Classifications

• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • 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
  • A61P31/04—Antibacterial agents
• • 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
  • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
  • G01N2333/21—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pseudomonadaceae (F)

Definitions

• the present invention relates generally to compounds, compositions and methods for the diagnosis and therapy of diseases in warm-blooded animals (e.g., in humans) involving infections with and colonization by Pseudomonas bacteria, including Pseudomonas aeruginosa in the lungs of patients with cystic fibrosis.
• the 0 invention relates more particularly to the use of one or more compounds selective for binding Pseudomonas bacteria. These compounds are useful for diagnosis and/or therapeutic intervention of the colonization of Pseudomonas bacteria, or may be linked to an agent(s) to target and effectively arrest or kill Pseudomonas bacteria.
• Pseudomonas infections occur in a variety of medical conditions and can be life threatening.
• Pseudomonas is an opportunistic bacterium. Examples of individuals at risk include cystic fibrosis patients and burn patients. Cystic fibrosis is described below as a representative example of a medical condition which can involve infection with Pseudomonas bacteria.
• Cystic Fibrosis CF is the most common lethal genetic disease among the Caucasian population. CF is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), which acts as a chloride channel.
• CFTR cystic fibrosis transmembrane conductance regulator
• the genetic mutations of CFTR which alter ion movements also affect the N- glycosylation of CFTR as well as other cell surface molecules. All of the exocrine 5 glands of the patients are affected; however, the lungs are the primary site of morbidity and mortality. The general change in glycosylation results in an increase in Lewis fucosylation and a decrease in sialylation.
• the salivary and respiratory musins from CF patients also contain higher levels of Lewis type oligosaccharides including sialylated and sulfated Lewis x/a structures.
• this invention provides compounds, compositions and methods for utilizing lectins expressed on Pseudomonas bacteria for the detection of Pseudomonas bacteria and the diagnosis and therapy of disease involving Pseudomonas bacteria, including human disease.
• glycomimetics of the Lewis structures that have high affinity binding to the lectins on P. aeruginosa will have a beneficial therapeutic effect on CF patients.
• these glycomimetics may be conjugated, for example, with strong antibiotics to increase the efficacy and lower the dose, thereby avoiding well known deleterious side effects of these potent antibiotics. Given that these binding sites are crucial for the colonization and pathogenicity of the bacterium, mutations in this target to become resistant to this conjugate therapy should result in non-pathogenic forms of the bacteria.
• One embodiment of the present invention provides a method of inhibiting Pseudomonas bacteria in a warm-blooded animal comprising administering to the animal in an amount effective to inhibit the bacteria a compound comprising a compound according to Figure 1 or Figure 2.
• the present invention provides a conjugate comprising a therapeutic agent linked to a compound according to Figure 1 or Figure 2.
• the present invention provides a method of detecting Pseudomonas bacteria comprising contacting a sample with a diagnostic agent linked to a compound comprising a compound according to Figure 1 or Figure 2, under conditions sufficient for the compound to bind to the bacteria if present in the sample; and detecting the agent present in the sample, wherein the presence of agent in the sample is indicative of the presence of Pseudomonas bacteria.
• the present invention provides a method of immobilizing Pseudomonas bacteria on a solid support comprising contacting, under conditions sufficient for binding, a sample containing Pseudomonas bacteria with a compound comprising a compound according to Figure 1 or Figure 2 that is immobilized on a solid support; and separating the sample from the solid support.
• the compounds and conjugates described herein may be used in the preparation of a medicament for the inhibition of Pseudomonas bacteria.
• Figures 1 A- IE show the structures of glycomimetic compounds.
• Figures 2A-2B show the structures of additional glycomimetic compounds.
• Figure 3 depicts the synthesis of representative glycomimetic compound 3.
• Figure 4 depicts the synthesis of glycomimetic compound 21.
• Figure 5 depicts the synthesis of representative glycomimetic compound 15.
• Figure 6 depicts the acylation of intermediate 21 to give a variety of representative glycomimetic compounds.
• Figure 7 depicts the synthesis of compound 22.
• Figures 8A-8B depict the synthesis of compounds 4, 6, 7, 8, 10, 12, 13, 14, 16, 17, 18 and 19.
• Figure 9 depicts the synthesis of compounds 22 and 23.
• Figure 10 depicts the synthesis of intermediates.
• Figure 11 depicts the synthesis of intermediates and compound 24.
• Figure 12 depicts the synthesis of intermediates xxxxiv and xxxxv.
• Figure 13 depicts the synthesis of compound 25.
• Figure 14 depicts the synthesis of compound 26.
• Figure 15 depicts the synthesis of intermediates xxxxxi and xxxxxii.
• Figure 16 depicts the synthesis of compound 27.
• Figure 17 depicts the synthesis of compound 28.
• Figure 18 depicts the binding of PA-IIL lectin to immobilized neutral carbohydrate structures.
• Figure 19 depicts the binding of PA-IIL lectin to immobilized acidic carbohydrate structures.
• Figure 20 depicts the determination of assay conditions for IC 5 o values of PA-IIL lectin inhibition.
• Figure 21 shows a schematic representation of the assay developed for determining IC 50 values of glycomimetic inhibitors of PA-IIL lectin.
• Figure 22 depicts the inhibition of PA-IIL lectin by glycomimetic compound 23.
• the present invention provides compounds and compositions that bind P. aeruginosa and may be used in the diagnosis and therapy of disease.
• Glycomimetic compound refers to a molecule that binds specifically to P. aeruginosa.
• the structures of the Glycomimetic compounds covered by this invention are shown in Figures 1 and 2, and also include the compounds disclosed herein except that the compounds do not contain the mimic for sialic acid which is shown as the terminal cyclohexyl lactic acid moiety. This is accomplished, for example, by eliminating the step involving the addition of intermediate E in certain of the reaction schemes. All compounds (or conjugates thereof) useful in the present invention include physiologically acceptable salts thereof.
• a diagnostic or therapeutic agent such as a drug to a Glycomimetic compound
• link a diagnostic or therapeutic agent such as a drug to a Glycomimetic compound
• therapeutic agent refers to any bioactive agent intended for administration to a warm-blooded animal (e.g., a mammal such as a human) to prevent or treat a disease or other undesirable condition or to enhance the success of therapies.
• therapeutic agents include antibiotics, hormones, growth factors, proteins, peptides, genes, non-viral vectors and other compounds.
• Glycomimetic compounds as described herein may be present within a pharmaceutical composition.
• a pharmaceutical composition comprises one or more Glycomimetic compounds in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
• Such compositions may comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.
• buffers e.g., neutral buffered saline or phosphate buffered saline
• carbohydrates e.g., glucose, mannose, sucrose or dextrans
• mannitol e.g., proteins, polypeptides or amino acids
• glycine
• compositions of the present invention may be formulated as a lyophilizate.
• Compositions of the present invention may be formulated for any appropriate manner of administration, including for example, aerosol, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous, or intramuscular administration.
• a pharmaceutical composition may also, or alternatively, contain one or more active agents, such as drugs, which may be linked to a Glycomimetic compound or may be free within the composition.
• active agents such as drugs
• the attachment of an agent to a Glycomimetic compound may be covalent or noncovalent.
• the compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule or sponge that effects a slow release of modulating agent following administration).
• a sustained release formulation i.e., a formulation such as a capsule or sponge that effects a slow release of modulating agent following administration.
• Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site.
• Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of modulating agent release.
• Glycomimetic compounds are generally present within a pharmaceutical composition in a therapeutically effective amount.
• a therapeutically effective amount is an amount that results in a discernible patient benefit, such as a measured or observed response of a condition associated with Pseudomonas infection.
• Glycomimetic compounds described herein may be used for achieving diagnostic and/or therapeutic results in disease (e.g., human disease) involving infection by Pseudomonas (e.g., P. aeruginosa) bacteria.
• disease e.g., human disease
• Pseudomonas e.g., P. aeruginosa
• Such diagnostic and/or therapeutic results may be achieved in vitro and/or in vivo in an animal, preferably in a mammal such as a human, provided that Pseudomonas (e.g., P. aeruginosa) is ultimately contacted with a Glycomimetic compound, in an amount and for a time sufficient to achieve a discernable diagnostic or therapeutic result.
• a therapeutic result would relate, for example, to the prevention of lung infections.
• therapeutic results would be associated with the inhibiting of Pseudomonas (including, for example, arresting the growth of or killing the bacteria or preventing colonization by the bacteria), such as P. aeruginosa.
• therapy or therapeutic results includes treatment or prevention.
• Glycomimetic compounds of the present invention may be administered in a manner appropriate to the disease to be treated or prevented. Appropriate dosages and a suitable duration and frequency of administration may be determined by such factors as the condition of the patient, the type and severity of the patient's disease and the method of administration. In general, an appropriate dosage and treatment regimen provides the modulating agent(s) in an amount sufficient to provide treatment and/or prophylactic benefit.
• a Glycomimetic compound may be administered at a dosage ranging from 0.001 to 1000 mg/kg body weight (more typically 0.01 to 1000 mg/kg), on a regimen of single or multiple daily doses. Appropriate dosages may generally be determined using experimental models and/or clinical trials. In general, the use of the minimum dosage that is sufficient to provide effective therapy is preferred. Patients may generally be monitored for therapeutic effectiveness using assays suitable for the condition being treated or prevented, which will be familiar to those of ordinary skill in the art.
• Glycomimetic compounds may also be used to target substances to Pseudomonas bacteria, e.g., P. aeruginosa.
• Such substances include therapeutic agents and diagnostic agents.
• Therapeutic agents may be a molecule, virus, viral component, cell, cell component or any other substance that can be demonstrated to modify the properties of a target cell so as to provide a benefit for treating or preventing a disorder or regulating the physiology of a patient.
• a therapeutic agent may also be a prodrug that generates an agent having a biological activity in vivo.
• Molecules that may be therapeutic agents may be, for example, polypeptides, amino acids, nucleic acids, polynucleotides, steroids, polysaccharides or inorganic compounds.
• Such molecules may function in any of a variety of ways, including as enzymes, enzyme inhibitors, hormones, receptors, antisense oligonucleotides, catalytic polynucleotides, anti-viral agents, anti-tumor agents, anti-bacterial agents, immunomodulating agents and cytotoxic agents (e.g., radionuclides such as iodine, bromine, lead, rhenium, homium, palladium or copper).
• cytotoxic agents e.g., radionuclides such as iodine, bromine, lead, rhenium, homium, palladium or copper.
• Diagnostic agents include imaging agents such as metals and radioactive agents (e.g., gallium, technetium, indium, strontium, iodine, barium, bromine and phosphorus-containing compounds), contrast agents, dyes (e.g., fluorescent dyes and chromophores) and enzymes that catalyze a colorimetric or fluorometric reaction.
• imaging agents such as metals and radioactive agents (e.g., gallium, technetium, indium, strontium, iodine, barium, bromine and phosphorus-containing compounds), contrast agents, dyes (e.g., fluorescent dyes and chromophores) and enzymes that catalyze a colorimetric or fluorometric reaction.
• therapeutic and diagnostic agents may be attached to a Glycomimetic compound using a variety of techniques such as those described above.
• a Glycomimetic compound may be administered to a patient as described herein.
• Glycomimetic compounds may also be used in vitro, e.g., within a variety of well known cell culture and cell separation methods.
• a Glycomimetic compound may be immobilized on a solid support (such as linked to the interior surface of a tissue culture plate or other cell culture support) for use in immobilizing Pseudomonas bacteria for screens, assays and growth in culture.
• Such linkage may be performed by any suitable technique, such as the methods described above, as well as other standard techniques.
• Glycomimetic compounds may also be used to facilitate cell identification and sorting in vitro, permitting the selection of such bacterial cells.
• the Glycomimetic compound(s) for use in such methods is linked to a diagnostic agent which is a detectable marker.
• Suitable markers are well known in the art and include radionuclides, luminescent groups, fluorescent groups, enzymes, dyes, constant immunoglobulin domains and biotin.
• a Glycomimetic compound linked to a fluorescent marker such as fluorescein, is contacted with the cells, which are then analyzed by fluorescence activated cell sorting (FACS).
• Such in vitro methods generally comprise contacting a sample (e.g., a biological preparation) with any one of the Glycomimetic compounds, and detecting the compound in the sample.
• a sample e.g., a biological preparation
• one or more wash steps may be added to a method. For example, subsequent to contacting a sample with a Glycomimetic compound but prior to detection of the compound, the sample may be washed (i.e., contacted with a fluid and then removal of the fluid in order to remove unbound Glycomimetic compound).
• a wash step may be added during the detection process.
• a Glycomimetic compound possesses a marker (a diagnostic agent) that can bind to a substance that is detectable
• detecting the compound (or agent) in the sample includes detecting the compound (or agent) while it is bound to the sample, or detecting the compound (or agent) which was bound to the sample but after it has been separated from the sample.
• the compound obtained previously is dissolved in THF (40 ml) and Pd (10%)/C (1/10 by mass) is added. The solution is degassed and an atmosphere of H 2 is generated. The reaction is allowed to proceed at RT until disappearance of starting material is confirmed by TLC. The solution is filtered thru a bed of celite and the filtrate is concentrated in vacuo giving the 4 and 6 OH compound. The compound is then dissolved in pyridine (25 ml) and cooled to 0°C. Ph 3 CCl (1.2 eq) is added dropwise and the reaction is allowed to proceed at RT for 6 hrs.
• TBDMS-C1 (1.52 g, 10.1 mmol) and imidazole (0.94 g, 13.8 mmol) are then added and the reaction allowed to proceed at RT for 1 hr.
• Ethyl acetate 250 ml is added and the solution washed with saturated NaHCO 3 (5 X 250 ml) and saturated NaCl (1 X 250 ml). The organic layer is then dried with Na 2 SO 4 and purified by silica gel chromatography giving compound I.
• the solution is diluted with ethyl acetate (500 ml) and washed with H 2 O (300 ml) and saturated NaCl (150 ml). The organic layer is dried with Na 2 SO 3 and evaporated to dryness.
• the compound is purified by silica gel chromatography. The purified product (12.2 g, 9.33 mmol) is then suspended in MeOH/H 2 O (200 ml/20 ml) solution and LiOH-H 2 O (5.1 g, 121.3 mmol) was added. The reaction is allowed to proceed at 65°C for 20 hrs. Ethyl ether (500 ml) was added and the solution is washed with saturated NaCl (200 ml). The organic layer is dried with Na 2 SO 4 and evaporated to dryness.
• Compound K is purified via silica gel chromatography.
• reaction is allowed to proceed for 3 hrs and is then evaporated taken up in CH 2 C1 2 and washed with 0.1M HC1, saturated NaHCO 3 , and saturated NaCl.
• the resultant compound is purified by silica gel chromatography giving compound L.
• Compound 3 (1.96 mmol) is stirred in DMF/dichloromethane (50 ml, 1 :5 mix) along with 4A molecular sieves, TBABr (968 mmol) and CuBr 2 (529 mmol) for 1 hr.
• Compound 4 (2.94 mmol) is dissolved in dichloromethane (5 ml) and added dropwise to the solution. The solution is allowed to react for 24 hrs, filtered, and washed with saturated sodium bicarbonate and water (50 ml each). The organic layer is isolated, dried over sodium sulfate, and evaporated to dryness.
• Compound 5 is purified via column chromatography (hexane/ethyl acetate, 4:1 mix).
• Compound 22 is obtained by dissolution of compound 5 in methanol followed by addition of 1M sodium methoxide solution. The solution is allowed to react for 3-5 hrs and is then neutralized with Amberlite IR-120 resin, filtered and concentrated in vacuo. Compound 22 is purified by column chromatography (dichloromethane/methanol. 20:1 mix).
• PA-IIL lectin Bound PA-IIL lectin is detected with anti-PA-IIL rabbit antisera followed by HRP-labeled anti-rabbit Ig and TMB reagent with color development by 1M H 3 PO 4 ( Figure 18). Structures and activities are presented in Table 1.
• Neu5Ac ⁇ 2 6 Neu5Ac ⁇ 2-3Gal ⁇ 1 -3GlcN Ac ⁇ 1 -3Gal ⁇ 1 -4Glc-R DiSialylLea
• oligosaccharides chemically coupled to albumin are coated in plastic microtiter wells. After blocking with BSA, the wells are then incubated and allowed to bind to purified PA-IIL lectin. Bound PA-IIL lectin is detected with anti-PA-IIL rabbit antisera followed by HRP-labeled anti-rabbit Ig and TMB reagent with color development by 1M H 3 PO 4 ( Figure 19). Structures and activities are presented in Table 2. Biotinylated polymers of Fucose (Fuc-PAA-biotin, GlycoTech Corp.) are allowed to couple HRP-labeled streptavidin (KPL labs) overnight at 4°C.
• PA-IIL lectin is immobilized in plastic microtiter wells and allowed to react with the HRP- labeled fucosylated polymer. Binding is detected by TMB substrate followed by color development with IM H 3 PO 4 ( Figure 20). Conditions chosen for the assay are coating with 3 ⁇ g/ml PA-IIL and incubation of inhibitor with 2 ⁇ g/ml of fucosylated polymer. Compound 23 inhibited PA-IIL with an IC 50 of 420 nM ( Figure 22), while native saccharide inhibitor, Mannose, inhibited PA-IIL lectin with an IC 50 of 95 ⁇ M and the negative control saccharide, Galactose, showed no inhibition (data not shown).

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