Composition for long-acting peptide analogs
09657078 ยท 2017-05-23
Assignee
Inventors
Cpc classification
A61P1/04
HUMAN NECESSITIES
A61P31/00
HUMAN NECESSITIES
A61P7/00
HUMAN NECESSITIES
A61K47/542
HUMAN NECESSITIES
C07K14/485
CHEMISTRY; METALLURGY
A61P7/04
HUMAN NECESSITIES
A61P9/02
HUMAN NECESSITIES
C07K14/57563
CHEMISTRY; METALLURGY
A61P1/16
HUMAN NECESSITIES
International classification
C07K17/06
CHEMISTRY; METALLURGY
C07K14/485
CHEMISTRY; METALLURGY
Abstract
The invention describes compositions of peptide analogs that are active in blood or cleavable in blood to release an active peptide. The peptide analogs have a general formula: A-(Cm).sub.x-Peptide (SEQ ID NO: 76), wherein A is hydrophobic moiety or a metal binding moiety, e.g., a chemical group or moiety containing 1) an alkyl group having 6 to 36 carbon units, 2) a nitrilotriacetic acid group, 3) an imidiodacetic acid group, or 4) a moiety of formula (Z.sub.yHis.sub.w).sub.p (SEQ ID NO: 50), wherein Z is any amino acid residue other than histidine, His is histidine, y is an integer from 0-6; w is an integer from 1-6; and p is an integer from 1-6; wherein if A has alkyl group with 6 to 36 carbon units x is greater than 0; and Cm is a cleavable moiety consisting of glycine or alanine or lysine or arginine or N-Arginine or N-lysine, wherein x is an integer between 0-6 and N may be any amino acid or none. The peptide analogs are complexed with polymeric carrier to provide enhanced half-life.
Claims
1. A composition comprising a peptide analog having a general formula: A-(Cm)x-peptide, wherein: a. Cm is independently selected from (i) Gly, (ii) Ala, (iii) Arg, (iv) Lys, (v) (N)q-Lys, wherein N is any amino acid, q is 0 or 1, and (vi) (N)q-Arg, wherein N is any amino acid, q is 0 or 1; b. x is an integer from 3-6; c. A is an alkyl group with 6 to 36 carbon units with a linker group selected from carbonyl and amino; and d. the peptide comprises glucagon-like peptide (GLP), leptin fragment, gastric inhibitory polypeptide (GIP), epidermal growth factor (EGF) receptor ligand, EGF, transforming growth factor alpha (TGF-alpha), gastrin/cholecystokinin receptor ligand, gastrin, cholecystokinin, auristatin, nisin, insulin, insulin-like growth factor, parathyroid hormone (PTH), atrial natriuretic factor, somatostatin, gonadotropin-releasing hormone, luteinizing-hormone-releasing-hormone, or vasoactive intestinal peptide (VIP).
2. The composition of claim 1, further comprising a polymeric carrier with a plurality of hydrophobic groups of 8-36 carbons each, wherein group A of the peptide analog is non-covalently bound to the plurality of hydrophobic groups of the polymeric carrier by hydrophobic interaction.
3. The composition of claim 2, wherein the peptide is atrial natriuretic factor, and A-(Cm)x- is attached to the N-terminus of the peptide.
4. The composition in claim 2, wherein the peptide is glucagon-like peptide (GLP) or atrial natriuretic factor.
5. The composition of claim 2, wherein the peptide is vasoactive intestinal peptide (VIP) or atrial natriuretic factor and A-(Cm)x- is attached to the N-terminus or the C-terminus of the peptide or a side chain.
6. The composition of claim 2, wherein A is a linear alkyl.
7. The composition of claim 3, wherein A is a linear alkyl.
8. The composition of claim 4, wherein A is a linear alkyl.
9. The composition of claim 5, wherein A is a linear alkyl.
10. The composition of claim 2, wherein A is a branched alkyl.
11. The composition of claim 3, wherein A is a branched alkyl.
12. The composition of claim 4, wherein A is a branched alkyl.
13. The composition of claim 5, wherein A is a branched alkyl.
14. The composition of claim 4, wherein the peptide is atrial natriuretic factor.
15. The composition of claim 1, wherein Cm is each independently selected from Ala, Arg, and Lys.
16. The composition of claim 1, wherein Cm is each independently selected from (i) Gly, (ii) (N)q-Lys, wherein N is any amino acid, q is 0 or 1, and (iii) (N)q-Arg, wherein N is any amino acid, q is 0 or 1.
17. The composition of claim 1, wherein the A-(Cm)x-(peptide) has lower biological activity in cell culture than a corresponding peptide lacking the A-(Cm)x in the absence of serum.
18. A composition comprising a peptide analog having a general formula: A-(Cm)x-peptide, wherein: a. Cm is independently selected from (i) Gly, (ii) Ala, (iii) Arg, (iv) Lys, (v) (N)q-Lys, wherein N is any amino acid, q is 0 or 1, and (vi) (N)q-Arg, wherein N is any amino acid, q is 0 or 1; b. x is an integer from 3-6; c. A is an alkyl group with 6 to 36 carbon units with a linker group selected from carbonyl, amino, and OCH(CH.sub.3)CO; wherein Cm has at least one Gly or one Ala linked directly to the peptide, wherein the A-(Cm)x-(peptide) has lower biological activity in cell culture than a corresponding peptide lacking the A-(Cm)x in the absence of serum, and wherein the peptide comprises glucagon-like peptide (GLP), leptin fragment, gastric inhibitory polypeptide (GIP), epidermal growth factor (EGF) receptor ligand, EGF, transforming growth factor alpha (TGF-alpha), gastrin/cholecystokinin receptor ligand, gastrin, cholecystokinin, auristatin, nisin, insulin, insulin-like growth factor, parathyroid hormone (PTH), atrial natriuretic factor, somatostatin, gonadotropin-releasing hormone, luteinizing-hormone releasing-hormone, or vasoactive intestinal peptide (VIP).
19. A method of making a peptide analog having a general formula: A-(Cm)x-peptide, wherein: a. Cm is independently selected from (i) Gly, (ii) Ala, (iii) Arg, (iv) Lys, (v) (N)q-Lys, wherein N is any amino acid, q is 0 or 1, and (vi) (N)q-Arg, wherein N is any amino acid, q is 0 or 1; b. x is an integer from 3-6; c. A is an alkyl group with 6 to 36 carbon units with a linker group selected from carbonyl and amino; and d. the peptide comprises glucagon-like peptide (GLP), leptin fragment, gastric inhibitory polypeptide (GIP), epidermal growth factor (EGF) receptor ligand, EGF, transforming growth factor alpha (TGF-alpha), gastrin/cholecystokinin receptor ligand, gastrin, cholecystokinin, auristatin, nisin, insulin, insulin-like growth factor, parathyroid hormone (PTH), atrial natriuretic factor, somatostatin, gonadotropin-releasing hormone, luteinizing-hormone releasing-hormone, or vasoactive intestinal peptide (VIP), the method comprising: step (i): forming a covalent bond between a resin and a reactive group on a first amino acid to provide a resin comprising a covalently bonded amino acid; step (ii): forming a covalent bond between the resin comprising the covalently bonded amino acid and a reactive group on a second amino acid; step (iii): repeating step (ii) to provide a (Cm)x-peptide; and forming a carbonyl or amino bond between the (Cm)x-peptide and an alkyl group with 6 to 36 carbon units to provide the peptide analog.
20. The composition of claim 1, wherein the peptide is an epidermal growth factor (EGF) receptor ligand.
21. The composition of claim 2, wherein the peptide is an epidermal growth factor (EGF) receptor ligand.
22. The composition of claim 18, wherein the peptide is an epidermal growth factor (EGF) receptor ligand.
23. The composition of claim 18, further comprising a polymeric carrier with a plurality of hydrophobic groups of 8-36 carbons each, wherein group A of the peptide analog is non-covalently bound to the plurality of hydrophobic groups of the polymeric carrier by hydrophobic interaction.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
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(22) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile[[u]]-Ala-Trp-Leu-Val-Lys-Gly-Arg (SEQ ID NO: 2); or
(23) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile[[u]]-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (SEQ ID NO: 3); or
(24) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile[[u]]-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-Gly (SEQ ID NO: 4); or
(25) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile[[u]]-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-Gly-Gly (SEQ ID NO: 5). The three letter codes are the amino acid representations known in the art of peptides.
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(27) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ileu-Ala-Trp-Leu-Val-Lys-Gly-Arg (SEQ ID NO: 2); or
(28) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ileu-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (SEQ ID NO: 3); or
(29) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ileu-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-Gly (SEQ ID NO: 4); or
(30) His-Ala-Glu-Gly-Thr-PHe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Gln-Phe-Ileu-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-Gly-Gly (SEQ ID NO: 5). The three letter codes are the amino acid representations known in the art of peptides.
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DETAILED DESCRIPTION OF THE INVENTION
(33) The present disclosure provides compositions of biologically active peptide complexes, formulations and methods of use of such compositions for the treatment of patients. The subject compositions, and methods of making and using the same, may achieve a number of desirable results and features. In one aspect, the present invention is directed to complexes of peptide analogs and polymeric carrier molecules that are configured to deliver, release and/or prolong the half-life of biologically-active peptides in a controlled fashion after administration to a patient. In another aspect, the present invention is directed to complexes of peptide analogs and polymeric carrier molecules that stabilize the peptides to create formulations suitable for administration to a patient. The present disclosure provides peptides with covalently attached binding moieties of hydrophobic and/or metal binding groups (peptide analogs) that can reversibly bind to polymeric carrier containing cores of hydrophobic polymeric carrier or polymeric carrier with covalently linked metal binding domains and chelated metal to form complexes suitable for administration to a patient. The present disclosure also provides peptides with alkyl groups that can form micelle or liposomes with or without cores of hydrophobic polymeric carrier. By way of a further embodiment, the peptide may optionally be covalently attached to hydrophobic or metal binding groups by a cleavable linker moiety that can be cleaved by host proteases in biological fluids or tissues, permitting the controlled and/or sustained release of active peptide from the delivery complex.
(34) U.S. Pat. No. 7,138,105 and United States patent publications 2005-0260259, 2007-0141145 and 2008-0015263 describe drug polymeric carrier molecules that reversibly bind hydrophobic and metal binding molecules, such as peptides. These polymeric carrier molecules can include a polymeric core (e.g., a backbone), such us a polyamino acid, such as polylysine. Protective groups, such as polyethylene glycol can be attached to the core. Also attached to the core are binding moieties that bind the molecule to be delivered. In certain embodiments, the binding moieties bind molecules with hydrophobic groups. In this case, the binding moiety can be a hydrophobic group such as an alkyl group. Alternatively, the binding moiety binds with molecules that, themselves, bind metals. Such binding moieties include metal binding groups, such as chelating groups, bound to a metal, such as a transition metal (e.g., Zn, Ni, Cu etc.).
(35) I. Peptide Analog Compositions
(36) This invention provides peptides that have been modified to include binding moieties, such as hydrophobic groups or metal binding groups, through which the peptides can bind to the polymeric carrier molecules. Modifications can include hydrophobic moieties or metal binding groups to create the peptide analogs of the invention. For example, a hydrophobic group can be an alkyl having 6-36 carbon atoms. A metal binding group can be, for example, a His tag (e.g., His.sub.6 (SEQ ID NO: 52)). In certain embodiments, the binding moiety is attached directly to the peptide to be delivered. In other embodiments, the binding moiety is attached to the peptide through a cleavable moiety, including sequences of amino acids such as Gly-Gly-Gly and other amino acid sequences susceptible to cleavage, as described more fully below. Thus, for the purpose of the present invention, peptide analog means a peptide with a covalently bound binding moiety, as well as a peptide with a covalently bound cleavable moiety and a covalently bound binding moiety. Cleavage of the cleavable moiety in the body releases the peptide from the polymeric carrier. Thus, peptide compositions of the invention have the general formula: Binding moietyOptional cleavable moietyPeptide, wherein the Binding moiety and Optional cleavable moiety can be bound, e.g., covalently, to the peptide at any appropriate location on the peptide. The cleavable moiety finds use when the binding moiety interferes with the biological activity of peptides.
(37) Accordingly, this invention also provides peptide complexes between a polymeric carrier and a modified peptide of this invention in which the modified peptide is reversibly bound to the polymeric carrier through hydrophobic attraction between alkyl groups of the modified peptide and polymeric carrier, or a metal ion binding interaction between a metal binding group of the modified peptide and metal binding groups and associated metal ions of the polymeric carrier. A. Peptides
(38) For the purpose of the present invention, peptide means biologically active polyamino acids ranging from 5 to 100 amino acids. In the past, polyamino acids larger than 51 amino acids wore termed proteins while 51 amino acid and smaller were termed peptides. The distinction between protein and peptide is only a matter of size. The synthesis of longer polyamino acids such as proteins has been challenging in the past due to some technology limitations of peptide synthesizers. This difficulty resulted in the distinction between peptide and proteins. The improvement in peptide synthesis technology, however, now allows for longer polypeptides up to 100 amino acids to be synthesized. It is also the intention of the present invention to include in the definition of peptide those polypeptides containing 5 to 100 amino acids since the technology now allows for the synthesis of those peptides in automated peptide synthesizer, making the old definition of peptide broader. Most large proteins are still made recombinant methods in biological systems while peptides are made using peptide synthesizers. The addition of alkyl groups, histidine, nitrilotriacetic acid, or iminodiacetic acid to a peptide can be easily done using a peptide synthesizer during peptide synthesis. For recombinant proteins, these alkyl groups, nitrilotriacetic acid, or iminodiacetic acid may be introduced in mature proteins but with limited reaction predictability from protein to protein. Peptide for the purpose of the present invention does not include homopolymers of amino acid such as polylysine, polyglutamic acid as they are not biologically active. The term biologically active for the purpose of the present invention means the peptide can bind to a cellular receptor that can send message inside the cell through a second messenger system that then causes a biological response. These are the peptides/proteins that when modified as outlined in the instant specification are the peptide of the present invention.
(39) The biologically active peptides suitable for use in the present invention include peptides that by themselves are susceptible to enzymatic cleavage in biological fluids such as plasma or are otherwise cleared by the body such that they have a short half-life. The present disclosure provides compositions containing peptides with biological activity that are suitable for treating a patient, such as vasopressin, terlipressin, glucagon like peptide (GLP), or analogs thereof. Other non-limiting examples of peptides include, but are not limited to leptin fragment, gastric inhibitory polypeptide (GIP), epidermal growth factor (EGF) receptor ligand, EGF, transforming growth factor alpha (TGF-alpha), gastrin/cholecystokinin receptor ligand, gastrin, cholecystokinin, lysostaphin, interferon, interferon gamma, interferon beta, interferon alpha, interleukin-1, interleukin-2, interleukin-4, interleukin-6, interleukin-8, interleukin-10, interleukin-12, tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, auristatin, nisin, insulin, insulin-like growth factor, growth hormone, nerve growth factor, brain-derived neurotrophic factor, endostatin, angiostatin, trombospondin, urokinase, streptokinase, blood clotting factor VII, blood clotting factor VIII, granulucyte-macrophage colony-stimulating factor (GM-CSF), granulucyte colony-stimulating factor (G-CSF), thrombopoetin, calcitonin, parathyroid hormone (PTH) and its fragments, erythropoietin, atrial natriuretic factor, somatostatin, adrenocorticotropin, gonadotropin releasing hormone, leutinizing-hormone-releasing-hormone, follicle stimulating hormone, glucoecrebrosidase, thrombopoietin, filgrastin, prostaglandins, epoprostenol, prostacyclin, desmopressin, or vasoactive intestinal peptide (VIP).
(40) The term derivative as used herein refers to a compound whose core structure is the same as, or closely resembles that of, a parent compound, but which has a chemical or physical modification, such as a different or additional groups. The term derivative includes co-polymers of parent compounds that can be linked to other atoms or molecules. The term derivative also includes peptides with at least 50% sequence identity with the parent peptide. The term derivative also include a peptide with additional groups attached to it, such as fatty acids and/or additional amino acids, but does not include binding moieties or cleavable moieties as they are defined herein. Vasopressin as use herein includes its derivatives such as terlipressin and other variations thereof. Glucagon like peptide (GLP) as used herein includes GLP and its derivatives such as GLP-1 (7-36), GLP-1 (7-37), and exenatide and other variation thereof. For clarity of the specification, a linear peptide, also referred to as polypeptide or polyamino acid, has a N-terminus and a C-terminus. The N-terminus refers to the alpha amino group of the terminal amino acid that is not used to form a peptide bond. The C-terminus refers to the alpha carboxyl group of the terminal amino acids that is not used to form a peptide bond. The N-terminus and C-terminus of a peptide is not in the R-group (known in the art) of any amino acid that made up the peptide. Alkyl group for the purpose of this invention is a chemical group that is made up of only carbon and hydrogen. The preferred alkyl group in this invention contains 6 to 36 carbon units with their covalently linked hydrogen atoms.
(41) Peptide analogs can be generated using standard techniques of peptide chemistry and can be assessed for activity either before or after incubation in serum. Particularly preferred peptides analogs of the invention are those peptides containing 1) an alkyl group of 6-36 carbon units at the N- or C-terminus of a linear peptide; or 2) iminodiacetic acid, which may or may not be a portion of nitrilotriacetic acid. The analogs can have a general formula: A-(Gly).sub.x-peptide (SEQ ID NO: 54) or peptide-(Gly).sub.x-A (SEQ ID NO: 55), wherein the left side of the peptide is N-terminal and the right side is C-terminal; Gly is Glycine; where x is an integer from 0-5; A can be any chemical group or moiety containing an alkyl group with 8 to 36 carbon units, a nitrilotriacetic acid group, a imidodiacetic acid group, or (His).sub.y (SEQ ID NO: 51); where y is an integer from 2-6. The peptide can be any sequence or chain of 5-100 amino acids. The amino acids can be any of the 20 naturally occurring amino acids or their derivatives. The attachment of A to the peptide may involve simple linker group such as lactate or glycine. Lactate is especially important if we want to attach the alkyl group using an ester bond to the amino terminus of a peptide. To attach an alkyl group to the carboxyl terminus of a peptide by amide bond, an amino-alkyl group will be used with the general formula CH.sub.3(CH).sub.nNH; where n=5-34.
(42) Analogs of peptides exemplified by vasopressin can be generated using standard techniques of peptide chemistry and can be assessed for calcium influx activity before and after incubation in serum, all according to the guidance provided herein. Preferred analogs of the invention are those based upon the sequence of vasopressin, as follows:
(43) TABLE-US-00001 (SEQIDNO:56) A-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-B-C-D
wherein:
(44) A is selected groups containing alkyl group with 3 to 36 carbon units, nitrilotriacetic acid, imidodiacetic acid or (Z.sub.xHis.sub.y).sub.p (SEQ ID NO: 50); where Z is an amino acid residue, His is histidine, x is an integer from 0-6; y is an integer from 1-6; and p is an integer from 1-6; Gly is Glycine Cys is Cystine or Cysteine Tyr is Tyrosine Phe is Phenylalanine Gin is Glutamine Asn is Aspargine Pro is proline B is Lysine or Arginine C is Glycine or Alanine D is NH.sub.2 or H A-Gly-Gly-Gly- subunit is covalently attached to any available amino group of the peptide by an amide bond.
(45) Many of the protected amino acid residues useful in synthesizing the preferred compounds of this invention are commercially available from many amino acid suppliers. Furthermore, all the peptide analogs that are the subject of the present invention can be synthesized by outside custom peptide providers such as Anaspec, San Jose Calif. USA. Polypeptide laboratories, Torrance, Calif., or ChemPep Inc, Miami, Fla. Synthesis of peptides according to the specification disclosed in this application can easily be done by those skilled in the art and preferably done using solid phase synthesis, especially if the modification is at a terminal residue of the peptides. For peptide with nitrilotriacetic acid at the carboxyl terminal (
(46) Once the desired analogs of peptides has been synthesized, cleaved from the resin and fully deprotected, the peptide is then purified to ensure the recovery of a single oligopeptide having the selected amino acid sequence with the desired functional group. Purification can be achieved using any of the standard approaches, which include reversed-phase high-pressure liquid chromatography (RP-HPLC) on alkylated silica columns, e.g. C.sub.4-18 silica. Such column fractionation is generally accomplished by running linear gradients, e.g., 10-90%, of increasing % organic solvent, e.g., acetonitrile, in aqueous buffer, usually containing a small amount (e.g., 0.1%) of pairing agent such as TFA or TEA. Alternatively, ion-exchange HPLC can be employed to separate peptide species on the basis of their charge characteristics. Column fractions are collected, and those containing peptide of the desired/required purity are optionally pooled with the guide of Tandem Mass spectrometry detector. In one embodiment of the invention, the vasopressin peptide is then treated in the established manner to exchange the cleaving acid (e.g., TFA) with a pharmaceutically acceptable acid anion and to allow intra-molecular disulfide bridge formation in dilute solution under a suitable oxidizing agent. This intra-molecular disulfide bridge formation can be confirmed by HPLC/MS analysis. B. Binding Moieties
(47) The present disclosure provides binding moieties of hydrophobic or metal binding groups that can be attached to the peptides and the polymeric carrier molecules of the embodiments that permit reversible binding to polymeric carrier to form the peptide complexes. 1. Hydrophobic Groups
(48) In one aspect, the binding moiety of the modified peptide is a hydrophobic group, e.g., an alkyl group of 6 to 36 carbon units. Such hydrophobic groups can non-covalently bind to hydrophobic groups in a carrier molecule. In one embodiment, the alkyl group of the binding moiety is a linear alkyl carbonyl group having a formula CH.sub.3(CH.sub.2).sub.nCO, where n is an integer between 4-34, or a corresponding ammo-alkyl group with 6 to 36 carbon units wherein the respective alkyl groups can be attached to the N- or C-terminus of the peptide or to a side chain of an amino acid of the peptide. In another embodiment, the alkyl group of the binding moiety is a branched alkyl carbonyl group or a branched amino-alkyl group with 6-36 carbon units attached, respectively, to the N- or C-terminus of the peptide or to a side chain of an amino acid or the peptide. The hydrophobic groups can also be ring compounds of 6-36 carbon units. 2. Metal Binding Groups
(49) In another aspect, the present disclosure provides binding moieties with a metal binding domain, such as those disclosed in U.S. patent application Ser. No. 11/112,879, which is incorporated herein by reference, in its entirety.
(50) The metal binding moiety can be any conformational arrangement of several chemical groups that is capable of forming a complex between the metal ion and the chemical groups by coordinate bonds. In certain embodiments, the metal binding moiety is a chelating group. Chelating groups posses a pair of unpaired electrons that are available for coordinate bonding with a metal ion. Bidentate-, tridentate- and tetradentate chelating groups are well known in the art.
(51) In one embodiment, a plurality of metal binding moieties with chelated metal ion are covalently attached to the core polymer of the polymeric carrier, a metal binding moiety is covalently to the peptide which forms a reversible coordinate bond to the metal ion. In general, the metal binding domains of the metal binding moieties used in the present invention contain a Lewis base fragment that is contemplated to encompass numerous chemical moieties having a variety of structural, chemical and other characteristics capable of forming coordination bonds with a metal ion. The types of functional groups capable of forming coordinate complexes with metal ions are known to those of skill in the art. For example, such moieties will generally include functional groups capable of interaction with a metal center, e.g., heteroatoms such as nitrogen, oxygen, sulfur, and phosphorus. Metal cations are almost always Lewis acidic and are therefore able to bind various moieties that may serve as Lewis bases. In preferred embodiments, the metal chelate ions include, but are not limited to, Zn.sup.2+, Ni.sup.2+, Co.sup.2+, Fe.sup.2+, Mn.sup.2+, and Cu.sup.2+.
(52) In general, a moiety serving as a Lewis base will be a strongly acidic group, e.g., with a pKa less than about 7, and more preferably less than 5, which may produce a conjugate base that, under the appropriate conditions, is a strong enough Lewis base to donate an electron pair to a metal ion to form a coordinate bond. The degree of this Lewis acid-to-Lewis base interaction is a function not only of the particular metal ion, but also of the coordinating moiety itself, because the latter may vary in the degree of basicity as well as in size and steric accessibility. Exemplary Lewis basic moieties which may be included in the metal binding domain include: amines (primary, secondary, and tertiary) and aromatic amines, amino groups, amido groups, nitro groups, nitroso groups, amino alcohols, nitrites, imino groups, isonitriles, cyanates, isocyanates, phosphates, phosphonates, phosphites, phosphines, phosphine oxides, phosphorothioates, phosphoramidates, phosphonamidites, hydroxyls, carbonyls (e.g., carboxyl, ester and formyl groups), aldehydes, ketones, ethers, carbamoyl groups, thiols, sulfides, thiocarbonyls (e.g., thiolcarboxyl, thiolester and thiolformyl groups), thioethers, mercaptans, sulfonic acids, sulfoxides, sulfates, sulfonates, sulfones, sulfonamides, sulfamoyls and sulfinyls.
(53) Illustrative of suitable metal binding domains include those chemical moieties containing at least one Lewis basic nitrogen, sulfur, phosphorous or oxygen atom or a combination of such nitrogen, sulfur, phosphorous and oxygen atoms. The carbon atoms of such moiety may be part of an aliphatic, cycloaliphatic or aromatic moiety. In addition to the organic Lewis base functionality, such moieties may also contain other atoms and/or groups as substituents, such as alkyl, aryl and halogen substituents. Preferred binding moieties are nitrilotriacetic acid and iminodiacetic acid groups, or his tags that consist of two to six linked histidine residues (SEQ ID NO: 51).
(54) In one embodiment, the metal binding moiety of the peptide analog is attached by a covalent amide bond to the N-terminus of the peptide. In another embodiment, the metal binding moiety of the peptide analog is covalently attached by a covalent amide bond to the C-terminus of the peptide. In another embodiment, the metal binding of the peptide analog is attached by a covalent bond to a side chain of an amino acid or the peptide. In further embodiments, a cleavable moiety is covalently bound and positionally located between the metal binding moiety and the peptide. In exemplary embodiments, the metal binding moiety is a his tag with cleavable moiety of the formula His-His-His-His-His-His-Gly-Gly-Gly (SEQ ID NO: 6), wherein the moiety is bound to either the N-terminus, or to the C-terminus, or to a side chain of an amino acid of the peptide. In a further embodiment, the metal binding moiety is a moiety of formula (Z.sub.yHis.sub.w).sub.p (SEQ ID NO: 50), wherein Z is an amino acid residue other than histidine, y is an integer from 0-6, w is an integer from 1-6, and p is an integer from 1-6, and is bound to a cleavable moiety. In another embodiment, the metal binding moiety is nitrilotriacetic acid and is bound to a cleavable moiety, e.g., Gly.sub.3, and the peptide. In another embodiment, the metal binding moiety is iminodiacetic acid and is bound to a cleavable moiety, e.g., Gly.sub.3, and the peptide.
(55) In certain embodiments, if peptide is GLP, A is attached to the peptide at a position other than the amino terminus.
(56) Other chelating groups include 1,4,7,10-tetraaza-cyclododecane-N,N,N-triacetic acid; 1,4,7-tris(carboxymethyl)-10-(2-hydroxypropyl)-1,4,7,10-tetraazocyclodecane, 1,4,7-triazacyclonane-N,N,N-tracetic acid; and 1,4,8,11-tetraazacyclotetra-decane-N,N,N,N-tetra acetic acid; diethylenetriamine-pentaacetic acid (DTPA); triethylenetetraamine-hexaacetic acid; ethylenediamine-tetraacelic acid (EDTA); EGTA; 1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid but preferably N-(hydroxyethyl)ethylenediaminetriacetic acid; nitrilotriacetic acid (NTA); and ethylene-bis(oxyethylene-nitrilo)tetraacetic acid, histidine, cysteine, oligoaspartic acid, oligoglutamic acid, S-acetyl mercaptoacetate and meractoacetyltriglycine. 3. Cleavable Moiety
(57) The present disclosure provides cleavable moieties that serve as linkers between the biologically active peptide and the binding moiety. The cleavable moiety may include an amino acid sequence that can serve as a substrate for a protease, usually an extracellular protease. In one embodiment, the binding moiety is attached to the peptide through a polyglycine cleavable moiety, such as Gly-Gly-Gly. In another embodiment, the binding moiety comprises one or more cysteine residues capable of forming a disulfide bond with corresponding cysteine residues incorporated into the polymeric carrier, which can be cleaved by action of a reducing agent. In such embodiments, cleavage of the cleavable moiety in the body releases the peptide from the polymeric carrier. The cleavable moiety may be (Gly).sub.x groups (SEQ ID NO: 58) and include, without limitation, Gly.sub.2, Gly.sub.3, Gly.sub.4 (SEQ ID NO: 63), Gly.sub.5 (SEQ ID NO: 64) and Gly.sub.6 (SEQ ID NO: 65). The cleavable moiety may also be (Ala).sub.x groups (SEQ ID NO: 59) and include without limitation Ala.sub.2, Ala.sub.3, Ala.sub.4 (SEQ ID NO: 66), Ala.sub.5 (SEQ ID NO: 67) and Ala.sub.6 (SEQ ID NO: 68). The cleavable moiety may also be (Lys).sub.x groups (SEQ ID NO: 60) and include without limitation Lys.sub.2, Lys.sub.3, Lys.sub.4 (SEQ ID NO: 69), Lys.sub.5 (SEQ ID NO: 70) and Lys.sub.6 (SEQ ID NO: 71). The cleavable moiety may also be (Arg).sub.x groups (SEQ ID NO: 61) and include without limitation Arg.sub.2, Arg.sub.3, Arg.sub.4 (SEQ ID NO: 72), Arg.sub.5 (SEQ ID NO: 73) and Arg.sub.6 (SEQ ID NO: 74). The cleavable moiety may also be (Ala-Arg).sub.n groups (SEQ ID NO: 62), where n is between 1-3. The cleavable moiety may also be a moiety of formula (N).sub.q-Arg, wherein N is any amino acid and q is 0 or 1. The cleavable moiety may also be a moiety of formula Arg-(N).sub.q, wherein N is any amino acid and q is 0 or 1. The cleavable moiety may also be a moiety of formula Lys-(N).sub.q, wherein N is any amino acid and q is 0 or 1. The cleavable moiety may also be a sequence of two amino acids in which one is Arg or Lys. Alternatively, the cleavable moiety may be any one from a group consisting of
(58) TABLE-US-00002 (SEQIDNO:7) Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr; (SEQIDNO:8) Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg; (SEQIDNO:9) Glu-Arg-Nle-Phe-Leu-Ser-Phe-Pro; (SEQIDNO:10) Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln; (SEQIDNO:11) Arg-Gly-Val-Val-Asn-Ala-Ser-Ser-Arg-Leu-Ala; (SEQIDNO:12) Glu-Val-Asn-Leu-Asp-Ala-Phe-Lys; (SEQIDNO:13) Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys, (SEQIDNO:14) Glu-Val-Lys-Val-Asp-Ala-Gln-Phe-Lys, (SEQIDNO:15) His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Lys; (SEQIDNO:16) Lys-Thr-Glu-Glu-Ile-Ser-Glu-Val-Asn-Leu-Asp-Ala- Glu-Phe; (SEQIDNO:17) Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu; (SEQIDNO:18) Pro-Gln-Gly-Leu-Glu; (SEQIDNO:19) Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys; (SEQIDNO:20) Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys; (SEQIDNO:21) Pro-Leu-Ala-Tyr-Trp-Ala-Arg; (SEQIDNO:22) Arg-Pro-Leu-Ala-Tyr-Trp-Met-Arg; (SEQIDNO:23) Arg-Pro-Ls-Pro-Lys-Pro-Leu-Ala-Nva-Trp-Lys; (SEQIDNO:24) Pro-Tyr-Ala-Tyr-Trp-Trp-Met-Arg; (SEQIDNO:25) Pro-Leu-Gly-Met-Trp-Ser-Arg; (SEQIDNO:26) Pro-Leu-Ala-Leu-Trp-Ala-Arg; (SEQIDNO:27) Pro-Leu-Gly-Leu-Trp-Ala-D-Arg; (SEQIDNO:28) Asp-Gln-Val-Asp; (SEQIDNO:29) Asp-Met-Gln-Asp; (SEQIDNO:30) Leu-Glu-Val-Asp; (SEQIDNO:31) Val-Glu-Ile-Asp; (SEQIDNO:32) Ile-Glu-Thr-Asp; and (SEQIDNO:33) Leu-Glu-His-Asp.
(59) II. Polymeric Carrier
(60) The polymeric carrier of the present invention include polymers and co-polymers of linear or branched structure or conjugates thereof, micelles, emulsions, colloids and solid surfaces, where the polymers may in addition self-organize in supramolecular structures including at least two polymers. The copolymers include as one of the main polymeric elements a backbone core polymer that contains a hydrophobic binding group or a metal binding group and, in certain embodiments, protective side chains, such as PEG or mPEG.
(61) The backbone can be a linear polymer, such as a polyamino acid; e.g., a homopolymer or a nucleic acid, or a branched polymer, such as a carbohydrate. In one example, a polymeric carrier composition of the present invention comprises the backbone linear homopolyamino acid core with a degree of polymerization in the range of 2-10,000 to which independently and covalently linked are polyglycol protective side chains, and chelating groups, where said chains and chelating groups are independently linked to the backbone core polymer. In another example, the degree of polymerization of the backbone core is in the range of 100-1,000. In still another example, the degree of polymerization is in the range of 100 to 300. Examples of polymeric backbone cores include carboxylated or carboxymethylated linear poly-l-lysine (PL) or poly-D-lysine, carboxylated or carboxymethylated poly-alpha,beta-(2-aminoethyl)-D,L-aspartamide; poly-aspartic acid, poly-glutamic acid, copolymers of histidine with positively or negatively charged aminoacids, carboxylated polyethyleneimines, i.e., polyethylene imines reacted with derivatives of carbonic acids. In preferred embodiments, the backbone linear core comprises poly-lysine.
(62) In another embodiment, the polymeric carrier further comprises protective side chains. In one embodiment, the protective side chain comprises polyethylene glycol (PEG). In a further embodiment, the protective side chain comprises alkoxy polyethylene glycol. In a further embodiment, the protective side chain comprises methoxy polyethylene glycol (MPEG). The protective side chains of the embodiments will have a mass of between 200 and 60,000 Daltons independent of the polymeric core weight, preferably a mass of between 1000 and 40,000 Daltons, and more preferably between 2,000 and 20,000 Daltons.
(63) The metal binding domains of the polymeric carrier may include polycarboxylic acids containing nitrogen where at least one of carboxylic groups may be utilized for covalent linking of the chelate to the carrier backbone polymer component of the composition of the invention. The addition of said metal ions to chelates included in the polymeric carrier compositions of the invention either at room temperature or at elevated temperatures results in the formation of coordinate complexes (metal-chelates). These metal-chelate complexes bind to the metal binding domain of peptide, added either in a purified state or in the presence of bulk protein or blood plasma proteins, with the formation of peptide complex compositions containing coordinate complexes formed between the metal-chelate of the polymeric carrier and the peptides. The amino acid sequence of the peptides of the invention may include one or more histidines or cysteines which increase the stability of the complex formed between the peptide and polymeric carrier metal-chelate complexes.
(64) Hydrophobic binding moieties attached to the carrier can include an alkyl group. In a further embodiment, the alkyl group comprises a linear or branched alkyl group. In a further embodiment, the alkyl group can be at least partially saturated. In a further embodiment, the alkyl group comprises an ethyl or propyl group. In a further embodiment, the alkyl group is a butyl, pentyl, or hexyl group. In a further embodiment, the alkyl group is CH.sub.3(CH.sub.2).sub.nCH.sub.2NH, CH.sub.3(CH.sub.2).sub.nCO, CH.sub.3(CH.sub.2).sub.nCH.sub.2O, CH.sub.3(CH.sub.2).sub.nCh.sub.2S, OC(CH.sub.2).sub.nCH.sub.2, OC(CH.sub.2).sub.nCH.sub.2NH, OC(CH.sub.2).sub.nCO, OC(CH.sub.2).sub.nCH.sub.2O, OC(CH.sub.2).sub.nCH.sub.2S, HNC(CH.sub.2).sub.nCH.sub.2, HNC(CH.sub.2).sub.nCH.sub.2, HNC(CH.sub.2).sub.nCH.sub.2NH, HNC(CH.sub.2).sub.nCO, HNC(CH.sub.2).sub.nCH.sub.2), HNC(CH.sub.2).sub.nCH.sub.2S, OCH.sub.2(CH.sub.2).sub.nCH.sub.2, OCH.sub.2(CH.sub.2).sub.nCH.sub.2NH, OCH.sub.2(CH.sub.2).sub.nCO, OCH.sub.2(CH.sub.2).sub.nCH.sub.2O, or OCH.sub.2(CH.sub.2).sub.nCH.sub.2S group, wherein n is 4-34, inclusive. In a further embodiment the present the hydrophobic chain is (CH.sub.2).sub.4NHCO(CH.sub.2).sub.nOCAOR.sub.3, (CH.sub.2).sub.4NHCO(CH.sub.2).sub.nNHCO(CH.sub.2), COAOR.sub.3, CH.sub.2OOC(CH.sub.2).sub.nOCAOR.sub.3, CH.sub.2OOC(CH.sub.2).sub.nNHCO(CH.sub.2).sub.yCOAOR.sub.3, CH(CH.sub.3)OOC(CH.sub.2).sub.nOCAOR.sub.3, CH(CH.sub.3)OOC(CH.sub.2).sub.nNHCO(CH.sub.2).sub.yCOAOR.sub.3, CH.sub.2COOC(CH.sub.2).sub.nCOAOR.sub.3, CH.sub.2COOC(CH.sub.2).sub.nNHCO(CH.sub.2).sub.yCOAOR.sub.3, CH.sub.2CONH(CH.sub.2).sub.nNHCOCH.sub.2CH.sub.2AOR.sub.3, CH.sub.2CONH(CH.sub.2).sub.nNHCO(CH.sub.2).sub.yCOAOR.sub.3, (CH.sub.2).sub.2COOC(CH.sub.2).sub.nCOAOR.sub.3, (CH.sub.2).sub.2COOC(CH.sub.2).sub.nNHCO(CH.sub.2).sub.yCOAOR.sub.3, (CH.sub.2).sub.2CONH(CH.sub.2).sub.nNHCOCH.sub.2CH.sub.2AOR.sub.3, (CH.sub.2).sub.2CONH(CH.sub.2).sub.nNHCO(CH.sub.2).sub.yCOAOR.sub.3, (C.sub.6H.sub.4)OCO(CH.sub.2).sub.nCOAOR.sub.3, and (C.sub.6H.sub.4)OCO(CH.sub.2).sub.nNHCO(CH.sub.2).sub.yCOAOR.sub.3, wherein n is 2-22; y is 2-6; R.sub.3 is H, (CH.sub.2).sub.pCH.sub.3 or (CH.sub.2).sub.pCOOH, wherein p is 0-7; and A is [OCH.sub.2CH.sub.2].sub.x, or [OCHCH.sub.3CH.sub.2].sub.x, wherein x is 17-250, or various combinations of [OCH.sub.2CH.sub.2] or [OCHCH.sub.3CH.sub.2] with a total or 17-250 units. In another embodiment, the hydrophobic group comprises an aromatic ring compound. In a further embodiment, the aromatic ring is phenyl. In a further embodiment, the aromatic ring is naphthyl. In a further embodiment, the aromatic ring compound is cholesterol. In a further embodiment, the aromatic ring compound is fluorescien and the carboxyl group in fluorescien will act as orienting molecule.
(65) The percent load capacity of the polymeric carrier for the peptide analog can vary, depending on the respective compositions of the carrier and the peptide analog. In one embodiment, the peptide analogs of the present invention are complexed with the polymeric carrier compounds by dissolving or suspending one hundred mg of the polymeric carrier in an appropriate solvent; e.g., phosphate buffered saline, saline, acetate buffer, water or other appropriate solvent known in the art to be compatible for parenteral drug administration, and mixing with 1-200 mg of peptide analog of the present invention in a final unit volume of 100 l to 1 ml until the peptide analog is bound to the carrier. The resulting formulation can be lyophilized for later reconstitution in appropriate volume for administration to a patient. The formulation can also be filter sterilized prior to lyophilization or prior to administration to a patient. Alternatively the carrier can be filter sterilized by passing through a filter (0.10 m to 0.22 m filter) prior to mixing with a peptide analog of the present invention that has been sterilized in similar manner.
(66) III. Peptide Formulations
(67) For administration to patients, the peptide complex compositions are provided in a pharmaceutically acceptable form. In one embodiment, the vasopressin analog peptide or its salt is provided in a pharmaceutically acceptable vehicle as a preparation that is sterile-filtered, e.g., through a 0.22 m filter, and is substantially pyrogen-free. Desirably, the vasopressin analog peptide to be formulated migrates as a single or individualized peak on HPLC, exhibits uniform and authentic amino acid composition and sequence upon analysis thereof, and otherwise meets standards set by the various national bodies which regulate quality of pharmaceutical products. The analogs with alkyl group of 6-36 carbon units can form micelle and can be administered in a suitable solvent. Alternatively, the analogs with alkyl group of 6-36 carbon units can be incorporated into a carrier with hydrophobic core such as those described in U.S. patent application Ser. No. 11/613,183. Alternatively, the alkyl containing analogs may be incorporated into micelles or liposomes. The analogs with 2-6 histidine residue (SEQ ID NO: 51) can be incorporated into metal containing carriers such as those described in U.S. Pat. No. 7,138,105. These carriers will further prolong the blood circulation half-life of these analogs by preventing their rapid activation which would be followed by rapid elimination from the blood.
(68) For therapeutic use, the chosen vasopressin- or other peptide-analog is formulated with a carrier (such as those described in U.S. Pat. No. 7,138,105 and U.S. application Ser. No. 11/613,183, herein incorporated by reference) and/or other pharmaceutically acceptable diluents or excipients that is appropriate for delivering the peptide by the chosen route of administration. Other suitable pharmaceutically acceptable diluents or excipients are those used conventionally with peptide-based drugs. Reference may be made to Remington's Pharmaceutical Sciences, 17th Ed., Mack Publishing Company, Easton, Pa., 1985, for guidance on drug formulations generally. In one embodiment of the invention, the compounds are formulated for administration by infusion or by injection, e.g., sub-cutaneously, intramuscularly or intravenously, and are accordingly utilized as aqueous solutions in sterile and pyrogen-free form and optionally buffered to physiologically tolerable pH, e.g., a slightly acidic or physiological pH. Thus, the compounds may be administered in a vehicle such as distilled water or, more desirably, in saline, phosphate buffered saline or 5% dextrose solution. Water solubility of the vasopressin/or other peptides analogs, especially those with alkyl group, may be enhanced, if desired, by incorporating a solubility enhancer. The vasopressin/or other peptide analogs of the invention may also be formulated as a slow release implantation device to further extend the duration of action. Examples of such sustained release formulations include composites of biocompatible polymers, such as poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen, and the like, preferably with covalently attached hydrophobic moiety or metal chelates. The structure, selection and use of degradable polymers in drug polymeric carriers have been reviewed in several publications, including, A. Domb et at., Polymers for Advanced Technologies 3:279-292 (1992). Additional guidance in selecting and using polymers in pharmaceutical formulation can be found in the text by M. Chasin and R. Langer (eds.), Biodegradable Polymers as Drug Delivery Systems, Vol. 45 of Drugs and the Pharmaceutical Sciences, M. Dekker, New York, 1990. Liposomes may also be used to further sustain the action of vasopressin analogs. Details concerning how to use and make liposomal formulations of drugs of interest can be found in, among other places. U.S. Pat. No. 4,944,948; U.S. Pat. No. 5,008,050; U.S. Pat. No. 4,921,706; U.S. Pat. No. 4,927,637; U.S. Pat. No. 4,452,747; U.S. Pat. No. 4,016,100; U.S. Pat. No. 4,311,712; U.S. Pat. No. 4,370,349; U.S. Pat. No. 4,372,949; U.S. Pat. No. 4,529,561; U.S. Pat. No. 5,009,956; U.S. Pat. No. 4,725,442; U.S. Pat. No. 4,737,323; U.S. Pat. No. 4,920,016. In one embodiment of the invention, the package contains the vasopressin/or other peptide analog (similarly altered as vasopressin analogs) with or without additional carrier, diluent and excipients as an administration-ready formulation. Alternatively, and according to another embodiment of the invention, the package provides the vasopressin/or peptide analog with or without carrier in a form, such as a lyophilized form, suitable for reconstitution in a suitable diluent or excipients, such as phosphate-buffered saline. In one embodiment, the package is a sterile-filled vial or ampoule containing an injectable solution which comprises an effective, active amount of vasopressin/or other peptide analog dissolved in an aqueous vehicle. As an alternative to injectable formulations, the vasopressin/or other peptide analog may be formulated for administration by other routes. Oral dosage forms, such as tablets, capsules and the like, can be formulated in accordance with standard pharmaceutical practice.
(69) IV. Methods of Use
(70) The novel compositions disclosed herein can be selected for use in methods of treatment of patients according to the combinations of peptide analogs and polymeric carriers provided and the underlying disease or physiologic condition of the patient and/or the molecular target and its location. The peptide analog compositions can be administered by any suitable means or route, including parenteral, intrapulmonary, and intranasal, and, if desired, for local injection. Parenteral administration routes include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
(71) The appropriate dosage of peptide analog will depend on the type of disease or condition to be treated, the severity and course of the disease, the patient's clinical history and response to the peptide analog, and the discretion ofthe attending physician. Peptide analogs can suitably be administered to the patient in a single dose, in divided doses, or over a series of treatments. Also, the present invention contemplates mixtures of more than one peptide analog, as well as use in combination with other therapeutic agents.
(72) In certain embodiments, the dosage of the subject compounds will generally be in the range of about 0.01 ng to about 1 g per kg body weight, specifically in the range of about 1 ng to about 0.1 g per kg, and more specifically in the range of about 100 ng to about 10 mg per kg.
(73) The peptide analog and polymeric carrier compositions will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of delivery of the peptide, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The therapeutically effective amount of a peptide composition and polymeric carrier complex to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat a disease or disorder.
(74) The precise time of administration and dosage of any particular compound that will yield the most effective treatment in a given patient will depend upon the activity, pharmacokinetics, and bioavailability of a particular compound, physiological condition of the patient (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage and type of medication), route of administration, and the like. The guidelines presented herein may be used to optimize the treatment, e.g., determining the optimum time and/or amount of administration, which will require no more than routine experimentation or will consist of monitoring the subject and adjusting the dosage and/or timing. An effective dose or amount, and any possible affects on the timing of administration of the dose, may need to be identified for any particular composition of the present invention. Dosages for the compounds of the present invention may be readily determined by techniques known to those of skill in the art. In one embodiment, the effective dose may be determined by routine experiment using one or more groups or animals (preferably at least 5 animals per group), or in human trials if appropriate. In another embodiment, the effectiveness of the composition and method of treatment or prevention may be assessed by administering the peptide analog and assessing the effect of the administration by measuring one or more indices associated with the disease or condition of interest, and comparing the post-treatment values of these indices to the values of the same indices prior to treatment. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. Accordingly, the method of treatment embodiments can include obtaining single or sequential blood or other body fluid samples from a patient after administration of the composition and quantitatively assaying for the free peptide by use of assays known in the art; e.g., HPCL, bioassay, mass spectronomy and the like. Resulting values can be compared to threshold values known in the art to correspond to therapeutically-effective concentrations; e.g., area under the curve (AUC), half-life, Cmax, and other pharmacokinetic parameters known in the art.
(75) Generally, alleviation or treatment of a disease or disorder involves the lessening of one or more symptoms or medical problems associated with the disease or disorder. Thus, one embodiment of the invention relates to a method of use of novel long-acting vasopressin peptide analogs in sustained release polymeric carrier complexes administered to a patient to increase blood perfusion to various organs in hypovolemic and hypotensive condition and/or increase the level of factor VIII and plasminogen activator in the blood. Exemplary disease or physiologic conditions in which the methods of treatment using the vasopressin peptide analogs of the present disclosure would have utility include, but are not limited to, hypovolemia, splanchnic vasodilation, systemic vasodilation, hypotension, esophageal variccal hemorrhage, hepatorenal syndrome (HRS), type 1 HRS, type 2 HRS, sepsis, liver cirrhosis, portal vein hypertension, esophageal varices, paracentesis-induced circulatory dysfunction, arterial hypotension induced by byproducts of bacteria, anesthesia-associated hypotension, cardiac arrest, and post-partum hemorrhage. Under these conditions a long acting vasoconstrictor such as the vasopressin analogs of the invention will be beneficial for these patients.
(76) In another embodiment, the invention related to a method of use of novel GLP peptide analog compositions. In preferred embodiments, the compositions are GLP-1 analogs with insulinotropic activity. The term insulinotropic activity relates to the ability of a substance to stimulate, or cause the stimulation of, the synthesis or expression of the hormone insulin. Thus, one embodiment ofthe invention relates to administration of novel long-acting GLP-1 analogs in sustained release polymeric carrier complexes to a patient to increase insulin levels in a patient, with concommitant reductions in circulating glucose levels. In one embodiment, the insulinotropic property of the GLP-1 analogs may be determined by administering the analog to a patient and monitoring the release of immunoreactive insulin (IRI) into the circulatory system. The presence of IRI is detected through the use of a radioimmunoassay which can specifically detect insulin or by other methods known in the art. In another embodiment, the therapeutic efficacy of the GLP-1 analogs can be determined by monitoring effects on circulating glycemic variables on single, repeated, or post-prandial or post-glucose-load blood samples and comparing the concentrations to values known in the art to correspond to normal glucose tolerance, impaired glucose tolerance, or diabetes-associated values for fasting and postload glucose and insulin, glycosylated hemoglobin (HbA1c), lipids, as well as insulin resistance parameters. In another embodiment, the invention related to a method of use of compositions of GLP-2 analogs having therapeutic utility in the treatment of diseases of the gastrointestinal tract. In particular, the GLP-2 analogs can act as trophic agents to enhance and maintain the functioning of the gastrointestinal tract and to promote growth of intestinal tissue. The methods and formulations of the present invention preferably provide about 0.1 to about 50 mg/ml of GLP-2 or a biologically active fragment thereof, preferably about 5 to about 40 mg/ml, more preferably about 7 to about 30 mg/ml, even more preferably about 10 to about 20 mg/ml, and most preferably about 20 mg/ml.
(77) The invention may be better understood with reference to the following examples. These examples are intended to be representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention as any peptide can be altered similarly as alteration made to vasopressin and the similar slow activation is expected to be observed on the presence of serum. Occasionally modified peptide depending on the peptide may be active after modification such as GLP-1 modification proposed here (Example 9;
(78) While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.
EXAMPLES
Example 1
(79) The synthesis of CH.sub.3(CH.sub.2).sub.10COGly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH.sub.2 (SEQ ID NO: 34) (also referred to as C12TerA which is a lysine vasopressin analog) with disulfide bonds between the two cysteine residues (Cys4 and Cys9) was done by Anaspec, San Jose, Calif., according to the inventor's specification. The known standard amino acids in the formula above are represented by three letter abbreviations know to those skilled in the art (See
Example 2
(80) The synthesis of CH.sub.3(CH.sub.2).sub.10CO-OHC(CH.sub.3)COGly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH.sub.2 (SEQ ID NO: 35) (also referred to as C12TerE) with disulfide bonds between the two cysteine residues (Cys4 and Cys9) was done by custom peptide supplier Anaspec, San Jose, Calif., according to the inventor's specification. The known standard amino acids in the formula above are represented by three letter abbreviations know to those skilled in the art (see
Example 3
(81) The synthesis of CH.sub.3(CH.sub.2).sub.6COGly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH.sub.2 (SEQ ID NO: 36) (also referred to as C8TerA) with disulfide bonds between the two cysteine residues was done by custom peptide supplier Anaspec, San Jose, Calif., according to the inventor's specification. The known standard amino acids in the formula above are represented by three letter abbreviation know to those skilled in the art (see
Example 4
(82) The synthesis of CH.sub.3(CH.sub.2).sub.6COOHC(CH.sub.3)COGly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH.sub.2 (SEQ ID NO: 37) (also referred to as C8TerE) with disulfide bonds between the two cysteine residues was done by Anaspec, San Jose, Calif., according to the inventor's specification. The known standard amino acids in the formula above are represented by three letter abbreviations know to those skilled in the art (see
Example 5
(83) The synthesis of His-His-His-His-His-His-Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH.sub.2 (SEQ ID NO: 38) (also referred to as His6Ter (His6 disclosed as SEQ ID NO: 52)) with disulfide bonds between the two cysteine residues was done by Anaspec, San Jose, Calif., according to the inventor's specification. The known standard amino acids in the formula above are represented by three letter abbreviations known to those skilled in the art (see
Example 6
(84) The synthesis of His-His-His-Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH.sub.2 (SEQ ID NO: 39) (also referred to as His3Ter) with disulfide bonds between the two cysteine residues was done by Anaspec, San Jose, Calif., according to the inventor's specification. The known standard amino acids in the formula above are represented by three letter abbreviations known to those skilled in the art (see
Example 7
(85) The synthesis of His-His-His-Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH.sub.2 (SEQ ID NO: 40) (referred to as His3Vas) with disulfide bonds between the two cysteine residues were done by custom peptide supplier Anaspec, San Jose, Calif., according to the inventor's specification. The known standard amino acids in the formula above are represented by three letter abbreviations know to those skilled in the art (see
Example 8
(86) The synthesis of His-His-His-His-His-His-Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH.sub.2 (SEQ ID NO: 41) (referred to as His6Vas (His6 disclosed as SEQ ID NO: 52)) with disulfide bonds between the two cysteine residues was done by custom peptide supplier Anaspec, San Jose, Calif., according to the inventor's specification. The known standard amino acids in the formula above are represented by three letter abbreviations know to those skilled in the art (see
Example 9
(87) The synthesis of the GLP-1 analogs SEQ ID NO: 2. SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 additionally containing iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA) at the C-terminal was done by Anaspec, San Jose, Calif., according to the inventor's specification and procedures (the attachment is as shown in
(88) TABLE-US-00003 (SEQIDNO:42) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Asp- Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu- Phe-Ile[[u]]-Ala-Trp-Leu-Val-Lys-Gly-Arg-IDA, (SEQIDNO:43) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Asp- Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu- Phe-Ile[[u]]-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-IDA, (SEQIDNO:44) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Asp- Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu- Phe-Ile[[u]]-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-Gly- IDA, (SEQIDNO:45) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Asp- Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu- Phe-Ile[[u]]-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-Gly- Gly-IDA; (SEQIDNO:46) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Asp- Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu- Phe-Ile[[u]]-Ala-Trp-Leu-Val-Lys-Gly-Arg-NTA; (SEQIDNO:47) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Asp- Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu- Phe-Ile[[u]]-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-NTA; (SEQIDNO:48) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Asp- Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu- Phe-Ile[[u]]-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-Gly- NTA; and (SEQIDNO:49) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Asp- Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu- Phe-Ile[[u]]-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-Gly- Gly-NTA;
(89) The first amino acid was attached to the secondary amino group of iminodiacetic acid or the primary amino group (after de-blocking) of N,N,bis(carboxymethyl)-lysine immobilized in the resin. This was followed by sequential addition of amino acids shown above according to the standard protocol known in the art. It should be noted that this example is not to limit the scope of the invention and that any GLP analog can be processed in a similar manner. It is also understood that in the present invention, the IDA and NTA can be attached to the R-group of any amino acids of GLP-1, except in the first six amino acids (His-Ala-Glu-Gly-Thr-Phe-Thr-Ser (SEQ ID NO: 75)) and in the N-terminal. In one particular example glycine was attached first. The number of glycines may vary from 0 to 3 before addition of arginine. This was followed by Arg, Gly, Lys and so on according to the above sequence. At the end of the synthesis the peptide was cleaved from the resin, deprotected, and purified. In this GLP-1 analog, iminodiacetic acid or nitrilotriacetic acid is at the C-terminal carboxyl group of the peptide. This also facilitates purification of the peptides using metal affinity column. More importantly, this provides additional metal binding domain to GLP-1 that can be used to attach this GLP-1 or its analogs to a protective carrier containing metal chelate. Examples of metal chelate covalently linked to polymeric carrier include, but are not limited to DTPA-Zn.sup.2+, NTA-Zn.sup.2+, DTPA-Ni.sup.2+, and NTA-Zn.sup.2+. Additional carriers with metal chelate are disclosed in U.S. Pat. No. 7,138,105 D2, which is herein incorporated by reference. The attachment to protective carrier provides a slow release of this GLP-1 or its analogs after administration to a patient.
Example 10
Testing of Biological Activity of Various Vasopressin Analogs
(90) Ca-influx after vasopressin analog stimulation of human umbilical chord artery endothelial cells (HUAEC) was used to test the biological activity of the various vasopressin analogs. The material used were: 75 cm.sup.2 tissue culture flasks, BD Bioscience, Bedford Mass. (catalogue number 137787) with 0.2 m vented blue plug seal caps (catalogue number 353136); human umbilical artery endothelial cells (HUAEC), Lonza, Walkersville, Md. (catalogue number CC-2520); EGM-2 fully supplement medium, Lonza, Walkersville, Md. (catalogue number CC-3162); Fura-2 AM, Invitrogen/Molecular Probes, Carlsbad, Calif. (catalogue number F-1221) (Fura-2 AM is a high affinity, intracellular calcium indicator that is ratiometric and UV light-excitable; the acetoxymethyl (AM) ester form is useful for noninvasive intracellular loading as it is cell permeable. Once inside the cell the ester is clipped which renders the Fura-2 molecule cell impermeable and therefore trapped inside; terlipressin derivatives custom made by Anaspec, San Jose Calif., according to inventors specifications; pooled human serum, Sigma, St. Louis, Mo. (catalogue number H4522); phosphate buffered saline (PBS), Fisher Scientific, Fair Lawn, N.J. (catalogue number BP399-500); trypsin-EDTA solution, 0.25%, Invitrogen/Gibco, Carlsbad, Calif. (catalogue number 25200-072); heat inactivated fetal bovine serum (FBS), Invitrogen/Gibco, Carlsbad, Calif. (catalogue number 10438-026); black polystyrene 96 well flat-bottom assay plates, Corning Inc., Corning, N.Y. (catalogue number 3916); Chameleon plate multilabel detection reader, Hidex, Turku, Finland, distributed by Bioscan, Washington, D.C.; dimethyl sulfoxide (DMSO), Fisher Scientific, Fair Lawn, N.J. (catalogue number D-136-1).
(91) The methods used for tissue or cell culture are as follows: a vial of HUAEC (>0.510.sup.6 cells) was thawed and seeded in two 75 cm.sup.2flasks with 15 ml EGM-2 medium each. Cells were grown over night and medium was changed. Media in the flasks were changed on a Monday, Wednesday, Friday schedule. Cells were split at approximately 80% confluency (using trypsin). Media were removed and the flasks were washed with 10 ml 1 PBS. The PBS was removed, 1.5 ml trypsin-EDTA solution was added and incubated for 5 min at 37 C. Three ml of PBS added and the cells were counted. The cells were centrifuged at 200 G for 5 min, resuspended in EGM-2 medium and distributed into new 75 cm2 flasks @3000-5000 cells per cm2.
(92) To prepare for the Ca2+ influx assay, cells were distributed into one black 96 well plate @105 cells/well. Cells were allowed to adhere overnight and the medium was then removed. Fifty g Fura-2 was dissolved in 50 l DMSO and added to 10 ml PBS/2%FBS. The cells were stained with 20 l of 5 g/ml Fura-2 in PBS/2% FBS for 2h, then 180 l EGM-2 medium supplemented with CaC12 and glucose was added (10 mM CaC12 and 11.1 mM glucose final concentrations).
(93) To prepare analogs for testing, 50 uM terlipressin (or derivative) stock solution in DMSO was prepared. Incubation of 30 ml of the drugs (500 nM final concentration) in 3 ml 100% human serum (NOT heat inactivated) for 0, 1, 2, and 24 h was performed at 37 C. (placed on ice after incubation).
(94) To measure Ca-influx, Fura-2 fluorescence was measured for 4 repetitions per well at 340 nm ex/510 nm cm and 420 nm ex/510 nm em (4 measurement before and 16 after injection of drug. At t=12 sec, 20 l of terlipressin or derivative was injected as a 500 nM solution (or serum) for a final concentration of 50 nM per well. Fura-2 fluorescence was measured for an additional 16 repetitions per well at 340 nm ex/510 nm em and 420 nm ex/510 nm em (4 measurements before and 16 after injection of drug). The controls included injected human serum alone, and terlipressin or derivative dissolved in PBS as negative controls. All measurements were done in hexuplicate.
(95) Data analysis was done using Graphpad Prism version 5.0 software (Graphpad software Inc. San Diego, Calif.). Data were normalized by subtracting the baseline readings (the average of the first 4 repetitions of Fura-2 fluorescence measurement before the injection of drugs) from the readings after injection. The results of the experiments are shown in
(96)
(97)
(98)
(99) The amount of C12TerA capable of being bound to a carrier was determined by varying the amount of the analog in the presence of 10 mg/ml hydrophobic group containing carrier PGC-HC18 or 20PLPEG555-C18. The carrier presented in the graph contains 20 kDa polylysine in which 55% of the epsilon amino group of polylysine was derivatized with 5 kDa methoxy PEG and 40% was derivator by stearic acid. As shown in
(100) Table 1 summarizes the results of the experiments in which the test samples were exposed to serum for the indicated periods of time and than assayed according to the methods described above. The results, expressed on a subjective scale as or no detectable activity and + to +++ for slight to high activity, respectively, indicate that terlipressin analogs with a C8 or C12 alkyl group bonded to the amide of terlipressin were able to protect the analog and maintain the biological activity of the activated terlipressin for at least 4h. The results indicated that the addition of the hydrophobic group to terlipressin would be able to prolong the biological half-life of the C12TerA and delay its rapid activation and degradation when administered to a patient.
(101) TABLE-US-00004 TABLE 1 Activity of Various Vasopressin Analogs 0 h* 1 h 2 h 4 h Terlipressin +++ + C8-Amide- +++ +++ +++ +++ Terlipressin C12-Amide- +++ +++ +++ +++ terlipressin C12-Ester- +++ + terlipressin *Time points refer to the amount of time the sample was pre-incubated in pooled human serum at 37 C. After the preincubation time the sample was placed on ice. 0 h time point contains the same amount of serum as the other samples. Samples without serum added showed no activity. Plain serum was used as negative control.
Example 11
Testing of Biological Activity of Vasopressin Analogs In Vivo
(102) Using a rat ear model, a vasopressin analog with metal binding moiety of the present invention, His6Ter (His6 disclosed as SEQ ID NO: 52), was evaluated for it ability to maintain vasoconstriction for an extended period of time. The analog was formulated at a 2% load in polymeric carrier containing 40PLPEG537-Zn-chelate. The peptide analog-polymeric complex was injected subcutaneously into the back of one set of rats, while terlipressin alone was also injected similarly in another set of rats. The ears were examined at baseline and at intervals up to 48h. The ears of rats injected with the peptide analog in polymeric carrier had longer pharmacologic efficacy than the known long acting analog terlipressin as evidenced by the finding that the His6Ter analog (His6 disclosed as SEQ ID NO: 52) plus polymeric carrier-treated rats had ears that remained pale at both the 6 and 24 h intervals, while the unformulated terlipressin-treated rats had ears that were pale at 6 h but had returned to a baseline color with grossly apparent normal blood flow at the 24 h interval.
(103) Using the same model, the effects of delayed release of biologically-active lysine vasopressin from the peptide analog His6Ter (His6 disclosed as SEQ ID NO: 52), with or without complexing in polymeric carrier, was tested. In the experiment, terlipressin was conjugated with a his tag metal binding moiety to result in the His6Ter peptide analog (His6 disclosed as SEQ ID NO: 52). A portion of the His6Ter analog (His6 disclosed as SEQ ID NO: 52) was formulated at 2% loading in 40PLPEG537-Zn-chelate. The three test materials were injected subcutaneously into groups of rats and the ears were examined after 6 and 24 h. Examination revealed that paling of ears was apparent for 24 h after subcutaneous injection of His6Ter (His6 disclosed as SEQ ID NO: 52) formulated m 40PLPEG537-Zn-chelate while His6Ter (His6 disclosed as SEQ ID NO: 52) alone and terlipressin alone show loss of paleness after 6 hours. The results demonstrate that the peptide analog complexed with polymeric carrier was able to result in sustained release of biologically-active lysine vasopressin for an extended period of time compared to terlipressin alone and to the Hs6Ter peptide analog (His6 disclosed as SEQ ID NO: 52) not formulated with polymeric carrier.
(104) Although the foregoing invention has been described in some detail by way of illustration and example for the purposes of clarity of understanding, one skilled in the art will easily ascertain that certain changes and modifications may be practiced without departing from the spirit and scope of the appended claims.
EQUIVALENTS
(105) Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.