KR20130081651A - Selective integrin inhibitors - Google Patents

Abstract

The present invention provides a method for selectively antagonizing α4β1 integrin heterodimers in a subject in need of selective antagonism of α4β1 integrin heterodimers, and preferably in a human subject. The methods of the present invention utilize a conjugate comprising two or more α4β1 small molecule antagonists covalently attached to a biocompatible polymer. These methods of selectively antagonizing α4β1 can be used to regulate both normal and pathological biological processes that are at least partially mediated through α4β1 activity.

Description

SELECTIVE INTEGRIN INHIBITORS

Cross Reference of Related Application

This application takes precedence over US Provisional Application 61 / 330,122, filed April 30, 2010, US Provisional Application 61 / 392,288, filed October 12, 2010, and US Provisional Application 61 / 516,845, filed April 8, 2011. Insisted, each of which is incorporated herein by reference in its entirety.

Field of invention

The present invention relates to selective inhibitors of integrin, more particularly selective inhibitors of α4β1.

In the following discussion certain documents and methods are described for background and introduction purposes. Nothing contained herein should be considered to be "recognized" by the prior art. Applicant reserves the right to explicitly state that the documents and methods referred to herein do not include prior art under applicable legal provisions.

Integrin receptors are transmembrane, non-covalently linked heterodimers consisting of one α-chain and one β-chain. In addition to performing structural cohesive functions, integrin receptors deliver extracellular signals across the plasma membrane. Integrin receptor α4β1 (also called VLA-4) mediates cell adhesion by binding to one of two protein ligands: Vascular Cell Adhesion Molecule-1 (VCAM-1) ( Osborn , L. et. al ., Cell , 1989, 59, 1203 ), or alternately conjugated fibronectin variants comprising type III connecting segments (CS-1) ( Wayner , EA et al. al ., Cell Biol ., 1989, 109, 1321 ). In contrast to the circular integrin receptors that recognize the Arg-Gly-Asp (RGD) tripeptide sequence in the corresponding ligands, α4β1 is Vln-1 in Fibronectin and Gln-Ile-Asp in Ile-Leu-Asp-Val (ILDV). Recognize Ser (QIDS). Although these sequences share Asp residues that are conserved with RGD, in other respects they are independent of each other.

α4β1 integrin receptors are expressed at high levels in mast cells, monocytes, eosinophils, macrophages, and basophils ( Adams , SP et al ., Ann . Rep . Med . Chem ., 1999, 34, 179 ). Binding of α4β1 to cytokine-induced VCAM-1 at the site of inflammation leads to leukocyte / endothelial adhesion followed by extravasation to these inflammatory tissues ( Chuluyan , HE et al. al ., Springer Semin . Immunopathol., 1995, 16, 391 ). The interaction of the α4β1 receptor with VCAM-1 also has an important effect on the adhesion of stem cells to myeloid mesenchymal cells ( Simmons , PJ et. al ., Blood , 1992, 80, 388 ).

Cell adhesion and migration through β1 integrins are important components of cell recruitment. Integrin α4β1 provides major costimulatory signals that support cell activation leading to growth factor and cytokine production and mediator release. Through recognition of the extracellular matrix, α4β1 increases the survival of activated cells by inhibiting apoptosis ( Yoshikawa , H. et. al ., J. Immunol ., 1996, 156, 1832 ).

Integrins can have a variety of α and β components, and therapeutic agents that affect one or the other of the α4β1 components can affect other integrin combinations. For example, a therapeutic agent that selectively binds to the α4 integrin subunit will bind to and regulate both α4β1 and α4β7 heterodimers. For many disease states, it would be desirable to have a therapeutic agent that selectively binds to these heterodimer combinations because it provides a more tailored treatment to the disease state without the possibility of side effects resulting from the binding of other heterodimers.

Accordingly, there is a need for compositions and methods that utilize compounds that selectively bind to the α4β1 integrin and have little effect on the process of signaling through α4β7 and / or using α4β7-mediated related pathways. The present invention relates to this need.

Summary of the Invention

This summary provides, in a simplified form, the concept of selection further described in the detailed description below. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to limit the scope of the claimed subject matter. Other features, details, uses, and advantages of the claimed subject matter will become apparent from the following detailed description, including the aspects described in the accompanying drawings, and those defined in the appended claims.

The present invention provides methods for selectively antagonizing α4β1 integrin heterodimers in a subject in need, preferably a human. The methods of the present invention utilize a conjugate comprising two or more α4β1 small molecule antagonists covalently attached to a biocompatible polymer. These methods of selectively inhibiting α4β1 may be used to regulate normal and / or etiological biological processes that are at least partially mediated through α4β1 activity.

The present invention therefore relates to a method of treating a disease, disorder or injury by selective inhibition of α4β1 heterodimers. Such diseases include, but are not limited to, inflammatory diseases, autoimmune diseases and cell-proliferative disorders. A therapeutically effective amount of such conjugates for use in the present invention may be provided to a subject through a variety of routes of administration, and optionally together with pharmaceutically acceptable carriers.

In certain aspects, the present invention includes compounds, such as those shown in formula (I), conjugates comprising such mixtures, such as racemic mixtures, diastereomers, and enantiomers, and related pharmaceutically acceptable salts Using conjugates to selectively inhibit α4β1:

Formula I

By varying the number of alkylene oxide-[CH ₂ -CH ₂ -O]-repeat units represented in formula (I), the total average molecular weight of the polymers generated by single or multiple polymer moieties in the conjugates is about 100 to 100,000; Preferably about 20,000 to 60,000, or about 20 to about 60 kiloDaltons (kDa); More preferably about 30,000 to about 50,000 (or about 30 to about 50 kDa). It is apparent to those skilled in the art that this type of synthetic organic polymer will be polydisperse. Polydispersity means that the polymer molecules contemplated herein are of different sizes, even if they are of the same type (chain length of a linear or multi-armed polymer). Thus, the average molecular weight will vary depending on the method of averaging. The common divisor of the polydispersity index (PDI), the molecular weight variability, is the ratio of the weight average molecular weight to the number average molecular weight. PDI is a measure of the distribution of molecular weight in a given polymer sample. The calculated PDI is the weight average molecular weight divided by the number average molecular weight. This represents the distribution of individual molecular weights in a polymer batch. In the case of the conjugates of the present invention, the PDI has a value of 1 or more, but if the polymer chains are close to a uniform chain length, the PDI is close to consistency (I). This represents the distribution of the individual molecular weights in the batch of polymers. The number average molecular weight is a method of measuring the molecular weight of a polymer. The number average molecular weight is a common average of the molecular weights of the individual polymers. This is determined by measuring the molecular weight of the n polymer molecules, sum the weights, and divide by n. The number average molecular weight of the polymer can be determined by osmometry, end-group titration, and colligative properties.

The weight average molecular weight can be measured by light scattering, small angle neutron scattering (SANS), X-ray diffraction, and settling velocity. The ratio of weight average to number average is called the polydispersity index. A theoretical polymer sample that does not have a dispersion will have a polydispersity index of 1. Preferred polydispersity index of the present invention is about 1.10 to about 1.05. More preferably, it is in the range of about 1.05 to the upper limit of commercially feasible synthesis, currently about 1.02. Conjugates of Formula I are also described in US Application No. 20060013799, which is incorporated herein by reference in its entirety.

Other conjugate compositions contemplated for use in the methods of the present invention include specific α4β1-selective conjugates comprising two or more α4β1-specific small molecule antagonists and a biocompatible polymer, which are individually US applications. number. Explained in 20060013799

In one aspect, the present invention utilizes a therapeutic dosage of formula (I) to inhibit α4β1-mediated leukocyte visit to the central nervous system (CNS) while preserving α4β7-mediated function and trafficking . In one aspect, the amount of formula (I) is typically from about 0.01 mg / kg to about 10 mg / kg, preferably from about 0.2 mg / kg to about 1.0 mg / kg. In another aspect, the amount is about 1.0 mg / kg to about 2.0 mg / kg. In a specific aspect, this dose is about 0.5 mg / kg. This dose is preferably administered weekly or monthly.

Another aspect of the invention provides the use of a therapeutically effective amount of a conjugate comprising two or more α4β1 small molecule antagonists covalently attached to a biocompatible polymer, wherein the conjugate is at least partially through α4β1 activity. The ability to direct α4β1 rather than α4β7 in the regulation of mediated biological processes is 20 to 100 times greater. Preferably, this conjugate, like the compound of formula (I), has 30 to 80 times greater ability to direct α4β1 than α4β7.

Conjugates and compounds for use in the present invention include a variety of α4β1-mediated biological processes, including but not limited to autoimmune diseases (eg, multiple sclerosis), inflammatory diseases, and cell proliferative disorders. Can be used for adjustment.

Thus, in one specific aspect of the present invention, the present invention provides the use of the conjugates and compounds of the present invention in the treatment of autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, asthma, psoriasis, inflammatory bowel disease and the like. To provide.

In another specific aspect of the invention, the invention provides the use of the conjugates and compounds of the invention in the treatment of inflammatory diseases such as restenosis and atherosclerosis.

In other specific aspects, the present invention provides the use of the conjugates and compounds of the present invention in the treatment of cell growth proliferative disorders, such as hematopoietic neoplasms, which are abnormal growth related cells of the hematopoietic lineage, in particular B cell malignancy.

In another specific aspect of the present invention, the present invention provides the use of the conjugates and compounds of the present invention for the promotion of immune suppression, such as transplant rejection or graft versus host disease (GVHD).

In still other aspects, the present invention provides the use of the conjugates and compounds of the present invention to induce neuroprotection after injury, such as spinal injury.

In some aspects, the invention provides the use of a therapeutically effective amount of a conjugate comprising two or more α4β1 small molecule antagonists covalently attached to a biocompatible polymer in the treatment of a patient with autoimmune disease, wherein In the conjugate is 20 to 100 times greater in its ability to direct α4β1 than α4β7.

In other aspects, the present invention provides a formula (I) for the treatment of a patient in need thereof comprising administering a dosage of formula (I) from about 0.5 mg / kg to about 2.0 mg / kg to the patient weekly or monthly Serves the purpose of.

In other aspects, the present invention provides a formula for the treatment of a patient comprising administering a dosage of formula (I) of about 0.5 mg / kg to about 2.0 mg / kg to a patient with an autoimmune disease weekly or monthly ) Uses.

These and other aspects and uses of the invention will be described in more detail in the written description, and are illustrated in the accompanying drawings.

1 is a line graph illustrating the competitive binding of compounds of formula (I) as compared to multivalent α4β1 ligands.
FIG. 2 is a line graph showing the ability of compounds of formula (I) to disrupt α4β1-dependent Jurkat ™ adhesion to rs-hu-VCAM-1.
3 is a line graph showing the ability of formula (I) to inhibit α4β1-dependent adhesion of human lymphocyte cells (Jurkat ™ cell line) to fibronectin (FN).
4 is a line graph showing the ability of formula (I) to inhibit the binding of human serum FN to activated α4β1 in Jurkat ™ cells.
5 is a line graph showing the concentration of formula (I) in the plasma of human subjects receiving various dosages of conjugates.
FIG. 6 is a line graph showing saturation levels of α4β1 and α4β7 in lymphocytes after various doses of Formula (I) are administered to human subjects.
7 is a bar graph showing mean receptor downregulation after 0.5 mg / kg administration of Formula (I) to a population of human subjects.
FIG. 8 is a bar graph showing individual receptor downregulation after administering 0.5 mg / kg of formula (I) to human subjects.
9 is a bar graph showing lymphocyte counts in patients at different dosages of formula (I).
FIG. 10 is a line graph illustrating the correlation of relative lymphocyte count with plasma concentration of formula (I) in a patient.
11 is a pair of line graphs illustrating receptor occupancy of α4β1 and α4β7 at various dosages of Formula (I).
12 is a line graph illustrating receptor saturation of α4β1 and α4β7 at a 0.5 mg / kg dose of Formula (I).
FIG. 13 is a line graph illustrating normalized receptor expression of α4β1 and α4β7 at a 0.5 mg / kg dose of Formula (I).
14 is a line graph illustrating receptor saturation of α4β1 and α4β7 at a 1.0 mg / kg dose of Formula (I).
15A and 15B are two line graphs illustrating the level of inhibition of α4β1 and α4β7 for both compounds of Formula (I) and ELN 476063.
16A-16F are a series of line graphs illustrating that Formula (I) differentially down-regulates soluble VCAM-1 (sVCAM-1) and soluble MadCAM-1 (sMadCAM-1).
17A and 17B are bar graphs showing the correlation of plasma sVCAM-1 and sMAdCAM-1 with plasma compound levels.
18A and 18B are line graphs illustrating the differential regulation of sVCAM-1 and sMAdCAM-1 by Formula (I) and ELN476063 compounds.
FIG. 19 is a line graph with square and circle measurements of radioactivity measurements of the compound of formula (I) (square) and ELN 476063 (circle) in various murine tissues.

Justice

The terms used herein have the same plain and common meanings as those skilled in the art understand. The following definitions are intended to assist the reader in understanding the present invention, but are not intended to limit or change the meaning of these terms unless otherwise indicated.

As used herein, the term "administering" includes the treatment of the various disorders described herein with a composition comprising the conjugates of the present invention. Also "administering" includes not only a single dose but also a dosing regimen provided with multiple doses at different times during the course of treatment. Dosage regimens of single or multiple doses may be administered in combination with other active agents.

As used herein, the term “α4β1-selective” refers to a compound that preferentially binds to and modulates α4β1 heterodimer rather than other integrin heterodimers, especially α4β7. Α4β1-selective molecules for use in the methods of the present invention are characterized by at least 20 to 100 times more potency on α4β1 than on α4β7, more specifically on at least 30 to 80 times more potency on α4β1 than on α4β7.

As used herein, the term “autoimmune disease” refers to diseases and disorders characterized by producing antibodies that react with host tissue or immune effector cells that are autoreactive to endogenous peptides.

As used herein, “cell proliferative disorders” refers to any disease or disorder associated with abnormal cell growth or cell activity, including but not limited to cell dedifferentiation or unregulated cell division. .

As used herein, "immunosuppression" refers to suppressing an immune response, eg, to prevent rejection of a graft or transplant or to control an autoimmune disease.

As used herein, the term “inflammatory disorders” refers to any disease or disorder characterized by a pathological process characterized by damage or destruction of tissue due to various cellular and chemical reactions.

As used herein, the term “subject” refers to an animal, preferably a mammal, most preferably a human.

As used herein, the term “therapeutically effective amount” means the amount of the conjugate of the invention that elicits the desired biological or medical response.

The terms “treat”, “treatment”, and the like refer to obtaining the desired pharmacological and / or physiological effect. As used herein, "treatment" encompasses any treatment to achieve the desired effect in mammals, especially humans, including: (a) prone to disease or physiological effects, Preventing the disease or physiological effects from developing in a subject who has not yet been diagnosed; (b) inhibit this disease or physiological effect, eg, inhibit development; And (c) degenerate to alleviate this disease or physiological effect, such as to completely or partially eliminate signs of the disease or physiological effect. The therapeutic effect may be a prophylactic aspect in the maintenance of a physiological condition that prevents side effects such as complete or partial prophylaxis of disease, physiological effects or signs thereof or tissue rejection. This effect may be therapeutic in terms of partial or complete cure in response to a disease or physiological effect and / or partial or complete cure of side effects resulting from the disease or physiological effect.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the techniques described herein utilizes common techniques and descriptions of organic chemistry, drug chemistry, polymer technology, cell biology, biochemistry, unless otherwise noted, and are within the skill of those skilled in the art. For specific description of suitable techniques, including those for the preparation of pharmaceutical combinations comprising the compositions of the present invention, may be referred to the description and examples herein. However, of course, other equivalent processes may also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Lehninger , Principles. of Biochemistry 3 rd Ed ., WH Freeman Pub ., New York, NY; and Berg meat al . (2002) Biochemistry , 5 th Ed ., WH Freeman Pub ., New Can be found in York , NY . Methods of formulating pharmaceutical compositions are described in many publications, such as Pharmaceutical Dosage Forms : Tablets , Second Edition , Revised and Expanded , Volumes 1-3, edited by Lieberman meat al ; Pharmaceutical Dosage Forms : Parenteral Medications, Volumes 1-2, edited by Avis meat al ; and Pharmaceutical Dosage Forms: Disperse Systems , Volumes 1-2, edited by Lieberman meat al ; Marcel Published by Dekker , Inc. A description of pharmaceutically acceptable carriers is given in The Handbook of Pharmaceutical Excipients , published by the American Pharmaceutical Association and the Pharmaceutical Society of Great You can find it in Britain . Each of which is incorporated herein by reference in its entirety for use in the present invention.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural unless the context clearly dictates. Thus, for example, "excipient" refers to one or more excipients, and "dosing regimen" includes equivalent steps and methods known to those skilled in the art.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing devices, formulations, and methods that may be associated with the presently described invention.

When a range of values is provided, it is to be understood that the values between each between the upper and lower limits of the range and any other stated or within the stated ranges also belong to the present invention. The upper and lower limits within these smaller ranges may be included separately in the smaller ranges, and may be specifically excluded from any of the stated ranges, which are also within the scope of the present invention. Where one or both of the limits are included in the stated ranges, the ranges in which either or both of these included limits are excluded also belong to the invention.

In the following description, for the purpose of providing a more thorough understanding of the present invention, numerous details are provided. However, it will be apparent to one skilled in the art that the present invention may be practiced without one or more of these specific details. In other instances the features and procedures known to those skilled in the art have not been described in order to avoid damaging the present invention.

The methods of the present invention comprise administering to a subject a therapeutically effective amount of an α4β1-selective inhibitor, such as specific α4β1-selective antagonists, disclosed in US Application No. 20060013799. Provide treatment. A preferred aspect of the present invention is a method of treating α4β1-mediated disorders in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula (I).

In specific aspects, the therapeutically effective daily amount of this compound is about 0.01 mg / kg / day to about 30 mg / kg / day.

Compositions of the invention comprising α4β1-selective conjugates include, but are not limited to, oral, nasal, pulmonary, sublingual, ocular, transdermal, rectal, vaginal and parenteral (eg, subcutaneous, intramuscular, intradermal, intravenous, etc.) Administration may be via any conventional route of administration that is not intended. Subcutaneous, intramuscular or intradermal routes of administration are preferred.

In preparing the compositions of the present invention in liquid dosage form for oral, topical and parenteral administration, any conventional pharmaceutical medium or excipient may be used. Thus, for liquid dosage forms such as suspensions (e.g., colloids, fluids and dispersions) and solutions, suitable carriers and additives are pharmaceutically acceptable humidifiers, dispersions, flocculants, thickeners, pH adjusting agents (e.g. Buffers), osmotic agents, coloring agents, flavoring agents, flavoring agents, preservatives (eg, to control microbial growth, etc.), and liquid vehicles may be used. For sterile parenteral formulations, non-pyrogenic liquid vehicles are preferred. Not all of the above components are necessary to prepare each liquid dosage form.

Oral solid combinations such as, for example, dry powders for reconstitution or inhalation, granules, capsules, caplets, gelcaps, pills and tablets, each including immediate release, timed release and delayed release formulations. Carriers and additives include, but are not limited to, diluents, granulators, lubricants, binders, glidants, disintegrators and the like. Due to their ease of administration, the tablets and capsules represent the most beneficial oral dosage unit form, where solid pharmaceutical carriers are reliably used. If desired, the tablet may be sugar coated using standard techniques, or may be gelatin coated, membrane coated or enteric.

Compositions comprising α4β1-selective conjugates will comprise the amount of conjugate necessary to deliver an effective dose per unit dosage form, eg, as described herein in tablets, capsules, powders, injections, teasticks and the like. .

Wherein the pharmaceutical composition is in a unit dosage form such as tablets, capsules, powders, sterile solutions or suspensions for injection, suppositories, and the like from about 0.01 mg / kg to about 300 mg / kg (preferably about 0.1 mg / kg). Kg to about 10 mg / kg; and more preferably, from about 0.5 mg / kg to about 2.0 mg / kg) and can provide a dosage that provides at least 80% α4β1 receptor saturation over a given period of time. Dosage will vary depending on the time interval between administrations.

Preferably, the method of treating α4β1 integrin-mediated disorders described herein using α4β1-conjugates will be administered weekly or monthly, and from about 0.01 mg / kg / week to about 1 mg / kg / compound of formula (I) of week; And more preferably, a conjugate of about 0.2 mg / kg / week to about 0.5 mg / kg / week, or about 0.5 mg / kg / month to about 3 mg / kg / month, and more preferably about 1 mg Dosage forms comprising a pharmaceutically acceptable carrier comprising a conjugate of / kg / month to about 2 mg / kg / month will be used and may be reconstituted in any form suitable for the mode of administration chosen. However, dosage forms may vary depending on the subject's requirements, the severity of the condition to be treated, and the time or titration procedure required for the particular dosage regimen. For example, when used for immunosuppression for a chronic disease or transplantation, such as an autoimmune disease, the dosage regimen may be acute or chronic administration, or both, and the pharmaceutical composition may be adapted to such long term administration and dosages and pharmaceuticals. Formulated with excipients.

Advantageously, the compositions for use in the methods of the invention are tablets, for administration by oral, nasal, sublingual, intraocular, transdermal, parenteral, rectal, vaginal, dry powder inhaler or other inhalation or spraying means. Units such as pills, capsules, reconstituted or inhaled dry powders, granules, lozenges, parenteral solutions or suspensions, prefilled syringes, metered aerosol or liquid sprays, drops, ampoules, autoinjectors or suppositories It can be in the liquid form. Parenteral solutions may also be provided in bulk ampoules or vials, wherein the desired amount of drug may be withdrawn for dosing at desired intervals. Alternatively, the composition may be provided in a form suitable for less frequent administration; For example, at two weeks or once a month or at six week intervals, the formulation can be adapted to provide a combination of depots for subcutaneous injection or intramuscular injection.

For the preparation of solid pharmaceutical compositions such as tablets for use in the methods of the invention, the main active ingredient is a pharmaceutical carrier, such as conventional tableting ingredients such as diluents, binders, adhesives, disintegrating agents, Mix with ingredients such as lubricants, antiadsorbents and flow improvers. Suitable diluents are starch (eg, hydrolyzable corn, wheat or potato starch), lactose (granulated, powder dried or anhydride), sucrose-suscrose-based diluent, dextrose, inositol, mannitol , Sorbitol, microcrystalline cellulose (eg, AVICEL ™ microcrystalline cellulose, FMC Corp. (Philadelphia, PA), dicalcium phosphate salt, calcium sulfate dihydrate, calcium lactate trihydrate and the like. Binders and tackifiers include acacia gum, guar gum, tragatan gum, sucrose, gelatin, glucose, starch and cellulose (e.g. methylcellulose, sodium carboxymethylcellulose, ethylcellulose, hydroxypropylmethylcellulose, hydroxypropyl Cellulose, and the like), water soluble or dispersible binders (eg, alginic acid and salts thereof, magnesium aluminum silicate) Hydroxyethylcellulose (e.g., TYLOSE ™, Hoechst Celanese, Dallas, Texas), polyethylene glycol, polysaccharide acid, bentonite, polyvinylpyrrolidone, polymethacrylate, pregelatinized specialty) and the like Suitable disintegrating agents include, but are not limited to, starch (corn, potato, etc.), starch sodium glycolate, pregelatinized starch, clay (magnesium aluminum silicate), cellulose (eg, crosslinked sodium carboxymethyl Cellulose and microcrystalline cellulose), alginate, pregelatinized starch (e.g. corn starch, etc.), gum (e.g., agar, guar, soybean, karaya, pectin and tragatan gum), crosslinked poly Including but not limited to vinylpyrrolidone and the like.

Suitable lubricants and antiadhesives are stearates (magnesium, potassium and sodium), stearic acid, talc wax, stearrowet, boric acid, sodium chloride, DL-leucine, carbowax 4000, carbowax 6000, sodium oleate , Sodium benzoate, sodium acetate, sodium lauryl sulfate, lauryl sulfate magnesium and the like.

Suitable rheology enhancers include, but are not limited to, talc, corn starch, silica (eg, CABO-SIL ™ silica (Cabot, Boston, Mass.) SYLOID ™ silica (W. R. Grace / Davison), and AEROSIL ™ Degussa) and the like. Sweeteners and flavoring agents may be added to the chewable solid dosage forms to improve the taste of the oral dosage forms. In addition, colorants and coatings may be added or applied to solid dosage forms for easy identification of the drug or for aesthetic purposes. These carriers are formulated with pharmaceutically active agents to provide accurate, appropriate dosages of pharmaceutical actives with therapeutic release profiles.

In general, these carriers are mixed with the conjugate to make a preformulation solid composition comprising a homogeneous conjugate mixture for use in the present invention. Generally made using one of three conventional methods: (a) wet granulation, (b) dry granulation and (c) dry blending. When referring to these homogeneous preformulation compositions, the active ingredient will be dispersed evenly throughout the composition so that the composition can be readily subdivided into equal effective amounts such as tablets, tablets and capsules. These monolithic solid compositions are subdivided into unit dosage forms of the type described above comprising from about 0.1 mg to about 500 mg of the active ingredient of the present invention. Tablets or pills comprising the novel compositions can also be formulated into multilayer tablets or pills to provide sustained release or to provide dual-release products. For example, a dual release tablet or pill can include an internal dosage component and an external dosage component, which will be in the form of an envelope of the internal component. The two components can be separated by an enteric layer, which functions to resist degradation in the gastrointestinal tract, allowing internal components to pass through the duodenum intact or to delay release. . A variety of materials can be used for such enteric layers or enteric shells, for example, cellulose, cellulose acetate (eg, cellulose acetate phthalate, cellulose acetate trimetllitate), polyvinyl acetate phthalate, hydroxypropyl methylcellulose Materials including a number of polymeric materials such as phthalate, hydroxypropyl methylcellulose acetate succinate, methacrylate and ethylacrylate copolymers, methacrylate and methyl methacrylate copolymers and the like can be used. Sustained release tablets may also incorporate active ingredients in low melt solids (wet granulation) in the form of a film coating or with some soluble or insoluble material in solution (for wet granulation acting as a binder), or in molten form. Can be made by wet granulation). These materials include natural and synthetic polymer waxes, hydrogenated oils, fatty acids and alcohols such as beeswax, brazilian wax, cetyl alcohol, cetylstearyl alcohol, and the like, esters of fatty acid metallic alkali salts, and granules. Other acceptable materials that can be used for ignition, coating, collection, or other materials that can be used to limit the solubility of the active ingredient to obtain prolonged or sustained release products.

Liquid forms comprising α4β1-selective conjugates for use in the present invention may be incorporated for oral or injection administration. These forms include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or oils, as well as elmuxir and similar pharmaceutical vehicles. One is not limited thereto. Suspending agents suitable for aqueous suspensions are synthetic and natural gums such as acacia, agar, alginate (eg propylene alginate, sodium alginate and the like), guar, karaya, soybean, pectin, tragatan and Xanthan gum, cellulose, such as sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose and hydroxypropyl methylcellulose, and combinations thereof, synthetic such as polyvinyl pyrrolidone, carbox Bomber (eg, carboxypolymethylene), and polyethylene glycol; Clays such as bentonite, hectorite, attachulgite or sepiolite; And other pharmaceutically acceptable suspending agents such as lecithin, gelatin or the like. Suitable surfactants are sodium docusate, lauryl sulfate sodium, polysorbate, octoxynol-9, nonoxynol-10, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate Bait 80, polyoxamer 188, polyoxamer 235, and combinations thereof. Suitable deflocculating or dispersing agents include pharmaceutical grade lecithin. Suitable flocculants include simple neutral electrolytes (eg sodium chloride, potassium chloride, and the like), highly charged insoluble polymers and polyelectrolyte species, water soluble divalent or trivalent ions (eg calcium salts, potassium alum or sulfates, citrate and phosphate). (can be used together in the formulation as pH buffer and flocculant.) Suitable preservatives include parabens (eg, methyl, ethyl, n-propyl, and n-butyl), sorbic acid, thimerosal, 4 Divalent ammonium salts, benzyl alcohol, benzoic acid, chlorohexidine gluconate, phenylethanol, and the like.There are many liquid vehicles available for liquid pharmaceutical dosage forms, however, The liquid vehicle used must be compatible with suspending agents, for example, nonpolar liquids such as fatty ester and oil liquid vehicles. It is best used with suspending agents, such as low Hydrophilic-Lipophile Balance (HLB) surfactants, steaalkonium hectorite, insoluble resins in water, insoluble film forming polymers in water, and the like. Polar liquids such as alcohols, polyols and glycols are best used with suspending agents such as high HLB surfactants, clay silicates, gums, water soluble cellulose, water soluble polymers and the like Sterile suspensions and solutions for parenteral administration Liquid forms useful for parenteral administration include sterile solutions, emulsions and suspensions If isotonic administration is desired, isotonic combinations are generally included, which include suitable preservatives Suitable parenteral preservatives are phenol, benzyl Alcohols, phenoxyethanol, methylparaben or propylparaben and combinations thereof.

In general, the compounds of formula (I) may be dissolved in sterile saline or buffered salt solutions, optionally with suitable preservatives, at a physiologically acceptable pH compatible with the tissue or fluid into which the drug solution is injected or injected. .

α4β1-selective conjugates can also be administered in the form of liposome delivery systems such as monolayer labelula vesicles, giant monolayer lamella vesicles, multilayer lamella vesicles and the like. Liposomes can be formed from various phospholipids such as cholesterol, stearylamine, phosphatidylcholine and the like.

A therapeutically effective amount of the conjugate drug is usually supplied at a dosage level of about 0.1 mg / kg to about 300 mg / kg per kg body weight per day. Preferably, this range is about 0.2 mg / kg to about 10 mg / kg per kg body weight per day; And most preferably, from about 0.5 mg / kg to about 1.0 mg / kg per kilogram of body weight per day. Advantageously, the compounds of the present invention may be administered once a week, two weeks, or once a month, or the total daily dose may be administered in divided doses of two, three or four times daily.

Appropriate dosages to be administered can be readily determined by one skilled in the art and will vary with the particular compound employed, the route of administration, the strength of the combination and the progress of the disease state. Or, due to factors associated with the particular subject to be treated, including the subject's age, weight, meal, and time of administration, it will be necessary to adjust the dosage to the appropriate level of treatment.

Overall synthesis methods

[Alpha] 4 [beta] 1-selective conjugates for use in the methods of the present invention can be synthesized as described in general synthesis methods and more specifically in Example 1. These examples are merely illustrative and the method of producing α4β1-conjugates for use in the present invention is not limited to the chemical reactions and conditions described herein.

The components of the α4β1-selective conjugates may be provided in the form of a pharmaceutically acceptable salt. For use as a drug, salts of the compounds are described in Ref . International J. Pharm ., 1986, 33, 201-217 and J. Pharm . "Pharmaceutically acceptable salts" as described in Sci ., 1997 ( January ), 66, 1,1) . However, other salts may be used in the preparation of the compounds used in the conjugates of the present invention.

Health Conditions Available for Treatment with Compounds of the Invention

The methods of the invention are useful for the treatment of various diseases, disorders and physiological effects at least partially mediated through α4β1 integrin signaling.

In certain aspects, the methods of the invention are used to treat autoimmune disorders. A significant minority of the population suffer from autoimmune diseases, which are often chronic, debilitating, and life-threatening. In women of all ages up to age 65, one of the top ten causes of death was estimated to be an autoimmune disease. Noel R. Rose and Ian R. MacKay , " The Autoimmune There are over 80 diseases caused by autoimmunity, as described in the Diseases " fourth edition (incorporated herein by reference). These diseases include, for example, asthma, Alzheimer's disease, atherosclerosis, AIDS, and dementia. , Diabetes (including acute onset diabetes), inflammatory bowel disease (including ulcerative colitis and Crohn's disease), multiple sclerosis, rheumatoid arthritis, tissue transplantation, tumor metastasis, meningitis, encephalitis, seizures and other brain traumatic injuries, nephritis, retinitis , Sjogren's disease, atopic dermatitis, psoriasis, myocardial ischemia, and acute leukocyte-mediated lung injury that occurs in adult respiratory distress syndrome.

In certain aspects, the methods of the present invention are used to treat multiple sclerosis. As used herein, the term "multiple sclerosis" refers to multiple forms of sclerosis (RFMS), including primary progressive multiple sclerosis (PPMS), secondary multiple sclerosis (SPMS), and relapsing-remitting multiple sclerosis and other recurrent forms of the disease. Including but not limited to.

In other aspects, the methods of the present invention are used to treat an inflammatory disease. Inflammatory diseases such as atherosclerosis and restenosis, both of which affect arterial vessels, are due to a chronic inflammatory response of the arterial wall. Atherosclerosis is a major cause of cardiovascular disease, and the accumulation and sustained replacement of leukocytes is associated with the development of "vulnerable" atherosclerotic plaques, which tend to rupture, leading to thrombosis, myocardial infarction or stroke attacks. It leads. Flock macrophages are responsible for most of the leukocytes in Flock and are thought to differentiate from monocytes recruited from circulating blood through α4β1-mediated interactions. Woollard KJ and Geissmann F, Nat Rev Cardiol . 2010 Feb ; 7 (2): 77-86. Epub 2010 Jan 12 . Restenosis, an inflammatory response after intervention, such as angiogenesis, causes the coronary arteries to narrow again. This usually occurs within 3-6 months in 40-50% of patients with angiogenesis and leads to severe pathologies including myocardial infarction and ischemia.

Both autoimmune and inflammatory diseases occur through abnormal adaptations of the human adaptive or innate immune system. In autoimmunity, the patient's immune system is activated against body specific cells and / or isolated antibodies. In inflammatory diseases this pathology is due to an overreaction of the immune system and its subsequent downstream signaling. These processes are mediated at least in part through α4β1 signaling, so selective inhibition of α4β1 has enormous use in therapeutic intervention.

Other exemplary autoimmune and / or inflammatory diseases that can be treated using the conjugates and compositions of the present invention include inflammatory conditions, such as nodular erythema, allergic conjunctivitis, optic neuritis, uveitis, allergic rhinitis, ankylosing myelitis, psoriatic arthritis , Vasculitis, Reiter's syndrome, systemic lupus erythematosus, progressive systemic sclerosis, multiple myositis, dermatitis, Wegner's granulomatosis, aoritis, sarcoidosis, lymphangiopathy, transient arteritis, pericarditis, myocarditis, congestive heart failure, nodular polyarthritis , Irritable syndrome, allergy, hypereosinophilic syndrome, Churg-Strauss syndrome, chronic obstructive pulmonary disease, irritable pneumonia, chronic active hepatitis, interstitial cystitis, autoimmune endocrine failure, primary biliary cirrhosis, autoimmune aplastic anemia, chronic persistent Including but not limited to hepatitis and thyroiditis.

In other aspects, the methods of the present invention are used to treat cell proliferative disorders. α4β1 integrins have been shown to promote homing of hematopoietic progenitor cells with A4B1 and fibronectin, which are both actively treated and found in tumor-associated blood vessels. These progenitor cells are attracted to active angiogenesis sites in tumors other than normal tissues due to the presence of α4β1 in tumor vessels. Blocking α4β1 with a composition according to the method of the present invention can prevent these progenitor cells from sticking to blood vessels, prevent migration to newly formed blood vessels, and prevent them from changing to different cell types. Thus, disruption of the α4β1-mediated homing process is useful for inhibiting new blood vessel formation in tumors.

Hematological disorders, in particular leukemia and multiple myeloma, can be ameliorated by treatment with the methods of the present invention. More specifically, cell proliferative disorders such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL).

In other aspects of the invention, methods are provided for reducing the level of damage due to damage associated with invasive leukocyte activity. For example, the extent of somatic disability due to spinal injury (SCI) is associated with secondary tissue destruction, partially caused by the influx of α4β1-mediated spinal cord of neutrophils and monocytes / macrophages after initial injury. Such methods of preventing neutrophils and monocyte / macrophage influx can significantly reduce oxidative activity in the injured spine and improve the recovery of function associated with left myelin-containing white matter. Fleming JC meat al , Exp Neurol . 2008 Dec; 214 (2): 147-59. Epub 2008 May 1. Inhibition of [alpha] 4 [beta] 1, especially after injury, the method of early intervention with the compositions of the present invention can be important as a neuroprotective strategy in neurology.

Example

The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the invention, but do not limit the scope of the invention to these inventions, or There is no intention to indicate or imply uniqueness. Those skilled in the art will recognize that various changes and / or modifications can be made to the invention, as indicated in specific embodiments, without departing from the scope or scope of the invention as broadly described herein. Accordingly, embodiments of the invention are illustrative in all respects, and are not intended to be limiting.

Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some experimental errors and deviations should be taken into account. Unless stated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example  1: of formula (I) Of conjugate  production

Compounds of formula (I) used in preferred methods of the invention were synthesized using the following components:

40 kDa 3-branched PEG alcohol (0.25 g, 0.00625 mmol, NOF Corp. Japan) and small molecule inhibitor 2- (3,3-dimethyl-1- (pyridin-3-yl sulfonyl) pyrrolidine-2- Carboxamido) -3- (4- (2-oxo-1H-imidazo [4,5-b] pyridin-3 (2H) -yl) phenyl) propanoic acid (0.04 g, 0.056 mmol) and triphenyl Phosphine (0.025 g, 0.094 mmol). These components were dried using azetropin distillation from toluene (5 mL). Half the volume was distilled off (2.5 mL) and the mixture was cooled to room temperature. CH ₂ Cl ₂ (0.5 mL) was added to make the reaction homogeneous. Diethylazodicarboxylate (0.015 mL, 0.094 mmol) was added dropwise and the reaction was stirred for 48 hours. HPLC method C showed complete loss of starting PEG alcohol. The reaction was concentrated in vacuo to give a t-butyl ester of a white solid. This resulting t-butyl ester is of formula (I).

Similar to ELN 476063, conjugates of pan alpha4 beta1 and alphabeta7 inhibitors are small molecule inhibitors under the conditions of Mitsonobu, 2- (2- (diethylamino)-, as described for compounds of formula (I) above. 5- (1-oxo-1- (pyrrolidin-1-yl) butan-2-yl) pyrimidin-4-yl amino) -3- (4- (2-oxo-1H-imidazo [4, 5-b] pyridin-3 (2H) -yl) phenyl) propanoic acid was conjugated.

Detailed methods of making small molecule inhibitors are provided in published US Application No. US20070037804. Detailed methods of making conjugates with drugs comprising Formula (I) and ELN 476063 are described in published US applications US20070021555 and US20080031848, each of which is incorporated herein by reference in its entirety.

Example  2: In vitro  Efficacy study

The efficacy of the compounds of formula (I), in particular the following, was determined using several assays that independently measure α4-dependent ligand interactions: 1) Formula (I) competing with the binding of α4β1-specific multivalent ligands Ability of the compound), 2) inhibition of α4β1-dependent adhesion by human lymphocytic cells (Jurkat ™ cell line) to human VCAM-1; 3) human lymphocytic cells (Jurkat ™ TM cell line) inhibit α4β1-dependent adhesion to fibronectin; And 4) inhibition of binding of human serum FN to α4β1 activated on Jurkat ™ cells. Each of these assays confirmed the efficacy of the compound of formula (I) based on α4β1 binding.

Multi competitive analysis ( Multivalent Competition  analysis)

The efficacy of the compound of formula (I) was assessed by lymphocyte binding competition assay using α4β1-specific multivalent ligands. This ligand consists of a nonspecific antibody that acts as a carrier of several small molecules with high affinity for α4β1. This polyvalent ligand was prepared as follows.

Non-specific antibodies were dissolved in non-nucleophilic buffers and treated with EDC and sulfo-NHS, thereby activating the aspartate and glutamate side chains on the surface of the antibody. Subsequently, a small molecule inhibitor of α4β1 integrins having a primary amino group in the region of this molecule that is not critical for binding to α4β1 integrins was added to this mixture. After allowing the conjugation reaction to proceed for an appropriate period of time, the reaction mixture was quenched by the addition of 2-hydroxyethylamine. Next, non-specific antibodies showing multiple copies of the small molecule α4β1 inhibitor were isolated from low molecular weight contaminants by dialysis. This multivalent ligand showed high affinity for Jurkat ™ cells that exhibit α4β1 integrins, and competition in this interaction is a means of distinguishing the efficacy of very potent α4β1-inhibiting compounds. Thus, this assay is a very rigorous assay used to characterize the efficacy of the molecule of formula (I). The binding of the compound of formula (I) to α4β1 was explained by a competitive binding assay demonstrating the ability of formula (I) to replace this polyvalent ligand in human lymphocytes.

Jurkat ™ cells were incubated for 30 min at room temperature in assay buffer with titration of formula (I) in the presence of a multivalent ligand diluted 1: 400. Unbound reagents were then removed by several washing steps, and cells were pelleted for 5 min in a Beckman tabletop centrifuge at 300 × g and then resuspended in fresh buffer. The remaining bound antibody multivalent ligand was incubated for 30 minutes at 4 ° C. with these cells with goat F (ab) ′ ₂ anti-mouse IgG (Fc) -phycoerythrin (Beckman-Coulter Inc., Brea CA), After washing it was detected by FACS analysis.

Compounds of formula (I) have been shown to significantly change the binding of this polyvalent ligand. This inhibition was reproducible and consistent between lots tested with this molecule and showed an IC 50 of 18.8 nM and 17.7 nM (see FIG. 1). This potency of formula (I) did not change in the presence of 100% serum of different species, demonstrating that formula (I) did not bind to serum isolated antibodies (data not shown).

α4β1-dependent adhesion: rs - hu - VCAM On -1 Jurkat ™ adhesive

Jurkat ™ lymphoma T cells express α4β1 integrin at high levels and strongly adhere to VCAM-1 in an α4 integrin-dependent manner. Assays evaluating the inhibition of this adhesion were also used to determine the efficacy of the molecule of formula (I). The ability of formula (I) to interfere with adhesion was compared with inhibition with formula (III) (a single small molecule component of formula (I)), and an anti-α4 antibody, 21/6.

96-well plates (Costar 3590) were coated with rs-hu-VCAM-1 (R & D Systems, Santa Cruz, CA) using 0.25 μl per well in PBS ++ (PBS + 1 mM CaCl ₂ + 1 mM MgCl ₂ ), Incubated overnight at 4 ° C. After incubation, the plates were blocked with assay buffer (20 mM Hepes, 140 mM NaCl, 1 mM CaCl ₂ , 1 mM MgCl ₂ and 0.3% BSA) for 30 minutes at room temperature, and washed three times with assay buffer. Fluorescently labeled Jurkat ™ cells (Calcene Fluorescent Dye, Molecular Probes, Carlsbad, Calif.) Were added in 96-well FlexiPlate (Falcon 353911) at 10 ⁵ cells per well in assay buffer (final volume: 100 μl per well). After addition of this conjugate, compound or antibody at various concentrations, these cells were incubated for 30 minutes at room temperature. Cells were then transferred to rs-hu-VCAM-1-coated plates and allowed to bind for 30 minutes at room temperature. This plate was washed three times with assay buffer and fluorescence was read (Applied Biosystems, CytoFluor series 4000, Foster City, CA).

Formula (I) strongly inhibited α4β1-dependent adhesion of Jurkat ™ cells to human VCAM-1 with an EC50 of 0.5 nM (FIG. 2). Interestingly, although there are three small α4β1 antagonist molecules per compound of formula (I), formula (I) appears to be at least 23 more potent than formula (III) (EC50 of 0.5 nM vs 11.6 nM, in turn).

α4β1-dependent adhesion: human Fibronectin Jurkat ™ cell adhesion

Jurkat ™ cells adhere to fibronectin (FN) in a μ4β1-dependent manner. Inhibition of this adhesion with Formula (I), Formula (III) and anti-α4 antibody, 21/6 was also used to determine the efficacy of the molecule of Formula (I).

96-well plates (Costar 3590) were coated with fibronectin (FN) (Calbiochem) at 0.6 μl per well in PBS ++ (PBS + 1 mM CaCl ₂ + 1 mM MgCl ₂ ) and incubated overnight at 4 ° C. This plate was then blocked with assay buffer (20 mM Hepes, 140 mM NaCl, 1 mM CaCl ₂ , 1 mM MgCl ₂ and 0.3% BSA) at this room temperature for 30 minutes, and washed three times with assay buffer. 10 μg of TS2 / 16 antibody against β1 activating α4β1 in assay buffer using Jurkat ™ cells fluorescently labeled at 10 ⁵ cells per well (calcined fluorescent dye, Molecular Probes, C-1430, according to the manufacturer's procedure) / Ml was added to 96-well FlexiPlate (Falcon 353911) (final volume: 100 μl per well). An inhibitory antibody against α5 integrin, 5 μg / ml (BD Pharmingen, Franklin Lakes, NJ) is added to the buffer to prevent α5-dependent adhesion to FN. After addition of compound or anti-α4 antibodies (21/6, GG5 / 3, or HP2 / 1) at various concentrations, these cells were incubated for 30 minutes on wet ice. Cells were then transferred to FN-coated plates and allowed to bind for 35 minutes at room temperature. This plate was washed three times with assay buffer and fluorescence was read (Applied Biosystems, CytoFluor series 4000).

The interaction of FN with α4β1 is less stringent than the VCAM-1 interaction, as illustrated by the ECCAM of 9.7 nM of 21/6 shifted to 1.1 nM in FN analysis in the VCAM-1 assay (see FIG. 3). Formula (I) inhibited FN adhesion with an EC 50 of 0.2 nM, similar to its small molecular component (0.1 nM). The two compounds of formula (I) and formula (III) are very potent compounds, thus reaching the sensitivity of this assay, so that the difference in efficacy can no longer be measured.

human In serum  α4β1- FN  Capture analysis

Α4β1 receptors on the Jurkat ™ cell surface can capture endogenous FN present in serum in solution. Inhibition of this monovalent interaction was also used to evaluate the efficacy of formula (I).

Log-phase Jurkat ™ cells were incubated in 96-well FlexiPlate for 30 minutes at room temperature under the following conditions: 50% human serum, 2x (20 μg / ml) of inhibitory antibodies to α5 integrin (BD Pharmingen, Franklin Lakes, NJ) and 10 ⁵ cells / 50 μL per well in assay buffer containing 20 × compound or antibody at various concentrations (20 mM Hepes, 140 mM NaCl, 1 mM CaCl ₂ , 1 mM MgCl ₂ , and 0.3% BSA). Next, an equal amount of assay buffer comprising 50% human serum, antibody 15/7 (2x, 20 μg / ml) and MnCl ₂ (2x, 3 mM) was added to each well. These reagents act as α4 integrin activating substances, allowing this receptor to capture FN from serum. This plate was then incubated for an additional 30 minutes at room temperature. Cells were then washed twice with cold assay buffer and incubated for 30 minutes with 15 μg / ml chicken anti-human FN antibody (American Research Products, Palos Verde, CA) in cold assay buffer on ice under dark conditions. . Cells were then washed once and resuspended with 300 μl of cold assay buffer for FACS analysis (Becton-Dickinson, Franklin Lakes, NJ).

Formula (I) inhibited FN capture with 0.05 nM EC50 in 50% human serum (FIG. 4). In this assay, formula (III) was less potent than formula (I), but there was only a 2.6-fold difference (unlike the 23 differences observed in the VCAM-1 adhesion assay). Formula (I) was about 6 times more potent than the anti-α4 antibody 21/6 (EC50 = 0.32 nM). This assay, which measures the interaction of this receptor with monovalent soluble ligands, has a lower stringency than the VCAM-1 cohesive assay involving multivalent interactions.

Example  3: In vitro  Selectivity study

To characterize its specificity, Formula (I) was tested in several cell adhesion assays dependent on integrins other than α4β1 and multivalent ligand assays using α9β1, the integrin most closely associated with α4β1. The specificity of formula (I) was assessed by examining its activity in four non-α4β1 integrin-dependent assays: 1) α4β7-dependent adhesion to α4β7; 2) αLβ2 (LFA-1) -dependent adhesion to ICAM-1; 3) α5β1-dependent adhesion to human fibronectin (FN); And 4) multivalent competition on α9β1 cells. Each of these is briefly described as follows.

α4β7-dependent adhesion: α4β7- Fc 8866 cell adhesion against

Wells of 96-well plates (Costar 3590) were incubated for 1 hour at room temperature with 1: 300 antibody dilution in H / S ++ (20 mM Hepes, 140 mM NaCl, 1 mM CaCl ₂ , 1 mM MgCl ₂ ) It was coated with mouse antibody against human Fc (Sigma. I-6260). This plate was blocked with assay buffer (20 mM Hepes, 140 mM NaCl, 1 mM CaCl ₂ , 1 mM MgCl ₂ and 0.3% BSA) for 1 hour at room temperature and then washed three times with the same buffer. Recombinant human α4β7-Fc in assay buffer (20 mM Hepes, 140 mM NaCl, 1 mM CaCl ₂ , 1 mM MgCl ₂ and 0.3% BSA) was added to the wells at 30 ng per well and incubated at 4 ° C. overnight. This plate was then washed three times with assay buffer. Fluorescently labeled 8866 cells (Calcene Fluorescent Dye, Molecular Probe. C-1430, used according to the manufacturer's procedure) were prepared in 96-well FlexiPlate (Falcon 353911) at 10 ⁵ cells per well (final volume: 100 μl per well) in assay buffer. Was added. After addition of compound or anti-α4 antibodies (21/6, GG5 / 3 or HP2 / 1) at various concentrations, these cells were incubated for 30 minutes at room temperature. Cells were then transferred to A4β7-Fc coated plates and allowed to bind for 30 minutes at room temperature. Plates were washed three times with assay buffer and fluorescence was read (Applied Biosystems, CytoFluor series 4000, Foster City, CA).

αLβ2-dependent adhesion: ICAM -One Fc 8866 cell adhesion to

96-well plates (Costar 3590) were coated with mouse ascites anti-Hu Fc (Sigma-Aldrich, St. Louis, Mo.) 1: 300 in H / S ++ for 1 hour at room temperature. The plate was blocked with assay buffer (20 mM Hepes, 140 mM NaCl, 1 mM CaCl ₂ , 1 mM MgCl ₂ and 0.3% BSA) for 1 hour at room temperature and washed three times with the same buffer. Rhuman-ICAM-1Fc (R & D Systems, Santa Cruz, Calif., 720-IC) was captured overnight at 4 ° C. at 40 ng per well in assay buffer, and the plates were washed again three times with assay buffer. Calcein-labeled 8866 cells at 10 ⁵ cells per well in assay buffer (final volume: 100 μl per well) were pre-incubated for 30 minutes at room temperature with 50 ng / mL PMA and a series of concentration range compounds in 96-well FlexiPlate. It was. Anti-αLβ2 (Pharmingen, 555381), 10 μg / ml, was also used as a positive control to inhibit αLβ2-dependent binding. Cells were then transferred to ICAM-1-Fc coated plates and allowed to bind for 35 minutes at 37 ° C., 5% CO ₂ (culture). This plate was washed three times with assay buffer and fluorescence was read (Applied Biosystems, CytoFluor series 4000.

α5β1-dependent adhesion: human FN For THP -1 cell adhesion

96-well plates (Costar 3590) were coated overnight at 4 ° C. with FN (Calbiochem, Gibbstown, NJ) at 0.6 μl per well in PBS ++ (PBS + 1 mM CaCl ₂ + 1 mM MgCl ₂ ). This plate was then blocked with assay buffer (20 mM Hepes, 140 mM NaCl, 1 mM CaCl ₂ , 1 mM MgCl ₂ , and 0.3% BSA) for 30 minutes at room temperature and washed three times with assay buffer. Calcein-labeled THP-1 cells in assay buffer (final volume: 100 μl per well) at 10 ⁵ cells per well were labeled with 10 μg / ml 21/6, 10 μg / ml TS2 / 16 and a series of concentration ranges in 96-well FlexiPlate. Pre-incubated at room temperature with compound of for 30 minutes. Anti-α5 antibody (Pharmingen Franklin Lakes, NJ), 5 μg / ml, was also used as a positive control to inhibit α5β1-dependent binding. Cells were then transferred to FN-coated plates and allowed to bind for 30 minutes at room temperature. This plate was washed three times with assay buffer and fluorescence was read (Applied Biosystems, CytoFluor series 4000.

Competitive Binding to α9β1

Log-phase SW480-expressing α9β1 cells were incubated in 96-well FlexiPlate (Falcon 353911) under the following conditions: assay buffer (20 mM Hepes, 140 mM NaCl, 1 mM CaCl ₂ , 1 mM MgCl ₂ and 0.3%). Small molecule that selectively binds 10 ⁵ cells / 100 μl per well in BSA) and α9β1 conjugated to an irrelevant mouse antibody. α9β1 binder was provided at its EC90 concentration (1: 400 dilution), and formula (I) was provided in a series of concentration ranges. Incubation was performed at room temperature for 30 minutes. Unbound reagents were then removed by two washes in assay buffer. The remaining bound α9β1 binding molecule was detected by goat Fab'2 anti-mouse IgG (Fc) -PE (Immunotech, Miami, FL) used at 1: 200 dilution in assay buffer for 30 minutes on ice in the dark. . Cells were then washed once and resuspended in 300 μl cold assay buffer for FACS analysis (Becton-Dickinson, Franklin Lakes, NJ).

result

Formula (I) did not have measurable activity against αLβ2 (LFA-1) and α5β1 integrins, and in α4β7 and α9β1 in turn possessed measurable activity of EC 50 of 38 nM and 51 nM. The efficacy of formula (I) for α9β1 was in the 3-fold range of efficacy for α4β1 (mean MV competition EC50 α4β1 = 16.5 nM vs EC50 α9β1 = 51 nM). Table 1 summarizes the results.

Formula (I) showed a> 75-fold preference for inhibiting the activity of α4β1 over α4β7; weak cross reactivity to α9β1, which is expected based on homology between α4 and α9 and between overlapping ligands; And did not retain activity against αLβ2 (LFA-1) and α5β1 integrins. Table 1 summarizes the specificity data:

Example  4: α4β1-selective Conjugate  Human Pharmacokinetics

The plasma pharmacokinetic behavior of the α4β1-selective compounds of formula (I) was evaluated.

Healthy human subjects between 18 and 45 years of age enrolled in this study. Thirty-nine healthy men and women between 18 and 45 years of age (including the limit) enrolled in this study. The subjects were divided into six groups and the subjects were dosed with 0.05, 0.1, 0.2, 0.5, 1.0 and 2.0 mg / kg of compound in turn. Peripheral venous blood samples were drawn from each subject into the collection tube via direct venipuncture. 4.0 mL of blood samples were collected in heparin sodium from each subject to determine plasma concentrations of the formula (I) conjugates at each of the following time periods: before dosing, 2 hours, 4 hours, 8 hours after dosing. , 12 hours, 16 hours, 24 hours, 30 hours, 36 hours, 48 hours, 72 hours, 96 hours, 120 hours, 168 hours, 240 hours, 336 hours, 504 hours, 672 hours, 1440 hours and 2160 hours. Liquid Chromatography Tandem Mass Spectrophotometer (LC / MS / MS) detection was used to determine the formula (I) concentration in plasma of subjects who received this conjugate. A summary of these results is provided in FIG. 5.

The lower limit of quantification of formula (I) (LLOQ) was 10.0 ng / mL, and the scale curve was acceptable in the range of 10.0-1000 ng / mL. Noncompartmental pharmacokinetic (PK) parameters were calculated using WinNonlin 5.2 software (Pharsight Corporation, Mountain View, CA). Table 2 is a summary of preliminary PK parameter estimates in the first five populations.

Abbreviation: AUC _{0 -} area under the curve from _inf = 0 to infinity; Area under the curve from AUC _{0 - last} = 0 to the last measurable concentration; CL / F = uncorrected removal for bioavailability; C _max = maximum concentration; hr = hour; Kg = kilograms; mL = milliliters; Μg = microgram; ND = not measured; T _max = time to maximum concentration observed; Vz / F = uncorrected distribution volume for bioavailability.

 At all times of the oral time of population 1 (0.05 mg / kg), the concentration of formula (I) was maintained below LLOQ of 10 ng / mL. No PK parameter evaluation was performed for this population. Formula (I) exposure was increased beyond that expected for the 0.1, 0.2, 0.5, 1.0 and 2.0 mg / kg dose groups. Elimination half-life is difficult to assess due to the non-linear nature of the concentration curve over time.

In summary, after subcutaneous (SC) administration of Formula (I), AUC increased beyond the dose proportional mode due to dose related extension in elimination half-life of Formula (I). C _max also increased beyond what was expected. The PK behavior of formula (I) was characterized by decreasing the apparent volume of systemic removal and distribution as the dosage was increased. This initial study supports the formula (I) preferred dosage range of 0.2-0.5 mg / kg weekly and 1.0-2.0 mg / kg monthly.

Example  5: Integrin  Receptor Saturation Assay

Peripheral blood lymphocytes were collected from blood samples from the six populations of humans described in Example 4 and analyzed for saturation of the α4β1 and α4β7 receptors and the total α4 receptor number.

α4β1 integrin saturation, α4β7 integrin saturation, and total α4 receptor number were measured in harvested circulating lymphocytes. 2 ml of lysis buffer was added to each tube and these tubes were vortexed for 2-4 seconds. The tube was again incubated for 13-17 minutes at 2-8 ° C. and covered with aluminum foil to reduce light exposure. At the end of this incubation, the cells were vortexed and centrifuged at 2200 rpm for 5 minutes at 4-15 ° C. The supernatant was aspirated off and the pellet was resuspended in 0.5 ml of Ortho fixed solution [PBS (phosphate-buffered saline solution), 1% formaldehyde in pH 7.6]. A 13X solution of Formula (I) and a 13X solution of 15/7 or 2G3 were provided in assay medium containing 5% volume of FBS in 1X PBS.

α4β1 integrin saturation assay was performed with ligand-induced binding site antibody, 15/7, and recognizes morphological changes in the β chain of integrins when formula (I) binds to heterodimers. These morphological changes reveal epitopes recognized by mouse-antihuman antibodies. Picoerythrin-labeled donkey anti-mouse IgG was used as a reporter. Flow cytometry was performed using a FACSCalibur ™ flow cytometer (Becton Dickinson, Franklin Lakes, New Jersey) to evaluate the percent saturation, and excess formula (I) was applied to different fractions of the blood sample to determine 100% saturation. Added. A commercially available murine monoclonal anti-human antibody against the β1 integrin chain, mAB13, was added to different blood fractions to obtain 0% saturation. mAb13 abolished 15/7 binding and was used to establish basal levels for α4β1 analysis.

α4β7 integrin saturation assay was performed with antibody 2G3 which selectively binds to the β7 chain of integrin. Whole blood was collected in heparin sodium tubes, and formula (I) was added to the blood fractions to a final concentration of 25 μg / ml to prepare saturated samples. Formula (I) was added to the blood aliquot so that the final concentration was 5 μg / ml to prepare a background sample. Test fractions (not containing formula (I)) were treated with 2G3 and binding of formula (I) to lymphocytes was detected using F (ab ') ₂ anti-mouse IgGPE. Monoclonal anti-human antibody, inhibitory monoclonal antibody FIB27 was added to different fractions of blood to commercially available β7 integrin chains to obtain 0% saturation. FIB27 abolished the binding of 2G3 and was used to establish the basal level for α4β7 analysis.

PE fluorescence of lymphocytes was analyzed using FACSCalibur ™ flow cytometry, and the mean fluorescence intensity (MFI) for test samples, background and saturated cases was measured in all of these assays.

Two saturation percentages of α4β1 and α4β7 were calculated by subtracting the background measurement determined from the test sample measurement, dividing it by (saturation level measurement—background measurement), and multiplying the divided number by 100.

At all doses, 70-100% saturation of β1 receptors occurred in populations 4-6, which lasted up to 14 days (FIG. 6). Correspondingly, the number of α4 receptors was reduced at all dose levels (data not shown). The saturation rate of α4β7 was investigated starting with population 4 (0.5 mg / kg). At this dose and at higher doses, the saturation of α4β7 was ≧ 80%. Receptor occupancy of α4β1 and α4β7 appears to be comparable at ≧ 0.5 mg / kg doses for at least 1 week. α4β1 saturation lasts longer than α4β7 saturation (at 0.5 mg / kg; lower doses were not tested for α4β7 saturation).

Example  6: after treating a human subject with formula (I) Integrin  Down regulation of receptors.

Peripheral blood lymphocytes were harvested from the human population who received 0.5 mg / kg as described in Example 4. 10 μl of mouse anti-α4 integrin antibody, 9F10-PE (Biolegend, San Diego, Calif.), Which cross reacts with human α4 integrin was added to the tube to measure overall anti-α4 downregulation, which was vortexed for about 2 seconds. It was. The tubes were then incubated at 2-8 ° C. for 30-50 minutes.

2 ml of lysis buffer was added to each tube and these tubes were vortexed for 2-4 seconds. The tube was again incubated for 13-17 minutes at 2-8 ° C. and covered with aluminum foil to reduce light exposure. At the end of this incubation, the cells were vortexed and centrifuged at 2200 rpm for 5 minutes at 4-15 ° C. The supernatant was aspirated off and the pellet was resuspended in 0.5 ml of Ortho fixed solution [PBS (phosphate-buffered saline solution), 1% formaldehyde in pH 7.6]. Mean fluorescence intensity (MFI) for 9F10-PE binding was measured and normalized to the 0 hour MFI (base level) of the same sample to calculate% receptor downregulation. MFIs of α4β1 and α4β7 were measured using standardized MFI of 15/7 or 2G3 antibody assays as described in Example 4.

Receptor downregulation results in human test populations receiving Formula (I) at a 0.5 mg / kg dose are shown in FIGS. 7 and 8. FIG. 7 shows the averaged downregulation within the population, and FIG. 8 shows the results of individuals receiving these doses. Total α4 and α4β1 receptor downregulation was relevant. No significant α4β7 receptor down regulation was observed.

Example  7: Dose effect of formula (I) on lymphocyte count and receptor occupancy.

The physiological effects of different dosages of formula (I) in human subjects were compared with the traditional α4 targeting therapeutics, Natalizumab (Tysabri ™). Natalizumab (Tysabri ™) is an anti-α4 antibody that binds to and regulates both α4β1 and α4β7.

As described in Example 4, peripheral blood lymphocytes were harvested from various human populations receiving a single dosage of 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, 1.0 mg / kg or 2.0 mg / kg, and Lymphocyte counts were measured as a percentage of basal level on day 6 post initial dosing. Peripheral venous blood was drawn into the collection tube by direct venipuncture from each subject. 4.0 ml blood samples were collected from each subject. Lymphocyte levels were measured by standard clinical protocols.

FIG. 9 is a bar graph showing patient lymphocyte counts at different dosages of formula (I), and FIG. 10 is a line graph illustrating the correlation between plasma concentration of formula (I) and relative lymphocyte counts in a patient. Each of these dose groups showed lower levels of lymphocyte counts compared to patients receiving comparable levels of Natalizumab (Tysabri ™).

Example  8: Effect of Formula (I) Dosage on Receptor Saturation

The effect of different dosages of formula (I) on α4β1 and α4β7 receptor saturation, occupancy and expression in lymphocytes was measured in dose escalation studies. Methods for determining receptor saturation were as shown in Example 5. These levels were followed for 91 days after initial dosing in different groups.

As shown in FIG. 11, 0.1 mg / kg −2 mg / kg of α4β1 receptors were completely saturated in lymphocytes for at least 1 week. In contrast, α4β7 receptor saturation returned to baseline 14 days after dosing. In certain populations, this measured saturation level appeared to be independent of the expression level, indicating a demonstration of selectivity. For example, as shown in FIG. 12, in group 4 the α4β1 saturation level remained high at 360 hours after dosing, while the saturation of α4β7 fell below 30%. As shown in FIG. 13, the expression level of α4β7 remained substantially higher than that seen for α4β1. Higher doses of Formula (I), 1.0 mg / kg, provided to population 5, resulted in higher saturation levels of both α4β1 and α4β7, as shown in FIG. 14. This explains that the saturation level of these receptors is dose dependent and can be adjusted to the appropriate dosage and regime.

Example  9: Dosage Selection

Preferred dosages of formula (I) are effective through their α4β1 regulation but minimize any side effects caused by α4β7. To assess the lowest known dose resulting in α4β1 receptor saturation of ˜90%, dosage ranges and dosage regimens that provide distinct levels and duration of α4β1 receptor saturation are determined. This assessment measures the lowest dose that is likely to result in acceptable α4β1 receptor saturation while reducing α4β7 saturation. The results of these studies are summarized in Table 3:

Example  10: α4β1 according to formula (I) Mediated  Selective inhibition of cell adhesion

Inhibition of α4β1 and α4β7-mediated cell adhesion by compounds of formula (I), in turn, for α4β1 and α4β7, respectively, was examined using a cell-based SRU BIND ™ cell adhesion assay (SRU Biosystems, Woburn, Mass.). Compounds of formula (I) were incubated with α4β1 + Jurkat cells or α4β7 + 8866 cells in a concentration range. For comparison purposes, the ELN476063 was also tested. Compound-cell mixtures were added to SRU BIND ™ assay plates coated with α4β1 or α4β7, and cell adhesion was measured by PVW shift.

Briefly, TiO ₂ SRU BIND ™ assay plates were coated with anti-human Fc antibody followed by human Fc-α4β1 or Fc-α4β7. Formula (I) and ELN476063, PEGylated small molecule α4-selective integrin inhibitors, were incubated with α4β1 + Jurkat cells or α4β7 + 8866 cells in a concentration range. Compound-cell mixtures were then added to SRU BIND assay plates to initiate cell adhesion and measured by PVW shift. 100% inhibition of adhesion is defined as the PWV shift observed in cells pre-treated with saturation concentrations of anti-α4 integrin blocking antibody, and 0% inhibition (maximal cell adhesion) refers to untreated Jurkat or 8866 cells α4β1 or α4β7 Defined as PWV shift observed by incubation in turn on plates.

Compounds of formula (I) inhibited adhesion of α4β1 + cells to VCAM-1 approximately 150 times stronger than the adhesion of α4β7 + cells to α4β7 (IC50 0.4 and 60 nM) (FIG. 15A). In contrast, ELN476063 equally inhibited α4β1- and α4β7-mediated cell adhesion, in turn, against α4β1 and α4β7 (IC50 1.7 and 1.2 nM) (FIG. 15B), wherein the compound of Formula (I) was at least four times more than ELN470603 4β1 adhesion was strongly inhibited.

Additionally, compounds of formula (I) inhibited α4β1-mediated adhesion more than four times more potent than ELN470603, which is a potent and selective inhibitor of α4β1-mediated adhesion, but It is not about the adhered adhesive.

Example  11: α4β7 Ligand Madcam Α4β1 rather than -1 Ligand VCAM Selective Down-regulation of Soluble Types of -1

Inhibition of α4 integrins was shown to be the cause of downregulation of the soluble circulating form of these ligands, VCAM-1 and MAdCAM-1 ( Millionig meat al ., J. Neuroimmunology Oct 8; 227 (1-2): 190-4. Epub 2010 Aug 23 (2010) ). Murine plasma levels of sVCAM-1 and sMAdCAM-1 were measured in a single dose experiment over a three week concentration range for compounds of formula (I) and ELN 476063. The sVCAM-1 and sMAdCAM-1 regulatory kinetics after a single, subcutaneous dosing of a compound of Formula (I) or ELN 476063 was calculated over 21 days.

Naive C57BL / 6 mice were treated with PBS (vehicle), a compound of formula (I) or ELN 476063 in a single, subcutaneous injection in the dosage range. Four mice were dosed at each level / time period. This was compared with the effect of non-selective conjugates. Both compounds of formula (I) and ELN 476063 downregulated sVCAM-1 and sMAdCAM-1 rapidly and dose-dependently, which recovered when drug levels were reduced.

The 0.5 mg / kg dose in mice was evaluated equivalent to the human 0.2 mg / kg dose (based on C _max ). A dose of 0.2 mg / kg in humans will result in a C _max value (0.57 μg / ml) comparable to the C _max (0.83 ng / mL) value obtained at the 0.5 mg / kg dose in mice. The 0.5 mg / kg dose in mice was sufficiently effective to block acute CNS cell trafficking. Using the same assay, a human dose of 0.5 mg / kg will result in a C _max value of 3.7 μg / ml, comparable to a mouse dose of 1 mg / kg (5.5-4.3 μg / ml). A human dose of 0.5 mg / kg is approximately equivalent to a mouse dose of 1.0 mg / kg (based on C _max value), which was also sufficiently effective to prevent acute CNS cell visits.

Figures 16A-16F show sVCAM-1 (Figures 16A and 16B), sMAdCAM-1 (Figures 16C and 16D), and plasma drug levels (Figures 16E and F) at various time points over a period of 4 hours to 21 days post dose. Indicates. 50% down-regulation of soluble adhesive molecules based on average levels in vehicle-treated animals is indicated by dotted lines in the graph of FIG. 16 as a reference point. These doses resulted in rapid, dose-dependent down-regulation of sVCAM-1 and sMAdCAM-1 and recovered when drug levels decreased (see graphs E and F).

 For each mouse used in the experiments summarized in FIG. 16, the compound of formula (I) and the plasma drug concentration of ELN 476063 versus sVCAM-1 or sMAdCAM-1 are indicated. Curves were fitted using variable-slope non-linear regression. Consistent with in vitro selectivity data, compounds of formula (I) more strongly down-regulated sVCAM-1, but also had the lowest effect on sMAdCAM-1 levels when compared to ELN 476063. Maximum inhibition of sVCAM-1 was seen at the plasma concentrations of compounds of formula (I), such as 3 nM, while sMAdCAM-1 was not sufficiently down-regulated even at maximum plasma concentrations (FIG. 17A). In contrast, sVCAM-1 and sMAdCAM-1 were similarly down-regulated at equivalent plasma concentrations of ELN 476063 (FIG. 17B).

Selective downregulation of the soluble form of the α4β1 ligand VCAM-1 over the α4β7 ligand MAdCAM-1 maintains in vitro selectivity in vivo through the selective regulation of soluble adhesive molecules, and in vivo through the regulation of sVCAM-1 over sMAdCAM-1. As shown, the compounds of formula (I) point to in vitro selectivity and potency for α4β1 over α4β7.

Example  12: Visiting cells by administration of formula (I) Trafficking ) Selective suppression

Radiolabeled splenocytes and lymph node cells were adopted for transfer into treated EAE mice and quantified total radioactivity in various organs. Gritty Briefly, were injected subcutaneously with _{35 -55} MOG peptide emulsified in CFA, followed by induction of experimental autoimmune encephalomyelitis (EAE) in C57BL / 6 mice by the intraperitoneal injection of pertussis toxin at the time 0 and 2. At 12 days post-induction, mice with clinical value 2 (affected by hind gait) were enrolled in this study and subcutaneously dosed with PBS, ELN476063, or Formula (I) in the dosage range. Splenocytes and mesenteric lymph node cells from naïve mice were labeled in vitro at ¹¹¹ indium and oceanicized overnight, and were transferred to EAE mice registered at day 13 of induction. Approximately 16 hours after cell-migration, organs were harvested, weighed and radioactivity quantified using a gamma counter.

Compounds of formula (I) were able to significantly inhibit trafficking of radiolabeled cells into the spinal cord and brain of EAE mice at doses between 0.5 and 3.0 mg / kg, but at 0.1 mg / kg and below It was not possible (Figures 19B, 19C, 19E and 19F). The compound of formula (I) did not have a significant effect on cellular trafficking with the Peyer patch of the small intestine, which was likely mediated in part by α4β7 interaction (FIGS. 19A and 19D). Inhibition of cell traffic into Peyer patches and CNS was observed at ELN 476063 at 10 and 1.0 mg / kg doses.

sVCAM-1 was down-regulated at the compound dosage of formula (I) which inhibits CNS visit but not downregulated at 0.1 mg / kg (FIG. 18A), indicating that sVCAM-1 is not only drug level but also drug activity Indicates a strong sign. Significant inhibition of cell-going to the CNS was seen in compounds of formula (I), which did not significantly affect sMAdCAM-1 levels, with compounds of formula (I) without interfering with α4β7-dependent immune cell navigation. Explain that CNS inflammation was controlled (FIG. 18B). In fact, as can be seen in FIG. 15B, the level of sMAdCAM in plasma (related to the level expressed in the bloodstream) remained at least 75% normal after dosing of Formula (I) at a dose up to 3 mg / kg. . Thus, administration of formula (I) does not affect signal generation through sMAdCAM at levels shown to have statistically significant downregulation of sVCAM-1 levels.

The foregoing merely illustrates the principles of the invention. Those skilled in the art will recognize that various arrangements that embody the principles of the present invention, although not explicitly described or shown herein, will be understood to fall within the scope and spirit of the present invention. Moreover, all of the embodiments and conditional terms mentioned herein are intended primarily to help the reader understand the principles of the present invention and the concepts contributed by the inventors and to the embodiments and conditions specifically mentioned. For, but not limited to. Moreover, all statements referring to the principles, aspects, and embodiments of the present invention, as well as specific embodiments thereof, are intended to include structural and functional equivalents thereof.

Claims (42)

Use of a therapeutically effective amount of formula (I) in a subject to treat a disease mediated by trafficking into the CNS of α4β1-mediated leukocytes while substantially protecting the α4β7-mediated visit. The use of claim 1, wherein the dosage is about 0.01 mg / kg to about 5 mg / kg. The use of claim 2, wherein the dosage is about 0.2 mg / kg to about 1.0 mg / kg. The use according to claim 3, wherein the dosage is about 0.2 mg / kg to about 0.5 mg / kg. The use of claim 1, wherein the dosage is about 1.0 mg / kg to about 2.0 mg / kg. Use according to any one of claims 2, 3, 4 or 5, wherein the compound of formula (I) is administered to the subject once a week. Use according to any one of claims 2, 3, 4 or 5, wherein the compound of formula (I) is administered to the subject once a month. The dosage of formula (I) is selected to maintain the subject's blood sMAdCAM level at 75% or higher after administration as compared to the subject's blood sMAdCAM level prior to administering formula (I). Usage. In the use of a therapeutically effective amount of a conjugate in a subject to modulate a biological process that is at least partially mediated through α4β1 activity, the conjugate may be capable of at least two α4β1 small molecule antagonists covalently attached to a biocompatible polymer. And wherein the conjugate has 20 to 100 times greater efficacy in directing α4β1 than α4β7. The use of claim 9, wherein the conjugate has 30 to 80 times greater efficacy in directing α4β1 than α4β7. Use according to claim 9 or 10, wherein the conjugate comprises a compound of formula (I). The use according to any one of claims 9, 10 or 11, wherein the conjugate is administered to a subject with autoimmune disease. The use of claim 12, wherein the conjugate is administered to a subject with multiple sclerosis. Use according to any one of claims 9, 10 or 11, wherein the conjugate is administered to a subject with an inflammatory disease. The use according to any one of claims 9, 10 or 11, wherein the conjugate is administered to a subject with a cell proliferative disorder. The use according to any one of claims 9, 10 or 11, wherein the conjugate is administered to a subject having a graft rejection or graft versus host disease. Use according to any one of claims 9, 10 or 11, wherein the conjugate is administered to the subject to promote neuroprotection after injury. The method of any one of claims 9, 10 or 11, wherein the dosage or interval of administration of the conjugate is 75% of the subject's blood sMAdCAM level after administration compared to the subject's blood sMAdCAM level prior to administration of Formula (I). Or selected to maintain more. In a therapeutically effective amount of a conjugate comprising two or more α4β1 small molecule antagonists covalently attached to a biocompatible polymer, the conjugate has the effect of directing α4β1 over α4β7 in the treatment of subjects with autoimmune diseases. Uses 20 to 100 times larger. The use of claim 19, wherein the autoimmune disease is multiple sclerosis. The use of claim 19, wherein the dosage is about 0.01 mg / kg to about 5 mg / kg. The use of claim 21, wherein the dosage is about 0.2 mg / kg to about 1.0 mg / kg. The use of claim 22, wherein the dosage is about 0.2 mg / kg to about 0.5 mg / kg. The use of claim 21, wherein the dosage is about 1.0 mg / kg to about 2.0 mg / kg. Use according to any one of claims 19, 20, 21, 22, 23 or 24, wherein the conjugate is administered to the subject once a week. Use according to any one of claims 19, 20, 21, 22, 23 or 24, wherein the compound of formula (I) is administered to the subject once a week. For the use of a therapeutically effective amount of a conjugate comprising two or more α4β1 small molecule antagonists covalently attached to a biocompatible polymer, the conjugate has an efficacy directed at α4β1 over α4β7 in the treatment of subjects with multiple sclerosis. 20 to 100 times larger. The use of claim 27, wherein the dosage is about 0.01 mg / kg to about 5 mg / kg. The use of claim 28, wherein the dosage is about 0.2 mg / kg to about 1.0 mg / kg. The use of claim 29, wherein the dosage is about 0.2 mg / kg to about 0.5 mg / kg. The use of claim 28, wherein the dosage is about 1.0 mg / kg to about 2.0 mg / kg. Use according to any one of claims 27, 28, 29, 30 or 31, wherein the conjugate is administered to the subject once a week. Use according to any one of claims 27, 28, 29, 30 or 31, wherein the compound of formula (I) is administered to the subject once a month. In the use of a therapeutically effective amount of a conjugate comprising two or more α4β1 small molecule antagonists covalently attached to a biocompatible polymer, the conjugate has an efficacy directed at α4β1 over α4β7 in the treatment of subjects with an inflammatory disorder. 20 to 100 times larger. The use of claim 34, wherein the dosage is about 0.01 mg / kg to about 5 mg / kg. The use of claim 35, wherein the dosage is about 0.2 mg / kg to about 1.0 mg / kg. The use of claim 35, wherein the dosage is about 0.2 mg / kg to about 0.5 mg / kg. The use of claim 35, wherein the dosage is about 1.0 mg / kg to about 2.0 mg / kg. Use according to any one of claims 34, 35, 36, 37 or 38, wherein the conjugate is administered to the subject once a week. Use according to any one of claims 34, 35, 36, 37 or 38, wherein the conjugate is administered to the subject once a month. Use for the treatment of a patient in need thereof comprising administering a dosage of Formula (I) from about 0.5 mg / kg to about 2.0 mg / kg to the subject once a week or once a month. Use for the treatment of a patient with autoimmune disease comprising administering a dosage of Formula (I) from about 0.5 mg / kg to about 2.0 mg / kg to the subject once a week or once a month.

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