United States Patent:  6,042,820

Assignee:  Connaught Laboratories Limited (North York, CA)

Filed:  December 20, 1996

Copolymers designed for use as particulate carriers containing functionalizable amino subunits for coupling with targeting ligand are described. The copolymers are polyesters composed of .alpha.-hydroxy acid subunits such as D, L-lactide and .alpha.-amino acid subunits such as serine or in the preferred embodiment, terpolymers of D,L-lactide and glycolide and .alpha.-amino acid subunits such as serine. Stable vaccine preparations useful as delayed release formulations containing antigen(s) or antigen(s) and co-adjuvants encapsulated within or physically mixed with ploymeric microparticles are described. The particulate carriers are useful for delivering agents to the immune system of a subject by mucosal or parenteral routes to produce immune responses, including antibody responses.

SUMMARY OF THE INVENTION

The present invention is directed towards the production of a novel and useful polymer that has properties suitable for manufacturing by various processes into microparticles and microspheres. In this invention, modifications of existing processing procedures results in significant improvement in encapsulation efficiencies.

This invention is further directed to the production of useful vaccine delivery systems for antigen(s) or antigen and co-adjuvant cocktails by various immunization routes which include parenteral, oral and intranasal.

In accordance with a first aspect of the invention, there is provided a novel biodegradable, biocompatible polymer having a molecular weight of about 5,000 to about 40,000 daltons and having the general formula: 

wherein; R₁, R₂, R₃, R₄ and R₅ are selected independently and are selected from H, linear or branched alkyl groups;

R₆ is selected from H, an amine protecting group, a spacer molecule or a biologically active species;

X is selected from an O or S group; and

x and y are integers such that at least about 95% of the polymer is comprised of .alpha.-hydroxy acid residues.

The novel polymers are derived by copolymerization of monomers comprising at least one .alpha.-hydroxy acid and at least one .alpha.-amino acid. The .alpha.-hydroxy acids are generally of the formula R₁ R₂ COHCO₂ H, where the R₁ and R₂ groups are H, linear or branched alkyl groups. The .alpha.-hydroxy acids may comprise a mixture of .alpha.-hydroxy acids, at least one of the mixture of .alpha.-hydroxy acids having R₁ and R₂ groups which are hydrogen and another .alpha.-hydroxy acid having an R₁ group which is CH₃ and R₂ which is H. The .alpha.-amino acids are generally of the formula R₅ CHNHR₆ CO₂ H, where the R₅ group is a hydroxyl methyl or methyl thiol group and R₆ is an amine protecting group.

The amine protecting groups may be carbobenzyloxy, benzyl, paramethoxybenzyl, benzyloxymethoxy, tert-butyloxycarbonyl or [9-fluorenylmethyloxy]carbonyl.

The .alpha.-hydroxy acids are generally selected from L-lactic acid, D,L-lactic acid, glycolic acid, hydroxy valeric acid and hydroxybutyric acid.

In a preferred aspect of the invention, the polymers are poly-D,L-lactide-co-glycolide-co-pseudo-Z-serine ester (PLGpZS) and poly-P,L-lactide-co-glycolide-co-pseudo-serine ester (PLGpS).

The polymers may contain biologically active moieties such as cell bioadhesion groups, macrophage stimulators, polyethylene glycol, poly amino acids and/or protected amino acid residues covalently bound to the polymer directly or through side groups.

In the preferred embodiment the bioactive substituents are linked to the polymer via the amino groups on the amino acid moieties directly or via a suitable spacer molecule. The spacer molecule can be selected from .alpha.-hydroxy acids represented by the formula R₇ R₈ COHCO₂ H, where R₇ or R₈ groups are independently selected from H, linear or branched alkyl units and .alpha.-amino acids represented by the formula R₉ CHNHR₁₀ CO₂ H, where the R₉ group is a hydroxyl methyl or methyl thiol group and R₁₀ is an amine protecting group.

In accordance with another aspect of the present invention, there is provided a process for making a biodegradable, biocompatible polymer of the general formula provided herein which comprises forming a mixture of monomers comprising at least one .alpha.-hydroxy acid and at least one .alpha.-amino acid with an organic solvent solution of an esterification catalyst under inert atmospheric conditions, copolymerizing the monomers and isolating the resultant polymer. The catalyst used is preferably stannous 2-ethylhexanoate.

The polymer formed by the process can be further deprotected by solid phase catalytic reduction or alternatively by acid catalysis using hydrogen bromide in acetic acid solution.

The process can also further comprise forming the polymer into a film or microparticles.

In accordance with another aspect of this invention, there is provided a particulate carrier for the delivery of biologically active materials to a host, the carrier comprising polymers having a molecular weight of about 5,000 to about 40,000 daltons and having the general formula: 

wherein; R₁, R₂, R₃, R₄ and R₅ are selected independently and are selected from H, linear or branched alkyl groups;

R₆ is selected from H, an amine protecting group, a spacer molecule or a biologically active species;

X is selected from an O or S group; and

x and y are integers such that at least about 95% of the polymer is comprised of .alpha.-hydroxy acid residues.

The particulate carrier generally has a particle size of about 1 to 10 .mu.M.

In a further aspect of the present invention is a process for making a particulate carrier for the delivery of at least one biologically active material to a host, the process comprising;

(a) mixing an organic solvent phase comprising an .alpha.-hydroxy acid polymer or copolymer with an aqueous composition comprising dispersed or dissolved biologically active material to form a first water-in-oil emulsion;

(b) dispersing the first water-in-oil emulsion into an aqueous detergent phase to form a second water-in-oil-in-water double emulsion;

(c) removing water from the second double emulsion to form microspheres; and

(d) collecting the microspheres and having the biological material entrapped therein.

The particulate carrier of the present invention can be used as a composition having a biologically active material mixed therewith or entrapped within. The biological materials used may be selected from those which elicit an immune response. Such materials may comprise Haemophilus influenzae proteins, such as a non-proteolytic Hin-47 analog, rD-15, P1, P2, and P6. Other biological material may include proteins (e.g. influenza viral protein), protein mimetics, bacteria, bacterial lysates, viruses (e.g. respiratory syncytial virus), virus-infected cell lysates, DNA plasmids, antisense RNA, peptides (e.g. CLTB-36 and M2), antigens, antibodies, pharmacological agents, antibiotics, carbohydrates, lipids, lipidated amino acids (e.g. tripalmitoyl cysteine), glycolipids, haptens and combinations and mixtures thereof.

The present invention also provides an immunogenic composition comprising the particulate carrier provided herein and a physiologically acceptable carrier therefor. The composition can be administered mucosally or parenterally. The immune response is an antibody response which is a local or serum antibody response. In accordance with this aspect of the invention, there is provided a controlled or delayed release vaccine preparation in stable particulate form and a method of making such a vaccine preparation. Said particles are microspherical and contain a matrix of biodegradable polymer and antigen(s) and/or antigen plus co-adjuvant containing regions.

Advantages of the invention include:

(a) fully biodegradable and biocompatible microparticle formulation;

(b) facilitated antigen presentation to the cells of the immune system resulting in improved antigen immunogenicity;

(c) improved formulating conditions which increase the bioavailability of the antigen.

Additional embodiments of the present invention include the use of the particulate carrier in diagnostic assays and for therapeutic strategies.

Claim 1 of 14 Claims

1. A biodegradable, biocompatible polymer having a molecular weight of about 5,000 to about 40,000 daltons and having the general formula: 

wherein: R₁, R₂, and R₅ are selected independently and are selected from H, linear or branched alkyl groups;

R₃ and R₄ are H;

R₆ is selected from H, an amine protecting group, a spacer molecule or a biologically active species;

X is selected from an O or S group; and

x and y are integers such that at least about 95% of the polymer is comprised of .alpha.-hydroxy acid residues.

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