hydrolysis of up to several days in aqueous environments. Although one would expect a prodrug to have been formed, most of the conjugate is eliminated prior to sufficient hydrolysis being achieved in vivo. It would therefore be preferable to provide prodrugs which have a linkage which allows more rapid hydrolysis of the polymer-drug linkage in vivo so as to generate the parent drug compound more rapidly.
It has also been surprisingly found that when only one or two polymers of less than 10,000 molecular weight (each) are conjugated to biologically-active compounds such as organic moieties, the resulting conjugates are often rapidly eliminated in vivo. In fact, such conjugates are often so rapidly cleared from the body that even if a substantially hydrolysis-prone ester linkage is used, not enough of the parent molecule is regenerated.
Even though previous prodrugs based on conjugates of a parent drug compound on a water soluble polymer have not been successful for a variety of reasons, including excessively slow hydrolysis of the linkage, work in this area has continued. There is still a need in improvements in polymer-based prodrugs and, in particular, ways of significantly increasing the payload of the polymer portion of the prodrug. The present invention addresses these shortcomings.
SUMMARY OF THE INVENTION
In one aspect of the invention, there are provided branched, substantially non-antigenic polymers corresponding to the formula:
(R)
n
L—A (I)
wherein (R) includes a water-soluble non-antigenic polymer;
(n)=2 or 3;
(L) is an aliphatic linking moiety covalently linked to each (R); and
(A) represents an activating functional group capable of undergoing nucleophilic substitution.
For example, (A) can be a group which is capable of bonding with biologically active nucleophiles or moieties capable of doing the same. In particularly preferred aspects of the invention, (R) includes a poly(alkylene oxide) PAO such as a poly(ethylene glycol) (hereinafter: PEG).
One preferred embodiment of the invention provides branched polymers containing a terminal carboxylic acid group which is useful in the formation of ester-based prodrugs. The branched polymers are of the formula:
(R)
n
L—COOH (Ia)
where (R), (n), and (L) are as defined above.
Another preferred embodiment of the invention includes branched polymers of the same formula set forth above, i.e.: (R)
n
L-A, except that (L) is selected from the group consisting of
where (a) is an integer of from about 1 to about 5;
X is O, NQ, S, SO or SO
2;
where Q is H, C
1-8
alkyl, C
1-4
branched alkyl, C
1-8
substituted alkyl, aryl or aralkyl;
(m) is 0 or 1;
(p) is a positive integer, preferably from about 1 to about 6;
(R) and (n) are as defined above; and
(A) is as defined above, including COOH as set forth in Formula (Ia).
These umbrella-like branched polymers of the present invention (U-PAO's or U-PEG's) react with biologically active nucleophiles to form conjugates. The point of polymer attachment depends upon the functional group (A). For example, (A) can be a succinimidyl succinate or carbonate and react with epsilon amino lysines. Alternatively, (A) can be a carboxylic acid which is capable of reacting with hydroxyl groups found on biologically-active nucleophiles to form ester-linked prodrugs. The branched polymers can also be activated to link with any primary or secondary amino group, mercapto group, carboxylic acid group, reactive carbonyl group or the like found on biologically-active materials. Other groups are apparent to those of ordinary skill in the art.
Other aspects of the invention include conjugates containing biologically-active materials and one or more of the branched polymers described above as well a
Greenwald Richard B.
Martinez Anthony J.
Enzon Inc.
Muserlian, Lucas & Mercanti, LLP
Russel Jeffrey E.
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