WO1997031938A1 - Oligopeptides for drug delivery - Google Patents

Abstract

The present invention relates to oligopeptides and compositions prepared from them. Theses compositions comprising an oligopeptide, and an active agent are useful in the delivery of a cargo to a target, and particularly in the oral delivery of biologically or chemically active agents. Methods for the preparation and for the administration of such compositions are also disclosed.

Description

OLIGOPEPTIDES FOR DRUG DELIVERY

FIELD OF THE INVENTION

The present invention relates to compositions prepared from oligopeptides. These compositions are useful in the delivery of a cargo to a target, and particularly in the oral delivery of biologically or chemically active agents. Methods for the preparation and for the administration of such compositions are also disclosed.

BACKGROUND OF THE INVENTION Conventional means for delivering active agents to their intended targets, such as human organs, tumor sites, etc., 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. Oral delivery to the circulatory system would be the route of choice for administration of many active agents to animals if not for physical barriers such lipid bilayers, and various organ membranes that are relatively impermeable to certain biologically active agents, but which must be traversed before an agent delivered via the oral route can reach the circulatory system. Additionally, oral delivery is impeded by chemical barriers such as the varying pH of the gastro-intestinal (Gl) tract and the presence of powerful digestive enzymes.

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. Patent No. 4,239,754; Patel et al. (1 976), FEBS Letters, Vol. 62, pg. 60; and Hashimoto et al. ( 1 979), 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) the systems are typically suitable only for low molecular weight cargos, (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. Patent 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. Further studies have demonstrated that cyclic peptides with an even number of alternating L- and D-amino acids were able to form organic nanotubes. (See, Whitesides et al., Science 1 991 , 254, 131 2, 1 31 9; Ghadiri, M.R. et al., Nature 1993, 366. 324-327.) Additionally, stabilized spherical micelles and tubular vesicles have been prepared from amphiphiles and bolamphiphiles. (See, Fuhrhop, J.H. et al., J. Amer. Chem. Soc , 1991 , 1 1 3, 7437, 7439; Frankel, D.A. et al. J. Amer. Chem. Soc , 1 991 , 1 13, 7436,- 7437; Fuhrhop, J.H. et al., J. Amer. Chem. Soc , 1 993, 1 1 5. 1600-1 601 .) L-Asp-diketopiperazines appended with amino acid subunits were found to self assemble into microspheres by Bergeron et al., J. Amer. Chem. Soc

(1 994) 1 16:8479-8484. This self assembly process was sensitive to solution pH and substrate concentration.

However, there is still a need in the art for simple, inexpensive delivery systems which are easily prepared and which can delivery a broad range of active agents.

SUMMARY OF THE INVENTION

The present invention provides structurally defined oligopeptides.

Several oligopeptides have been synthesized. The oligopeptides are useful for the delivery of active agents. The oligopeptides have from 2 to about 1 1 amino acid residues. They can be linear (all σ-bonding) or branched (a- and side chain bonding) peptides.

One type of oligopeptide useful in practicing the invention is a pγroglutamic acid initiated oligopeptide having the formula: PyGlu(X)_n I where each X is an amino acid residue and n is an integer from 1 to about

10.

Another type of oligopeptide useful in practicing the invention is a proline initiated oligopeptide having the formula: Pro(X)_n II where each X is an amino acid residue and n is an integer from 1 to about

10.

The oligopeptides of the present invention may be combined with active agent(s). Also contemplated are methods for administering compositions that includes an active agent.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a scheme illustrating the synthesis of branched tri- peptides starting from pyroglutamic acid. Figure 2 is a scheme illustrating the synthesis of branched tetra- peptides starting from pyroglutamic acid.

Figure 3 is a scheme illustrating the synthesis of tripeptides and tetrapeptides starting from L-Proline. Figure 4 is a scheme illustrating the synthesis of tripeptides using a hydrobromide dipeptide and DPPA as a coupling agent.

Figure 5 is a ¹H NMR spectrum of DiBzOCOGIuTsOH. Figure 6 is an IR spectrum of DiBzOCOGIuTsOH. Figure 7 is a ¹H-NMR spectrum of TsOH-GluBz. Figure 8 is a 'H NMR spectrum of DiBzOCOAspTsOH. Figure 9 is an IR spectrum of DiBzOCOAspTsOH.

Figure 10 s a 'H NMR spectrum of BzOCOPheTsOH. Figure 1 1 s an IR spectrum of BzOCOPheTsOH. Figure 12 s a H-NMR spectrum of TsOH-D-PheBz. Figure 13 s a ¹H NMR spectrum of BzOCOTyrTsOH. Figure 14 s an IR spectrum of BzOCOTyrTsOH. Figure 15 s a 'H-NMR spectrum of TsOH-AlaBz. Figure 16 s a 'H-NMR spectrum of TsOH-L-LeuBz. Figure 17 s a ¹H-NMR spectrum of D-diBzOCOGIu-TsOH. Figure 18 s a 'H NMR spectrum of BzOCO-NHAsp-0 Bz. Figure 19 s a IR spectrum of BzOCO-NHAsp-0 Bz. Figure 20 s a 'H NMR spectrum of BzOCO-NHAsp. Figure 21 s a 'H NMR spectrum of BzOCO-NHGIu. Figure 22 s an IR spectrum of BzOCO-NHGIu. Figure 23 s a 'H NMR spectrum of BzOCO-NHPhe. Figure 24 s an IR spectrum of BzOCONH-Phe. Figure 25 s a H-NMR spectrum of t-Boc-L-Glu. Figure 26 s a H-NMR spectrum of BzOCO-L-Proline. Figure 27 s a ¹ H NMR spectrum of PyGluGlu. Figure 28 s a ¹H NMR spectrum of PyGluAsp. Figure 29 s a ¹H NMR spectrum of PyGlu-Phe. Figure 30 s an IR spectrum of PyGlu-Phe. Figure 31 s a ¹H-NMR spectrum of PyGlu-σ-Phe-σ-Phe. Figure 32 s an IR spectrum of PyGlu-σ-Phe-σ-Phe. Figure 33 s a 'H-NMR spectrum of PyGlu-Glu-( -Phe). Figure 34 s a ¹H-NMR COSSY spectrum of PyGlu-Glu-( -Phe). Figure 35 s an IR spectrum of PyGlu-Gly-( -Phe). Figu re 36 H-NMR spectrum of PyGlu-Asp-(yff-Phe). Figu re 37 an IR spectrum of PyGlu-Asp-(yS-Tyr) Figu re 38 a ¹ H NMR spectrum of PyGlu-Asp-(#-Tyr). Figu re 39 a H-NMR spectrum of PyGlu-Glu- -Gly. Figu re 40 a H-NMR spectrum of PyGlu-Glu-y-Ala. Figu re 41 a H-NMR spectrum of PyGlu-Glu- -Leu. Figu re 42 a H-NMR spectrum of PyGlu-Asp-ø-Gly. Figu re 43 is an IR spectrum of PyGlu-Asp- ?-Gly. Figu re 44 is a H-NMR spectrum of PyGlu-Asp-/?-Ala. Figu re 45 is an IR spectrum of PyGlu-Asp- ?-Ala. Figu re 46 is a H-NMR spectrum of PyGlu-Glu-σ-Phe- -Phe. Figu re 47 s a H-NMR spectrum of PyGlu-Asp-σ-Gly-ff-Gly. Figu re 48 s an IR spectrum of PyGlu-Asp-σ-Gly- .-Gly. Figu re 49 is a H-NMR spectrum of PyGlu-Asp-σ-Ala- ?-Ala. Figu re 50 s a H-NMR spectrum of PyGlu-σ-Phe-σ-Phe-σ-Tyr. Figu re 51 s a H-NMR spectrum of PyGlu-L-Phe-L-Phe-L-Glu. Figu re 52 is an IR spectrum of PyGlu-L-Phe-L-Phe-L-Glu. Figu re 53 s a H-NMR spectrum of PyGlu-L-Phe-L-Phe-D-Glu. Figu re 54 s a H-NMR spectrum of PyGlu-Phe-Phe-Asp. Figu re 55 is a H-NMR spectrum of PyGlu-Glu-( -Phe)-σ-Phe. Figu re 56 is a H-NMR spectrum of PyGlu-Asp-(/?-Phe)-σ-Phe. Figu re 57 is a H-NMR spectrum of PyGiu-Glu-( -Ala)-σ-Ala. Figu re 58 is a H-NMR spectrum of PyGlu-Glu-( -Gly)-σ-Gly. Figu re 59 s a H-NMR spectrum of PyGlu-Asp-( ?-Tyr)-σ-Tyr. Figu re 60 s a H-NMR spectrum of PyGlu-Glu-( ,D-Phe)-σ,D-Phe. Figu re 61 is an IR spectrum of PyGlu-Glu-( ,D-Phe)-σ,D-Phe. Figu re 62 is a H-NMR spectrum of Py-L-Glu-D-Glu( -Phe)-σ-Phe. Figu re 63 s a H-NMR spectrum of PyGlu-Glu-( -Phe)-σ-Gly. Figu re 64 is an IR spectrum of PyGlu-Glu-( -Phe)-σ-Gly. Figu re 65 s a ¹ H-NMR spectrum of PyGlu-Glu-( -Phe)-σ-Ala. Figu re 66 s a 'H-NMR spectrum of PyGlu-Asp-(#-Tyr)-σ-Phe. Figu re 67 an IR spectrum of PyGlu-Glu-( -Phe)-σ-Phe. Figu re 68 is a ¹H-NMR spectrum of PyGlu-Glu-( -Phe)-σ-Phe. Figure 69 is a ¹H-NMR spectrum of PyGlu-Asp-(#-Phe)-σ-Phe. Figure 70 is a ¹ H-NMR spectrum of PyGlu-Asp-( ?-Tyr-Phe)-σ-Tyr- Phe.

Figure 71 s a 'H-NMR COSSY spectrum of L-Pro-L-Glu- -Phe. Figure 72 s a 'H-NMR of L-Pro-L-Glu- -Phe. Figure 73 s a 'H-NMR of L-Pro-L-Asp-yff-Phe. Figure 74 s an IR spectrum of L-Pro-Glu- -Phe. Figure 75 s a 'H-NMR spectrum of L-Pro-Glu-y-Phe. Figure 76 s a 'H-NMR spectrum of L-Pro-Glu. Figure 77 s a 'H-NMR spectrum of BzOCO-Pro-Asp. Figure 78 s a 'H-NMR spectrum of BzOCO-Pro-Glu. Figure 79 s a 'H-NMR spectrum of BzOCO-ProNHS. Figure 80 s a 'H-NMR spectrum of L-Pro-Glu-σ-Phe-y-Phe. Figure 81 s an IR spectrum of L-Pro-Glu-σ-Phe- -Phe. Figure 82 s a 'H-NMR spectrum of L-Pro-Glu-σ-Phe- -Phe. Figure 83 s a 'H NMR COSSY spectrum of L-Pro-L-Asp-σ-Gly- -

Gly.

Figure 84 is a 'H NMR spectrum of L-Pro-L-Asp-σ-Gly- ?-Gly. Figure 85 is a 'H-NMR spectrum of L-Pro-L-Asp-σ-Phe- . -Phe. Figure 86 is a 'H-NMR spectrum of L-Pro-L-Giu-σ-Gly- -Gly. Figure 87 is a 'H NMR COSSY spectrum of L-Pro-L-Glu-σ-Gly-y-

Gly.

Figure 88 is a 'H-NMR spectrum of L-Pro-L-Glu-σ-Tyr- -Tyr.

Figure 89 is an IR spectrum of L-Pro-L-Glu-σ-Tyr-y-Tyr.

Figure 90 is a 'H-NMR spectrum of L-Pro-L-Glu-σ-Leu- -Leu.

Figure 91 is a 'H-NMR spectrum of BzOCONPhe-PheBz.

Figure 92 is a 'H-NMR spectrum of BrH₃N⁺-Phe-PheBz.

Figure 93 is a 'H-NMR spectrum of (L-Asp- ?-Glu).

Figure 94 is a 'H-NMR spectrum of a mixture of (L-Glu- -Asp) and L-Glu-σ-Phe- -Asp.

Figure 95 is a 'H NMR COSSY spectrum of L-Glu- -Asp and L-

Glu-σ-Phe- -Asp.

Figure 96 is a 'H-NMR spectrum of Br H₃N⁺-L-Glu-a-Phe-y-Phe. Figure 97 is a Η-NMR of spectrum CϊΗ₃N⁺-Glu-σ-Phe-Bz- -Phe-

Bz.

Figure 98 is a Η-NMR spectrum of CIΗ₃N⁺-L-Glu-σ-Phe- -Phe.

Figure 99 is a graphic illustration of the results of oral gavage testing in rats using heparin with PyGlu-Glu- -Phe carrier.

Figure 100 is a graphic illustration of Hydrophobicity (Partition Coefficient) of Oligopeptides.

Figure 101 is a graphic illustration of Binding Affinity of Oligopeptides to Heparin at pH = 2.27.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides structurally defined oligopeptides. Several oligopeptides have been synthesized. The oligopeptides are useful for the delivery of active agents. The oligopeptides have from 2 to about 1 1 amino acid residues. They can be linear (all σ-bonding) or branched (a- and side chain bonding) peptides.

One type of oligopeptide useful in practicing the invention is pyroglutamic acid initiated oligopeptides having the formula:

PyGlu(X). I where each X is an amino acid residue and n is an integer from 1 to about 10.

Another type of oligopeptide useful in practicing the invention is proline initiated oligopeptides having the formula:

Pro(X)_n II where each X is an amino acid residue and n is an integer from 1 to about 10.

Each X independently is an amino acid radical or a poly amino acid radicals.

In a preferred embodiment the oligopeptides can have from 4 to about 6 amino acid residues and n is from about 3 to about 5.

It has been found that, delivery of heparin, a hydrophilic drug, is enhanced when mixed with an oligopeptide having at least one hydrophobic and at least one hydrophilic amino acid. Thus, oral delivery can be effected. Preferably, the hydrophobic amino acid has at least one aromatic group.

An amino acid is any carboxylic acid having at least one free amine group and includes naturally occurring and synthetic amino acids. An amino acid radical is an amino acid in which either one hydrogen atom of a free amine group or the hydroxyl from the carboxyl group has been removed such as by, for example, a condensation reaction in the formation of the oligopeptide.

Amino acid radicals are derived from naturally occurring or synthetic amino acids. Amino acid radicals are preferably derived from σ- amino acids, and most preferably from naturally occurring σ-amino acids. Many amino acids and amino acid esters are readily available from a number of commercial sources such as Aldrich Chemical Co. (Milwaukee, WI, USA); Sigma Chemical Co. (St. Louis, MO, USA); and Fluka Chemical Corp (Ronkonkoma, N.Y. USA). Representative, but not limiting, amino acids from which amino acid radicals suitable for use in the present invention may be derived are generally of the formula

O

II H - N (R¹) - (R² - C) - OH III

wherein: R' is hydrogen, C C₄ alkyl, or C₂-C₄ alkenyl;

R² is C C₂₄ alkyl, C₂-C₂₄ alkenyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, phenyl, naphthyl, (C C₁₀ alkyl) phenyl, (C₂-C₁₀ alkenyl) phenyl, (C^C^ alkyl) naphthyl, (C₂-C₁₀ alkenyl) naphthyl, phenyl (C C₁₀ alkyl), phenyl (C₂-C₁₀ alkenyl), naphthyl (C C₁₀ alkyl), or naphthyl (C₂-C₁₀ alkenyl); R² being optionally substituted with C C₄ alkyl, C₂-C₄ alkenyl, C_rC₄ alkoxy, -OH, -SH, -CO₂R³, 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, (C C₁₀ alk)aryl, ar(C,-C₁₀ alkyl) or any combination thereof; R² being optionally interrupted by oxygen, nitrogen, sulfur, or any combination thereof; and R³ is hydrogen, C_rC₄ alkyl, or C₂-C₄ alkenyl. The naturally occurring amino acids useful in practicing the invention are alanine, arginine, asparagine, aspartic acid, ci trulline, cysteine, cystine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, proline, hydroxy proline, -carboxyglu tamate, phenylglycine, or O-phosphoserine. The non-naturally occurring amino acids useful in practicing the invention are ? -alanine, σ-amino butyric acid, y-amino butyric acid, y- (aminophenyl) butyric acid, σ-amino isobutyric acid, citrulline, e-amino caproic acid, 7-amino heptanoic acid, . -aspartic acid, aminobenzoic acid, aminophenyl acetic acid, aminophenyl butyric acid, -9lutamic acid, cysteine (ACM), e-lysine, e-lysine (A-Fmoc), methionine sulfone, norleucine, norvaline, ornithine, d-ornithine, p-nitro-phenylalanine, hydroxy proline, 1 ,2,3,4,- te trahydr oisoquinoline-3-carboxylic acid, and thioproline.

The preferred amino acids are pyroglutamic, glutamic, aspartic, σ-alanine,

Hβucine, lysine, σ-phenylalanine, ? -phenylalanine, -phenylalanine, σ-tyrosine, -tyrosine, tryptophan, proline, and ^-valine. Poly amino acids can be used to form the oligopeptides. Typically, 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. Poly amino acids can be homo- or hetero- poly amino acids, and can include natural amino acids, synthetic amino acids, or any combination thereof. Poly amino acids can be homo- or hetero- poly amino acids, and can include natural amino acids, synthetic amino acids, or any combination thereof. Poly amino acid radicals are poly amino acids in which at least one, and preferably one, hydrogen atom of a free amine group has been removed such as by, for example, a condensation reaction in the formation of the oligopeptide.

Peptides are two or more amino acids joined by a peptide bond. Peptides can vary in length from di-peptides with two amino acids to polypeptides with several hundred amino acids. See, Walker, Chambers Biological Dictionary. Cambridge, England: Chambers Cambridge, 1 989, page 215.

Active Agents Active agents suitable for use in the present invention include biologically active agents and 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 gastro-intestinal 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.

The methods and compositions of the present invention may combine one or more active agents.

In a typical liquid (solution) composition, the final solution can contain from about 10 mg to about 2000 mg of oligopeptide per ml of solution, preferably between about 20 to about 500 mg of oligopeptide per ml of solution, and most preferably from about 20 to about 200 mg per ml. Optionally, the mixture is heated to a temperature between about 20° C and about 60° C, preferably about 40°C, until the oligopeptide dissolves. Particulates remaining in the solution may be filtered out by conventional means such as gravity filtration over filter paper.

Additives The compositions may optionally contain additives such as stabilizing additives. The presence of such additives promotes the stability and dispersability of any active 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 buffer salts, gum acacia, gelatin, methyl cellulose, polyethylene glycol, polypropylene glycol, and polylysine. The preferred stabilizing agents are gum acacia, gelatin, and methyl cellulose.

The oligopeptides may be used directly as an active agent carrier by simply mixing one or more oligopeptides with the active agent(s) prior to administration.

The compositions of the present invention may be formulated into dosage units by the addition of one or more excipient(s), diluent(s), disintegrant(s), lubricant(s), plasticizer(s), colorant(s), or dosing vehicle(s). Preferred dosage unit forms are oral dosage unit forms. Most preferred dosage unit forms include, but not limited to, tablets, capsules, or liquids. The dosage unit forms can include biologically, pharmacologically, therapeutically, or chemically effective amounts of the active agent or can include less than such an amount if multiple dosage unit forms are to be used to administer a total dosage of the active agent. Dosage unit forms are prepared by methods conventional in the art.

The 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. Patent No. 5,206,384. Actinonin derivatives have the formula:

wherein R'² is sulfoxymethyl or carboxyl or a substituted carboxy group selected from carboxamide, hγdroxγaminocarbonγl and alkoxycarbonyl groups; and R'³ is hydroxyl, alkoxy, hydroxyamino or sulfoxyamino group. Other enzyme inhibitors include, but are not limited to, aprotinin (Trasylol) and Bowman-Birk inhibitor.

Administration The 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 chemical or biologically active agents which would otherwise be destroyed or rendered less effective by conditions encountered before the composition reaches 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 compositions of the present invention are useful in orally administering active agents, especially those which are not ordinarily orally deliverable.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples illustrate the invention without limitation. All reagents were purchased either from the Aldrich

Chemical Co. or the Sigma Chemical Co. and were used without further purification. Silica gel 40 mm, obtained from J.T. Baker, was used for flash column chromatography. 'H NMR spectra were recorded at 300 MHz and ¹³C NMR were recorded at 75 MHz. Chemical shifts are given in parts per million downfield from an internal tetramethylsilane standard.

All amino acids used were in the L-configuration unless otherwise indicated. All intermediates in the synthesis were measured by ¹H NMR in D_β-DMSO and the structures were confirmed. The final products were confirmed by Η NMR, ¹H-¹³C Hetcor NMR, ES/MS and RHPLC.

Chromatography: Reversed-Phase HPLC

Reversed-phase HPLC (RHPLC) was carried out on a Waters-600 HPLC system (Delta-Pak C₁₈ Column, 3.9 x 1 50 mm) by using the 50% (v/v) CH₃OH in deionized water plus 0.002% TFA (pH = 2.30) as the mobile phase at a flow rate of 0.5 mL/min and detection at λ = 230 nm. The following standards were used to calibrate the partition coefficients of the oligopeptides: pyridine, aniline, benzyl alcohol, benzoic acid, benzene. K' = (V _.-V₀)/V₀ , V₀ = 2.81 mL (Kl was used as a non-retained reference compound). The linear equation to fit the standards is: Log P_oct = 1 .43 + 0.85 Log K'

Preparation of σ-Amino Acid Benzyl Esters EXAMPLE 1 A:

Synthesis of L-Glutamic σ. -Di(benzyl Ester) p-Toluensulfonate. (DiBzGluTsOH)

L-Glutamic acid ( 150 mmol 22.07 g) and p-toluenesulfonic acid (monohydrate, 32 g 162.8 mmol) were added to a mixture of freshly distilled benzyl alcohol (220 ml) and toluene (100 ml) in a 500 ml round-bottom flask. The mixture was heated to reflux (1 10-120°C) and the water formed in the reaction was trapped in a Dean-Stark receiver. When no more water appeared in the distillate (about 5 h) the mixture is allowed to cool to room temperature and precipitated with either ( 1000 ml). The crystalline p- toluenesulfonate of glutamic σ, -di(benzyl ester) was collected on a filter, washed with 200 ml of ether and dried in air. After recrystallization from methanol-ether 67.37g (90%) of DiBzGluTsOH were obtained. M.P. 1 37°C. The ¹H NMR spectrum is shown in Figure 5. The IR spectrum is shown in Figure 6.

EXAMPLE 2A

Synthesis of Glvcyl Benzyl Ester of p-Toluenesulfona te (TsOH-Glv-Bz) Glycine (18.8 g, 250 mmol) and p-toluenesulfonic acid (monohydrate; 48.5 g, 255 mmol) were added to a mixture of freshly distilled benzyl alcohol (100 mL) and benzene or toluene (50 mL) in a 500 mL round- bottom flask. The mixture was heated to reflux. The water formed was collected in a Dean-Stark receiver for about 5 hours. When no more water was formed in the distillate, the mixture was allowed to cool to room temperature. The reaction mixture was then diluted with ether (500 mL) and refrigerated for 2 hours.

The crystalline product, the p-toluenesufonate glycine benzyl ester, was filtered, washed with ether (200 mL), and air-dried. After recrystallization from methanol/ether, 30.4 g (yield 90%) was obtained. M.P. 132-134°C.

The ¹H NMR spectrum is shown in Figure 7.

EXAMPLE 3A

Synthesis of L-Aspartic σ, ?-Di(benzyl Ester) p-Toluenesulfonate, (DiBzAsp.TsOH)

The product was obtained by following the procedure described in Example 1 A, above (63.3 g, 87%). M.P. 1 54°C.

The 'H NMR spectrum is shown in Figure 8. The IR spectrum is shown in Figure 9.

EXAMPLE 4A

Synthesis of L-Phenylalanine Benzyl Ester p-Toluenesulfonate, ⁽BzPhe.TsOH⁾ The product was obtained by following the procedure described in Example 1 A, above, with 150 mmol of phenylalanine, 75 ml of benzyl alcohol, 75 ml of toluene and 32 g p-toluenesulfonate acid monohydrate (51.4 g, 80%). M.P. 159-160°C.

The ¹H NMR spectrum is shown in Figure 10. The IR spectrum is shown in Figure 1 1.

Synthesis of D-Phenylalanine Benzyl Ester p-Toluenesulfonate. (Bz-D- Phe.TsOH)

The product was obtained by following the procedure described above. M.P. 157-158°C.

The ¹H NMR spectrum is shown in Figure 12.

EXAMPLE 5A

Synthesis of L-Tyrosine Benzyl Ester p-Toluenesulfonate, (BzTyr.TsOH) The product was obtained by following the procedure described in Example 1 A, above, with 150 mmol of tyrosine, 75 ml of benzyl alcohol,

75 ml toluene, and 32 g p-toluenesulfonate acid monohydrate (50 g, 75%).

M.P. 166-168°C.

The ¹H NMR spectrum is shown in Figure 13. The IR spectrum is shown in Figure 14.

EXAMPLE 6A

S^ynthesis of Alanine Benzyl Ester of p-Toluenesulfonate (TsOH-Ala-Bz)

The product was obtained by following the procedure as described in Example 1 A, above (yield 88%). M.P. 1 10-1 1 1 °C.

The 'H NMR spectrum is shown in Figure 15. EXAMPLE 7A Synthesis of Leucyl Benzyl Ester of p-Toluenesulfonate (TsOH-L-Leu-Bz)

The product was obtained starting with 19.67 g, 150 mmol, of leucine, by following the procedure in Example 1A, above (30.4 g, 90% yield). M.P. 197-198°C.

The IR spectrum is shown in Figure 16.

EXAMPLE 8A

Synthesis of D-Glutamic Di(Benzyl Ester) of p-Toluenesulfonate (D-DiBz-Glu- TsOH)

The product was obtained by following the procedure as described in Example 1A, above. M.P. 130-131 °C.

The 'H NMR spectrum is shown in Figure 17.

A. Synthesis N-Benzyloxycarbonyl and N-ferf-Butyloxycarbonyl Amino Acids

EXAMPLE 1 B

Synthesis of N-Benzyloxycarbonyl L-Aspartic Acid /.-Benzyl Ester

(BzOCO-N-Asp-_?Bz) L-Aspartic acid .-benzyl ester (150 mmol, 1 1.86 g) was dissolved in hot water (750 ml). The solution was allowed to cool to 60°C and then treated with NaHCO₃ (125 mmol, 10.5 g) and benzyl chlorocarbonate (60 mmol, 9 ml) and this solution was allowed to cool slowly to room temperature under vigorous stirring. Stirring was continued for about 3 h. The solution was extracted with ether (2 X 250 ml) and acidified to Congo Blue with 5 N HCl. After several hours in the refrigerator the material was collected on a filter, washed with water, and dried in vacuo. N- Benzyloxycarbonyl /? -benzyl L-aspartic acid was obtained after recrystallization from CCI₄; (13.0 g, 73.6%). M.P. 107-108°C. The 'H NMR spectrum is shown in Figure 18.

The IR spectrum is shown in Figure 19. EXAMPLE 2B

Synthesis of N-Benzyloxycarbonyl L-Asoartic Acid (BzOCO-N-Asp)

A solution of L-aspartic acid (13.3 g 100 mmol) in 2N NaOH (1 50 ml) was cooled in an ice-water bath and stirred with a magnetic stirrer. Benzyl chlorocarbonate (1 5.8 ml, 1 10 mmol) and 2N NaOH (250 ml) were added alternately in about ten portions each, (the reaction of the mixture was maintained distinctly alkaline). The temperature of the reaction mixture was maintained between 5 and 10°C for about 30 minutes, and controlled by the rate of addition of the reactants. The ice-water bath was replaced and vigorous stirring was continued at room temperature for 45 min. The alkaline solution was extracted four times with ether (75 ml each). The aqueous layer was acidified to Congo Blue by the addition 5N HCl. An oil separated. After the removal of water by decantation. The product solidified in the refrigerator after about 6 hours (18.7 g, 70%). M.P. 1 14-1 15°C. The 'H NMR spectrum is shown in Figure 20.

EXAMPLE 3B

Synthesis of N-Benzyloxycarbonyl L-Glutamic Acid (BzOCO-N-Glu)

The product was obtained by following the procedures in Examples 2A and 2B. The amount of product obtained was 19 g, 70%. M.P. 1 16-1 1 8°C.

The 'H NMR spectrum is shown in Figure 21 .

The IR spectrum is shown in Figure 22.

EXAMPLE 4B

Synthesis of N-Benzyloxycarbonyl L-Phenylalanine (BzOCO-N-Phe)

The product was obtained by following the procedures in Examples 2A and 2B (25 g, 83.3%). M.P. 86-88°C.

The Η NMR spectrum is shown in Figure 23. The IR spectrum is shown in Figure 24. EXAMPLE 5B

Synthesis of N-tert-Butyloxycarbonyl Glutamic Acid (t-Boc-Glu)

A solution of 100 mmol of glutamic acid in a mixture of dioxane

(200 mL), water ( 100 mL), and 2N NaOH ( 100 mL) was stirred and cooled in an ice-water bath. Di-tert-butylpyrocarbonate (24 g. 1 10 mmol) was added and stirring was continued at room temperature for ¹/_ hour. The solution was concentrated in vacuo to about 100 mL, cooled in an ice-water bath, covered with a layer of ethyl acetate (300 mL) and acidified with a dilute solution of KHSO₄ to pH 2-3. The aqueous phase was extracted with ethyl acetate (1 50 mL), and the extraction was repeated. The ethyl acetate extracts were combined, washed with water (2 X 200 mL), and dried over anhydrous sodium sulfate. The t-Boc-Glu was recrystallized from ethyl acetate-hexane.

The 'H NMR spectrum is shown in Figure 25.

EXAMPLE 6B

Synthesis of N-Benzyloxycarbony L-Proline (BzOCO-N-Pro)

A solution of L-proline ( 1 1 .5 g, 100 mmol) in 2N NaOH (50 mL) was cooled in an ice-water bath and stirred with a magnetic stirrer. Benzylchlorocarbonate ( 18.7 g, 1 10 mmol) and 2N NaOH (55 mL) were added in about 10 portions alternatively. The reaction mixture was maintained distinctly alkaline by addition of more of 2N NaOH, if necessary.

The temperature of reaction mixture was kept between 5°C and 10°C by the rate of the reactants addition during 1 ¹/_ hours. The ice-water bath was replaced and the stirring was continued for ¹ hour at room temperature. The alkaline solution was extracted with ether (4 X 50 mL). The aqueous layer was removed and then acidified to pH 2-3 by the addition of 5N HCl. An oil was separated, washed with water, and dried in vacuo. The 'H NMR spectrum is shown in Figure 26

B. Preparation of Sequential Peptides Initiated with PvGlu

A typical synthesis procedure for the PyGlu initiated peptide is described as follows: EXAMPLE 1 C Synthesis of Pyroolutamyl Glutamic acid dipeptide

(PyGluGlu):

To a stirred mixture of the PyGlu (0.65 g, 5.0 mmol) and DiBzGlu (2.5 g, 5 mmol) in dimethylformamide (DMF (10 mL) was added to diphenylphosphoryl azide (DPPA) (1 .41 mL, 6.5 mmol) in 5 mL of DMF at 0°C followed by the addition of triethylamine (TEA) (2.32 mL, 16.5 mmol) in 5 mL of DMF at 0°C during 5-10 min. period. The mixture was stirred at room temperature overnight. The mixture was then diluted with 300 mL of benzene/ethyl acetate (1 :2 in volume). The solution was washed with 5% of HCl aqueous solution (2 x 35 mL) aqueous solution, 5% NaHCO₃ solution (2 x 35 mL), water (35 mL), and finally a saturated NaCl solution (2 x 35 mL). After drying over Na₂SO₄ the solution was filtered, followed by removal of the solvent under reduced pressure. Yield 1 .9 g (60.3%). Removal of the ester protecting groups: The benzyl ester groups were removed by hydrogenation:

1 .8 g of the dipeptide in 200 mL of the methanol/tetrahydrofuran (THF) (1 : 1 ) was stirred at room temperature. To this solution was added 0.18 g. Pd/C (10 w/w%). Hydrogen gas was introduced into the system from a H₂ balloon. The mixture was stirred overnight. The catalyst was removed by filtration. After removal of the solvent, the residue was recrystallized from chloroform to give 1 .1 g (100%) of the unprotected dipeptide. The 'H NMR spectrum is shown in Figure 27.

EXAMPLE 2C. Synthesis of Pyroolutamyl Aspartic acid (PvGlu-Asp)

The product was obtained by following the procedure as described in Example 1 C above.

The 'H NMR spectrum is shown in Figure 28.

EXAMPLE 3C. Synthesis of Pyroolutamyl Phenylalanine (PvGlu-

Phe)

The product was obtained by following the procedure as described in Example 1 C(a) above.

The ¹H NMR spectrum is shown in Figure 29. The IR spectrum is shown in Figure 30.

EXAMPLE 4C. Synthesis of Pyroolutamyl Phenylananyl

Phenylalanine (PvGlu-Phe-Phe):

(PyGlu-Phe-Phe) (mp 203-204) was obtained by condensation of PyGlu-Phe with TsOH-PheBz at a molar ratio of 1 :1 in the presence of DPPA as a catalyst followed by the removal of the protecting ester group by hydrogenation.

The Η NMR spectrum is shown in Figure 31 . The IR spectrum is shown in Figure 32.

EXAMPLE 5C. Synthesis of Pyroglu tamv! Glutamyl y-Phenyla anine (PvGlu-Glu- -Phe):

Pyroglutamylglutamyl- -phenylalanine (PyGlu-Glu-κ-Phe) was obtained by condensation of PyGlu-Glu and TsOH-Phe-Bz at a molar ratio of 1 : 1 using DPPA as a catalyst, followed by the removal of the protecting group by hydrogenation. The 'H NMR spectra are shown in Figure 33 and Figure 34.

The IR spectrum is shown in Figure 35.

EXAMPLE 6C. Synthesis of Pyroglutamyl Asoartyl fl-Phenylalanine

(PvGlu-Asp- ?-Phe): Pyroglutamylaspartyl β-phenylalanine (PyGlu-Asp- .-Phe) was obtained following the procedure described above in Example 1 C(b). The 'H NMR spectrum is shown in Figure 36. The IR spectrum is shown in Figure 37.

EXAMPLE 7C. Synthesis of Pyroglutamyl Asoartyl . -Tyrosine

(PvGlu-Aso-ff-Tyr):

Pyroglutamylaspartyl ? -tyrosine (PyGlu-Asp- ?-Tyr) was obtained following the procedure described above in Example 1 C. M.P. 208-210°C. The ¹H NMR spectrum is shown in Figure 38.

Additional tripeptides were obtained according to the general route in the schemes disclosed in Figures 1 and 2. All tripeptides obtained by these routes were isolated after hydrogenation by freeze-dry technique.

EXAMPLE 8C. Synthesis of Pyroolutamyl Glutamyl Glvcine (PvGlu-Glu- -

GMI

The trimer PyGlu-Gly- -Gly was synthesized following the schemes disclosed in Figures 1 and 2.

The 'H NMR spectrum is shown in Figure 39.

EXAMPLE 9C. Synthesis of Pyroolutamyl Glutamyl Alanine (PvGlu-Glu-.--

A a): The trimer PyGlu-Glu- -Ala was synthesized following the schemes disclosed in Figures 1 and 2.

The ¹H NMR spectrum is shown in Figure 40.

EXAMPLE 10C. Synthesis of Pyroolutamyl Glutamyl Leucine (PvG u-Glu-^- Leu):

The trimer PyGlu-Glu- -Leu was synthesized following the schemes disclosed in Figures 1 and 2.

The 'H NMR spectrum is shown in Figure 41 .

EXAMPLE 1 1 C. S^ynthesis of Pyroolutamyl Asoartyl Glvcine (PvGlu-Asp-fr- GJyi:

The trimer, PyGlu-Asp-/?-Gly, was synthesized following the schemes disclosed in Figures 1 and 2.

The ¹H NMR spectrum is shown in Figure 42. The IR spectrum is shown in Figure 43. EXAMPLE 1 2C. Synthesis of Pyroolutamyl Asoartyl Leucine (PVGIU-ASP- ?- Ala):

The trimer, PyGlu-Asp- . -Ala were synthesized following the schemes disclosed in Figures 1 and 2. The 'H NMR spectrum is shown in Figure 44.

The IR spectrum is shown in Figure 45.

EXAMPLE 1 3C. Synthesis of Pyroglutamyl Glutamyl Phenylalanyl Phenylalanine (PvGlu-Glu-σ-Phe-y-Phe): 1 g (3.9 mmol) of the dipeptide PyGluGlu was dissolved in

10 mL of DMF followed by the addition of 3.32 g (7.8 mmol) of BzPhe. To this were added 2.19 mL of DPPA in 5 mL DMF and 3.59 mL of TEA in 5 mL of DMF at 0°C. The mixture was stirred at room temperature for 1 day. After completion, the reaction product was purified as described above. The yield was 1 .5 g (56.4%) .

The ¹H NMR spectrum is shown in Figure 46.

EXAMPLE 14C. General Synthesis of Tetrapeptides:

Tetrapeptides may be classified as linear (all σ-bonding) and branched (σ- and side chain bonding) tetrapeptides. The structures are illustrated in Table 2.

Linear tetrapeptides (σ) were obtained by sequentially repeating the condensation and deprotection procedures. The following linear σ-tetrapeptides were prepared: pyroglutamylphenylalanyltyrosyl tyrosine (Py- Glu-σ-Phe-σ-Phe-σ-Tyr); pyroglutamylphenylalanylphenylalanyl glutamic acid

(Py-σ,L-Glu-σ,L-Phe-σ,Phe-σ,L-Glu); Py-σ,L-Glu-σ,L-Phe-σ,L-Phe-σ,D-Glu, and

PyGlu-Phe-Phe-Asp.

Tetrapeptides, such as a,γ and a,β, with identical amino acids were obtained by the condensation of PyGlu-Glu or PyGlu-Asp, respectively, with TsOH-Phe-Bz, TsOH-Ala-Bz, and TsOH-Glu-Bz at a molar ratio of 1 :2.3.

DPPA was used as a catalyst. The condensation was followed by the removal of the protecting group by hydrogenation. The following compounds were synthesized: PyGlu-σ-Glu-σ-Phe- -Phe; PyGlu-Asp-σ-Phe- ?-Phe; PyGlu- Glu-σ-Ala- -Ala; PyGlu-σ-Glu-σ-Gly- -Gly; PyGlu-Glu-σ,D-Phe- ,D-Phe, PyGlu- σ,D-Glu-σ-Phe-y-Phe, and PyGlu-Asp-σ-Tyr-/?-Tyr. Additional purification was used for PyGlu-Glu-σ-Phe-y-Phe and for PyGlu-σ-Asp-σ-Phe-yff-Phe. The purification was carried out by suspending oligopeptides in acetone, stirring for 30 min., and filtering.

Tetrapeptides, such as a,y and a,β, with different amino acids in positions a,γ or a,β, were obtained from tripeptide and a benzyl ester tosylate of the corresponding amino acid. Tetrapeptide PyGlu-Glu-y-Phe-σ-Gly was synthesized by the condensation of PyGlu-Glu- -Phe-Bz with TsOH-GlyBz, followed by hydrogenation. The similar procedure was used to obtain PyGlu- σ-Glu- -Phe-σ-Ala and PyGlu-Asp- ? -Tyr-σ-Phe.

Tetrapeptides, such as γ,γ and β,β, were obtained by condensation of PyGlu-Glu and PyGlu-Asp with Phenylalanyl phenylalanine benzyl ester hydrobromide at a molar ratio of 1 : 1 , using DPPA as a catalyst. The following oligopeptides were synthesized: PyGlu-Glu-y-(Phe-Phe), and PyGlu-Asp-/_^*-(Phe-Phe) .

The tetrapeptides, PyGlu-Asp-σ-Gly-ff-Gly and PyGlu-Asp-σ-Ala-/?- Ala were prepared as described above.

The oligopeptide structures were determining using 'H-NMR and IR. The 'H NMR and IR spectra are shown in Figures 47 to 69

The Hexapeptide PyGlu-Asp-/?-(Tyr-Phe)-σ-(Tyr-Phe) was obtained by the condensation of PyGlu-Asp-σ-Tyr-£-Tyr with TsOH-PheBz, followed by the removal of the protecting groups. The ¹H NMR is shown in Figures 70.

The oligopeptides prepared using PyGlu initiation according to the procedures described above are tabulated in Tables 1 and 2, below. The oligopeptides in table 1 have had molecular weights of the products determined by GPC using a low molecular weight column (M_w. range, 50- 1500); Ultrastyragel Column 100A° (Waters). TABLE 1

Peptides Initiated With PyGlu

Sequence Yield (%) M(obs.) M(calc)

PyGlu-Glu 60.3 464 438

PyGlu-Asp 65.0 399 424

PyGlu-Glu-σ-Phe- -Phe 56.4 803 432

PyGlu-Glu-Tyr- -Tyr 53.2 1006 764

PyGlu-Asp-Phe-0-Phe 60.3 718 718

PyGlu-Asp-Tyr- . -Tyr 55.1 873 750

PyGlu-Asp-Phe-0-Tyr 49.0 529 734

PyGlu-Glu-Phe- -Tyr 51.6 933 748

TABLE 2 OLIGOPEPTIDES INITIATED WITH PYROGLUTAMIC ACID

EXAMPLE D

Synthesis of Oligopeptides Initiated by L-Proline:

EXAMPLE 10 Synthesis of N-benzyloxycarbonyl-L-proline-N'- hydroxysuccinimide ester:

N-hydroxysuccinimide (4.2 g, 0.036 mol) was added to a solution of N-benzyloxycarbonyl-L-proline (9.1 5 g, 0.0366 mol, prepared in Example 5B) in THF. The mixture was cooled in an ice-water bath, and dicyclohexylcarbodiimide (7.6 g, 0.0368 mol) was added, with stirring for 24 hours. The separated N,N'-dicycIohexylurea was removed by filtration and the solvent was evaporated in vacuo. The crude product was recrystallized from isopropanol.

The 'H NMR spectrum is shown in Figure 78.

EXAMPLE 2D. Synthesis of BzOCO-Prolyl Glutamic acid with z-proline-N-hvdroxysuccinimide ester:

L-Glutamic acid (0.0224 mol) was dissolved in aqueous, 5.7% NaHCO₃. The solution was treated with a solution of benzyloxy- carbonylproiine (0.024 mol) in absolute methanol. The solution was stirred for 1 hour at room temperature and acidified to pH 2 with 5N HCl. The separated solid was washed with water and dried in vacuo. The 'H NMR spectrum is shown in Figure 77.

EXAMPLE 3D. Synthesis of BzOCO-Prolyl Aspartic acid with z-proline-N-hvdroxysuccinimide ester:

Aspartic acid (0.0224 mol) was dissolved in aqueous, 5.7% NaHCO₃. The solution was treated with a solution of benzyloxy- carbonylproline (0.024 mol) in absolute methanol. The solution was stirred for 1 hour at room temperature and acidified to pH 2 with 5N HCl. The separated solid was washed with water and dried in vacuo. The 'H NMR spectrum is shown in Figure 77. EXAMPLE 4D. Synthesis of L-Prolyl glutamic acid (L-Pro-Glu)

Dimer:

L-Prolinyl glutamic acid (L-Pro-Glu) was obtained by condensation of z-Pro with diBz-Glu-TsOH using DPPA as a condensation agent, followed with reduction with H₂, Pd/C.

The 'H NMR spectrum is shown in Figure 76.

EXAMPLE 5D. Synthesis of L-Prolyl Glutamyl-.'-Phenylalanine acid

(L-Pro-Glu- -Phe): Pro-Glu-y-Phe was synthesized using the ester activating procedure, DPPA and TEA, followed by removal of the protecting groups as depicted in the reaction scheme in Figure 4.

The 'H NMR spectrum is shown in Figure 75. The IR spectrum is shown in Figure 74.

EXAMPLE 6D. Synthesis of L-Prolyl Aspartyl-_?-Phenylalanine acid

(L- Pro- ASP- ?- Phe):

Pro-Asp- .-Phe was synthesized using the ester activating procedure, DPPA and TEA, followed by removal of the protecting groups as depicted in the reaction scheme in Figure 4.

The ¹H NMR spectrum is shown in Figure 73.

EXAMPLE 7D. Syntheses of L-Prolyl L-Glutamyl y-Phenylanaline acid (L-Pro-L-Glu-y-Phe) Trimer: L-Pro-L-Glu- -Phe was obtained by condensation of z-Pro with -phenylalanine benzyl ester of glutamic acid bromohydride (HBr ^■ NH₂- Glu- -Phe-Bz) using DPPA as a condensation agent followed by the removal of the protecting groups by hydrogenation (Pd/C) following the procedure in Figure 3. The 'H NMR spectra are shown in Figures 71 and 72.

Other oligopeptides initiated using proline were prepared following the procedures described herein. The oligopeptide structures were determining using 'H-NMR and IR.

The ¹H NMR and IR spectra are shown in Figures 74 to 90.

The oligopeptides prepared using Proline initiation according to the procedures described above. The results are tabulated in Table 3, below.

TABLE 3 Oligopeptides Initiated with Proline

Dipeptides Tripeptides Linear Tetrapeptides

Pro-Glu Pro-Glu-y-Phe Pro-Glu-σ-Phe- -Phe

Pro-Asp Pro-Asp-yPhe Pro-Asp-σ-Phe-/?-Phe

Pro-Glu-σ-Phe Pro-Glu-σ-Gly-y-Gly

L-Pro-L-Glu-σ-Leu- -Leu

Pro-Glu-σ-Tyr- Tyr

Pro-Asp-σ-Gly-^-Gly

EXAMPLE E Other Oligopeptides

EXAMPLE 1 E(a). Synthesis of Phenylalanyl Phenylalanine Benzyl Ester Hydrobromide (Br-H,N⁺-Phe-Phe-Bz):

BzOCONPhe-PheBz was prepared by condensation of N-benzyloxycarbonyl L-phenylalanine with TsOH-PheBz in DMF and TEA with DPPA as a catalyst (yield 84%, M.P. 1 54.5-155°C) following the procedure illustrated in the scheme in Figure 2.

The 'H NMR spectrum is shown in Figure 91 .

EXAMPLE 1 E(b). Selective Acidolvsis:

Br-H₃N⁺-Phe-Phe-Bz was obtained by selective acidolysis of the N-benzyloxycarbonyl L-phenylalanyl phenylalanine benzyl ester (BzOCONPhe- PheBz).

A solution of HBr (30 wt.%) in acetic acid (30mL) was placed in a 250 mL round-bottom flask. N-benzyloxycarbonyl L-phenylalanyl phenylalanine benzyl ester (BzOCONPhe-PheBz) ( 10 mmol) was added with stirring. The flask was closed with a cotton-filled drying tube. The reaction mixture was stirred to allow for a complete dissolution of the protected dipeptide, with a vigorous evolution of CO₂. When the gas evolution ceased (after about 25 min.), dry ether (200 mL) was added with stirring. The reaction mixture was kept in a refrigerator for several hours. The precipitate formed was collected on a filter, washed with ether (50 mL), and dried over NaOH pellets in vacuo. The product phenylalanyl phenylalanine benzyl ester hydrobromide was recrystallized from methanol/ether. The Η NMR spectrum is shown in Figure 92.

EXAMPLE 2E. Synthesis of L-Asoartyl .-Glutamic Acid (L-ASP- ?-GIU) :

L-Aspartyl .-Glutamic Acid (L-Asp-/?-Glu) was obtained by condensation of N-(carbobenzyloxy)-L- aspartic acid with DiBzOCO-Glu-TsOH with DPPA as a catalyst followed by the removal of the protecting groups by hydrogenation.

The 'H NMR spectrum is shown in Figure 93.

EXAMPLE 3E. Synthesis of a mixture of . -Glutamyl-v-Aspartic acid (L-

Glu-y-L-Asp) and L-G utamy -σ-Phenylalanine-y-L-Aspar tic acid (L-Glu-σ-Phe-,'- L-ASP) : .-Glutamyl- -aspartic acid (L-Glu- -L-Asp) was obtained in a mixture with L-glutamyl-σ-phenylalanine- -L-aspartic acid (L-Glu-σ-Phe- -L- Asp) by condensation of N-(carbobenzyloxy)-L-glutamic acid with DiBzOCO- Asp-TsOH with DiBzOCO-Glu-TsOH with DPPA as a catalyst. After purification the product was reacted by condensation with PheBzOCO-TsOH followed by the removal of the protecting groups by hydrogenation. The mixture of (L-Glu- -L-Asp) and (L-Glu-σ-Phe-y-L-Asp) was obtained at a molar ratio of 80:20, determined by NMR.

The 'H NMR spectrum is shown in Figure 94.

The 'H NMR COSSY spectrum is shown in Figure 95. EXAMPLE 4E. Synthesis of a mixture of L-Glutamyl-σ-Phenyla anine-.'-

Phenylalanine (L-Glu-σ-Phe-y-Phe):

L-Glutamyl-σ-Phenylalanine- -Phenylalanine (L-Glu-σ-Phe- -Phe) was obtained by two procedures. (a) N-(carbobenzyloxy)-L-glutamic acid was reacted with

PheBzOCO-TsOH using DPPA as a catalyst. The products BzOCO-Glu-σ-Phe- -Phe-Bz and BzOCO-Glu- -Phe-σ-Phe)-Bz were obtained. The protecting groups were removed by hydrogenation (Pd/C).

The 'H NMR spectrum is shown in Figure 96. (b) The protecting groups were removed selectively using

HCI/dioxane (4 M; room temperature, 1 hour) from BzOCO-Glu-σ-Phe-Bz- - Phe-Bz. The product was isolated by precipitation with ether. The structure was determined by NMR. The benzyl ester groups were removed by hydrogenation. The ¹H NMR spectrum of the dibenzyl ester is shown in Figure

97.

The ¹H NMR spectrum of the free acid is shown in Figure 98. The other oligopeptides prepared according to the procedures described above are tabulated in Tables 4, below. TABLE 4

Other Oligopeptides

Dipeptides Tripeptides

L-Asp-/?-L-Glu L-Glu- -Asp-σ-Phe

L-Glu-y-L-Asp L-Glu-σ-Phe-y-Asp

Phe-Phe L-Glu-σ-Phe- -Phe

L-Glu- -Asp-σ-Phe Example F Delivery of Heparin to rats Example 1 F Preparation of Dosing solutions.

The oligopeptide, PyGlu-Glu-y-Phe, was dissolved in distilled water and adjusted to pH 7.2-8.0. A solution containing heparin was prepared. Heparin was dissolved in a solution of 1 .7 N citric acid and 0.5% gum arabic. The solutions were warmed to about 40°C and mixed. Two samples were prepared the first sample had a carrier concentration of 100 mg/mL. The heparin concentration was 33.3 mg/mL.

Following a similar procedure a second sample having a carrier concentration of 200 mg/mL and a heparin concentration of 33.3 mg/mL was prepared.

Example 2F HEPARIN In Vivo EXPERIMENTS IN RATS

For each sample a group of fasted rats were anesthetized. The rats were administered, by oral gavage, one of the heparin/carrier dosages prepared in Example 1 F. In the first group each rat was administered a dosage of 100 mg/kg of heparin and 300 mg/kg of carrier. In the second group each rat was administered a dosage of 100 mg/kg of heparin and 600 mg/kg of carrier. Blood samples were collected serially from the tail artery.

Heparin activity was determined by utilizing the activated partial thromboplastin time (APTT) according to the method of Henry, J.B., Clinical Diagnosis and Management bv Laboratory Methods: Philadelphia, PA; WB Saunders ( 1 979). The results of the test are illustrated in Figure 99.

It can be clearly seen from Example F that the oligopeptides of the invention are capable of delivering active agents to a target preferably through the Gl tract.

Example G

HEPARIN BINDING AFFINITY EXPERIMENTS Syntheses

The following oligopeptide series were synthesized according to the procedures described herein and the synthesis routes were optimized. (The numbers in the brackets represent the code number of the oligopeptides.) These groups of peptides were tested to determine their binding affinities with heparin.

Group 1 Group 2 Group 3

1 ) PyGluGlu-σ-Phe (101 ) PyGluGlu- -Phe ( 102) PyGluGlu-σ-Phe-y-Phe ( 103)

2) PyGluAsp-σ-Phe (201 ) PyGluAsp-0-Phe (202) PyGluAsp-σ-Phe- ?-Phe (203)

3) PyGluGlu-σ-Tyr (301 ) PyGluGlu- -Tyr (302) PyGluGlu-σ-Phe- -Tyr (303) 4) PyGluGlu-σ-Ala (401 ) PyGluGlu- -Ala (402) PyGluGlu-σ-Phe-y-Ala (403)

5) PyGluGlu-σ-Val (501 ) PyGluGlu- -Val (502) PyGluGlu-σ-Phe- -Val (503)

6) PyGluGlu-σ-Leu (601 ) PyGluGlu- -Leu (602) PyGluGlu-σ-Phe-y-Leu (603)

7) ProGlu-σ-Phe (701 ) ProGlu- -Phe (702) ProGlu-σ-Phe- )Phe(703) All amino acids used were in the L-configuration.

Since glutamic acid is hydrophilic, it is essential to have hydrophobic amino acids such as phenylalanine, tyrosine, alanine, valine, leucine as the terminal amino acid for the oligopeptides to balance the hydrophobicity in order to enhance delivery of the active agents. These oligopeptides were purified on a preparative reversed-phase HPLC column (Delta-Pak C₁₈, Waters). The relative purity of the oligopeptides was measured on an analytical reversed-phase HPLC column (Delta-Pak C₁₈, Waters).

Example 1 G Circular Dichroism

Circular dichroism studies were carried out on a Jasco-600 for PyGluGlu-σ-Phe- -Phe with the heparin fraction #1 which is the first fraction (high molecular weight) obtained by preparative Bio-Rad P-30 GPC chromatography. Samples were prepared in a phosphate buffer with variable concentrations of oligopeptide from 0.01 mg/mL to 0.05 mg/mL and 0.5 mg/mL of heparin at pH 7.0 and ultrasonicated for 20 minutes before scanning. Example 2G Differential Scanning Calorimetrv

Differential scanning calorimetry was carried out on a Microcal MC-2 scanning calorimeter. Samples were prepared in the same manner as above. Circular Dichroism experiments did not provide any CD pattern changes at neutral pH even though the differential scanning calorimetry data indicated that the addition of PyGluGlu-σ-Phe- -Phe, PyGluAsp-σ-Phe-£-Phe or PyGluGlu-σ-Phe- -Tyr eliminates the thermal transition peak for heparin. Possible explanations are that either at neutral pH, the oligopeptides do not bind to heparin or heparin polymer conformation is not as sensitive to such weak binding.

Example 3G Equilibrium Dialysis

Equilibrium dialysis was carried out by using Spectra/Por CE (cellulose ester) membrane -MWCO = 1000) to estimate the binding parameters. The heparin fraction #1 solution with constant concentration was contained inside the membrane, which is immersed into the oligopeptide solution at specific concentrations. The oligopeptide concentration was monitored by UV absorption (λ = 257 nm for PyGluGlu- -Phe and PyGluGlu-σ- Phe- -Phe; λ = 274 nm for PyGluGlu-σ-Phe- -Tyr) until equilibrium was reached. The buffer composition was 100 mM NaCl in 25 mM phosphate plus 0.005% NaN₃ at pH = 6.85 or 100 mM NaCl in 25 mM acetate buffer plus 0.005% NaN₃ at pH = 2.27. By assuming n equivalent and independent binding sites between heparin and oligopeptides, the dissociation constants and binding numbers were determined by the application of the following equation: r / [P] = 1 / n + Kd / (n x [P]), r = [P]_bound/[D]_tolal where [P] is the concentration of unbound or free oligopeptides, Inbound ^{is tne} difference of total oligopeptide concentration and free oligopeptide concentration and [D]_t01al is the total heparin concentration. The binding number is n and Kd is the dissociation constant.

The partition coefficient P_oct between octanol and water is one of the most effective physical parameters of bioactive compounds for predicting their biological activities in the study of quantitative structure-activity relations, K. Miyake et al., Chem. Pharm. Bull. , 1987, 35(1 ), 377-388. It was reported that P_oct has a linear relationship with reversed phase HPLC index K'. The hydrophobicity of these oligopeptides, evaluated by analytical RHPL, is shown in Figure 100. It was found that the tetramers have higher hydrophobicity than either the σ-series trimers or the

trimers. The hydrophobicity tendency for the tetramers is in agreement with the hydrophobicity of the amino acids themselves except that PyGluGlu-σ-Phe- - Tyr has much lower hydrophobicity than PyGluGlu-σ-Phe- -Phe even though Phe and Tyr have almost similar hydrophobicity characteristics.

Example 4G Heparin Affinity Chromatography

Heparin affinity chromatography was carried out on a SigmaChrom™ AF-Heparin affinity HPLC column. The mobile phase was 100 mM NaCl in 25 mM phosphate buffer plus 0.005% NaN₃ at pH = 6.85 or 100 mM NaCl in 25 mM acetate buffer plus 0.005% NaN₃ at pH = 2.27 with and without organic additives such as isopropanol or ethylene glycol. The flow rate was 1 .0 mL/min. K' = (V-V₀) / V₀, V₀ = 2.25 (NaNO₂ was used as the non-retained reference).

It was found that the binding affinity of these oligopeptides to heparin increased with decreasing pH. Oiigomers PyGluGlu-σ-Phe- -Phe, PyGluAsp-σ-Phe-^-Phe, PyGluGlu-σ-Phe- -Tyr, and ProGlu-σ-Phe- -Phe, which are tetramers and contain Phe or Tyr, bind to heparin better at low pH ~ 2.3 [Figure 101 ] than at higher pH ~ 6.8 (actually there was no apparent retention at neutral pH). The other oligopeptides do not bind to heparin at either the lower ( ~ 2) or the higher pH ( ~ 7). Upon addition of different concentrations of isopropanol (which was used to prevent hydrophobic interaction) to the mobile phase, the retention of oligopeptides to heparin is decreased. The addition of ethylene glycol (which was used to reduce the hydrogen-bonding) to the mobile phase also decreased the retention of oligopeptides on heparin. These data indicate that the binding affinities are due to hydrophobicity and hydrogen-binding. Correlation between heparin affinity and hydrophobicity data indicates that the hydrophobic aromatic ring plays a role in the binding since PyGluGlu-σ-Phe- -Leu, which has high hydrophobic character with leucine as the terminal groups, has very low retention on the heparin column.

The results are illustrated in Figure 101 .

Correspondingly, the binding affinities of oligopeptides to heparin were also evaluated by equilibrium membrane dialysis to estimate the binding parameters. Both natural and commercially available heparin have a broad molecular weight distribution. Therefore, fractionation of commercial heparin was carried out on a Bio-Rad Gel SEC (size exclusion chromatography) column to obtain a high molecular weight fraction which was anticipated to have some degree of secondary structure.

Table 5 Affinity Parameters of Oligopeptides to Heparin Fraction #1

PyGluGlu- -Phe PyGluGlu-σ-Phe- -Phe PyGluGlu-σ-Phe-y-Tyr

n 5.7 5.5 87.0* 3.3

Kd 1.42 x 10^"4 1.46x10 8.0x10^'3* 1.89 x 10^"4

The results, shown in Table 5 indicate that at neutral pH, the oligopeptides PyGluGlu- -Phe, PyGluGlu-σ-Phe- -Phe,and PyGluGlu-σ-Phe- - Tyr have a weaker binding affinity to the high molecular weight heparin fraction. The estimated binding number was 3-6 and the dissociation constant is 10⁴. At pH ~ 2, PGIuGlu-σ-Phe- -Phe has a slightly weaker binding to heparin. However, a higher binding number (87) was found at this lower pH. These data indicate that the interactions are weak, which is not surprising since these oligopeptides do not contain a basic amino acid. However, it has been reported that basic amino acid containing oligopeptides such as KWK-COOH and RWR-COOH have similar order interaction with heparin (K_a = 3.2 or 4.5 x 10^"3 respectively), D. P. Mascotti and T. M. Lohman, Biochemistry, 1995, 34, 2908(291 5. All patents, patent applications, literature publications and test methods cited herein are hereby incorporated by reference.

Many variations of the present invention will suggest themselves to those skilled in the art in light of the above detailed disclosure. All such modifications are within the full intended scope of the appended claims.

Claims

CLAIMS:

1 1 . A composition comprising:

.2 (a) an oligopeptide having the formula:

3 Y(X)_n

^'4 wherein Y is PyGlu or Pro,

5 each X is an independent amino acid residue,

6 and n is an integer from 1 to about 10; and

7 (b) an active agent.

1 2. A composition as defined in claim 1 , wherein said

2 oligopeptide has the formula:

3 PyGlu(X)_n

4 wherein each X is an independent amino acid residue and

5 n is an integer from 1 to about 10.

1 3. A composition as defined in claim 1 , wherein said

2 oligopeptide has the formula:

3 Pro(X)_n

4 wherein each X is an independent amino acid residue and

5 n is an integer from 1 to about 10.

1 4. A composition as defined in claim 1 , wherein n is from

2 about 3 to about 5.

1 5. A composition as defined in claim 1 , wherein each X is a

2 naturally occurring amino acid independently selected from the group

3 consisting of alanine, arginine, asparagine, aspartic acid, citrulline, cysteine,

4 cystine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,

5 ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine,

6 valine, proline, hydroxy proline, -carboxyglutamate, phenylglycine, or

7 O-phosphoserine. 6. A composition as defined in claim 1 , wherein each X is a non-naturally occurring amino acid independently selected from the group consisting of . -alanine, σ-amino butyric acid, γ-am no butyric acid, y- (aminophenyl) butyric acid, σ-amino isobutyric acid, citrulline, e-amino caproic acid, 7-amino heptanoic acid, ?-aspartic acid, aminobenzoic acid, aminophenyl acetic acid, aminophenyl butyric acid, -g'utamic acid, cysteine (ACM), e-lysine, e-lysine (A-Fmoc), methionine sulfone, norleucine, norvaline, ornithine, d-ornithine, p-nitro-phenylalanine, hydroxy proline, 1 ,2,3,4,- tetrahydroisoquinoline-3-carboxylic acid, and thioproline.

7. A composition as defined in claim 1 , wherein each X is an amino acid independently selected from the group consisting of pyroglutamic, glutamic, aspartic, σ-alanine, y-alanine, Hβucine, lysine, σ-phenylalanine, β- phenylalanine, -phenylalanine, σ-tyrosine, y-tyrosine, tryptophan, proline, and -valine.

8. A composition as defined in claim 1 , wherein said active agent is selected from the group consisting of a biologically active agent, a chemically active agent, or a combination thereof.

9. A composition as defined in claim 8, wherein said biologically active agent comprises at least one peptide, mucopolysaccharide, carbohydrate, or lipid.

10. A composition as defined in claim 8, wherein said biologically active agent is selected from the group consisting of human growth hormone, bovine growth hormone, growth hormone-releasing hormone, an interferon, interleukin-l, insulin, heparin, low molecular weight heparin, calcitonin, erythropoietin, atrial naturetic factor, an antigen, a monoclonal antibody, somatostatin, adrenocorticotropin, gonadotropin releasing hormone, oxytocin, vasopressin, cromolyn sodium, vancomycin, desferrioxamine (DFO), antimicrobials, anti-fungal agents, or any combination thereof.

1 1 1 . A composition as defined in claim 8, wherein said

2 biologically active agent comprises heparin. 3

, 1 12. A composition as defined in claim 8, wherein said

2 biologically active agent comprises low molecular weight heparin. 3

1 13. A composition as defined in claim 1 , further comprising a

2 stabilizing additive. 3

1 14. A composition as defined in claim 1 3, wherein said

2 stabilizing additive is selected from the group consisting of buffer salts, gum

3 acacia, gelatin, methyl cellulose, polyethylene glycol, polypropylene glycol,

4 and polylysine. 5

1 15. A composition as defined in claim 14, wherein said

2 stabilizing additive is selected from the group consisting of gum acacia,

3 gelatin, and methyl cellulose. 4

1 1 6. A dosage unit form comprising 2 (A) a composition as defined in claim 1 ; and 3 (B) (a) an excipient 4 (b) a diluent, 5 (0 a disintegrant, 6 (d) a lubricant, 7 (e) a plasticizer, 8 (f) a colorant, 9 (g> a dosing vehicle, or

10 <h) any combination thereof. 1

: 1 1 7. A dosage unit form as defined in claim 1 6, comprising a

2 tablet, a capsule, or a liquid. 3

1 18. A composition as defined in claim 1 , further comprising an enzyme inhibitor.

1 9. A composition as defined in claim 1 8, wherein said enzyme inhibitor comprises actinonin or derivatives thereof, epiactinonin or derivatives thereof, aprotinin (TRASYLOL), or Bowman-Birk inhibitor.

20. A method for administering an active agent to an animal in need of said agent, said method comprising administering orally to said animal a composition as defined in claim 1 .