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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
  • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
  • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
• • 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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
• • 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
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
  • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

• the invention generally relates to Cysteine-X-Cysteine Chemokine Receptor 4 ("CXCR4" or "CXC chemokine receptor 4"), and more particularly, to binding compounds for CXC chemokine receptor 4.
• Methods of the invention are useful for the treatment of disease by identifying and preparing binding compounds for CXC chemokine receptor 4.
• Chemokines comprise a family of structurally related secreted proteins of about 70-110 amino acids that share the ability to induce migration and activation of specific types of blood cells. See Proost P., et al. (1996) Int. J. Clin. Lab. Rse. 26: 211-223; Premack, et al. (1996) Nature Medicine 2: 1174-1178; Yoshie, et al. (1997) J.
• Leukocyte Biol. 62: 634-644 Over 30 different human chemokines have been described to date. While they are primarily responsible for the activation and recruitment of leukocytes, they vary in their specificities for different leukocyte types (neutrophils, monocytes, eosinophils, basophils, lymphocytes, dendritic cells, etc.), and in the types of cells and tissues where the chemokines are synthesized. Further analysis of this family of proteins has shown that it can be divided up into two further subfamilies of proteins. These have been termed CXC or ⁇ - chemokines, and the CC or ⁇ -chemokines based on the spacings of two conserved cysteine residues near the amino terminus of the proteins.
• Chemokines are typically produced at sites of tissue injury or stress, where they promote the infiltration of leukocytes into tissues and facilitate an inflammatory response. Some chemokines act selectively on immune system cells such as subsets of T-cells or B lymphocytes or antigen presenting cells, and may thereby promote immune responses to antigens. In addition, some chemokines have the ability to regulate the growth or migration of hematopoietic progenitor and stem cells that normally differentiate into specific leukocyte types, thereby regulating leukocyte numbers in the blood.
• chemokines are mediated by cell surface receptors that are members of a family of seven transmembrane ("7TM"), G-protein coupled receptors ("GPCR"). At least twelve different human chemokine receptors are known, including CCRl, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CXCRl, CXCR2, CXCR3, and CXCR4. These receptors vary in their specificities for specific chemokines. Some receptors bind to a single known chemokine, while others bind to multiple chemokines.
• chemokine receptors such as CXCR4
• HIN Human Immunodeficiency Nirus
• Chemokines are important in medicine because they regulate the movement and biological activities of leukocytes in many disease situations, including, but not limited to: allergic disorders, autoimmune diseases, ischemia/reperfusion injury, development of atherosclerotic plaques, cancer (including mobilization of hematopoietic stem cells for use in chemotherapy or myeloprotection during chemotherapy), chronic inflammatory disorders, chronic rejection of transplanted organs or tissue grafts, chronic myelogenous leukemia, and infection by HIN and other pathogens
• CXCR4 in particular, has been implicated in diseases such as glioblastoma multiforme tumor, hepatocellular carcinoma, colon cancer, esophageal cancer, gastric cancers, breast cancer metastasis, pancreatic cancer and in renal allograft rejection.
• Antagonists of chemokine receptors may be of benefit in many of these diseases by reducing excessive inflammation and immune system responses.
• chemokines and antagonists that bind to HIV co-receptors may have utility in inhibiting viral entry into cells.
• HIV causes Acquired Immune Deficiency Syndrome ("AIDS"), which is one of the leading causes of death in the United States and throughout the world.
• AIDS Acquired Immune Deficiency Syndrome
• According to the Center for Disease Control at least 30.6 million people world- wide have been infected with HIV. HIV attacks the immune system and leaves the body vulnerable to a variety of life- threatening illnesses. Common bacteria, yeast, and viruses that would not cause disease in people with a fully functional immune system often cause these illnesses in people affected with HIV.
• CD4 + lymphocyte count a patient having a count of less than 200 cells/ ⁇ l is considered to have AIDS.
• the CD4 protein is a glycoprotein of approximately 60,000 molecular weight and is expressed on the cell membrane of mature, thymus-derived (T) lymphocytes, and to a lesser extent on cells of the monocyte/macrophage lineage.
• CD4 cells appear to function by providing an activating signal to B cells, by inducing T lymphocytes bearing the reciprocal CD8 marker to become cytotoxic/suppressor cells, and/or by interacting with targets bearing major histocompatibility complex (MHC) class II molecules.
• MHC major histocompatibility complex
• the host immune system will produce antibodies targeted against various antigenic sites, or determinants, of gpl20. Some of those antibodies will have a neutralizing effect and will inhibit HIV infectivity. It is believed that this neutralizing effect is due to the antibodies' ability to interfere with HIV's cellular attachment. It is also believed that this effect may explain in part, the rather long latency period between the initial seroconversion and the onset of clinical symptoms.
• T-tropic T cell tropic
• M-tropic macrophage tropic
• CXCR4 CXCR4
• the second extracellular loop is the region most required for the entry of HIV into the cell; however, the N-terminus and the third extracellular loop are also involved.
• CXCR4 CXCR4-binding protein
• T22 and T140 small molecule inhibitors
• ALX40-4C and T134 small molecule inhibitors
• AMD 3100 a heterocyclic bicyclam (one positive charge on each of two rings) has been reported as well. See e.g., Tamamura H, et. al, Bioorg Med Chem 6(7):1033-41(1998); Tamamura H, et. al, Biochem. Biophys. Rs. Comm. 252:877-82 (1998); Doranz BJ, et. al., JExp Med 186(8): 1395-400 (1997); Arakaki R, et. al, J. Virol.
• PBMCs Peripheral Blood Monocytes
• bicyclams may interfere with other CXCR4-like receptors. See e.g., Schols D, et. al, J. Exp. Med. 186(8):1383-1388 (1997).
• T22 inhibits calcium mobilization, therefore interfering with CXCR4's natural required signaling.
• HIV uses the CXCR4 as a co-receptor for cellular entry that can be blocked by its natural ligands and this makes a high affinity ligand for CXCR4 an important therapeutic target.
• GPCRs in general, and CXCR4 in particular, are very difficult to solubilize and purify because they normally need to fold and be maintained in the presence of the native lipids of the cell membrane.
• the present invention provides binding compounds for CXCR4 and methods for identifying those binding compounds.
• screening methods are provided to identify binding motifs for CXCR4, as well as ligands capable of binding to CXCR4.
• the invention comprises the design and identification of therapeutic peptides, peptidomimetics, or small molecules suitable for use in the prevention or treatment of HIV and AIDS.
• methods of the invention provide for the synthesis and purification of linear and cyclic peptide libraries useful for screening and identifying a binding motif for CXCR4, as well as screening for potential ligands thereof.
• Methods of the invention provide for the incorporation of unnatural amino acids and amino acids of the D configuration into linear or cyclic peptides for use in such libraries. Libraries comprising peptides having such amino acids demonstrate enhanced binding affinity and duration of action in vivo resulting from resistance to proteolysis.
• the invention provides for the use of highly diverse libraries of peptide (linear and cyclic, natural and unnatural amino acids), peptidomimetic, and small molecule compounds for the lead ligand identification step.
• ligands may be directly or indirectly agonistic or antagonistic to CXCR4 binding activity.
• the invention provides for the use of phage display methods for the identification of preliminary motif information, followed by additional rounds of affinity purification with purified receptor preparations of the invention and highly diverse libraries.
• phage display technology is combined with the use of cyclic peptide and/or peptidomimetic libraries.
• computer-aided design technology is used to virtually screen, identify, design, or validate lead compounds for agonistic or antagonistic potential with regard to CXCR4 activity.
• Such technology uses computer-generated, three- dimensional images based upon molecular and structural information of both the CXCR4 and the potential binding partners by virtually aligning the protein with the binding partners.
• potential leads are identified by prior screening of an actual library or through some other means.
• One embodiment of the invention involves the screening of biologically appropriate drugs that relies on structure based rational drug design.
• a three dimensional structure of the protein (or similar family member), peptide or molecule is determined and potential agonists and/or antagonists are designed with the aid of computer modeling.
• the drug is contacted with CXCR4, whereby a binding complex is formed between the potential drug and CXCR4.
• Methods of contacting the drug to CXCR4 are generally understood by anyone having skill in the art of drag development.
• the present invention provides for the use of partially purified CXCR4 receptor protein as the agent for carrying out the selection, identification, and improvement of tight binding ligands in identifying therapeutically useful compounds.
• the invention comprises the use of tagging methods to generate a modified CXCR4 receptor protein that functions to facilitate purification and identification steps involved in the screening methods.
• the invention comprises a nucleic acid sequence corresponding to the receptor CXCR4 fused to tag sequences (i.e., GST, FLAG, 6xHis, dual tagged with FLAG-GST, C-MYC, MBP, V5, Xpress, CBP, HA) with appropriate specific protease sites engineered into the vector.
• methods of the invention provide for solubilization and immobilization of CXCR4 to facilitate ligand selection methods provided herein.
• CXCR4 may be derived from any source, including without limitation: inactive, precipitated protein preparations; cell membrane preparations; and, whole cell preparations.
• the invention provides for a method of screening combinatorial libraries directly for general affinity determination using membranes from baculo virus expression systems or any other appropriate expression system.
• partially purified CXCR4 is used in carrying out the selection, identification, and improvement of tight binding ligands.
• partially purified, tagged CXCR4 is used in a sequestered form to screen diverse libraries (focused or highly diverse) for the affinity purification of a tight binding ligand.
• the conditions for solubilization and immobilization of the appropriate ligand provide for the use of low salt, such as, for example, low magnesium or calcium conocentrations; and no sodium chloride (“NaCl”) (O.OnM NaCl).
• low salt such as, for example, low magnesium or calcium conocentrations
• NaCl no sodium chloride
• the invention comprises the step of eluting bound components of the libraries from the immobilized protein with specific N-terminally blocked peptides or other non-sequencable analogs.
• the invention comprises the step of binding combinatorial libraries to a resin-immobilized protein.
• the invention comprises a purified polypeptide with tag sequences, which may be immobilized onto an appropriate affinity resin for assay.
• a further embodiment comprises the step of releasing or eluting tagged protein with its bound library with specific N-terminally blocked peptides or other non-sequencable analogs.
• a method of the invention comprises the step of cleaving a tag from a protein of interest using a specific protease (as designed into the protein/vector) after immobilization onto an affinity resin and after the combinatorial library is bound to release the complex.
• the target ligand is selected from a linear peptide library, a peptidomimetic library, a cyclic peptide library, or a focused library developed using an initial motif identified by phage display techniques or a library combining any of the foregoing.
• a target ligand is eluted from the receptor preparation using a peptide or other ligand, or by using pH change or chaotropic agents, such as urea or guanidine hydrochloride, that can disrupt the hydrogen bonding structure of water and denature proteins in concentrated solutions by reducing the hydrophobic effect.
• ligands for CXCR4 identified using the methods disclosed herein.
• protein sequencing techniques are used for the determination of the structure of the ligand identified by the affinity purification step.
• the invention comprises therapeutic agents, such as, for example, a small molecule antagonist of CXCR4 binding that are identified using methods of the invention appropriate for the treatment of a disease or disorder, such as, for example, HIV infection or AIDS.
• a patient infected with HIV is treated with a therapeutic agent comprising a compound identified using methods of the invention, or a small molecule antagonist of CXCR4 binding.
• a patient infected with HIV is treated through the use of combinations of therapeutics that include, for example, CXCR4 inhibitors and reverse transcriptase and protease inhibitors.
• Figure 1 shows a peptide library with a fixed, non-degenerate lysine or arginine and eight degenerate positions consisting of eighteen amino acids in approximately equal proportion.
• Figure 2 shows a peptide library screening using binding domains.
• Figure 2a shows a SDS-PAGE of cell lysate containing CXCR4 and of purified CXCR4 stained with coomassie.
• Figure 3 shows an isolated human CXCR4 cDNA sequence.
• Figure 4 shows a baculo virus transfer vector for CXCR4-HIS.
• Figure 5 shows a baculovirus transfer vector for CXCR4-FLAG.
• Figure 6 shows a baculovirus transfer vector for CXCR4-GST.
• Figure 7a is a chart showing the radioligand saturation binding studies using membrane preparations of GST-CXCR4 (High Five).
• Figure 7b is a chart showing the displacement curves for the radioligand binding studies using membrane preparations of GST-CXCR4 (High Five).
• FIG. 8 shows the immobilization of GPCRs for affinity purification from libraries.
• Figure 9 is a chart showing the displacement curves for CXCR4.
• Figure 10 is a chart showing the high-throughput ligand-binding inliibition for CXCR4.
• Figure 11 is a chart showing the ICso's for CXCR4 analogs.
• Figure 12a and Figure 12b are charts showing the inhibition of HIV infection by CXCR4 peptide inhibitors.
• methods of the invention provide for the determination of a binding motif for CXCR4. Further, methods of the invention provide for the identification of agonists or antagonists of the interaction of CXCR4 with its natural ligand, thereby providing for the identification of therapeutic lead compounds.
• Methods for library design and synthesis, and library screening that are particularly useful in the invention are described in the following patent and patent applications, the disclosure of each of which is incorporated by reference herein: Cantley et al, U.S. Patent No. 5,532,167; Cantley, et al, U.S.S.N. 08/369,643, filed December 17, 1998; Cantley, et al, U.S.S.N.
• CXCR4 is cloned and expressed, and tested for activity.
• the CXCR4 may be tagged on the C-terminus or on the N-terminus to facilitate the determination of the character of the CXCR4's ligand-binding properties.
• Exemplary tags include, without limitation, 6xHis, FLAG, GST, V5, Xpress, c-myc, HA, CBD, and MBP.
• the tagged CXCR4 is used in screening of libraries comprising, for example, linear and/or cyclic peptides having natural and/or unnatural amino acids, peptidomimetics and/or small molecules.
• Such peptidomimetics and small molecules may comprise any natural or synthetic compound, composition, chemical, protein, or any combination or modification of any of the foregoing that is used to screen for binding compounds of CXCR4.
• an oriented degenerate peptide library method useful in methods of the invention uses soluble peptide libraries consisting of one or more amino acids in non-degenerate positions, known or suspected to be important for ligand binding, and eighteen amino acids in approximately equal proportions in degenerate positions. Cysteine and tryptophan may be omitted to avoid certain analytical difficulties on sequencing.
• a library is shown in Figure 1, where X represents a degenerate position consisting of any of eighteen amino acids and a lysine or arginine is fixed at a non-degenerate position.
• arginine or lysine as an orienting residue is based on the fact that basic residues of gpl20 are important determinants in binding to CXCR4.
• Another aspect of the invention involves the selection of any amino acid as an orienting residue. Additional residues can be added to the N-terminal of the sequence shown in Figure 1 because there are often interfering substances present in the first and second sequencing cycles. Additional residues can be added at the C-terminal end to provide amino acids to better anchor the peptide to the filter in the sequencer cartridge.
• Another aspect of the invention provides for the use of highly diverse libraries of peptide (linear and cyclic, natural and unnatural amino acids), peptidomimetic, and small molecule compounds for the lead identification step.
• these ligands can be agonistic or antagonistic in their function on the receptor.
• the invention uses partially purified CXCR4 as the agent for carrying out the selection, identification, and improvement of tight binding ligands as a route to therapeutically useful compounds.
• the invention provides for the development and use of solubilization and immobilization procedures that facilitate efficient ligand selection methods provided herein.
• the optimal conditions for the solubilization and immobilization for efficient ligand selection comprise the use of low salt, such as, for example, low or no magnesium or calcium concentrations, and no NaCl concentrations (O.OnM NaCl).
• Low salt such as, for example, low or no magnesium or calcium concentrations, and no NaCl concentrations (O.OnM NaCl).
• Ligand selection methods using, for example, inactive, precipitated protein, cell membrane preparations, and whole cell preparations are further provided herein.
• the screening step may comprise phage display technology.
• phage display systems have been used to screen peptide libraries for binding to selected target molecules and to display functional proteins with the potential of screening these proteins for desired properties. More recent improvements of the display approach have made it possible to express enzymes as well as antibody fragments on the bacteriophage surface thus allowing for selection of specific properties by selecting with specific ligands. See e.g., Smith SF, et. al, Methods Enzym. 217:228-257 (1993). Phage display methods may be used for the identification of preliminary motif information, and followed by additional rounds of affinity purification with purified receptor preparations of the invention and highly diverse libraries, especially cyclic peptide and peptidomimetic libraries.
• the phage display methods allow the identification of motifs of natural amino acids.
• Information derived from phage display can be taken into affinity purification methods using, for example, synthetic libraries containing novel amino acid analogs or cyclic peptides to select ligands that have enhanced pharmaceutical characteristics.
• the use of initial, secondary and tertiary libraries allows a more complete definition of the specificity of the binding site. Secondary libraries may be sequenced incorporating information from the initial library. With the first library, some degenerate positions may yield high preferences for specific amino acids and these may become non- degenerate positions consisting of the preferred amino acid in a second library. See e.g., Wu R, J Biol Chem 271(27):15934-41 (1996).
• computer-aided design technology may be used in the screening and/or designing of peptides, peptidomimetics, and small molecules.
• computer aided design technology may virtually screen, identify, design and validate potential compounds with regards to their CXCR4 activity.
• Computer programs that may be used to aid in the design of appropriate peptides, peptidomimetics and small molecules include, for example, Dock, Frodo and Insight.
• An example of a method for screening of biologically appropriate drags relies on structure based rational drug design.
• a three dimensional structure of the protein, peptide or molecule is determined (or modeled after a close family member) and potential agonists and/or antagonists are designed with the aid of computer modeling.
• potential agonists and/or antagonists are designed with the aid of computer modeling. See e.g., Butt et al, Scientific American, December 92-98 (1993); West et al, TIPS, 16:67-74 (1995); Dunbrack et al, Folding & Design, 2:27-42 (1997).
• the drug is contacted with CXCR4, wherein a binding complex forms between the potential drag and CXCR4.
• Methods of contacting the drag to CXCR4 are generally understood by anyone having skill in the art of drug development.
• the screening step may be performed in solution phase, or with the CXCR4 immobilized on affinity columns.
• other forms of sequestration can be used to perform the affinity purification of select ligands from libraries. These include, but are not limited to the following examples.
• the receptor and bound library components can be separated from non-bound library components using equilibrium dialysis.
• the tagged receptor can be bound to specific affinity membranes, which are in the form of plates or are separate.
• the libraries can then be incubated with the membrane and easily washed to remove non-specific binding components. Size exclusion methodology can be used to separate a purified receptor bound library complex from unbound components after pre-incubating the receptor with the library.
• micellar complex containing the receptor (which may or may not incorporate lipids as well as detergent) can be separated after binding select affinity components from a library by differential centrifugation.
• the high affinity ligand can be released using low pH or high salt conditions and the structure identified by sequencing as described herein.
• peptide libraries were screened to determine each library's respective inhibition binding. In general, a greater than 10% inhibition at 100 ⁇ M was significant for continued evaluation of the sequence via affinity purification.
• specific peptides are synthesized by the same methods as employed for library synthesis.
• a high preference value is greater than 1. The value is determined by subtracting the control value from the sample value and dividing by the reference value. In a preferred embodiment of the invention, the preference value is greater than 1.2.
• the preference value is greater than 2.
• HPLC High Performance Liquid Chromatography
• MALDI-TOF MS Matrix-Assisted Laser Desorption Ionization- Time of Flight Mass Spectrometer
• Edman Sequencing Generally, relative affinities may be measured by modifying the radiolabel binding assay used in receptor purification.
• the bound components of the libraries can be eluted from the immobilized protein with specific N-terminally blocked peptides or other non-sequencable analogs.
• the release/elution of the tagged CXCR4 with its bound library can be accomplished using specific N-terminally blocked peptides or other non-sequencable analogs. This can be done using acetylated FLAG peptide to elute CXCR4-FLAG receptor from the resin.
• the tag from CXCR4 may be cleaved using a specific protease (as designed into the protein/vector; either enterokinase or thrombin) after immobilization onto the affinity resin and after the combinatorial library is bound to release the complex.
• libraries can be prescreened for their ability to bind to the receptor (using significantly less protein) by a binding assay using CXCR4-containing membranes from, for example, Sf9 (Spodoptera frugiperd ⁇ ) or High Five (Trichoplusia n ⁇ ) cells (both obtained from Invitrogen) in a single assay or in an array assay. This screening may be performed using CXCR4 and a number of linear and cyclic libraries to determine their effectiveness in inhibiting the natural ligand to bind.
• Methods of the invention further comprise the design of therapeutic agents comprising peptides, peptidomimetics, and/or small molecules that are antagonistic to CXCR4 activity appropriate for the treatment of patients with a disease, such as AIDS.
• Binding compounds for CXCR4 and the identification of optimal synthesis and purification thereof provides for an effective treatment of AIDS and HIV infection.
• the small peptide ligand binding compounds of the invention both cyclic and linear peptide ligands, demonstrate enhanced binding affinity and action, and are resistant to proteolysis as identified, for example, in Table 1.
• Amino acids and peptides are abbreviated and designated following the rales of the IUPAC-IUB Commission of Biochemical Nomenclature in J Biol. Chem. 247, 977-983 (1972). Amino acid symbols denote the L-configuration unless indicated otherwise.
• amino acids from the residues of gpl20 that are crucial for viral uptake have been used to specify fixed, or non-degenerate positions in the peptide libraries that have been designed for use in the oriented peptide library method described below and in U.S. Patent 5,532,167, the disclosure of which is incorporated by reference herein. See e.g., Rizzuto CD, et. al, Science 280(5371):1949-53 (1998).
• EXAMPLE 1 Preparation of Tagged CXCR4; Screening Of Linear Peptide Libraries
• Various CXCR4 vectors were prepared for the baculovirus expression system containing epitope tags using standard techniques known by those skilled in the art that allowed for easier purification of the receptor.
• Tags may be incorporated at the N- or C-terminus of proteins.
• tags were incorporated at the C-terminus of the receptor to determine the character of the receptor's ligand-binding properties that are in the N-terminal region of the molecule to allow easier purification of the receptor.
• tags were incorporated at the C-terminus of the receptor to determine the receptor's ligand-binding properties at the N-terminal region of the molecule to allow easier purification of the receptor. Tags were placed at the N-terminus of proteins. For CXCR4, tags may be incorporated either at the N- or C-termini of the receptor.
• C-terminal tags There were no commercially available baculovirus transfer vectors with C-terminal tags.
• C terminal 6xHis tagged and C-terminal FLAG constructs are provided below as examples.
• Alternative tags may include, for example, GST, V5, Xpress, c-myc, HA, CBD, and MBP. These constructs were made by analogous procedures using standard techniques known by those skilled in the art.
• the 6xHis tag enables a one-step purification using nickel chelation.
• the cDNA for CXCR4 was isolated from a spleen cDNA library using Polymerase Chain Reaction ("PCR") and primers for the 3' and 5' ends of CXCR4, as well as to the middle of the gene.
• PCR Polymerase Chain Reaction
• CXCR4 was subcloned into an E. coli vector, pET30a, with a C-terminal 6xHis tag.
• the newly created CXCR4-6xHis was then excised and ligated into pBlueBac, a baculovirus transfer vector (Invitrogen, Carlsbad, CA).
• the construct was analyzed using both restriction digest and sequencing, and fransfected into Sf9 insect cells (Pharmmgen, San Diego, CA) for expression as typically done by those skilled in the art of protein expression.
• a C-terminal bacterial FLAG construct was available from Sigma (pFLAG-CTC). A similar strategy using standard techniques was employed for the construction of this vector.
• the CXCR4 was subcloned into the pFLAG-CTC plasmid, excised with the C-terminal FLAG tag and then ligated into the digested pBlueBac vector. The construct was analyzed using both restriction digest and sequencing, and fransfected into Sf9 or High Five insect cells for expression.
• the pBlueBac vector containing the CXCR4 insert was cotransfected with Bac-N-Blue DNA using cationic liposome mediated transfection using standard techniques.
• the CXCR4 was inserted into the baculovirus genome by homologous recombination. Cells were monitored from 24 hours posttransfection to 4-5 days. After about 72 hours, the transfection supernatant was assayed for recombinant plaques using a standard plaque assay. Cells which have the recombinant virus produce blue plaques when grown in the presence of X-gal (5-bromo-4-chloro-3-indoyl- ⁇ -D-galactoside).
• plaques were purified and the isolate was verified by PCR for correctness of recombination using standard techniques. From this, a high-titer stock was generated and infection performed from this stock for expression work using standard techniques. Controls for transfection include cells only and transfer vector. Sf9 or High Five cells were maintained both as adherent and suspension cultures using standard techniques known to those skilled in the art. The adherent cells were grown to confluence and passaged using the sloughing technique at a ratio of 1 :5. Suspension cells were maintained in spinner flasks with 0.1% pluronic F-68 (to minimize shearing) for 2-3 months by sub-culturing to a density of 1 x 10 6 cells/ml.
• FIG. 1 A time course after infection with recombinant virus was used to define optimal growth conditions for expression using standard techniques. Aliquots of cells from spinner flasks were taken for this time course, centrifuged at 800 x g for 10 minutes at 4°C and both supernatant and pellet assayed by SDS-PAGE/Western blot analysis.
• Figure 2a shows the SDS-PAGE/Western Blots of cell lysate containing CXCR4 and purified CXCR4 stained with coomassie. The CXCR4 was expected to be in the membrane fraction (pellet). All viable systems were assayed in this fashion for levels of expression. The systems with the best expression levels was assayed for activity using a standard binding assay on a membrane preparation using SDF-1 (Chemicon) and [ 125 I]-SDF-1 (New England Nuclear, "NEN", Boston, MA).
• the membrane fraction was isolated by first pelleting the whole Sf9 cells (800 x g for 10 minutes at 4°C), then resuspending the pellet in a lysis buffer with homogenization.
• Typical lysis buffer is around neutral pH and contains a cocktail of protease inhibitors, all of which are standard techniques for those skilled in the art. Membranes were pelleted. Solubilization was also conducted using varying NaCl concentrations. Despite conventional thinking, the step of solubilization using low salt, for example, low calcium and magnesium concentrations substantially in the absence of NaCl provided unexpected optimal conditions for solubilization when compared for quantity and activity.
• CXCR4 was purified from the membrane fraction.
• the exact purification scheme will depend on the construct chosen, which is subject to activity and ease of solubilization.
• the membrane fraction was loaded onto a Ni-NTA column (Qiagen, Valencia, CA) in the presence of detergent, washed extensively, and eluted with imidazole.
• Purification of the FLAG-tagged CXCR4 was performed using the anti-FLAG M2 affinity matrix (Sigma, St. Louis, MO) in the presence of NP-40 and eluted with glycine. The purification was performed in the presence of NP-40 in the experiment described above. Activity of the purified receptor was assessed using a standard binding/displacement assay using SDF-1 and [ 125 I]-SDF-1.
• tBu was used for the protection of side-chains of Asp, Glu, Ser, Thr, and Tyr, tert.-butyloxycarbonyl ("Boc") for Lys and Trp, 2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl ("Pbf ') for Arg, and triphenylmethyl ("trityl", "Tit”) for Cys, His, Asn and Gin.
• the scale of the synthesis was 0.20 mmol.
• the resin was initially washed with N-methylpyrrolidinone ("NMP") followed by a 1 x 3 minutes and 1 x 7.6 minutes treatment of piperidine:NMP (1 :4) for N ⁇ -Fmoc removal.
• NMP N-methylpyrrolidinone
• ⁇ BTU N-[(lH-benzotriazol- 1 -yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide
• DMF N-[(lH-benzotriazol- 1 -yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide
• DMF N-[(lH-benzotriazol- 1 -yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide
• the resin was filtered and rinsed six times with a total of 90 ml of NMP and the cycle was repeated.
• a batch of resin was allowed to react with mixtures of the combinatorial amino acids without any partitioning of the resin.
• Adjusting the concentrations of the amino acids in the starting mixture controls the relative coupling rates, thereby ensuring equal incorporation of the amino acids in the library.
• the optimization of a mixture of natural Boc and Fmoc protected amino acids for the one pot synthesis has been previously described (see e.g., U.S. Patent No. 5,225,533; Ivanetich, et. Al, Combinatorial Chemistry, vol 267, Academic Press, San Diego, CA USA, p 247-260 (1996); Buettner, et al.
• Fmoc-Asp(OH)-ODmab (Dmab, 4-[N-(l-(4,4-dimethyl-2,6- dioxoxcyclohexylidene)-3-methylbutyl)amino]-benzyl) was side-chain anchored to Rink amide resin followed by chain elongation as described above. Following linear assembly, removal of the Dmab and Fmoc group was accomplished by treatments with hydrazine:DMF (1 :49) for 7 minutes and piperidine:NMP (1 :4) for 6 x 3 minutes, respectively. The resin was transferred to a syringe containing a polypropylene frit for manual cyclization.
• On-resin head-to-tail cyclization was performed using 7-azabenzotriazol- 1 -yloxy)-tris(pyrrolidino)phosphonium hexafluorophosphate ("PyAOP"):DIEA (1:2, 4 equiv) in a solution containing 1% Trition X in NMP:DMF:dichloromethane, methylene chloride, DCM) (1:1:1) for 2 hours at 55°C.
• the unreacted linear precursor was treated with Fmoc-Nva-OH/PyAOP/DIEA ("Nva", "norvaline”)(l:l:2, 4 equiv) in DMF for 1 x 18 hours and 1 x 3 hours.
• the initial libraries synthesized had single, non-degenerate orienting amino acids (i.e., M-X-X-X-X-R-X-X-X-X-A, where X is a degenerate equimolar mixture of all amino acids except cysteine). Cyclic libraries (head-to-tail) were also prepared with single, non-degenerate orienting amino acids. Through the use of these initial libraries, the optimal residues at some degenerate positions become defined and secondary libraries were made fixing these positions.
• the head to tail cyclized library cyclo(M-X-X-X-X-R-X-X-X-N) indicated that the -4 position (from the fixed R) should be lysine, the -2 position should be aspartate, the -1 position should be histidine, and the +3 position should be lysine so the secondary library was cyclo(M-K-X-D-H-R-X-X-K-N).
• An oriented linear peptide library was applied to a column containing immobilized
• FIG. 1 A schematic diagram showing the peptide library using binding domains can be seen in Figure 2. After washing, bound peptides were eluted from the column. Next, bound peptides and the entire library applied to the column were submitted individually to Edman degradation, to determine the distribution of amino acids as a function of position. Finally, the preferences of amino acids at the degenerate positions was determined. For example, if serine was 5% of the amino acids at position +1 in starting library but 15% of the amino acids in position +1 in the high affinity peptides, there would be a selection for serine at the +1 position. A preference value of 3 at that position would be obtained. Table 2 provides a selective review of the use of the peptide library method with binding domains.
• the initial library used a single, non-degenerate basic amino acid (i.e., M-A-X-X-X-X-R- X-X-X-X-K-K-K). Secondary libraries were made fixing optimal residues found at some degenerate positions. For example, M-A-X-X-X-X-W-X-X-X-X-A-K-K-K may indicate that the -4 position should be arginine, -1 should be isoleucine, and +1 should be arginine so the secondary library would be M-A-R-X-X-I-W-R-X-X-X-A-K-K-K.
• the receptor was exposed to the library, and separation of free and bound peptides was accomplished by pelleting the membranes by centrifugation.
• the 6xHis-tagged CXCR4 purified receptor was incubated with a peptide library, about 1 ⁇ mole of peptide and about 1 nmole of binding sites. After incubation, receptor with bound peptide was separated from unbound peptides by centrifugation (receptor*peptide complex in the pellet, unbound peptide in the supernatant). Nonspecifically bound peptides were removed by exhaustive washing, and resuspension of the pellet in low pH ( ⁇ 2.5) was used to remove the bound peptide.
• This peptide was sequenced to determine the consensus sequence.
• the receptor was immobilized on an anti-FLAG M2 affinity matrix (St. Louis, MO).
• An additional purification approach used the CXCR4-GST construct and immobilized glutathione (Pierce, Rockford, IL).
• Both the bound peptide mixture and the starting peptide library were sequenced using standard techniques. The amounts of each amino acid, as a function of position, were determined. Preference values for each amino acid at each position were calculated by comparing the amounts of amino acids present in the starting library and bound fraction of peptides.
• phage display technology is also used to identify preliminary binding motifs.
• the phage display method provides for the identification of motifs of natural amino acids.
• Phage display technology involves the insertion of DNA sequences into a gene coding for one of the phage coat proteins. The gene is inserted in a particular location so that the expressed protein insert can interact with other molecules. As a result, the encoded peptide or protein sequence will be presented on the surface of the phage and exposed for binding. By inserting degenerate nucleotides, each phage can express a different peptide sequence ("a phage library").
• Relative affinities were measured by modifying the radiolabel binding assay used in receptor purification. Therefore, the ability of these peptides to displace [ 125 I]-SDF-1 from purified CXCR4 membranes was measured.
• CXCR4 Cloning and Expression CXCR4 was isolated from a spleen cDNA library in two halves and spliced together. These two fragments were isolated using PCR technology and primers to the 3' and 5' ends and the middle of the CXCR4 gene. A full-length clone was not isolated with the 3' and 5' primers; however, two halves were isolated and ligated together using a unique BamHl site in the gene. The identity of the constract was confirmed by sequencing. The sequence of the isolate is in Figure 3. An alternate splice shorter form was also isolated, which is called CXCR4s. Tags were added to the C-terminus of the receptor for use in immobilizing them for affinity purification assays using standard techniques. The following are specific examples from experiments using the tagging method. Construction ofCXCR4 with C-Terminal Histidine Tag (Insect Select Expression System)
• CXCR4 A previous construct containing the gene for GnRHR (gonadotropin releasing hormone receptor) was used to make the first CXCR4 constract.
• the gene for GnRHR was spliced out and replaced by the isolated cDNA for CXCR4.
• This vector was originally the pet30a vector with the 6xHis tag at the C-te ⁇ ninus.
• Construction ofCXCR4 construct with C-terminal FLAG tag PCR was performed using the primers 5' BspEl CXCR4 and 3' Bgl CXCR4 engineered with unique sites for ligation of CXCR4 in frame with the FLAG tag of pFLAG-CTC (a bacterial expression vector) from Sigma. This constract is called CXCR4-FLAG-CTC.
• CXCR4-FLAG was then removed by digestion and filled in with Klenow fragment.
• the fragment containing CXCR4-FLAG was ligated into pBluebac 4.5 that was first digested then blunted with Klenow. This final construct is called CXCR4-FLAG.
• the constract was confirmed by restriction digestion and sequencing using standard techniques. This constract has been used for expression and has been determined to be expressed sufficiently and in active form for use in affinity purification screening.
• CXCR4 Construct with C-terminal GST tag The newly constructed CXCR4-FLAG cDNA was removed from the CTC vector and subcloned into another constract, CCR5-GST, in place of the CCR5 (using Bgl and BspEl). This created the vector for CXCR4- GST using one step. The construct was confirmed by restriction digestion and sequencing using standard techniques. This construct has been used for expression and has been determined to be expressed sufficiently and in active form for use in affinity purification screening. Construction ofCXCR4 with N-terminal 6xHis tag: This construct was prepared by subcloning the CXCR4 into the commercially available vector, pBluebacHis2b (Invitrogen).
• the construct was confirmed by restriction digestion and sequencing using standard techniques. Plasmid maps for these vectors are found in Figures 4-6.
• the vectors for the three new constracts (for CXCR4-FLAG, CXCR4-GST, and CXCR4- HIS) were used to co-transfect Sf9 cells for the production of a viral stock of each. These viral stocks were purified using a standard plaque assay and then used in experiments to infect for the optimization of expression of CXCR4 with its various C-terminal tags. High Five cells (Invitrogen) were also fransfected with these CXCR4 tagged constracts and tested for expression of CXCR4. All constracts were determined to express the appropriately tagged receptor.
• CXCR4-FLAG, CXCR4-GST, and CXCR4-HIS were used to co-transfect Sf9 and High Five cells, as described in Example 1.
• Whole cells from Sf9 and High five cell lines were lysed using hypotonic buffers (10 mM Tris, pH 7.4, 5 mM EDTA), and membrane preparations were made by homogenization and centrifugation using standard techniques known to those skilled in the art.
• Membrane preparations for CXCR4-GST, CXCR4-FLAG, and CXCR4-HIS were assayed using a standard radioligand binding assay.
• the radioligand [ 125 I]-SDF-l ⁇ was incubated with membranes (0.5 ⁇ g) in binding buffer at 27°C for 1 hour with and without unlabelled SDF-1. For filtration and washing, the reaction was transferred to Millipore Multiscreen filter plates (HATF 0.45 ⁇ m; pre-blocked with 10% BSA), filtered using vacuum, washed 4-5 times with 200 ⁇ L ice-cold buffer, and radioactive counts bound were detected using scintillation counting. All points were done in triplicate. Uninfected cells were used as a control for this experiment. To determine the K D , saturation binding was measured using increasing concentrations of [ I]-SDF-l ⁇ (from 0.5 nM to 10 nM).
• Nonspecific binding was measured in the presence of 400 nM unlabelled SDF-1 ⁇ .
• Competitive binding assays were performed by incubating CXCR4-containing membranes with 0.5 nM [ 125 I]- SDF-l ⁇ and serial dilutions of unlabelled SDF-1 ⁇ or peptide ligand.
• Analyses of K D and IC 50 were performed using non-linear curve fitting in Kaleidagraph.
• Figure 7 generally shows charts exemplifying radioligand binding studies using membrane preparations of GST-CXCR4 (High Five). Saturation binding demonstrated a K D of 3.27 nM as seen in Figure 7a.
• Solubilization of the tagged versions of CXCR4 have been performed using many different combinations of detergents (NP-40, Triton X- 100, ⁇ -D-maltoside, n-octylglucoside, CYMAL, Zwittergents, Tween-20, lysophosphatidyl choline, CHAPS, etc.), salts (NaCl, CaCl 2 , MgCl 2 , MnCl 2 , KC1, etc.), buffers (Tris, Hepes, Hepps, Pipes, Mes, Mops, acetate, phosphate, imidazole, etc.), and various pH's (range 6.8-8.2).
• detergents NP-40, Triton X- 100, ⁇ -D-maltoside, n-octylglucoside, CYMAL, Zwittergents, Tween-20, lysophosphatidyl choline, CHAPS, etc.
• salts NaCl
• solubilization was found using Zwittergent 3-14 and low salt, e.g. low magnesium and calcium, but no NaCl (O.OnM NaCl) and buffered at pH 8.1.
• at least 20% of the solubilized, immobilized protein is active.
• at least 30%, 40%, 50% and 75% of the solubilized, immobilized protein is active.
• CXCR4-GST were immobilized onto affinity columns for purification and as an active protein ready for use in screening of peptide libraries.
• a schematic diagram showing the immobilization of GPCRs for affinity purification from libraries is shown in Figure 8.
• CXCR4-GST was bound and immobilized onto glutathione-agarose (Pierce) and glutathione-sepharose (Amersham Pharmacia Biotech).
• the immobilization of the functional protein was accomplished by first solubilizing the receptor in 0.3% NP-40, 10 mM Hepes (or Pipes), pH 7.5, then binding it to the glutathione-sepharose resin in 0.3% NP-40, 10 mM Hepes (or Pipes), pH 7.5, 3 mM CaCl 2 , 15 mM MgCl 2 .
• the activity of the immobilized receptor was determined by incubation for 1 hour at 4°C with the radiolabeled SDF-1 ⁇ (as with the membrane assay above) and competition with cold SDF-1 . Uninfected cells were used as controls for this activity, as well as the column alone.
• FIG 9 is a chart showing the displacement curves for CXCR4. Binding displacement experiments were performed both on CXCR4-containing membranes and on the immobilized receptor with radiolabeled SDF-1 displaced by increasing concentrations of cold SDF-1.
• FIG. 10 is a chart showing the high-throughput ligand-binding inhibition for CXCR4.
• CXCR4-containing membranes were incubated in the presence of 100 ⁇ M library and radiolabeled SDF-1. Percent inhibition was calculated to determine the effect of each library. Peptide libraries with the highest percent inhibition were assayed first.
• the sequence for the consensus motif for the specific ligand was identified from this screening to be M-A-R-S-L-I-W-R-P-A-K-A-K-K-K.
• the affinity for the receptor was determined using standard radioligand displacement methodology as performed by those skilled in the art.
• This ligand was determined to have an IC 50 of ⁇ 60 ⁇ M.
• Figure 11, for example, is a chart showing the IC 50 for CXCR4 analogs. Selective analogs increased binding inhibition.
• CPI- 1221, a 15-mer was the original peptide. Analogs of this peptide were made to increase specificity of binding as demonstrated by the curves in Figure 11. Rational analoging of this peptide was performed.
• Minimal length of the peptide sequence was determined by deleting from both the N- and C-termini. Approaches to protect against metabolism were conducted using synthetic analogs. Insertion of a rigid segment of the peptide was accomplished with cyclic amino acids such as azetidine-2-carboxylic acid, tetrahydroisoquinoline (Tic), pipecolic acid (Pip), thiazolidine-4-carboxylic acid (Thz), 1-amino- 1-cyclopentane-carboxylic acid and 1 -amino- 1-cyclohexanecarboxylic acid (Sawyer, 1995) as well as ⁇ , ⁇ -dialkyl residues such as aminoisobutyric acid (Aib) and diethylglycine (Deg).
• cyclic amino acids such as azetidine-2-carboxylic acid, tetrahydroisoquinoline (Tic), pipecolic acid (Pip), thiazolidine-4-carboxylic
• Hydrophobic unnatural amino acid such as naphthylalanine and cyclohexylalanine substitutions were used successfully.
• Table 3 is a summary of the substitutions made at the various positions in the peptide ligand which were synthesized and screened for activity using inhibition of [ 125 I]-SDFl ⁇ binding and toxicity on Jurkat and CEM cells.
• all positions were individually substituted with Ala, several positions were substituted with D-amino acids, the ⁇ -terminus of several peptides were acetylated, deletions of the ⁇ - and C-termini were made, and several retro-inverso peptides were synthesized.
• CPI-1500 (ARSLI(2- ⁇ al)R(Tic)ARR(2- ⁇ al)RR) also demonstrated specificity for the X4 virus Illb over the R5 virus 9881.
• Figure 12 (b) shows that other CXCR4 peptide inhibitors demonstrated inhibition of the X4 virus, with specificity, as well.
• CXCR4 was used to isolate phage which bind to CXCR4 using standard techniques known to those skilled in the art. Subtraction of the background from the glutathione-sepharose beads, BSA, and SDFl ⁇ used in the assay was performed by incubation of the phage library with the mixture of these components. After incubating subtracted phage libraries (i.e., NEB PhD C7C) with the receptor, bound phage were eluted both with the natural CXCR4 ligand (SDFl ⁇ ) and with glycine, pH 2.2.
• PhD C7C is a particular phage library with 7 random amino acids between disulfides, and may be obtained from New England Biolabs ("NEB"). Multiple rounds of screening were performed. Both conditions have provided specific sequences (see Table 4) which bind to CXCR4 and inhibit ligand binding.
• Table 4 Phage sequences isolated from CXCR4 screening. Standard one-letter abbreviations are used for the 20 natural amino acids.
• Methods of preventing and treating AIDS and HIV infection comprise the step of administering a composition comprising such a compound capable of inhibiting CXCR4 binding as described herein.
• Administration may be by any compatible route.
• administration may include oral or parenteral, including intravenous and intraperitoneal routes of administration.
• a particularly preferred method is by controlled- release injection of a suitable formulation.
• administration may be by periodic injections of a bolus of a composition, or may be made more continuous by intravenous or intraperitoneal administration from a reservoir that is external (e.g., an intravenous bag) or internal (e.g., a bioerodable implant).
• Therapeutic compositions contemplated by the present invention may be provided to an individual by any suitable means, directly (e.g., locally, as by injection, implantation or topical administration to a tissue locus) or systemically (e.g., parenterally or orally).
• composition may comprise part of an aqueous or physiologically compatible fluid suspension or solution.
• the carrier or vehicle is physiologically acceptable so that in addition to delivery of the desired composition to the patient, it does not otherwise adversely affect the patient's electrolyte and/or volume balance.
• Useful solutions for parenteral administration may be prepared by any of the methods well known in the pharmaceutical art, described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES (Gennaro, A., ed.), Mack Pub., 1990.
• Formulations of the therapeutic agents of the invention may include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable, origin, hydrogenated naphthalenes, and the like.
• Formulations for direct administration in particular, may include glycerol and other compositions of high viscosity to help maintain the agent at the desired locus.
• Biocompatible, preferably bioresorbable, polymers including, for example, hyaluronic acid, collagen, tricalcium phosphate, polybutyrate, lactide, and glycolide polymers and lactide/glycolide copolymers, may be useful excipients to control the release of the agent in vivo.
• the concept of a controlled release injectable formulation for peptide drugs is well-accepted and offers several advantages.
• bioavailabilities are high.
• treatment regimens can consist of once per month or per three months (like Abbott's Leupron®), or once per year (e.g. Alza's Viadur®).
• controlled release injectable formulations substantially reduces the doses that can be used (the Leupron injection dose is 1 mg/day but the 90 day formulation uses is 11.25 mg total). Also, increased efficacy can be achieved if the therapeutic is present continuously to prevent infectivity. This consideration is particularly important in view of the need to approach a cure for this disease by preventing the reformation of slow-to-clear deposits of infection such as the memory T cell compartment. See e.g., Lee, V., ed. Peptide and Protein Drug Delivery. Marcel Dekker, Inc., NY (1991).
• parenteral delivery systems for these agents include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
• Formulations for inhalation administration contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
• Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or cutric acid for vaginal administration.

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