JP2003508501A - Pulmonary delivery for biological binding - Google Patents

Abstract

  (57) [Summary] The present invention relates to the field of therapeutic and diagnostic agents in medicine. In particular, the present invention relates to the field of delivery of therapeutic and diagnostic agents, particularly pulmonary delivery, wherein the agent has increased bioavailability and increased therapeutic and diagnostic benefits for a given agent. It can be covalently attached to a site of interest in vivo to provide a mechanical duration. Disclosed are methods and compositions for pulmonary delivery of a therapeutic agent that can form a covalent bond at a site of interest or that forms a covalent bond with a pulmonary solution protein. Therapeutic agents useful in the present invention include wound healing agents, antibiotics, anti-inflammatory agents, anti-inflammatory agents, anti-reproductive agents, immunosuppressants, anti-infectives, and anti-cancer agents.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to the field of therapeutic and diagnostic agents in medicine. In particular, the present invention relates to the field of therapeutic and diagnostic agent delivery, particularly pulmonary delivery, wherein the agent is
It may be covalently linked to the site of interest in vivo to provide increased bioavailability and increased pharmacodynamic duration of therapeutic and diagnostic benefit for a given drug.

BACKGROUND OF THE INVENTION Peptide and protein drugs are increasingly used in major research and development programs in the pharmaceutical industry, and this is also important for therapeutic agents because of their advantages in genetic and biotechnology. It is a class. However, systemic delivery of these macromolecular and other therapeutic and diagnostic agents is largely due to their widespread presystemic elimination when taken orally, parenterally. Limited to the route. Faced with this dilemma of systemic delivery of these macromolecules with their unique conformational complexity of therapeutic activity, pharmaceutical scientists frequently seek alternative parenteral routes of administration as an alternative. Evaluate to.

Despite the tremendous efforts to address this problem, most have achieved only limited success with small peptides. An alternative non-invasive means for systemic delivery of therapeutic and diagnostic agents is via the pulmonary arterial system. Delivery through the pulmonary artery system is advantageous because it provides a large but very thin absorbable mucosa for increased absorption and delivery to the bloodstream. Pulmonary delivery of therapeutic and diagnostic agents, however, depends on the complexity of the human respiratory anatomy; the effect of breathing on drug deposition and the instability of the drug resulting from degradation in either the lungs or plasma. It's complicated.

Accordingly, there exists a need for improved and increased delivery of therapeutic and diagnostic agents, particularly for pulmonary delivery of therapeutic and diagnostic agents via increasing drug stability and blood absorption. To do.

SUMMARY OF THE INVENTION To meet these needs, the present invention relates to therapeutic and diagnostic agents capable of forming covalent bonds ex vivo or in vivo to proteins and other components of blood and lung fluids. The therapeutic agents of the invention have a long duration of action for disease management. The present invention provides for ex vivo and in vivo bioconjugation of therapeutic agents to proteins (eg, albumin), as well as dramatically increasing the half-life of therapeutic agents and avoiding the need for parenteral administration. For the in vivo pulmonary bioconjugation of therapeutic agents to endogenous pulmonary fluid proteins or other components.

The present invention reflects the ability to bioconjugate selected therapeutic agents to albumin-containing blood and lung fluid proteins for treatment as particles for pulmonary drug delivery. Pulmonary fluid protein conjugates are targeted to provide stable drugs that retain biological activity over time. The present invention provides long-term, local retention of therapeutic agent activity in the respiratory tract for use of selected therapeutic agents in the management of lung disease.

The invention further provides methods for facilitating systemic drug delivery of protein-therapeutic agent bioconjugates and proteins in vivo via pulmonary delivery with subsequent transcellular transport across the alveoli and lung mucosa. To a drug capable of forming a bioconjugate. The invention further relates to a method of facilitating systemic drug delivery of a protein-therapeutic agent bioconjugate via pulmonary delivery of the drug capable of forming a bioconjugate in vivo, the agent comprising:
It traverses the epithelium of the alveoli or lung mucosa, either via diffusion or via receptor-mediated transport, for conjugation with blood proteins. The methods of the invention result in a long-acting systemic therapy that is stabilized by ex vivo or in vivo conjugation to lung fluid proteins and / or blood proteins.

The present invention further relates to site-specific and protein-specific bioconjugation of therapeutic agents to albumin. Albumin has a unique nucleophilic moiety, particularly a thiol functional group on cysteine 34, which can nucleophilically attack electrophilic moieties present on therapeutic agents modified according to the present invention. This selective covalent bond formation allows bioconjugation with extracellular as well as intracellular albumin for long-term retention and bioavailability of the therapeutic agent.

The present invention further relates to the use of reaction chemistry including: N-hydroxysulfosuccinimide, maleimido-benzoyl-succinimide, γ-maleimido-
Butyryloxysuccinimide ester, maleimide propionic acid, isocyanate, thiol ester, thionocarboxylic acid ester, imino ester, and carbodiimide anhydride. Maleimidopropionic acid is the preferred reaction chemistry,
The present invention also contemplates the selection of the above and similar reaction chemistries as electrophilic moieties for bioconjugation with albumin or other proteins.

The invention further relates to the use of the composition for the manufacture of a medicament, wherein the composition comprises a derivative of an antihistamine and analogs thereof, wherein the derivative is a stable covalent bond. To react with amino, hydroxyl, or thiol groups on blood components, which reactive functional groups in the treatment of the human body to provide an antihistamine effect. For use it is selected from N-hydroxysuccinimide groups, N-hydroxy-sulfosuccinimide groups and maleimide groups.

The modified antihistamine can be cetirizine, loratadine and its analogs.

The invention further relates to the use of the composition for the manufacture of a medicament, wherein the composition comprises a derivative of an anti-angina agent and its analog, wherein the derivative is a stable covalent bond. To form an amino group, a hydroxyl group on the blood component,
Or a reactive functional group that reacts with a thiol group, which reactive functional group is an N-hydroxysuccinimide group, N-hydroxy-sulfo group for use in treating the human body to provide an anti-angina effect. It is selected from succinimide groups and maleimide groups.

The modified anti-angina agent can be tirofiban or an analog thereof.

The invention further relates to the use of the composition for the manufacture of a medicament, wherein the composition comprises a derivative of an antihypertensive agent and its analogs, wherein the derivative is
It contains a reactive functional group that reacts with an amino group, a hydroxyl group, or a thiol group on blood components to form a stable covalent bond, which reactive functional group of humans to provide an antihypertensive effect. It is selected from N-hydroxysuccinimide groups, N-hydroxy-sulfosuccinimide groups and maleimide groups for use in the treatment of the body.

[0015]   The antihypertensive agent can be enalapril or an analog thereof.

The invention further relates to the use of the composition for the manufacture of a medicament, wherein the composition comprises a derivative of an antiarrhythmic agent and analogs thereof, wherein the derivative is
It contains a reactive functional group that reacts with an amino group, a hydroxyl group, or a thiol group on a blood component to form a stable covalent bond, which reactive functional group of human to provide an antiarrhythmic effect. It is selected from N-hydroxysuccinimide groups, N-hydroxy-sulfosuccinimide groups and maleimide groups for use in the treatment of the body.

[0017]   The antiarrhythmic agent can be capovenic acid or an analog thereof.

The invention further relates to the use of the composition for the manufacture of a medicament, wherein the composition comprises a derivative of an antidepressant and its analogs, wherein the derivative is a stable covalent bond. To react with amino, hydroxyl, or thiol groups on blood components, which reactive functional groups in the treatment of the human body to provide antidepressant effects. For use it is selected from N-hydroxysuccinimide groups, N-hydroxy-sulfosuccinimide groups and maleimide groups.

[0019]   The antidepressant can be fluoxetine or its analogs.

The present invention further relates to the use of the composition for the manufacture of a medicament, wherein the composition comprises a derivative of a bronchodilator and its analogs, wherein the derivative is a stable covalent bond. To form an amino group, a hydroxyl group on the blood component,
Or a reactive functional group that reacts with a thiol group, the reactive functional group being an N-hydroxysuccinimide group, N-hydroxy-sulfo group for use in the treatment of the human body to provide a bronchodilatory effect. It is selected from succinimide groups and maleimide groups.

The bronchodilator may be theobromine acetamine or an analog thereof.

The invention further relates to the use of the composition for the manufacture of a medicament, wherein the composition comprises a derivative of opioid and analogs thereof, wherein the derivative is
It contains a reactive functional group that reacts with an amino group, a hydroxyl group, or a thiol group on blood components to form a stable covalent bond, which reactive functional group allows the human body to provide an analgesic effect. Are selected from N-hydroxysuccinimide groups, N-hydroxy-sulfosuccinimide groups and maleimide groups.

[0023]   The opioid can be fentanyl or an analog thereof.

The invention further relates to the use of the composition for the manufacture of a medicament, wherein the composition comprises a derivative of an anti-inflammatory agent and its analogs, wherein the derivative is a stable covalent bond. To react with amino, hydroxyl, or thiol groups on blood components, which reactive functional groups in the treatment of the human body to provide anti-inflammatory effects. For use it is selected from N-hydroxysuccinimide groups, N-hydroxy-sulfosuccinimide groups and maleimide groups.

[0025]   The anti-inflammatory agent can be loxoprofen and its analogs.

The invention further relates to the use of the composition for the manufacture of a medicament, wherein the composition comprises a derivative of an antithyroid deficiency drug and its analogs, wherein the derivative is a stable covalent compound. It contains a reactive functional group that reacts with amino, hydroxyl, or thiol groups on blood components to form a bond, which reactive functional group of the human body to provide an antithyroid deficiency effect. It is selected from N-hydroxysuccinimide groups, N-hydroxy-sulfosuccinimide groups and maleimide groups for use in treatment.

[0027]   The antithyroid deficiency agent can be thyroxine or an analog thereof.

The present invention further relates to compositions comprising one or more compounds selected from the group consisting of: 2- [2- [4-[(4-chlorophenyl) phenylmethyl [-1]
-Piperazinyl] ethoxy] -maleimidopropionyl acetamide; 11- (N
-Maleimidopropionyl-4-piperidylidene) -8-chloro-6,11-dihydro-5H-benzo- [5,6] -cyclohepta- [1,2-b] -pyridine; N- (1 (S) -ethoxy Carbonyl-3-phenylpropyl) -L-alanyl-L-prolinyl maleimidopropionylamide; maleimidopropynamyl-
ε- (3,4,5-trimethoxybenz-amide) -caproicamide; maleimidopropionamyl-1-theobromine acetamide; maleimidopropamyl 2- [
4- (2-oxocyclopentan-1-ylmethyl) phenyl] propionamide; N-maleimidopropionyl-N-methyl-3- (p-trifluoromethylphenoxy) -3-phenylpropylamine; 4-anilino-1- (2 -Phenethyl) piperzine and maleimidopropionamyl-3,5-3 ', 5' tetraiodothyroninamide.

The present invention further relates to an aerosol composition for delivery of a therapeutic agent to the pulmonary system of a host, the composition comprising an aerosolized aqueous solution containing the modified therapeutic agent bound to a blood protein.

The invention further relates to the use of a particulate formulation for delivery of a therapeutic agent to the pulmonary system of a host, the formulation comprising a dispersible dry powder containing the modified therapeutic agent, the modified The therapeutic agent comprises a therapeutic agent and a reactive group that reacts with an amino, hydroxyl or thiol group on the lung component to form a stable covalent bond, where the therapeutic The agent covalently binds to blood proteins.

DETAILED DESCRIPTION OF THE INVENTION Definitions In order to ensure a thorough understanding of the invention, the following definitions are provided: Therapeutic Agent: A therapeutic agent is a drug that has a therapeutic effect. Includes peptide and non-peptide organic molecules. Therapeutic agents include, but are not limited to, wound healing agents, antibiotics, antiinfectives, antioxidants, chemotherapeutic agents, anticancer agents, antiinflammatory agents, and antiproliferative agents. Therapeutic agents also include: abortion drugs, ace-inhibitors, alpha-adrenergic agonists, beta-adrenergic agonists, alpha-adrenergic blockers, beta-adrenergic blockers,
Corticosteroids, corticosteroids (suppressants), adrenocorticotropic hormones, alcohol inhibitors (deterrent), aldose reductase inhibitors, aldosterone antagonists, 5-alpha reductase inhibitors, anabolic agents (anabolics), analgesics, androgens , Anesthetics, angiotensin-protecting enzyme inhibitors, appetite suppressants, antacids, anthelmintics, anti-acne drugs, anti-allergic drugs, anti-hair loss drugs, anti-amebic drugs, anti-androgen drugs, anti-angina drugs, anti-arrhythmic drugs Agents, anti-arteriosclerotic agents, anti-arthritic / rheumatic agents, anti-asthma agents, antibacterial agents, aminoglycosides, amphenicol (amphenico)
l), ansamycin, β-lactam, lincosamide (
lincosamide), macrolide antibiotics, polypeptides, tetracyclines, antibacterial agents, 2,4-diaminopyrimidines, nitrofurans, quinolones and analogs, sulfonamides, sulfones, antibiotics, anti-gallstones (antic)
anti-cholesterolemia, anticholinergic, anticoagulant, anticonvulsant, antisuppressant, hydrazide / hydrazine, pyrrolidone, tetracycline, antidiabetic, biguanide, hormone, sulfonylurea derivative, antidiarrheal , Anti-drug, antidote, anti-dyskinesia, anti-eczema drug, anti-emetic drug, antispasmodic drug, anti-estrogen drug, anti-fibrotic drug, anti-bloom drug, anti-fungal drug, polyene, allylamine, imidazole, Triazole, and anti-glaucoma drug.

Other therapeutic agents include: anti-viral agents, anti-fusogenic agents, blood-brain barrier peptides (BBB).
Peptide), RGD peptide, glucagon-like peptide, anti-gonadotropin drug,
Anti-gout, anti-bleeding and anti-histamines; alkylamines (alkylmai)
ne) derivatives, aminoalkyl ethers, ethylenediamine derivatives, piperazine and tricyclic antidepressants, antihypercholesterolemic agents, antihyperlipidemic agents, antihyperlipidemic and antihyperlipoproteinemic agents, aryloxyalkanes Acid derivatives, bile acid sequestrants, hmg coa reductase inhibitors, nicotinic acid derivatives, thyroid hormones / analogs, antihyperphosphatasemia agents, antihypertensive agents, arylethanolamine (arlethanolamine) derivatives,
Aryloxypropanolamine (arloxypropanolamine)
) Derivatives, benzothiadiazine derivatives, n-carboxyalkyl derivatives, dihydropyridine derivatives, guanidine derivatives, hydrazine / phthalazine, imidazole derivatives, quaternary ammonium compounds, quinazolinylpiperazine derivatives, reserpine derivatives, sulfonamide derivatives, antithyroid hyperactivity Drugs, antihypertensive drugs, antihyperthyroid drugs, antiinfectives, antiinflammatory drugs, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives and arylcarboxylic acids.

Therapeutic agents also include: arylpropionic acid derivatives, pyrazole, pyrazolone, salicylic acid derivatives, thiazinecarboxamides, antileptic agents,
Anti-leukemia drug, anti-lipemic drug (antilipemic, antilipidemi)
c), antimalarial drugs, antimanic drugs, antimethemoglobinemia drugs, antimigraine drugs, antifungal drugs, antiemetic drugs, antitumor drugs and alkylating agents, antimetabolites, enzymes, androgens,
Antiadrenals, antiandrogens, antiestrogens, I
h-rh analogues, progestogens, adjuvants, folate supplements (replenish)
er), uroprotective agents and anti-osteoporosis agents.

Therapeutic agents also include: anti-Paget's disease agents, anti-Parkinson's disease agents, anti-peristaltic agents, anti-pheochromocytoma agents, anti-pneumocystis agents, anti-prostatic hypertrophy agents, antiprotozoal agents, anti-prozoal (Antiprozoal), antipruritic, antipsoriatic and antipsychotic, butyrophen, phenothiazine, thioxanthene, antipyretic,
Anti-rheumatic drug, anti-rickettsia drug, antiseborrheic drug
And antiseptics / bactericides, alcohols, aldehydes, pigments, guanidine, halogens / halogen compounds, mercury compounds, nitrofurans, peroxides / permanganates, phenols, quinolines, silver compounds, others, antispasmodics, antisyphilis drugs , Antithrombotic drug, anti-nodule drug,
Antitumor, antitussive, antiulcer, antiurine, antitoxin, antivertigo, and antiviral, purine / pyrimidinome, anxiolytic, arylpiperazine, benzodiazepine derivative, carbamate, astringent, Benzodiazepine antagonist, β-blocker, bronchodilator, ephedrine derivative,
Calcium channel blocker, arylalkylamine, dihydropyridine derivative, piperazine derivative, calcium regulator, calcium supplement, cancer chemotherapeutic agent, capillary protective agent, carbonic anhydrase inhibitor, cardiac depressant, cardiotonic agent,
Laxative, cation exchange resin, cck antagonist, central stimulant (central
stimulant), cerebral vasodilator, chelating agent, cholecystokine (c
holecystokinn) antagonist, gallstone solubilizer (choleith)
lytic agent), bile secretagogue, cholinergic agent, cholinesterase inhibitor, cholinesterase reactivating agent, cns stimulant, cognitive activator, contraceptive, drug for controlling intraocular pressure, converting enzyme inhibitor, coronary vasodilator , A cell protective agent, a debriding agent,
Decongestant, depigmenting agent, dermatitis herpes suppressant (dermatitis her)
preformis supplement), diagnostic aid, digestive aid,
Diuretics, bentothiadiazine derivatives, organic mercury compounds, pteridines, purines, steroids, sulfanamide derivatives, uracils, others, dopamine and receptor agonists.

Therapeutic agents also include: dopamine receptor antagonists, ectoparasiticides, electrolyte replenishers, emetics, enzymes, digestive agents, mucolytics, penicillin quenchers, proteolytic agents. , Enzyme inducers, estrogen antagonists, expectorants, gastric and pancreatic secretion stimulants, gastric proton pump inhibitors, gastric secretion inhibitors, glucocorticoids, α-glucosidase inhibitors, gonadotropins, gonadotropins, gout suppressors,
Growth hormone inhibitor, growth hormone releasing factor, growth stimulant, hematopoietic agent, hemolytic agent, hemostatic agent (demostatic), heparin antagonist, hepatoprotective agent, histamine h ₁ -receptor antagonist, histamine h ₂ -receptor antagonist, hmg coa reductase inhibitor , Hypnotics, low cholesterol and antihyperlipidemic agents.

Therapeutic agents also include: hypotensive agents, immunomodulators, immunosuppressants, inotropic agents, keratolytic agents, lactating hormones, laxatives / cathargin (catharg).
ic), Ih-rh agonist, fat increasing agent, local anesthetic, lupus erythematosus inhibitor, potent tranquilizer, mineralocorticoid, palliative tranquilizer, miotic, monoamine oxidase inhibitor, mucolytic, muscle relaxant , Mydriatic agents, anesthetics; analgesics, anesthetic antagonists, nasal decongestants, neuroleptics, neuromuscular blockers, neurotrophic agents, NMDA antagonists, notropic agents, NS
AID agents, opioid analgesics, oral contraceptives and ovarian hormones.

Therapeutic agents also include: uterine contractile agents, blood-brain barrier proteins,
GP-41 peptide, insulinotropic
Peptide parasympathomimetics, pediculicides, pepsin inhibitors, peripheral vasodilators,
Peristaltic stimulant, pigmentation agent, plasma expander (volume expander)
), Potassium channel activators / stomach fixators, pressors, progestogens, prolactin inhibitors, prostaglandins / prostaglandin analogs, protease inhibitors, proton pump inhibitors, 5a-reductase inhibitors, replenishers / replacement Agents, respiratory stimulants, reverse transcriptase inhibitors, scabicides, sclerosants, sedatives / hypnotics, acyclic ureides, alcohols, amides, barbituric acid derivatives, benzodiazepine derivatives, bromides, carbamates, chloral derivatives, quinazolones Derivatives and piperidinediones.

Therapeutic agents also include: serotonin receptor agonists, serotonin receptor antagonists, serotonin uptake inhibitors, skeletal muscle relaxants, somatostatin analogs, antispasmodics, stool softeners, succinylcholine synergists, sympathomimetics. , Thrombogen, thyroid hormone, thyroid inhibitor, thyroid hormone, preterm birth preventative, topical protectant, uric acid excretion agent, vasodilator, venous blood pressure agent, vasoconstrictor, vitamin / vitamin source, antichtic,
Anti-scurvy and anti-eyeball agents, enzyme cofactors, hematopoietic agents, proteinogenic agents and xanthene oxidase inhibitors.

Diagnostic Imaging Agents: Diagnostic imaging agents are agents useful in imaging the vascular system of mammals, and are proton emission tomography (PET) agents, computed tomography (CT) agents, magnetic resonance. Agents such as imaging (MRI) agents, nuclear magnetic imaging agents (NMI), fluoroscopy agents and ultrasound contrast agents. Iodine (
I) (including ¹²³ I, ¹²⁵ I, ¹³¹ I, etc.), barium (Ba), gadolinium (
Gd), technetium (Tc) (including ⁹⁹ Tc), phosphorus (P) (including ³¹ P),
Radioisotopes of elements such as iron (Fe), manganese (Mn), thallium (Tl), chromium (Cr) (including ⁵¹ Cr), carbon (C) (including ¹⁴ C), fluorescently labeled A compound etc. are mentioned.

Wound remedy: A wound remedy is an agent that facilitates the treatment of wounds. Wound therapeutic agents include integrins, cell adhesion molecules (eg, ICAM, ECAM, ELAM
Etc.), antibiotics, growth factors (eg EGF, PDGF, IGF, bFGF,
aFGF and KGF), fibrin, thrombin, RGD peptide and the like.

Anti-proliferative agents: Anti-proliferative agents include anti-metabolites, topoisomerase inhibitors, folate antagonist-like methotrexate, purine antagonist-like mercaptopurines, azathioprine, and pyrimidine antagonist-like fluorouracil, cytarabine, and the like.

Antioxidants: Antioxidants are agents that prevent oxidative damage to tissues, and include aspartates, orotates, tacophenol derivatives (vitamin E), and free radical scavengers (eg, SOD). , Glutathione, etc.).

Mammalian cells are continuously exposed to activated oxygen species such as superoxide, hydrogen peroxide, hydroxyl radicals, and singlet oxygen. These reactive oxygen intermediates respond to aerobic metabolism, catabolism of drugs and other xenobiotics,
Produced in vivo by cells in the ultraviolet and X-ray irradiation, and in respiratory bursts of phagocytic cells (eg, white blood cells) to kill invading bacteria (eg, bacteria introduced through a wound). For example, hydrogen peroxide is produced during respiration of most living organisms, especially by stressed and damaged cells.

Reactive oxygen species can damage cells. An important example of such damage is the peroxidation of lipids involved in the oxidative degradation of unsaturated lipids. Lipid peroxidation is very detrimental to membrane structure and function and can cause many cytopathic effects.
Cells protect against lipid peroxidation by producing radical scavengers such as superoxide dismutase, catalase, and peroxidase. Damaged cells have a reduced ability to produce radical scavengers. Excess hydrogen peroxide can react with DNA, causing stem damage, mutation generation, and altered and free bases. Hydrogen peroxide also reacts with pyrimidine,
The 6-double bond can be cleaved and this reaction inhibits pyrimidine's ability to hydrogen bond to complementary bases (Hallenderer et al. (1971)). Such oxidative biochemical damage results in loss of cell membrane integrity, reduced enzymatic activity, alterations in transport kinetics, changes in membrane lipid content, and leakage of potassium ions, amino acids, and other cellular material. Can happen.

Antioxidants have been shown to inhibit damage associated with reactive oxygen species. For example, pyruvate and other α-keto acids have been reported to react rapidly and stoichiometrically with hydrogen peroxide to protect cells from cytolytic effects (O'Do.
nnell-Tormey et al. Exp. Med. , 165,500-514
P. (1987)).

Anti-infective: An anti-infective is an agent that inhibits infection and includes antiviral, antifungal, and antibiotic agents.

Antiviral agents: Antiviral agents are agents that inhibit the virus and include vidarabine, acyclovir and trifluorothymidine.

Antifungal agent: An antifungal agent is an agent that inhibits fungal growth. Examples of antifungal agents include amphotericin B, miconazole, terconazole, econazole, isoconazole, thioconazole, and bifonazol.
le), clotrimazole, ketoconazole, butoconazole (butacon)
azole), itraconazole, oxyconazole, fenticonazole (p
lenticonazole), nystatin, naphthyfene (naphthy)
and fluconazole.

Antibiotics: Antibiotics are relatively low molecular weight, naturally occurring chemicals produced by various microbial species, such as bacteria (including Bacillus spp.), Radiobacilli (including Streptomyces) and fungi. Which inhibits the growth of or destroys other microorganisms. Agents with similar structure and type of action can be chemically synthesized or natural compounds can be modified to produce semi-synthetic antibiotics. These biosynthetic and semisynthetic derivatives are also effective as antibiotics. The main classes of antibiotics are: (1) β-lactam (penicillin,
(Including cephalosporins and monobactams); (2) aminoglycosides (eg, gentamicin, tobramycin, netilmycin, and amikacin);
(3) Tetracycline; (4) Sulfonamide and Trimethoprim; (5)
Fluoroquinolones (eg, ciprofloxacin, norfloxacin, and ofloxacin); (6) vancomycin; (7) macrolides, which include, for example, erythromycin, azithromycin, and clarithromycin;
And (8) other antibiotics such as polymyxin, chloramphenicol and lincosamide.

Antibiotics achieve their antibacterial effects through several mechanisms of action, which can be generally classified as: (1) agents that act on the cell wall of bacteria (eg bacitracin, Cephalosporins, cycloserine, fosfomycin, penicillin, ristocetin, and vancomycin); (2) agents that affect cell membranes or exert a washing effect (eg, colistin, novobiocin, and polymyxin); (3) its effect on ribosomes. Agents that influence cell replication machinery, signal transduction, and protein synthesis, such as aminoglycosides, tetracyclines, chloramphenicol, clindamycin, cycloheximide,
Fucidin, lincomycin, puromycin, rifampicin, other streptomycin, and macrolide antibiotics (eg, erythromycin and oleandomycin); (4) agents that affect the metabolism of nucleic acids (
For example, fluoroquinolones, actinomycin, ethambutol, 5-fluorocytosine, griseofulvin, rifamycin; Salicylic acid). Some drugs may have more than one primary mechanism of action, especially at high concentrations. Furthermore, secondary changes in the structure or metabolism of bacterial cells often occur after the primary effects of antimicrobial agents.

Anticancer agent: An anticancer agent (chemotherapeutic agent) is a natural or synthetic molecule that is effective against one or more forms of cancer. This definition includes molecules that by their mechanism of action are cytotoxic (anticancer chemotherapeutic agents), which stimulate the immune system (immunostimulators) and modulators of angiogenesis. The result in both cases is to slow the growth of the cancer cells.

Anti-cancer therapy is used to treat polycythemia vera and ³² P used in chronic leukemia
Including radioactive isotopes such as. Radioactive phosphorus has a biological half-life in humans of about 8 days. It emits β rays, which have a destructive effect on rapidly increasing cells. ³² P is usually administered at a dose of about 1 mc daily for 5 days.
Either the oral or the intravenous route can be used, and the doses do not vary widely. Radioactive iodine ¹³¹ I, radioactive gold ¹⁹⁸ Au, and other isotopes are not as useful as ³² P. Nevertheless, ¹³¹ I has some limited uses in metastatic thyroid cancer. Other radioisotopes are ⁵¹ Cr, ⁵² Mn, ⁵² M
g, ⁵⁷ Ni, ⁵⁵ Co and ⁵⁶ P, ⁵⁵ Fe, ¹⁰³ Pd, ¹⁹² Ir, ⁶⁴ Cu and ⁶⁷ C
As a complex of a radioactive metal such as u or (BFC), 6- [p- (bromoacetamido) benzyl] -1,4,8,11-tetraazacyclotetradecane-
1,4,8,11-Tetraacetic acid (BAT), 6- [p- (isothiocyanato) benzyl] -1,4,8,11-tetraazacyclotetradecane-1,4,8,11-
Tetraacetic acid (SCN-TETA), 4-[(1,4,8,11-tetraazacyclotetradec-1-yl) methyl] benzoic acid (CPTA), and 1-[(1,4,4.
7,10,13-Pentaazacyclopentadeca-1-yl) methyl] benzoic acid (
It can be used with our technique, either as a chelate of these metals using a bifunctional chelating agent such as PCBA).

Many drugs within the category of chemotherapeutic agents useful in the treatment of neoplastic disease are acceptable to the embodiments of the present application. Such agents induced using this technique may include: antimetabolites such as methotrexate (folate derivative), fluorouracil, cytarabine, mercaptopurine, thioguanine, petostatin (pyrimidine and purine). Analogs or inhibitors), various natural products (eg, vincristine and vinblastine (vinca alkaloids)), etoposide and teniposide.
de)), various antibiotics (eg mitomycin, plicamycin (plic)
amycin), bleomycin, doxorubicin, danorubicin (danor)
ubicin), ductomycin (dactomycin); various biological response modifiers (including interferon-α); various miscellaneous drugs and hormone modulators (cisplatin, hydroxyurea, mitoxantrone, procarbazine, amino) Glutethimide
Ethide), prednisone, progestin, estrogen, anti-estrogen (eg tamoxifen), androgen steroids, anti-androgen agents (eg flutamide), gonadotropin releasing hormone analogues (eg leuprolide), matrix metalloprotease inhibitors (MMPI) and Anti-cancer agents (including Taxol (paclitaxel)), and related molecules collectively referred to as taxoids, taxins, or taxanes.

The definition of “taxoid” includes various modifications and attachments to basic ring structures (taxoid nuclei), which may be shown to be effective for the reduction of cancer cell growth, and which Can be produced by organic chemistry techniques known to those skilled in the art.

Chemotherapeutic agents include podophyllotoxin and its derivatives and analogs. Another important class of chemotherapeutic agents useful in the present invention is camptothecin.

Another preferred class of chemotherapeutic agents useful in the present invention is anthracyclines (adriamycin and daunorubicin).

Another important class of chemotherapeutic agents is the compounds drawn from the following list: Taxotere, Amonafide, Illudin S, 6-hydroxymethylacrylfulvene Bryostatin 1, 26-succinylbryostatin 1, Palmitoyl Rhizoxin, DUP 941, M
itomycin B, Mitomycin C, Penclomedine,
Angiogenesis inhibitor compounds, Cisplatin hydrophobic complex (eg, 2-
Hydrazino-4,5-dihydro-1H-imidazole and platinum chloride, and 5-hydrazino-3,4-dihydro-2H-pyrrole and platinum chloride, vitamin A, vitamin E and its derivatives), especially tocopherol succinate.

Other compounds useful in the present invention include: 1,3-bis (2-chloroethyl) -1-nitrosourea (“carmustine” or “BCNU).
)), 5-fluorouracil, doxorubicin (“adriamycin”), epirubicin, aclarubicin, Bisantrene (bis (2-imidazolene-
2-ylhydrazone) -9,10-anthracene dicarboxaldehyde (an
thracenedicarboxaldehyde), mitoxantrone, methotrexate, edatrexate, muramyl tripeptide, muramyl dipeptide, lipopolysaccharide, vidarabine and its 2-fluoro derivatives, resveratrol and retinoic acid and retinol, retinol, retinol, retinol, and retinol. .

Other chemotherapeutic agents useful in the application of the present invention include: D
ecarbazine, Lonamine, Piroxantrone, A
nthrapyrazoles, Etoposide, Camptotheci
n, 9-aminocamptothecin, 9-nitrocamptothecin, camptothecin-
11 ("Irinotecan"), Topotecan, Bleomycin
, Vinca alkaloids and their analogs [Vincristine, Vi
norelbine, Vindesine, Vintripol, Vinxal
tine, Ancitabine], 6-aminochrysene, and Navelb
ine.

Other compounds useful in the application of the present invention are taxol, eleuterobin (e
leutherobin, sarcodictin, discodermolide and epothilone (ep)
It is a mimicry of othiolone).

Anti-neoplastic-Antineoplastic agents include fluoropyrimidines, pyrimidine nucleosides,
Purines, platinum analogs, anthracyclines / anthracenediones, podophyllotoxins, camptothecins, hormone and hormone analogs, enzymes, proteins and antibodies, vinca alkaloids, taxanes, antihormones
agent, antifolate, anti-microtubule inhibitor, alkylating agent (classical and nonclassical), antimetabolite, antibiotic, topoisomerase inhibitor,
Antiviral agents, and miscellaneous cytotoxic agents (eg hydroxyurea, mitotane,
Anti-cancer agents such as fusion toxins, PZA, bryostatin, retinoids, butyric acid and derivatives, pentosan, fumagillin) and the like. The purpose of all anti-neoplastic drugs is to eliminate cancer cells (curing) or to delay the growth and spread of cancer cells (remission). Most of the anti-neoplastic agents listed above explore this end by possessing primary cytotoxic activity that affects the direct killing of cancer cells. Other anti-neoplastic drugs stimulate the body's natural immunity, which affects the death of cancer cells.

Matrix Metalloprotease Inhibitor (MMPI) This is also known as matrix metalloproteinase inhibitor, MMPI is an inhibitor of matrix metalloprotease. This metalloprotease is a family of enzymes containing zinc in the active site, which facilitates the catalytic hydrolysis of various protein substrates. A subfamily of the metalloprotease family is the matrix metalloprotease (MMP
). Because these enzymes can degrade the major components of joint cartilage and underlying membranes. These matrix metalloproteases include, inter alia, stromelysin, collagenase, matrilysin and gelatinase. The activity of matrix metalloproteases is inhibited by the MMPI used in the preparation of the derivatized MMPI of the present invention. Some characterized MMPs and their preferred substrates are illustrated in the table below.

The nomenclature used to describe the interactions of proteases and their substrates is widely used in the protease literature. In this system,
The binding site for the polypeptide substrate on the protease is envisioned as a series of substrates. Each subsite interacts with a substrate amino acid residue. By convention, the amino acid residue of this substrate is called P (peptide); the subsite on the protease that interacts with the substrate is called S (subsite). This subsite resides in the catalytic or active site of proteases. The amino acid residues on the amino-terminal site of the scissile bond (the bond that is cleaved on the substrate) are called P ₁ , P ₂ , P _3, etc., and the residues on the carboxy-terminal site of the scissile bond are P ₁ ', P _2', is referred to as P ₃ '. This residue can be numbered up to P ₆ on each site of the scissile bond. Subsites on the protease are called S ₃ , S ₂ , S ₁ , S ₁ ', S ₂ ', S ₃ ', etc.,
Captures residues of substrates that interact with the enzyme.

[0064]

[Table 1] In this application, the term MMPI should be understood to include matrix metalloprotease inhibitors and analogs thereof. Furthermore, the term M
MPI refers to optical isomers and diastereomers; and racemic and resolved enantiomerically pure R and S stereoisomers; and R
And S stereoisomers and other mixtures of pharmaceutically acceptable salts thereof.

Oxytocin-Oxytocin is a hormone involved in the enhancement of lactation, uterine contractions, and prenatal pelvic relaxation. Oxytocin secretion in child-rearing women is stimulated by direct neural feedback obtained by stimulation of the teats during drinking. Its physiologic effects include contraction of mammary myoepithelial cells (inducing milk ejection from the mammary gland) and stimulation of uterine smooth muscle contraction leading to birth. Oxytocin causes the myoepithelial cells that surround the secretory acini of the mammary gland to contract, pushing milk through the ducts. In addition, it stimulates the release of prolactin, which in turn nourishes the breast and stimulates the formation of milk in the acini.

Cholecystokinin (CCK) -CCK is a 33 amino acid polypeptide originally isolated from porcine small intestine that stimulates gallbladder contraction and bile flow and increases secretion of digestive enzymes from the pancreas. It exists in multiple forms, including CCK-4 and CCK-8, with the octapeptide representing the dominant molecular species with maximum activity. It belongs to the CCK / gastrin peptide family and is distributed centrally in the nervous system and peripherally in the gastrointestinal system. It has many biological roles, including pancreatic secretions, gallbladder contractions and intestinal motility in the gastrointestinal tract, as well as possible mediation of satiety and pain stimuli.

Antihypertensive agents-Antihypertensive agents are a variety of agents that can be used to treat hypertension, including, but not limited to, enalapril, acebutolol, and doxazosin. Enalapril
Is a prodrug that is activated against angiotensin converting enzyme (ACE) inhibitor (enalaprilate). This prodrug exerts its antihypertensive effect by inhibiting the conversion of angiotensin I to angiotensin II and by suppressing the renin-angiotensin-aldosterone system. Acebutolol is a class of drugs called beta blockers, these are the class of drugs called blockers, which affect the heart and circulatory system. Acebutolol is used to lower blood pressure, reduce heart rate, and reduce angina (chest pain). Doxazosin is a member of the alpha blocker family of drugs used to lower blood pressure in people with hypertension. Doxazosin is also used to treat the symptoms of benign prostatic hyperplasia (BPH). Doxazosin acts by relaxing blood vessels, so that blood passes more easily through the blood vessels, thereby helping to lower blood pressure.

Methylprednisolone-Methylprednisolone is a synthetic steroid that suppresses acute and chronic inflammation. In addition, it stimulates gluconeogenesis, increasing protein catabolism and free fatty acid translocation. In addition, it may synergize vascular smooth muscle relaxation with β-adrenergic agonists and alter airway hyperactivity. It is also a potent inhibitor of inflammatory responses.

GP-41 Peptide-GP-41 is an HIV transmembrane protein that has been shown to be essential for the virus to fuse and infect healthy cells.

Anti-viral and anti-fusogenic peptides: Anti-viral peptides include, for example, cell-cell fusion or free.
) A peptide that inhibits viral infection of cells by inhibiting viral infection. The infectious pathway may involve membrane fusion, as occurs in the case of enveloped viruses, or some other fusion event involving viruses and cellular structures. Peptides that inhibit viral infection by a particular virus can be referred to for the particular virus (eg, anti-HIV peptide, anti-RSV peptide, etc.). Anti-spindle forming peptides inhibit the level of membrane fusion events between two or more entities (eg virus-cell or cell-cell) relative to the level of membrane fusion that occurs in the absence of these peptides. Or a peptide showing the ability to reduce.

In particular, as antiviral peptides and anti-spindle forming peptides, DP107
Peptides and DP178 peptides and analogs thereof, as well as amino acid sequences from other (non-HIV) viruses corresponding to the gp41 region of HIV from which DP107 and DP178 are derived, and other (non-HIV) ) Amino acid sequences derived from viruses. These peptides are effective against not only HIV but also other viruses, including human RS virus (RSV), human parainfluenza virus (HPV), measles virus (MeV) and simian immunodeficiency virus (SIV). It may exhibit viral activity.

In particular, anti-HIV peptides refer to peptides that show antiviral activity against HIV, and these activities include inhibition of CD-4 + cell infection by free virus and / or infected CD-4 + cells. Inhibition of HIV-induced syncytium formation with uninfected CD-4 + cells. Anti-SIV peptides are peptides that show antiviral activity against SIV, and these activities include inhibition of cell infection by SIV virus and inhibition of syncytium formation between infected and non-infected cells. To be Anti-RSV peptides are peptides showing antiviral activity against RSV, and these activities include inhibition of mucosal cell infection by free RSV virus and inhibition of syncytium formation between infected and non-infected cells. Is mentioned. Anti-HPV peptides are peptides that show antiviral activity against HPV, and these activities include inhibition of infection with free HPV virus and inhibition of syncytium formation between infected and uninfected cells. To be Anti-MeV peptides are peptides that show anti-viral activity against MeV, and these activities include inhibition of infection by free MeV virus and inhibition of syncytium formation between infected and non-infected cells. To be

Blood-Brain Barrier (BBB) Peptide-The "blood-brain barrier" is the layer of cells that controls substances that can penetrate the systemic circulation into the brain. The BBB protein may go through this barrier by protein translocation. Small sections of these proteins (10-16 residues long)
(Ie the BBB protein) is responsible for this transition.

RGD Peptide: An RGD peptide for conjugation to tissue or immobilized endogenous protein according to the invention has the following formula: R ₁ -Arg-Gly-Asp-R ₂ , an amino acid, preferably Naturally occurring L-amino acids and glycine)
Contains an array of. In this formula, R ₁ and R ₂ represent an amino acid or a sequence of more than one amino acid or a derivatized amino acid or a chemically modified amino acid or a sequence of more than one derivatized amino acid or a chemically modified amino acid. .

Insulin affinity peptide: Insulin affinity peptide (ITP) is a peptide that has insulin affinity activity. The insulinotropic peptide stimulates or causes the synthesis or expression of the hormone insulin. Such peptides include glucagon-like peptides, exendin
) 3 and exendin 4 and precursors, analogs, fragments of peptides such as other peptides having insulinotropic activity.

Glucagon-like peptides: Glucagon-like peptides (GLP) and GLP derivatives are intestinal hormones, which generally stimulate insulin secretion during hyperglycemia, suppress glucagon secretion and produce (pro) insulin. It stimulates synthesis and slows gastric emptying and acid secretion. As disclosed in US Pat. No. 5,574,008, which is incorporated herein by reference, some GLPs and G
LP derivatives promote glucose uptake by cells but do not stimulate insulin expression.

Pulmonary Condition: Pulmonary condition is a disease that affects lung function. Such a condition can be due to a deficiency in gene (s) associated with lung function (eg, cystic fibrosis), pneumonia, allergies, immune disorders or autoimmune disorders, microbial infections (eg, bacterial infections, viral infections, Fungal or parasitic infections), or mechanical damage to the lungs.

Exemplary pulmonary conditions contemplated by the present invention include cystic fibrosis, asthmatic bronchitis, tuberculosis, bronchitis, bronchiectasis, laryngotracheobronchitis, bronchiolitis, emphysema, bronchi. Pneumonia, allergic bronchial pneumonia, viral pneumonia, whooping cough, diphtheria, spastic croup, pulmonary tuberculosis, encephalitis with retention secretions, and pulmonary edema. Pulmonary conditions that develop as a result of injury or surgery (eg, after tracheotomy) as well as other pulmonary conditions associated with insufficient surfactant secretion in the lungs of premature infants are also contemplated by the present invention. Pulmonary conditions that are sensitively affected by treatment with the methods of the present invention also include inhalation of particulate matter (eg, smoking), exposure to architectural or other high dust areas, occupational occupations associated with inhalation of particles. Hazardous, environmental particles (eg smog, pollen, asbestos, silica powder (sil
Ionics)), pulmonary delivery of pharmaceutical agents (eg, bronchodilators), or activity associated with inhalation of cocaine.

Other pulmonary conditions include: diffuse parenchymal lung disease when infectious (eg cytomegalovirus pneumonia or miliary tuberculosis), drug-induced lung disease (penicillin, nitrofuranto). Infusion), neoplastic lung disease with a pattern of lymphangitis spread or bronchoalveolar cell carcinoma, chronic granulomatosis (infectious or non-infectious), hypersensitivity pneumonitis, histoplasmosis, tuberculosis, idiopathic ( idiophatic
) Pulmonary fibrosis (also known as fibrotic alveolitis of unknown cause), hereditary lung disorders (eg, alveolar microlithiasis and bronchiectasis), eosinophilic granuloma, lymphangiomyomatosis (ly
mpphangioleimyomatosis), and alveolar proteinosis.

Pulmonary Condition Symptoms: Pulmonary condition symptoms are symptoms associated with any of the above pulmonary conditions. Classic symptoms associated with such pulmonary conditions may include cough, exertional dyspnea, wheezing, chest pain and purulent sputum formation. Other components of the syndrome that may be associated with pulmonary conditions include hypoxia, carbon dioxide intoxication, hyperventilation, decreased expiratory volume, and decreased lung volume.

Pulmonary fluid: Pulmonary fluid is a fluid that covers the apical surface of the lung epithelium, particularly the alveolar epithelium, and comprises fixed and fluid lung fluid components.

Pulmonary Delivery Agents: Pulmonary delivery agents are agents that can be delivered to the lungs. Such agents include therapeutic agents.

Fixed lung component: The fixed lung component is a non-fluid lung component, which is a tissue, membrane receptor, interstitial protein, fibrin protein, collagen, platelet, endothelial cell, epithelial cell and its associated membrane and membrane receptors. , Somatic cells, skeletal muscle cells and smooth muscle cells, neuronal components, osteocytes and osteoclasts.

Fluid lung component: A fluid lung component is a lung component that does not have a fixed position for any extended period of time (generally not more than 5 minutes, more usually 1 minute). Flowable lung components are components of lung or lung fluid and include soluble proteins such as immunoglobulins, serum albumin, ferritin, transferrin, and the like.

Blood components: Blood components can be either fixed or flowable. Fixed blood components are non-fluid blood components and include: tissue, membrane receptors, interstitial proteins, fibrin proteins, collagen, platelets, endothelial cells, epithelial cells and their associated membrane and membrane receptors, body. Body tissues associated with cells, skeletal and smooth muscle cells, neuronal components, osteocytes and osteoclasts, and especially the circulatory and lymphatic systems. A fluid blood component is a blood component that does not have a fixed position for any extended period of time (typically no more than 5 minutes, more usually 1 minute). These blood components are not membrane bound and are present in blood for extended periods of time and are present at a minimum concentration of at least 0.1 μg / ml. Liquid blood components include serum albumin, transferrin, ferritin and immunoglobulins (eg,
IgM and IgG). The half-life of the fluid blood component is at least about 12 hours.

Inhaler Device: An inhaler device is any device useful for administering the inhaled medicament of the present invention. Examples of inhalation devices are nebulizers, metered dose inhalers, dry powder inhalers, intermittent positive pressure breathing apparatus, hygroscopic devices, bubble enviroments.
), an oxygen chamber, an oxygen mask, and a ventilator.

Reactive group: A reactive group is a chemical group capable of forming a covalent bond. Such reactive groups are linked or attached to therapeutic or diagnostic agents. The reactive group is
Generally stable in an aqueous environment and usually a carboxy group, a phosphoryl group, or a convenient acyl group (either as an ester or mixed anhydride), an imidate or a maleimide, thereby providing a target site on the lung component. Can form a covalent bond with a functional group such as an amino group, a hydroxy group or a thiol group in. For the most part, the esters include phenolic compounds or are thiol esters, alkyl esters, phosphate esters and the like. Preferably, the reactive group is a maleimide group.

Functional group: A functional group is a group on the lung component to which a reactive group on a therapeutic agent that has been modified reacts to form a covalent bond. As the functional group, a hydroxyl group for binding to an ester reactive entity; a thiol group for binding to a maleimide group, an imidate group and a thioester group; an amino group for binding to a carboxy group, a phosphoryl group or an acyl group; and A carboxyl group for bonding to an amino group may be mentioned.

IC ₅₀ : concentration of enzyme inhibitor at which 50% of enzyme activity is inhibited.

Protecting group: A protecting group is a chemical moiety utilized to protect a reactive entity from reacting with other functional groups. Various protecting groups are disclosed in US Pat. No. 5,493,007, which is incorporated herein by reference. Such protecting groups include acetyl, fluorenylmethyloxycarbonyl (FMOC), t-butyloxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and the like. For small organic molecules, tetrahydropyranyl (T
HP), all silyl derivatives, acetals, thioacetals and the like are suitable.

Linking group: A linking group is a chemical moiety that links or connects a reactive group to a therapeutic agent. The linking group is an alkyl moiety, a cycloalkyl moiety, a polycyclic moiety, an aryl moiety,
It may include polyaryl moieties, substituted aryl moieties, heterocyclic moieties, and one or more alkyl, alkoxy, alkenyl, alkynyl, or amino moieties substituted with a substituted heterocyclic moiety. The linking group can also be a polyethoxyamino acid (eg, AEA ((2-amino) ethoxyacetic acid) or the preferred linking group AEE.
A ([2- (2-amino) ethoxy)] ethoxyacetic acid).

Sensitive Functional Group: A sensitive functional group is a group of atoms that represents a potential reactive site on a therapeutic agent. When present, sensitive functional groups can be selected as attachment points for linking group-reactive group modifications. Sensitive functional groups include, but are not limited to, carboxyl groups, amino groups, thiol groups, and hydroxyl groups.

Modified Therapeutic and Diagnostic Agents-Modified therapeutic and diagnostic agents are agents that have been modified by attaching a reactive group. The reactive group can be attached to the therapeutic agent either through a linking group or optionally without a linking group.
The modified therapeutic and diagnostic agents may be administered in vivo such that conjugation with a blood or lung component occurs in vivo, or first in vitro conjugated with a blood or lung component. And the resulting conjugated therapeutic agent (as defined below) may be administered in vivo.

Conjugated Therapeutic Agents and Diagnostic Agents-Conjugated therapeutic agents and diagnostic agents include a reactive group of the modified therapeutic agent and a functional group of the lung component, with or without a linking group. Modified therapeutic and diagnostic agents conjugated to blood or lung components via a covalent bond formed between the two. As used throughout this application, the term "
A “conjugated therapeutic agent” can be more specialized as it refers to a specific conjugated therapeutic agent, eg, a “conjugated antihistamine.”

In light of these definitions, the present invention relates to modified therapeutic and diagnostic agents capable of reacting via covalent bonds with available functional groups on the lung or blood components. The invention also relates to methods of making these modified agents and uses thereof. The modified therapeutic agents of the present invention may react in vivo to form a conjugate with lung and / or blood components (eg, lung proteins or blood proteins),
Thereby, the half-life of the therapeutic agent can be extended and the bioavailability improved without adversely altering the therapeutic effect of the agent. In a preferred embodiment of the invention, the functional group on the protein is a thiol group and the reactive group on the modified therapeutic agent is a maleimide-containing group (eg, γ-maleimido-butyramide (GMBA)). , Maleimidopropionic acid (MPA) or maleimido-benzoyl-succinimide (MBS)).

The invention in one aspect contemplates the delivery of modified agents to the blood of the host for conjugation to blood components, including blood proteins. Pulmonary administration is further described as a route of administration, but the present invention is not limited to such a route of administration, and is intravenous (IV), intraarterial (IA), intramuscular (IM), subcutaneous (SC). Administration of the modified agent into the bloodstream of a patient using other methods is also contemplated, including parenteral routes such as.

For pulmonary delivery, a wide variety of devices and carrier molecules have been utilized to enhance pulmonary drug delivery and can be used with the modified agents of the invention. These devices and methods include metered doses, liposomes (M
eisner et al., 1989), lactide / glycolide copolymer (PLGA) nanospheres (Niwa et al., 1995), albumin microspheres (Feinstein et al., 1990), and other physical forms of aerosol or nanoparticle-forming physical technology. Be done.

A new type of inhalation aerosol, characterized by low mass density and large size particles, has enabled highly efficient delivery of inhaled therapeutic agents (eg insulin, testosterone) to the systemic circulation. Particles having a mass density of less than 0.4 grams per cubic centimeter and an average diameter of greater than 5 micrometers are:
It has been reported to circumvent the natural clearance mechanism of the lung and provide higher bioavailability than conventional inhaled therapeutic particles (Edwards et al., 1997). For most of these therapeutics, aerosols are small spherical droplets or mass densities of about 1 g / cm ³ and average geometries of between about 1 and 3 microns suitable for particle penetration into the respiratory tract or lung periphery. Designed to include particles of a geometric diameter.

Studies conducted primarily with liquid aerosols have shown that inhalation aerosols of these characteristics provide optimal therapeutic effects for both local and systemic therapeutic agent delivery. Inefficient drug delivery results from excessive particle agglomeration in the inhaler, deposition in the oral cavity and throat, and excessively rapid removal of particles from the lungs by a mucocillary or phagocytic clearance mechanism. It can still occur.

To address these issues, particle surface chemistry and surface roughness have traditionally been addressed. Recent data indicate that most improvements in aerosol particle performance can also be achieved by decreasing particle mass density and increasing particle size. Larger porous particles are less prone to agglomeration than (conventional) smaller non-porous particles. Also, large porous particles inhaled into the lungs can potentially release long-term therapeutic agents by escaping phagocyte clearance from the lung periphery, which can range from hours to many days. Enables therapeutic action for a period of time (Ed
wards et al., 1998).

Specific transport receptors for albumin (GP60, albongin (a
has been previously reported to reside in the endothelium, which functions as a unique albumin carrier (US Pat. No. 5,254,342). These transcytosis proteins promote the migration of albumin and albumin carriers across the respiratory tract wall, and result in high plasma levels of these proteins or protein carriers. As further described, agents modified according to the present invention are prepared to react with albumin, and upon pulmonary delivery, the resulting conjugates pass through the bloodstream through such carriers. obtain.

Pulmonary drug delivery is also advantageous for local treatment of the lung, where it promotes increased drug retention time in the lungs and, more importantly, reduced extrapulmonary side effects, It produces a constant enhanced therapeutic efficacy (Shek, 1994). Important benefits of pulmonary delivery include reduced systemic toxicity and increased drug concentration at the site of action (eg, site of infection or inflammation) (Stout and Der.
endorf, 1987).

In Vivo or Ex Vivo Conjugates Related to Pulmonary Drug Delivery (bioco
The use of njugation) includes the following non-limiting advantages. Retention of the drug at the site of deployment is enhanced due to the covalent attachment of the drug to the respiratory tract site. Furthermore, in situ conjugation to soluble proteins for localized intrapulmonary activity allows for a long duration of drug action both in the lung as well as systemic absorption and conjugation to blood proteins.

Drug stability is improved both locally and systemically as conjugation provides protection against enzymatic degradation that occurs in lung mucus or plasma. Also, in the deep lung, alveolar macrophages rapidly deposit particles; the reactivity of the modified agents of the invention with epithelial cells allows localized retention of the agent.

Localized delivery to lung tissue also resulted in a first liver-pass effect.
The absence of any s liver effect reduces toxicity and reduces systemic exposure. Systemic delivery is also due to, for example, liver, central nervous system (CNS) limitations, eg, reduced extravascular side effects due to conjugation to albumin or limited clearance of albumin to these organs. Shows reduced renal toxicity.

Pulmonary delivery of the modified agents of the present invention also provides the advantage of improved patient compliance due to the long duration of action of the modified agent. Cost benefits can then be achieved, for example, by reducing the cost of goods per course of treatment due to the long duration of action, and outpatient use of drugs with otherwise limited or complex dose titration schedules. Can be done. Pulmonary delivery may also reduce difficulties associated with oral dosing, including low solubility, food interaction, and low bioavailability.

A further advantage of pulmonary delivery of the modified agents of the invention is that due to infusion, due to large macromolecular drugs such as insulin, growth hormone, β-interferon, calcitonin, and their large size and instability. The ability to deliver other drugs that are typically delivered systemically. The present invention provides an alternative or convenient route of administration for these drugs. Moreover, many drugs and therapeutic peptides are more stable in solid, dry form than in solution. The dry lung formulation of the drug thus modified according to the present invention provides a more stable form of the drug and the convenience of pulmonary administration.

1. Therapeutic Agents A wide variety of therapeutic agents are contemplated for use in the present invention, including peptide therapeutic agents and small organic molecules, where they can be modified as described.

In addition to the therapeutic agents discussed above, the following therapeutic agents are within the scope of the invention. Sympathomimetic compounds mimic the effects of endogenous catheter amines (adrenergic-like neurotransmitters) on peripheral sympathetic neurons in addition to the CNS effects.
The adrenergic system controls important physical functions such as blood pressure regulation and alertness. A huge variety of compounds have been developed that selectively fine-tune these systems. These include agonists used as decongestants and therapeutic agents for asthma, and antagonists used as antihypertensive agents.

[0110]

[Chemical 1] Selective agents include alpha agonists such as phenylephrine (neo-synephrine) and more lipophilic agents such as naphazoline. Clonidine, an antihypertensive drug, is sometimes used in ethanol withdrawal; and has been tried in smoking cessation. This is the autoreceptor
Probably has a net antagonistic effect through the presynaptic receptor, which detects excess adrenergic agonists and reduces norepinephrine release. Ivopamine, a cardiotonic, has diuretic and dopamine agonist activity.

Beta agonists are general sedative sedatives. Isoproterenol and dobutamine are fairly selective for the β1 receptor; the latter are used as cardiotonics.

[0112]

[Chemical 2] Alpha blockers such as phentolamine and prazosin are also used as antihypertensive agents. Yohimbine, a selective α2 blocker, is a popular libido-promoting drug for men aimed at prolonging and enhancing erections. The α2 receptor is inhibitory and consequently blocks the development of stimulatory effects. The α2 and α3 receptors are also present at fat storage sites; antagonizing them may provide a way to promote the catabolism of fat to central stimulants without re-storing.

Β-antagonists include propranolol, which has been used for some time to calm nerves during event-specific anxiety (graded fear), as well as for its traditional role in hypertension. It was Acebutolol is a cardioselective beta blocker used in angina.

Antidepressants Antidepressants act by altering the concentrations of catecholamines and / or serotonin in CNS neurons that emanate from the limbic system to the frontal lobe. Elevated levels of catecholamines (excitatory adrenergic neurotransmitters) cause irritation. Elevation of serotonin, an inhibitory neurotransmitter, produces a sedative effect and then upregulation of the catecholamine system as a mechanism of habit.

[0115]   (Selective serotonin reuptake inhibitor)

[0116]

[Chemical 3] Selective serotonin reuptake inhibitors (SSRIs) inhibit serotonin reuptake without significantly affecting the adrenergic system. The side effect profile is much less than that of tricyclic. This is because muscarinic binding, histamine binding and adrenaline binding are significantly reduced.

The mood effects of these serotonin agents have led to a more complex theory of how antidepressants work. According to one hypothesis, the noradrenaline system (dopamine, noradrenaline), which is the basis of the serotonin system, upregulates, ie, by increasing the number of receptors on, individual postsynaptic nerve surfaces, the inhibitory serotonin function. Respond to the increase in. This increase in adrenergic neuronal function, in turn, accounts for antidepressant activity that was delayed for weeks by the onset of inhibition of serotonin reuptake. Another possibility is that serotonin receptors take some time to register excess serotonin and respond to similar mechanisms. Furthermore, recent evidence suggests that, for example, transcriptase-mediated interaction with DNA increases neurotransmitter production by producing more synthase enzymes.

Anti-Histamines, Anti-Asthma Agents and Histamine Agonists Anti-histamines are compounds that block histamine from activated histamine receptors. Because histamine functions to mediate allergic reactions, blocking histamine at the H1 receptor abolishes the body's characteristic responses (ie, inflammation and vasoconstriction). H2 receptors in the stomach regulate the release of gastric acid; therefore, novel classes of H2 blockers (eg, Zantac and Tagamet) selectively block H2 receptors without affecting H1 receptors involved in allergic reactions. To stop the secretion of acid.

Histamine is concentrated in mast cells. The function of mast cells is
A cell that is essentially responsible for releasing histamine and immune globin when tissue damage occurs. Receptors on the cell surface lyse (pry open (br
eaking open)) and release these allergic agents. Mast cells are parts of the body that are often damaged (eg, fingers and toes),
Or parts of the body that frequently come into contact with the environment (eg, mucous membranes such as lips, nose).
Especially in.

Histamine is also a neurotransmitter in the CNS, and a typical problem with some antihistamines is lethargy. Attempts to create compounds that do not enter the brain have worked.

[0121]   (Antihistamine)

[0122]

[Chemical 4] Fenethazine represents a tricyclic antihistamine that is very similar to Thorazine (a potent antipsychotic dopamine blocker). Cyproheptadin (which also acts on serotonin receptors) is similar to the currently widespread Claritin. Peria
Ctin has been psychiatrically prescribed for anxiety. Benadryl is
Probably the best known class; Benadryl has potent sedative properties. Hydroxyzin has also been prescribed as a sedative. Dimenhydrinate has been marketed as Dramamine as an antiemetic drug. Cetirizine (
Cetirizine) (Zyrtec) is a metabolite of hydroxyzine; since hydroxyzine is available as a generic, it is cheaper and as effective as newer drugs.

Azelin has a novel structure, but also makes both H1 and H2 receptors. Cromolyn acts by another mechanism; cromolyn prevents the release of histamine (binding to mast degranulation) after binding of immunoglobin on mast cells.

[0124]   (Histamine blocker (H2 blocker))

[0125]

[Chemical 5] H2-selective antihistamines have become popular as a treatment for excess gastric acid.
These histamine blockers are structurally and mechanistically very similar. All four have about the same bioavailability, half-life, and antagonist activity.

[0126]   (Proton pump inhibitor)

[0127]

[Chemical 6] Omeprazole (Prilosec) and Lansoprazole (Prevac)
id) belongs to the class of antisecretory compounds (substituted benzimidazoles), which show no anticholinergic properties or histamine H2 receptor antagonist properties, but of the (H +, K +)-ATPase enzyme system at the secretory surface of gastric parietal cells. By specific inhibition, it suppresses the secretion of gastric acid. These proton pump inhibitors are
It has emerged as a therapeutic alternative to histamine agonists for the treatment of gastric disorders, especially acid reflux disease ("heartburn").

[0128]   (Histamine agonist)

[0129]

[Chemical 7] It is possible to find drugs that act as mimetics or agonists for any receptor system. With respect to histamine receptors, these drugs do not find much clinical utility, but nonetheless their listing is (to some extent) satisfactory. 2-Methylhistamine acts as a selective agonist at the H1 receptor; 4-methylhistamine is relatively selective at H2. Impromidine antagonizes the H2 receptor but functions as an agonist in H3.

Anti-Asthma Agents Asthma essentially emphasizes allergic reactions to the environment (ie, autoimmune disorders). The process of allergic reaction is complex and this gives many points of attacking the problem. First, immune globins and antigens bind to the surface of mast cells. Mast cells then release histamines, leukotrienes, peptides, which bind to tissue receptor sites and control various chemical functions (eg, blood pressure regulation, smooth muscle tone, fluid disposal, etc.). To change. Compounds that inhibit any of these steps may be used to treat asthma and allergies that begin with the antihistamines listed above.

[0131]

[Chemical 8] Probably the oldest way to reduce asthma symptoms is bronchodilation with methylxanthine compounds such as caffeine and theophylline. These are now obsolete due to other compounds such as β2-adrenergic agonists (β agonists), which perform the same function more selectively. Methylxanthine is cAM
It acts by inhibiting the enzyme that performs P-degradation (phosphodiesterase) and by antagonizing adenosine, which causes bronchoconstriction. Beta agonists, like metaproterenol, isoethalin, isoproterenol, terbutaline, and albuterol, mimic adrenaline in a subset of adrenergic receptors, which control smooth muscle tone such as bronchial tone. Another older drug exemplified by atropine (an antimuscarinic drug)
Enjoyed some historical uses.

Zafirlukast (Accolate) represents a line of attack on leukotriene compounds released from mast cells with histamine. Leukotrienes are histamine-like mediators that bind receptors on tissue cells and signal allergic reactions. By blocking these, they block the signal to the (bronchial) cells and thus the unwanted response.

The final mechanism that can be attacked is the slow immune response to binding by leukotrienes and / or histamine. The body uses glucocorticoid steroids to control inflammation, and fluticasone (Flona).
Se) and beclomethasone (Beclonase) are synthetic mimetics.

Antihyperlipidemic Agents Antihyperlipidemic agents reduce atherosclerosis by lowering blood cholesterol levels and inhibiting plaque formation on the inside of arteries and vessels. It is a relatively new drug that helps prevent it. The formation of this plaque depends on the proportion of different types of blood fat, in particular the ratio of high density lipoprotein (HDL) to low density lipoprotein (LDL). This rate is then influenced by genetics and the amount of certain substances in the diet, especially cholesterol and saturated fat. Cholesterol is important in the formation of VLDL (a large lipoprotein produced by the liver); versus catalysis by lipoprotein lipase, VLDL is a smaller LDL (this is the so-called "bad cholesterol"). HDL), and HDL is commonly referred to as "good cholesterol". Due to the complexity of producing these lipoproteins, several points can be targeted for action by various drugs.

[0135]   (HMG-CoA reductase inhibitor ("statin drug"))

[0136]

[Chemical 9] The most commonly used antihyperlipidemic agents are simvastatin analogues, especially simvastatin (Zocor) itself. These drugs, known as HMG-CoA reductase inhibitors, reduce cholesterol production by inhibiting the first step of sterol synthesis involved in the production of the mevalonate ion from CoA-S-mevalonate. . Reduced cholesterol formation results in a decrease in VLDL and thus a decrease in LDL. Pravastatin is an active metabolite of mevastatin; lovastatin and simvastatin are inactive prodrugs that act via their hydroxyl derivatives obtained by similar ring opening.

These drugs also stimulate receptor-mediated clearance of LDL. L
DL receptors undergo upregulation (increased receptor density),
And the VLDL catabolism is increased.

Recent studies suggest a reduction in osteoporosis as a result of treatment with statin drugs.

[0139]   (Clofibrate analog)

[0140]

[Chemical 10] Other drugs such as clofibrate and gemfibrozil increase the activity of lipoprotein lipase and decrease the level of VLDL. This enzyme is an extracellular species found in blood and intestine. The effects of other lipase enzymes can cause common nausea with this drug. Cholesterol production in the liver is reduced, probably as a side effect due to lower VLDL levels, but fecal cholesterol excretion is increased. Clofibrate is quite toxic and can be carcinogenic.

Antihypertensive Agents The body controls blood pressure by a complex feedback mechanism between baroreceptors and effector nerves, which are primarily predominantly adrenergic. This system is regulated by the peptide system (angiotensin / renin).

Pharmacological control of blood pressure works via four basic mechanisms. Sympathoplegic agents reduce peripheral vascular tolerance, inhibit cardiac function, and increase venous retention by many mechanisms involved in the adrenergic nerves.

Direct acting vasodilators reduce blood pressure by increasing the volume of blood. Veins and arteries are in the form of smooth muscle, and relaxing muscles produce greater volume and lower pressure. Angiotensin antagonists act on the peptide system with effects on smooth muscle. Finally, diuretics reduce sodium content, reduce blood volume and thus lower blood pressure. Since the body responds to exogenous drugs by regulating homeostasis, the simultaneous use of several drugs acting on different mechanisms is often used rather than simply increasing the dose of a single drug.

[0144]   (Adrenergic receptor drug)

[0145]

[Chemical 11] Sympathetic paralytics or sympathetic blockers antagonize the function of adrenaline compounds. β-blockers block β-1 receptors in myocardium,
Reduces cardiac output. As a β blocker, propranolol (Inder
al), metoprolol (Lopressor), labetalol (Normod)
yne), nadolol (Corgard), atenolol (Tenormin)
, And acebutolol (Spectrol). Some adrenergic receptor activating compounds have a hypotensive effect by acting centrally on the alpha receptor. Adrenergic receptor activating compounds include clonidine (Catapres), guanfacine (Tenex), and guanabenz (Wy
tensin), and methyldopa. They probably act by activating presynaptic autoreceptors and decreasing norepinephrine (agonist) release.

Compounds that peripherally block the α-1 receptor in blood vessels include prazosin (Minipress) and terazosin (Hytrin).

Reserpine blocks amine uptake, while guanethidine (Ismelin) and guanadrel (Hylorel) block sympathetic nerve endings.

Propranolol, which was previously the most prescribed class of drugs, leads to the accumulation of bradykinin, which contributes to the hypotensive effect.

[0149]   (Colinergic drug)

[0150]

[Chemical 12] Some anticholinergic drugs (ganglion blockers) are effective as antihypertensive agents, but they are less popular because of side effects. These anticholinergics include trimetaphane (Arfonad) and mecamylamine (Inversin).
e) can be mentioned.

[0151]   (Direct vasodilator)

[0152]

[Chemical 13] Diazoxide acts by opening potassium channels and relaxing venous and arterial smooth muscle. Despite structural similarities, diazoxide does not have diuretic properties. Diazoxide has a relatively long half-life of about 24 hours and is often used parenterally (by injection) in emergencies. This drug also inhibits insulin release and is used in diabetes. Minoxidil is mainly used orally and also opens potassium channels.

Hydralazine and minoxidil dilate arteries but not veins.
Nitroprusside (as the sodium salt) expands both by mechanisms involved in the activation of guanylyl cyclase, resulting in the formation of cGMP and relaxation of smooth muscle.

[0154]   (Calcium channel blocker)

[0155]

[Chemical 14] Calcium channel blockers are a popular tool for controlling hypertension. Smooth muscle contraction controls muscle tone, depending on calcium influx. The reaction pathway is complex and involves peptide calmodulin and myosin light chain kinase (M
LCK) is included. When the latter enzyme is activated, it phosphorylates myosin, which works with actin, causing muscle contraction. Blocking this channel statistically prevents calcium ion influx and reduces tone in vascular smooth muscle. Skeletal muscle depends on intracellular calcium ions and is unaffected by these drugs. Myocardium is highly dependent on the action of calcium channels.

Calcium channels are also present at presynaptic nerve endings in adrenergic neurons. These are voltage-gated ion channels embedded in the neuronal membrane at the ends of adrenergic neurons. When a voltage pulse arrives at the end of the neuron (transmitted by the continuous firing of sodium channels), calcium channels detect changes in voltage and allow Ca ⁺⁺ ion influx. It binds vesicles and co-transmitters (eg peptides, A
Trigger the release of vesicular adrenaline, noradrenaline or dopamine with TP). One of the acid tests on whether a substance is a neurotransmitter is whether its release is calcium dependent.

Verapamil is the oldest and prototype calcium channel blocker. It is highly bound to plasma proteins and undergoes 80% liver first pass elimination upon oral administration. This is because most of the drugs that are absorbed through the intestines
It is meant to be removed by the kidneys before reaching the target tissues (heart and major blood vessels). Nifedipine is much less active at cardiac sites than diltiazem or verapamil, and is also very plasma-bound (plasma-bo).
und). Diltiazem has very low plasma binding. Despite plasma binding, all three drugs have fairly short half-lives (3-6 hours).

Ion channel blockers generally load into open channels.
And work by blocking that channel. The similarity in ion channels means that some sodium channel blockade occurs with calcium channel blockers. This is a problem that is more associated with verapamil than other drugs. In addition to treating hypertension, these drugs are used to treat angina, migraine and atherosclerosis.

[0159]   (Diuretic)

[0160]

[Chemical 15] Diuretics reduce the volume of blood by causing drainage of water through the kidneys. This reduces blood pressure very efficiently. The drug used for this purpose is typically thiazide and the main problem is potassium deficiency.
Potassium-sparing diuretics have also been developed.

[0161]   (Angiotensin antagonist and ACE inhibitor)

[0162]

[Chemical 16] Recent studies have shown that ACE inhibitors are very safe drugs. ACE is an enzyme that converts angiotensin I into its active form, angiotensin II. Angiotensin receptors regulate tone of smooth muscle, including venous and arterial tissues. Inhibiting this enzyme reduces the amount of active peptide remaining in body tissues.

Antipsychotics, Lithium, Mood Stabilizers and Dopamine Agonists Antipsychotics are used to calm mania or racing thought, to control aggression, or to fake thoughts in schizophrenia ( spu
Used to block riotous things, including hallucinations (hearing). They exert their sedative effect by blocking the excitatory neurotransmitter dopamine at the postsynaptic terminal. Dopamine neurons are abundant in the limbic system and its projections into the cerebrum, especially the origin in the substantia nigra. Blocking the adrenergic, histamine and serotonin receptors also contributes to the CNS effects of these drugs.

Although these are powerful psychotropic drugs, these drugs are generally not abused. This is because they inhibit the cerebral pleasing pathway from the limbic system to the frontal lobe.

[0165]   (Thorazine analog)

[0166]

[Chemical 17] Tricyclic compounds such as Thorazine (known as phenothiazine) are the oldest compounds and the least selective, blocking some subtypes of dopamine receptors. Thorazine was discovered incidentally during the search for better antihistamines. This shows efficacy in blocking the effects of LSD, confirming the drug's dopamine agonist activity.

[0167]   (Haloperidol analog)

[0168]

[Chemical 18] A newer compound in the haloperidol class (butyrophenone
phenone)) (first synthesized in the late 1950s) is more selective for the D2 subreceptor.

[0169]   (Sulpiride analog)

[0170]

[Chemical 19] Clevopyrido and sulpiride analogs represent another structural class of antipsychotic drugs with similar effects. The binding profiles for all of these groups (actually each compound) are slightly different.

[0171]   (Clozapine analog and novel neuroleptic drug)

[0172]

[Chemical 20] Clozapine analogs represent another structural type with different neurological profiles. These are not as selective for D2 as the Haldol class, but may be more effective in controlling some forms of psychosis. These drugs show significant 5-HT2 receptor blockade and may be more selective for the limbic dopaminergic system (in contrast to drugs involved in motor control) and are common to antipsychotics. Reduces extrapyramidal syndrome (EPS) and motor abnormalities. Newer drugs, such as Zyprexa and Seroquel, do not appear to give clozapine the general granulocytopenia.

Clozapine is well metabolized and some of its metabolites exhibit anti-AIDS activity.

Another novel structural class that functions as neuroleptic agents is butaclamol (bu).
It is represented by taclamol) (pentacyclic) and tetrabenazine. The latter is a dopamine depletor, a drug that can chemically induce depression.

[0175]   (Mood stabilizer)

[0176]

[Chemical 21] The use of antispasmodic medicines for psychotropic purposes has recently developed mainly for prophylactic agents against manic-depressive syndrome and / or panic syndrome. Phenytoin and carbamazepine presumably act by affecting the ion barrier system (especially sodium channels) in excitable membranes. Phenytoin is structurally similar to barbiturate, while carbamazepine has a tricyclic antidepressant-like tricyclic structure.

Gabapentin and valproic acid probably act on the GABA system.
The latter inhibits the enzyme responsible for degrading GABA at high concentrations, but probably acts by other mechanisms at relatively low therapeutic levels. Lamotrigine is another antispasmodic used as a mood stabilizer. This reduces glutamate (excitatory amino acid) release. Topamax (fructose derivative) is GABA
It enhances the system while inhibiting glutamate.

Propanolol is a beta adrenergic blocker prescribed for motion phobia or stage startle. Clonidine is an alpha agonist sometimes used, for example, to sedate peripheral tremor in the case of alcohol withdrawal. Verapamil (calcium channel blocker) is also used for this purpose.

[0179]   (Dopamine agonist)

[0180]

[Chemical formula 22] Pergolide and naxagolide are dopamine agonists used for tremor paralysis, which results from reduced dopamine function in the age-related CNS. Apomorphine is a selective D2 agonist, while ibopamine is
It provides agonist function for both dopamine and adrenergic receptors.

Cholinergic Drugs Acetylcholine neurons carry sensory information to the brain and control muscle tone, which includes peristaltic and motor control. Cholinergic neurons
It is dominant in the inhibitory activity intrinsic to so-called parasympathetic neurons, which are dopamine / norepinephrine-based neurons of complement in parallel sympathetic nerve structures. Two cholinergic receptor subtypes (nicotinic and muscarinic) have been identified by selective agonists. At least two subtypes of muscarinic receptors (M1 and M2) have been identified.

In addition to direct agonists, selective antagonists, enzyme inhibitors and antidotes to enzyme inhibitors have been developed. Corinoceptor (chol
inoculator) also acts as a heteroreceptor, presynaptically governing the release of norepinephrine and other neurotransmitters.

[0183]   (Corinoceptor agonist)

[0184]

[Chemical formula 23] Nicotine is a selective agonist at the nicotine receptor: it defines this subset of cholinergic receptors. Muscarinic defines another subset, with additional discrimination of pre-existing M1 and M2 (at least). Muscarine is produced in trace amounts in fly agaric mushrooms. Other species of fungi produce larger amounts. Fly Agaric also contains a muscarinic antagonist (atropine) and a GABA agonist (muscimol). Atropine was used before the role of muscimol was elucidated and applied as an antidote to the toxic effects of muscarine in this fungus.

N-hydroxymethylamide of nicotinic acid is also active as an agonist at nicotinic corinoceptors. Carbachol is used ophthalmically (ie, dilates the pupil) as a miotic.
Carbachol is also used primarily in the relaxed gastrointestinal tract in large animals.
Because its positive positive charge prevents carbachol from entering the brain and limits its absorption in the digestive tract. In addition to receptor action, carbachol probably enhances acetylcholine release. Racecin is a selective muscarinic agonist.

Guanidine exists as a guanidinium ion at physiological pH; guanidine is used as a procholinergic, antiviral, antifungal, antipyretic and muscle excitatory agent. Betanicol activates the M1 and M2 subreceptors, releases IP3 (inositol triphosphate), and activates guanylyl cyclase. In addition, as a fourth order positively charged species, betanicol is primarily used to mimic acetylcholine in the gastrointestinal tract. It is sometimes provided to reduce antimuscarinic constipation caused by tricyclic antidepressants or other drugs. Pilocarpine is a cholinergic agent that also increases gastric acid secretion.

GABA Drug GABA (γ-aminobutyric acid) is the most important inhibitory neurotransmitter in the CNS. By passing the negative chloride ion (CI-) through the barrier into nerve cells, GABA inhibits the presynaptic release of neurotransmitters due to positive voltage polarity pulses. Such inhibition is quite common: GABA receptors can be found in 60-80% of CNS neurons.

The GABA receptor subtype can be activated by the mushroom toxin muscimol (in the A subtype) as well as the antispasmodic amino acid baclofen (B subtype). These drugs directly mimic the action of GABA at the receptor.

Allosteric facilitation of GABA receptors occurs at several different sites; compounds that bind to these are used as sedatives and anxiolytics. These compounds bend the receptor open and indirectly facilitate GABA binding.

[0190]   (GABA agonist / promoter)

[0191]

[Chemical formula 24] Progabide is a prodrug that degrades to GABA in the CNS. this is,
GABA itself crosses the blood-brain barrier, a non-crossing dipolar ion (a doubly ionized amino acid). Vigabatrin (γ-vinyl-GABA) is GABA
Inhibits aminotransferase (GABA-T), which is responsible for the degradation of GABA at synapses. It therefore prolongs the temporary retention of GABA molecules and thus promotes binding.

Depakote (valproic acid) appears to act on the neuronal membrane and reduce its susceptibility to stroke. At high doses it acts like vigabalin and inhibits GABA-T. Gabapentine is another recently marketed antispasmodic (Neurontin), which has also found psychiatric application as a mood stabilizer. The neurological basis for this application is that panic attacks (or mania in bipolar disorder) are characterized by a pre-panic "excitation" phenomenon, characterized by repetitive neurological flare, leading to clinical stages. It is similar to epilepsy in terms of guiding. Gabapentin may facilitate the production of GABA or prevent GABA degradation. Lamotrigine probably acts by reducing the release of glutamate, an excitatory neurotransmitter normally dominated by inhibitory GABA.

Novel GABA drugs represent one of the most active areas of psychotropic research.
For example, riluzole is a GABA uptake inhibitor with anticonvulsant and hypnotic properties; it also blocks sodium channels and inhibits glutamate release.

Opium Anesthetics Opium agents derived from poppy plants include alkaloids that activate the brain's endogenous endorphin receptors to produce analgesia, euphoria, and respiratory depression.
Quessiah opiates possess a polycyclic phenanthrene core with various substituents that determine their adaptation to the receptor. Morphine-like compounds have been found in mammalian brain tissue, but it is generally accepted that enkephalins and endorphins represent endogenous compounds that mimic poppy components.

A diverse array of opiate receptors is responsible for the major pharmacological effects. These subtypes are the Greek name μ (analgesia, euphoria), σ (discomfort, cardiac stimulation).
, Κ (sedation, spinal analgesia, miosis), Δ, etc. Antitussive properties, vomiting (
Vomiting) and anticholinergic (constipation) effects also occur, which exhibit a wide variety of receptor types and effects. The σ receptor is presumed here to be associated with glutamate function.

The opiate receptor acts on synaptic transmission by regulating the release of neurotransmitters presynaptically, including acetylcholine, norepinephrine, dopamine, serotonin, and substance P. The latter compound is a peptide neurotransmitter involved in nociceptive (pain-related) neurons. The opiate receptor is a G peptide (postsynaptic intracellular enzyme (eg,
Adenylyl cyclase) or ion channels (eg, K ⁺ , Ca ⁺⁺ ) linked transmembrane macromolecules. At high doses, opiates cause a generalized CNS depression sufficient for surgical anesthesia.

[0197]   (Phenathrene)

[0198]

[Chemical 25] (Phenylheptylamine)

[0199]

[Chemical formula 26] (Phenylpiperidine)

[0200]

[Chemical 27] Codeine is a mild analgesic, which retains agonist activity at other receptor subtypes including respiratory regulation, peristalsis and euphoria. Morphine is among the most potent phenanthrene type. Heroin, which is rarely amphoteric, crosses the blood-brain barrier more easily, but is once broken down there to morphine. Oxycodone, the main active ingredient in Percodan and Percocet, is somewhat less potent.

Meperidine (Demerol) is a synthetic drug, which has almost the same analgesic activity as morphine. Methadone (invented by Nazis and originally named dolofine) is well known for its use in alleviating heroin withdrawal syndrome. Its half-life is substantially longer than that of heroin, while it binds to newly administered heroin-blocking receptors. This analgesic activity is also about equal to morphine but gives less euphoria.

Fentanyl is the smallest and most potent synthetic propoxyphene (Darvon).
). Methoxy compounds, such as codeine and oxycodone, are largely insensitive to early passage reactions, typically associated with glucuronide, and thus have relatively high oral-parenteral rates. Less amphoteric compounds (compounds with more defined acid or basic properties) cross the blood-brain barrier more easily.

[0203]   (Partial agonist-antagonist)

[0204]

[Chemical 28] Modification of the phenanthrene skeleton produces a drug with mixed agonist / antagonist properties at the opiopeptin subreceptor. These drugs are used variously as analgesics, heroin and even withdrawal aids from alcohol addiction, and (illegal) to increase physical fitness of athletes. Stadol is used intranasally to reduce migraine headaches. Although mixed agonists retain their analgesic properties, they often give an unpleasant effect.

[0205]   (Anesthetic antagonist)

[0206]

[Chemical 29] Anesthetic antagonists are particularly useful in the case of overdose, which can reverse the CNS depression caused by opiate agonists. Naloxan is the most frequently used, most effective, and prototypical anesthetic antagonist. Naloxane, nalmefene, and nadide are among some other compounds used to antagonize morphine receptors.

Naltrexone has been used recently (like ReVia) to reduce craving for alcohol during recovery from alcohol and heroin poisoning.

Nootropics and Smart Drugs Nootropics (also known as smart drugs or cognitive activators) are drugs that enhance mental functioning. Several mechanisms that affect neural function can be attacked. The compounds used by the body to manufacture neurotransmitters make up one group (precursors). Inhibitors of reuptake and degradation form another. Excitatory neurotransmitter mimetics and inhibitory neurotransmitter agonists
Both can stimulate nerve function. Antianoxic enhances the ability of neurons to burn glucose. Phospholipid compounds affect the neuronal lipid excitable membrane, which is responsible for transporting depolarizing pulses down the dendrites and axons. Steroid compounds also affect membrane chemistry. CN
Vasodilatory drugs that act in S increase blood supply to brain cells. Still other drugs increase the adaptability of red blood cells, so that they may gain more frequent access to more neurons. All these effects are theoretically used to enhance neurological function in the CNS.

[0209]   (Precursors and mimetics)

[0210]

[Chemical 30] The glycine system performs an inhibitory function in the CNS. Enhancement of these pathways provides anxiolytic effects and stabilizes mood. Glycine itself is a zwitterion and does not adequately cross the blood-brain barrier. Dimethylglycine is stabilized by a methyl group; its greater lipophilicity provides good transport to the CNS, where dimethylglycine is converted to glycine. Mirasemide is CN
Glycinamide in S, then decomposes to glycine (via MAO-B)
It is a pro compound.

Glutamate and aspartate are another group of excitatory neurotransmitters prominent in the CNS. Because they are acidic amino acids, they have difficulty crossing the blood-brain barrier, but standard tricks can be used to deliver them to the CNS. Making amides other than carboxylic acids is one of these (as in glutamine and aceglutamide); and the more radical method is, as is the case in calcium-N-carbamoyl aspartate, with calcium. Making a covalent salt.

Carnitine is a catabolic (destroying) amino acid that acts as a neuroprotectant at the NMDA receptor, a subset of glutamate / alpartate receptors. Acetylation of the hydroxy group is the result of ALC (which also
Has the effect of promoting transport to S).

[0213]   (steroid)

[0214]

[Chemical 31] Several steroids have been used to support mental function and libido. Both testosterone and estrogen have been historically administered to increase vitality and sexual drive as a person ages (replacement therapy). DHE
Precursors of estrogen and androgenic steroids such as A and pregnenolone have recently been marketed as nutritional drugs. These steroids do not have significant estrogenic or androgenic properties until they are converted into their active form by the body. As with all precursors, we rely on the body's homeostasis function to regulate the formation of active molecules by rate limiting processes, competitive mechanisms, and tachyphylaxis.

[0215]   (Mood stabilizer)

[0216]

[Chemical 32] The use of antispasmodic medicines for psychotropic purposes has recently developed mainly for prophylactic agents against manic-depressive syndrome and / or panic syndrome. Phenytoin and carbamazepine probably act by affecting the ion-gate system in excitable membranes. Phenytoin is structurally similar to barbiturate, while carbamazepine has a tricyclic antidepressant-like tricyclic structure. Propanolol (β blocker) has been used to quench peripheral responses to stress (eg, stage startle).

Propanolol is beta prescribed for motion phobia or stage startle
It is an adrenergic blocker. Clonidine is an alpha agonist sometimes used, for example, to sedate peripheral tremor in the case of alcohol withdrawal.
Verapamil (calcium channel blocker) is also used for this purpose.

[0218]   (Anti-anoxia)

[0219]

[Chemical 33] The piracetam group of anti-anoxia compounds acts by several functions to stimulate nerve function. By supplying glutamate analogs to the Krebs ring, they use glucose in aerobic respiration, which is the main means by which animal cells extract chemical energy from sugars through ATP formation.
To enhance. This in turn raises phospholipid cAMP levels and enhances dopamine and acetylcholine neuron function. Furthermore, they function as antioxidants (compared to the structure for vitamin C) and delay lipofuscin formation. Experimentally, piracetam has been shown to increase locomotor performance, reverse alcohol-induced brain degradation, and has been tested as a treatment for dyslexia.

[0220]   (Cerebral vasodilator and anti-anticoagulant coagulant)

[0221]

[Chemical 34] Methylxanthine is used as a bronchodilator (typically theophylline) in the treatment of asthma and in combination with analgesics to treat headaches. Pentoxifylline and propentofylline have central and peripheral vasodilatory properties. The increased blood supply to brain tissue also explains whatever the anotropic properties they have. Pentoxifylline also increases the elasticity of red blood cells, allowing them to be better compressed through the contracted capillaries. Such drugs are called anti-ischemic agents, and ischemia refers to a lack of blood supply to tissues.

Pytirinol is another vasodilator that has been used for the elderly with dementia (senility). Idebenone is similar to ubiquinone, a compound that catalyzes mitochondrial metabolic processes. It promotes secretion of nerve growth factor (NGF) and may also protect the cell membrane against lipid oxidation. Ergocryptine is an ergot alkaloid that has been used to combat age-related memory loss and Alzheimer's disease. This extends the defect by blocking the α-adrenergic receptor. It has been used in accident victims to increase blood flow to the brain after trauma to prevent tissue damage due to anoxia. Vinpocetine and vincamine are two alkaloids from the vinca plant (also,
It has an anticoagulant effect and a vasodilatory effect).

Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) Nonsteroidal anti-inflammatory drugs (NSAIDs) are mild drugs of choice to relieve pain and reduce fever (antipyretics). They prevent the formation of prostaglandins by inhibiting the enzyme cyclooxygenase, which closes the bond on arachidonic acid (essential oil). Periodic prostaglandin compounds are potent short-lived mediators of the inflammatory response. They are not stored in cells but are synthesized when required in response to injury or inflammation. Interfering with their production peripherally blocks the inflammatory response in the body.

Aspirin and other NSAIDs may also act at sites in the CNS. NS
Some of the AIDs (eg ketoprofen) inhibit other enzymes (eg lipoxygenase), further delaying the allergic / inflammatory response.

[0225]

[Chemical 35] Aspirin (acetylsalicylic acid) has been used for hundreds of years with its related product (methylsalicylic acid). The latter, known as the oil of Pomegranate and often used topically, is even more toxic than overdose of aspirin. Aspirin inhibits platelet aggregation and delays blood coagulation.
This property probably explains its use in the long-term prevention of heart attack.

Recently, drugs such as irbuprofen and ketoprofen have become available over the counter. These more lipophilic drugs (eg, Naproxen (Alev
e, Naprosyn), ketoprofen (Actron) and nabumetone (
Nabumetone) (Relefen)) has a long half-life and requires less frequent dosing, but they are probably not effective analgesics the same as ibuprofen. Liver, kidney and GI problems of varying severity, which are proportional to medication history, are common. Penicillamine has been used as a long-acting NSAID, but it is quite toxic, causing reduced health and autoimmune hosts, and histological disease.

[0227]   (Cyclooxygenase inhibitor)

[0228]

[Chemical 36] A new class of NSAIDs inhibits cyclooxygenase-2, the enzyme responsible for the internal conversion of prostaglandins. These COX-2 inhibitors, which tend to maintain the formation of cytoprotective prostaglandins, target inhibition of compounds responsible for pain and inflammation, reduce gastritis, and Celebrex (celecoxib) It is one of these drugs. Vioxx is another new drug in this class.

[0229]   (Non-anesthetic analgesics)

[0230]

[Chemical 37] (Glucosamine and chondroitin)

[0231]

[Chemical 38] Recent studies have shown that a pair of aminated sugar compounds can assist in repairing damage to cartilage in osteoarthritis. Glucosamine (monomer) and chondroitin (polymer) are commercially available as nutrients for this purpose. In cartilage, the sugar polymer forms a flexible binding matrix (composite material) around the tough protein chains in cartilage.

[0232]   (Sedatives and alcohol)   (Benzodiazepine)

[0233]

[Chemical Formula 39] Some benzodiazepine sedatives are Valium, to name a few.
Librium, Halcion, Xanax, Ativan, Serax and Klonopin. In addition to their potential effects on lipophosphate neural membranes, these drugs act by allosterically enhancing the effects of the inhibitory neurotransmitter GABA at postsynaptic receptors. That is,
These slightly "bend" the GABA molecules so that they bind and activate their receptors more effectively and more frequently.
Barbiturates (eg seconal, nembutal and phenobarbital)
Their main advantage over is that they do not act directly on the chloride channels to open.

Serotonin Drugs Serotonin is an inhibitory neurotransmitter that complements the excitatory sympathetic nervous system, like adrenaline and dopamine in the CNS. Like "battle or flight" adrenergic compounds, serotonin is not released only at specific synaptic sites, but from a set of "diffusing" neurons that spread from the emotional center in the limbic system to the frontal lobe. It is also released to brain tissue in a broadcast fashion. This diffuse release sets the biochemical tone of large areas of neural function, controlling mood and motivation. However, the inhibitory effect of serotonin is GA
It is more complex and selective than the action of BA sedatives (like Valium or Xanax), which acts more globally. Due to these effects on mood, serotonin-active agents are antidepressant and anxiolytic agents.
s) (anti-anxiety).

[0235]   (Serotonin antagonist)

[0236]

[Chemical 40] Ondansetron is a selective 5-HT3 antagonist. This receptor subtype is found in cholinergic neurons; when ondansetron is activated, it inhibits the release of acetylcholine. Therefore, its chemical relationships (eg, granisetron and zatoset).
In addition to ron)), this may be useful in restoring memory function in the elderly. Grasetron, a selective 5-HT2 antagonist, is also used as an antiemetic drug (Kytril) in chemotherapy.

Ketanserin, a selective 5-HT2 antagonist, also acts on the alpha-1 adrenergic receptor and lowers blood pressure. Mescaline, a hallucinogenic drug,
It antagonized the HT2 terminus and was attempted as an alternative to dopamine blocking antipsychotics (with little success; it promotes dopamine function). Oxetrone, like pizotyline, is a relatively new antagonist used for migraine. Cyproheptadine is an older serotonin antagonist and antihistamine. Mirtazapine (Remeron) causes serotonin release but blocks the 5-HT2 and 5-HT3 subreceptors, effectively increasing serotonin action at the 5-HT1 receptor. Mianserin and homochlorcyclidine also antagonize serotonin receptors.

[0238]   (Serotonin agonist)

[0239]

[Chemical 41] Sumatriptan activates the 5-HT1d terminus and has the trade name Imitre
Used under x for migraine. Zoimitriptan
an) (Zomig) and Rizatriptan (Ma)
xal), a recently approved similar anti-migraine serotonin drug, buspirone, ipsapirone and jepirone enjoy 5-HT1 antagonist properties with only a weak D2 blocking effect. Buspirone is used for anxiety as an alternative to GABA-mimicking sedatives.
8-Hydroxy-DPAT acts selectively at the 5-HT1a receptor, while 2-methylserotonin activates the 5-HT3 terminus.

Steroids and Reproductive Drugs Steroids are lipolytic (lipophilic) hormones with a tetracyclic-based structure. Steroidal structures are synthesized from smaller structures called terpenes as precursor molecules, which then undergo extensive and subtle changes for a wide variety of applications: meiosis, carbohydrate metabolism, fat storage, muscle. To control systems as diverse as growth, immune function and neuronal membrane chemistry, often in adipose tissue. Due to their high lipophilicity, they can cross the cell membrane, which is the fat bilayer, and affect DNA transcription, thereby altering protein synthesis. By binding to specific sites in DNA, they are a type of molecular "boot".
Releases (hsp90), which locks the DNA in the absence of steroids,
And it prevents short sequences from being expressed into proteins. The action of enzymes and activating peptides produced by the activation of DNA can be long lasting, which means that
Explain the effects of many steroids over time.

Steroids are sites of synthesis in the ovary or testis for gonadal diversity,
And depending on the site of synthesis in the adrenal cortex for glucocorticoids can be classified into a wide range of gonadal compounds and glucocorticoids. They can also be separated according to function, the common names being androgens, estrogens and progesterones (usually for gonadal hormones), and anabolic and catabolic effects (usually for glucocorticoids).

However, it is important to understand that these terms are not totally exclusive. In other words, even the gonadal hormone testosterone is synthesized in small amounts by the adrenal cortex and transmits an anabolic property different from its effect on gonadal function. Moreover, all hormones act in combination with other compounds to produce a net result.

[0243]   (Gonadal steroid)

[0244]

[Chemical 42] Testosterone is the prototype of the androgen group of gonadal steroids. Androgens convey the traits symbolized by males of the mammalian species. These include morphological features such as protruding eyeline ridges, robust bone structures, and large canines. It is also responsible for aggression and libido. It also acts as an anabolic effect, helping muscle formation in response to exercise. On the one hand, estradiol is a prototypical estrogen, transmitting feminine characteristics such as breast growth and storage of subcutaneous fat. Estrogens also prevent heart disease (women statistically suffer less heart disease than men).

Oral contraceptives reorganize the body's steroid chemistry, mimicking the steroid chemistry of pregnancy and inhibiting ovulation. This is usually done according to a 28-day menstrual cycle, but it is possible to delay ovulation of the body for longer periods. This is achieved with a mixture of estrogen and progestogen. The most common preparations are norethindrone (progesterone) and ethinyl estradiol (estrogen). This combination, which is massively taken up just after sexual intercourse without contraception, may also prevent pregnancy by the same mechanism. Testosterone for men, and estrogen / progesterone for women (retained ProV
era) and more recently also replacement therapy for gonadal steroids in the form of testosterone has been challenged to combat the symptoms of aging, including reduced sexual drive. In men, testosterone may support libido and improve cardiovascular fitness and general efficacy. In women, postmenopausal loss of estrogen conveys some changes, but this decline is relatively modest (about 20%) compared to that with progesterone, which indicates that osteoblasts Causes cells to create new bone tissue and inhibits cancer cell formation. Estrogen, an anabolic or tissue-forming compound, can promote cancer cell growth, so modern compensatory regimens should be more compensated for progesterone than estrogen. Non-steroidal soybean estrogens are currently receiving attention as a treatment for menopausal hot flashes, as are testosterone creams and tablets that enhance sexual drive.

[0246]   (Glucocorticoid)

[0247]

[Chemical 43] Cholesterol is highly contained in the membrane in metabolic chemistry. One of its major uses in the body is to reduce the permeability of phospholipid cell walls to ionic species (eg Na ⁺ , K ⁺ and Ca ⁺⁺ ). Recently, cholesterol has been implicated in helping to build up plaques on the inner surface of veins and veins. Animal meat is usually rich in cholesterol. Through natural selection, carnivores (eg,
(Cats and dogs) have different and improved chemistries applied to the treatment of higher amounts of cholesterol, cholestanol (saturated form) and other steroids, which, in terms of meat, are less than these for us. Make food safer for animals. Cholesterol is excreted via bile (a fat digestive substance secreted by the liver). It is present in large amounts in gallstones. Bile is also used to excrete lipolytic substances such as bilirubin (from the heme population in lysed red blood cells). Cholesterol can also be used endogenously to synthesize vitamin D.

Cortisone, another prototypical glucocorticoid, controls the healing process associated with the immune system, in addition to regulating other functions. Hydrocortisone (also known as cortisol) acts similarly, inhibits histamine-mediated allergic reactions, and regulates the body's response to stress by altering neuroexcitatory membrane chemistry. Prednisone is a synthetic compound used in the present formula in place of cortisone. Dexamethasone has been used to diagnose depression (ie, in the dexamethasone suppression test, where the body is “stressed” by the introduction of steroids, and its response is measured).

[0249]   (Non-steroidal estrogen)

[0250]

[Chemical 44] Hexestrol and diethylstilbestrol are two examples of polycyclic, non-steroidal compounds that activate the estrogen receptor. The local structure in the bond formation is similar to that of steroids. The other major category of non-steroidal estrogens is isoflavones.

Environmental estrogens have become a health concern as cows normally eat estrogen due to their anabolic (weight gain) trait. Therefore, confiscation of meat is considered equivalent to absorbing some estrogen. Some pesticides are nonsteroidal estrogens, such as THC, which is the major psychoactive constituent of marijuana. Compounds such as diethylstibestrol have been shown to be carcinogens, but this is not due to their action on DNA.

[0252]   (Tamoxifen and Raloxifene)

[0253]

[Chemical formula 45] Recently, two nonsteroidal estrogens have shown great medical promise in some women's health problems. These selective estrogen receptor modulators (SERMs) mimic the effects of estrogen in some tissues, but some do not.

Tamoxifen has been used for several years after the detection of breast cancer. By blocking the estrogen receptor, this blunts tumor growth. New studies have shown that this protects against breast cancer, probably by the same mechanism. However, the benefits of this prevention penalize the increased risk of uterine cancer and other potential risks.

Raloxifene retains the ability to promote bone maintenance and prevent osteoporosis; it cuts the risk of breast cancer by 60% and reduces LDL (“malignant” cholesterol) levels.

[0256]   (Finasteride (Propecia))

[0257]

[Chemical formula 46] Finasteride (Proscar, Propecia) is currently in the spotlight as a systemic (oral) anti-bald drug with a mechanism different from that of minoxidil, a local vasodilator that stimulates vesicle activity with improved blood flow. Has been done. It acts by inhibiting the formation of 5-α-dihydrotestosterone (potent androgen) from the less potent parent compound (testosterone). This is also
It has been used to treat benign prostatic hypertrophy (enlargement of the prostate).

The side effects of reduced androgens in the body can basically be said to be feminization (male gonadal atrophy, increased breasts, decreased aggressive behavior, increased risk of osteoporosis, etc.).

[0259]   (Plant steroid)

[0260]

[Chemical 47] Steroids are not limited to the animal kingdom, but are similarly synthesized by plants. The known cardiotonic drug (digitalis) is derived from the foxglove plant that synthesizes some glucoside steroids (ie, steroids linked to sugar moieties).

The oleander shrub produces a number of steroids that have similar effects on cardiac conduction, including oleandrin and oleandrigenin.

[0262]   (Viagra and Gossypol)

[0263]

[Chemical 48] Sildenafil (Viagra) is an erection enhancer. Erection depends on the interaction of adrenergic and cholinergic neurons, where muscles must relax to direct blood to the erectile tissue. The presence of nitric oxide (NO) species is involved, and Viagra can affect the enzyme responsible for the production of NO. Viagra also uses cGMP phosphodiesterase (phop).
It is a selective inhibitor of HO, which acts on some GI vascular smooth muscles. Caffeine, which has a central ring structure similar to that of Viagra, also acts on a broader range of cAMP phosphodiesterases (a broader second messenger system). Yohimbine, a selective α2-adrenergic blocker, has also been taught to be capable of prolonging or enhancing erections.

Gossypol (isolated from cotton plants) has the ability to inhibit viable sperm production in men. This damages the epithelial lining of semiferous vesicles and inhibits spermatogenesis. It also impairs the oxygen carrying capacity of blood.

Peptide therapeutic candidates include peptides that exhibit anti-neoplastic activity (eg, RGD peptides comprising peptide SEQ ID NOs: 1-3).

Peptides active against HIV are also included, including the GP-41 peptides, which include peptide SEQ ID NOs: 4-6.

Antiviral peptides that exhibit the ability to disrupt fusion induction commonly occur in viral infections. These include RSV peptides that exhibit the ability to treat or prevent acute immunodeficiency syndrome (AIDS) caused by respiratory syncytial virus (RSV) infection as well as human immunodeficiency virus (HIV) infection. Such peptides include peptide SEQ ID NOs: 7-9.

Also included are GLP-1 peptides, including those peptides set forth in SEQ ID NOS: 10-11.

Kringle containing these polypeptides set forth in SEQ ID NOs: 12-13
Or K5 peptides are also included; BBB peptides (TAT) that include these peptides set forth in SEQ ID NOs: 14-15 and analgesic peptides that include SEQ ID NO: 16 (eg, dynorphin) are also useful.

(3. Modified therapeutic agent) The modified therapeutic agent of the present invention includes a therapeutic agent modified by adding a reactive group. The reactive group can be attached therapeutically via a linking group or optionally without a linking group. The modified therapeutic agent can react with covalent bonds with available functional groups on the blood or lung components or blood components. The invention also relates to such modifications, combinations with such lung or blood components, and methods for their use. These methods involve prolonging the effective therapeutic longevity of the conjugated therapeutic agent as compared to administration of the unconjugated therapeutic agent.

A wide variety of active carboxyl groups, especially esters, can be used as reactive groups to form covalent bonds with functional groups on proteins, where the hydroxyl moiety is used to modify the therapeutic agent. It is physiologically acceptable at the required level.
Many different hydroxyl groups can be used, but most conveniently N-hydroxysuccinimide (NHS), N-hydroxy-sulfosuccinimide (sulfo-NHS), maleimide, maleimidic acid (maleimidopropionic acid (MPA).
) And maleimide esters).
In a preferred embodiment of the invention, the functional group on the blood component is a thiol group and the reactive group is maleimide.

Primary amines are the primary targets of NHS esters. The accessible α-amine group present at the N-terminus of the protein reacts with the NHS ester. But,
The α-amino group on the protein may not be desired or available for NHS coupling. Five amino acids have a nitrogen in their side chains, but only the ε-amine of lysine reacts significantly with NHS esters. The amide bond is formed when the NHS ester conjugation reaction reacts with a primary amine to release N-hydroxysuccinimide as shown in the scheme below.

[0273]

[Chemical 49] In a preferred embodiment of the invention, the functional group on the protein is a thiol group and the chemically reactive group is a maleimide-containing group (eg MPA or GMBA (γ-maleimido-butyl aramid)). Is. Maleimide groups are most selective for sulfhydryl groups on peptides when the pH of the reaction mixture is maintained between 6.5 and 7.4. At pH 7.0, the rate of reaction of maleimide groups with sulfhydryls is 1000 times faster than reaction with amines. A stable thioester bond is formed between the maleimido group and the sulfhydryl, which cannot be cleaved under physiological conditions, as shown in the scheme below.

[0274]

[Chemical 50] A. Specific Labeling Preferably, the modified therapeutic agents of the present invention are designed to specifically react with thiol groups on lung proteins or mobile blood proteins. Such a reaction is preferably a pulmonary protein (eg intracellular albumin or extracellular albumin) or a mobile blood protein (eg serum albumin or IgG).
A) Covalent attachment of a therapeutic agent modified with a maleimide bond (eg, prepared from GMBS, MPA or other maleimide) to the thiol group above.

[0275] Under certain circumstances, specific labeling with maleimide can be achieved by functional groups such as NHS.
And sulfo-NHS) to provide some advantages over non-specific labeling of proteins. Thiol groups are less abundant in vivo than amino groups. Thus, the maleimide-modified therapeutic agents of the invention (ie, maleimide therapeutic agents) are covalently attached to fewer proteins. For example, albumin (the most abundant blood protein) has only one thiol group. Thus, the therapeutic agent-maleimide-albumin conjugate tends to include a molar ratio of therapeutic agent to albumin of about 1: 1. In addition to albumin, IgG molecules (class II) also
Has one thiol. For systemic delivery, IgG molecules and serum albumin
Since they make up the majority of soluble proteins in blood and they also make up the majority of free thiol groups in blood, they are available for covalent attachment to maleimide-modified therapeutic agents.

[0276]   Furthermore, even between free thiol-containing blood proteins (including IgG),
The specific labeling of maleimide is due to the unique features of albumin itself,
It leads to the preferential formation of the therapeutic agent-maleimide-albumin conjugate. Albumin single
The free thiol group of C. cerevisiae (which is highly conserved among species) is associated with amino acid residue 34 (Cys ³⁴ ). Albumin Cys³⁴However, other free thiol-containing tampers
It has recently been shown to have an increased reactivity compared to free thiols on the matrix.
Came. This is albumin Cys³⁴To a very low pK value of 5.5 for
Dependently. This value is a typical pK value for common cysteine residues (
Which is typically about 8). Positive due to this low pK
Under normal physiological conditions, albumin Cys³⁴Exists in a predominantly ionized form
Present, which dramatically increases its activity. Cys³⁴In addition to the low pK value of
Cys³⁴Another factor that enhances the reactivity of is its position, which is
It is located in a recess close to the surface of one loop in the V region of the camera. This position is for all species
Cys against a class of ligands³⁴Is very available, and this is
Cys as a Caltrap and Free Thiol Scavenger³⁴Biological role of
It is an important factor in comparison. These properties are³⁴Maleimide-therapeutic agent
It is very reactive towards, and the acceleration of the reaction rate is
1000 times higher than the rate of reaction of maleimide-therapeutic agents with proteins
obtain.

Another advantage of the therapeutic agent-maleimide-albumin conjugate is the reproducibility associated with a 1: 1 loading of therapeutic agent on albumin, especially at Cys ³⁴ . Other technologies (
For example, free amines such as glutaraldehyde, DCC, EDC and other chemical activations) lack this selectivity. For example, albumin contains 52 lysine residues, 25-30 of which are located on the surface of albumin and are therefore accessible for conjugation. Activating these lysine residues or modifying the therapeutic agent to bind through these lysine residues results in a heterogeneous population of conjugates. Even if a 1: 1 molar ratio of therapeutic agent to albumin is used, the product consists of multiple conjugate products, some containing 0, 1, 2 or more therapeutic agents per albumin, And each has 25
Having therapeutic agents randomly coupled at any one or more of the ˜30 available lysine sites. Given the large number of possible combinations, the correct composition characteristics and the nature of each conjugate batch becomes difficult, and batch-to-batch reproducibility is all but impossible. Have made such conjugates which are less desired as therapeutic agents. Furthermore, although it has been found that conjugates via lysine residues of albumin have at least the advantage of delivering more therapeutic agent per albumin molecule, studies have shown that a ratio of therapeutic agent to albumin of 1: 1: It has been shown to be preferable. Stehle et al., "The
Loading Rate Determines Tumor Target
ing prototies of Methoterxae-Albumi
n Conjugates in Rats ", Anti-Cancer Dr
In the article by Ugs, Vol. 8, 677-685 (1988), which is incorporated herein in its entirety, the authors describe the anticancer methotrexate to albumin conjugated via glutaraldehyde. We have reported that a 1: 1 ratio gives the most promising results. These conjugates are preferably taken up by tumor cells, while the conjugates have a 5: 1 to 20: 1 methotrexate molecule, which alters the HPLC profile and this is in vivo by the liver. Captured quickly. At these higher ratios, the structure changes such that albumin diminishes its effectiveness as a therapeutic agent carrier.

Specific labeling of albumin and IgG can be controlled in vivo via controlled administration of the maleimide therapeutic agent in vivo. For systemic delivery via pulmonary administration, 80 local doses of administered maleimide-therapeutic agent reach the bloodstream
~ 90% is labeled albumin and less than 5% is labeled IgG. Slight labeling of free thiols (eg glutathione) also occurs. Such specific labeling is preferred for in vivo use because it allows accurate calculation of the estimated half-life of the administered drug.

In addition to providing controlled and specific in vivo labeling, maleimide-therapeutic agents may provide labeling of albumin or other proteins ex vivo.
Such ex vivo labeling involves the addition of a maleimide-therapeutic agent to a saline solution containing albumin or other proteins. Once the conjugate with the maleimide-therapeutic agent occurs ex vivo, the saline solution can be administered via pulmonary administration for in vivo treatment.

In contrast to NHS-therapeutic agents, maleimide-therapeutic agents are generally very stable in the presence of aqueous solutions and in the presence of free amines. Protecting groups are generally not necessary to prevent the maleimide-therapeutic agent from reacting with itself, as the maleimide-therapeutic agent reacts only with the free thiol. Additionally, the increased stability of the modified therapeutic agent allows the use of additional purification steps (eg, HPLC) to prepare stable purified products for in vivo use. Finally, the increased chemical stability provides the product with a longer shelf life.

B. Non-Specific Labeling The therapeutic agents of the present invention may also be modified for non-specific labeling of lung or blood components. Coupling to amino groups is also used, inter alia, in the formation of amide bonds for non-specific labeling. It can be used as a chemically reactive group (a wide range of active carboxyl groups, especially esters) to form such bonds. Here, the hydroxyl moiety is pharmaceutically acceptable at the required level. Multiple different hydroxyl groups can be used in these linking agents, but the most convenient ones are N-hydroxysuccinimide (NHS) and N-hydroxy-sulfosuccinimide (sulfo-NHS).

Other linking groups that may be utilized are described in US Pat. No. 5,612, which is incorporated herein by reference.
034.

The various sites to which the chemically reactive group of the modified therapeutic can react in vivo include cells, particularly alveolar cells and capillary endothelial cells (alveoli in the lungs and red blood cells in the blood itself (red). blood cell) (erythrocyte)
) And produced from platelets). The agent also includes lung proteins (including membrane bound receptors, intracellular and extracellular albumin, immunoglobulins, ferritin, and transferrin), as well as blood serum proteins (eg, immunoglobulins (including IgG and IgM), serum. Albumin, ferritin, steroid binding protein, transferrin, thyroxine binding protein,
α-2-macroglobulin, etc.). Those receptors that react with the modified therapeutic agent, which is not long-lived, are generally cleared from the human host within about 3 days. The above proteins (including cellular proteins) can be maintained for at least 3 days, and especially for half-life, and can be maintained for 5 days or more (usually not more than 60 days, more usually, based on blood levels). Not exceed 30 days).

For the most part, it reacts with mobile components in the blood, especially blood proteins and cells, more particularly blood proteins and red blood cells, for systemic delivery of therapeutic agents. “Moveable” means that this component is not in a fixed position for any extended period (generally not more than 5 minutes, more usually 1 minute), but some blood components remain relatively immobile for the extended period. possible. First, there is a relatively homogenous group of functionalized proteins and cells. However, for the most part, depending on the half-life of the functionalized protein in the bloodstream, within a few days the group changes substantially from the initial group. Therefore, usually, within about 3 days or more, IgG becomes a predetermined functionalized protein in the bloodstream.

Typically, by 5 days after administration, IgG, serum albumin and red blood cells are at least about 60 mole%, and usually at least about 75 mole% of the binding components in blood, IgG, IgM (substantially lesser range) and Serum albumin is at least about 50 mole% of non-cell-bound components, usually at least about 75 mole%.
, More usually at least about 80 mole%.

The desired binding of the non-specific modified therapeutic agent to the blood component can be prepared in vivo by pulmonary administration of the modified therapeutic agent to the patient. The patient is a human or other mammal. If desired, this conjugation can also be prepared ex vivo by combining a carrier protein or protein solution with the modified therapeutic agent of the invention to covalently attach the modified therapeutic agent to functionalize the protein, The conjugated mixture can then be administered to the host via pulmonary delivery.

(3. Diagnostic Agent and Modified Diagnostic Agent) The diagnostic agent is a useful agent for imaging the vascular system of a mammal, and the diagnostic agent includes a positron emission tomography (PET) agent. , Computer-linked tomography (
CT) agents, magnetic resonance imaging (MRI) agents, nuclear magnetic resonance tomography (NMI) agents, flurocopy agents and ultrasound contrast agents.

[0288]   The modified diagnostic agent of the present invention has, for the most part, the following formula: XYZ.

In this formula, X is a diagnostic agent selected from PET agents, CT agents, MRI agents, NMI agents, fluoscopy agents and ultrasound contrast agents. The diagnostic agent of interest is a radioisotope of the following element: iodine (including ¹²³ I, ¹²⁵ I, ¹³¹ I, etc.)
, Barium (Ba), gadolinium (Gd), technesium (Tc) (including ⁹⁹ Tc), phosphorus (P) (including ³¹ P), iron (Fe), manganese (Mn), thallium (TI), chromium ( Cr) (including ⁵¹ Cr), carbon (C) (including ¹⁴ C) and the like, and fluorescent labeling compounds and the like.

In this formula, Y is 0 to 30 atoms, more usually 2 to 12 atoms, preferably 4 to 12 atoms, especially carbon, oxygen, phosphorus and nitrogen, more especially carbon and Is a linking group of oxygen, where oxygen is preferably present as an oxyether, wherein Y can be alkylene, oxyalkylene, or polyoxyalkylene, wherein the xylene group is 2 ~ 3 carbon atoms and the like. A linking group of 0 atoms is preferred when it is desired to place X as close to Z as possible.

In this formula, Z is a reactive entity (eg carboxy, carboxyester), wherein the ester group is 1-8, more usually 1-6 carbon atoms, especially A physiologically acceptable leaving group that activates carboxycarbonyl for reaction with an amino group in an aqueous system, such as N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (sulfo-NHS), maleimide , Maleimide ester, maleimide acid, maleimide-benzoyl-succinimide (MBS), γ-maleimide-butyryloxysuccinimide ester (
GMBS) and maleimidopropionic acid (MPA), N-hydroxysuccinimide isocyanate, isothiocyanate, thiol ester, thionocarboxylic acid ester, imino ester, mixed anhydride (for example, carbodiimide anhydride,
Carbonate ester etc.) and the like. The reactive entity Z is covalently attached for functionalization in vivo or ex vivo.

4. Synthesis of Peptide Therapeutic Agents A. Peptide Synthesis Therapeutic agents according to the present invention that are peptides can be synthesized by standard methods of solid phase peptide chemistry known to those skilled in the art. For example, the peptides are Steward and Y
oung (Steward, JM and Young, JD Solid.
Phase Peptide Synthesis, Second Edition, Pierce C
chemical Company, Rockford, III. , (1984)
Applied Bio by solid-phase chemistry techniques according to the procedure described by
It can be synthesized using the system synthesizer. Similarly, multiple peptide fragments can be synthesized and then ligated together to form a larger peptide. These synthetic peptides can also be made with amino acid substitutions at specific positions.

For a solid phase peptide synthesis, an overview of many techniques can be found in J. Am. M. Stewart and J.M. D. Young's Solid phase Peptide Synt
hesis, W.C. H. Freeman Co. (San Francisco)
1963 and J. et al. Meiehofer, Hormonal Protei
ns and Peptide, Volume 2, p. 46, Academic Press (
New York), 1973. For classical solution synthesis, see G. Schroder and K.S. Lupke, The Petides, 1st
Vol., Academic Press (New York). Generally, these methods involve the sequential addition of one or more amino acids or appropriately protected amino acids to the growing peptide chain. Usually, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. This protected or derivatized amino acid is then added to the next amino acid in a sequence with an appropriately protected complementary (amino or carbonyl) group under suitable conditions to form an amide bond. Either bound to an inert solid support or utilized in solution. The protecting group is then removed from the newly added amino acid residue and the next amino acid (suitably protected) is added and the process is repeated.

After linking all desired amino acids with the appropriate sequence, any remaining protecting groups (and any solid support) are removed sequentially or simultaneously to yield the final polypeptide. With a simple modification of this general procedure, it is possible to add more than one amino acid to the growing chain at one time. For example, a protected tripeptide (under conditions that will not racemize the chiral center) is coupled with an appropriately protected dipeptide to form the pentapeptide after deprotection.

A particularly preferred method of preparing compounds of the present invention involves solid phase peptide synthesis wherein the amino acid α-N-terminus is protected by an acid or base sensitive group. Such protecting groups have the property of being stable to the conditions of peptide bond formation, but are easily removed without increasing peptide chain breakage or racemization of any chiral centers contained therein. Should be. Such protecting groups include 9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc).
), Benzyloxycarbonyl (Cbz), biphenylisopropoxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, α, α
-Dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl and the like. This 9
The -fluorenyl-methyloxycarbonyl (Fmoc) protecting group is particularly preferred for the synthesis of the peptides of the invention. Other preferred side chain protecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, for side chain amino groups such as lysine and arginine. 4-Methoxybenzene-sulfonyl, Cbz, Boc, and adamantyloxycarbonyl; for tyrosine benzyl, o-bromobenzyloxycarbonyl, 2,6-
Dichlorobenzyl, isopropyl, t-butyl (t-Bu), chlorohexyl,
Chloropenyl and acetyl (Ac); t-butyl, benzyl and tetrahydropyranyl for serine; trityl, benzyl, Cbz, p- for histidine.
Toluenesulfonyl and 2,4-dinitrophenyl; formyl for tryptophan; benzyl, and t-butyl for aspartic acid and glutamic acid, and triphenylmethyl (trityl) for cysteine.

In the solid phase peptide synthesis method, the α-C-terminal amino acid is attached to a suitable solid support or resin. Suitable solid supports useful for the above synthesis are materials that are inert to the agents of the step concentration-deprotection reaction and the reaction conditions and are insoluble in the medium used. A preferred solid support for the synthesis of α-C-terminal carboxy peptides is 4-hydroxymethylphenoxymethyl-
It is copoly (styrene-1% dimethylbenzene). The preferred solid support for the α-C-terminal amide peptide is 4- (2 ′, 4′-dimethoxyphenyl-
Fmoc-aminomethyl) phenoxyacetamide ethyl resin (Applied)
Biosystems (available from Foster City, Calif.)). This α-C-terminal amino acid is 4-dimethylaminopyridine (DMA
P), 1-hydroxybenzotriazole (HOBT), benzotriazole-
N, N′-dicyclohexylcarbodiimide (DCC) with or without 1-yloxy-tris (dimethylamino) phosphonium-hexafluorophosphate (BOP) or bis (2-oxo-3-oxazolidinyl) phosphine chloride (BOPCl) ), N, N′-diisopropylcarbodiimide (DIC) or O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyluronium-hexafluorophosphate (HBTU), like dichloromethane or DMF. It is bound to the resin by mediated coupling in a suitable solvent at a temperature of 10 ° C to 50 ° C for about 1 to about 24 hours.

When the solid support is 4- (2 ′, 4′-dimethoxyphenyl-Fmoc-aminomethyl) phenoxy-acetamidoethyl resin, the Fmoc group is
It is cleaved with a secondary amine (preferably piperidine) prior to coupling with the α-C-terminal amino acid as described above. A preferred method for coupling the deprotected 4- (2 ', 4'-dimethoxyphenyl-Fmoc-aminomethyl) phenoxy-acetamidoethyl resin is O-benzotriazole-1 in DMF.
-Yl-N, N, N ', N'-tetramethyluronium hexafluoro-phosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOB
T, 1 equivalent). Convenient coupling of protected amino acids can be carried out on an automated polypeptide synthesizer as is well known in the art. In this preferred embodiment, the α-N-terminal amino acid of the growing peptide chain is Fmoc protected. Removal of the Fmoc protecting group from the increasing α-N-terminal side chain of the peptide is accomplished by treatment with a secondary amine (preferably piperidine). Each protected amino acid is then introduced in about 3-fold molar excess and the coupling is preferably carried out in DMF. This coupling agent is usually O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyluronium hexafluorophosphate (HBTU, 1 eq) and 1-hydroxybenzotriazole (HOBT, 1 Equivalent).

For the purpose of solid phase synthesis, the polypeptide is removed from the resin and deprotected, either successfully or in a single operation. Removal and deprotection of the polypeptide can be accomplished in a single operation by treating the resin-bound polypeptide with a cleavage reagent that includes thioanisole, water, ethanediol and trifluoroacetic acid. In this case, if the α-C-terminus of the polypeptide is an alkylamide, the resin is cleaved by ammonolysis with an alkylamine. Alternatively, the peptide can be removed by a transesterification reaction (eg with methanol) and removed by ammonolysis or direct transesterification reaction. The protected peptide can be purified at this point or taken directly to the next step. Removal of side chain protecting groups is accomplished using the cleavage cocktail described above. The fully deprotected peptide is purified by an arrangement of chromatographic steps using any or all of the following types: weakly basic (acetate form) ion exchange; hydrophobicity of underivatized polystyrene-divinylbenzene. Absorption chromatography (eg Amberlite XAD); silica gel absorption chromatography; carboxymethylcellulose ion exchange chromatography; partition chromatography (eg Sephadex G-25, LH-20.
Or countercurrent partitioning); high performance liquid chromatography (HPLC), especially reverse phase HPLC in an octyl or octadecylsilyl-silica bonded phase column package.

The molecular weight of these ITPs is determined using fast atom bombardment (FAM) mass spectrometry.

(1) N-terminal protecting group As mentioned above, the term “N-protecting group” protects the α-N-terminal of an amino acid or peptide, otherwise during synthetic procedures. Refers to groups intended to protect the amino group or amino groups of peptides against undesired reactions. Commonly used N-protecting groups are Greene “Protective Groups”
In Organic Synthesis "(John Wiley & S
ons, New York (1981)), which is incorporated by reference. In addition, protecting groups can be used as prodrugs that are easily cleaved in vivo (eg, enzymatic hydrolysis releases a biologically active parent). The α-N-protecting group is a lower alkanoyl group (eg, formyl, acetyl (“Ac”), propionyl, pivaloyl, t-).
Butylacetyl etc.); other acyl groups (2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, -chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl and the like); a sulfonyl group (for example,
Benzenesulfonyl, p-toluenesulfonyl, etc.); carbamate-forming groups (eg, benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-
Methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2
-Nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3
, 4-dimethoxyoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-ethoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4 , 5-trimethoxybenzyloxycarbonyl, 1- (p
-Biphenylyl) -1-methylethoxycarbonyl, α, α-dimethyl-3,
5-dimethoxybenzyloxycarbonyl, benzhydroxycarbonyl, t-
Butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopripyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-. Methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like); arylalkyl group (for example, benzyl, triphenylmethyl, benzyloxymethyl, 9-fluorenylmethyloxycarbonyl (Fmoc ) And silyl groups (eg, trimethylsilyl, etc.).

(2) Carboxy Protecting Group As mentioned above, the term “carboxy protecting group” refers to a carboxylic acid protecting ester or amide group used to block or protect a carboxylic acid functional group,
Reactive groups containing other functional sites of this compound are preferred. The carboxy protecting group is Gr
eene "Protective Groups In Organic Sy"
nthesis ", pages 152-186 (1981)), which is incorporated by reference. In addition, the carboxy protecting group can be used as a prodrug, which allows the carboxy protecting group to be readily cleaved in vivo (eg, by enzymatic hydrolysis, the biologically active parent).
) Is released). Such carboxy protecting groups are well known in the art and include penicillin and cephalo as disclosed in US Pat. Nos. 3,840,556 and 3,719,667, which are incorporated by reference. Widely used for the protection of carboxyl groups in the field of sporins. A typical carboxy protecting group is C ₁
-C ₈ lower alkyl (eg, methyl, ethyl, or t-butyl); arylalkyl (eg, phenethyl or benzyl and substituted derivatives thereof (eg, alkoxybenzyl group or nitrobenzyl group)); arylalkenyl (eg, Phenylethenyl etc.); aryl and substituted derivatives thereof (eg 5-indanyl etc.); dialkylaminoalkyl (eg dimethylaminoethyl etc.); alkanoyloxyalkyl groups (eg acetoxymethyl,
Butyryloxymethyl, valeryloxymethyl, isobutyryloxymethyl, isovaleryloxymethyl, 1- (propionyloxy) -1-ethyl, 1- (pivaloyloxyl) -1-ethyl, 1-methyl- 1- (propionyloxy)-
1-ethyl, pivaloyloxymethyl, propionyloxymethyl and the like); cycloalkanoyloxyalkyl group (for example, cyclopropylcarbonyloxymethyl, cyclobutylcarbonyloxymethyl, cyclopentylcarbonyloxymethyl, cyclohexylcarbonyloxymethyl and the like); aroyloxy Alkyl (eg, benzoyloxymethyl, benzoyloxyethyl, etc.); arylalkylcarbonyloxyalkyl (eg, benzoylcarbonyloxymethyl, 2-benzylcarbonyloxyethyl, etc.); alkoxycarbonylalkyl or cycloalkyloxycarbonylalkyl (eg, Methoxycarbonylmethyl, cyclohexyloxycarbonylmethyl, 1-methoxycarbonyl-
1-ethyl etc.); alkoxycarbonyloxyalkyl or cycloalkyloxycarbonyloxyalkyl (eg, methoxycarbonyloxymethyl,
t-Butyloxycarbonyloxymethyl, 1-ethoxycarbonyloxy-1
-Ethyl, 1-cyclohexyloxycarbonyloxy-1-ethyl, etc.); Aryloxycarbonyloxyalkyl (eg, 2- (phenoxycarbonyloxy) ethyl, 2- (5-indanyloxycarbonyloxy) ethyl, etc.)
Alkoxyalkylcarbonyloxyalkyl (for example, 2- (1-methoxy-2-methylpropane-2-oiloxy) ethyl and the like; arylalkyloxycarbonyloxyalkyl (for example, 2- (benzyloxycarbonyloxy) ethyl and the like); Arylalkenyloxycarbonyloxyalkyl (such as 2- (3-phenylpropen-2-yloxycarbonyloxy) ethyl); alkoxycarbonylaminoalkyl (such as t-butyloxycarbonylaminomethyl); alkylaminocarbonylaminoalkyl (Eg, methylaminocarbonylaminomethyl, etc.); Alkanoylaminoalkyl (eg, acetylaminomethyl, etc.); Heterocyclic carbonyloxyalkyl (eg, 4-methylpipera) Such alkenyl carbonyloxy-methyl); dialkylaminocarbonyl alkyl (e.g., dimethylaminocarbonyl-methyl, diethylamino-carbonyl methyl, etc.); (5- (lower) -2-oxo-1,3-Okisoren -
4-yl) alkyl (such as (5-t-butyl-2-oxo-1,3-dioxolen-4-yl) methyl); and (5-phenyl-2-oxo-1,
3-Dioxolen-4-yl) alkyl (for example, (5-phenyl-2-oxo-1,3-dioxolen-4-yl) methyl and the like).

Representative amidocarboxy protecting groups are aminocarbonyl groups and lower alkylaminocarbonyl groups.

Preferred carboxy protected compounds of the present invention are those wherein the protected carboxy group is a lower alkyl, cycloalkyl or arylalkyl ester (eg methyl ester, ethyl ester, propyl ester, isopropyl ester, butyl ester, sec-butyl ester). , Isobutyl ester, amyl ester, isoamyl ester, octyl ester, cyclohexyl ester, phenylethyl ester, etc.) or alkanoyloxyalkyl, cycloalkanoyloxyalkyl, aroyloxyalkyl or arylalkylcarbonyloxyalkyl ester. A preferred amidocarboxy protecting group is a lower alkylaminocarbonyl group. For example, aspartic acid can be protected at the α-C-terminus with an acid labeling group such as t-butyl, and β-C-.
It may be protected at the end with a hydrogenation labeling group such as benzyl and then selectively deprotected during the synthesis.

B. Peptide Modifications The modalities for producing the modified peptides of the present invention will vary widely depending on the nature of the various elements comprising the peptide. The synthetic procedures are chosen to be simple, to provide high yields, and to allow very well purified and stable products. Typically, chemically reactive groups are created at the final stages of synthesis (eg, esterification with a carboxyl group to form an active ester). Specific methods for the production of the modified peptides of the invention are described below.

In particular, the selected peptides are modified with a linking group only at either the N-terminus, the C-terminus or the interior of the peptide. The therapeutic activity of this modified peptide linking group is then assayed. If the therapeutic activity is not dramatically reduced (ie, not less than 10-fold), then the stability of the modified peptide connector is measured by its in vivo lifespan. If stability is not improved to the desired level, the peptide is modified at alternative sites and the procedure is repeated until the desired level of treatment and stability is achieved.

More specifically, each peptide selected to carry out the modification with a linking group and a reactive group was modified according to the following criteria: terminal carboxyl.
oxylic) groups are available in the peptide and are not essential for retention of therapeutic activity, and if no other sensitive functional groups are present in the peptide, the carboxylic acid is a Is selected as the connecting point.
Any other sensitive functional group that is not essential for retention of therapeutic activity when the terminal carboxyl group is involved in therapeutic activity, or when the carboxyl group is not available, is for modifying the linking group reactive entity. Is selected as the connection point of. If some sensitive functional groups are available for the peptide, the combination of protecting groups is
After addition of linking group / reactive entity and deprotection of all protected sensitive functional groups,
Retention of therapeutic activity is used in the manner still available. If sensitive functional groups are not available for the peptide, or if a simpler modification route is desired, the result of the synthesis will allow modification of the native peptide such that retention of therapeutic regimen is maintained. In this case, the modification occurs at the opposite end of the peptide.

NHS derivatives can be synthesized from carboxylic acids in the absence of other sensitive functional groups in peptides. Specifically, such peptides react with anhydrous CH ₂ Cl ₂ and N-hydroxysuccinimide in EDC, and the product is
It is purified by chromatography or recrystallized from a suitable solvent system to give the NHS derivative.

Alternatively, NHS derivatives can be synthesized from peptides containing amino and / or thiol groups and carboxylic acids. Free amino group or free thiol group,
If present in the molecule, it is preferred to protect these sensitive functional groups prior to carrying out the addition of the NHS derivative. For example, if the molecule contains a free amino group,
Transformation of the amine into Fmoc or preferably the transformation of the amine into a tBoc protected amine is required before carrying out the above chemistry. The amine functional group is NHS
It is not deprotected after preparation of the derivative. Therefore, this method applies only to compounds where the amine group does not need to be liberated to induce the desired therapeutic effect. If the amino group needs to be liberated to retain the original properties of this molecule, another form of the chemistry described below must be carried out.

Further, the NHS derivative is. It can be synthesized from peptides containing amino or thiol groups and no carboxylic acids. If the molecule selected does not contain a carboxylic acid, an array of bifunctional linking groups can be used to convert the molecule into a reactive NHS derivative. For example, triethylamine (having a 10: 1 ratio to EGS) dissolved in ethylene glycol-bis (succinimidyl succinate) (EGS) and DMF and added to the free amino containing molecule,
Produces a mono NHS derivative. N-[-maleimidobutyryloxy] succinimide ester (GMBS) and triethylamine can be used in DMF to produce NHS derivatives from thiol derivative molecules. Maleimide group
Reacts with the free thiol and the NHS derivative is purified from the reaction mixture by chromatography on silica or by HPLC.

NHS derivatives can also be synthesized from peptides containing multiple sensitive functional groups.
Each case must be analyzed and resolved in a different way. However, thanks to the large array of protecting groups and bifunctional linking groups that are commercially available, the present invention preferably provides one chemical step, or two steps (above all), which only modifies the peptide (as above). Applied prior to the protection of sensitive groups) or 3 steps (protection, activation and deprotection) to any peptide. Only in exceptional circumstances, multiple steps (more than these steps) of synthesis are required to transform the peptide into active NHS or maleimide derivatives.

Maleimide derivatives can also be synthesized from peptides containing free amino groups and free carboxylic acids. N- [γ-maleimidobutyryloxy] succinimide ester (GMBS) for the production of maleimide derivatives from amino derivatized molecules
And triethylamine can be used in DMF. The succinimide ester group reacts with the free amino and the maleimide derivative is purified from the reaction mixture by crystallization, or by chromatography on silica, or by HPLC.

Finally, maleimide derivatives can be synthesized from peptides containing multiple other sensitive functional groups and no free carboxylic acids. If the molecule of choice does not contain a carboxylic acid, an array of bifunctional cross-linking reagents can be used to convert the molecule into a reactive NHS derivative. For example, maleimidopropionic acid (MPA
) Is coupled to the free amine to give HBTU / HOBt / in DMF.
DIIEA activity can be used to produce maleimide derivatives via reaction of carboxyl groups of MPA with free amines.

Many other commercially available heterobifunctional cross-linking reagents can be used alternatively, if needed. A large number of bifunctional compounds are available for linking to entities.
Exemplary reagents include: azidobenzoyl hydrazide, N- [
4- (p-azidosalicylamino) butyl] -3 '-[2'-pyridyldithio)
Propionamide), bis-sulfosuccinimidyl suberate, dimethyl adipimidate, disuccinimidyl tartrate, Ny-maleimidobutyryloxysuccinimide ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate , N-succinimidyl [4-azidophenyl] -1,3'-
Dithiopropionate, N-succinimidyl [4-iodoacetyl] aminobenzoate, glutaraldehyde, and succinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate.

5. Synthesis of Modified Organic Therapeutic Agents Similar to the procedures for modified peptide therapeutics, synthetic procedures used to prepare modified organic therapeutic agents are also straightforward. , Which provides high yields and allows for very well purified product. Normally, the chemically reactive group is made in the final stage of the synthesis, eg esterification with a carboxyl group to form an active ester is the final step in the synthesis. Methods for the production and / or modification of the organic therapeutic agents of the present invention are described in the examples below.

Each organic therapeutic agent selected to perform derivatization with a linking group and a reactive group is modified according to the following criteria: Generally, the therapeutic agent is commercially available. If not commercially available, they can be synthesized by procedures well known in the art. As a first step, the therapeutically active region of the therapeutic agent is identified. The therapeutic agent is then modified at a site sufficiently distant from the active moiety to prevent potential interference between the modified drug and the drug's target site, such that the modifying agent modifies its therapeutic activity. Are substantially retained (ie, therapeutic activity is reduced by less than 10-fold). Finally, retaining the constant site of chemical modification optimizes the biological activity of the modifier by modifying the length and nature of the linking group.

Carboxyl groups that are not essential for retention of pharmacological activity are available in the native molecule, and when no other reactive functional group is present on the molecule, the carboxylic acid is a linking group reactive entity. Selected as the point of attachment for modification. If no carboxylic acid is available, any other functional group that is not essential for retention of pharmacological activity is selected as the point of attachment for the linking group reactive entity modification. When some functional groups are available in the therapeutic agent, a combination of protecting groups allows retention of pharmacological activity after addition of linking group / reactive entity and deprotection of all protected functional groups. Is still available. In the absence of a functional group available in the therapeutic, the synthetic approach is to retain biological activity and retain receptor or target specificity,
Expect to modify the original parent drug.

Derivatized therapeutic agents of the invention have derivatized enzyme inhibitors that have substantially lower IC _50's , typically in the range of about 0.5 to 0.01 of the IC ₅₀ of the parent molecule. , Generally have. Desirably, the IC ₅₀ should be 0.05 or higher, preferably about 0.1 or higher. The amount of derivatized therapeutic agent administered will also vary, taking into account the varying IC ₅₀ .

The determination of the nature and length of linking groups is performed empirically through optimal phases and is measured by retention or deletion of biological activity. For example, due to the interaction of a given inhibitor enzyme, repeated modification of the nature and length of the linking group and measurement of biological enzyme activity may be necessary to determine the most preferred linking group length. Preferably, easily synthesized hydrophilic 4-12 atom linking groups are advantageous for initiating the iterative process.

In the case of radiolabeled therapeutics, a minimum distance from the target must be adhered to, based on isotopic and osmotic properties. The length and nature of the linking group is less important than for the enzyme inhibitor combination. For example, β such as ³² P
The line-emitting isotopes have a limited effect or have a maximal effect (99%) from the target, with no effect resulting from small changes in the nature and length of the linking group. Should be located within 5 mm.

6. Synthesis of Modified Diagnostic Agents The methods for producing the modified diagnostic agents of the invention vary widely depending on the nature of the various elements, including the molecule. The synthetic procedure is simple, provides high yields, and allows for very well purified products. Typically, the chemically reactive group is created at the final stage of the synthesis, for example esterification to form an activated ester with a carboxyl group is the final step in the synthesis. The method for producing the diagnostic agent of the present invention is described below.

7. Lung Formulations and Methods of Delivery A further aspect of the present invention is the ability to form covalent bonds with pulmonary fluid proteins to aerosol compositions for the treatment of symptoms of pulmonary conditions. Targeted with possible therapeutic agents. Alternatively, the therapeutic agent can be first conjugated to solution protein in the lung prior to administration.

The aerosol composition comprises at least 2.5% by weight (more preferably between about 3% and 10% by weight, and most preferably at least about 5% by weight) derivatized therapeutic agent. Can consist of an aqueous solution suitable for inhalation. Aerosol droplets should be no more than 10μ to maximize deposition in the alveoli of the lungs, rather than in the throat or upper respiratory tract.

The invention also features a suction device for the administration of the suction composition (or medicament) of the invention. In one aspect of the invention, the aspiration device includes a housing defining a chamber containing dry powder. Dry powders consist of therapeutic compound present in an amount that binds to lung protein upon administration. Alternatively, the drug is covalently bound to pulmonary proteins ex vivo prior to administration. At least 50%
(Preferably at least 70%, and more preferably at least 90%)
The powder of 1) consists of major particles having a diameter of 10 μm or less, and major particles that assemble into larger particles and agglomerate, or agglomerate that easily breaks into major particles upon aspiration from the device. The chamber has an opening through which the medication can be inhaled by suction by the patient.

In another aspect of the invention, the aspiration device comprises a tube containing an inhalable medicament in the form of an aqueous solution suspended in a propellant gas that is pressurized or melted. An aqueous solution of at least 2.5% by weight (preferably about 3%, more preferably at least 4%, even more preferably at least 5% and most preferably between 6% and 10%) comprises a pH raising interfering compound. Is.

The aspiration device may also have a housing defining a port in which the tube is provided, a lumen in communication with the port, and a mechanism for controllably releasing the propellant from the tube into the lumen, whereby Suspended medication may be released from the tube to the lumen. A lumen is formed to route the drug suspended in the propellant into the patient's respiratory system.

8. Therapeutic Uses of Modified Therapeutic Agents The modified therapeutic agents of the present invention find numerous uses, as listed below.

A. Therapeutic Uses of Modified Anti-Tumor Agents Anti-tumor agents of the present invention, including but not limited to those specified in the examples, possess anti-angiogenic activity. As modified anti-neoplastic agents having anti-pulse related activity, the compounds of the present invention are useful in the treatment of the following various diseases (eg, the following primary and metastatic solid tumors and carcinomas (breast) : Colon; rectum; lung; oropharynx; hypopharynx; esophagus; stomach; pancreas; liver; gallbladder; bile duct; small intestine; ureter including kidneys; bladder and urothelium; cervix, uterus, endometrium, ovary, Female genital diseases including choriocarcinoma and festive trophoblasts; male reproductive tract tumors including prostate, seminal vesicles, testes and germ cells; endocrine glands including thyroid, adrenal gland and pituitary gland; hmangiomas, Melanoma, sarcomas originating from soft tissue bone and Karposi's sarcoma, Wilms tumor, rhabdomyosarcoma; astrocytoma, glioma, glioblastoma, retinoblastoma, neuroma Tumors of the head and neck, brain, nerves, eyes, and meninges, including neuroblastoma; tumors of the bone marrow and of hematopoietic origin, solid tumors arising from malignant tumors of hematopoietic origin (eg leukemia), and green Solid tumors, including tumors, plasmacytomas, disease and mycosis fungoides and cutaneous T-cell lymphoma / leukemia tumors; acute lymphocytic leukemia, acute granulocytic leukemia and chronic granulocytic leukemia; Hodgkin lymphoma and non-Hodgkin lymphoma Lymphomas including; Prevention of autoimmune diseases including rheumatoid arthritis, immunoarthritis and osteoarthritis; Eye diseases including diabetic retinopathy; Retinopathy of prematurity; Corneal graft rejection, post lens fibroplasia, angiogenesis Retinal neovascularization due to glaucoma, rubeosis, macular degeneration and hypoxia; abnormal neovascularization of the eye; skin disorders including psoriasis; Vascular diseases, including hemangiomas and capillary proliferation within plaques of sclerosis; myocardial angiogenesis; plaque neovascularization status; hemophiliac joints; hemangiofibromas; wound granulation; intestinal adhesions, Having angiogenesis as a result of abnormalities including Crohn's disease, atherosclerosis, scleroderma and diseases characterized by excessive or abnormal stimulation of endothelial cells including scleroderma and ulcers (Hericobacterium pilori) Diseases; rheumatoid arthritis, osteogenic sarcoma, osteoarthritis, osteopenia (eg, osteoporosis, periodontitis, gingivitis, corneal epidermal ulceration or gastric ulceration), and tumor metastasis, invasion and proliferation, retinopathy , Wound healing (eye inflammation, soft tissue and bone tissue disease, gingivitis / periodontal disease), vascular disease (restenosis),
annuerysm inflammation, autoimmune diseases, and rare cancers (eg, choriocarcinoma).

The compounds of the present invention may also be used as described above, either alone or in combination with other chemotherapeutic treatments conventionally administered to patients to treat radiotherapy and / or angiogenic disorders. It may be useful for preventing tumor-derived metastases. For example, when used to treat solid tumors, the compounds of the invention may be administered the following chemotherapeutic agents (eg, α-interferon, COMP (cyclophosphamide), vincristine, methotrexate). And prednisone), etoposide, mBAC
OD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine and dexamethasone), PROMACE / MOPP (prednisone, methotrexate, doxorubicin, cyclophosphamide, taxol, etoposide / methylethamine (mechloetamine).
), Vincristine, prednisone and procarbazine), vincristine,
Vinblastine, angioinhibin, TNP-
470, pentosan polysulfate, platelet factor-4, angiostatin (a
niostatin), LM-609, SU-101, CM-101, techgalan, thalidomide, SP-PG, etc.). Other chemotherapeutic agents include the following: alkylating agents such as nitrogen mustards (mechloethamine, melphanchloambucil, cyclophosphamide (cyc).
lophaosphamide) and ifosfamide (ifosfami)
derosion); nirosrosdureas (including carmustine, lomustine, semustine and streptozocin); alkyl sulphonates containing busulfan; triazines containing dacarbazine; thiotepana hexamethylmelane (thiotepa na dhexamethylmethymethyl).
fine analogs containing methotrexate; 5-FU, cytosine arabinoside; purine analogs containing 6-mercaptopurine and 6-thioguanine;
Antitumor antibiotics including actinomycin D; doxorubicin, bleomycin,
Anthracycline containing mitomycin C and mithramycin
raqcyclines); hormones and hormone antagonists, including tamoxifen and corticosteroids, and cisplatin and brechner (
(brequinar) -containing myoscellaneous
Agents; fragments of plasminogen (Kringle-5) as well as fragments from other integrin binding substances. For example, using tumors, surgery, radiation or chemotherapy and administration of modified anti-tumor agents to cause tumors to prolong the resting of micrometastases and to inhibit the growth of any remaining primary tumors. It can be treated conventionally.

B. Therapeutic Uses of Modified Matrix Metalloprotease Inhibitors MMPIs of the invention, including but not limited to those identified in the examples, possess anti-angiogenic activity. As modified matrix metalloprotease inhibitors having anti-angiogenic activity, the compounds of the present invention are useful in the treatment of a variety of diseases, such as the following primary and metastatic solid tumors and carcinomas: (Breast: colon; rectum; lung; oropharynx; hypopharynx; esophagus; stomach; pancreas;
Liver; gallbladder; bile duct; small intestine; ureter including kidney; bladder and urothelium; female genital diseases including cervix, uterus, ovary, choriocarcinoma and festive trophoblast; prostate, seminal vesicle, testis And reproductive tract tumors including germ cells; endocrine glands including thyroid gland, adrenal gland and pituitary gland; mangiomas
), Melanoma, sarcoma and Karposi sarcoma arising from soft tissue bone; including astrocytoma, glioma, glioblastoma, retinoblastoma, neuroma, neuroblastoma, nrain, Tumors of the brain, nerves, eyes, and meninges; tumors of the bone marrow and tumors of the hematopoietic system, solid tumors resulting from malignant tumors of the hematopoietic system (eg, leukemia), and chloroma, solid tumors, plasmacytomas, illness and Solid tumors including mycosis fungoides and cutaneous T-cell lymphoma / leukemia tumors; lymphomas including Hodgkin lymphoma and non-Hodgkin lymphomas; prevention of autoimmune diseases including rheumatoid arthritis, immunoarthritis and osteoarthritis; diabetic Ocular diseases including retinopathy; retinopathy of prematurity; corneal graft rejection, post lens fibroplasia, neovascular glaucoma, rubeosis, macular degeneration and hypoxia Retinal neovascularization; abnormal neovascularization of the eye; skin diseases including psoriasis; vascular diseases including hemangiomas and capillary proliferation within atherosclerotic plaques; myocardial angiogenesis; plaque neovascularization; Hemophilia connection (hemophil)
angiofibroma; wound granulation; intestinal adhesions, Crohn's disease, atherosclerosis, scleroderma, and diseases and ulcers characterized by excessive or abnormal stimulation of endothelial cells, including hyperplastic scars ( Hericobacter
diseases with angiogenesis as a result of abnormalities including piori); rheumatoid arthritis, osteoarthritis, osteopenia (eg osteoporosis, periodontitis, gingivitis, corneal epidermal ulceration or gastric ulceration), and Tumor metastasis, invasion and proliferation, retinopathy, wound healing (eye inflammation, soft tissue and bone tissue disease, gingivitis / periodontal disease), vascular disease (restenosis), anneurysm inflammation, and autoimmune disease.

The compounds of the present invention may also be used as described above, either alone or in combination with other chemotherapeutic treatments conventionally administered to patients to treat radiation therapy and / or angiogenic disorders. It may be useful for the prevention of tumor-derived metastases. For example, when used in the treatment of solid tumors, the compounds of the invention can be administered, for example, the following chemotherapeutic agents (eg, α-interferon, COMP (cyclophosphamide, vincristine, methotrexate (meth)). methotraxate) and prednisone), etoposide,
mBACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine and dexamethasone), PROMACE / MOP
P (prednisone, methotrexate, doxorubicin, cyclophosphamide,
Taxol, etoposide / methylethamine (mechloeta)
min), vincristine, prednisone and procarbazine), vincristine, vinblastine, angioinhibin,
TNP-470, pentosan polysulfate, platelet factor-4, angiostatin, LM-609, SU-101, CM-101.
, Techgalan, thalidomide, SP-PG, etc.). Other chemotherapeutic agents include: alkylating agents such as nitrogen mustards (mechloethamine, melphanchloambucil, cyclophosphamide, and ifosfamide (ifosfamide).
(including family)); niros doureas (
Carmustine, lomustine, semustine, and streptozocin); Busulfan-containing alkyl sulfonates; Dacarbazine-containing triazines; Thiotepana hexamethylmelanin
Ethylenimines including melanin; folate analogs including methotrexate; pyrimidine analogs including 5-FU, cytosine arabinoside; purine analogs including 6-mercaptopurine and 6-thioguanine; antitumor antibiotics including actinomycin D Anthracyclines (a including doxorubicin, bleomycin, mitomycin C, and mithramycin;
hormones and hormone antagonists, including tamoxifen and corticosteroids, and myoscellaneos, including cisplatin and brequinar.
Us) agents; fragments of plasminogen (Kringle-5) as well as fragments derived from other integrin binding substances. For example, tumors have traditionally been treated with surgery, radiation or chemotherapy and modified MMPI molecules of the invention to prolong the resting of micrometastases and to inhibit the growth of any remaining primary tumor. Can be treated.

Hydroxamic acid MMPI can inhibit the production of the cytokine tumor necrosis factor (TNF) (Mohler et al., Nature, 1994, 370, 218-220).
Gearing AJH et al., Nature 1994, 370, 555-55.
7; McGeehan GM et al., Nature 1994, 370, 558-5.
61). Compounds that inhibit the production or activity of TNF are believed to be potentially useful for the treatment or prevention of many inflammations, infections, immunological or malignant diseases. These include, but are not limited to: septic shock, haemodynamic shock and sepsis syndrome, post-ischemic reperfusion injury, malaria, Crohn's disease, mycobacterial infection, Meningitis, psoriasis, congestive heart failure, fibrotic disease, cachexia, graft rejection, cancer, autoimmune disease, rheumatoid arteritis, multiple sclerosis, radiation damage, immunosuppressive monoclonal antibody ( For example, toxicity and hyperoxic alveolar injury after administration of OKT3 or CAMPATH-1). Since excess TNF production is noted in some diseases or conditions and is characterized by MMP-mediated tissue degradation, compounds that inhibit both MMP and TNF production are treated or prevented for diseases or conditions that are involved in both mechanisms. In particular, it can have particular advantages.

The compounds of the present invention are compounds of the matrix metalloproteinase family (eg,
It inhibits a variety of enzymes from collagenase, stromelysin (protoglycanase), and gelatinase that initiate collagen degradation and is therefore useful in the treatment of matrix metalloendoproteinase diseases. Rheumatoid arthritis (Arthritis and Rheumatimsm, 20, 1231-1239
, 1977) and there is evidence to involve collagenase as one of the key enzymes in the degradation of articular cartilage and bone. The strong promoters of collagen and other metalloproteases involved in tissue degradation are useful in the treatment of rheumatoid arthritis and related diseases in which collagenolytic activity is important. Therefore, inhibitors of this type of metalloprotease can be used to treat or prevent conditions associated with tissue degradation; therefore, they are arthritis, dermatological conditions, bone resorption, inflammatory diseases and tumor invasion. It is useful in the treatment of and the promotion of wound healing. In particular, the compounds of the present invention may be useful in the treatment of osteopenia (eg osteoporosis), rheumatoid arthritis, osteoarthritis, periodontitis, gingivitis, corneal ulceration and tumor invasion.

C. Therapeutic Uses of Oxytocin Conjugated oxytocin can be used to help prenatal lactation and to help relax the pelvis. Also, after delivery (post par)
can be used to prevent uterine bleeding.

D. Therapeutic Uses of Cholecystokinin (CCK) Conjugated CCK was found in diagnostic studies of the gallbladder or in chronic cholecystitis (c).
It can be used in

[0335]   (E. Therapeutic Use of Antihypertensive Drug)   Antihypertensive drugs are used to treat hypertension.

F. Therapeutic Uses of Methylprednisolone Methylprednisolone is used to treat a wide range of disorders such as asthma and arthritis. In gastroenterology, it is effective in the treatment of several inflammatory conditions, such as ulcer and microscopic colitis, Crohn's disease and autoimmune hepatitis. A newer use is for the reduction of traumatic posterior spinal cord edema.

G. Therapeutic Uses of GP-41 Peptide GP-41 and HIV transmembrane proteins can be used to make therapeutic and diagnostic agents for HIV. For example, antibodies can be constructed to recognize the epitope of gp41. The structure of this antibody provides important information about antibody / antigen interactions, guides scientists in selecting superior antigenic peptides for HIV detection, and future recombination with genetically engineered antibodies. Provides important information for the experiment.

H. Therapeutic Uses of Blood-Brain Barrier (BBB) Peptides Since BBB peptides can cross the blood-brain barrier via protein transduction, these peptides are compound, peptide, antisense peptide nucleic acids. Alternatively, covalently linked to 40 nm iron beads or as an in-frame fusion with the full-length protein, these compounds may be able to enter any cell type in a receptor- and transporter-independent manner. It efficiently delivers these compounds across the blood brain barrier.

I. Therapeutic Uses of Modified Cell Adhesion (RGD) Peptides The RGD peptides of the present invention and their derivatives and analogs are useful for treating neoplastic diseases and inflammatory diseases such as rheumatoid arthritis, lupus. Find multiple uses, including as.

(1. Antineoplastic Treatment) The modified cell adhesion peptide of the present invention or a derivative or analog thereof directly targets cancer cells via a peptide-specific receptor. It has been shown that receptors for these peptides are expressed at elevated levels on the surface of tumor cells. Thus, modified peptides or their derivatives or analogs can be used to preferentially target drugs to metastatic tumor cells. Accordingly, the modified cell adhesion peptides or their derivatives or analogs are useful as agents for the treatment of various types of cancer such as breast cancer, melanoma, and fibrosarcoma.

The use of an effective amount of modified cell adhesion peptides or their derivatives or analogs as a treatment for cancer has the advantage of being more potent than unmodified cell adhesion peptides. Since modified cell adhesion peptides or their derivatives or analogs are more stable in vivo, smaller amounts of the molecule can be administered for effective treatment.

Derivatives and conjugates of modified cell adhesion peptides and their analogs can be used in a number of different ways to achieve a number of different purposes. As mentioned above, these substances can be used in place of typical cell adhesion peptide drugs as anti-adhesive agents. Compared to currently available cell adhesion peptide drugs, the agents of the invention may reduce clot formation, have fewer side effects, and are substantially longer in time than conventionally administered cell adhesion peptide drugs. , Available to reduce clot formation. Furthermore, the induced cell adhesion peptides of the present invention may be used with various other anti-adhesion or anti-cancer therapies (US Pat. Nos. 5,443,827; 5,43.
9,88 and 5,433,940 and PCT application number WO / 97.
/ 01093, which are hereby incorporated by reference). Such anti-cancer treatments include the use of radiation or, for example, vinca alkaloids, alkylating agents, doxorubicin, etoposide, methotrexate, tamoxifen, vinblastine, asparaginase, vicurtamide (bic).
lutamide), bleomycin, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel (docetaxel), floxuridine, fludarabine (fludurabin).
darabine), fluorouracil, gemcitabine (gemcitabine)
e), hydroxyurea, idarubicin, ifosfamide, interferon α
, Irnotecan, leuprolide, mechlorethamine, megestrol, melphalan, mercaptopurine, mitomycin, mitozantrone, paclitaxel, plicamycin, porfilmer, procarbazine, streptozocin, teniposide.
teniposide), thioguanine, thiotepa, topotecan
can), trastuzumab, vincristine, vinorelbine, and the like for use in treatment with antineoplastic agents.

The present invention also provides a method for treating an individual cancer, the method comprising providing a modified cell adhesion peptide in an amount sufficient to treat the cancer, the composition Include modified cell adhesion peptides.

2. Treatment of Inflammatory Diseases The modified cell adhesion peptides of the invention and their derivatives and analogs also find use as anti-inflammatory agents. In one aspect of the invention, there is provided a method of using the modified cell adhesion peptides of the invention or a derivative or analog thereof to treat a mammalian subject having an abnormality that results in increased inflammation of a joint or tissue. To be done. The method involves administering to the subject a modified cell adhesion peptide or derivative or analog thereof in an amount sufficient to produce an anti-inflammatory effect on the subject. The modified cell adhesion peptide is intraventricular, oral, subcutaneous,
It can be administered intramucosally or intravenously.

The peptides, their derivatives, analogs, and conjugates of the present invention can be used to treat acute or chronic inflammatory disorders including ischemia, infection, tissue swelling and / or bone and cartilage degradation. Inflammatory disease refers to the situation where activation of white blood cells leads to impaired normal physiological function. Examples of such situations include acute and chronic inflammation such as osteoarthritis, sepsis, ARDS, immune and autoimmune disorders, rheumatoid arthritis, IBD (inflammatory bowel disease), lupus, MS, transplant rejection. , Cirrhosis, sarcoidosis, granulomatous injury, periodontitis / gingivitis, graft-versus-host disease, contact dermatitis, etc. Autoimmune disorders that can be treated using the methods of the invention include chronic active hepatitis, Graves' disease, insulin-dependent diabetes mellitus (type I), and Hashimoto's thyroiditis. Inflammatory disorders that may be treated using the methods of the invention include inflammatory brain disease, inflammatory demyelinating disease, inflammatory vasculitis, inflammatory myopathy, osteomyelitis, Crohn's disease and interstitial cystitis. Can be mentioned. Further examples of inflammatory diseases include myocardial disease, infectious disease, lung disease and transplant rejection.

(J. Modified Insulinotrophins such as GLP-1
pic) Therapeutic Uses of Peptides) Modified insulinotropic peptides (ITPs) such as GLP-1 of the present invention are
Use in the treatment of diabetes, sedatives, nervous system disorders, use to induce anxiolytic effects on the CNS, use to activate the CNS, post-surgical treatment, and use as a treatment for insulin resistance. Find multiple uses, including.

1. Diabetes Treatment The modified ITPs of the present invention generally normalize hyperglycemia via glucose-dependent, insulin-dependent and insulin-independent mechanisms. As such, the modified ITP is useful as a primary agent for the treatment of type II diabetes and as an additional agent to the treatment of type I diabetes.

The use of an effective amount of modified ITP as a treatment for diabetes has the advantage of being more potent than unmodified ITP. Because the modified ITPs migrate stably in vivo, smaller amounts of the molecule can be administered for effective treatment. The present invention relates to diabetes (types I and II) in that the action of the peptide is dependent on blood glucose concentration and thus the risk of hypoglycemic side effects is greatly reduced over the risk of using current treatment methods. It is particularly suitable for the treatment of patients with both).

The present invention also provides a method for treating individual diabetes, the method comprising providing a modified ITP in an amount sufficient to treat diabetes, wherein the composition comprises
Includes modified ITP.

2. Treatment of Nervous System Disorders The modified ITPs of the present invention also find use as sedatives. In one aspect of the invention, there is provided a method of sedating a mammalian subject having an abnormality that results in increased activation of the central or peripheral nervous system using a modified ITP of the invention.
The method involves administering to the subject a modified ITP in an amount sufficient to produce a sedative or anxiolytic effect on the subject. The modified ITP can be administered intracerebroventricularly, orally, subcutaneously, intramucosally, or intravenously. Such methods are useful for treating or ameliorating nervous system conditions such as anxiety, movement disorders, aggression, psychosis, seizures, panic attacks, hysteria and sleep disorders.

In a related aspect, the invention includes a method of increasing the activity of a mammalian subject, wherein the modified ITP is added to the subject in an amount sufficient to produce an activating effect on the subject. The step of administering is included. Preferably, the subject has a condition that results in decreased activity of the central or peripheral nervous system. Modified ITP is associated with depression, schizoaffective disorder, sleep apnea, attention deficit syndrome with poor concentration, memory loss, amnesia,
And narcolepsy, which finds particular use in treating or ameliorating narcolepsy, which merely lists some conditions in which central nervous system arousal may be beneficial.

The modified ITPs of the invention are used to introduce wakefulness to treat or ameliorate depression, schizoaffective disorder, sleep apnea, poorly focused attention deficit syndrome, memory loss, amnesia, and narcolepsy. Can be done. The therapeutic efficacy of modified ITP can be monitored by patient interviews to assess a patient's condition, by psychological / neurologic testing, or by amelioration of symptoms associated with these conditions. For example, treatment of narcolepsy can be assessed by monitoring the occurrence of narcolepsy attacks. As another example, the effect of a modified ITP on a subject's ability to concentrate, or the effect of a modified ITP on memory ability, can be tested using any of a number of diagnostic tests well known to those of skill in the art.

3. Post-Surgery Treatment The modified ITP of the present invention can be used for post-surgical treatment. The patient may be treated about 1 to 16 hours before surgery is performed on the patient, during surgery on the patient, and about 5 hours after surgery on the patient.
For up to a day, the modified ITP of the present invention is required.

The modified ITP of the invention is administered from about 16 hours to about 1 hour before surgery begins. The length of time before surgery that the compounds used in the present invention should be administered to reduce catabolic effects and insulin resistance depends on many factors. These factors are generally known to physicians in the field and most importantly, the patient is fasting during the preoperative preparatory period, or glucose infusion, glucose drink or some other form. Including whether you are nutritionally supplemented. Other important factors include
Patient's sex, weight, age, inability to control blood sugar, original cause of inability to control blood sugar, expected severity of trauma caused by surgery, route of administration and bioavailability, physical continuity, The formulation, as well as the potency of the compound administered, can be mentioned. The preferred time interval for initiating administration of the modified ITP used in the present invention is from about 1 hour to about 10 hours before surgery begins. The most preferred interval to begin administration is 2-8 hours before surgery begins.

Insulin resistance following certain types of surgery, selective peritoneal surgery, may be deepest on the day after the first surgery, last for at least 5 days, and take 3 weeks to normalize. Thus, post-surgical patients may be required to administer the modified ITP used in the present invention for the duration of time following surgical trauma, depending on factors that are understood and determinable by one of ordinary skill in the art. Among these factors, whether the patient is fasting after surgery or is supplemented with glucose infusion, glucose drink or some other form of maintenance, as well as, but not limited to, the patient's sex, weight, age, blood glucose. Uncontrollable severity, the original cause of inability to control blood sugar, the actual severity of trauma caused by surgery, the route and bioavailability of the administration, body continuity, prescription, and efficacy of the administered compound. is there. The preferred period of administration of the compounds used in the present invention is 5 days or less after surgery.

4. Insulin Resistance Treatment The modified ITPs of the present invention can be used to treat insulin resistance independent of their use in post-surgical treatment. Insulin resistance may result from decreased binding of insulin to cell surface receptors or altered intracellular metabolism. The first type, characterized as reduced insulin sensitivity, can typically be overcome by increasing insulin concentration. The second type, characterized by reduced insulin responsiveness, can be overcome by high doses of insulin. Insulin resistance following trauma can be overcome by a dose of insulin that is proportional to the degree of insulin resistance and is therefore clearly caused by a decrease in insulin sensitivity.

The dose of modified ITP effective to normalize a patient's blood glucose levels will depend on a number of factors including, but not limited to, the patient's sex, weight, age, severity of inability to control blood glucose,
It depends on the original cause of the inability to regulate blood glucose, whether glucose or another source of carbohydrate is co-administered, or the route and bioavailability of the administration, body duration, prescription, and efficacy).

K. Therapeutic Uses of Modified Kringle 5 Peptides As described above, angiogenesis is associated with tissue neovascularization, including "sprouting", angiogenesis or vascular swelling. Including various processes including. With the exception of traumatic wound healing, corpus leuteum formation, and embryogenesis, most processes of angiogenesis are thought to be associated with disease processes, and thus the use of the therapeutic methods of the present invention is directed toward disease. It is selective and has no harmful side effects.

There are a variety of diseases in which angiogenesis appears to be important, and these may be treatable with the modified peptides of the invention. These diseases include, but are not limited to, disorders associated with inflammatory disorders such as immune and non-immune inflammation, rheumatoid arthritis and psoriasis, inappropriate or untimely invasion of the vasculature such as diabetic retinopathy. ,
Neovascular glaucoma, restenosis, capillary proliferation and osteoporosis in atherosclerotic plaques, and cancer-related disorders such as solid tumors, solid tumor metastases, angiofibromas, postlens fibroplasias, hemangiomas, Kaposi's sarcoma and tumors Included are similar cancers that require neovascularization to support growth.

The modified kringle 5 peptides of the present invention find use in methods that inhibit angiogenesis in diseased tissues, ameliorate the symptoms of the disease, and, depending on the disease, may contribute to the cure of the disease. The modified peptides of the invention are more stable in vivo and smaller amounts of the modified peptides can be administered for effective treatment. In one embodiment, the present invention contemplates inhibiting angiogenesis by itself in tissue. The degree of angiogenesis in tissues, and thus the degree of inhibition achieved by the methods of the invention, was assessed by immunohistochemistry by various methods for detecting ∀ ₅ # ₃ immunopositive immature and early vasculature. obtain.

As described herein, any of a variety of tissues, or organs composed of organized tissues, includes skin, muscle, intestine, connective tissue, joints, bones and similar tissues ( Can support angiogenesis in disease states, including blood vessels that can be invaded by neovascular stimulation.

In one related embodiment, the tissue treated with the modified kringle 5 peptides of the invention is inflamed tissue and the angiogenesis inhibited is the presence of neovascularization of the inflamed tissue. Inflammatory tissue angiogenesis. In this class, the method contemplates inhibition of angiogenesis in joint tissue, such as patients with rheumatoid arthritis, immune or non-immune inflammatory tissue, psoriatic tissue, and the like.

The patient treated in the present invention in many of its embodiments is preferably a human patient, but the principles of the present invention contemplate that the present invention is included in all mammals (the term “patient”). To be effective. In this relationship,
It is understood that mammals include any mammalian species in which treatment of diseases associated with angiogenesis is desirable, particularly agricultural and domestic mammalian species.

In another related embodiment, the tissue treated with the modified kringle 5 peptide of the invention is retinal tissue of a patient having diabetic retinopathy, macular degeneration or neovascular glaucoma and is inhibited. Angiogenesis is retinal tissue angiogenesis in which there is angiogenesis of retinal tissue.

In a further related embodiment, the tissue treated with the modified kringle 5 peptides of the invention is of a patient with a solid tumor, metastases, skin cancer, breast cancer, hemangiomas or angiofibromas and similar cancers. Angiogenesis that is tumor tissue and is inhibited is tumor tissue angiogenesis in which there is neovascularization of the tumor tissue. Representative solid tumor tissues treatable by the methods of the invention include lung, pancreas, breast, colon, larynx, ovary,
And similar organizations.

Inhibition of tumor tissue angiogenesis is a particularly preferred embodiment because of the important role of neovascularization in tumor growth. In the absence of neovascularization of the tumor tissue, the tumor tissue lacks the necessary nutrients, grows slowly, ceases to grow further, recedes, and eventually becomes a necrosis that causes tumor killing. .

Accordingly, the invention provides a method of inhibiting tumor neovascularization by inhibiting tumor angiogenesis according to the methods of the invention using the modified kringle 5 peptides of the invention. Similarly, the invention provides a method of inhibiting tumor growth by performing a method of inhibiting angiogenesis. This method is also particularly effective for the formation of metastases. Because (1) their formation requires angiogenesis of primary tumors so that metastatic cancer cells can exit the primary tumor, and (2) their establishment at secondary sites supports the growth of metastases. Because it requires neovascularization.

In a related embodiment, the present invention contemplates the practice of the invention with conventional chemotherapy for solid tumors and for the control of the establishment of metastases. Administration of the modified kringle 5 peptides of the invention, typically during or after chemotherapy, induces angiogenesis in which the tumor tissue is restored by blood and nutrient supply to the tumor tissue. Sometimes it is preferable to inhibit angiogenesis after a chemotherapy regimen when responding to a toxic assault. Furthermore, if solid tumors are removed as a prophylaxis against metastases, it is preferable to administer the modified kringle 5 peptide after surgery. As long as the method of the invention is applied to the inhibition of tumor neovascularization,
This method can also be applied to inhibition of tumor tissue growth, inhibition of tumor metastasis formation, and regression of established tumors using the modified kringle 5 peptides of the invention.

Restenosis is a process of smooth muscle cell (SMA) migration and proliferation at the site of percutaneous transluminal coronary angioplasty that prevents successful angioplasty. Migration and proliferation of SMCs during restenosis can be considered a process of angiogenesis that is inhibited by the modified kringle 5 peptides of the invention. Thus, the present invention also contemplates inhibition of restenosis by inhibiting angiogenesis in a patient following angioplasty surgery. For inhibition of restenosis, the modified kringle 5 peptide is typically administered for about 2 to about 28 days after angioplasty, more typically for the first 14 days following this surgery.
Is administered.

[0370] The method of the invention for inhibiting angiogenesis in a tissue comprises contacting a tissue with or at risk of angiogenesis with a composition comprising a therapeutically effective amount of a modified kringle 5 peptide. The step of causing is included. The dosage range for administration of the modified Kringle 5 peptide depends on the form of the peptide, as described herein, and its potency, with angiogenesis and disease symptoms mediated by angiogenesis. An amount sufficient to produce the desired effect that is improved. The dosage should not be so large as to cause adverse side effects (eg hyperviscosity syndrome, pulmonary edema, congestive heart failure, etc.). Generally, the dosage will vary with the age, condition, sex and extent of disease of the patient and can be determined by one skilled in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

As an angiogenesis inhibitor, such modified kringle 5 peptides are found in both primary and metastatic solid tumors and cancers of the following: breast; colon; rectum; lung; oropharyngeal; pharyngeal larynx; Esophagus; stomach; pancreas; liver; gallbladder; bile duct; small intestine; urinary tract (including kidney, bladder and urothelium); female reproductive tract (including cervical, uterine, ovarian, choriocarcinoma and trophoblastic pregnancy); Male reproductive tract (including prostate, seminal vesicles, testes and germ cell tumors); Endocrine glands (including thyroid, kidneys and pituitary gland); Skin (including hemangiomas, melanoma, sarcomas and Kaposi's sarcomas arising from bone or soft tissue) ); Brain, nerve, ocular and meningeal tumors (including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas, schwannomas and meningiomas) Solid tumors arising from hematopoietic malignancies (eg , Leukemia and chloroma, plasmacytoma, plaque and mycosis fungoides tumors and cancerous T-cell lymphoma / leukemia); lymphoma (including both Hodgkin lymphoma and non-Hodgkin lymphoma); autoimmune disease (chronic joints) Prevention of rheumatism, including immunoarthritis and osteoarthritis; ocular diseases (diabetic retinopathy, precocious retinopathy, corneal transplant rejection, post lens fibroplasia, neovascular glaucoma, rubeosis, retinal neovascularization ( (Due to macular degeneration and hypoxia)); abnormal neovascularization of the eye; skin disorders (including psoriasis); vascular disorders (blood glomeruli (hema)
gio)) and capillary growth within atherosclerotic plaques); Osler
-Webber syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophilic joints; angiofibromas; wound granules; diseases characterized by excessive or abnormal stimulation of endothelial cells (intestinal Diseases with adhesions, Crohn's disease, atherosclerosis, scleroderma and hyperplastic scars (ie keloids) and angiogenesis as a pathological consequence (cat scratch disease (Rochele minalia q
uintosa) and ulcers (including Helicobacter pylori). Another use is as a birth control drug that inhibits ovulation and placenta establishment.

The modified kringle 5 peptides of the present invention may also be used alone or with radiation therapy and / or
Or when used in conjunction with other chemotherapeutic treatments conventionally administered to patients to treat angiogenic diseases, it may be useful in preventing metastases from the tumor. For example, when used in the treatment of solid tumors, the modified kringle 5 peptides of the invention include alpha interferon, COMP (cyclophosphamide, vincristine, methotrexate and prednisone), etoposide, mBACOD (methotrexate, bleomycin, doxorvidin, cyclophosphine). Famide, vincristine and dexamethasone), PRO-MACE / MOPP (prednisone,
Methotrexate (w / leucovin rescue), doxorubicin, cyclophosphamide, taxol, etoposide / mechlorethamine, vincristine,
Prednisone and procarbazine), vincristine, vinblastine, angioinhibin, TNP-470, pentosan, polysulfate, platelet factor 4, angiostatin (angiostati).
n), LM-609, SU-101, CM-101, Techgalan, thalidomide, SP-PG and the like. Other chemotherapeutic agents include alkylating agents (eg, mechloethamine).
ine), a nitrogen mustard containing melphalan, chlorambucil, cyclophosphamide and ifosfamide); nitrosoureas (including carmustine, lomustine, semustine and streptozocin); alkyl sulfonates (including busulfan) ); Triazine (including dacarbazine); ethyleneimine (including thiotepa and hexamethylmelamine); folic acid analog (including methotrexate); pyrimidine analog (including 5-fluorouracil, cytosine arabinoside); purine analog (6-mercapto) (Including purines and 6-thioguanine); antitumor antibiotics (
Actinomycin D); Anthracyclines (including Doxorubicin, Bleomycin, Mitomycin C, and Mitramycin); Hormones and hormone antagonists (including tamoxifen and corticosteroids and miscellaneous drugs, including cisplatin and brequinar). For example, tumors have traditionally been treated with surgery, radiation or chemotherapy and kringle 5 administration, along with kringle 5 administration to extend quiescence of micrometastases and stabilize and inhibit the growth of any remaining primary tumor. Can be treated.

L. Therapeutic Uses of Modified Opioid Molecules and Analgesic Agents Derivatives and conjugates of opioid molecules and analgesic agents can be used in a number of different ways to achieve a number of different purposes. These agents may be used in place of typical antinociceptive agents to improve pain. Compared to currently available drugs, the agents of the invention may improve pain without centrally mediated side effects or addiction or loss of efficacy, reducing pain for substantially longer times than traditionally administered drugs. Is available for. The opioid derivatives and conjugates of the present invention may also be utilized as anti-inflammatory and / or anti-stimulant agents or generally to inhibit vascular leakage from tissues (US Pat. No. 5,482,93).
No. 0). In addition, as is known in the art, these agents may be used to treat hosts that are or become resistant to morpholine (or to treat patients undergoing a methadone treatment program), Generally, discontinuation of anesthesia is performed.

M. Therapeutic Uses of Modified Immunosuppressive Agents Various such as cyclosporine and derivatives, corticosteroids, sulfasalazine, thalidomide, methotrexate, OKT3, peptide-T, or agents that inhibit T cell activation or adhesion. Immunosuppressants are useful prior to transplantation to mask immune responsiveness and organ rejection. Such agents may be applied at the time of tissue collection (eg, heart, lung, liver collection) or shortly before restoration of blood flow in the recipient. Such immunosuppressive agents interfere with the recognition of foreign antigens from the donor tissue, facilitating short-term acceptance and facilitating the host's longer-term ability to fit the transplanted organ.

[0375]   (N. Therapeutic Use of Modified Antibiotics)   The modified antibiotics of the present invention find use for treating infections.

[0376]   (O. Therapeutic use of modified antidepressant)   The modified antidepressants of the present invention are useful in treating depression.

(P. Modified anti-virus and anti-fusogenic peptides HIV)
And Therapeutic Uses of Anti-HIV Peptides The human immunodeficiency virus (HIV), responsible for the acquired immunodeficiency syndrome (AIDS), is a member of the lentivirus family of retroviruses. Two
There are two prevalent forms of HIV, HIV-1 and HIV-2, each of which various strains have been identified. HIV targets CD-4 + cells, and viral influx is dependent on the binding of HIV protein gp41 to the CD-4 + cell surface receptor.

The modified antiviral or antifusogenic peptides of the present invention may be used as therapeutic agents in the treatment of patients suffering from HIV infection, according to the methods described below and other methods known in the art. Can be administered. Effective therapeutic dosages of modified peptides can be determined by procedures well known to those of skill in the art and will be considered in terms of the potential toxicity of the peptide.

The modified peptides can also be administered prophylactically to individuals who have not been previously infected. This may be advantageous if the individual is subjected to a high risk of viral exposure,
Where an individual is at high risk of viral transmission, it can occur when in contact with an infected individual. This may be particularly advantageous if no cure for the virus is known, such as the HIV virus. As an example, prophylactic administration of a modified anti-HIV peptide may be used in situations where health care workers are exposed to blood from HIV-infected individuals,
Or it is advantageous in other cases where the individual is engaged in a high risk activity that potentially exposes the individual to the HIV virus.

1. SIV and anti-SIV peptides: Simian immunodeficiency virus (SIV) is a lentivirus that causes acquired immunodeficiency syndrome (AIDS) -like disease in susceptible monkeys. The modified antiviral peptides according to the invention can be used for the treatment of infected animals or as a prophylactic agent in a similar manner against HIV.

2. RSV: The RS virus (RSV) is a respiratory pathogen that is particularly dangerous in infants and children where it can cause bronchiolitis (small airway inflammation) and pneumonia. RSV is a negative-sense single-stranded RNA virus,
It is also a member of the Paramyxoviridae family of viruses. The route of infection of RSV is typically through the mucous membranes by the respiratory tract (ie, nose, throat, trachea, and bronchi and bronchioles). The antiviral peptides according to the invention can be used for the prevention and treatment of RSV-related diseases.

3. HPV: Like RSV, human parainfluenza virus (HPIV or HPV) is another major cause of respiratory tract disease, and like RSV, P
It is a negative-sense single-stranded RNA virus that is a member of the aramyxoviridae family of viruses. HPIV --- HPIV-1,
There are four recognized serotypes, HPIV-2, HPIV-3 and HPIV-4. HPIV-1 is the major cause of croup in children, and H
Both PIV-1 and HPIV-2 cause upper and lower respiratory tract disease. HPIV-3 is often associated with bronchiolitis and pneumonia. The antiviral peptides according to the invention can be used for the treatment of HPV-related diseases.

4. MeV: measles virus (MV or MeV) is Paramyxovir
It is an enveloped negative single-stranded RNA virus belonging to the viruses of the idae family. MeV as well as RSV and HPV
Causes respiratory illness and also produces immunosuppression, which is responsible for additional opportunistic infections. In some cases, MeV can establish brain infections that lead to serious neurological complications. The antiviral peptides according to the invention can be used for the treatment of RSV related diseases.

Q. Therapeutic Uses of Modified Antihistamines Modified antihistamines find use in treating excess histamine formed in body tissues, including allergic reactions.

R. Therapeutic Uses of Modified Anti-Angina Agents Modified anti-angina agents include choking and suffocat.
ing) find use in treating angina, including the treatment of sensations.

Angina results from an inadequate blood supply to the heart and is often caused by blockages in the arteries that supply the heart muscle (coronary stenosis due to atherosclerosis). An “unstable” angina condition can develop an acute coronary syndrome (ACS), including myocardial infarction. Anti-angina treatments include treatment with nitroglycerin and the use of aspirin and heparin.

Platelet activation and aggregation play an important and fundamental role in the formation of intracoronary thrombosis in acute arterial syndrome (ACS). Glycoprotein IIb / II
Ia receptor inhibitors are currently used in ACS with heparin and aspirin. Glycoprotein IIb / IIIa receptor inhibitors block the final steps for platelet aggregation and fibrinogen binding,
Therefore, it prevents thrombus formation. Tirofiban is a potent synthetic non-peptide and specific glycoprotein llb / Illa receptor inhibitor, and ischemic complication, non-Q-wave myocardial infarction in patients with well-tolerated and unstable angina And when used in combination with aspirin and heparin have been shown to reduce the risk of high risk patients to undergo revascularization. Other GP IIb / IIIa receptor inhibitors include abciximab and eptifibabat.
ide).

S. Use of Modified Thyroxine Molecules Thyroxine (thyroid amino acid (Merck Index, 1989, 93
48: 1483)) and thyroxine analogs are well known in the art. Thyroid hormones, especially thyroxine T3 and T4, are well established in the literature to have two different types of biological effects: one on cell metabolism and the second on cell differentiation and development (Jorgensen). , 1978, "
Thyroid Hormones and Analogues II. S
structure-Activity Relationships ", Hor
monal Proteins and Peptides, Volume VI, 107
-204, C.I. H. Li, ed., Academic Press, New Yo
rk). For example, thyroxine suppresses iodine uptake by the thyroid (M
oney et al., 1959, “The Effect of Variant Th.
yroxine Analogues on Suppression of
locline-131 Uptake by the Rat Thyroi
d ", Endocrinology 64: 123-125) and induce cell differentiation as studied by tadpole metamorphosis (Money et al., 1).
958, "The Effect of Change in Chemica"
l Structure of Some Thyroxine Analog
ues on the Metamorphosis of Rana Pip
iens Tadpoles ", Endocrinology 63: 20-2
8). In addition, thyroxine and certain thyroxine analogs suppress the growth of non-malignant mouse pituitary thyroid-stimulated tumors (Kumaoka et al., 1960, “.
The Effect of Thyroxine Analogues on
a Transplantable Mouse Pituitary Tu
Mor, "Endocrinology 66: 32-38; Grinber.
g., et al., 1962, "Studies with Mouse Pituitar".
y Thyrotropic Tumors. V. Effect of Var
ioous Thyroxine Analogs on Growth and
"Secretion", Cancer Research 22: 835-8.
41).

The structural requirements of thyroxine and thyroxine analogs for metabolic stimulation and induction of cell differentiation are not identical (Jorgensen, 1978, "Th.
yroid Hormones and Analogues II. Stru
culture-Activity Relationships ", Hormon
al Proteins and Peptides, Volume VI, page 150, C
． H. Li, ed., Academic Press, New York). For example, Money et al. Found that there was no correlation between suppression of iodine uptake in the thyroid and induction of tadpole metamorphosis (Money et al., 1958, “.
The Effect of Change in Chemical Str
ucture of Some Thyroxine Analogues o
n the Metamorphosis of Rana Pipiens
Tadpoles ", Endocrinology 63: 20-28). Based on these observations, currently unidentified cellular responses can be altered or induced by specific thyroxine analogs that do not exhibit any mode of action (metabolic or differentiation) exhibited by thyroxines T3 and T4. Was thought.

Defects in thyroid activity (whether spontaneous or caused by surgical removal of the thyroid), diminished function following thyroiditis or pituitary degeneration result in clinical hypothyroidism. Whatever the cause, the condition is treated by replacement therapy using the modified thyroxine molecules of the invention.

The present invention also relates to a method for the treatment of anemia associated with rheumatoid arthritis and anemia present in a patient having a viral or bacterial infection, wherein the symptoms of rheumatoid arthritis are modified according to the invention. The presence of thyroxine molecules is also present.

In accordance with the present invention, associated anemia characterized as moderately hypopigmentary or normocytic increases cytoxin in the bloodstream and thereby matures from bloodstream stem cells. Treated by administering to the patient in need of treatment a composition to increase the upper limit of red blood cell count. The composition includes the presence of an anti-inflammatory agent to treat existing inflammation and reduce any pain.

T. Uses of Modified Bronchodilators and Anti-Asthma Agents Anti-asthma agents find use in the treatment of asthma and other lung diseases. Such anti-asthma agents include albuterol (Proventil or Ventoli).
n) and bronchodilators such as maleimidopropionamyl-1-theobromineacetamide.

U. Uses of Modified Diagnostic Agents The diagnostic agents used and the targeted vascular protein (s) are
Depending on whether it is desirable to diagnostically image anatomical fractions over a long period of time, preferentially image only specific cell types or fractions, or both . The applications of covalently linking diagnostic agents of interest to long-lived vascular proteins for long-term diagnostic imaging of the vascular space, and detecting abnormalities in blood flow throughout the mammalian vascular system Includes enhancing capacity, which includes detection of internal damage that causes abnormal bleeding, or the presence of thrombosis. For example, while they occur, it may be desirable to image the vascular space over an extended period of time to detect the effects of certain treatments (ie, detection of embolism disappearance, cessation of internal bleeding, etc.). obtain.

Diagnostic imaging of the vascular space over time also allows detection of various diseases associated with the vascular system (ie, arterial blockage in the heart). Therefore, diagnostic imaging of the vascular space over time can be used to non-invasively detect consistently diminished blood flow to the heart. Such a method also
It provides a means for quantitatively measuring cardiac efficiency and ventricular stroke volume over a long period of time (ie, such as during prolonged exercise).

Other applications for such methods include anatomy of the mammalian vasculature and their anatomy over time of administration of various drugs (eg, vasodilators, vasoconstrictors, etc.). It results from the ability to visualize effects on structures non-invasively. These may allow early detection of developing vascular abnormalities, injuries, etc.

Further applications resulting from the ability to diagnostically image the vascular space over a long period of time include functional assessment of the cardiovascular system, such as the conventional utilization of nuclear medicine for single measurements.

Applications for preferential binding of a diagnostic agent of interest to specific protein (s) present in the vasculature for diagnostic imaging of specific cell types or fractions only. There are also many. For example, the ability to preferentially direct a diagnostic agent of interest to a particular cell type in the vasculature allows for the non-invasive and early detection of lesions or various tumors associated with the mammalian vasculature. It may be possible by directing the functional anchor molecule to a tumor-specific cell surface protein.

In addition, diagnostic agents may be directed to cell surface proteins of particular cell types that are primarily associated with particular anatomical fractions and may preferentially diagnostically image such fractions as: : Lymph nodes, Peyer's patches, kidney glomeruli, liver, pancreas, tonsils, or any other organ in which mobile cells in the vasculature migrate.

Other applications for preferentially diagnostic imaging of specific cell types or fractions of the vasculature are stenotic or plaque, vascular short re-endothelialization due to tissue growth (
This includes the diagnosis and treatment of organ rejection due to reendothelialization) or short deficiency, or tissue migration.

The diagnostic agents of the invention can be delivered to local sites via local delivery devices. Delivery devices include catheters, needles, trocars and endoscopes.
Delivery of the drug to a local site allows for imaging of specific delivery areas. Agents that find particular use in localized delivery are non-specific diagnostic agents (eg, NHS-derivatives).

[0402]   The present invention can be more clearly illustrated by the following non-limiting examples.

Example 1 Preparation of Modified RGD Peptide AGYKPEGKRGDAK The RGD peptide AGYKPEGKRGDAK (SEQ ID NO: 1) was synthesized and modified to include a linking group and a maleimido group according to the synthetic scheme described below.

Solid phase peptide synthesis on a 100 μmol scale was performed using manual solid phase synthesis, Symph.
ony Peptide Synthesizer and Fmoc Protected Rama
It was carried out using ge resin. The following protected amino acids were sequentially added to the resin: Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-A.
sp (tBu) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf)
-OH, Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmo
c-Glu (tBu) -OH, Fmoc-Asp (tBu) -OH, Fmoc-
Pro-OH, Fmoc-Lys (Boc) -OH, Fmoc-Tyr (tBu
) -OH, Fmoc-Gly-OH, Fmoc-Ala-OH. These are N, N
-Dissolved in dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DI.
Activated using EA). Removal of the Fmoc protecting group was achieved using a solution of 20% (V / V) piperidine in N, N-dimethylformamide (DMF) for 20 minutes (step 1). In the final extension step, the synthesis was re-automated for the addition of 3-maleimidopropionic acid (step 2). Between each coupling, the resin was washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. This peptide, 85% TFA / 5%
Cleaved from the resin using TIS / 5% thioanisole and 5% phenol,
It was then precipitated with dry ice cooled Et ₂ O (step 3). The product is
arian (Rainin) preparative two-component HPLC system (Phenomen
ex Luna 10 μ Phenyl-hexyl, 21 mm × 25 cm column and UV detector at λ 214 and 254 nm (Varian Dynamax U
VD II) 30-55% B (H ₂ O) for 180 minutes at 9.5 mL / min.
0.045% TFA in (A) and 0.045% TFA in CH ₃ CN (B)
Purification by preparative reverse-phase HPLC using (gradient elution)) to give the desired molecule in> 95% purity as determined by RP-HPLC.

[0405]

[Chemical 51] (Example 2) (Preparation of modified RGD peptide KRGDACEGDSGGPFC) The RGD peptide KRGDACEGDSGGPFC (SEQ ID NO: 2) was synthesized and modified to include a linking group and a maleimide group according to the synthetic scheme described below.

Solid phase peptide synthesis on the 100 μmol scale was performed using manual solid phase synthesis, Symph.
ony Peptide Synthesizer and Fmoc Protected Rama
It was carried out using ge resin. The following protected amino acids were continuously added to the resin: Fmoc-Cys (Acm) -OH (C), Fmoc-Phe-OH, Fmo.
c-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmo
c-Ser (tBu) -OH, Fmoc-Asp (tBu) -OH, Fmoc-
Gly-OH, Fmoc-Glu (tBu) -OH, Fmoc-Cys (Acm
) -OH (C), Fmoc-Ala-OH, Fmoc-Asp (tBu) -OH
, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-L
ys (Boc) -OH. These were dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N '.
, N'-Tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA) were used for activation. Fmo
Removal of the c-protecting group was achieved by adding 20% (V / V) in N, N-dimethylformamide (DMF).
V) Achieved using a solution of piperidine for 20 minutes (step 1). C is a cyclized cysteine. Cyclization was achieved by treatment with Tl (TFA) ₃ (3 equiv on a 175 ummol scale) once the coupling was terminated at the last lysine residue (step 2). In the last extension step after cyclization, the synthesis was re-automated for the addition of 3-maleimidopropionic acid (step 3). During each coupling, the resin was loaded with N, N-dimethylformamide (DMF).
) And 3 times with isopropanol. The peptide was cleaved from the resin using 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, then precipitated with dry ice cold Et ₂ O (step 4). The product is a Varian (Rainin) preparative binary HP
LC system (Phenomenex Luna 10μ phenyl-hexyl,
21 mm x 25 cm column and UV detector at λ 214 and 254 nm (V
arian Dynamax UVD II) at 9.5 mL / min 18
30-55% B (0.045% TFA (A) in H ₂ O and CH ₃ CN in 0 minutes)
Purification by preparative reverse phase HPLC (gradient elution of 0.045% TFA (B)) in to give the desired protein in> 95% purity as determined by RP-HPLC.

[0407]

[Chemical 52] Example 3 (Preparation of Modified RGD Peptide KRGDACEGDSFFPFC) The RGD peptide KRGDACEGDSFFPFC (SEQ ID NO: 3) was synthesized and modified to include a linking group and a maleimide group according to the synthetic scheme described below.

Solid-phase peptide synthesis on the 100 μmol scale was performed using manual solid-phase synthesis, Symph.
ony Peptide Synthesizer and Fmoc Protected Rama
It was carried out using ge resin. The following protected amino acids were continuously added to the resin: Fmoc-Cys (Acm) -OH (C), Fmoc-Phe-OH, Fmo.
c-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmo
c-Ser (tBu) -OH, Fmoc-Asp (tBu) -OH, Fmoc-
Gly-OH, Fmoc-Glu (tBu) -OH, Fmoc-Cys (Acm
) -OH (C), Fmoc-Ala-OH, Fmoc-Asp (tBu) -OH
, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-L
ys (Boc) -OH, Fmoc-AEEA-OH, Fmoc-AEEA-OH
. These are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and It was activated using diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is done with N
Achieved using a solution of 20% (V / V) piperidine in N, N-dimethylformamide (DMF) for 20 minutes (step 1). C is a cyclized cysteine. The cyclization is initiated with Tl (T1 if the coupling is stopped once at the last lysine residue.
Achieved by cyclization by treatment with FA) ₃ (3 eq on 175 ummol scale) (step 2). In the last extension step after cyclization, the synthesis was re-automated for the addition of linking group and 3-maleimidopropionic acid (step 3).
Between each coupling, the resin was washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide was cleaved from the resin using 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, then precipitated with dry ice cold Et ₂ O (
Step 4). The product was loaded onto a Varian (Rainin) preparative two-component HPLC system (Phenomenex Luna 10μ phenyl-hexyl, 21 mm.
× 25 cm column and UV detector at λ 214 and 254 nm (Varia
n Dynamax UVD II) 30~55% of 9.5 mL / min at 180 minutes using the B (H ₂ O 0.045% in TFA (A) and CH ₃ 0 in CN.
Purification by preparative reverse phase HPLC (gradient elution of 045% TFA (B))) gave the desired peptide with> 95% purity as determined by RP-HPLC.

[0409]

[Chemical 53] (Example 4) (Modified GP-41A peptide YTSLIHSLIEESQNQQEKNEQE
Preparation of LLELDKWASLWNWF) GP-41A peptide YTSLIHSLIEESQNQQEKNEQELLE
LDKWASLWNWF (SEQ ID NO: 4) was synthesized and modified to include a linking group and a maleimide group according to the synthetic scheme described below.

Solid phase peptide synthesis on a 100 μmol scale was performed using manual solid phase synthesis, Symph.
ony Peptide Synthesizer and Fmoc Protected Rama
It was carried out using ge resin. The following protected amino acids were sequentially added to the resin: Fmoc-Phe-OH, Fmoc-Trp (Boc) -OH, Fmoc-A.
sn (Trt) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Leu
-OH, Fmoc-Ser (tBu) -OH, Fmoc-Ala-OH, Fmo
c-Trp (Boc) -OH, Fmoc-Lys (Boc) -OH, Fmoc-
Asp (tBu) -OH, Fmoc-Leu-OH, Fmoc-Glu (tBu
) -OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Gl
u (tBu) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Glu (
tBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Bo
c) -OH, Fmoc-Glu (tBu) -OH, Fmoc-Gln (Trt)
-OH, Fmoc-Gln (Trt) -OH, Fmoc-Asn (Trt) -O
H, Fmoc-Gln (Trt) -OH, Fmoc-Ser (tBu) -OH,
Fmoc-Glu (tBu) -OH, Fmoc-Glu (tBu) -OH, Fm
oc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ser (tBu)-
OH, Fmoc-His (Boc) -OH, Fmoc-Ile-OH, Fmoc
-Leu-OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tB
u) -OH, Fmoc-Tyr (tBu) -OH. These are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and It was activated using diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was accomplished by N, N-dimethylformamide (
A solution of 20% (V / V) piperidine in DMF) was used for 20 min (
Step 1). In the final extension step, the synthesis was automated for the addition of 3-maleimidopropionic acid (step 2). Between each coupling, the resin was washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide was cleaved from the resin using 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, then precipitated with dry ice cold Et ₂ O (step 3). The product was converted into Varian (Ra
inin) Preparative two-component HPLC system (Phenomenex Luna)
10 μ Phenyl-hexyl, 21 mm × 25 cm column and λ 214 and 2
30-55% B (0.045% in H ₂ O in 180 min at 9.5 mL / min using a UV detector (Varian Dynamax UVD II) at 54 nm.
TFA (A) and (gradient elution of 0.045% TFA (B)) in CH ₃ CN) by preparative reverse phase HPLC with purity> 95% as determined by RP-HPLC. The desired molecule was obtained.

[0411]

[Chemical 54] (Example 5) (Modified GP-41B peptide YTSLIHSSLIEESQNQQEKNEQE
Preparation of LLELDKWASLWNWF) GP-41B peptide YTSLIHSLIEESQNQQEKNEQELLE
LDKWASLWNWF (SEQ ID NO: 5) was synthesized and modified to include a linking group and a maleimido group according to the synthetic scheme described below.

Solid phase peptide synthesis on a 100 μmole scale was performed using manual solid phase synthesis, Symph.
ony Peptide Synthesizer and Fmoc Protected Rama
It was carried out using ge resin. The following protected amino acids were sequentially added to the resin: Fmoc-Phe-OH, Fmoc-Trp (Boc) -OH, Fmoc-A.
sn (Trt) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Leu
-OH, Fmoc-Ser (tBu) -OH, Fmoc-Ala-OH, Fmo
c-Trp (Boc) -OH, Fmoc-Lys (Aloc) -OH, Fmoc
-Asp (tBu) -OH, Fmoc-Leu-OH, Fmoc-Glu (tB
u) -OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-G
lu (tBu) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Glu
(TBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (B
oc) -OH, Fmoc-Glu (tBu) -OH, Fmoc-Gln (Trt
) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Asn (Trt)-
OH, Fmoc-Gln (Trt) -OH, Fmoc-Ser (tBu) -OH
, Fmoc-Glu (tBu) -OH, Fmoc-Glu (tBu) -OH, F
moc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ser (tBu)
-OH, Fmoc-His (Boc) -OH, Fmoc-Ile-OH, Fmo
c-Leu-OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (t
Bu) -OH, Fmoc-Tyr (tBu) -OH.

These are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyl-uronium hexafluorophosphate ( HBTU) and diisopropylethylamine (DIEA) were used for activation. Removal of the Fmoc protecting group is done with N
Achieved using a solution of 20% (V / V) piperidine in N, N-dimethylformamide (DMF) for 20 minutes (step 1). Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of C ₆ H ₆ CHCl ₃ (1: 1): 2.5% N
MM (v: v): 3 equivalents of Pd (PPh ₃ dissolved in 5% AcOH (v: v).
Achieved by treating the resin with the solution of ₄ for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% AcOH in DCM (6 × 5 mL).
mL), DCM (6 x 5 mL) and DMF (6 x 5 mL).

In the final extension step, the synthesis was automated for the addition of 3-maleimidopropionic acid (step 3). Between each coupling, the resin was washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide was cleaved from the resin using 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, then dry ice cooled E.
Precipitated with t ₂ O (step 4). The product was transformed into Varian (Rainin
) Preparative binary HPLC system (Phenomenex Luna 10μ phenyl-hexyl, 21 mm x 25 cm column and λ 214 and 254 nm).
Using a UV detector (Varian Dynamax UVD II) at 9
． 5 mL / min for 180 minutes _{30~55% B (H 2 O 0.045} % in TFA (
Purification by preparative reverse-phase HPLC using a A) and CH ₃ CN 0.045% in TFA (B) gradient elution)), as determined by RP-HPLC,
The desired molecule with purity> 95% was obtained.

[0415]

[Chemical 55] (Example 6) (Modified GP-41C peptide YTSLIHSLIEESQNQQEKNEQE
Preparation of LLELDKWASLWNWF) GP-41C peptide YTSLIHSLIEESQNQQEKNEQELLE
LDKWASLWNWF (SEQ ID NO: 6) was synthesized and modified to include a linking group and a maleimido group according to the synthetic scheme described below.

Solid phase peptide synthesis on a 100 μmol scale was performed using manual solid phase synthesis, Symph.
ony Peptide Synthesizer and Fmoc Protected Rama
It was carried out using ge resin. The following protected amino acids were sequentially added to the resin: Fmoc-Phe-OH, Fmoc-Trp (Boc) -OH, Fmoc-A.
sn (Trt) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Leu
-OH, Fmoc-Ser (tBu) -OH, Fmoc-Ala-OH, Fmo
c-Trp (Boc) -OH, Fmoc-Lys (Aloc) -OH, Fmoc
-Asp (tBu) -OH, Fmoc-Leu-OH, Fmoc-Glu (tB
u) -OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-G
lu (tBu) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Glu
(TBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (B
oc) -OH, Fmoc-Glu (tBu) -OH, Fmoc-Gln (Trt
) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Asn (Trt)-
OH, Fmoc-Gln (Trt) -OH, Fmoc-Ser (tBu) -OH
, Fmoc-Glu (tBu) -OH, Fmoc-Glu (tBu) -OH, F
moc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ser (tBu)
-OH, Fmoc-His (Boc) -OH, Fmoc-Ile-OH, Fmo
c-Leu-OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (t
Bu) -OH, Fmoc-Tyr (tBu) -OH. These are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and It was activated using diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was achieved using a solution of 20% (V / V) piperidine in N, N-dimethylformamide (DMF) for 20 minutes (step 1).

Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL C ₆
H ₆ CHCl ₃ (1: 1): 2.5% NMM (v: v): 5% AcOH (v: v
This is accomplished by treating the resin with a solution of 3 equivalents of Pd (PPh ₃ ) ₄ dissolved in 2) for 2 hours (step 2). The resin was then treated with CHCl ₃ (6 × 5 mL)
, 20% AcOH in DCM (6 x 5 mL), DCM (6 x 5 mL) and DM
Wash with F (6 x 5 mL).

In the final extension step, the synthesis was automated for the addition of Fmoc-AEEA-OH and finally 3-maleimidopropionic acid (step 2). Between each coupling, the resin was washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. This peptide is
Cleaved from the resin using% TFA / 5% TIS / 5% thioanisole and 5% phenol, then precipitated with dry ice cold Et ₂ O (step 3).
. The product was analyzed by a Varian (Rainin) preparative two-component HPLC system (P
henomenex Luna 10μ Phenyl-hexyl, 21mm x 25c
m column and UV detector at λ 214 and 254 nm (Varian Dy
Namax UVD II) at 9.5 mL / min for 180 minutes 30-5
5% B (0.045% TFA (A) in H ₂ O and 0.045% in CH ₃ CN
TFA (B)) gradient elution) and purified by preparative reverse phase HPLC, RP
-Obtained the desired molecule with> 95% purity as determined by HPLC.

[0419]

[Chemical 56] (Example 7) (Modified RSV peptide VYPSDEYDASISQVNEEINQALAYI
Preparation of RKADELLENV) RSV peptide VYPSDEYDASISQVNEEINQALAYIRKA
DELLENV (SEQ ID NO: 7) was synthesized according to the synthetic scheme described below and was modified to include a linking group and a maleimido group.

100 μmol scale solid phase peptide synthesis, manual solid phase synthesis, Sympho
ny Peptide Synthesizer and Fmoc Protected Rink
Performed using Amide MBHA. The following protected amino acids were sequentially added to the resin: Fmoc-Lys (Aloc) -OH, Fmoc-Val-OH, F.
moc-Asn (Trt) -OH, Fmoc-Glu (tBu) -OH, Fmo
c-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu (tBu) -O
H, Fmoc-Asp (tBu) -OH, Fmoc-Ala-OH, Fmoc-
Lys (Boc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Il
e-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ala-OH, Fm
oc-Leu-OH, Fmoc-Ala-OH, Fmoc-Gln (Trt)-
OH, Fmoc-Asn (Trt) -OH, Fmoc-Ile-OH, Fmoc
-Glu (tBu) -OH, Fmoc-Glu (tBu) -OH, Fmoc-A
sn (Trt) -OH, Fmoc-Val-OH, Fmoc-Gln (Trt)
-OH, Fmoc-Ser (tBu) -OH, Fmoc-Ile-OH, Fmo
c-Ser (tBu) -OH, Fmoc-Ala-OH, Fmoc-Asp (t
Bu) -OH, Fmoc-Try (tBu) -OH, Fmoc-Glu (tBu
) -OH, Fmoc-Asp (tBu) -OH, Fmoc-Ser (tBu)-
OH, Fmoc-Pro-OH, Fmoc-Tyr (tBu) -OH, Fmoc
-Val-OH. These were dissolved in N, N-dimethylformamide (DMF) according to the sequence, and O-benzotriazol-1-yl-N, N, N ', N'-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine ( Activated using DIEA). Removal of the Fmoc protecting group
Achieved in 20 minutes using a solution of 20% piperidine (V / V) in N, N-dimethylformamide (DMF) (step 1).

The amino group of the final amino acid was replaced with O-benzotriazol-1-yl-N, N, N
', N'-Tetramethyl-uronium hexafluorophosphate (HBTU)
And acetylated using acetic acid activated with diisopropylethylamine (DIEA). Selective deprotection of the Lys (Aloc) group was performed with 5 mL of CH
3 equivalents of Pd (PP dissolved in Cl ₃ : NMM: HOAc (18: 1: 0.5)
This is done manually by treating the resin with the solution of h ₃ ) ₄ for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOA in DCM.
Wash with c (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The synthesis is then re-automated for the addition of 3-maleimidopropionic acid (step 3).

Between each coupling, the resin is washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. 85% TFA / 5% TIS /
The peptide was cleaved from the resin using 5% thioanisole and 5% phenol, then precipitated with dry ice cold Et ₂ O (step 4). This product was subjected to preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system (Phenomenex Luna 10μ Phenyl-hexyl at 9.5 mL / min at 180 mL for 30-55 min.
% B (0.045% TFA (A) in H ₂ O and 0.045% TF in CH ₃ CN)
Gradient elution of A (B), 21 mm x 25 cm column, and λ 214 and 25
UV detector at 4 nm (Varian Dynamax UVD II))
The desired molecule was obtained in> 95% purity (determined by RP-HPLC).

[0423]

[Chemical 57] (Example 8) (Modified RSV peptide VYPSDEYDASISQVNEEINQALAYI
Preparation of RKADELLENV) RSV peptide VYPSDEYDASISQVNEEINQALAYIRKA
DELLENV (SEQ ID NO: 8) was synthesized and modified to include a linking group and a maleimide group to produce the modified peptide shown below.

100 μmol scale solid phase peptide synthesis, manual solid phase synthesis, Sympho
ny Peptide Synthesizer and Fmoc Protected Rink
Performed using Amide MBHA. The following protected amino acids were sequentially added to the resin: Fmoc-Val-OH, Fmoc-Asn (Trt) -OH, Fm.
oc-Glu (tBu) -OH, Fmoc-Leu-OH, Fmoc-Leu-
OH, Fmoc-Glu (tBu) -OH, Fmoc-Asp (tBu) -OH
, Fmoc-Ala-OH, Fmoc-Lys (Boc) -OH, Fmoc-A
rg (Pbf) -OH, Fmoc-Ile-OH, Fmoc-Tyr (tBu)
-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Ala
-OH, Fmoc-Gln (Trt) -OH, Fmoc-Asn (Trt) -O
H, Fmoc-Ile-OH, Fmoc-Glu (tBu) -OH, Fmoc-
Glu (tBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Va
1-OH, Fmoc-Gln (Trt) -OH, Fmoc-Ser (tBu)-
OH, Fmoc-Ile-OH, Fmoc-Ser (tBu) -OH, Fmoc
-Ala-OH, Fmoc-Asp (tBu) -OH, Fmoc-Try (tB
u) -OH, Fmoc-Glu (tBu) -OH, Fmoc-Asp (tBu)
-OH, Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmo
c-Tyr (tBu) -OH, Fmoc-Val-OH, Fmoc-Lys (A
loc) -OH. These were dissolved in N, N-dimethylformamide (DMF) according to the sequence, and O-benzotriazol-1-yl-N, N, N ', N'-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine ( Activated using DIEA). Removal of the Fmoc protecting group
Achieved in 20 minutes using a solution of 20% piperidine (V / V) in N, N-dimethylformamide (DMF) (step 1). The amino group of the final amino acid was replaced with O-benzotriazol-1-yl-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (
Acetylated using acetic acid activated using DIEA). Lys (Al
oc) group selectively deprotected with 5 mL of CHCl ₃ : NMM: HOAc (18: 1).
: 0.5) by manual treatment by treating the resin with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ for 2 hours. The resin was then treated with CHCl ₃ (6 × 5
mL), 20% HOAc in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The synthesis is then re-automated for the addition of 3-maleimidopropionic acid (step 3). Between each coupling, the resin is washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. 85% TFA / 5% TIS / 5% thioanisole and 5%
The peptide was cleaved from the resin using phenol and then precipitated with dry ice cold Et ₂ O (step 4). This product was converted into Varian (Ra
in) Preparative Reversed-Phase HPLC Using a Preparative Binary HPLC System
(Using Phenomenex Luna 10μ Phenyl-hexyl.
30 mL of 55% B (0.045% in H ₂ O over 180 minutes at 5 mL / min.
Gradient elution of TFA (A) and 0.045% TFA (B) in CH ₃ CN, 21
Purification by mm × 25 cm column and UV detector at λ 214 and 254 nm (Varian Dynamax UVD II) gave the desired molecule in> 95% purity (determined by RP-HPLC).

[0425]

[Chemical 58] (Example 9) (Modified RSV peptide VYPSDEYDASISQVNEEINQALAYI
Preparation of RKADELLENV) RSV peptide VYPSDEYDASISQVNEEINQALAYIRKA
DELLENV (SEQ ID NO: 9) was synthesized according to the synthetic scheme described below and was modified to include a linking group and a maleimido group.

100 μmol scale solid phase peptide synthesis, manual solid phase synthesis, Sympho
ny Peptide Synthesizer and Fmoc Protected Rink
Performed using Amide MBHA. The following protected amino acids were sequentially added to the resin: Fmoc-Val-OH, Fmoc-Asn (Trt) -OH, Fm.
oc-Glu (tBu) -OH, Fmoc-Leu-OH, Fmoc-Leu-
OH, Fmoc-Glu (tBu) -OH, Fmoc-Asp (tBu) -OH
, Fmoc-Ala-OH, Fmoc-Lys (Aloc) -OH, Fmoc-
Arg (Pbf) -OH, Fmoc-Ile-OH, Fmoc-Tyr (tBu
) -OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Al
a-OH, Fmoc-Gln (Trt) -OH, Fmoc-Asn (Trt)-
OH, Fmoc-Ile-OH, Fmoc-Glu (tBu) -OH, Fmoc
-Glu (tBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-V
al-OH, Fmoc-Gln (Trt) -OH, Fmoc-Ser (tBu)
-OH, Fmoc-Ile-OH, Fmoc-Ser (tBu) -OH, Fmo
c-Ala-OH, Fmoc-Asp (tBu) -OH, Fmoc-Try (t
Bu) -OH, Fmoc-Glu (tBu) -OH, Fmoc-Asp (tBu
) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fm
oc-Tyr (tBu) -OH, Fmoc-Val-OH. These were dissolved in N, N-dimethylformamide (DMF) according to the sequence, and O-benzotriazol-1-yl-N, N, N ', N'-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine ( Activated using DIEA). Removal of the Fmoc protecting group was accomplished by removing N, N-dimethylformamide (DM
Achieved in 20 minutes using a solution of 20% piperidine (V / V) in F) (Step 1). The amino group of the final amino acid was replaced with O-benzotriazole-1-N, N, N ′.
, N'-Tetramethyl-uronium hexafluorophosphate (HBTU) and acetic acid activated with diisopropylethylamine (DIEA) is used for acetylation. Selective deprotection of the Lys (Aloc) group was performed with 5 mL of CHC.
Done by hand by treating the resin with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ in L ₃ : NMM: HOAc (18: 1: 0.5) for 2 hours. The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6 × 5 m).
L), DCM (6 x 5 mL), and DMF (6 x 5 mL). The synthesis is then re-automated for the addition of 3-maleimidopropionic acid (step 3). Between each coupling, the resin is washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. 85% TFA / 5% TI
The peptide was cleaved from this resin using S / 5% thioanisole and 5% phenol, then precipitated with dry ice cold Et ₂ O (step 4).
This product was purified by preparative reverse phase HPLC (Phenomenex Luna 10μ using a Varian (Rainin) preparative binary HPLC system.
Using phenyl-hexyl at 9.5 mL / min for 180 min, 30-
55% B (0.045% TFA (A) in H ₂ O and 0.045% in CH ₃ CN
Gradient elution of TFA (B), 21 mm x 25 cm column, and UV detector at λ 214 and 254 nm (Varian Dynamax UVD I).
I)) to give the desired molecule in> 95% purity (RP-HP
Determined by LC).

[0427]

[Chemical 59] (Example 10) (Modified GLP-1 peptide HAEGTFTSDVSSYLEGQAAKEFI
Preparation of AWLVKGRK) GLP-1 peptide HAEGFTSDVSSYLEGQAAKEFIAWL
VKGRK (SEQ ID NO: 10) was synthesized according to the synthetic scheme described below and was modified to include a linking group and a maleimide group.

100 μmol scale solid phase peptide synthesis, manual solid phase synthesis, Sympho
NY Peptide Synthesizer and Fmoc protection Ramag
e Resin was used. The following protected amino acids were sequentially added to the resin: Fmoc-Lys (Aloc) -OH, Fmoc-Arg (Pbf) -OH.
, Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH, Fmoc-V
al-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, F
moc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, F
moc-Glu (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmo
c-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -O
H, Fmoc-Gly-OH, Fmoc-Glu (tBu) -OH, Fmoc-
Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu
) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fm
oc-Asp (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc
-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tB
u) -OH, Fmoc-Gly-OH, Fmoc-Glu (tBu) -OH, F
moc-Ala-OH, Fmoc-His (Boc) -OH. These are N, N-
Dissolved according to sequence in dimethylformamide (DMF) and using O-benzotriazol-1-yl-N, N, N ', N'-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Activated. Removal of the Fmoc protecting group is accomplished by removing N, N-dimethylformamide (D
Achieved in 20 minutes using a solution of 20% piperidine (V / V) in MF) (step 1). Selective deprotection of the Lys (Aloc) group was performed with 5 mL of C ₆ H ₆ CHCl ₃
Manual by treating the resin with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ in (1: 1): 2.5% NMM (v: v): 5% AcOH (v: v) for 2 hours. And achieve (step 2). The resin was then treated with CHCl ₃ (6 × 5 mL),
20% AcOH in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF
Wash with (6 x 5 mL). The synthesis is then re-automated for the addition of 3-maleimidopropionic acid (step 3). During each coupling, the resin is N, N-
Wash 3 times with dimethylformamide (DMF) and 3 times with isopropanol. Peptides were cleaved from this resin using 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, then dry ice cooled Et _2.
Precipitated with O (step 4). This product was converted into Varian (Rainin
Preparative Reversed-Phase HPLC (Phe) using the Preparative Binary HPLC System of
9.5 mL / using nomenex Luna 10μ phenyl-hexyl
Min over 180 minutes, 30 to 55% of B (H ₂ O in 0.045% TFA (
With a gradient solvent of A) and CH ₃ CN in 0.045% TFA (B)), 21mm × 2
5 cm column, and UV detector at λ 214 and 254 nm (Var
yan Dynamax UVD II)) to give the desired molecule 9
Obtained with a purity higher than 5% (determined by RP-HPLC).

[0429]

[Chemical 60] (Example 11) (Modified GLP-1 peptide HAEGTFTSDVSSYLEGQAAKEFI
Preparation of AWLVKGRK) GLP-1 peptide HAEGFTSDVSSYLEGQAAKEFIAWL
VKGRK (SEQ ID NO: 11) was synthesized as described below and modified to include a linking group and a maleimide group.

100 μmol scale solid phase peptide synthesis, manual solid phase synthesis, Sympho
NY Peptide Synthesizer and Fmoc protection Ramag
e Resin was used. The following protected amino acids were sequentially added to the resin: Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-.
Lys (Boc) -OH, Fmoc-Val-OH, Fmoc-Leu-OH,
Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Il
e-OH, Fmoc-Phe-OH, Fmoc-Glu (tBu) -OH, Fm
oc-Lys (Aloc) -OH, Fmoc-Ala-OH, Fmoc-Ala
-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmo
c-Glu (tBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (t
Bu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Ser (tBu
) -OH, Fmoc-Val-OH, Fmoc-Asp (tBu) -OH, Fm
oc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc
-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH
, Fmoc-Glu (tBu) -OH, Fmoc-Ala-OH, Fmoc-H
is (Boc) -OH. These were dissolved in N, N-dimethylformamide (DMF) according to the sequence, and O-benzotriazol-1-yl-N, N, N ', N'-
It was activated using tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was accomplished by 20% piperidine (V / V) in N, N-dimethylformamide (DMF).
Was achieved in 20 minutes using the solution in (Step 1). Selective deprotection of the Lys (Aloc) group was performed by adding 5 mL of C ₆ H ₆ CHCl ₃ (1: 1): 2.5% NMM (v:
v): accomplished manually by treating the resin with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ in 5% AcOH (v: v) for 2 hours (step 2). Then
This resin was treated with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6 × 5 mL).
), DCM (6 × 5 mL), and DMF (6 × 5 mL). The linking group, Fmoc-AEEA-OH, was added and then Fmoc was removed in the usual manner. This procedure was repeated again to add the second AEEA linking group.

The synthesis was then re-automated for the addition of 3-maleimidopropionic acid (step 3). During each coupling, the resin was loaded with N, N-dimethylformamide (D
Wash 3 times with MF) and 3 times with isopropanol. 85% TFA /
The peptide was cleaved from this resin using 5% TIS / 5% thioanisole and 5% phenol, then precipitated by dry ice cold Et ₂ O (step 4).

This product was subjected to preparative reverse phase HPLC (Phenomenex Luna 10) using a Varian (Rainin) preparative binary HPLC system.
30 using μphenyl-hexyl at 9.5 mL / min for 180 min
55% of B (H ₂ O 0.045% TFA (A) and CH ₃ CN in 0.045
% TFA (B)) gradient elution, 21 mm × 25 cm column, and UV detector at λ 214 and 254 nm (Varian Dynamax UVD).
II)) to give the desired molecule in> 95% purity (RP-H
Determined by PLC).

Example 12 Preparation of Modified K5 Peptide PRKLYDYK The K5 peptide PRKLYDYK (SEQ ID NO: 12) was synthesized according to the synthetic scheme described below and modified to include a linking group and a maleimido group. did.

The following protected amino acids were serially linked to Link A using automated peptide synthesis.
Added to mide MBHA resin (0.48 mmol / mg) (250 μmol scale): Fmoc-Lys (Aloc) -OH, Fmoc-Tyr (tB.
u) -OH, Fmoc-Asp (tBu) -OH, Fmoc-Tyr (tBu)
-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmo
c-Arg (Pbf) -OH, Fmoc-Pro-OH. 2 for each coupling
Achieved using equivalents of amino acid, 1 equivalent of HBTU, and 2 equivalents of DIEA, and performed twice in 30 minutes. The Fmoc group of the N-terminal amino acid (Pro) is changed to 2
Removed using 0% piperidine / DMF (3 x 10 min).

This resin was added to 6 × 4 mL DMF, 3 × 3 mL EtOH, and 6 × 4 mL.
It was washed successively with DMF. N-terminal acetylation was performed by adding 4 mL of 1 in DMF.
It was achieved manually with Symphony by adding 5 eq of HOAc, 2 eq of DIEA and 4 eq of HBTU. 2x acetic acid capping
I went for 30 minutes. This resin was added to 3 × 4 mL CH ₂ Cl ₂ , 6 × 4 mL 0.5%
It was washed successively with DIEA / CH ₂ Cl ₂ , 3 × 4 mL EtOH and 6 × 4 mL DMF. Selective deprotection of the Lys (Aloc) group was performed with 2.5% NMM (v
/ V) and CHCl ₃ : benzene (1: 1) with 5% HOAc (5 mL)
By treating the resin with a solution of 3 equivalents of Pd (PPh ₃ ) ₄ dissolved in
Performed manually for 2 hours in ymphony. The resin is then treated with CHCl ₃
(6 × 5 mL), 0.5% DIEA in CH ₂ Cl ₂ (6 × 5 mL), 0.
02M Sodium diethylthiocarbaminsanate (6 x 5 mL), EtOH (3
X 4 mL) and DMF (6 x 5 mL).

[0436]   Coupling of 3-maleimidopropionic acid (MPA) is based on Symphony
By restarting the automation of 2 equivalents of MPA, 2 equivalents of DIE
Includes delivery of A and 1 equivalent of HBTU to the reaction vessel. 30 this coupling
I went twice in a minute. 6 × 4 mL DMF, 3 × 3 mL EtOH, and 6 × 4 m
Washing was done using L DMF. Use 10 mL of the following cleavage mixture to
Cleavage from the resin was accomplished by mobilization: 85% TFA / 5% Tri
Isopropylsilane / 5% thioanisole / 5% phenol. 2 peptides
After cutting from the resin in time, the resin is treated with TFA and CH.₂Cl₂Washed with. Combined
Rotate the cut and washed aliquots soaked under gentle heating (30 ° C).
Concentrate to 1-2 mL using an evaporator, and remove the peptide with Et. ₂ Precipitated with O 2. The precipitate was filtered using a SPPS manifold.
Collected, and 10 mL ethyl acetate and 30 mL Et.₂Washed with O
. The precipitate was purified by chromatography in water (0.04% TFA) for purification.
% CH₃It was continuously dissolved in water (10 mL) containing CN (0.04% TFA).

[0437]

[Chemical formula 61] (Example 13) (Preparation of modified K5 peptide RNPDGDVGGPWAWTTAPRKLYDY) K5 peptide RNPDGDVGVGWAWTTAPRKLYDY (SEQ ID NO: 1)
3) was synthesized according to the synthetic scheme described below and was modified to include a linking group and a maleimide group.

The following protected amino acids were linked to the Link Amide using automated peptide synthesis.
Added continuously to MBHA resin (0.48 mmol / mg) (100 μmol scale): Fmoc-Tyr (tBu) OH, Fmoc-Asp (tBu).
-OH, Fmoc-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc
-Lys (Boc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-P
ro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Thr (tBu)
-OH, Fmoc-Thr (tBu) -OH, Fmoc-Tyr (tBu) -O
H, Fmoc-Ala-OH, Fmoc-Trp-OH, Fmoc-Pro-OH,
Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val-OH,
Fmoc-Asp (tBu) -OH, Fmoc-Gly-OH, Fmoc-As
p (tBu) -OH, Fmoc-Pro-OH, Fmoc-Asn (Trt)-
OH, Fmoc-Arg (Pbf) -OH, MPA.

Each coupling has 5 equivalents of amino acid, 1 equivalent of HBTU, and 2 equivalents of D.
Achieved using IEA and done twice in 30 minutes. Cleavage from the resin was done by automation using 10 mL of the following cleavage mixture: 85%
TFA / 5% triisopropylsilane / 5% thioanisole / 5% phenol. After cleaving the peptide from the resin in 2 hours, the resin was treated with TFA and CH ₂
Wash with Cl ₂ .

The combined cut and washed aliquots were concentrated to 1-2 mL using a rotary evaporator with gentle heating (30 ° C.), and the peptide was precipitated with Et ₂ O. The precipitate was collected by filtration using SPPS manifold, and washed with Et ₂ O to ethyl acetate and 30mL of 10 mL. The precipitate was washed with water (0.04% TFA) for chromatographic purification.
It was continuously dissolved in water (10 mL) with 5% CH ₃ CN (0.04% TFA) in it. Purification All the peptides are, Phenomenex Luna 10 [mu] phenyl - hexyl, water / TFA mixture (H ₂ O in 0.045% TFA; solvent A) was performed using a column of 21 mm × 250 mm equilibrated with.

10-30% acetonitrile gradient (0.03 in CH ₃ CN over 60 minutes
Elution was performed at 18 mL / min by eluting with 45% TFA; solvent B). Peptides were detected by UV absorbance (Varian Dynamax UVD II) at λ 214 and 254 nm. Fractions were collected in 9 mL aliquots. Fractions containing the desired product were identified by mass after direct injection on LC / MS. Selected fractions were subsequently subjected to HPLC (10-40 for 20 minutes).
% Solvent B; Phenomenex Luna 5 [mu] Phenyl-hexyl, 1
Fractions with> 95% purity for the pool were identified by analysis by 0 mm x 250 mm column, 0.5 mL / min). The pool was lyophilized using dry ice and acetone, followed by lyophilization for at least 2 days to give a white powder.

[0442]

[Chemical formula 62] Example 14 Preparation of Modified BBB Peptide YGRKKRRRQRRRL BBB peptide YGRKKRRRQRRRL (SEQ ID NO: 14) was synthesized according to the synthetic scheme described below and modified to include a linking group and a maleimido group.

100 μmol scale solid phase peptide synthesis, manual solid phase synthesis, Sympho
NY Peptide Synthesizer and Fmoc protection Ramag
e Resin was used. The following protected amino acids were sequentially added to the resin: Fmoc-Lys (Aloc) -OH, Fmoc-Arg (Pbf) -OH.
, Fmoc-Arg (Pbf) -OH, Fmoc-Arg (Pbf) -OH, F
moc-Gln (Trt) -OH, Fmoc-Arg (Pbf) -OH, Fmo
c-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, Fmoc-
Lys (Boc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gl
y-OH, Fmoc-Tyr (tBu) -OH. These were dissolved in N, N-dimethylformamide (DMF) according to the sequence, and O-benzotriazol-1-yl-N, N, N ', N'-tetramethyluronium hexafluorophosphate (
HBTU) and diisopropylethylamine (DIEA) were used for activation. Removal of the Fmoc protecting group was performed in N, N-dimethylformamide (DMF) 20.
Achieved in 20 minutes using a solution of% piperidine (V / V) (step 1). After deprotection of tyrosine, biotin was immobilized at the N-terminus via regular activation and coupling conditions. Selective deprotection of the Lys (Aloc) group was performed by adding 5 mL of C ₆ H ₆ CHCl ₃ (1: 1): 2.5% NMM (v: v): 5% AcOH (v:
This is accomplished manually by treating the resin with a solution of 3 equivalents of Pd (PPh ₃ ) ₄ in v) for 2 hours (step 2). The resin was then treated with CHCl ₃ (6
X5 mL), 20% AcOH in DCM (6 x 5 mL), DCM (6 x 5 mL),
And wash with DMF (6 x 5 mL). The synthesis is then re-automated for the addition of 3-maleimidopropionic acid (step 3). Between each coupling, the resin is washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide was cleaved from this resin using 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, then precipitated by dry ice cold Et ₂ O (step 4). This product was converted into Varian (
Rainin) preparative reverse phase HP using a preparative binary HPLC system
LC (Phenomenex Luna 10 μ Phenyl-hexyl at 9.5 mL / min for 30 min over 30 min at 55% B (0.04 in H ₂ O) over 180 min.
5% TFA (A) and 0.045% TFA (B) in CH ₃ CN),
Purification by 21 mm × 25 cm column and UV detector at λ 214 and 254 nm (Varian Dynamax UVD II) gave the desired molecule in> 95% purity (determined by RP-HPLC).

[0444]

[Chemical formula 63] Example 15 Preparation of Modified BBB Peptide YGRKKRRRQRRRL BBB peptide YGRKKRRRQRRRL (SEQ ID NO: 15) was synthesized according to the synthetic scheme described below and modified to include a linking group and a maleimido group.

100 μmol scale solid phase peptide synthesis, manual solid phase synthesis, Sympho
NY Peptide Synthesizer and Fmoc protection Ramag
e Resin was used. The following protected amino acids were sequentially added to the resin: Fmoc-Lys (Aloc) -OH, Fmoc-Arg (Pbf) -OH.
, Fmoc-Arg (Pbf) -OH, Fmoc-Arg (Pbf) -OH, F
moc-Gln (Trt) -OH, Fmoc-Arg (Pbf) -OH, Fmo
c-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, Fmoc-
Lys (Boc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gl
y-OH, Fmoc-Tyr (tBu) -OH. These were dissolved in N, N-dimethylformamide (DMF) according to the sequence, and O-benzotriazol-1-yl-N, N, N ', N'-tetramethyluronium hexafluorophosphate (
HBTU) and diisopropylethylamine (DIEA) were used for activation. Removal of the Fmoc protecting group was performed in N, N-dimethylformamide (DMF) 20.
Achieved in 20 minutes using a solution of% piperidine (V / V) (step 1). Ly
Selective deprotection of the s (Aloc) group was performed with 5 mL of C ₆ H ₆ CHCl ₃ (1: 1):
2.5% NMM (v: v): 3 equivalents of P dissolved in 5% AcOH (v: v)
Done by hand, by treating the resin with a solution of d (PPh ₃ ) ₄ for 2 hours (step 2). This resin was then treated with CHCl ₃ (6 × 5 mL), 20 in DCM.
% HOAc (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL)
) Wash with. After deprotection of aloc, biotin was immobilized on the ε-N-terminus of deprotected lysine by regular activation and coupling conditions. Removal of N-terminal Fmoc was then achieved using standard conditions. The synthesis was then re-automated for the addition of 3-maleimidopropionic acid at the end (step 3).
. The resin was washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol between each coupling. 85% TFA / 5% TIS /
The peptide was cleaved from the resin using 5% thioanisole and 5% phenol, then precipitated with dry ice cold Et ₂ O (step 4). This product was subjected to preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system (Phenomenex Luna 10μ Phenyl-hexyl at 9.5 mL / min at 180 mL for 30-55 min.
% B (0.045% TFA (A) in H ₂ O and 0.045% TF in CH ₃ CN)
A (B)) gradient melt, 21 mm x 25 cm column, and λ 214 and 2
UV detector at 54 nm (Varian Dynamax UVD II)
) To give the desired molecule in> 95% purity (RP-HPLC).
Determined by).

Example 16 Preparation of Modified Dinorphin Peptide YGGFLRRRIRPKLK The diinorphin peptide YGGFLRRRIRPKLK (SEQ ID NO: 16) was synthesized according to the synthetic scheme described below and was prepared to include a linking group and a maleimido group. Qualified.

100 μmol scale solid phase peptide synthesis, manual solid phase synthesis, Sympho
NY Peptide Synthesizer and Fmoc protection Ramag
e Resin was used. The following protected amino acids were sequentially added to the resin: Fmoc-Lys (Aloc) -OH, Fmoc-Leu-OH, Fmoc.
-Lys (Boc) -OH, Fmoc-Pro-OH, Fmoc-Arg (Pb
f) -OH, Fmoc-Ile-OH, Fmoc-Arg (Pbf) -OH, F
moc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-Phe
-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Boc-Tyr (
tBu) -OH. These were dissolved in N, N-dimethylformamide (DMF) according to the sequence, and O-benzotriazol-1-yl-N, N, N ', N'-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine ( Activated using DIEA). Removal of the Fmoc protecting group
Achieved in 20 minutes using a solution of 20% piperidine (V / V) in N, N-dimethylformamide (DMF) (step 1). The amino group of the final amino acid was replaced with O-benzotriazol-1-yl-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (
Acetylated using acetic acid activated using DIEA). Lys (Al
oc) group selectively deprotected with 5 mL of CHCl ₃ : NMM: HOAc (18: 1).
: 0.5) by manual treatment by treating the resin with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ for 2 hours. The resin was then treated with CHCl ₃ (6 × 5
mL), 20% HOAc in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The synthesis is then re-automated for the addition of 3-maleimidopropionic acid (step 3). Between each coupling, the resin is washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. 85% TFA / 5% TIS / 5% thioanisole and 5%
The peptide is cleaved from the resin using phenol and then precipitated with dry ice cold Et ₂ O (step 4). This product was converted into Varian (Ra
in) Preparative Reversed-Phase HPLC Using a Preparative Binary HPLC System
(Using Phenomenex Luna 10μ Phenyl-hexyl.
30 mL of 55% B (0.045% in H ₂ O over 180 minutes at 5 mL / min.
Gradient elution of TFA (A) and 0.045% TFA (B) in CH ₃ CN, 21
Purification by mm × 25 cm column and UV detector at λ 214 and 254 nm (Varian Dynamax UVD II) gave the desired molecule in> 95% purity (determined by RP-HPLC).

Example 17 (2- [2- [4-[(4-chlorophenyl) phenylmethyl [1-piperazinyl] ethoxy] -maleimidopropionylacetamide (modified Cetiriz
ine)) A mixture of 1-[(4-chlorophenylmethyl)]-piperazine 1, methyl (2-chloroethoxy) -acetate 2 and sodium carbonate in anhydrous xylene is stirred well as illustrated below. While heating to reflux. The reaction mixture is then cooled, filtered, and the solid washed with benzene and the washed solid discarded. The filtrate was evaporated to dryness and the evaporated residue was washed with a column of silica (eluent: chloroform: methanol 97: 3 v / v).
) And chromatograph. This results in methyl 2- [2- [4-[(4
-Chlorophenyl) phenylmethyl] -1-piperazinyl] ethoxy] -acetate 3 was produced. This compound is dissolved in absolute ethanol. Then 1N ethanolic solution of potassium hydroxide is added to this and the reaction mixture is heated to reflux for 4 hours. It is cooled and the precipitate is filtered off and then washed with diethyl ether. The filtrate was evaporated to dryness and the evaporated residue was triturated with diethyl ether, leaving crystals. The compound, potassium 2- [2- [4-[(4-chlorophenyl) phenylmethyl] -1-piperazinyl] ethoxy] -acetate, is then obtained. The potassium salt is dissolved in water and 10
Adjust to pH 4 with% HCl. The solution is extracted with chloroform and the organic phase is dried over anhydrous magnesium sulphate and then evaporated to dryness. The evaporated residue was triturated with diethyl ether and left with crystals, 2
-[2- [4-[(4-Chlorophenyl) phenylmethyl [-1-piperazinyl] ethoxy] acetic acid 4 is produced. 2- [2- [4-[(4-chlorophenyl) phenylmethyl [-1-piperazinyl] ethoxy] -acetic acid 4 was then placed in DMF and O- (benzotriazol-1-yl) -N, N '. , N ', N'-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). To this mixture is added 3-maleimidopropylamine. The reaction is stirred for 3 hours. The organic phase was then washed with water and brine, dried over MgSO _4, leaving the crystals were triturated with cold ether. This last step produces the modified antihistamine molecule of 5- [2- [4-[(4-chlorophenyl) phenylmethyl [-1-piperazinyl] ethoxy] -maleimidopropionylacetamide] of 5.

[0449]

[Chemical 64] Example 18 (11- (N-maleimidopropionyl-4-piperidylidene) -8-chloro-6,11-dihydro-5H-benzo- [5,6] -cyclohepta- [1,2-
b] -Pyridine (modified Loratidine) 11- (N-8-chloro-4-piperidylidene) -6,11-dihydro-5H
-Benzo- [5,6] -cyclohepta- [1,2-b] -pyridine 1 was placed in DMF and then O- (benzotriazol-1-yl) -N, N as illustrated below. Activate with ', N', N'-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA).
3-Maleimidopropionic acid is added to the reaction mixture. The reaction is stirred for 3 hours. The organic phase is then washed with water and brine and dried over MgSO ₄ , chromatographed and triturated with cold ether to leave crystals, 11- (
N-maleimidopropionyl-4-piperidylidene) -8-chloro-6,11-
The modified antihistamine molecule of dihydro-5H-benzo- [5,6] -cyclohepta- [1,2-b] -pyridine 2 is obtained.

[0450]

[Chemical 65]   (Example 19)   (Modified Tirofiban)   Mechanical stirrer, cooler, nitrogen inlet, HCl trap, heating unit and
A 4-neck round bottom flask with a thermometer probe was purged with nitrogen overnight and then
L-tyrosine 1, CH₃CN, N, O-bis-trimethylsilyl-trifluor
Add romethyl-acetamide. Gently reflux this suspension and heat for 2 hours
To do. As shown in the scheme below, the resulting clear solution O, O'-bis-
Cool Limethylsilyl- (L) -Tyrosine 2, Pyridine and n-BuSO ₂ Cl is added slowly over 30 minutes. The reaction mixture is then stirred at room temperature
It Almost all solvent was removed in a batch concentrator and the resulting oily residue was removed with 15% KH
SO_FourAnd stir vigorously for 1 hour. This mixture was added to i-propylacetate.
To extract. Treating the combined organic layers with Ecosorb ™ S-402,
Then stir overnight at room temperature. Removing the Ecosorb ™ by filtration, and
The filter cake is washed with i-propyl acetate. Evaporate the filtrate to dryness.
And dissolve the resulting yellow oil in hot EtOAc. Add hexane
The resulting slurry is stirred overnight at room temperature. This solid
Are collected by filtration and the filter cake is washed with EtOAc / hexane
. After drying under reduced pressure, it is obtained as a white solid.

A 4-neck round bottom flask equipped with a mechanical stirrer, condenser, nitrogen inlet and thermometer probe was charged with Nn-butanesulfonyl- (L) -tyrosine 3,4- (4.
-Pyridinyl) -butyl chloride. Charge HCl 4 and DMSO and, with vigorous stirring, add aqueous 3N KOH solution over 15 minutes.

The temperature is maintained in the range of 30-40 ° C. with cooling water during this operation. Potassium iodide is added and the mixture is heated for 36 hours. After cooling to room temperature, the mixture is diluted with 0.25N NaOH and extracted once with t-butyl methyl ether. The aqueous layer was Ecosorb S-402 and Nuchar SA.
And mechanically stir the resulting mixture for 1 hour. The mixture is filtered through a coarse porosity sinter funnel and the filter cake is washed with water. Place the combined filtrate in a container equipped with a pH meter probe and a mechanical stirrer. Add NaCl with vigorous stirring, stir for 30 minutes, then add 50% aqueous acetic acid wash to pH 4.80 and continue stirring for 2-3 hours. The resulting slurry is filtered through a coarse porosity sinter funnel and the cake washed with water. The crude product is dried under positive nitrogen pressure under house-vacuum to give a beige solid 5 with a wt% purity of 95%.

Selective hydrogenation of the pyridine ring to the piperidine ring was performed with 5% by weight of 10% in AcOH.
Achieved by using% Pd / C at 60 ° C., apparently the phenol ring is not reduced to give the target product. Filtration of the reaction mixture, evaporation of acetic acid, followed by crystallization of product 6 from 6% AcOH / water.

RB flask equipped with thermometer probe and dropping funnel was charged with crude and 0.2
Add 5N NaOH. After the dilution is complete, the solution is cooled to room temperature and adjusted to pH 7 by slowly adding 1N HCl. The solution is lowered to pH 5.5 by slowly adding an additional 0.5 HCl. Stirring is continued for 1 hour, then the slurry is filtered through a coarse funnel containing a piece of sharkskin paper and polypropylene pad (10 mu m), and the cake washed with water. The solid is dried under house vacuum with nitrogen flushing to give a beige solid. This compound was then placed in DMF and O- (benzotriazol-1-yl) -N, N ', N', N ',-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine (DI.
Activate with EA). To the reaction mixture is added 3-maleimidopropylamine. The reaction is stirred for 3 hours. The organic phase is then washed with water and brine, MgS
The modified t was dried over O ₄ , triturated with cold ether and left to crystallize.
yields irofiban 7. Tirofiban is an anti-angina agent.

[0455]

[Chemical formula 66]   (Example 20)   (N- (1 (S) -ethoxycarbonyl-3-phenylpropyl) -L-ara
Nyl-L-prolinylmaleimidopropionylamide (modified enalapril
))   As shown in the scheme below, ethyl 2-oxo-4-phenylbutyrate 1
And L-alanyl-L-proline 2 are dissolved in a 1: 1 ethanol-water solvent.
It Sodium cyanoborohydride in ethanol-water solution at room temperature for 2 hours
Drop it. When the reaction is complete, absorb the product on a strong acid ion exchange resin and
And elute with 2% pyridine in water. Lyophilize the rich cuts of the product to obtain crude N
-(1-Ethoxycarbonyl-3-phenylpropyl) -L-alanyl-L-p
Lorin 4 was obtained and this compound was purified by chromatography to give the desired compound.
Get the sex. Compound 4 was then placed in DMF and the O-benzotriazol
(L-1-yl) -N, N ', N', N ',-tetramethyluronium hexaflu
Orophosphate (HBTU) and diisopropylethylamine (DIEA)
Activate with. 3-Maleimidopropylamine is added to the reaction mixture.
The reaction system is stirred for 3 hours. The organic phase is then washed with water and brine, MgSO _Four Dried at room temperature, triturated with cold ether, left to crystallize and then 5 N- (1 (S
) -Ethoxycarbonyl-3-phenylpropyl) -L-alanyl-L-prolyl
Generates nylmaleimidopropionylamide, a modified antihypertensive drug.

[0456]

[Chemical formula 67] Example 21 Maleimidopropynamyl- [epsilon]
-3,4,5-Trimethoxybenz-amide) -capronamide (modified capobenic acid)) 3,4,5-trimethoxybenzoyl chloride 1 was added to amino-hexanoic acid as shown in the scheme below. Add with 2 into 1 N NaOH solution.
The resulting solution is preferably decolorized by treatment with bone charcoal, the bone charcoal is filtered and the filtrate is neutralized with dilute HCl to the end of the Congo red indicator. The precipitate formed is separated by filtration, washed with water, dried and then recrystallized from ethanol to give 3.
This compound was then placed in DMF and with O- (benzotriazol-1-yl) -N, N ', N', N ',-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Activate. 3-Maleimidopropylamine is added to the reaction mixture. Reaction system 3
Stir for hours. The organic phase is then washed with water and brine, dried over MgSO ₄ , triturated with cold ether and left to crystallize to give 4 maleimidopropynamyl-ε- (3,4,5-trimethoxybenz- Amide) -capronamide, an anti-arrhythmic agent.

[0457]

[Chemical 68] Example 22 Maleimidopropionamyl-1-theobromineacetamide (modified 1
-Theobromine acetic acid)) 1-Theobromine acetic acid 1 was placed in DMF and O- (benzotriazol-1-yl) -N, N ', N', N ', as shown in the scheme below.
Activate with tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). 3-Maleimidopropylamine is added to the reaction mixture. The reaction system is stirred for 3 hours. The organic phase is then washed with water and brine, dried over MgSO _4, and chromatographed,
It is triturated with cold ether and left to crystallize to yield 2 maleimidopropionamyl-1-theobromine acetamide, a modified bronchodilator.

[0458]

[Chemical 69] Example 23 (4-anilino-1- (2-phenethyl) piperidine (modified fentanyl)) 1-phenylethyl-4-piperidone 1 in aniline 2, sodium cyanoborohydride in 1,2-dichloroethane. And reflux for 18 hours. The reaction is then cooled to room temperature and the reaction is extracted with brine to give 3 as shown in the scheme below. Then finally the compound is placed in DMF and O- (benzotriazol-1-yl) -N, N ', N', N '
, -Tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). To this reaction mixture, 3-
Add maleimidopropionic acid. The reaction system is stirred for 3 hours. The organic phase is then washed with water and brine, dried over MgSO ₄ , triturated with cold ether and left to crystallize to give 4 (modified analgesic (opioid molecule)).

[0459]

[Chemical 70] Example 24 (Maleimidopropamyl 2- [4- (2-oxocyclopentan-1-ylmethyl) phenyl] propionamide (modified loxoprofen)) Ethyl 2-cyclopenta, as shown in the scheme below. Non-carboxylate 1 and ethyl 2- (4-iodomethylphenyl) propionate 2 are placed in N, N-dimethylformamide with potassium hydroxide. The solution is stirred at room temperature for 5 hours and at 50 ° C. for 1 hour. The reaction is cooled and acidified with acetic acid to remove N, N-dimethylformamide under reduced pressure. The residue is extracted with ether and the organic phase is washed with water and dried over Mg ₂ SO ₄ 3
To get Then finally compound 3 is placed in DMF and O- (benzotriazol-1-yl) -N, N ', N', N ',-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine ( DIE
Activated in A). 3-Maleimidopropylamine is added to the reaction mixture. The reaction system is stirred for 3 hours. The organic phase is then washed with water and brine, Mg
Dried over SO ₄ , chromatographed, triturated with cold ether and left to crystallize to give 4 maleimidopropamyl 2- [4- (2-oxocyclopentan-1-ylmethyl) phenyl] propionamide. The resulting modified anti-inflammatory drug is obtained.

[0460]

[Chemical 71] (Example 25) (N-maleimidopropionyl-N-methyl 3- (p-trifluoromethylphenoxy) -3-phenylpropylamine (modified fluoxetine)) β-dimethylaminopropiophenone hydrochloride 1 was added to sodium hydroxide. Converted to the corresponding free base by the action of aqueous solution. The free base liberated is dissolved in ether, the ether layer is separated and dried, and the ether is removed therefrom under reduced pressure. The residual oil containing β-dimethylaminopropiophenone is dissolved in tetrahydrofuran and the resulting solution is added dropwise with stirring to a solution of diborane in tetrahydrofuran. The reaction mixture is stirred at room temperature overnight. Then add hydrochloric acid,
Decomposes excess diborane that is present. Tetrahydrofuran is removed by evaporation. The acidic solution is extracted twice with benzene and the benzene extract is discarded. The acidic solution is then made basic with excess 5N sodium hydroxide. The basic solution is extracted 3 times with benzene. Separate the benzene extracts and combine,
The combined extracts are then washed with saturated aqueous sodium chloride solution and then dried 2
give. N, N-dimethyl 3-phenyl-3-hydroxypropylamine 2
The chloroform solution containing is saturated with dry gaseous hydrogen chloride. Thionyl chloride is then added to the chloroform solution at a rate sufficient to maintain reflux. The solution is refluxed for another 5 hours. Evaporation of chloroform and other volatile components under reduced pressure gave N, N-dimethyl-3-phenyl-3-chloropropylamine hydrochloride 3, which was collected by filtration and the filter cake was washed twice with acetone. To do. p-Trifluoromethylphenol 4, solid sodium hydroxide and methanol are placed in a round bottom flask equipped with a magnetic stirrer, condenser and drying tube. The reaction mixture is stirred until the sodium hydroxide has dissolved. Next, hydrochloric acid N
, N-dimethyl 3-phenyl-3-chloropropylamine is added. The resulting reaction mixture is refluxed for about 5 days, then cooled. The methanol is then removed by evaporation and the resulting residue is dissolved in a mixture of ether and 5N aqueous sodium hydroxide solution. The ether layer is separated and washed twice with 5N aqueous sodium hydroxide solution and three times with water. The ether layer is dried and the ether is removed by evaporation under reduced pressure to give N, N-dimethyl 3- (p-trifluoromethylphenoxy) -3-phenylpropylamine 5 as a residue. A solution of cyanogen bromide in benzene and toluene is placed in a 3-neck round bottom flask equipped with a thermometer, dropping funnel, drying tube and nitrogen inlet tube. The solution is cooled and nitrogen gas is bubbled through the solution. Next, N, N-dimethyl 3- (
A solution of p-trifluoromethylphenoxy) 3-phenylpropylamine 5 dissolved in benzene is added dropwise. The temperature of the reaction mixture is slowly warmed to room temperature and stirring is continued overnight at this temperature while maintaining a nitrogen atmosphere. The reaction mixture is washed twice with water, once with 2N sulfuric acid and then with water until neutral. The organic layer is dried and the solvent removed therefrom by evaporation under reduced pressure,
N-methyl-N-cyano 3- (p-trifluoromethylphenoxy) -3-phenylpropylamine 6 is obtained. A solution of potassium hydroxide, water, ethylene glycol and N-methyl-N-cyano 3- (p-trifluoromethylphenoxy) -3-phenylpropylamine in a 3-neck round bottom flask equipped with a magnetic stirrer and condenser. Put in. The reaction mixture is heated to reflux temperature for 20 hours and then cooled. The reaction mixture is extracted with ether. The ether extracts are combined and the combined extracts are washed with water. Discard the water wash. The ether solution is then contacted with 2N hydrochloric acid. Separate the acidic water layer. Make a second aqueous acidic extract with 2N hydrochloric acid, then three times with water and once with saturated sodium chloride. All the aqueous layers are combined and made basic with 5N aqueous sodium hydroxide. N-methyl 3
-(P-Trifluoromethylphenoxy) 3-phenylpropylamine 7 (formed in the above reaction) is insoluble in basic solution and separates out. The amine is extracted into ether. The ether extracts are combined and the combined extracts are washed with saturated aqueous sodium chloride solution and then dried. By evaporating the ether under reduced pressure, N-methyl 3- (p-trifluoromethylphenoxy)
-3-Phenylpropylamine 7 is obtained. Then finally compound 7 is placed in DMF and O- (benzotriazol-1-yl) -N, N ', N', N '
, -Tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). To this reaction mixture, 3-
Add maleimidopropionic acid. The reaction system is stirred for 3 hours. The organic phase is then washed with water and brine, dried over MgSO _4, and chromatographed,
Triturate with cold ether and leave to crystallize to give 8 to obtain the modified antidepressant molecule.

[0461]

[Chemical 72] (Example 26) (Maleimidopropionamyl-3,5-3 ', 5' tetraiodothyroninamide (modified tyrosine)) Nt-Boc-3,5-3 ', 5' tetraiodothyronine 1 Was placed in DMF and the O- (benzothiazole-1-
Yl) -N, N ', N', N ',-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). To this reaction mixture was added 3-maleimidopropylamine. The reaction was stirred for 3 hours. The organic phase is then washed with water and brine, M
Dry over gSO ₄ , triturate with cold ethanol and crystallize to yield 2. Finally, the compound was placed in a 25% TFA solution (CH ₂ Cl ₂ ) for 15 minutes,
Then CH ₂ Cl ₂ is removed under reduced pressure. The oily residue is then lyophilized to give the desired compound 3, a modified thyroxine for the treatment of thyroid deficiency, an antithyroid deficient.

[0462]

[Chemical formula 73] (Example 27) (2S-hydroxy-3R- [1S- (MEEA-EDA-carbonyl) -2]
, 2-dimethyl - a propylcarbamoyl] -5-methyl hexanoate hydroxamic acid in (modified MMPI)) Compound 1 and 3,4-dihydro -2H- pyran (CH ₂ Cl ₂ and pyridinium p- toluenesulfonate (sulfornate)) , Stir at room temperature for 12 hours as shown in the scheme below. This solution is then treated with EtO.
Dilute with Ac and wash with half saturated brine to remove catalyst. The solvent was evaporated and the residue was triturated with NaOH (1N) and EtOH.
Process for 0 minutes. The solution was acidified with AcOH and the product was washed with EtO.
Extract with Ac. The EtOAc solution is dried and evaporated to give THP ether 2. The compound 2, DCC and HOBT (in CH ₂ Cl ₂ ) are stirred at room temperature for 60 minutes. Then, MEEA-EDA HCl (N- (2-
Aminoethyl) [2- (2-maleimidoethoxy) ethoxy] acetamide) and N-methylmorpholine were added. The reaction was stirred for 2 hours, then Ac
Quench by adding OH. This precipitate is removed by filtration. The filtrate was washed with dilute HCl, NaHCO ₃ and dried. The crude product is used in the next step. The crude product was treated with 2N HCl H ₂ O / EtOH 1: 1 at 30
Treat for minutes. Evaporate EtOH. The product is extracted with CH ₂ Cl ₂ . The combined CH ₂ Cl ₂ phases are washed with NaHCO ₃ and dried. Evaporation of the solvent gives a residue which is purified by flash column chromatography to give 3. This compound can be further purified by HPLC on a reverse phase column and lyophilized to give the modified MMPI.

[0463]

[Chemical 74] Example 28 (Preparation of Rhodamine NHS Ester) Rhodamine Green ^™ -X, succinimidyl ester, and mixed chloride isomers were prepared as described below in Molecular Probes (Eugen).
e Oregon).

[0464]

[Chemical 75] (Example 29) (In vivo addition of NHS-rhodamine) New Zealand rabbits (2 kg) (male or female) were treated with xylazine (20 kg).
mg / kg), ketamine (50 mg / kg) and acepromazine (0.75 m)
g / kg) followed by intramuscular anesthesia, after which the left carotid artery was surgically exposed. Both carotid arteries were isolated and blood flow was measured. A catheter (22G) was inserted into the arterial segment and rinsed with 0.9% sodium chloride through the catheter until the blood in the segment was invisible.

A 1 cm incubation chamber was created by ligation of this segment area. The incubation chamber was flushed 3 times with 1 mL of 0.9% sodium chloride. A solution of 100 μl of 500 μM NHS-rhodamine was prepared and incubated in the incubation chamber for 3 minutes. Excess rhodamine was removed with a 1 mL syringe. The incubation chamber was washed once again with 100 mL of 0.9% sodium chloride three times. The incubation chamber was then removed from the rabbit and
Three pieces were cut and dipped in 10% formalin for further evaluation.
NHS rhodamine-treated arteries showed dramatic fluorescence levels, whereas rhodamine-only treated arteries showed little fluorescence over background. These results demonstrated that rhodamine was covalently attached to the local delivery site.

[0466] (Example 30) ^([3 H] -NHS- propionate Preparation of) ^[3 H] -NHS- propionate, Amersham Canada L
Td. (Oakville, Ontario, Canada) and can be prepared from tritiated propionic acid via the art-known condensation of N-hydrosuccinimide in the presence of EDC (in DMF or methylene chloride).

Example 31 In Vivo Pharmacokinetic Study of [ ³ H] -NHS-Propionate New Zealand rabbits (2 kg) (male or female) were treated with xylazine (20 mg).
mg / kg), ketamine (50 mg / kg) and acepromazine (0.75 m)
g / kg) followed by intramuscular anesthesia, after which the left carotid artery was surgically exposed. Carotid 1
The 0 mm segment was transiently isolated by temporary ligation and rinsed with 0.9% sodium chloride via cannula until blood components were invisible.

A catheter (18G) is inserted into the arterial segment and an angioplasty balloon (Boston Scientific Inc., diameter 2. over the wire).
5 mm) was introduced. Vascular injury (angiogenesis) was performed on the isolated segments to remove the layer of endothelial cells. The angioplasty balloon was continuously inflated at different (4, 6, 8 and 10) atmospheres for 1 minute with a 45 second delay between inflations. Balloon traction at 4 atmospheres
) Was performed 5 times and 1000 U / kg heparin was infused into the blood circulation.

The angioplasty balloon was then retrieved from the artery and the catheter reintroduced. The arterial segment was rinsed 3 times with saline and 100 [mu] M [ ³ H] -NHS-propionate was added to the isolated arterial segment 30 times.
Incubated for 3 seconds or 30 minutes respectively. Finally, excess incubation fluid was collected from the arteries and the segments were rinsed 5 times with saline. The treated arteries were immediately harvested and the dispersal of [ ³ H] -labeled compounds within the arteries was assessed by scintillation counting. Incubation 3
After 0 seconds, the inventor recorded a binding efficiency of 2.55%. At 3 and 30 minutes, we recorded a binding efficiency of 5.5% and 6.5%, respectively. The inventor has determined that a 3 minute incubation time is sufficient to treat the artery in an efficient manner.

[0470] When evaluating the retention level, ^[3 H] -NHS-propionate or of ^[3 H] 100 [mu] M - propionate, and incubated the artery for 3 minutes, after which the segments using the five saline I rinsed. The catheter is then removed and the arteriotomy closed with a microsuture, thus restoring blood flow within the carotid artery. Finally, the cervical wound was closed with sutures and the animal allowed to recover. Three days after treatment, the animals were sacrificed by sodium pentobarbital overdose, the carotid segment was removed and tested for the presence of the compound by scintillation counting. The retention of 10.94% of [ ³ H] -NHS-propionate was monitored 3 days after 3 minutes of incubation based on residual radiography in the arteries. After 3 minutes incubation, the difference in retention efficiency between covalent and non-covalent propionate was determined. A significant 12-fold increase in retention was observed, favoring NHS-propionate (0.6% of total amount incubated versus 0.046% in non-covalent binding). This means that the binding of compounds to tissues was dramatically enhanced by covalent binding in vivo. Subsequent recovery of blood flow demonstrated retention of [ ³ H] -NHS-propionate in approximately 10% of the material 72 hours after trauma. This allows the literature on standard non-covalent drugs (Cir)
Excess tissue retention is demonstrated using a reagent-embodied technique that is significantly greater than that found in all drug delivery techniques such as those exemplified in 1994 89 (4) 1518-1524).

Example 32 Synthesis of [ ³² P] NHS Derivatives Protected R and R ′ (both R and R ′ can be alkyl, phenyl, or alkoxy groups, and X is O or S, where alkoxy, alkyl and any other functional groups are stable under these conditions) phosphodiester (0.1 mmol) and N-hydroxysuccinimide (0.2 mm
ol), diisopropylethylamine (0.11 mmol) was added,
Subsequently, HBTU (0.22 mmol) is added. The reaction mixture is allowed to cool to room temperature at 36
Stir for hours. DMF was removed by vacuum evaporation and the residue was removed from MeOH.
Dissolve in (10 mL). The MeOH solution is filtered to remove insolubles, the filtrate is concentrated under reduced pressure and the residue is dissolved in the minimum amount of MeOH. Water is then added to induce precipitation and the precipitate is dried under reduced pressure to give the desired compound.

[0472]

[Chemical 76] The obtained reaction product is usually used as an ED as a coupling agent as will be described below.
It can be improved by using C. R and R'phosphodiesters (0.
054 mmol) and N-hydroxysuccinimide (0.115 mmol)
To (in anhydrous DMF (3 mL)) add EDC (31 mg, 0.162 mmol). The solution is stirred at room temperature for 24 hours. DMF is removed by vacuum evaporation and the residue is further dried under high vacuum. The residue is then dissolved in a minimum amount of MeOH (0.12 mL) and H ₂ O (3.2 mL) is added to induce precipitation. The precipitate was washed with _{H 2 O (3 × 0.8mL)} , and dried under reduced pressure to give a solid product.

[0473]   Any protected phosphorous acid derivative can undergo similar transformations.

Example 33 New Zealand rabbits (2 kg) (male or female) were treated with xylazine (20 kg).
mg / kg), ketamine (50 mg / kg) and acepromazine (0.75 m)
g / kg) was anesthetized intramuscularly, after which the left carotid artery was surgically exposed. The carotid artery was surgically dissected and a segment approximately 10 mm long was isolated. The vessel was cannulated and rinsed with 0.9% sodium chloride until the blood components were invisible.

A catheter (18G) is inserted into the arterial segment and an angioplasty balloon (Boston Scientific Inc., diameter 2. over the wire).
5 mm) was introduced. Vascular injury (angiogenesis) was performed on the isolated segments to remove the layer of endothelial cells. The angioplasty balloon was continuously inflated at different (4, 6, 8 and 10) atmospheres for 1 minute with a 45 second delay between inflations. Balloon traction at 4 atmospheres
) Was performed 5 times and 1000 U / kg heparin was infused into the blood circulation.

The angioplasty balloon was then retrieved from the artery and the catheter reintroduced. The arterial segment was rinsed 3 times with saline and 100 μM [ ³² P] -NHS- [linking group] was incubated within the isolated arterial segment for 3 minutes. Finally, excess incubation fluid was collected from the arteries and the segments were rinsed 5 times with saline. Blood vessels were sutured closed, blood flow was restored, and surgical wounds were repaired. Animals were returned to the farm for up to 4 weeks. Tissue retention of [ ³² P] -NHS- [linking group] was evaluated using whole animal radiography at selected times after injury.

[0477] (Example 34) ^([131 I] Synthesis of -NHS derivative) is protected with protected amino ^[131 I] - iodotyrosine (0.1 mmol
) And N-hydroxysuccinimide (0.2 mmol) was added diisopropylethylamine (0.11 mmol), followed by HBTU (0.22).
mmol) is added. The reaction mixture is stirred at room temperature for 12 hours. DMF is removed by vacuum distillation and the residue is dissolved in MeOH (10 mL).
The MeOH solution is filtered to remove insolubles, the filtrate is concentrated under reduced pressure and the residue is dissolved in the minimum amount of MeOH. Water is then added to induce precipitation and the precipitate is dried under reduced pressure to give the desired compound.

The yield of this reaction is, as illustrated below, EDC as the coupling reagent.
Can be usually improved. [ ¹³¹ I] in anhydrous DMF (3 mL)
-Iodotyrosine (0.54 mmol) and N-hydroxysuccinimide (
To a solution of 0.115 mmol) EDC (31 mg, 0.162 mmol) is added. The solution is stirred at room temperature for 24 hours. DMF was removed by vacuum distillation,
Then, the residue is further dried under high vacuum. The residue is then removed with a minimum amount of Me.
Dissolve in OH (0.12 mL) and add water (3.2 mL) to induce precipitation. The precipitate is washed with H ₂ O (3 x 0.8 mL) and dried under reduced pressure to give a solid product.

[0479]

[Chemical 77] (Example 35) (In vivo pharmacology of ¹³¹ I derivative) New Zealand rabbits (2 kg) (male or female) were treated with xylazine (20 kg).
mg / kg), ketamine (50 mg / kg) and acepromazine (0.75 m)
g / kg) was anesthetized intramuscularly, after which the left carotid artery was surgically exposed. The carotid artery was surgically dissected and a segment approximately 10 mm long was isolated. The vessel was cannulated and rinsed with 0.9% sodium chloride until the blood components were invisible.

A catheter (18G) is inserted into the arterial segment and an angioplasty balloon (Boston Scientific Inc., diameter 2. over the wire).
5 mm) was introduced. Vascular injury (angiogenesis) was performed on the isolated segments to remove the layer of endothelial cells. The angioplasty balloon was continuously inflated for 1 minute at different atmospheric pressures (4, 6, 8 and 10) with a 45 second delay between inflations. Balloon traction was performed 5 times at 4 atmospheres and 1000 U / kg heparin was infused into the blood circulation.

The angioplasty balloon was then retrieved from the artery and the catheter reintroduced. The arterial segment was rinsed three times with saline and 100 μM [ ¹³¹ I] -NHS- [linking group] was incubated within the isolated arterial segment for 3 minutes. Finally, the excess incubation fluid is recovered from the artery,
The segments were then rinsed 5 times with saline. Blood vessels were sutured closed, blood flow was restored, and surgical wounds were repaired. Animals were returned to the farm for up to 4 weeks. Tissue retention of [ ¹³¹ I] -NHS- [linking group] was evaluated using whole animal radiography at selected times after injury.

Example 36 (2- [2- [4-[(4-chlorophenyl) phenylmethyl [-1-piperazinyl] ethoxy] -maleimidopropionylacetamide (2- [2- [4-
[(4-chlorophenyl) phenylmethyl [-1-pipe
razynyl] ethoxy] -maleimidopropionylac
Pulmonary Delivery of Etamide (Modified Cetirizine) Working with Bird Mark 7 Respirator (in line w)
ith) In a Bird Micronebulizer, 5-10 ml of a solution of 12 mg / ml of 2- [2- [4-[(4-chlorophenyl) phenylmethyl [-1-piperazinyl] ethoxy] -maleimidopropionylacetamide in mannitol / phosphate buffer. Can be filled. Then, this Micronebulizer
Use, 22 cm H ₂ O at 1.8 mg / min for 30 minutes, may be simultaneously venting and administered to the patient. At this pressure, the patient should vent with approximately normal inspiratory volume. The patient should be exhaled as usual after each breathed breath. In addition, the patient should be in a supine position for administration. After the first dosing period, the patient should be allowed to breathe an additional 20 minutes normally. After this 20 minutes, the second dose should be administered in the same way as the first dose. Plasma samples should be taken at the beginning of the first dose and then monitored for levels of 2- [2- [4-[(4-chlorophenyl) phenylmethyl [-1-piperazinyl] ethoxy] -maleimidopropionylacetamide. Is.

Example 37 (2- [2- [4-[(4-chlorophenyl) phenylmethyl [-1-piperazinyl] ethoxy] -maleimidopropionylacetamide (modified cetirizine) using the Spiros DPI system Pulmonary Delivery Spiros DPI is an aerosol generation system that is largely independent of inspiratory flow, and its use is described in US Pat. No. 6,060,069.

A modified beclomethasone dipropionate (BDP) formulation was prepared by first micronizing via conventional means (eg, jet mill) to produce a range of particle sizes that appear to sediment in the human respiratory tract. You can Generally, a diameter of 0.5-
Fine particles in the 5.8 micron range are believed to settle between the oropharynx and bronchioles. Particles within this general size category are considered to be in the "respirable range." Such finely divided materials have an excess of surface free energy and as a result tend to adhere strongly to many surfaces, most especially to themselves.

Lactose particles in the 20-100 micron size range can be mixed with smaller diameter micronized drug particles to produce a uniform mixture. Each lactose particle generally binds to a large number of smaller drug particles in the mixture. This mixture flows more easily during the filling and dosing process.

The formulation may then be filled into cassettes, each containing 30 individual doses. The cassette can then be filled into a sealed foil pouch.

The following steps using the Spiros DPI system may be used to deliver a metered dose of inhaled drug: Spiros DPI does not need to be primed for water in advance; 2. 2. Remove the blue plastic cap from the mouthpiece; Keep the inhaler level; 4. 4. Open the DPI lid as far back as possible (this lid will snap when the correct angle is reached); Then close the lid completely; 6. Before taking the inhaler to the mouth, the patient exhales,
Do not exhale into the inhaler; 7. Carry the inhaler horizontally to the mouth; 8
． 8. Completely close lips around mouthpiece with no gap between mouthpiece and lips; The patient breathes through the mouth for about 4 seconds, preferably at a flow rate of about 20 LPM. 10. Actuate motor and patient may taste / feel drug when inhaled; 10. Patients hold their breath for up to 10 seconds whenever possible. 11. S
The piros DPI is kept level during loading and administration.

[Sequence list]

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) A61K 31/401 A61K 31/401 4C206 31/454 31/454 31/4545 31/4545 31/496 31/496 31/522 31/522 38/00 47/42 38/22 47/48 38/55 A61P 3/10 47/42 5/10 47/48 5/14 A61P 3/10 5/38 5/10 9/00 5/14 9/06 5/38 9/10 9/00 9/12 9/06 11/00 9/10 11/06 9/12 11/08 11/00 25/00 11/06 25/04 11 / 08 25/24 25/00 29/00 25/04 31/00 25/24 31/12 29/00 31/18 31/00 35/00 31/12 37/06 31/18 41/00 35/00 43 / 00 105 37/06 111 41/00 113 43/00 105 C07D 207/452 111 401/12 113 401/14 // C07D 207/452 473/10 401/12 A61K 37/02 401/14 37/64 473 / 10 37/24 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I , LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM) , KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD , GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, S , SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Robituille, Martin Canada J 7V 5 S 1 Kebek, Terrasse-Vaudruille, 2 Im Boulevard 108 (72) Inventor Milner, Peter G. United States California 94022, Los Altos Hills, Manuela Road 14690 (72) Inventor Brydon, Dominickie P. Canada H2V2B2Kevek, Woutslemont, Schumencote Sainte-Catherine, 243F Term (Reference) 4C063 AA01 AA03 BB01 BB07 BB09 CC10 CC17 DD04 DD10 EE01 4C069 AD08 BB02 BB38 BB52 4C076 AA24 AA24 A CC01 CC03 CC04 CC05 CC07 CC11 CC15 CC16 CC26 CC27 CC29 CC30 CC31 CC35 CC41 EE41 EE59 FF34 FF68 4C084 AA02 AA03 BA01 BA08 BA17 BA18 BA19 BA26 BA32 BA41 CA01 CA59 DA01 DB01 DB05 DB34 DB57 MA13 MA43 MA56 NA10 NA31ZA01 NA14 NA11 NA01 NA12 NA14 NA12 NA08 ZA36 ZA42 ZA59 ZA61 ZB31 ZB33 ZC03 ZC04 ZC06 ZC13 ZC20 ZC35 ZC55 4C086 AA01 AA02 BC08 BC21 BC50 CB07 GA07 GA08 MA01 MA04 MA05 MA13 MA43 NA10 NA11 NA13 Z36C31 Z36C31 Z36C31 Z36C31 Z36C31 Z36C31 Z13C31 ZA04 ZA33 ZC33 ZC04 AA01 AA02 FA21 GA07 GA36 KA01 MA01 MA04 MA33 MA76 NA10 NA12 NA13 NA15 ZA12 ZA36

Claims (65)

[Claims] 1. A modified therapeutic agent, comprising: a therapeutic agent, and a reactive group, which reacts in vivo with an amino group, a hydroxyl group or a thiol group on a lung component or a blood component to form a stable covalent bond. The therapeutic agent comprises a GP-41 peptide, a BBB peptide, an anticancer agent, an antihistamine, a bronchodilator, an antihypertensive agent, an anti-angina agent, an opioid, an analgesic, an antidepressant,
And a modified therapeutic agent selected from the group consisting of a hypothyroid drug. 2. The modified therapeutic agent according to claim 1, wherein the reactive group is a succinimidyl group or a maleimide group. 3. The modified therapeutic agent according to claim 1, wherein the reactive group is a maleimide group reactive with a thiol group on a mobile lung component. 4. The modified therapeutic agent according to claim 1, wherein the reactive group is a maleimide group reactive with a thiol group on a fixed lung component. 5. The modified therapeutic agent according to claim 1, wherein the reactive group is a maleimide group reactive with a thiol group on a mobile blood component. 6. The modified therapeutic agent according to claim 1, wherein the reactive group is a maleimide group reactive with a thiol group on albumin. 7. The modified therapeutic agent according to claim 1, wherein the reactive group is a maleimide group reactive with a thiol group on a fixed blood component. 8. The modified therapeutic agent according to claim 1, wherein the therapeutic agent is an antihistamine. 9. The modified therapeutic agent according to claim 1, wherein the therapeutic agent is a hypothyroid agent. 10. The modified therapeutic agent according to claim 9, wherein the therapeutic agent is loratidine. 11. The modified therapeutic agent according to claim 9, wherein the therapeutic agent is cetirizine. 12. An aerosol composition for delivery of a therapeutic agent to the pulmonary system of a host, the aerosol composition comprising an aerosolized aqueous solution containing a modified therapeutic agent, the modified therapeutic agent comprising: An aerosol composition comprising a therapeutic agent and a reactive group which reacts with an amino group, a hydroxyl group or a thiol group on a lung component or a blood component to form a stable covalent bond. 13. The aerosol according to claim 12, further comprising a pharmaceutically acceptable carrier. 14. The aerosol according to claim 12, wherein the modified therapeutic agent is 2.5% to 10% by weight. 15. The aerosol according to claim 12, wherein the therapeutic agent is an antihistamine. 16. The aerosol according to claim 15, wherein the therapeutic agent is loratidine. 17. The aerosol according to claim 15, wherein the therapeutic agent is cetirizine. 18. A particulate formulation for delivery of a therapeutic agent to the pulmonary system of a host, the particulate formulation comprising a dispersible dry powder containing a modified therapeutic agent, the modified therapeutic agent. A particulate formulation comprising a therapeutic agent and a reactive group that reacts with an amino group, a hydroxyl group or a thiol group on the lung component to form a stable covalent bond. 19. The particulate formulation of claim 18, wherein at least 50% of the dry powder is in the form of particles having a diameter of 10 μm or less. 20. The particulate formulation of claim 18, wherein the therapeutic agent is an antihistamine. 21. The particulate formulation of claim 20, wherein the therapeutic agent is loratidine. 22. The particulate formulation of claim 20, wherein the therapeutic agent is cetirizine. 23. A method of delivering a therapeutic agent to a host, the method comprising: a step of obtaining a modified therapeutic agent, wherein the modified therapeutic agent is: a therapeutic agent, and a reactive group. A reactive group that reacts in vivo with an amino group, a hydroxyl group or a thiol group on a lung component or a blood component to form a stable covalent bond; and the modified therapeutic agent in the pulmonary system of the host. The method comprises the step of administering to. 24. The method of claim 23, wherein said administering step further comprises aerosolizing said modified therapeutic agent for inhalation by said host. 25. The method of claim 23, wherein said administering step further comprises dispersing a dry formulation of said modified therapeutic agent for inhalation by said host. 26. The method of claim 23, wherein said administering step further comprises the step of instilling said modified therapeutic agent into said pulmonary system of said host. 27. The method according to claim 23, wherein the reactive group is a succinimidyl group or a maleimide group. 28. The method of claim 23, wherein the reactive group is a maleimide group that is reactive with thiol groups on mobile lung components. 29. The method of claim 23, wherein the reactive group is a maleimide group that is reactive with a thiol group on a fixed lung component. 30. The method of claim 23, wherein the reactive group is a maleimide group that is reactive with thiol groups on mobile blood components. 31. The method of claim 23, wherein the reactive group is a maleimide group that is reactive with thiol groups on fixed blood components. 32. The method of claim 23, wherein the reactive group is a maleimide group that is reactive with a thiol group on human serum albumin. 33. The method of claim 23, wherein the therapeutic agent is an antihistamine. 34. The method of claim 33, wherein the therapeutic agent is loratidine. 35. The method of claim 33, wherein the therapeutic agent is cetirizine. 36. Use of a composition for the manufacture of a medicament for use in the treatment of the human body to provide an antihistamine effect, said composition comprising an antihistamine drug and a derivative thereof. Here, the derivative contains a reactive functional group that reacts with an amino group, a hydroxyl group, or a thiol group on a blood component to form a stable covalent bond, and the reactive functional group is an N-hydroxy group. Use, selected from succinimide group, N-hydroxy-sulfosuccinimide group and maleimide group. 37. Use of a composition according to claim 36, wherein the antihistamine is selected from cetirizine, loratidine and analogs thereof. 38. The use of the composition according to claim 36, wherein the antihistamine is selected from cetirizine and its analogs. 39. Use of a composition according to claim 36, wherein the antihistamine is selected from loratidine and its analogs. 40. Use of a composition for the manufacture of a medicament for providing an anti-angina effect for use in the treatment of the human body, the composition comprising an anti-angina agent and a derivative thereof. Here, the derivative contains a reactive functional group that reacts with an amino group, a hydroxyl group, or a thiol group on a blood component to form a stable covalent bond, and the reactive functional group is an N-hydroxy group. Use, selected from succinimide group, N-hydroxy-sulfosuccinimide group and maleimide group. 41. Use of a composition according to claim 40, wherein the anti-angina agent is tirofiban. 42. Use of a composition for the manufacture of a medicament for use in the treatment of the human body to provide an antihypertensive effect, said composition comprising an antihypertensive agent and its analog derivatives. Here, the derivative contains a reactive functional group that reacts with an amino group, a hydroxyl group, or a thiol group on a blood component to form a stable covalent bond, and the reactive functional group is an N-hydroxy group. Use, selected from succinimide group, N-hydroxy-sulfosuccinimide group and maleimide group. 43. Use of a composition according to claim 42, wherein the antihypertensive agent is enalapril. 44. Use of a composition for the manufacture of a medicament for providing an antiarrhythmic effect for use in the treatment of the human body, the composition comprising an antiarrhythmic agent and a derivative of an analog thereof. Here, the derivative contains a reactive functional group that reacts with an amino group, a hydroxyl group, or a thiol group on a blood component to form a stable covalent bond, and the reactive functional group is an N-hydroxy group. Use, selected from succinimide group, N-hydroxy-sulfosuccinimide group and maleimide group. 45. Use of a composition according to claim 44, wherein the arrhythmic agent is capovenic acid. 46. Use of a composition for the manufacture of a medicament for use in the treatment of the human body to provide an antidepressant effect, said composition comprising an antidepressant and a derivative of an analog thereof. Here, the derivative contains a reactive functional group that reacts with an amino group, a hydroxyl group, or a thiol group on a blood component to form a stable covalent bond, and the reactive functional group is an N-hydroxy group. Use, selected from succinimide group, N-hydroxy-sulfosuccinimide group and maleimide group. 47. Use of a composition according to claim 46, wherein the depressant is fluoxetine. 48. Use of a composition for the manufacture of a medicament for use in the treatment of the human body to provide a bronchodilator effect, the composition comprising a bronchodilator and its analog derivatives. Here, the derivative contains a reactive functional group that reacts with an amino group, a hydroxyl group, or a thiol group on a blood component to form a stable covalent bond, and the reactive functional group is an N-hydroxy group. Use, selected from succinimide group, N-hydroxy-sulfosuccinimide group and maleimide group. 49. Use of a composition according to claim 48, wherein the bronchodilator is theobromine acetamine and analogs thereof. 50. Use of a composition for the manufacture of a medicament for providing an anti-inflammatory effect for use in the treatment of the human body, the composition comprising an anti-inflammatory agent and a derivative of an analog thereof. Here, the derivative contains a reactive functional group that reacts with an amino group, a hydroxyl group, or a thiol group on a blood component to form a stable covalent bond, and the reactive functional group is an N-hydroxy group. Use, selected from succinimide group, N-hydroxy-sulfosuccinimide group and maleimide group. 51. Use of a composition according to claim 50, wherein the anti-inflammatory agent is loxoprofen and its analogs. 52. Use of a composition for the manufacture of a medicament for providing an antithyroid deficiency effect for use in the treatment of the human body, said composition comprising an antithyroid deficiency agent and a derivative thereof. Wherein the derivative contains a reactive functional group that reacts with an amino group, a hydroxyl group, or a thiol group on a blood component to form a stable covalent bond, the reactive functional group being N A use selected from a hydroxysuccinimide group, an N-hydroxy-sulfosuccinimide group and a maleimide group. 53. Use of a composition according to claim 52, wherein the antithyroid deficiency agent is thyroxine and analogs thereof. 54. A composition, which comprises: 2- [2- [4-[(4-chlorophenyl) phenylmethyl [-1-piperazinyl] ethoxy] -maleimidopropionylacetamide; -(N-maleimidopropionyl-4-piperidylidene) -8-chloro-
6,11-Dihydro-5H-benzo- [5,6] -cyclohepta- [1,2-b
] -Pyridine; N- (1 (S) -ethoxycarbonyl-3-phenylpropyl) -L-alanyl-L-prolinylmaleimidopropionylamide; Maleimidopropynamyl-ε- (3,4,5-trimethoxybenz- Amide)
-Caproicamide; maleimidopropionamyl-1-theobromine acetamide; maleimidopropamyl 2- [4- (2-oxocyclopentan-1-ylmethyl) phenyl] propionamide; N-maleimidopropionyl-N-methyl-3- ( p-trifluoromethylphenoxy) -3-phenylpropylamine; from the group consisting of 4-anilino-1- (2-phenethyl) piperzine and maleimidopropionamyl-3,5-3 ', 5' tetraiodothyroninamide. A composition comprising a selected compound. 55. The compound is maleimide propionamyl-3,5-3 ′.
55. The composition of claim 54, which is 5,5 'tetraiodothyroninamide. 56. An aerosol composition for delivery of a therapeutic agent to the pulmonary system of a host, the aerosol composition comprising a modified therapeutic agent bound to a blood protein.
An aerosol composition comprising an aerosolized aqueous solution. 57. The composition of claim 56, wherein the protein is albumin. 58. The aerosol of claim 56, wherein the therapeutic agent is an antihistamine. 59. The aerosol of claim 56, wherein the therapeutic agent is loratidine. 60. The aerosol of claim 56, wherein the therapeutic agent is cetirizine. 61. A particulate formulation for delivery of a therapeutic agent to the pulmonary system of a host, the particulate formulation comprising a dispersible dry powder containing a modified therapeutic agent, the modified therapeutic agent. Include the following: a therapeutic agent, and a reactive group that reacts with an amino group, a hydroxyl group or a thiol group on a lung component to form a stable covalent bond, wherein the treatment is The agent is a particulate formulation that covalently binds to blood proteins. 62. The formulation of claim 61, wherein the blood protein is albumin. 63. The formulation of claim 61, wherein the therapeutic agent is an antihistamine. 64. The formulation of claim 61, wherein the therapeutic agent is loratidine. 65. The particulate formulation of claim 61, wherein the therapeutic agent is cetirizine.