United States Patent:  6,022,955

Assignee:  Abbott Laboratories (Abbott Park, IL)

Filed:  December 10, 1991

This invention discloses a method for chemically modifying a pulmonary surfactant protein or polypeptides with various fatty acids. These conjugates are useful in preparing formulations for the treatment of respiratory disease.

DISCLOSURE OF THE INVENTION

There is disclosed a composition of matter comprising a covalently linked compound consisting of two parts and having the structural formula FA-SP-C wherein -SP-C is a protein selected from the group comprising human, porcine, canine, bovine surfactant associated protein C (-SP-C) either naturally derived or produced by peptide synthesis or recombinant DNA means and wherein FA- is a fatty acid.

There is further disclosed a composition of matter comprising a compound having the structural formula FA-SP-C wherein SP-C is a protein selected from the group comprising human, porcine, canine, bovine surfactant associated protein C (-SP-C) either naturally derived or produced by peptide synthesis or recombinant DNA means and wherein -FA is a fatty acid selected from the group comprising fatty acids having a carbon chain length from two (2) to about twenty (20) carbon atoms.

This novel entity comprises a fatty acid (FA) covalently attached to the SP-C protein (or analogs thereof). These covalent SP-C adducts or conjugates (FA-SP-C) exhibit improved solubility and enhanced surfactant activity when admixed with phospholipids either alone or in combination with SP-B and/or fragments of SP-B. The SP-B fragment being that portion of the SP-B protein which contains at least the terminal amino acid sequence and substitution, deletion and addition analogs thereof.

There is also disclosed a composition of matter comprising a fatty acid/SP-C conjugate (or analogs thereof) in combination with at least one lipid. The lipid is selected from the group consisting of synthetic phospholipids, naturally occurring phospholipids, neutral lipids, cholesterol, cholesterol esters, phosphatidylcholine, disaturated phosphatidylycholine, phosphatidylglycerol, dipalmitoyl phosphatidylcholine, phosphatidylihisotyl and mixtures thereof.

The most preferred lipids are a mixture comprising dipalmitoyl-sn-phosphatidylcholine (DPPC), egg phosphatidylglycerol (PG) and palmitic acid (PA).

Also disclosed is a method for the treatment of pulmonary surfactant deficient states (e.g. hyaline membrane disease) and/or abnormal surfactant states (e.g. respiratory distress syndrome), said method comprising the administration of an effective amount of a surfactant composition to a patient in need of treatment, said surfactant composition comprising a FA-SP-C conjugate and at least one lipid. The FA-SP-C conjugate may be employed alone or in combination with full length SP-B or a SP-B fragment, said fragment contains at least a terminal amino acid sequence; and at least one lipid.

Further disclosed is a method for pulmonary drug delivery, said method consisting of administering to a patient in need, a therapeutically effective amount of a composition comprising: 1) a FA-SP-C conjugate either alone or in combination with fragment of the SP-B protein that contains at least a terminal amino acid sequence; 2) at least one lipid; and 3) an appropriate therapeutic agent.

Also disclosed is a method for the preparation of polyclonal antibodies exhibiting specificity for the antigenic determinants on natural SP-C, said method comprising immunizing with an effective amount of a composition comprising the FA-SP-C conjugate with suitable carriers and/or adjuvants.

According to the present invention, novel, non-naturally occurring peptide-fatty acid conjugates are disclosed which demonstrate improved solubility in some organic solvents and which have the ability to markedly enhance the surfactant-like activity of natural and/or synthetic phospholipids. The novel chemical entities of this invention comprise the conjugates of fatty acids covalently attached to the known sequences of naturally occurring SP-C. This conjugate which may be combined with phospholipids alone, or combined with natural, synthetic or recombinant SP-B and/or SP-A, or combined with one or more SP-B fragments, said fragment being that portion of the SP-B protein which contains at least a terminal amino acid sequence and substitution, deletion, replicate and addition analogs thereof. The FA-SP-C conjugates of the invention are readily and economically produced via chemical means and may be formulated with natural and/or synthetic phospholipids.

The fatty acids useful in preparing the conjugates of this invention are the fatty acids of 2 to about 20 carbon atoms.

The preferred FA-SP-C conjugates of the invention are:

C-14 (Myristic acid)-SP-C;

C-16 (Palmitic acid)-SP-C.

C-20 (Arachadic acid)-SP-C

The most preferred fatty acids useful in this invention are the fatty acids of 14-16 carbon atoms. Furthermore, it is also contemplated that the preferred fatty acids (of certain chain lengths, both saturated and unsaturated) may also include lipase resistant analogs such as the ether, phosphoryl, and/or sulfur derivatives of these fatty acids.

It is contemplated that the SP-C polypeptide useful in this invention may include addition analogs (wherein one or more amino acid-residues which are not naturally present in a given SP-C sequence are provided in the synthetic polypeptide at terminal or intermediate locations), deletion analogs (wherein one or more residues are deleted from a natural sequence), substitution analogs (wherein one or more residues are replaced by other amino acid or modified residues) and replicate analogs (wherein one or more residues are repeated, replicated, in a natural sequence). Specifically comtemplated are interspecies hybrid analogs comprising composite replicas of more than one species (i.e. human, canine, bovine, porcine, etc.) of naturally occurring SP-C proteins and those analogs wherein D-forms of amino acids replace the naturally occurring L-forms. The polypeptides useful in this invention preferably retain the overall hydrophobic character of the SP-C protein and are also expected to retain substantial elements of secondary and tertiary conformation.

Unlike unmodified SP-C (native, synthetic or recombinant) the FA-SP-C conjugates of this invention are readily formulated with either natural or synthetic phospholipids to yield admixtures which are useful in the treatment of pulmonary surfactant deficient (eg. hyaline membrane disease) and/or abnormal (eg. respiratory distress syndrome, RDS) surfactant states, and for pulmonary drug delivery systems. The FA-SP-C conjugates of this invention are also expected to have considerable use in the preparation of polyclonal and monoclonal antibodies exhibiting specificity for the antigenic determinants occurring on natural SP-C and which would therefore be useful inter alia in immunopurification and/or quantitative assessment of the SP-C protein in clinical immunodiagnosis.

Other aspects and advantages of the invention will be apparent upon consideration of the following detailed description of the invention and are intended to be illustrative embodiments thereof and not limitative.

Best Mode for Carrying Out the Invention

A number of fatty acids of varying chain length were covalently attached to SP-C synthesized by means of solid phase peptide synthesis. However, identical and/or similar SP-C could also be produced by known recombinant methodologies and the like.

The fatty acids of various chain lengths (C₂ to C₂₀) were covalently attached to the N-terminal end of SP-C by means of an symmetric anhydride reaction which is well known in the art. The fatty acids may be saturated or unsaturated entities. It will be readily appreciated by one skilled in the art of organic and/or protein chemistry that several alternative coupling strategies could be employed to achieve the same result. Furthermore, the fatty acids could also be attached to alternative sites on the SP-C molecule, e.g. the carboxyl terminal, .epsilon.-amino terminal group of lysine residues, SH groups of cysteine, etc. Fatty acid chain lengths greater than two (2) carbon atoms were found to yield enhanced solubility of the corresponding FA-SP-C conjugates and improved surfactant activity of the resultant FA-SP-C/phospholipid admixtures.

Thus, the scope of the present invention includes all SP-C regardless of origin and all chemical methods available for coupling saturated or unsaturated fatty acids to the SP-C polypeptide. The resulting FA-SP-C conjugates when combined with phospholipids exhibit enhanced surfactant activity as compared to unmodified SP-C. These conjugates can be facilely produced by relatively standard organic chemistry techniques.

As a result of formulation experiments and biophysical testing thereof (as hereinafter described) the inventors have determined that certain of these FA-SP-C conjugates exhibit an unexpected, unpredicted, unusual and surprising ability to facilitate enhanced surface activity of the resultant phospholipid admixtures.

Claim 1 of 16 Claims

1. A purified composition of matter consisting of a covalently linked compound in physical admixture with at least one lipid, wherein said covalently linked compound consists of two parts and has the structural formula: FA-SPC, wherein -SP-C is protein selected from the group comprising human, porcine, canine and bovine surfactant associated protein C (SP-C) produced by chemical or enzymatic synthesis or recombinant DNA means, wherein said SP-C protein comprises a sequence of thirty-four (34) amino acids, said sequence containing eleven (11) valine amino acid residues, nine (9) of said valine residues being contained in two (2) adjacent polyvaline stretches, said polyvaline stretches having a first stretch of six adjacent valine amino acid residues and a second stretch of three adjacent valine amino acid residues separated from the first stretch by two hydrophobic amino acid residues, said SP-C protein enhances surfactant-like activity of phospholipids in lungs of an animal, and is substantially resistant to protease, endoglycosidase F and collagenase enzymes, and wherein FA- is a single fatty acid selected from the group comprising fatty acids having a carbon chain length from two(2) to about twenty(20) carbon atoms and wherein said fatty acid is covalently attached to the amino end terminal amino acid residue of said SP-C.

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