The present invention relates to compounds for delivering active agents, and particularly biologically active agents such as, for example, bioactive peptides and the like.  These compounds are used as carriers to facilitate the delivery of a cargo to a target.  The carriers are modified amino acids and are well suited to form non-covalent mixtures with biologically-active agents for oral administration to animals.  Methods for the preparation and for the administration of such compositions are also disclosed. 
Conventional means for delivering active agents are often severely limited by biological, chemical, and physical barriers.  Typically, these barriers are imposed by the environment through which delivery occurs, the environment of the target for delivery, or the target itself. 
Biologically active agents are particularly vulnerable to such barriers.  For example in the delivery to animals of pharmacological and therapeutic agents, barriers are imposed by the body.  Examples of physical barriers are the skin and various organ membranes that must be traversed before reaching a target.  Chemical barriers include, but are not limited to, pH variations, lipid bi-layers, and degrading enzymes. 
These barriers are of particular significance in the design of oral delivery systems.  Oral delivery of many biologically active agents would be the route of choice for administration to animals if not for biological, chemical, and physical barriers such as varying pH in the gastro-intestinal (GI) tract, powerful digestive enzymes, and active agent impermeable gastro-intestinal membranes.  Among the numerous agents which are not typically amenable to oral administration are biologically active peptides, such as calcitonin and insulin; polysaccharides, and in particular mucopolysaccharides including, but not limited to, heparin; heparinoids; antibiotics; and other organic substances.  These agents are rapidly rendered ineffective or are destroyed in the gastro-intestinal tract by acid hydrolysis, enzymes, or the like. 
Earlier methods for orally administering vulnerable pharmacological agents have relied on the co-administration of adjuvants (e.g., resorcinols and non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecylpolyethylene ether) to increase artificially the permeability of the intestinal walls, as well as the co-administration of enzymatic inhibitors (e.g., pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymatic degradation. 
Liposomes have also been described as drug delivery systems for insulin and heparin.  See, for example, U.S. Pat.  No.  4,239,754; Patel et al. ( 1976), FEBS Letters, Vol.  62, pg.  60; and Hashimoto et al. ( 1979), Endocrinology Japan, Vol.  26, pg.  337. 
However, broad spectrum use of such drug delivery systems is precluded because: (1) the systems require toxic amounts of adjuvants or inhibitors; (2) suitable low molecular weight cargos, i.e. active agents, are not available; (3) the systems exhibit poor stability and inadequate shelf life; (4) the systems are difficult to manufacture; (5) the systems fail to protect the active agent (cargo); (6) the systems adversely alter the active agent; or (7) the systems fail to allow or promote absorption of the active agent. 
More recently, microspheres of artificial polymers of mixed amino acids (proteinoids) have been used to deliver pharmaceuticals.  For example, U.S. Pat.  No.  4,925,673 describes drug-containing proteinoid microsphere carriers as well as methods for their preparation and use.  These proteinoid microspheres are useful for the delivery of a number of active agents. 
There is still a need in the art for simple, inexpensive delivery systems which are easily prepared and which can deliver a broad range of active agents. 
Compounds useful in the delivery of active agents are provided.  These compounds include 
or salts thereof. 
Compositions comprising at least one biologically active agent and at least one of the compounds above are also provided.  Further contemplated by the present invention are dosage unit forms that include these compositions. 
Also contemplated is a method for preparing these compositions which comprises mixing at least one active agent with at least one compound as described above, and optionally, a dosing vehicle. 
In an alternative embodiment, these non-toxic compounds are orally administered to animals as part of a delivery system by blending or mixing the compounds with an active agent prior to administration. 
The specific compounds of the present invention or salts thereof such as, for example, sodium salts, may be used to deliver various active agents through various biological, chemical, and physical barriers.  These compounds are particularly suited for delivering active agents which are subject to environmental degradation.  The compounds and compositions of the subject invention are particularly useful for delivering or administering biologically-active agents to any animals such as birds; mammals, such as primates and particularly humans; and insects. 
Other advantages of the present invention include the use of easy to prepare, inexpensive raw materials.  The compositions and the formulation methods of the present invention are cost effective, simple to perform, and amenable to industrial scale up for commercial production. 
Amino acids, poly amino acids, and peptides, in modified form, may be used to deliver active agents including, but not limited to, biologically active agents such as for example, pharmacological and therapeutic agents. 
An amino acid is any carboxylic acid having at least one free amine group and includes naturally occurring and synthetic amino acids. 
Poly amino acids are either peptides or two or more amino acids linked by a bond formed by other groups which can be linked, e.g. an ester, anhydride, or an anhydride linkage.  Special mention is made of non-naturally occurring poly amino acids and particularly non-naturally occurring hetero poly amino acids, i.e. polymers of mixed amino acids. 
Peptides are two or more amino acids joined by a peptide bond.  Peptides can vary in length from dipeptides with two amino acids to poly peptides with several hundred amino acids.  See Chambers Biological Dictionary, editor Peter M. B. Walker, Cambridge, England: Chambers Cambridge, 1989, page 215.  Special mention is made of di-peptides, tri-peptides, tetra-peptides, and penta-peptides. 
The terms modified amino acids, modified poly amino acids, and modified peptides are meant to include amino acids which have been modified or poly amino acids and peptides in which at least one amino acid has been modified by acylating at least one free amine group with an acylating agent which reacts with at least one of the free amine groups present. 
Modified Amino Acids
Several of the compounds of the present invention are broadly represented by one of formula XLVI or XLVII below:
Arxe2x80x94Yxe2x80x94(R1)nxe2x80x94OHxe2x80x83xe2x80x83XLVI
wherein Ar is a substituted or unsubstituted phenyl or naphthyl;
Y is 
or xe2x80x94SO2xe2x80x94, R1 has the formula 
wherein:
R2 is C1 to C24 alkyl, C1 to C24 alkenyl, phenyl, naphthyl, (C1 to C10 alkyl) phenyl, (C1 to C10 alkenyl) phenyl, (C1 to C10 alkyl) naphthyl, (C1 to C10 alkenyl) naphthyl, phenyl (C1 to C10 alkyl), phenyl (C1 to C10 alkenyl), naphthyl (C1 to C10 alkyl), and naphthyl (C1 to C10 alkenyl);
R2 is optionally substituted with C1 to C4 alkyl, C1 to C4 alkenyl, C1 to C4 alkoxy, xe2x80x94OH, xe2x80x94SH and xe2x80x94CO2R4 or any combination thereof;
R4 is hydrogen, C1 to C4 alkyl or C1 to C4 alkenyl;
R2 is optionally interrupted by oxygen, nitrogen, sulfur or any combination thereof; and
R3 is hydrogen, C1 to C4 alkyl or C1 to C4 alkenyl; or 
wherein: R5 is (i) C3-C10 cycloalkyl, optionally substituted with C1-C7 alkyl, C2-C7 alkenyl, C1-C7 alkoxy, hydroxy, phenyl, phenoxy or xe2x80x94CO2R8, wherein R8 is hydrogen, C1-C4 alkyl, or C2-C4 alkenyl; or
(ii) C1-C6 alkyl substituted with C3-C10 cycloalkyl;
R6 is hydrogen, C1-C4 alkyl, or C2-C4 alkenyl;
R7 is C1-C24 alkyl, C2-C24 alkenyl, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, phenyl, naphthyl, (C1-C10 alkyl) phenyl, (C2-C10 alkenyl) phenyl, (C1-C10 alkyl) naphthyl, (C2-C10 alkenyl) naphthyl, phenyl (C1-C10 alkyl), phenyl (C2-C10 alkenyl), naphthyl (C1-C10 alkyl) or naphthyl (C2-C10 alkenyl);
R7 being optionally substituted with C1-C4 alkyl, C2-C4 alkenyl, C1-C4 alkoxy, xe2x80x94OH, xe2x80x94SH, xe2x80x94CO2R9, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, heterocycle having 3-10 ring atoms wherein the hetero atom is one or more of N, O, S or any combination thereof, aryl, (C1-C10 alk)aryl, ar(C1-C10 alkyl), or any combination thereof;
R7 being optionally interrupted by oxygen, nitrogen, sulfur, or any combination thereof; and
R9 is hydrogen, C1-C4 alkyl, or C2-C4 alkenyl. 
Special mention is made of compounds I-XLV above. 
The modified amino acids of compounds I-XLV may be prepared by reacting single amino acids, mixtures of two or more amino acids, amino acid esters, or amino acid amides, with an amine modifying agent which reacts with free amino moieties present in the amino acids to form amides. 
Modified amino acids are typically prepared by modifying the amino acids or an ester thereof.  Many of these compounds are prepared by acylation with acylating agents having the formula 
wherein: R10 is the appropriate radical to yield the modification indicated in the final product as would be within the skill of the art based upon the detailed disclosure herein, and Y is a leaving group.  Typical leaving groups include, but are not limited to, halogens such as, for example, chlorine, bromine, and iodine.  Additionally, the corresponding anhydrides are suitable acylating agents. 
Many of the compounds of the present invention can be readily prepared and modified by methods within the skill of those in the art based upon the present disclosure.  For example, the modified amino acid compounds above may be prepared by reacting the single amino acid with the appropriate acylating agent or an amine modifying agent which reacts with free amino moiety present in the amino acids to form amides.  Protecting groups may be used to avoid unwanted side reactions as would be known to those skilled in the art. 
For example, the amino acid can be dissolved in aqueous alkaline solution of a metal hydroxide, e.g., sodium or potassium hydroxide, and heated at a temperature ranging between about 5xc2x0 C. and about 70xc2x0 C., preferably between about 10xc2x0 C. and about 40xc2x0 C., for a period ranging between about 1 hour and about 4 hours, preferably about 2.5 hours.  The amount of alkali employed per equivalent of NH2 groups in the amino acid generally ranges between about 1.25 and about 3 mmole, preferably between about 1.5 and about 2.25 mmole per equivalent of NH2.  The pH of the solution generally ranges between about 8 and about 13, preferably ranging between about 10 and about 12. 
Thereafter, the appropriate amino modifying agent is added to the amino acid solution while stirring.  The temperature of the mixture is maintained at a temperature generally ranging between about 5xc2x0 C. and about 70xc2x0 C., preferably between about 10xc2x0 C. and about 40xc2x0 C., for a period ranging between about 1 and about 4 hours.  The amount of amino modifying agent employed in relation to the quantity of amino acid is based on the moles of total free NH2 in the amino acid.  In general, the amino modifying agent is employed in an amount ranging between about 0.5 and about 2.5 mole equivalents, preferably between about 0.75 and about 1.25 equivalents, per molar equivalent of total NH2 group in the amino acid. 
The reaction is quenched by adjusting the pH of the mixture with a suitable acid, e.g., concentrated hydrochloric acid, until the pH reaches between about 2 and about 3.  The mixture separates on standing at room temperature to form a transparent upper layer and a white or off-white precipitate.  The upper layer is discarded, and the modified amino acid is collected from the lower layer by filtration or decantation.  The crude modified amino acid is then dissolved in water at a pH ranging between about 9 and about 13, preferably between about 11 and about 13.  Insoluble materials are removed by filtration and the filtrate is dried in vacuo.  The yield of modified amino acid generally ranges between about 30 and about 60%, and usually about 45%. 
If desired, amino acid esters, such as, for example methyl or ethyl esters of amino acid compounds, may be used to prepare the modified amino acids of the invention.  The amino acid ester, dissolved in a suitable organic solvent such as dimethylformamide or pyridine, is reacted with the appropriate amino modifying agent at a temperature ranging between about 5xc2x0 C. and about 70xc2x0 C., preferably about 25xc2x0 C., for a period ranging between about 7 and about 24 hours.  The amount of amino modifying agent used relative to the amino acid ester is the same as described above for amino acids. 
Thereafter, the reaction solvent is removed under negative pressure and the ester functionality is removed by hydrolyzing the modified amino acid ester with a suitable alkaline solution, e.g. 1N sodium hydroxide, at a temperature ranging between about 50xc2x0 C. and about 80xc2x0 C., preferably about 70xc2x0 C., for a period of time sufficient to hydrolyze off the ester group and form the modified amino acid having a free carboxyl group.  The hydrolysis mixture is then cooled to room temperature and acidified, e.g. aqueous 25% hydrochloric acid solution, to a pH ranging between about 2 and about 2.5.  The modified amino acid precipitates out of solution and is recovered by conventional means such as filtration or decantation. 
The modified amino acid may be purified by recrystallization or by fractionation on solid column supports.  Suitable recrystallization solvent systems include acetonitrile, methanol and tetrahydrofuran.  Fractionation may be performed on a suitable solid column supports such as alumina, using methanol/n-propanol mixtures as the mobile phase; reverse phase column supports using trifluoroacetic acid/acetonitrile mixtures as the mobile phase; and ion exchange chromatography using water as the mobile phase.  When anion exchange chromatography is performed, preferably a subsequent 0-500 mM sodium chloride gradient is employed. 
Active Agents
Active agents suitable for use in the present invention include biologically active agents, chemically active agents, including, but not limited to, fragrances, as well as other active agents such as, for example, cosmetics. 
Biologically active agents include, but are not limited to, pesticides, pharmacological agents, and therapeutic agents.  For example, biologically active agents suitable for use in the present invention include, but are not limited to, peptides, and particularly small peptides; hormones, and particularly hormones which by themselves do not or only pass slowly through the gastrointestinal mucosa and/or are susceptible to chemical cleavage by acids and enzymes in the gastro-intestinal tract; polysaccharides, and particularly mixtures of muco-polysaccharides; carbohydrates; lipids; or any combination thereof.  Further examples include, but are not limited to, human growth hormones; bovine growth hormones; growth releasing hormones; interferons; interleukin-1; insulin; heparin, and particularly low molecular weight heparin; calcitonin; erythropoietin; atrial naturetic factor; antigens; monoclonal antibodies; somatostatin; adrenocorticotropin, gonadotropin releasing hormone; oxytocin; vasopressin; cromolyn sodium (sodium or disodium chromoglycate); vancomycin; desferrioxamine (DFO); anti-microbials, including, but not limited to anti-fungal agents; or any combination thereof. 
Delivery Systems
The compositions of the present invention may include one or more active agents. 
In one embodiment, compounds I-XLV or poly amino acids or peptides that include at least one of these compounds may be used directly as a drug delivery carrier by simply mixing one or more compound, poly amino acid or peptide with the active ingredient prior to administration. 
In an alternative embodiment, the compounds, poly amino acids, or peptide may be used to form microspheres containing the active agent.  These compounds, poly amino acids, or peptides are particularly useful for the oral administration of certain biologically-active agents, e.g., small peptide hormones, which, by themselves, do not pass or only pass slowly through the gastro-intestinal mucosa and/or are susceptible to chemical cleavage by acids and enzymes in the gastrointestinal tract. 
If the modified amino acids, poly amino acids, or peptides are to be converted into microspheres, the mixture is optionally heated to a temperature ranging between about 20 and about 50xc2x0 C., preferably about 40xc2x0 C., until the modified amino acid(s) dissolve.  The final solution contains between from about 1 mg and to about 2000 mg of compound, poly amino acid, or peptide per mL of solution, preferably between about 1 and about 500 mg per mL. The concentration of active agent in the final solution varies and is dependent on the required dosage for treatment.  When necessary, the exact concentration can be determined by, for example, reverse phase HPLC analysis. 
When the compounds, poly amino acids, or peptides are used to prepare microspheres, another useful procedure is as follows: Compounds, poly amino acids, or peptides are dissolved in deionized water at a concentration ranging between about 75 and about 200 mg/ml, preferably about 100 mg/ml at a temperature between about 25xc2x0 C. and about 60xc2x0 C., preferably about 40xc2x0 C. Particulate matter remaining in the solution may be removed by conventional means such as filtration. 
Thereafter, the compound, poly amino acid, or peptide solution, maintained at a temperature of about 40xc2x0 C., is mixed 1:1 (V/V) with an aqueous acid solution (also at about 40xc2x0 C.) having an acid concentration ranging between about 0.05 N and about 2 N, preferably about 1.7 N. The resulting mixture is further incubated at 40xc2x0 C. for a period of time effective for microsphere formation, as observed by light microscopy.  In practicing this invention, the preferred order of addition is to add the compound, poly amino acid, or peptide solution to the aqueous acid solution. 
Suitable acids for microsphere formation include any acid which does not
(a) adversely effect the modified amino acids, poly amino acids, or peptides e.g., initiate or propagate chemical decomposition;
(b) interfere with microsphere formation;
(c) interfere with microsphere incorporation of the cargo; and
(d) adversely interact with the cargo. 
Preferred acids for use in this aspect include acetic acid, citric acid, hydrochloric acid, phosphoric acid, malic acid and maleic acid. 
A microsphere stabilizing additive may be incorporated into the aqueous acid solution or into the compound or cargo solution prior to the microsphere formation process.  With some drugs the presence of such additives promotes the stability and/or dispersibility of the microspheres in solution. 
The stabilizing additives may be employed at a concentration ranging between about 0.1 and 5% (w/v), preferably about 0.5% (w/v).  Suitable, but non-limiting, examples of microsphere stabilizing additives include gum acacia, gelatin, methyl cellulose, polyethylene glycol, and polylysine.  The preferred stabilizing additives are gum acacia, gelatin and methyl cellulose. 
Under the above conditions, the compound molecules, poly amino acids, or peptides form hollow or solid matrix type microspheres wherein the cargo is distributed in a carrier matrix or capsule type microspheres encapsulating liquid or solid cargo.  If the compound, poly amino acid, or peptide microspheres are formed in the presence of a soluble material, e.g., a pharmaceutical agent in the aforementioned aqueous acid solution, this material will be encapsulated within the microspheres.  In this way, one can encapsulate pharmacologically active materials such as peptides, proteins, and polysaccharides as well as charged organic molecules, e.g., antimicrobial agents, which normally have poor bioavailability by the oral route.  The amount of pharmaceutical agent which may be incorporated by the microsphere is dependent on a number of factors which include the concentration of agent in the solution, as well as the affinity of the cargo for the carrier.  The compound, poly amino acid, or peptide microspheres do not alter the physiological and biological properties of the active agent.  Furthermore, the encapsulation process does not alter the pharmacological properties of the active agent.  Any pharmacological agent can be incorporated within the microspheres.  The system is particularly advantageous for delivering chemical or biological agents which otherwise would be destroyed or rendered less effective by conditions encountered within the body of the animal to which it is administered, before the microsphere reaches its target zone (i.e., the area in which the contents of the microsphere are to be released) and for delivering pharmacological agents which are poorly absorbed in the gastrointestinal tract.  The target zones can vary depending upon the drug employed. 
The particle size of the microsphere plays an important role in determining release of the active agent in the targeted area of the gastro-intestinal tract.  The preferred microspheres have diameters between about xe2x89xa60.1 microns and about 10 microns, preferably between about 0.5 microns and about 5 microns.  The microspheres are sufficiently small to release effectively the active agent at the targeted area within the gastro-intestinal tract such as, for example, between the stomach and the jejunum.  Small microspheres can also be administered parenterally by being suspended in an appropriate carrier fluid (e.g., isotonic saline) and injected directly into the circulatory system, intramuscularly or subcutaneously.  The mode of administration selected will vary, of course, depending upon the requirement of the active agent being administered.  Large amino acid microspheres ( greater than 50 microns) tend to be less effective as oral delivery systems. 
The size of the microspheres formed by contacting compounds, poly amino acids, or peptides with water or an aqueous solution containing active agents can be controlled by manipulating a variety of physical or chemical parameters, such as the pH, osmolarity or ionic strength of the encapsulating solution, size of the ions in solution and by the choice of acid used in the encapsulating process. 
The administration mixtures are prepared by mixing an aqueous solution of the carrier with an aqueous solution of the active ingredient, just prior to administration.  Alternatively, the carrier and the biologically active ingredient can be admixed during the manufacturing process.  The solutions may optionally contain additives such as phosphate buffer salts, citric acid, acetic acid, gelatin and gum acacia. 
Stabilizing additives may be incorporated into the carrier solution.  With some drugs, the presence of such additives promotes the stability and dispersibility of the agent in solution. 
The stabilizing additives may be employed at a concentration ranging between about 0.1 and 5% (W/V), preferably about 0.5% (W/V).  Suitable, but non-limiting, examples of stabilizing additives include gum acacia, gelatin, methyl cellulose, polyethylene glycol, and polylysine.  The preferred stabilizing additives are gum acacia, gelatin and methyl cellulose. 
The amount of active agent is an amount effective to accomplish the purpose of the particular active agent.  The amount in the composition typically is a pharmacologically or biologically effective amount.  However, the amount can be less than a pharmacologically or biologically effective amount when the composition is used in a dosage unit form, such as a capsule, a tablet or a liquid, because the dosage unit form may contain a multiplicity of carrier/biologically active agent compositions or may contain a divided pharmacologically or biologically effective amount.  The total effective amounts can then be administered in cumulative units containing, in total, pharmacologically or biologically active amounts of biologically or pharmacologically active agent. 
The total amount of active agent, and particularly biologically active agent, to be used can be determined by those skilled in the art.  However, it has surprisingly been found that with some biologically active agents, the use of the presently disclosed carriers provides extremely efficient delivery.  Therefore, lower amounts of biologically active agent than those used in prior dosage unit forms or delivery systems can be administered to the subject, while still achieving the same blood levels and therapeutic effects. 
The amount of carrier in the present composition is a delivery effective amount and can be determined for any particular carrier or biologically active agent by methods known to those skilled in the art. 
Dosage unit forms can also include any of excipients; diluents; disintegrants; lubricants; plasticizers; colorants; and dosing vehicles, including, but not limited to water, 1,2-propane diol, ethanol, olive oil, or any combination thereof. 
Administration of the present compositions or dosage unit forms preferably is oral or by intraduodenal injection. 
The delivery compositions of the present invention may also include one or more enzyme inhibitors.  Such enzyme inhibitors include, but are not limited to, compounds such as actinonin or epiactinonin and derivatives thereof.  These compounds have the formulas below: 
Derivatives of these compounds are disclosed in U.S. Pat.  No.  5,206,384.  Actinonin derivatives have the formula: 
wherein R12 is sulfoxymethyl or carboxyl or a substituted carboxy group selected from carboxamide, hydroxyaminocarbonyl and alkoxycarbonyl groups; and R13 is hydroxyl, alkoxy, hydroxyamino or sulfoxyamino group.  Other enzyme inhibitors include, but are not limited to, aprotinin (Trasylol) and Bowman-Birk inhibitor. 
The compounds and compositions of the subject invention are useful for administering biologically active agents to any animals such as birds; mammals, such as primates and particularly humans; and insects.  The system is particularly advantageous for delivering chemically or biologically active agents which would otherwise be destroyed or rendered less effective by conditions encountered before the active agent its target zone (i.e. the area in which the active agent of the delivery composition are to be released) and within the body of the animal to which they are administered.  Particularly, the compounds and compositions of the present invention are useful in orally administering active agents, especially those which are not ordinarily orally deliverable. 
The following examples illustrate the invention without limitation.