WO1994020136A1

WO1994020136A1 - Cyclodextrin-peptide compositions

Info

Publication number: WO1994020136A1
Application number: PCT/US1994/001847
Authority: WO
Inventors: Peter K. Chiang; Dennis L. Butler; Nesbit D. Brown
Original assignee: Department Of The Army, United States Government
Priority date: 1993-03-08
Filing date: 1994-02-28
Publication date: 1994-09-15
Also published as: CA2134753A1

Abstract

This invention provides improved compositions containing cyclodextrin complexes of peptides, particularly synthetic peptides and peptides of ≤ 40 amino acids. Such peptides are particularly useful for administration as receptor agonists, receptor antagonists, and as vaccines. The compositions of the invention provide improved means for delivery of such peptides.

Description

CYCL0DE5CTRIN-PEPTIDE CC OSITIONS

Field of the Invention:

/ This invention relates to a method of presenting pharma¬ ceutically active peptides, particularly receptor bloc ers and i munogenic peptides, in cyclodextrin compositions.

10 Background of the Invention;

Cyclodextrins are cyclic molecules containing six or more α-D-glucopyranose units linked together at the 1,4 positions. The 2-hydoxypropyl-β-cyclodextrin (HPCD) has been used for stabilization and solubilization of various compounds,

15 including proteins and steroids. Brewster, et al. described use of cyclodextrins in solubilizing proteins to prevent aggregation , precipitation, and loss of biopotency. ("Appli¬ cation of 2-hydroxypropyl beta cyclodextrin to Proteins", Minutes Int. Symp. Cyclodextrins. 5th. 1990. pp 440-444) The

20 proteins studied therein were interleukin-3, and insulin, two large regulatory proteins. There is no suggestion that the 2- hydroxypropyl beta cyclodextrin would be useful in formulating peptides for use as receptor blockers or immunogens. The Brewster article suggests that the improved potency of the

25 proteins is due to the avoidance of hydrolysis, deamidation, racemization, oxidation and disulfide bond exchange, and changes in dimensional protein structure related to folding of the protein. There is no suggestion that the cyclodextrins can be useful for formulations containing synthetic peptides, nor

30 is there any suggestion that the preparations disclosed therein can be administered by application to the mucosa.

Josef Pitha, in U.S. patent 4,727,064, which is incorpo¬ rated herein by reference, suggests the use of cyclodextrin in solubilizing medicinals including steroids and vitamins, but

35 does not disclose the solubilization of peptides in cyclodex¬ trin.

Szejtli, et al., in U.S. patent 4,380,626, teach the use of cyclodextrins in preparations of plant growth regulators including 2-chloroethylphosphonic acid. No use of cyclodex¬ trins for preparation of peptides is taught or suggested therein. Gideon Goldstein, in U.S. Patent 5,140,010, which is incorporated herein by reference, teaches the stabilization of aqueous formulations of synthetic peptides corresponding to position 32-36 of thymopoietin and known as thymopentin or TP-5 (Arg-Lys-Asp-Val-Tyr) in glycine. Goldstein does not disclose or suggest use of cyclodextrin for stabilization of peptides. TP-5 is effective in blocking the stimulation of smooth muscle contraction caused by the neurotoxin (+)-anatoxin-a (ANTX) . ANTX is a bicyclic a ine exotoxin produced by the blue-green algae, Anabaena flos-aσuae, and has been found to cause death to livestock and waterfowl. The toxin acts by depolarizing blockade of neuromuscular transmission. Such depolarization results in respiratory paralysis. The action of ANTX has been ascribed to its potent nicotinic cholinergic agonist activities in skeletal muscle and mammalian skeletal muscle and the central nervous system. ANTX can also cause cardiovascular aberrations by activation of nicotinic receptors in the adrenal medulla and sympathetic ganglia. The antagonist effect of TP-5 has been attributed to its ability to block nicotinic receptors in a noncompetitive manner. summary of the Invention;

This invention provides improved compositions containing cyclodextrin complexes of peptides, particularly synthetic peptides and peptides of £ 40 amino acids. Such peptides are particularly useful for administration as receptor agonists, receptor antagonists, and as vaccines. The compositions of the invention provide improved means for delivery of such peptides.

Many peptides, especially peptides of about three to 20 amino acids, are unstable in low concentrations and tend to loose biological activity. While Brewster describes the value of preparing formulations of cyclodextrin and regulatory proteins to avoid conformational changes, there is no sugges¬ tion therein that cyclodextrin would be useful for increasing stability of small peptides such as thymopentin.

The instant invention improves methods of administration of peptides to the ucosa of mammals in need of treatment with effective peptides. Detailed Description of the Invention;

The invention provides a means of formulating peptides to avoid loss of efficacy and to facilitate delivery of the active peptides to the reactive site. The method has been exemplified using the synthetic peptides corresponding position 32-36 of thymopoietin and known as TP-5 (Arg-Lys-Asp-Val-Tyr) . While the hydroxypropyl cyclodextrin has been exemplified, other cyclodextrins, including mixed cyclodextrins, may be used in the method of the invention.

One problem in use of peptides is their instability in aqueous solution, especially very dilute compositions. Furthermore, many of the solvents used to provide stable, soluble compositions for treatment of other mammals can not be used in man. At present, there is no known compatible solvent for TP-5 in which the peptide is stable and easily adminis- tered. This is a significant problem because the instability will hinder acceptance for prophylactic and/or therapeutic applications. Materials and Methods;

The 2-hydroxypropyl-β-cyclodextrin used in the examples was purchased from Pharmatec in Alachua, Florida. TP-5 (10"² M) was synthesized as describe in Chiang, et al, Life Sci 49; (1991) PL13-19 and was made up in various percentages of HPCD dissolved in sterile water. Mixtures were stirred for about one hour. The solutions were then maintained at room tempera- ture. Control solutions of TP-5 dissolved in sterile water without HPCD were also prepared in the same manner. The two sets of solutions were stored at ambient room temperatures (25°C) for 14 months. Aliquots were removed monthly for stability testing. The stability study was performed by assaying the ability of the TP-5 solutions to counteract the stimulation of contraction of guinea pig ileum by ANTX. Guinea pig ileum contraction stimulated by ANTX was performed as reported in Chiang, et al. (supra) . The final concentration of TP-5 for use was obtained by diluting with Krebs-Ringer buffer. EXAMPLE I Aqueous solution of 2-hydroxy-β-cyclodextrin (HPCD) were prepared at concentration of 2.5%, 5.0%, 10%, 15%, 20%, 25% and 30% (w/v) . TP-5 was added in sufficient amounts to provide a final molarity of 10^"2 molar solution of TP-5. Further dilution to provide final dosage was made using Krebs-Ringer buffer. The solutions were then stored at ambient temperature for 14 months, after which activity of the cyclodextrin solutions was compared to freshly made solutions. As a control a 10^"2 solution without cyclodextrin was prepared. After storage at ambient temperature (25^eC) for four weeks the control solution showed no activity. EXAMPLE II

Evaluation of Anatoxin-A Response alone and in conjunction with TP-5 was carried out in accord with standard procedures as disclosed in U.S. Patent 4,973,734 issued November 27, 1990, which is incorporated herein by reference. Results:

IC₅₀ values of the inhibition by TP-5 of guinea-pig ileum contraction stimulation by ANTX at 3 x 10^"5 N was compared using freshly made 10_₂ molar solutions of TP-5 and similar concentrations of TP-5 in 5%, 15% and 20% solutions of HPCD which had been stored for 14 months at ambient temperature to determine relative activity. The results are shown as mean + s.e. of four separate experiments as indicated in Table I TABLE I

HPCD (%) IC₅₀ ( X 10^"5 M)

0 (freshly made) 3.9 + 1.9

5% 4.1 + 3.1

15% 4.9 + 4.4 20% 3.3 + 3.0 EXAMPLE III

A composition containing 0.5 mg TP-5 is administered intraperitoneally to rabbits to provide protection against ANTX. Formulations may be administered for up to 4 days. Dosage range for thymopentin may vary from 1 μg/kg/day to 1 g/kg/day. It is, of course, understood that smaller animals will require higher dosage per kilogram than larger mammals.

Formulations of active agents in HPCD for administration may be prepared using any pharmaceutically appropriate solvent, including water, isotonic saline, glucose, or saline. The formulations may be administered orally in the form of liquid bolus, or may be administered as lyophilized powders or tablets. When provided as lyophilized powders, many of the compositions may be administered nasally for inhalation. Compositions of the invention may be administered parenterally by, for example intramuscular, subcutaneous or intraperitoneal routes. Solutions of the cyclodextrin inclusion complexes can be administered to the mucosa by any means appropriate such as by nasal spray, buccal tablet or sublingually as drops. The site of administration will be governed, in many instances, by the site of effective response. For example, it is often advantageous to administer immunogenic peptides to the mucosa.

Many other peptides could be formulated in a similar manner. Such peptides include splenopentin (SP-5) having the structure Arg-Lys-Glu-Val-Tyr. This peptide is effective for inducing T-cell differentiation and for modulation of neuromus- cular transmission. (Proc. Natl. Acad. Sci. USA 81; 2847-2847 (1984)) Others include a nine amino acid sequence known as delta sleep inducing peptide (DSIP) of the structure Trp-Ala- Gly-Gly-Asp-Ala-Ser-Gly-Glu (Neurosci. Biobehav. Rev. ; 83-93 (1984)), vasoactive intestinal peptide (VIP) or biotinyl-VIP from human, porcin, chick, rat or other sources, having the sequence His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu- Arg-Lys-Gln-Met-Ala-Val-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH_j for prevention for cell killing by human immunodeficiency virus (Nature 335; 639-642 (1984)) and for pharmacological treatment of tissues involving neuromuscular transmission (Arch, int. Pharmacodvn 305. 14-24 (1990)). The peptide HG165-178 representing the sequence 165.-178 of gpl20 is represented by the sequence Asn-Ile-Ser-Thr-Ser-Ile-Arg-Gly-Lys-Val-Gln-Lys- Gln-Lys-Glu-Tyr, which is analogous to sequences in snake neurotoxins and rabies virus glycoprotein is conjugated to a keyhole limpet hemocyanin (KLH) and can be, thereafter, encapsulated in cyclodextrin to prevent the binding of viruses, toxins, viral coatings and gpl20 to cells. (See FEBS Letters 311; 115-118 (1992)). The methods of the invention should be particularly considered to stabilize peptides containing asparytyl, asparaginyl and glycine residues.

Claims

1. A pharmaceutically effective composition comprising an 5 effective amount of a receptor agonist, antagonist or

\ immunogenic peptide of 3 to 40 amino acids in a cyclo¬ dextrin inclusion complex in a pharmaceutically accept¬ able diluent.

10 2. A composition of claim 1 wherein the immunogenic peptide is TP-5.

3. A composition of claim 1 wherein cyclodextrin is present at a concentration of .5% to 30%. 15

4 A composition of claim 3 wherein a cyclodextrin is 2- hydroxypropyl-β-cyclodextrin.

5. A composition of claim 1 wherein the active peptide is an 20 immunogen.

6. A composition of claim 1 wherein the peptide is spleno- pentin.

25 7. A composition of claim 1 wherein the peptide is delta sleep-inducing peptide.

8. A composition of claim 1 wherein the peptide is vasoac- tive intestinal peptide.

30

9. A composition of claim 1 wherein the peptide is HG 165- 178.

10. A method of administering an immunogen to an animal by 35 administering an immunogenic effective amount of a pharmaceutical composition of claim 5.

11. A method of claim 10 wherein the pharmaceutical composi¬ tion is administered directly to the mucosa.

12. A method of claim 11 wherein the pharmaceutical composi¬ tion is administered sublingually.

13. A method of claim 11 wherein the pharmaceutical composi¬ tion is administered to the nasal mucosa by inhalation.