Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/10—Tetrapeptides
  • C07K5/1002—Tetrapeptides with the first amino acid being neutral
  • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
  • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5082—Supracellular entities, e.g. tissue, organisms
  • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the invention relates to applying appropriate criteria for identifying molecules that have organ or tissue specificity for use as drugs and/or in targeting therapeutic or diagnostic compounds to desired locations in vivo. More specifically, it concerns a method for establishing the targeting specificity of these molecules and the use of this method to rapidly generate an optimized ligand.
• a second major aspect of the invention relates to compositions consisting of a brain targeting ligand which has therapeutic utility in neuroinflammatory disorders.
• the candidate molecule In addition to exhibiting a desired targeting ratio for the target organ or tissue as compared to other organs or blood, the candidate molecule must have a satisfactory "selectivity index" - i.e., accumulation in the target organ as compared to other non-selective compounds. Furthermore, its clearance from the target organ must be delayed as compared to its clearance from blood and other organs and its clearance as compared to unrelated molecules in the target organ must be substantially slower. Finally the targeting pharmacokinetic profile should be dose responsive.
• peptides bind to a binding site on ⁇ v ⁇ 3 integrin that is distinct from other binding sites that exist on the ⁇ v ⁇ 3 integrin molecule for other ligands, such as the Adam-23 binding site described by Cal, S., et al, Mol. Biol Cell (2000) 11 : 1457-1469.
• Additional examples of the progression from general RGD sequence with broad specificity to a variety of RGD-binding integrin receptor family members, to a constrained peptide with higher affinity and improved specificity to a single family member, to therapeutics are found in Blackburn, B. K., et al., J. Med. Chem. (1997) 40:717-729; Abraham, D. G., et al, Mol. Pharmacol. (1997) 52:227-236; Egbertson, M. S., et al., J. Med. Chem. (1994) 37:2537-2551.
• U.S. patents 5,767,071 and 5,780,426 describe 7-mer and 5-mer cyclic peptide inhibitors of ⁇ v ⁇ 3 . These compounds are based on extrapolation from the RGD motif and include the cyclic peptide RCGGDSMCY. Additional peptides are also described.
• PCT publication WO 01/51508 published 19 July 2001 describes the design of peptide inhibitors of LFA-l/ICAM-1 interaction. These interactions are described in detail by Stanley, P., et al, Biochem. J. (2000) 351:79-86, and the transfer of the structural characteristics to small molecules is described by Gadek, T. R., et al, Science (2002) 295:1086-1089.
• the above examples represent antagonists of the integrin superfamily whose utility is designed to block the blood cell-vascular interactions that underlie responses to intrinsic (e.g., pathological angiogenesis, thrombosis, atherosclerosis or autoimmune responses) or extrinsic (infections, wounds) pathologies.
• This family of ligand mimetics provides a well characterized example of how subtle differences in the presentation of key chemical elements can evolve a low affinity ligand with broad specificity to multiple family members to a ligand with a high affinity and high degree of specificity to one family member.
• Capture and binding of leukocytes and platelets to activated endothelium in an inflamed tissue under normal blood flow involves multiple interactions between receptors expressed on the circulating blood cell and members of the integrin, selectin and immunoglobulin cell adhesion molecules (CAM) superfamilies.
• CAM immunoglobulin cell adhesion molecules
• the generally accepted sequence of events is that the initial rolling and tethering of platelets or leukocytes under conditions of flow is mediated by the selectins, and subsequent firm adhesion is mediated by the integrin receptor interactions with their cognate CAMs within the post capillary venules, most notably, ICAM-1, VCAM-1 and MAdCAM (Carlos, T.M., and Harlan, J.M., Blood (1994) 84:2068-101; Springer, T.A., Annu Rev Physiol (1995) 57:827-872; von Andrian, U.H., N Engl J. Med. (2000) 343: 1020-1034).
• platelet integrins exhibit a multiplicity of interactions that regulate both thrombotic events as well as the pro-inflammatory response of the activated platelet (Plow, E.F. and Ginsberg, M.H. (2000) in Hoffman, R., et al. (Eds.) Hematology: Basic Principles and Practice, 3rd Ed., Churchill Livingstone, New York, NY, pp 1741-1752; McEver, R.P., Thromb. Haemost. (2001) 86:746-756).
• the major challenge in developing therapies that antagonize these reactions is to determine which aspect (i.e., which specific interaction or set of binding sites) most critically impacts clinical aspects of disease.
• immune modulators designed to block leukocyte adhesion and extravasation may show efficacy in peripheral inflammatory disorders, but have had limited effect on neuroinflammatory disorders because of the differing architecture and the nature of the integrated immune response at the blood brain barrier (Davson, H, Segal, M.B., (1998) Physiology of the CSF and blood-brain-barriers. CRC press, Boca Raton; Miller, D.W., J. Neurovirol. (1999) 5:570-578).
• the present invention can identify ligands that recognize the relevant endothelial binding sites as they exist in vivo and may not necessarily be predicted by rational in vitro screening methods currently employed by those skilled in the art.
• the methods of the invention facilitate the discovery of peptidic ligands and ligand mimetic molecules.
• these peptides are useful for the targeting of a variety of therapeutic molecules by direct coupling to a drug entity itself or to a delivery vehicle that encapsulates incorporates or binds the drug.
• Such delivery vehicles might be liposomes, nanoparticles, microspheres, linear or branched polymers, dendrimers, dextran, polylysine, polyglutamatic acid, polyaspartic acid, mixed amino acid polymers, poloxamers, polyethylene glycol, polysaccharides, lipids, glycolipids, phospholipids, neutral lipids, proteins, glycosylated proteins, hyaluronic acid, chondroitin sulfate, polyvinyl alcohol, polyvinylpyrrolidone, poly N-(2-hydroxypropyl) methacrylamidefPHMPA], polystyrene-maleic anhydride copolymer[SMA], polylactic acid, polyglycolic acid, poly(lactic acid-glycolic acid) copolymer [PLGA], cyclodextrin, cyclodextrin derivatives, methacryloylglycinamide [MAG], polyanhydrides, polyorthoesters, polycaprolact
• Such polymers include but are not limited to linear or branched polymers, dendrimers, dextran, polylysine, polyglutamatic acid, polyaspartic acid, mixed amino acid polymers, poloxamers, polyethylene glycol, polysaccharides, lipids, glycolipids, phospholipids, neutral lipids, proteins, glycosylated proteins, hyaluronic acid, chondroitin sulfate, polyvinyl alcohol, polyvinylpyrrolidone, poly N-(2- hydroxypropyl)methacrylamide[PHMPA], polystyrene-maleic anhydride copolymer[SMA], polylactic acid, polyglycolic acid, poly(lactic acid-glycolic acid) copolymer [PLGA], cyclodextrin, cyclodextrin derivatives, methacryloylglycinamide [MAG], polyanhydrides, polyorthoesters, polycaprolactones, polycarbonates, polyf
• a second accepted method for increasing the plasma half-life and thereby exposure of the target tissue to a therapeutic peptide is to couple the peptide to another peptidic moiety by standard molecular recombinant methodologies to generate a fusion peptide or protein with increased molecular weight, since hepatobiliary and renal clearance rate is inversely proportional to molecular weight.
• certain proteins have inherently long plasma half-lives.
• Frir, et al, J. Exp. Med (1967) 126:207 were the first to attribute the long plasma half-life of IgGs to the Fc portion of the molecule.
• the invention in one aspect, relates to an improved method for designing peptide candidates for specific tissue targeting. This method involves providing for randomization of translated nucleotide sequences so that a multiplicity of peptides is obtained.
• the invention is directed to a method to provide a multiplicity of peptide candidates which method comprises effecting expression of a nucleotide sequence putatively designed to encode a peptide, preferably a cyclic peptide of the formula CX n C, optionally extended at the N- and/or C-terminus with additional codons (preferably ⁇ 10 each), wherein C represents cysteine, X represents any gene encoded amino acid, and n is an integer of 2-25, wherein the nucleotide sequence encoding X n is randomized.
• the nucleotide sequence "putatively" designed to encode amino acid sequence X n may include codons which encode additional cysteine residues and/or may include codons which are termination codons.
• the resultant will be a series of cyclic peptides of varying ring size with optional N- and/or C-terminal extensions of additional amino acid sequence of one or more amino acids.
• peptide libraries can be designed to display motifs with terminal acidic or basic functionality. This method may be used to generate a set of preferred candidate compounds for use in further aspects of the invention.
• the invention is directed to a method to validate the targeting ability of a candidate compound which specifically targets an organ or tissue which method comprises administering said compound in vivo to a model animal and determining its pharmacokinetic parameters in accordance with the following criteria; at a minimum, the compound must satisfy at least three of the first four.
• the compound should have
• a selectivity index (SI) for the target organ i.e., the ratio of accumulation of the candidate compound in the target organ as compared to a compound known to be nonspecific for accumulation in that organ
• SI selectivity index
• SI specificity index
• any display platform can be used for this technology, including but not limited to filamentous phage and the T family of phage
• the preferred method is to use a platform with relatively rapid clearance from blood, such as the T7 phage exemplified herein.
• the invention is directed to a method to ascertain structure/activity relationships among compounds which contain at least one variable subunit, such as an amino acid, and wherein said activity is characterized by ability to target a specific organ or tissue
• a method comprises administering to a model animal one variant or a mixture of variants of said compounds wherein the variants contain certain patterns of variation of said subunit, and assessing the pharmacokinetics and tissue or organ retention characteristics of the various compounds in the mixture, preferably according to the foregoing criteria.
• This method enables a ranking of the chemical structural features that are necessary for targeting an organ-specific binding site as it is presented in vivo.
• a representative 3D model of the basic pharmacophore structure can be derived.
• the invention is directed to a compound having the charge/space/hydrophobicity conformational characteristics, i.e., the electronic shape, of the cyclic peptide CAGALCY in at least a portion of said molecule.
• charge/space/hydrophobicity conformational characteristics i.e., the electronic shape
• the compounds included within the scope of these variants are themselves cyclic peptides.
• the invention relates to compounds of the formula Z — A 1 X 1 X 2 X 3 X 4 A 2 X 5 (1) wherein Z represents a non-interfering substituent; X*-X 5 represent independently selected amino acids; and wherein each of A 1 and A 2 represents an amino acid or amino acid derivative or cyclizing moiety wherein A 1 and A 2 are coupled by a covalent bond, wherein said compounds specifically target at least one tissue, most preferably brain.
• Z can represent the coat protein of a bacteriophage or a linker coupled to a drug payload of interest to be directed to the target site.
• the peptide can be synthesized by standard solid support methodologies known by those skilled in the art and any linker modality can be added at the amino terminus of the disclosed peptide during synthesis to facilitate coupling to the drug or therapeutic agent of choice.
• Such linkers can be rationally designed to include flexible spacer linkers (for example additional glycine or alanine residues, short linear aliphatic linkers), and can include additional functional groups to facilitate coupling to the desired therapeutic payload or imaging agent.
• Z can represent a variety of compositions to improve the plasma stability/plasma half-life of the peptide. These compositions include but are not limited to water soluble polymers or the carboxy terminus of proteinacious supports including, but not limited to, Fc or antibody fusion proteins or glutathione-S-transferase.
• the invention is directed to a subset of cyclic peptides of formula (1) having formula (2) wherein X'-X 3 are independently hydrophobic aliphatic residues, the amino acid residue at the X 4 position is N, M or L, and the amino acid at the X 5 position is Y or F.
• X'-X 3 are independently hydrophobic aliphatic residues
• the amino acid residue at the X 4 position is N, M or L
• the amino acid at the X 5 position is Y or F.
• Z A 1 -X 1 -X 2 -X 3 -(N /L)-A ⁇ -(Y F) (2) where Z, A 1 and A 2 are as defined above, and X'-X 3 are independently hydrophobic aliphatic residues, preferably A, G, L, M, V or I.
• these peptides are useful as therapeutics for the treatment of neuroinflammatory disorders when formulated in a suitable pharmaceutical vehicle. More generally, these compositions are useful to treat any inflammatory disorder that involves pathological interactions of activated platelets or leukocytes with vascular endothelium.
• the invention relates to the use of compounds of formula (2) in the treatment of inflammatory disorders and to pharmaceutical compositions of the compound of formula (2).
• Figure 1 shows the selectivity index of the T7-A3 phage isolate for brain compared to two reference organs, lung and liver.
• the T7-A3 phage displays the peptide CAGALCY, that was enriched by in vivo panning to brain.
• Figure 2 shows yield of T7-CAGALCY (originally denoted T7-A3 or just A3) and control phage T7-lacZ from the brain, lung, liver, kidney, spleen and blood at different time intervals following administration (yield is defined as the ratio of the number of phage recovered from a given organ or tissue to the number of phage injected).
• Figure 3 shows the dose response of the selectivity index of T7-CAGALCY for various tissues (the ratio between the yield of T7-A3 to control phage T7 -lacZ obtained from the brain and the reference tissues).
• Figure 4 shows the ability of a GST-fusion protein expressing the A3 peptide at the carboxy terminus to effectively compete with T7-A3 specific binding to brain tissue.
• Figure 5 shows selectivity index for various organs plotted for A3, the A3 variant which is Y7F, the A3 variant C6S, the A3 variant L5P and the variant lacking Y7 ( ⁇ Y7).
• Figure 6 shows graphs of frequencies of the 20 phage variants where position 5 is filled by any one of 20 amino acids in the initial phage mixture (input) compared with the frequency of sequences in phage accumulating in the target organ, brain at 30 minutes and 4 hours.
• One hundred and seven (107) sequences were determined in the phage pool administered (input), and 88 and 94 individual phage from the pools recovered from brain at 30 minutes or 4 hours after administration, respectively.
• Figure 7 shows the distribution among samples analyzed for amino acid at position 7 in a mixture of phage in which all 20 amino acids are provided at this position.
• One hundred eighty-four (184) sequences were determined in the phage pool administered (input), and 173 each of phage pools recovered from brain at 30 minutes or 4 hours after administration to assess the relative shifts in frequency within the target organ.
• Figure 8 shows a head to head comparison of the relative selectivity of the three preferred CAGAXCY variants (L5, N5 and M5) identified by the kSAR analysis presented in Figure 6 to validate their relative targeting activity with respect to an internal standard, the control phage (SI values represent accumulation of the test phage relative to the control phage) over the six hour time period following administration of the phage.
• Figure 9 shows a minimized three dimensional (3D) structure of cyclic acetyl A3 using the PM3 Hamiltonian technique.
• Figure 10 shows a comparison between the structure of Figure 9 and a similarly constructed structure of the cyclic acetyl A3 variant L5P.
• Figure 11 shows a comparison between the cyclic acetyl A3 structure of Figure 9 with a comparably constructed model of a cyclic acetyl A3 variant where the alanine at position 2 is deleted.
• Figure 12 shows a comparison between the cyclic acetyl A3 model of Figure 9 and a similarly constructed cyclic acetyl A3 variant model where alanine at position 2 is replaced by glycine.
• Figure 13 shows the effect of a fusion protein of the A3 ligand on the adhesion of platelets and platelet thrombi in the LPS-induced model of sepsis.
• a first aspect relates to validation of an initial candidate preferably, but not necessarily, selected by in vivo panning wherein the candidate has been identified by its elevated presence in a desired location after one or several rounds of selection.
• in vivo panning a mixture of compounds, generally containing a retrievable tag, is administered to an animal model and allowed to circulate for a predetermined period of time. At that time, the animal is sacrificed and the target site harvested and compounds that have accumulated at that site are recovered. Depending on the initial complexity of the library, the recovered compounds are then either analyzed directly or administered to model animals and the process is repeated as needed. After several rounds of selection, the contents of the desired site with regard to the administered compounds are analyzed and compounds that appear to accumulate at the target site are used as candidates in the method of the invention.
• the candidate compounds are peptides and the retrievable tags used in the methods of the invention are phage. This has the advantage of pennitting ready amplification of peptides that are harvested from particular sites.
• the variety in the candidate library within desirable bounds can be enhanced by generating a set of cyclic peptides where the cyclic peptides are encoded by a nucleotide sequence having codons putatively corresponding to the form CX n C where C is cysteine, X is any amino acid, and n is preferably less than 25, more preferably less than 20, more preferably less than 15, and more preferably 10 or less, but in any event at least 2 and preferably at least 4.
• the codons for these amino acids may be extended by an arbitrary number of codons at the N- or C-terminus upstream of the first or downstream of the second cysteine codon.
• the number of codons in any such extension may typically be 15 or less, preferably 10 or less.
• X is usually an amino acid codon, it may also be a termination codon.
• a multiplicity of other cyclic forms may be generated due to the presence of cysteine codons and/or termination codons at various positions of X.
• a nucleotide sequence of the form CXioC yields a multiplicity of cyclic peptides including truncated peptides with free carboxy termini as a result of the degeneracy of the X codons. Because of enrichment due to successful targeting, the most frequently retrieved peptide from in vivo testing for brain targeting in the example presented below is of the form CX 4 CX.
• the candidate peptides in phage for in vivo panning is very convenient, with respect to the validation testing, in principle, any compound could be used and a retrievable tag employed to recover this compound and analyze it.
• the compounds administered might be labeled with fluorescent beads, including magnetic beads, which could then be recovered and analyzed. If additional amounts of compound are required, depending on the nature of the compound, additional compound might be supplied simply by synthesis.
• the candidate compound must satisfy at least three of four pharmacokinetic parameters that verify its ability to target a particular site.
• These pharmacokinetic parameters include 1) a satisfactory selectivity index (SI); 2) a satisfactory specificity index (SPI); 3) a satisfactory measure of retention designated the area under the curve (AUC); 4) a satisfactory half-life, a measure of the clearance rate from blood or a particular organ.
• the compound also displays dose responsive kinetics.
• the "selectivity index" refers to the level of compound bound to the tissue as compared to the level of a non-specific or irrelevant compound with similar blood pharmacokinetics.
• SI selectivity index
• compound A is the candidate compound, and it is understood that compound B distributes nonspecifically, the ratio of A to B in a desired site at a specified time after administration is the selectivity index of compound A.
• compound A In order to fulfill this criterion, compound A must have a selectivity index > 5, preferably > 10, preferably > 20, preferably > 100 at any time period after administration such as 30 minutes or 1 hour or 2 hours or 4 hours. Satisfaction of this criterion at any of these time points is sufficient.
• the concentration is determined by any convenient method depending on the manner in which the compound is administered. If the compound is a polypeptide and is administered by phage display, formation of plaques can be used as an index of concentration. Other methods can also be used, depending on the manner in which the compound is labeled as will be further described below.
• the "specificity index" (SPI) of a compound for an organ or tissue refers to the ratio of the SI of the compound that is found in the target tissue at a specific time after administration as compared to the SI of that compound in other organs and tissues.
• the specificity index for the candidate must be > 5, preferably > 10, preferably > 20, and more preferably > 100 at any time period after administration such as 30 minutes, 1 hour, 2 hours and 4 hours after administration. Satisfaction of this criterion at any one of these time points is sufficient. With respect to the reference organ for which the value of the ratio is obtained, this is arbitrarily chosen among those organs which do not appear to harbor high concentrations of the compound.
• the area under the curve refers to the area beneath the curve generated when the time after administration is plotted on the x axis of a graph and the concentration of the compound in the relevant tissue or organ at these time intervals is plotted on the y axis.
• the AUC offers a measure of the retention of the material in a particular measured location over a chosen interval of time and provides a measure of the total exposure of the tissue of interest to a compound integrated over time. If the area under the curve is large, the compound is shown to be retained at the target organ. If, however, the compound is rapidly cleared from the location in question, the area under the curve will be small.
• the area under the curve may be enhanced by selective concentration at the location, or simply a reduced rate of clearance.
• both parameters need to be assessed before a conclusion of selective retention at the target site can be drawn.
• specific times after administration must be designated, and again, satisfaction at any one of the time points mentioned above is satisfactory.
• One measure of the "clearance rate” is the "half-life" of the compound from or in blood or an organ or tissue which is defined as the time required for the concentration to be reduced to half. As this parameter is essentially a measure of rate, a specific time point need not be chosen.
• dose responsiveness refers to the dependence of the measured selectively index on the dosage level.
• a satisfactory dose response relationship will be that wherein the selectivity index increases as the level of dosage increases to a maximal plateau, representative of saturable binding.
• AUC area under the curve
• the differential between the clearance rate from the target organ compared to the clearance rate from blood must be substantially slower than for a non-targeting molecule.
• the selectivity index be responsive to dosage. That is, the selectivity index should increase as the level of dosage increases and exhibit hallmarks of saturable binding.
• a candidate compound is administered to an animal model such as a mammalian or avian model of either a normal or disease state and the criteria above determined in order to validate its status as a successful targeting molecule.
• a second aspect of the invention relates to optimizing a targeting molecule with respect to its activity in vivo.
• the kinetic technique of in vivo panning can be used to determine structure activity relationships in an efficient way.
• the effect of variation of certain features of the molecule can be rapidly explored using one of a number of approaches.
• the compound has specific structural subunits or features. If the compound is a polysaccharide, the subunit is typically an individual sugar; if the compound is a peptide, a typical subunit is an amino acid.
• the structural feature may be a functional group or a subset of atoms that is chosen for its spatial or charge contour characteristics.
• this method defines structure activity relationships in a kinetic setting, it can be referred to as a kinetic structure activity relationship (kSAR) determination.
• variants are constructed where one or more of the defined subunits is altered to obtain a single variation of this subunit or to obtain multiple variants thereof which can be assessed simultaneously.
• a single subunit may be replaced by a variant form - e.g., glycine might be replaced by alanine, or a carbonyl group may be replaced by a carboxyl group or a benzene ring replaced by a cyclohexane.
• several such subunits are varied at the same time - i.e., a variant compound obtained where two or more of the subunits are each replaced by a single altered form.
• G could be replaced by S and P by V, or in the peptide C'AGALC ⁇ Y, C 1 might be replaced by homocysteine and L by V.
• C 1 might be replaced by homocysteine and L by V.
• a single subunit is varied but multiple compounds are simultaneously assessed where a multiplicity of replacements for the varied subunit is employed.
• a particular subunit glycine is replaced in one molecule by alanine, in another by serine, in another by tryptophan, and so on, or a carbonyl group is replaced by a carboxylic acid in one molecule, a carboxylic ester in another molecule, an amide moiety in another, and so on.
• kSAR kinetic in vivo panning
• the "space/charge/hydrophobicity conformation" of a molecule or of a portion of a molecule refers to the contours of the atoms and bonds forming the molecule in three- dimensional space (i.e., the space component) and the location of electron density in three- dimensional space (the charge + hydrophobicity components).
• a particularly important feature of the space/charge/hydrophobicity conformation is the bulk emplacement of any hydrophobic regions.
• the overall conformation is dictated by the ability of the peptide to attain a cyclic configuration through disulfide bonding of the two cysteines and the proximity of an aromatic moiety with a free carboxyl group provided by the Y residue.
• Other features of the space/charge/hydrophobicity conformation of this molecule will be apparent from the description below.
• the ability to translate a predetermined space/charge/hydrophobicity conformation into a series of "small molecule" compounds is understood in the art, e.g., as set forth above and is exemplified, for example, by the generation of LFA-1 antagonists using an ICAM-1 immunoregulatory epitope as a model.
• a third aspect of this invention relates to applying the kSAR technique to known peptide mimetics to optimize for the required pharmaceutical properties (such as target specificity, pharmacokinetics) for in vivo functionality.
• Another aspect of the invention relates to the use of cyclic peptides of formula (1), i.e., Z-A 1 X 1 X 2 X 3 X 4 A 2 X 5 and in particular the subset wherein X ! -X 3 are independently aliphatic residues, X 4 is N, M or L , and X 5 is Y or F for targeting a drug or therapeutic agent to brain or lung, to increase exposure at the target site while minimizing exposure of other organs.
• Z can represent a range of substituents without impairing the targeting ability of the parent peptide.
• Z can represent the coat protein of a bacteriophage, and thus facilitate the targeting of a phage particle to the brain, a tag that facilitates imaging of the peptide location, or a polypeptide sequence.
• peptides can be readily made by standard solid phase synthesis methods where an additional functional group can be incorporated to facilitate coupling to the carrier, drug or imaging agent of choice.
• Z can represent a linker, including but not limited to additional glycine or alanine residues or a short chain aliphatic molecule, such as aminocaproic acid, to provide a flexible linker/spacer, functional groups such as amino, carboxyl, sulfhydryl, esters, aldehyde, hydroxyl, etc., to facilitate conjugation of said peptide to a drug entity or any desired a water-soluble polymer formulated in a suitable pharmaceutical vehicle.
• a linker including but not limited to additional glycine or alanine residues or a short chain aliphatic molecule, such as aminocaproic acid, to provide a flexible linker/spacer, functional groups such as amino, carboxyl, sulfhydryl, esters, aldehyde, hydroxyl, etc.
• Such polymers include but are not limited to linear or branched polymers, dendrimers, dextran, polylysine, polyglutamatic acid, polyaspartic acid, mixed amino acid polymers, poloxamers, polyethylene glycol, polysaccharides, lipids, glycolipids, phospholipids, neutral lipids, proteins, glycosylated proteins, hyaluronic acid, chondroitin sulfate, polyvinyl alcohol, polyvinylpyrrolidone, poly N-(2-hydroxypropyl)methacrylamide[PHMPA], polystyrene-maleic anhydride copolymer[SMA], polylactic acid, polyglycolic acid, poly(lactic acid-glycolic acid) copolymer [PLGA], cyclodextrin, cyclodextrin derivatives, methacryloylglycinamide [MAG], polyanhydrides, polyorthoesters, polycaprolactones, polycarbonates, polyfum
• the peptide structure can be modified to generate a more stable molecule.
• the amino terminus can be modified by electrophiles to diminish proteolytic degradation.
• the amino terminus can be transformed by various reactions including but not limited to alkylation, acylation, oxidation, and carbamylation
• the intramolecular disulfide bond can be modified or replaced to generate a more stable cyclic structure.
• One or both sulfur atoms could be replaced by other moieties including but not limited to alkyl, amide, ketone, hydroxyalkyl, alkene, alkyne, imine, amine, alkylamine, ether, ester, urea, carbamide, carboxamide, or carbamate.
• the drug agent can include, but is not limited to any from the following groups: chemotherapeutic agents, immune modulators, 5HT agonists and antagonists, NMDA antagonists, neuralgesics including opioid analgesics, steroids, psychotropic agents and anxiolytics.
• imaging agents could also be coupled through a linker for diagnostic purposes.
• the agents can be selected from the following non-limiting examples; vasoconstrictors, vasodilators, bronchoconstrictors, bronchodilators, anti-neoplastic agents, surfactants, steroids, antibiotic agents, antioxidants and antiproteases, or a combination thereof.
• Pulmonary specificity can be readily enhanced by formulation in a suitable carrier for nebulizer, dry powder inhaler or metered dose inhaler mediated delivery to the airways.
• Another aspect of the invention relates use of the candidate targeting molecule, A3, predicted from the modeling of the targeting peptide and evaluation of its structural similarities or differences to known families of ligand mimetics for receptor superfamilies. Specifically, as will be shown in the examples below, an aspect of the invention relates to the use of cyclic peptides of formula (1) or the subset of formula (2), as therapeutics in the treatment of neuroinflammatory disorders when formulated in a suitable pharmaceutical vehicle.
• Such applications include but are not limited to hemorrhagic shock, ischemic reperfusion injury, stroke, vascular dementia, Alzheimer's Disease, multiple sclerosis, cerebral malaria, meningitis as well as other brain disorders secondary to septicemia or trauma, traumatic brain injury, cerebral edema, and inflammatory sequelae of epilepsy.
• Pulmonary inflammatory indications include asthma, chronic obstructive pulmonary disease, pulmonary embolism, pulmonary edema and acute lung injury. More broadly these compositions may have applications in the treatment of any inflammatory disorder that involves pathological interactions of activated platelets and platelet-complexes with vascular endothelium.
• the invention includes the peptides sequences disclosed formulated on a carrier such water-soluble polymer formulated in a suitable pharmaceutical vehicle as described above.
• the disclosed peptides can be formulated in a variety of agents for controlled release from slowly dissolving or eroding formulations including but not limited to alginates, polylactic-co-glycolic acid [PLGA], polylactic acid, polyglycolic acid, polyanhydrides, polyorthoesters, polycaprolactones, polycarbonates, polyfumarates, liposomes, nanoparticles, microspheres, cyclodextrin, or hydrogels.
• a fusion protein expressing the peptide at the carboxy terminus such that the ligand is appropriately presented can be generated by standard molecular cloning techniques. These can include Fc domains or other antibody derivatives in a suitable pharmaceutical vehicle.
• Dosage ranges are dependent on the nature of the condition, the susceptibility of the subject, and the judgment of the practitioner. Suitable subjects include human and veterinary subjects, both avian and mammalian. Routes of administration are those generally suitable for peptides or small molecules designed based on the space/charge/hydrophobicity conformation of the compounds of formula (2), and more specifically on the space/charge/hydrophobicity conformation based on compound A3 - i.e., CAGALCY in cyclic form.
• Suitable formulations for various administration routes may be found, for example, in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA.
• Routes of administration include transdermal, transmucosal, oral, intravenous, intramuscular injection, subcutaneous injection and the like. Formulations can readily be designed for any of these administration routes.
• the in vivo panning techniques used in this example are those described, for example, in U.S. patent 6,068,829 referenced above and incorporated herein by reference. Briefly, in order to isolate peptides that target brain vasculature, including peptides that enable T7 phage to cross the blood-brain barrier, peptides displayed on the coat proteins of T7 phage are administered to the tail vein of murine subjects and the phage allowed to circulate for a suitable time period, typically 1 hour, to allow time for sufficient clearance of non-binding phage from the circulation for a more accurate determination of specific binding events. After that time, the mice are sacrificed and the brain tissue harvested and homogenized.
• Phage are recovered from the brain, amplified and re-injected for succeeding rounds of selection. After a suitable number of rounds of selection, phage present in the harvested brain tissue are recovered and sequenced. The presence of multiple phage plaques containing similar or identical sequences is evidence that the enriched sequence is successful in targeting the brain vasculature.
• a "CX ⁇ oC-T7" phage library was constructed so that the peptides were displayed on each of the 415 copies of the 10B co-protein of the T7 phage surface.
• oligonucleotides with suitable redundancies in the genetic code were designed to encode a 12 amino acid peptide containing cysteines at the N- and C-termini.
• additional peptides could be obtained.
• codons would be present at various locations which encode additional cysteines and which encode termination codons.
• oligonucleotides were gel purified and ligated to T7 (415-1) vector and mixed with packaging extract to obtain a library.
• the library was amplified in the BLT-5615 bacterial host strain in the presence of 1 mM IPTG.
• the resulting library was purified using standard purification techniques and then used for panning studies.
• An aliquot of the phage library was injected into the tail veins of female FVB/N mice, allowed to circulate for varying lengths of time, before anesthetizing the mice and perfusing the organs with PBS to remove the excess unbound phage.
• the brains were harvested, rinsed with cold PBS, and homogenized in 500 ⁇ l PBS containing 0.5% NP-40 and 2% BSA with a Dounce homogenizer.
• Plaque assays were used to determine the relative amounts of phage contained in blood and brain. For the blood assays, 300 ⁇ l of blood was drawn into 300 ⁇ l of 100 mM sodium citrate before perfusion. Plasma was recovered and saved for plaque assay. The phage content was assessed by mixing aliquots of the tissue homogenate or plasma with the bacterial BLT-5615 host, then plating on agar plates.
• a total of 76 phage were picked from the fifth round of selection and the inserts amplified by PCR and sequenced. Of these, a total of 30 represent the sequence CAGALCY.
• a mixture of the two phage (10 9 pfu of each phage) was injected into the tail veins of non-anesthetized BALB/c mice.
• FIG. 1 shows the ratio of A3 to control phage (the selectivity index) obtained in the brain compared to two reference tissues, lung and kidney (particulates can readily marginate in lung and kidney is one of the sites of clearance for small blood borne particulates). This indicates that A3, which exhibited a slightly shorter half-life than control phage targets brain selectively. Another measure of selective retention is to compare target organ to blood levels of the test phage. A3 showed a high brain to blood ratio (» 100-fold after normalization to control phage) compared to other organs.
• the peptide CAGALCY has a high selectivity index when compared to non-specific, LacZ-T7, a high specificity index for brain with respect to other organs or blood, and clears from brain much more slowly than from blood, but clears from blood substantially at the same rate as L ⁇ cZ-T7.
• the control phage clears from brain with similar pharmacokinetics as its clearance from blood. This selectivity is dose-responsive.
• A3-T7 was amplified in the host strain BLT-5615 in the presence of 1 mM IPTG and purified.
• T7-l ⁇ cZ phage were amplified by large scale plating on LB plates. The plates were incubated at 25°C overnight, and phage were eluted with phage extraction buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 6 mM MgSO 4 ) overnight at 4°C with gentle rocking. The extracted phage were pooled and purified.
• phage extraction buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 6 mM MgSO 4 ) overnight at 4°C with gentle rocking. The extracted phage were pooled and purified.
• three mice were used per time point, co-injected with 1 x 10 10 pfu of each phage. Time points were 2 min, 10 min, 30 min, 1 hr, 2 hr, 4
• FIG. 2 shows yield/g for each organ and yield/mL obtained in blood.
• T7-A3 is rapidly cleared from the blood, with a half-life of 8.9 min.
• the T7 -lacZ control phage shows a slightly longer half-life of 14.7 min.
• T7 -lacZ was cleared faster from all the organs examined compared to the T7-A3 phage, indicating the T7-A3 phage has a greater tissue retention than the control T7-/ ⁇ cZ phage in these organs. However, the most marked accumulation was in brain.
• the T7-A3 phage also appeared to accumulate slightly in the first 30 min, before steadily declining. In the spleen and liver, the amount of both T7- A3 and T7 -lacZ phage declined steadily after the first time point (2 min). At every time point examined, more T7-A3 phage than T7-lacZ control phage were found in all organs examined in this study. However, the greatest differential retention was observed in the brain with a selectivity index (SI) of 51 at 2 hr and 280 at 4 hr. This compared to SI values ranging from 1.98 in spleen to 16.0 in lung at 2 hr, and from 4.62 in liver to 54.8 in lung at 4 hr.
• SI selectivity index
• evaluation of the SI value for brain compared to lung at the 4 hour time point provides a value of 288 for brain and 47 for lung, which indicates the relative specificity of the T7-A3 isolate for brain is 6-fold.
• arbitrarily picking a single time point especially when plasma levels of phage are high, can be misleading in the evaluation of organ targeting properties.
• T7-A3 phage and T -lacZ control phage were injected into FVB female mice: 1 x 10 7 , 1 10 s , 1 10 9 , 1 x 10 10 , and 1 10 11 pfu of each phage.
• 3 mice were injected.
• phage were recovered from the brain, lung, liver, kidney, colon, and blood.
• Figure 3 shows the effect of phage dose on the selectivity index values of the T7-A3 phage for various organs. This was measured as A3-T7 phage yield per gram tissue from various organs normalized by the yield/g tissue of the control phage.
• the selectivity index (SI) given in Table 2 represents the ratio of the yield of the targeting phage, T7-A3 to the yield of control phage, T7-lacZ.
• Table 3 shows the organ specificity index (SPI) which represents the ratio of SI values of brain to the various reference tissues.
• Selectivity index values for the brain and various reference tissues mean + SEM values of the data resented in Fi ure 3
• SI values for T7-A3 in brain increased from 13.2 for 1 x 10 7 pfu/phage and 10.5 for 1 x 10 8 pfu/phage to 121 for 1 x 10 9 pfu/phage and 150 for 1 x 10 10 pfu/phage.
• Targeting to lung, kidney, liver and colon tissues appears to be dose-dependent in the doses ranging from 1 10 7 pfu/phage to 1 x 10 9 pfu/phage.
• These changes in SI values were all due to changes in the yield/g tissue of the test A3-T7 phage.
• the yield/g tissue of the control phage T7-lacZ recovered from the various organs in contrast, was independent of the dose in all the organs examined.
• SPI values i.e., ratio of SI
• SPI value for brain/liver, brain/kidney, and brain/colon were greater than 10 at doses of 1 x 10 10 pfu and 1 10 11 pfu.
• SPI value for brain/liver at the 1 x 10 9 pfu dose was 26, the only SPI value greater than 10 at this dose.
• the SPI values for lung, liver, kidney or colon compared to the other organs were all lower than 10 at all doses tested.
• T7-A3 phage appeared to be retained in the brain throughout the time course examined from 2 min to 24 hr, and only showed a very slow decline after 2 hr post injection. At this time point, the number of T7-A3 phage in the blood had decreased more than 3 logs. Retention of T7-A3 phage in the brain may be the result of both interaction of T7-A3 phage with brain and translocation of T7-A3 phage into the brain tissue
• T7-A3 phage was cleared more slowly than T -lacZ control phage in all tissues examined, T7-A3 phage exhibits the longest tissue half-life in the brain (498 min vs. 43 min for T7-lacZ phage).
• the half-lives of T7-A3 phage in other tissues are 330, 78, 468, 348 min for the lung, liver, kidney, and spleen respectively.
• the corresponding half- lives oTT7-lacZir ⁇ these tissues are 18, 50, 270, 221 min.
• Targeting of T7-A3 to the brain was dose-dependent.
• the brain SI values increased significantly from 10 for the 1 x 10 8 pfu dose to 121 and 150 for the 1 x 10 9 pfu and the 1 x 10 10 pfu dose respectively. Further increasing the dose to 1 x 10 11 pfu did not increase the SI value, indicating the binding sites in the brain tissue may be saturated at this dose.
• the binding of T7-A3 phage to the various reference organs also appeared to be dose-dependent at lower doses, but, the SI values are all lower than for brain.
• the ability of the displayed peptide to recapitulate the brain targeting properties outside the context of the T7 phage particle was tested.
• the first approach was to generate a glutathione (GST)-A3 -fusion protein, where the peptide was displayed at the carboxy terminus of GST.
• the insert encoding the CAGALCY sequence was subcloned into the pGEX-5x-3 vector (Pharmacia Biotech), transfected into the JM 109 host bacteria and individual clones amplified to confirm the construct by sequencing.
• the BL21 strain was used for protein expression. Overnight cultures were diluted 1:100 in 2xYT broth and incubated at 30 C/250 rpm till the OD 60 o reached 0.5.
• IPTG was added to the culture to a final concentration of 1 mM to induce GST fusion protein expression and the culture incubated for a further 3 hrs.
• the cell pellets were suspended in PBS containing 100 ⁇ g/mL lysozyme and 10 ⁇ g/mL of DNase I. The suspensions were sonicated for 30 min in an ice-water bath.
• GST fusion proteins in the supernatants were purified using a Glutathione Sepharose 4B gel column according to the manufacturer's instructions (Pharmacia Biotech, but washing the column extensively with 1% detergent buffer to minimize endotoxin contamination). Fusion proteins were eluted with reduced glutathione, and dialyzed against PBS overnight.
• mice were dosed with either 50 or 500 ⁇ g of GST-A3-fusion or GST control protein followed by administration of 1 x 10 10 pfu T7-A7 plus T7 -lacZ control phage. Tissues were harvested after 2 hours and the content of each phage type determined by plaque analysis. While the GST fusion protein had no effect at either dose on the total yield of T7-A3 phage or SI value, the presence of 50 ⁇ g GST-A3 reduced the yield of T7-A3 recovered from brain by 90% and the 500ug dose reduced the yield of T7-A3 phage to the level of control phage as reflected by SI values of 1.
• Curve fit analysis indicated that monomeric GST- A3 fusion protein competed binding of the multivalent phage with an apparent ED 50 of 450 nM, confirming that the brain targeting activity resided within the displayed A3 peptide. This together with additional information presented below, indicates that the brain targeting activity resides entirely within the displayed peptide, and suggests that a variety of constituents can be substituted for the phage coat protein at the amino terminus without impairing this activity.
• a control peptide lacking the terminal essential tyrosine was used as a negative control. After 1 hour, the brain tissue was harvested and phage content analyzed by plaque analysis. The results indicated that the free peptide reduced T7-A3 by 90% to levels approaching those of the control T7-lacZ phage, i.e., the free synthetic peptide could also compete for T7-A3 phage binding to brain tissue. Taken together the data demonstrate that the activity of the A3 ligand can be readily transferred to any moiety providing the free aromatic-acid functionality is maintained.
• CAGALCY-X (8-12) destroys targeting activity indicating the necessity of a free carboxyl group at this position for targeting activity.
• distorting the general shape of the cyclic peptide results in loss of targeting.
• deletion of the tyrosine residue at position 7 destroys the brain targeting activity, since similar peptides lacking this residue fail to have high selectivity indices in brain.
• cysteines is changed to serine, thus preventing cyclization (CAGALSY) or when proline is introduced at position 5 (CAGAPCY) brain targeting capacity is lost.
• mice were anesthetized, perfused and blood and brain were harvested.
• a total of 107 individual phage from the initial mixture used for dosing were sequenced to verify the relative abundance of the individual phage within the administered dose and these were compared with the relative frequency of the different phage recovered from brain after 30 minutes or 4 hours of circulation. There was a marked and rapid shift in frequency even by 30 minutes, where there was a marked enrichment of phage expressing N at the X5 position, (from 7% in the initial pool to 49%), M (from 5% to 24%) or L (2.8% to 12%) at 30 minutes. By 4 hours, these were the only three phage recovered from brain at a frequency of 55%, 37% and 7.5% for the N, M and L variant, respectively.
• Figure 7 shows the results. As demonstrated, only the tyrosine and phenylalanine variant showed any accumulation within the brain. All other variants except the histidine and tryptophan variants were rapidly eliminated from the target site. These data indicate that phenylalanine is a reasonably successful substitute for tyrosine at position 7, but no other amino acid in this group is able to replace the tyrosine successfully, and that a carboxy-terminal aromatic residue displaying a free acid is required at this position for brain targeting activity.
• a CXtCY library was prepared and tested. 1 x 10 10 pfu of the library phage were injected into 6 BALB/c mice (3 mice/time point). At 30 min and 4 hr post-injection, brain was harvested and processed as described above. Similar numbers of plaques were picked individually, and the insert sequence amplified by PCR and sequenced. Alignment analysis of the sequence data was performed with a version of Clustral/W.
• Table 5 shows the distribution of peptides recovered. There were no enriched phage with the A3 peptide or with CAGANCY or CAGAMCY within the 83 sequences sampled from the brain at 4 hrs post phage injection. However, phage expressing sequences of CXGXLCY, where X is predominantly valine, were enriched. Since the CXGXLCY motif, where X represents a hydrophobic amino acid, consistent with the structural requirements determined in the previous examples for brain targeting activity, accounts for 57% of phage recovered from the brain, whereas only 2% of the same motif was found in the unselected library, it can be concluded that the selection of brain targeting phage in vivo is highly effective even with just one round of panning. Table 5
• MOPAC Technique a semi-empirical molecular orbital methodology optimized for calculating heat of formation energies for small to medium size molecular systems.
• Figure 12 shows a comparison similar to that of Figures 10 and 11 for acetyl cyclic A3 with acetyl cyclic A3 analog A2G. While these structures appear similar, as shown above, this variant fails to exhibit brain targeting capability.
• Platelets were isolated from blood of source animals by differential centrifugation at 200g for 10 minutes, to pellet erythrocytes and leukocytes. The supernatant, designated platelet-rich plasma contained less than 0.5% leukocytes as determined by the number of DAPI-positive nucleated cells. The platelets were pelleted by spinning at 500g, resuspended in Tyrode's buffer and incubated for 20 min with 5-,-6-carboxyfluorescein diacetate, succinimidyl ester (CFDA, or CFSE). lxlO 8 platelets in 0.1 ml were injected slowly over 5 minutes via a carotid catheter into anesthetized mice.
• CFDA succinimidyl ester
• the body temperature was maintained at 37°C by a heating pad with a rectal thermometer. Platelet rolling and adhesion was recorded for off-line analysis onto videotape or onto hard-drive of the computer via an intensified camera (Cascade, Roper Scientific) on a Leica DM-FSA upright microscope. The pial vessels were visualized by surgically developing a small window in the skull. Leukocytes are fluorescence-labeled in vivo by injecting Rhodamine 6G via a carotid catheter and analyzing the movement of the labeled cells on brain endothelium.
• a fusion protein of the CAGALCY ligand or GST-null control protein For the platelet studies a known antagonist of platelet-platelet interactions, IntegrilinTM (Cor Therapeutics) was also evaluated.
• a neutralizing anti-ICAM-1 antibody was used for the labeled leukocyte studies.
• the fusion protein expressing the CAGALCY ligand effectively inhibited platelet adhesion in this DIC model of sepsis (PO.0001).
• the GST control protein had no effect, and Integrilin, administered at the clinical dose per mouse (40 mg/kg), showed a slight antagonistic effect but this was not statistically significant. Indeed at higher doses Integrilin exacerbated DIC and adhesion of microaggregates to the brain vasculature.
• the A3-GST fusion protein had no effect on the adhesion of labeled leukocytes to the pial vessels. Only the anti-ICAM-1 antibody was able to completely reverse leukocyte adhesion. [0123] P.
• mice results in marked platelet and leukocyte adhesion in the pial vessels on day-6 of infection the time point when infected mice develop impaired consciousness and are moribund. Similar effects of the GST- A3 fusion protein but not the GST-null protein on blocking platelet and microthrombi adhesion to the pial vessels were observed.
• Example 10 A3 Functions as an Antithrombotic
• FeCl 3 ferric chloride induced thrombosis model.
• a 5% Fe Cl 3 solution was applied to the lower surface of a carotid artery, to which a modified flow probe had been attached. Flow was monitored at 1 minute intervals and time to occlusion measured using a Transonic System. Groups of 6 mice were treated with either GST control protein or the GST- A3 fusion protein at a dose of 1.25 mg/kg just before applying the FeCl solution.

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