Title:  Lanthionine antibiotic compositions and method

United States Patent:  6,521,596

Issued:  February 18, 2003

Inventors:  Caufield; Page W. (Birmingham, AL); Novak; Jan (Birmingham, AL)

Assignee:  The UAB Research Foundation (Birmingham, AL)

Appl. No.:  151203

Filed:  September 10, 1998

Certain bacteria indigenous to humans produce antimicrobial substances called bacteriocins which inhibit other bacteria, including members of their own species. Mutacins are a class of antibiotic substances made by Streptococcus mutans. Disclosed is the purification and biochemical characterization of a novel lanthionine-containing mutacin peptide from S. mutans. The purified peptide is pH- and temperature-stable and its amino acid composition indicates the presence of lanthionine and .beta.-methyllanthionine. Also provided are methods of making and using the purified polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

The literature concerning lantibiotics and other bacteriocin-like substances supports two main themes. First, there appears to be as many antibiotic substances as there are species. Even within the same species, several different antibiotics can be distinguished. Even though many of these substances exhibit similar properties (phenotypes), their genetic components (genotypes) may differ. Second, in every lantibiotic system characterized genetically thus far, and without exception, each locus consists of multiple genes arranged as contiguous coding sequences and in many cases, coordinately regulated. The genes and their products involved in mutacin expression are likely to lie within a cluster, making identification and transfer of the complete system to a non-producer strain feasible.

The present application describes the isolation and characterization of a lanthionine-containing antibiotic (lantibiotic) from human-derived strains of S. mutans. The lantibiotic will be referred to herein as "mutacin" with the understanding that other mutacin-like antibacterials are associated with various Streptococcus strains. Lantibiotics are polycyclic peptides with several intrachain sulfide bridges, consisting of the thioether amino acids lanthionine and .beta.-methyllanthionine (Jung, 1991). In addition, lantibiotics contain a .beta.-unsaturated amino acids such as didehydroalanine and didehydroaminobutyric acid, post-translationally modified via dehydration of serine and threonine residues (Dodd, et al., 1990). The dehydrated, unsaturated serine or threonine residues can remain as such or form thioether bridges with neighboring cysteine.

Perhaps the best studied, and clearly the oldest known polypeptide antibiotic is nisin (Hurst, 1981). Produced by Lactococcus lactis, nisin is widely used in European countries as a food preservative. More recently, nisin has been tested as an anti-plaque agent in beagle dogs (Howell, et al., 1993). Nisin is a 3.4 kDa peptide that exhibits a fairly narrow spectrum of activity mainly against gram-positive bacteria including most streptococci, staphylococci, clostridia including C. botulinum, and Mycobacterium tuberculosis, among others. Nisin is not effective against most gram-negative bacteria (neisseria is an exception), fungi or yeast (Hurst, 1981). Because of its unique cyclic thioether ring structure and small size, the nisin molecule is extremely heat stable, a property that enhances its suitability for the food industry. Sensitive to pancreatic enzymes such as trypsin and chymotrypsin, however, nisin is readily inactivated when ingested.

Nisin and other lantibiotics of type A exert their antibacterial activity by interacting with cell membranes, resulting in a decrease of t h e membrane potential gradient with ensuing membrane depolarization, disruption and rupture leading to irreversible cell lysis (Sahl, 1991). For this reason, lantibiotics are bactericidal. As highly charged cationic peptides, the membrane active component of lantibiotics resides in its amphipathic amino acid composition, arranged into both hydrophobic and hydrophilic domains. Type B lantiobiotics are enzyme inhibitors. In addition, the lantibiotics are preceded by a leader sequence arranged in a n .alpha.-helical conformation, contributing to its amphipathic properties and putative function as an escort/processing "handle."

Other gene products necessary for posttranslational modification, cleavage, and export of nisin are known; their location, genetic determinants and some aspects of their regulation have been recently reported (Kaletta and Entian, 1989; van der Meer, et al., 1993). The coding sequence of the nisin structural gene is part of a much larger transcript, somewhere between 2 and 5 kb, indicating the nisin gene is part of a polycistronic cluster (Steen, 1991). An insertion of a transposon such as Tn916 anywhere within this 2 to 5 kb transcript is likely to result in the inactivation of the nisin gene and yield a nisin negative phenotype.

Prior to its genetic characterization, nisin was thought to be non-ribosomally synthesized because it contained non-protein amino acids including lanthionine and .beta.-methyllanthione, among others. A complete understanding evolved after the amino acid sequence derived from the DNA sequence was compared with the actual amino acid sequence. In the place of expected serines and threonines were the lanthionine and .beta.-methyllanthiones; other residues were apparently dehydrated it thus became clear that the mature nisin molecule was the result of posttranslational modifications, thereby prompting a search for these otherwise unknown processing enzymes began.

Functions ascribed to the gene products from the nisin cluster include: nisA, the structural gene coding the precursor of nisin; nisB, a membrane-located enzyme involved with processing; nisT, an ABC-translocator whose function includes the export of peptides; nisC, post-translational modification of the polypeptide; nisI, a lipoprotein involved with immunity; nisP, a membrane-located serine protease; and nisR, a DNA binding protein involved with regulation of nisin expression (van der Meer, et al., 1993). Analogs to most of the nisin genes are within the subtilin locus (Schnell, et al., 1992) and the epidermin locus (Schnell, et al., 1992). Bac/Hly from E. faecalis may also be a member of the lantibiotic family as the A factor is a protease capable of not only activating the Bac molecule just prior to its export, but also serves as an "immunity" factor by proteolytic cleavage of incoming Bac from other Bac-producing cells. This dual role for the A component appears unique at the present but may turn out to be a common theme in other lantibiotic producers. The polycistronic nature of the Hly locus is reminiscent of the nisin cluster as recently presented by Booth and Gilmore (1993).

The entire locus of at least some of the lanthionine antibiotics can be transferred en bloc to a non-producer strain. This is true for nisin (Horn, et al., 1991), subtilin (Liu and Hansen, 1991), and epidermin (Schnell, et al., 1992). In fact, the plasmid-borne epidermin locus (14 kb) was recently expressed across species (Augustin, et al., 1992). In the case of nisin, transfer has evidently occurred in nature since the coding sequences were found on both large plasmids and on chromosomal sites. The nisin locus was a part of a transposon Tn5301 (Horn, et al., 1991) (Tn5276) along with the sucrose metabolism locus, sac. The relationship between nisin and the locus for sucrose catabolism is interesting. It is not known whether there is a parallel in S. mutans and mutacin, especially considering that sucrose metabolism is a major virulence factor in mutans streptococci.

The development of a growth medium which permitted the purification of mutacin from liquid culture was an important factor in the purification of the protein. Additional steps employing ultrafiltration and selective precipitation were critical to the purification process. A single, biologically active protein with an M_r of 2,500 was the final product as measured by size exclusion high-performance liquid chromatography. Other distinct characteristics of the present purified mutacin are the molecular weight as determined by size exclusion HPLC and ion spray mass spectroscopy, trypsin, pH and temperature sensitivity, solubility in water-soluble solvents, and migration pattern on TLC. The temperature resistance, size, and biological activity found for purified mutacin are dissimilar to previously reported properties of other mutacins in situ in agar plates and as measured by thin-layer chromatography (Parrot, et al., 1990, and as summarized in Table 1).
 

Based on its amino acid composition and isoelectric focusing point, mutacin demonstrates characteristics of a basic protein. This is consistent with its mobility in agar gels as detected by gel overlay with a sensitive indicator strain.

The present polypeptide contains lanthionine and most likely contains a related amino acid, B-methyllanthionine. This new group II mutacin is a member of the lanthionine-containing polypeptide family. Other lanthionine-containing peptides produced by gram-positive species include nisin from Lactococcus lactis, epidermin from Staphylococcus epidermidis and subtilin from Bacillus subtilis (Allgaier, et al., 1985; Gross and Kiltz, 1973; Gross and Morell, 1971).

The N-terminal amino acid sequence (Asn Arg Trp Trp Gln Gly Val Val) (SEQ ID NO:1) of mutacin as determined by Edman degradation was compared with known sequences in protein databases and found to be unique. Additional cycles did not reveal any PTH-amino acid derivatives. Both lanthionine and methyllanthionine are expected to be suitable for cleavage from protein with the Edman procedure. However, they were not detected as bis-PTH derivatives, apparently because of their low solubility compared to mono-amino acid derivatives. Edman cleavage of one residue contributing to lanthionine or methyllanthionine is expected to result in a blank cycle but sequencing should continue uninterrupted. Additional modifications of mutacin may explain the failure to remove further amino acids. The absence of methionine at the N-terminus suggests that mutacin is processed from a precursor form, as reported for other lantibiotics (Buchman, et al., 1988; Kaletta, et al., 1989; van der Meer, et al., 1993).

Biologically Functional Equivalents

Modifications and changes may be made in the structure of the mutacin compositions of the present invention and still obtain molecules having like or otherwise desirable characteristics. For example, it is well known in the art that certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, bacteria, components of the immune system, and the like. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like or even countervailing properties (e.g., antagonistic vs. agonistic). Various changes may be made in the sequence of mutacin proteins or peptides (or underlying DNA) without appreciable loss of their biological utility or activity.

It is also well understood by the skilled artisan that, inherent in the definition of a biologically functional equivalent protein or peptide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity. Biologically functional equivalent peptides are thus defined herein as those peptides in which certain, not most or all, of the amino acids may be substituted. I n particular, where the mutacin polypeptides are concerned, it is contemplated that only about one to three, or more preferably, one or two, amino acids may be changed within a given peptide. Of course, a plurality of distinct proteins/peptides with different substitutions may easily be made and used in accordance with the invention.

It is also well understood that where certain residues are shown to be particularly important to the biological or structural properties of a protein or peptide, e.g., residues in active sites, such residues may not generally be exchanged. In the present invention, for example, one will likely not choose to change the lanthionine or b-methyllanthionine residues which are particularly characteristic of this type of antibiotic.

In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982). Certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, including substrates, receptors, and cellular entities such as bacteria. It is known in the art that an amino acid may be substituted by another amino acid having a similar hydropathic index and still obtain a biological functionally equivalent protein. In such changes, the substitution of amino acids whose hydropathic indices are within .about.2 is preferred, those which are within .about.1 are particularly preferred, and those within .about.0.5 are even more particularly preferred.

Substitution of like amino acids is also made on the basis of hydrophilicity, particularly where the biological functional equivalent protein or peptide thereby created is intended for use in immunological embodiments. U.S. Pat. No. 4,554,101, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0_-- 1); glutamate (+3.0_-- 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5_-- 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). An amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within .about.2 is preferred, those which are within .about.1 are particularly preferred, and those within .about.0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes may be effected by alteration of the encoding DNA; taking into consideration also that the genetic code is degenerate and that two or more codons may code for the same amino acid.

Epitopic Core Regions

U.S. Pat. No. 4,554,101 (Hopp) also teaches the identification and preparation of epitopes from primary amino acid sequences on the basis of hydrophilicity. Through the methods disclosed in Hopp, one of skill in the art would be able to identify epitopes from within an amino acid sequence such as the mutacin sequences disclosed herein and those sequences obtained by cloning according to the methodology herein. The regions of sequence thus identified are also referred to as "epitopic core regions".

Numerous scientific publications have been devoted to the prediction of secondary structure, and to the identification of epitopes, from analyses of amino acid sequences (Chou & Fasman, 1974a,b; 1978a,b, 1979). Any of these may be used, if desired, to supplement the teachings of Hopp in U.S. Pat. No. 4,554,101. Moreover, computer programs are currently available to assist with predicting antigenic portions and epitopic core regions of proteins. Examples include programs based upon the Jameson-Wolf analysis (Jameson & Wolf 1988; Wolf et al., 1988), and also new programs for protein tertiary structure prediction (Fetrow & Bryant, 1993).

Nucleic Acid Hybridization

DNA sequences derived from the N-terminal amino acid sequence (SEQ ID NO:1) disclosed herein, and functional equivalents thereof, have particular utility as probes and primers in nucleic acid hybridization embodiments. As such, oligonucleotide fragments corresponding to such sequences for stretches of between about 10 nucleotides to about 20 nucleotides will find particular utility. Oligonucleotides with other 10 or 20 or so nucleotide long sequences which correspond to the amino sequences in SEQ ID NO:1 and ID NO:2, therefore, have particular utility.

The ability of such nucleic acid probes to specifically hybridize to mutacin-encoding sequences will enable them to be of great use in, for example, detecting the presence of complementary sequences in a given sample. For example, the gene encoding the mutacin may be cloned and sequenced using the complementary nucleotide probes as disclosed herein. Furthermore, the area immediately surrounding the structural gene for mutacin may be characterized to identify regulatory regions and other genes related to the regulation, production, and processing of the mutacin polypeptide. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions. This i s particularly important in the preparation of second generation mutacin products, as described herein.

Nucleic acid molecules having stretches of 10, 15 or 20 or so nucleotides designed from a consideration of the amino acid sequences disclosed herein will have utility as hybridization probes for use in Southern and Northern blotting in connection with analyzing mutacin-like structural or regulatory genes in other organisms and in various bacterial strains. The total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in such hybridization embodiments.

The use of a hybridization probe of about 10 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 10 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to 20 nucleotides, or even longer where desired. Such fragments are readily prepared by, e.g., directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR technology of U.S. Pat. No. 4,603,102 or by introducing selected sequences into recombinant vectors for recombinant production. Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of mutacin genes or cDNAs. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and.backslash.or high temperature conditions, such as provided by 0.02M-0.15M NaCl at temperatures of 50^oC. to 70^oC. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating mutacin genes. Single specific primer PCR is one possible method; reverse PCR is another possible method when one amino acid sequence is known.

Of course, for some applications, for example, where one desires to prepare engineered, i.e., intelligently designed, second generation mutacin products, e.g., by site-specific mutagenesis, or where one seeks to isolate mutacin-encoding sequences from related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as 0.15M-0.9M salt, at temperatures ranging from 20^oC. to 55^oC. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, mutacin-hybridization conditions can be readily manipulated, according to the results desired.

In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with a n appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred embodiments, one will likely desire to employ a fluorescent label or a n enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, calorimetric indicator-substrates are known which can be employed to provide a means visible to the human eye o r spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to-specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label.

Longer DNA segments will often find particular utility in the recombinant production of mutacin and mutacin peptides. The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is contemplated that a mutacin-encoding or mutacin peptide-encoding nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments may be prepared in accordance with the present invention which are up to 10,000 base pairs in length, with segments of 5,000 or 3,000 being preferred and segments of about 1,000 base pairs in length being particularly preferred.

As discussed above, it will be understood that this invention is not limited to the particular nucleic acid and amino acid sequences of SEQ ID NOS:1-2. Therefore, DNA segments prepared in accordance with the present invention may also encode biologically functional equivalent proteins or peptides which have variant amino acids sequences. Such sequences may arise as a consequence of codon redundancy and functional equivalency which are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, mutacin proteins or peptides, including second generation mutacin variants, may be created via the application of recombinant DNA technology, in which changes in the protein structure are engineered, based on considerations of the properties of the amino acids being exchanged.

DNA segments encoding a mutacin gene may be introduced into recombinant host cells and employed for expressing a mutacin protein or peptide. Alternatively, through the application of genetic engineering techniques, subportions or derivatives of selected mutacin genes may be employed. Equally, through the application of site-directed mutagenesis techniques, one may re-engineer DNA segments of the present invention to alter the coding sequence, e.g., to introduce improvements to the antibiotic actions of the resultant protein or to test such mutants in order to examine their structure-function relationships at the molecular level. Where desired, one may also prepare fusion peptides, e.g., where the mutacin coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for immunodetection purposes (e.g., enzyme label coding regions).

Pharmaceutical Compositions and Formulations

Pharmaceutical compositions comprising the disclosed mutacins may be orally administered, for example, with an inert diluent or with an assimilable edible carrier or they may be enclosed in hard or soft shell gelatin capsules or they may be compressed into tables or may be incorporated directly with the food of the diet.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

For oral prophylaxis the polypeptide may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.

The active compounds may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active compounds in such therapeutically useful compositions results in a suitable dosage.

The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such a s magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

The active compounds may also be administered parenterally, e.g., formulated for intravenous, intramuscular, or subcutaneous injection. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Claim 1 of 8 Claims

What is claimed is:

1. "A pharmaceutical composition comprising a polypeptide having an amino acid sequence comprising SEQ ID NO: 14 dispersed in a pharmacologically acceptable carrier".
 

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