RU2417100C2 - Methods, compositions and preparations for treating thyroid-associated ophthalmopathy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: there are offered compositions and methods for reducing orbital fat accumulation in a patient while treating a thyroid-associated ophthalmopathy, proptosis and related conditions (e.g. endocrinous ophthalmopathy). The compositions contain a beta-adrenergic agonist in the form of a crystalline microparticle suspension (e.g., prolonged release) for systemic injections or local introduction, and/or methylprednisolone acetate, fluticasone propionate in the form of the crystalline microparticle suspension as well. The methods involve the introduction of a compound for reducing beta-adrenergic receptor desensitisation (e.g. a corticosteroid) prior to the introduction or combined with the introduction of the beta-adrenergic agonist.

EFFECT: reduced weight of fat bodies and decreased volume of orbital fat after applying the declared compositions and methods.

39 cl, 7 dwg, 7 tbl, 6 ex

Description

CROSS REFERENCE

This application sets forth the priority of provisional patent application US No. 60/852221, filed October 17, 2006; 60/898009, filed January 29, 2007, and 60/919011, filed April 13, 2007, and these applications are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Graves' disease is a common disorder with an incidence in women of 1/1000 of the population per year. In addition to hyperthyroidism, 25-50% of individuals with Graves disease develop clinical eye damage, i.e. thyroid ophthalmopathy. Graves Ophthalmopathy (OG) is a typical form of thyroid ophthalmopathy. Although some patients with hypertension experience only moderate eye discomfort, 3-5% suffer from intense pain and inflammation with double vision or even loss of vision.

Clinical symptoms and signs of hypertension can be explained mechanically by an increase in tissue volume expressed within the orbit. Enlarged orbital tissues cause the eyeball to move forward and worsen venous and lymphatic outflow from the orbit. These changes, combined with the local production of cytokines and other inflammatory mediators, lead to proptosis, periorbital edema, conjunctival erythema and chemosis (Figure 1).

SUMMARY OF THE INVENTION

Compositions, preparations, methods and systems for treating thyroid ophthalmopathy are described herein by contacting a targeted fat deposition in the eye with a composition comprising a long-acting beta-2 adrenergic receptor agonist and a compound that reduces beta-adrenergic receptor desensitization in the target tissue on long-acting beta-2 adrenergic receptor agonist. Embodiments of this composition are administered, for example, by retrobulbar (ocular) injection and / or transocularly. Glucocorticosteroid has the additional effect of reducing the number of inflammatory cells and the release of inflammatory cytokines present in the orbit and in the adipose tissue of the orbit.

Accordingly, in one aspect, there is provided a method of reducing ocular fat accumulation in a patient in need thereof (for example, in a subject suffering from thyroid ophthalmopathy) by administering to said patient a therapeutically effective amount of at least one beta adrenergic agonist and a therapeutically effective amount of at least one compound to reduce desensitization of the beta adrenergic receptor. In some embodiments, a patient in need of the treatment just described suffers from Graves ophthalmopathy. In some embodiments, a patient in need of treatment suffers from an increase in the external muscles of the eye. In some embodiments, said patient suffers from proptosis.

In some embodiments, the administration of at least one beta adrenergic agonist or at least one compound to reduce desensitization is parenteral, oral, intraocular, intraocular, intrathoracic, ophthalmic, retrobulbar, periorbital, local, intramuscular, transdermal, sublingual, intranasal or Airways.

In some embodiments, at least one beta adrenergic receptor desensitizing compound is administered to (for example, from about 3 days to about 7 days to) at least one beta adrenergic agonist. In some embodiments, the at least one compound is administered by the intraocular, intraocular, ocular, periorbital, retrobulbar, or intrathoracic routes of administration. In some embodiments, at least one compound is administered in the form of a suspension of crystalline microparticles.

In some embodiments, at least one compound is administered orally and at least one beta adrenergic agonist is administered to the eye. At least one beta adrenergic agonist administered to the eye is administered in a crystalline microparticle preparation.

In some embodiments, the at least one beta adrenergic agonist to be administered to a patient comprises a long acting beta adrenergic agonist. In some embodiments, the at least one compound is a glucocorticosteroid and the long-acting beta-adrenergic agonist is salmeterol, formoterol, or a combination thereof. In some embodiments, the beta adrenergic agonist to be administered is a beta adrenergic agonist that is selective for the beta-2 adrenergic receptor. In some embodiments, the at least one beta adrenergic agonist comprises salmeterol, formoterol, or any combination thereof. In some embodiments, the at least one beta adrenergic agonist contains salmeterol, and the therapeutically effective amount of salmeterol is from about 0.01 μg / day to about 100 μg / day (e.g., from about 1 μg / day to about 100 μg / day, from about 10 μg / day to about 100 μg / day, or from about 50 μg / day to about 100 μg / day) of salmeterol. In other embodiments, the at least one beta adrenergic agonist contains formoterol, and the therapeutically effective amount of formoterol is from about 0.001 μg / day to about 50 μg / day (e.g., from 0.01 μg / day to about 1.0 μg / day , from about 0.1 μg / day to about 10 μg / day, from about 1 μg / day to about 20 μg / day, or from about 5 μg / day to about 40 μg / day).

In some embodiments, the at least one compound for reducing desensitization of the beta adrenergic receptor is a glucocorticosteroid, antihistamine, or any combination thereof. In some embodiments, the at least one beta adrenergic receptor desensitizing compound comprises dexamethasone, prednisolone, methylprednisolone, fluticasone propionate, budesonide, ketotifen, or any combination thereof.

In some embodiments, before administering at least one beta adrenergic agonist and at least one compound to reduce desensitization of the beta adrenergic receptor, a therapeutically effective amount of an immunosuppressant is administered to the patient via the intraocular, intraocular, ophthalmic, periorbital, retrobulbar or intrathoracic routes of administration. In some embodiments, the immunosuppressant is administered in the form of a suspension of crystalline microparticles.

In another aspect, a method for treating proptosis by administering to a patient in need of treatment a composition comprising a therapeutically effective amount of at least one beta adrenergic agonist is provided.

In some embodiments, the at least one beta adrenergic agonist comprises a long acting beta adrenergic agonist. In some embodiments, the at least one beta adrenergic agonist comprises a beta adrenergic agonist that is selective for the beta 2 adrenergic receptor. In some embodiments, the at least one beta adrenergic agonist comprises salmeterol, formoterol, bambuterol ,formoterol, isoproterenol, albuterol, or fenoterol. In some embodiments, the composition comprises a mixture of at least one long-acting beta adrenergic agonist and at least one short-acting beta adrenergic agonist. In some embodiments, the composition also comprises a therapeutically effective amount of hyaluronidase.

In some embodiments, the composition comprises salmeterol, and a therapeutically effective amount of salmeterol is administered to the patient from about 0.01 μg / day to about 100 μg / day. In other embodiments, the composition comprises formoterol, and a therapeutically effective amount of formoterol is administered to the patient from about 0.001 μg / day to about 50 μg / day.

In some embodiments, the administration of the composition is parenteral, oral, intraocular, intraocular, periorbital, ophthalmic, retrobulbar, intrathoracic, local, intramuscular, transdermal, sublingual, intranasal or airway.

In another aspect, there is provided a method of reducing ocular fat accumulation in a patient in need of treatment by administering to said patient a therapeutically effective amount of one or more compounds that stimulate the pathway associated with the adrenergic receptor and a therapeutically effective amount of at least one compound to reduce desensitization of the beta adrenergic receptor. In some embodiments, one or more than one adrenergic receptor pathway stimulating compound comprises a catecholamine, an alpha adrenergic antagonist, forskolin, aminophylline, or analogs thereof.

In yet another aspect, an ophthalmic pharmaceutical composition is provided comprising an ophthalmologically acceptable excipient and a therapeutically effective amount of methylprednisolone acetate or fluticasone propionate in the form of a suspension of crystalline microparticles. In some embodiments, the ophthalmic pharmaceutical composition further comprises solubilized methylprednisolone acetate or solubilized fluticasone propionate. In some embodiments, the ophthalmic pharmaceutical composition further comprises a therapeutically effective amount of at least one long-acting beta-2 agonist in the form of a suspension of crystalline microparticles.

In another aspect, an ophthalmic pharmaceutical composition is provided comprising an ophthalmologically acceptable excipient and a therapeutically effective amount of at least one long-acting beta-2 agonist in the form of a suspension of crystalline microparticles. In some embodiments, the at least one long-acting beta-2 agonist comprises salmeterol or formoterol. In some embodiments, the ophthalmic pharmaceutical composition further comprises a therapeutically effective amount of at least one solubilized, long-acting beta-2 agonist. In some embodiments, the ophthalmic pharmaceutical composition further comprises a therapeutically effective amount of at least one compound to reduce desensitization of the beta adrenergic receptor in the form of a suspension of crystalline microparticles.

In another aspect, the use of at least one beta adrenergic agonist and at least one compound for reducing desensitization of a beta adrenergic receptor for the manufacture of a medicament for the treatment of an disease including accumulation of orbital fat is provided.

In another aspect, the use of at least one beta adrenergic agonist and at least one compound for decreasing (desensitizing) a beta adrenergic receptor for use in a method of treating a disease comprising accumulating orbital fat is provided.

TURNING ON THE LINK

All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

The new features of the invention are specifically set forth in the appended claims. A better understanding of the distinguishing features and advantages of the present invention will be obtained by reference to the following detailed description, which sets forth illustrative embodiments in which the municipalities of this invention are used, and to the accompanying graphic materials in which:

Figure 1 presents a schematic illustration of lipolysis in adipocytes.

FIG. 2 presents a histogram illustrating a dose-dependent induction of lipolysis in cultured adipocytes using a long-acting beta-2 agonist formoterol after a three-hour incubation.

FIG. 3 is a bar graph illustrating a dose-dependent induction of lipolysis in cultured adipocytes using a long-acting beta-2 agonist of salmeterol after a three-hour incubation.

FIG. 4 is a bar graph illustrating a dose-dependent induction of lipolysis in cultured adipocytes using the budesonide glucocorticosteroid after a short incubation period (three hours) and inhibition of lipolysis after longer incubation periods (18 hours).

FIG. 5 is a bar graph illustrating the dose-dependent inhibition of lipolysis in cultured adipocytes by a long-acting beta-2 agonist with salmeterol, which was given alone for 18 hours, and the dose-dependent induction of lipolysis with salmeterol after 18 hours, when given in combination with budesonide glucocorticosteroid.

FIG. 6 is a bar graph illustrating the average value of the differences in the mass of the epididymal fat body in an animal (left fat body compared to the right fat body) in fat bodies in which a carrier solution (2% PEG (polyethylene glycol)), one formoterol or formoterol were injected plus budesonide for a three-day treatment period.

FIG. 7 is a bar graph illustrating a dose-dependent reduction in fat body mass for two different dose combinations of the formoterol beta-2 agonist and budesonide glucocorticosteroid over a three-day treatment period.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Computed tomography images show that most patients with hypertension have an increase in both eye fat and the external muscles of the eye, while others appear to have only damage to the adipose tissue or external muscles of the eye. The cells of the external muscles of the eye are intact in an early, active disease, which shows that they themselves are not targets for an autoimmune attack. Rather, an increase in the bodies of the external muscles of the eye occurs due to the accumulation of hydrophilic mucopolysaccharides in the perimisial connective tissues, including, in particular, hyaluronan. At a later stage of the disease, the absorbable inflammatory process in the muscles can leave them fibrous and displaced.

An increase in the volume of fat / connective tissue in the orbit appears to be more likely to contribute to a general increase in the volume of orbit tissue than an increase in the external muscles of the eye. Studies using computed tomography show that in these patients, proptosis rates most closely correlate with the volume of the fat compartment. This increased volume of adipose tissue, apparently, is due to both the accumulation of hyaluronan in concomitant edema and the appearance of newly differentiated fat cells in these tissues.

A histological examination of the tissues of the eye socket in OG reveals that characteristic changes occur mainly due to the accumulation of hyaluronan in edema, an increase in the fat compartment, and the infiltration of these tissues with T-lymphocytes. The orbital fat cells in this condition are apparently differentiated, although smaller than other fat cells in the body (for example, subcutaneous or omental). Higher levels of PPAR-gamma, adiponectin, and leptin transcripts are expressed in the eye socket fat cells during hypertension. In addition, eye socket fat cells may contain less than 11-beta-hydroxysteroid dehydrogenase type 1, an enzyme involved in the conversion of cortisone to its active form, cortisol.

Studies using cells derived from these tissues have shown that orbital fibroblasts are orbital cells that can participate in these various cellular processes. These cells are particularly sensitive to stimulation by cytokines and other immuno-mediators, responding by increasing expression of CD40, synthesizing large amounts of hyaluronan and secreting inflammatory cytokines. In addition, a preadipocytic subpopulation of fibroblasts is able to differentiate into mature fat cells that exhibit high levels of TSHR. Fibroblasts have also been shown to exhibit IGF-1R (insulin-like growth factor receptor 1). When binding to IgG from patients with Graves' ophthalmopathy, these receptors initiate the lower stages of signal transmission, which leads to the production of RANTES (Regulated on Activation, Normal T-cell Expressed and Secreted - a cytokine that is activated upon activation, expressed and secreted by normal T cells) and IL-16 (interleukin-16) and leads to local lymphocytic infiltration. The relative site-specificity of the involvement of the orbit in Graves' disease can be explained by both the relative sensitivity of these fibroblasts to immuno-mediators and the unique anatomical features of these sites, which apparently predispose them to compress lymphatic and venous channels of low pressure.

Adipose tissue is the basis of energy storage tissue of the body. Fat cells or adipocytes store this energy in the form of triglycerides. Triglycerides are mobilized from fat stores to provide energy to the body through hormonal induction of triglyceride hydrolysis. This process releases free or unesterified fatty acids and glycerin into the blood for use by other body tissues. The breakdown of triglycerides from fat stores is called lipolysis. There is also an increase in new adipocytes called lipogenesis.

Catecholamines are the main regulators of adipose tissue through adrenergic receptors. Adipose tissue has beta-1, 2 and 3 adrenergic receptors and alpha-2-adrenergic receptors. The binding of beta agonists to beta receptors in adipose tissue can lead to lipolysis of adipocytes, while the binding of alpha receptor agonists can inhibit lipolysis. Beta receptor activation can also inhibit lipogenesis. In humans, the beta-2 receptor is often the most common on the surfaces of fat cells and is the main mediator of beta receptor stimulated lipolysis. Stimulation of lipolysis by beta-agonists is mediated by adenylate cyclase and increased formation of cyclic adenosine monophosphate (cyclic AMP, cAMP). Alpha-2 receptors reduce lipolysis in mature fat cells. Alpha-2 adrenergic receptors may be involved in the proliferation of preadipocytes. Glucocorticosteroids can have a permissive effect on adipose tissue and increase the response of adipocytes, such as lipolysis, to stimulation with catecholamines. This permissive effect may be due to the activation of beta-adrenergic receptors and other components included in the secondary intracellular messengers.

Treatment of exhaust gas would inevitably have the goal of reducing the volume of increased adipose tissue of the orbit. Therefore, a drug to reduce adipocyte volume by lipolysis may be useful for this condition. In addition, the reduction of the inflammatory process and inflammatory cells in the orbit, which may be associated with an increase in adipose tissue, can further reduce the volume of orbital tissue. In addition, treatment of hyaluronan accumulation in the orbit may provide additional volume reduction and relief of ophthalmopathy. Finally, inhibition of lipogenesis can improve this condition.

It has been suggested and shown that the delivery of adrenergic active ingredients into the subcutaneous tissue of both beta-agonists and alpha-2 antagonists leads to a loss of regional fat and to a better view of regional fat accumulations. For example, it was shown that isoproterenol 11 and yohimbine 8 reduce the femoral circumference. Since these lipolytic agents, especially beta-agonists, act briefly and can be quickly removed from adipose tissue, it is likely that lipolysis occurred only within a short time after injection, thereby reducing the potential magnitude of the effect, despite numerous injections. In addition, the prolonged exposure of beta-agonists to adipocytes leads to desensitization and downregulation of receptors and to a loss of lipolytic activity. Means to reduce or prevent these effects on the receptor can also improve therapy. However, a strategy for treating adipocytes in hypertension using adrenergic agents and glucocorticosteroids to induce lipolysis and inhibit lipogenesis can provide an effective reduction in the tissue mass responsible for clinical signs and symptoms.

Embodiments of pharmaceutical compositions, preparations, methods and systems for the treatment of thyroid ophthalmopathy (eg, Graves' ophthalmopathy or "OG") by reducing eye tissue mass are described herein. Decreasing eye tissue mass can reduce proptosis, restore or prevent vision loss and diplopia, and reduce pain. This reduction in tissue mass can be accomplished by doing at least one of the following, including: decreasing the mass of orbital fat, reducing inflammation (e.g., inflammatory cells and cytokines), and decreasing the accumulation of glycosaminoglucans (GAGs). Fat reduction can be achieved by modulating the adrenergic system. As used and / or cited here, the term “modulation” is commonly used in its general sense and more specifically refers to adrenergic receptor agonism, adrenergic receptor antagonism and / or changes in receptor-associated signaling pathways. One example of a change in receptor-associated signaling pathways includes an increase (concentration) of cyclic AMP, for example, as schematically illustrated in FIG. 1. In some embodiments, “modulation” refers to upregulation of the receptor or to an increase in the number of adrenergic receptors, to a decrease in deactivation or sequestration of the receptor, to changes in receptor activity (eg, increase in activity) and / or to changes in receptor affinity. Adrenergic receptor modulation can preferably be done with a glucocorticosteroid or antihistamine, which will also work to reduce inflammation. Similarly, the accumulation of glycosaminoglycan can be reduced using a glucocorticosteroid and can be further reduced using an enzyme for the degradation of hyaluronic acid, such as recombinant human hyaluronidase.

The decrease in the mass of fat in the tissue of the eye socket is preferably achieved by a non-ablation method, initiating lipolysis, inhibiting lipogenesis, or reducing lipid accumulation. Ablative methods of reducing fat, such as phosphatidylcholine or deoxycholate, can be problematic for use behind the eye, where non-selective tissue destruction can damage nerves or muscles and can cause scarring and fibrosis. Stimulation of lipolysis, inhibition of lipogenesis and a decrease in lipid accumulation can be achieved through stimulation of beta-adrenergic receptors. Beta-adrenergic receptor stimulation can also counteract some cellular transcripts that are known to be activated in the fat cells of the eye socket in Graves disease, such as PPAR-gamma, adiponectin and leptin. For example, stimulation of the beta adrenergic receptor can reduce the expression of PPAR gamma and adiponectin in differentiated adipocytes. It is believed that some embodiments of prolonged modulation of adrenergic receptors in adipose tissue result in some combination of prolonged lipolysis, reduced adipocyte lipid content, reduced adipocyte cell size, reduced adipose tissue mass, and / or reduced fat accumulation. Some embodiments provide a selective reduction in the accumulation of adipose tissue and adipocytes in the orbit through prolonged adrenergic modulation. In some embodiments, prolonged adrenergic modulation leads to prolonged inhibition of fat cell proliferation (lipogenesis).

Various embodiments of the disclosed pharmaceutical compositions include at least one selective beta-2 adrenergic receptor agonist (e.g., a long-acting selective beta-2 agonist) in combination with at least one compound that reduces desensitization of beta-adrenergic receptors, e.g. desensitization target tissues on beta-adrenergic receptor agonist (s), such as glucocorticosteroids or ketotifen, or with their analogs, and also reduces inflammation. The term "desensitization" includes both short-term desensitization (tachyphylaxis) and long-term desensitization, as well as desensitization over other periods of time. Beta-2 adrenergic receptor agonists are also referred to herein as "beta-2 agonists" and "beta-2 receptor agonists." Unless otherwise indicated, references to beta-2 adrenergic receptor agonists also include their analogs, physiologically acceptable salts and / or solvates. In some embodiments, these compositions comprise from about 100: 1 to about 1: 100 long-acting selective beta-2 agonist for a glucocorticosteroid.

As discussed above, it is believed that lipolytic activity, inhibition of adipocyte proliferation and a decrease in lipid accumulation are mediated by modulation of adrenergic receptors in adipose tissue and / or on adipocytes. In some embodiments, reduction therapy is enhanced by prolonged exposure or prolonged activity of one or more adrenergic receptor agonists and / or compounds that stimulate the (signal) pathway associated with the receptor, for example, catecholamines, beta agonists, alpha antagonists, forskolin, aminophylline , their analogues or combinations.

Some embodiments provide sustained adrenergic modulation through the use of pharmaceutical compositions containing one or more essentially selective long-acting beta-2 receptor agonists. In some embodiments, a prolonged-acting pharmaceutical composition comprises one or more than one suitable, selective, long-acting beta-2 agonist, for example, salmeterol 1, formoterol 2, bambuterol 3 ,formoterol, their physiologically acceptable salts or solvates, or combinations thereof.

Prolonged adrenergic modulation may not be observed with typical adrenergic compositions, since the adrenergic compound is usually quickly removed from adipose tissue through the blood and / or lymph, in particular due to their hydrophilicity. In addition, it is believed that the prolonged exposure of adrenergic agents to adipose tissue, in particular beta-receptor agonists, leads to desensitization of the receptor by phosphorylation and sequestration of the receptor. It is believed that these effects limit the ability of the adrenergic modulating composition to treat adipose tissue and lead to tachyphylaxis, a condition in which the body exhibits a rapidly waning response to the agonist after administration of the initial doses with respect to the desired lipolytic and antilipogenic effect. Accordingly, the therapeutic effect is short-term.

Short-acting beta-2 agonists often lead to tachyphylaxis, as discussed above. However, since preferred embodiments of selective long-acting beta-2 agonists have substantially selective beta-2 receptor activity and high lipophilicity, long-acting beta-2 agonist activities are retained in adipose tissue for longer periods of time compared to beta 2 short-acting agonists. The activity of the partial beta-2 receptor antagonist that is observed when salmeterol is used can prevent some desensitization, which can occur with the continuous exposure of complete adrenergic agonists to adipocytes. In addition, salmeterol may not fully activate the arrestin signaling pathway, which leads to internalization and degradation of the receptor and to long-term downregulation of the receptor. Compared to short-acting beta-2 agonists, lipolysis also occurs for a longer time after administration, since long-acting selective beta-2 agonists have longer half-lives. The combination of longer half-lives and activities may reduce the frequency of administration of pharmaceutical compositions. Therefore, in some embodiments, daily administration of the composition or its administration more than once a day is not required. In addition, preferred embodiments of selective long-acting beta-2 agonists also exhibit greater selectivity for beta-2 receptors, providing substantially the same therapeutic effects of the compound compared to short-acting beta-2 agonists at a lower dosage. In addition, more selective beta-2 activity can limit side effects on the heart, which are often induced by stimulation of beta-1 receptors in the heart.

As discussed above, lipolysis and / or inhibition of lipogenesis and lipid accumulation are stimulated by beta-1, 2 or 3 receptor subtypes. Thus, agonists to one, two and / or all three receptors are able to stimulate lipolysis and / or inhibit lipogenesis. In humans, beta-2 receptor activity is believed to be more important for stimulating lipolysis, especially in the presence of an anti-inflammatory steroid or glucocorticosteroid.

Long-acting selective beta-2 agonists, for example, salmeterol 1 (± 2- (hydroxymethyl) -4- [1-hydroxy-2- [6- (4-phenylbutoxy) hexylamino] ethyl] phenol, CAS reg. No. 94749- 08-3) and formoterol 2 (± N- [2-hydroxy-5- [1-hydroxy-2- [1- (4-methoxyphenyl) propan-2-ylamino] ethyl] phenyl] methanamide, CAS reg. 73573-87-2) are preferred in some embodiments. In some embodiments, these compositions contain one or more long-acting selective beta-2 agonists in the form of physiologically acceptable salts or solvates, for example, salmeterol xinafoate and / or formoterol fumarate. In many cases, salts and / or solvates of beta-2 agonists will have the desired activity. Accordingly, unless otherwise indicated, references to the active ingredient, for example, salmeterol 1, formoterol 2, isoproterenol 4, albuterol 5, phenoterol and forskolin include the compounds themselves, as well as their physiologically acceptable analogues, salts and / or solvates, or combinations thereof .

Some preferred long acting beta-agonists exhibit high intrinsic adenylate cyclase activity, which enhances cAMP synthesis. For example, some embodiments include formoterol 2 as a beta-2 selective long-acting agonist that exhibits some combination of higher activity, reduced systemic effects, high intrinsic activation of adenylate cyclase and / or increased (level) cyclic AMP, a lipolysis mediator.

In some preferred embodiments, formoterol 2 is present in the form of a physiologically acceptable salt and / or solvate thereof. Suitable physiologically acceptable salts of formoterol 2 include, for example, acid addition salts derived from inorganic and organic acids such as hydrochloride, hydrobromide, sulfate, phosphate, maleate, fumarate, tartrate, citrate, benzoate, 4-methoxybenzoate, 2-hydroxybenzoate, 4 -hydroxybenzoate, 4-chlorobenzoate, para-toluenesulfonate, methanesulfonate, ascorbate, salicylate, acetate, succinate, lactate, glutarate, gluconate, tricarballylate, hydroxynaphthalene carboxylate, oleate, combinations thereof and the like. Preferred embodiments comprise formoterol 2 in the form of its fumarate salt and / or in the form of a dihydrate. A suitable concentration of formoterol 2 in adipose tissue is from about 1 pM to about 100 μM, more preferably from about 0.1 nM to about 10 μM, for example, from about 1 nM to about 1 μM, from about 40 nM to about 3 μM, from about 0.1 μM to about 1 μM, or any other concentration of formoterol in the tissue from about 0.1 nM to about 10 μM.

In some embodiments, salmeterol is used in the compositions and methods described herein. Salmeterol 1 exhibits a partial agonistic activity, which is believed to reduce receptor desensitization and may limit the arrestin signaling pathway, leading to less downregulation of the receptor. In some embodiments, salmeterol 1 is present in the form of its physiologically acceptable salt and / or solvate. Suitable physiologically acceptable salts of salmeterol 1 include, but are not limited to, acid addition salts derived from inorganic and organic acids such as hydrochloride, hydrobromide, sulfate, phosphate, maleate, tartrate, citrate, benzoate, 4-methoxybenzoate, 2-hydroxybenzoate, 4- hydroxybenzoate, 4-chlorobenzoate, para-toluenesulfonate, methanesulfonate, ascorbate, salicylate, acetate, fumarate, succinate, lactate, glutarate, gluconate, tricarballylate, hydroxynaphthalene carboxylate, 1-hydroxy-2-naphthalene-2-naphthalene carboxylate, arboxylate, oleate, combinations thereof and the like. In some embodiments, salmeterol 1 is provided as a 1-hydroxy-2-naphthalene carboxylate salt (hydroxy naphthoate).

In some embodiments, a suitable concentration of salmeterol 1 in adipose tissue varies from about 1 pM to about 100 μM, preferably from about 1.0 nM to about 1 μM, for example, from about 10 nM to about 1 μM, from about 40 nM to about 3 μM, from about 0.1 μM to about 1 μM, or is any other tissue salmeterol concentration from about 1.0 nM to about 10 μM.

In some embodiments, the long-acting selective beta-2 agonist to be administered is formoterol, and the therapeutically effective amount of formoterol is from about 0.001 to about 100 μg / day, for example, from about 0.001 to about 50, from 0.01 to about 1.0, from about 0.1 to about 10, from about 1 to about 20, from about 5 to about 40, from about 25 to about 75, from about 50 to about 100 μg / day formoterol, or any other dose of formoterol from about 0.001 μg / day to about 100 μg / day.

In some embodiments, the selective long-acting beta-2 agonist to be administered is salmeterol, and the therapeutically effective amount of salmeterol to be administered is from about 0.01 μg / day to about 1000 μg / day, for example, from about 0, 1 μg / day to about 100 μg / day, from about 1 μg / day to about 100 μg / day, from about 10 μg / day to about 100 μg / day, from about 50 μg / day to about 100 μg / day, or any other dose of salmeterol from about 0.01 mcg / day to about 1000 mcg /day.

As used herein, the term “therapeutically effective amount” refers to a sufficient amount of an administered agent (eg, a long-acting beta-2 agonist) or compound that will somewhat alleviate one or more symptoms of a disease or condition that is being treated. The result may be a reduction and / or alleviation of the signs, symptoms or causes of the disease, or any other desired change in the biological system. For example, an “effective amount” for therapeutic applications is the amount of a composition containing a compound, as disclosed herein, necessary to provide a clinically significant reduction in disease symptoms without undesirable harmful side effects. A suitable “effective amount” in each case can be determined using methods such as dose increase studies. The term “therapeutically effective amount” includes, for example, a prophylactically effective amount. An “effective amount” of a compound disclosed herein, such as a selective beta-2 agonist, used alone or in combination with other compounds (eg, with a compound to reduce desensitization of the beta-2 adrenergic receptor), is an amount effective to achieve the desired pharmacological effect or therapeutic improvement without undesirable side effects. It is understood that an “effective amount” or “a therapeutically effective amount” may vary from subject to subject due to varying metabolism of beta-2 agonists and compounds used in combination with beta-2 agonists (eg, glucocorticosteroids), age, weight, total the condition of the subject, the condition that is being treated, the severity of the condition that is being treated, and the decisions of the practitioner.

Some embodiments include optically pure isomers of the beta-adrenergic (s) agonist (s), which can improve lipolysis and inhibition of lipogenesis and reduce potential side effects. In some embodiments, these optically pure isomers make it possible to obtain preparations containing high loads of the active ingredient, for example, by eliminating one or more isomers without a physiological effect, having a lower physiological effect, a negative effect and / or an unclear physiological effect. Removing undesirable bonds from the racemic mixture allows one to isolate the active isomer, or eutomer, thus providing a large load of the eutomer in this preparation by removing inactive components.

Two stereogenic centers in a molecule usually form two diastereomers, referred to herein (R *, R *) and (R *, S *), and their enantiomers. Diastereomers are stereoisomers that are not enantiomers, i.e. the mirror image of one diastereomer does not overlap with another diastereomer. Enantiomers are stereoisomers that are mirror images of each other. The racemate is a 1: 1 mixture of enantiomers. Enantiomers of (R *, R *) - diastereomers are called (R, R) - and (S, S) -enantiomers, which are mirror images of each other and, therefore, have some common chemical and physical properties, for example, melting points. Similarly, (R, S) - and (S, R) -isomers are enantiomers of the (R *, S *) -diastereomer. For example, formoterol 2 is available as a racemate of (R, R) - and (S, S) -isomers in a 1: 1 ratio, typically in the form of a fumarate salt dihydrate. Some embodiments include the (R, R) enantiomer, (R, R) -formoterol, which is more active as a long-acting beta-2 agonist. Some embodiments include optically pure isomers of other beta-2 agonists, for example, (R) -salmeterol.

In addition, in some embodiments, the at least one long-acting selective beta-2 agonist is highly lipophilic, thereby providing a pharmaceutical composition with prolonged activity in adipose tissue. High lipid solubility is believed to extend the residence time of the beta-2 agonist in adipose tissue, thereby eliminating or reducing the need for a carrier for sustained and / or controlled release in some embodiments. In formulations containing a sustained release carrier, such as a sustained release polymer, the high lipophilicity of the beta-2 agonist facilitates incorporation into the sustained release carrier, as discussed in more detail below.

Salmeterol 1 and formoterol 2 have high solubility in lipids, which prolongs their residence time in adipose tissue and / or in one or more than one fat cell. In some embodiments, the composition comprises a highly lipophilic beta agonist that reduces or eliminates the need for a carrier for sustained or controlled release due to distribution and isolation in adipose tissue, thereby prolonging the effect of treatment. In some embodiments, beta agonists are used with an oil-water partition coefficient of at least about 1000 or at least about 10,000 to 1. For example, salmeterol 1 is at least 10,000 times more lipophilic than albuterol 5, a hydrophilic beta agonist short-term action. Additionally, salmeterol 1 and formoterol 2 have anti-inflammatory properties used in the treatment of GO, as discussed below. In some embodiments, they also stimulate beneficial changes in the extracellular matrix and reduce fluid accumulation, which improves the treatment of exhaust gas and the accumulation of orbital fat.

Prolonged beta-adrenergic activity is further enhanced by the prevention of desensitization (tachyphylaxis), which can occur with continuous exposure of adrenergic agonists to adipocytes, as discussed above. “Compounds that reduce the desensitization of beta adrenergic receptors” (for example, reduce the desensitization of the target tissue with respect to the beta agonist) include all suitable compounds that reduce the tolerance of the target tissue to beta adrenergic receptor agonists, including glucocorticosteroids and suitable antihistamines for example ketotifen, and thyroid hormones, for example T3 and T4. Glucocorticosteroids are also referred to herein as “anti-inflammatory steroids”, “glucocorticosteroids” and / or “corticosteroids”. It is believed that glucocorticosteroids sensitize the accumulation of ocular fat by increasing the number of beta-2 receptors, thereby favoring lipolysis or a decrease (content) of fat compared to storing fat. Glucocorticosteroids can also reduce the number of alpha-2 receptors. Glucocorticosteroids can also stabilize or reduce downregulation of receptors, especially when given concurrently with a beta agonist. It is noteworthy that Graves' disease and GO are more common in women than in men. In women, estrogen can induce the expression of alpha-2 adrenergic receptors in the subcutaneous adipose tissue, leading to a ratio of beta-2 receptor and alpha-2 receptor less than 1. It is believed that the ratio of beta-2 receptors and alpha-2 receptors more than 1 leads to a decrease in fat rather than the accumulation of fat in adipocytes. In some embodiments, a composition comprising one or more than one glucocorticosteroid is effective in treating areas of fat containing a reduced number of beta-2 receptors and / or an increased number of alpha-2 receptors that are resistant to beta-adrenergic stimulation of lipolysis or inhibition of lipogenesis, for example, in subcutaneous adipose tissue, especially in women.

Thus, it is believed that glucocorticosteroids or other compounds to reduce desensitization of beta adrenergic receptors improve lipolysis, inhibition of lipogenesis and / or regional reduction in fat content during exposure to the beta agonist. In some embodiments, treating the adipocytes with a glucocorticosteroid that increases lipolytic activity supports and / or increases both lipolytic activity and the number of beta receptors in the target tissue. Examples of suitable corticosteroids include dexamethasone 6, fluticasone propionate 7, budesonide 8, prednisolone 9, methylprednisolone 10 and their analogs. In some embodiments, the glucocorticosteroid is dexamethasone. In some embodiments, the corticosteroid is methylprednisolone 6, (9-fluoro-11,17-dihydroxy-17- (2-hydroxyacetyl) -10,13,16-trimethyl-6,7,8,11,12,14,15, 16-octahydrocyclopenta [a] phenanthren-3-one, CAS reg. No. 50-02-2) and / or fluticasone propionate 7.

As discussed above, in some embodiments, a suitable compound for reducing desensitization of beta receptors is ketotifen 11, which is also useful as an antihistamine. In some embodiments, the composition comprises one compound that reduces the desensitization of adipose tissue to a beta-2 agonist.

In some embodiments, a variety of compounds are used to reduce desensitization of beta receptors, for example, a variety of glucocorticosteroids. Some preferred embodiments comprise at least one glucocorticosteroid and a ketotifen antihistamine or ketotifen analog.

In some embodiments, the activity or density of at least one beta-2 receptor in response to administration of an anti-inflammatory steroid or ketotifen is increased in human eye socket adipocytes, especially in the presence of a beta agonist. In some embodiments, the increasing activity and / or density of beta-2 receptors potentiates the effect of long-term or short-acting beta-2 agonists. Thus, in some embodiments, a glucocorticosteroid can sensitize eye fat to the effects of stimulating beta-2 receptors, lipolysis, inhibiting lipogenesis and / or apoptosis, and / or increasing the ratio of beta-2 adrenergic receptors to alpha-2 adrenergic receptors, thereby shifting the balance in adipose tissue from fat accumulation to fat loss. In some embodiments, the number of beta-2 receptors is increased or maintained, especially by a glucocorticoid, ketotifen, or thyroid hormone, especially when co-administered with a beta-2 adrenergic agonist.

The addition of an anti-inflammatory agent, such as a glucocorticosteroid or antihistamine, has the additional effect of decreasing the number of inflammatory cells and the inflammatory response in the orbit. The orbit and orbital fat in OG have a diffuse lymphocytic infiltrate containing lymphoid aggregates or inclusions. Other eye socket tissues, such as the external muscles of the eye, have a similar white blood cell infiltrate. Mast cells may also be present in large quantities. The secretion of cytokines by these lymphocytes is consistent with both cell and humoral-mediated responses. Glucocorticosteroids reduce leukocyte accumulation, cytokine secretion and can induce leukocyte apoptosis. These effects may be enhanced by long-acting selective beta-2 agonists. In addition, long-acting selective beta-2 agonists can activate, stabilize and stimulate translocation of the glucocorticosteroid receptor to the nucleus in leukocytes and even in adipocytes, additionally potentiating anti-inflammatory effects. A long-acting glucocorticosteroid and beta-agonist can stabilize mast cells and can work additively or synergistically. Ketotifen can also stabilize mast cells and can also inhibit TNF-alpha (tumor necrosis factor-alpha), a known cytokine involved in lymphocytic cell-mediated inflammatory responses.

Suitable tissue glucocorticosteroid concentrations used for the therapeutic methods described herein can vary from about 0.001 μM to about 10 mm, for example, from about 1.0 μM to about 5 mm, from about 40 μM to about 3 mm, from about 100 μM to about 1 mM, or may be any other concentration of glucocorticosteroid in the tissue from about 10 μM to about 10 mm.

In some embodiments, the glucocorticosteroid to be administered is budesonide, and a pharmaceutically effective amount of budesonide is from about 1.0 to about 320 μg / day, for example, from about 80 to about 300, from about 100 to about 280, from about 120 to about 260; from about 140 to about 240; from about 160 to about 220; from about 180 to about 200; from about 185 to about 195 μg / day of budesonide; or any other dose of budesonide from about 60 to about 320 μg / day.

In some embodiments, the glucocorticosteroid to be administered is fluticasone, and a therapeutically effective amount of fluticasone is from about 1.0 to about 500 μg / day, for example, from about 120 to about 480, from about 140 to about 460, from about 160 to about 440, from about 180 to about 420, from about 200 to about 400, from about 220 to about 380, from about 240 to about 360, from about 260 to about 340, from about 275 to about 310, from about 290 to about 300 mcg / day of fluticasone, or any other dose fluticasone from about 100 to about 500 mg / day.

In some embodiments, the glucocorticosteroid to be administered is methylprednisolone at a dose of from about 1.0 μg / day to 10,000 μg / day or more, for example, from 50 to 5000, from 100 to 5000, from 500 to 5000, from 700 to 3000 , from 800 to 2500, from 1000 to 2000, or in any other dose from about 1.0 to 10,000 μg / day. In some embodiments of methylprednisolone, succinate can be solubilized in a suspension of crystalline microparticles of methylprednisolone acetate or administered in conjunction with a suspension of crystalline microparticles of methylprednisolone acetate to provide a dosage for immediate administration or a dose for prolonged administration.

In some embodiments, the composition comprises additional possible ingredients. For example, the orbit of the eye and accumulation of orbital fat, especially in the case of GO, contain large amounts of glycosaminoglycans, consisting essentially of hyaluronic acid. In some situations, it is useful to subject this hyaluronic acid to degradation, for example, to improve the diffusion of the beta-adrenergic agonist drug and glucocorticosteroid through the orbit. In addition, the degradation of hyaluronic acid can further reduce the mass of the tissues of the orbit and reduce swelling of the orbit, thereby improving the state of proptosis and GO. In some embodiments, the composition comprises an enzyme, such as hyaluronidase (e.g., recombinant human hyaluronidase, Hylenex, Halozyme Therapeutics, San Diego, CA), which breaks down hyaluronic acid.

Some embodiments of this composition include one or more antilipolytic blocking agents, for example, a selective alpha-2 receptor antagonist such as phentolamine 12 (CAS reg. No. 73-05-2) or yohimbine 13 (CAS reg. No. 146-48- 5), which blocks antilipolytic effects during regional accumulation of fat. Anti-lipolytic effects in adipocytes and adipose tissue are usually observed in subcutaneous and regional areas of fat accumulation. For example, with beta-agonist exposure, subcutaneous fat has a lower lipolysis rate than visceral fat. The action of antilipolytic blocking agents on orbital fat in some embodiments can improve lipolytic activity.

In some embodiments, the composition comprises other adrenergic agents that enhance the effect of the long-acting selective beta-2 agonist. For example, aminophylline 14 (1,3-dimethyl-7H-purine-2,6-dione, diethylamine CAS reg. No. 317-34-0) and theophylline 15 (CAS reg. No. 58-55-9) are lipolytic agents that block the destruction of cyclic AMP.

Other possible ingredients enhance the secondary signals produced by the binding of beta agonists. For example, in some embodiments, the composition comprises Forskolin 16 (CAS reg. No. 66575-29-9), which stimulates adenylate cyclase, thereby increasing the synthesis of cyclic AMP initiated by a long-acting beta-agonist. An increased concentration of cyclic AMP helps maintain lipolytic activity.

In some embodiments, the composition comprises growth hormone in combination with a long-acting beta-agonist and a glucocorticosteroid, which appears to stimulate lipolysis.

In other embodiments, the composition further comprises one or more non-selective beta agonists, such as isoproterenol 4, and / or a short-acting selective beta-2 agonists, such as terbutaline. Some compositions contain at least one alpha-2 antagonist or physiologically acceptable salts or solvates thereof.

Embodiments of this composition are formulated for administration by any suitable method, for example, as described in Remington: The Science And Practice Of Pharmacy (21 ^st ed., Lippincott Williams & Wilkins). Exemplary routes of administration include, but are not limited to, parenteral, oral, intraorbital, periorbital, ophthalmic, retrobulbar, topical, intramuscular, transdermal, sublingual, intranasal, or airway administration. In some embodiments, the composition is formulated for injection into an area in which treatment is desired, such as an orbit or an orbital fat deposit.

In some embodiments, beta-agonists, compounds for preventing beta-receptor desensitization, or both, are prepared in the form of suspensions of crystalline microparticles to prolong release and thereby further support adrenergic modulation.

Excipients may be used for injectable preparations. Typically, the excipient is ophthalmologically acceptable. In other words, the excipient essentially does not have a lasting or permanent harmful effect on the eye into which it is administered. Examples of ophthalmologically acceptable carriers include water (distilled or deionized), saline and other aqueous media. The compounds of the invention are preferably soluble in a carrier that is used for their administration, so that these compounds are administered to the eye in the form of a solution. Alternatively, you can also use a suspension of the active compound or compounds (for example, a suspension of crystalline microparticles) in a suitable carrier. In some embodiments, one or more beta-2 receptor agonists or glucocorticosteroids are formulated in a liquid carrier, for example, as a solution, suspension, gel and / or emulsion. Some embodiments include any suitable lipophilic, for example, modified oils (e.g. Cremophor® BASF), soybean oil, propylene glycol, polyethylene glycol, derivatives of polyesters, combinations thereof and the like. Some embodiments include a carrier in the form of microparticles and / or nanoparticles for at least one of the beta-2 receptor agonists and / or glucocorticosteroids, as will be discussed in more detail below. Some embodiments include one or more than one sustained or controlled release carrier or agent, for example polymeric microspheres. Some embodiments include excipients suitable for stable suspensions of micronized particles of beta-2 receptor agonists or glucocorticosteroids.

Injectable drugs are administered by any means, including the use of a single needle, multiple needles and / or using a needleless injection device. In some embodiments, a dose of the active ingredients for administration to the tissue is formulated in a suitable vehicle delivered by injection. In some embodiments, the delivery includes a single needle injection. In some embodiments, the delivery includes injection using a multi-needle kit, which in some embodiments provides a wide distribution of the drug in the target tissue. In some embodiments, the drugs are injected in a manner that distributes in a suitable layer of orbital fat. In some embodiments, the drug is injected in small aliquots to reduce the additional volume added to the orbit, and their volume can vary from about 0.1 ml to about 0.5 ml. In some embodiments, the device used for injection has a curved needle to facilitate delivery to the retroorbital or intra-corneal space.

In some embodiments, a beta-2 agonist and a compound that reduces desensitization are administered, for example, injected as separate preparations or, alternatively, administered in different ways (e.g., a glucocorticosteroid is administered orally followed by a long-acting beta-2 agonist) . In some embodiments, a compound that reduces desensitization is administered to a beta-2 agonist. In other embodiments, the beta-2 agonist is administered to a compound that reduces desensitization.

The interval between the administration of a compound that reduces desensitization and the administration of a beta-2 agonist can be an interval from about 5 minutes up to 7 days, for example, 30 minutes, 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, or any other time interval from about 5 minutes to about 7 days. In a preferred embodiment, a compound that reduces desensitization (e.g., a corticosteroid) is administered orally up to 7 days, for example, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days before beta -2-agonist (for example, by topical application of an ophthalmic drug to the eyes).

In other embodiments, the beta-2 agonist is co-administered (e.g., as part of the same preparation) with a compound that reduces beta-receptor desensitization (e.g., with a glucocorticosteroid).

In some embodiments, the drug is given to a subject to be treated as a depot preparation that contains one or more agents for sustained or controlled release to provide sustained or controlled release of a beta-2 agonist or compound (e.g., glucocorticosteroid) to inhibit desensitization beta receptors. In such preparations, a beta-2 agonist, a compound for decreasing beta-receptor desensitization, or both are encapsulated in an agent or carrier for sustained or controlled release, coupled and / or conjugated to an agent or carrier for slow or controlled release. In some embodiments, sustained or controlled release biocompatible, biodegradable drugs provide local tissue activity for weeks to months. Suitable delayed or controlled release agents or carriers include polymers, macromolecules, active ingredient conjugates, hydrogels, combinations thereof and the like. Some embodiments of a sustained release carrier in a target fat are, for example, liposomes. Preferably, the sustained release substances are selected to facilitate the delivery of a substantially equal amount of active substance per unit time, in particular for at least about 3 days, more specifically for at least about 4 days, up to one year or more. For treatment of one area, several cycles of injection of the drug with a slow release can be carried out. In some embodiments, sustained release occurs due to the preparation of a beta-2 agonist or compound to reduce the desensitization of the beta receptor, or both, in the form of a suspension of crystalline microparticles of the drug.

In some embodiments, the sustained release agent comprises a polymer, for example, polylactides, polyglycolides, poly (lactide glycolides), polylactic acids, polyglycolic acids, polyanhydrides, polyorthoesters, polyesters based on ethers and esters, polycaprolactones, polyetheramides, polycarbonates, polycyanoacrylates, polycyanoacrylates and the combinations, mixtures or copolymers (polymers) given above that are used to encapsulate, bind or conjugate to the active ingredient (s) (e.g., beta-agon istami and / or glucocorticosteroids). In some preferred embodiments, sustained release polymers comprise polyethylene glycol groups to which one or more active ingredients are conjugated. In some preferred embodiments, the sustained release agent comprises a poly (lactide glycolide) copolymer 17 (PLGA, lactic and glycolic acid copolymer)).

In some embodiments, the sustained release agent comprises one or more hydrogels, including modified alginates. Examples of suitable modified alginates include alginates disclosed in WO 98/12228. In some embodiments, the sustained release agent comprises an albumin-based carrier or excipient in the form of nanoparticles.

In some embodiments, a preparation containing a prepolymer solution is injected into a region of the target tissue, where it is then polymerized (e.g. by photopolymerization) or cured (e.g. using temperature-sensitive gelling agents) in vivo.

In some embodiments, controlled release substances have release characteristics developed for a particular tissue reduction application. In some embodiments, a sustained release or controlled release agent is prepared in the form of microparticles, such as microspheres, which are prepared as an injectable solution and / or gel. In some embodiments, the size of the microparticles varies from about 10 microns to about 100 microns in diameter, and they are usually uniform in size. In some embodiments, formulations are provided comprising alginates and / or copolymers of poly (lactide glycolides) 17, in the form of an injectable gel, or processed into microspheres. In other embodiments, a beta-2 agonist or corticosteroid (or other compound to reduce beta-receptor desensitization) is prepared in the form of crystalline microparticles. Other examples of suitable injectable biodegradable biocompatible substances suitable for microparticle formation include chitosan, dextran, hydroxyapatite and silicon.

Microspheres and / or microparticles are formed using any method, including evaporation of the solvent and / or polymerization of the emulsion. In some embodiments, the microspheres have an average diameter of from about 5 microns to about 60 microns, preferably about 20 microns. In some embodiments, PLGAs are formulated with varying ratios of lactide to glycolide, depending on the desired release rate of the active ingredient (s). Since the degradation rate of this copolymer is proportional to its crystallinity and the proportion of glycolide in this preparation, non-racemic mixtures of lactide and / or glycolide increase crystallinity and decrease the rate of degradation. Higher glycolide ratios increase the rate of degradation. In some embodiments, a ratio of about 65% -75% lactide to about 25% -35% glycolide provides release of the active ingredients within about 2 weeks to about 45 days. In other embodiments, the ratio of lactide to glycolide is from about 0: 100 to about 100: 0, thus providing other rates of release.

In some embodiments, the microspheres or microparticles have a hollow and / or porous internal structure. In some embodiments, these microspheres comprise a solid or porous outer shell,

In some embodiments, preparations containing porous outer shells and / or microspheres exhibit a biphasic release profile of the active ingredient (s) with an initial explosive release of the active ingredient (s) and subsequent sustained release associated with degradation of the polymer microspheres. An initial burst loads the tissue with an effective lipolytic / lipogenesis inhibitory and anti-inflammatory concentration of the active ingredient (s), with a subsequent slower release maintaining the desired concentration. In some embodiments, various microsphere structures and release profiles of the active ingredient optimize the effect of treating adipose tissue of the eye socket and adipocytes and, through modulation of the adrenergic receptor, the effect on leukocyte infiltrate to reduce inflammation. In some preferred embodiments, tissue-supported local concentrations of long-acting selective beta-2-adrenergic agents, such as salmeterol 1 and / or formoterol 2, are concentrations from about 1.0 pM to about 10 μM, for example, from about 0.01 μM to about 10 μM, from about 0.1 μM to about 5 μM, from 0.5 μM to about 4 μM, or any other concentration from about 0.001 μM to about 10 μM. Supported local tissue concentrations of glucocorticosteroids can range from about 0.01 μM to about 10 mM.

In some embodiments, one or more than one active ingredient is encapsulated, bound and / or conjugated to the polymer in a ratio of about 10-12% by weight compared to polymer microspheres. The amount of active ingredient in the form of a mass percentage relative to the carrier (for example, microparticles or microspheres) is referred to herein as “active ingredient load”. As used herein, the terms “loaded” and “loading” refer to active ingredients that are substantially encapsulated, bound and / or conjugated to a carrier. In some embodiments, the load of the active ingredient is up to about 75%. Thus, some preferred preparations contain one or more beta-2-adrenergic active ingredients, such as salmeterol 1, formoterol 2 and / or their physiologically acceptable salts and solvates, loaded on polymer microspheres in a concentration of from about 1 mg to about 20 mg of active ingredient in about 10 to about 200 milligrams of polymer. In some embodiments of the drug with this load of active ingredient, it is sufficient to provide from about 15 days to about 45 days of release of the active ingredient in a concentration suitable for lipolysis and / or inhibition of lipogenesis. Similarly, to obtain the anti-inflammatory effect, budesonide and fluticasone glucocorticosteroids in pharmaceutically acceptable forms can be loaded at a concentration of about 1 mg to about 20 mg of the active ingredient with about 10 to about 200 mg of the polymer.

In some embodiments, two or more active ingredients are loaded into the same microparticle, for example, a liposome or PLGA. Thus, in some embodiments, a polymer encapsulating a glucocorticosteroid in an adrenergic compound is delivered to adipose tissue simultaneously. Alternatively, the two active ingredients are loaded into separate microparticles. The two types of microspheres are then mixed to obtain a drug with the desired ratio of beta receptor agonist and glucocorticosteroid, then they are administered simultaneously. Alternatively, two types of microparticles are administered sequentially.

Microspheres containing the active ingredient (s) are suspended in about 0.5 ml to 10 ml of a suitable physiologically acceptable liquid carrier. In some embodiments where separate microspheres with active ingredients are used, these microspheres are mixed together in a liquid carrier. In other embodiments, each type of microspheres is mixed separately with the liquid carrier. In some embodiments, the liquid carrier may contain a pharmaceutically acceptable form (1.0 to 15 IU / ml) and an amount of hyaluronidase. As used herein, an amount of “1 ME hyaluronidase” produces the same reduction in turbidity in a mixture of hyaluronic acid and albumin as 1 ME (International Unit) of the standard hyaluronidase preparation. See, for example, Mathews et al. (1966), Methods Enzymol 8, 654-662. In some embodiments, the suspension of microspheres is then injected into the orbit or retrobulbar space with 0.1-0.5 ml aliquots. Alternatively, the injections, as described above, are performed separately and sequentially in the same areas using two microsphere preparations encapsulating each active ingredient.

In some embodiments, a glucocorticosteroid, such as dexamethasone 6, budesonide 8 and / or fluticasone propionate 7, also acts as an anti-inflammatory agent, thereby reducing inflammation caused by the administration of this drug, for example, caused by polymers, polymer microspheres and / or liposomes in a drug with slow release.

Microspheres from PLGA 15 encapsulate hydrophobic compounds more easily than hydrophilic compounds. To increase the load of hydrophilic active ingredients, in some embodiments, the microspheres are modified with polyethylene glycol units, as discussed above. Microspheres of a certain size are not substantially absorbed into the blood or removed by lymph, thereby providing release of the active ingredient (s) in the desired area. For example, in some embodiments, the microspheres have a diameter of from about 20 microns to about 200 microns. In some embodiments, the size of the microspheres also affects the release profile of the active ingredient (s) into the tissue. In general, larger microspheres tend to provide a longer and more uniform release profile.

In an exemplary embodiment, the sustained release preparation contains from about 0.5 milligrams to about 7.5 mg (for example, about 0.7; 1; 1.5; 2.0; 2.5; 3; 3.5; 4; 4.5; 5; 5.5; 6; 6.5, 7 or any other amount from about 0.5 mg to about 7.5 mg) of salmeterol 1 and / or formoterol 2, and from about 1.5 mg to about 7.5 mg (e.g., about 2; 2.5; 3; 3.5; 4; 4.5; 5; 5.5; 6; 6.5; 7 or any other amount from about 1.5 to approximately 7.5 mg) of dexamethasone 6, fluticasone propionate 7 and / or budesonide 8, encapsulated in about 100 milligrams of polylactide glycolide microspheres (PLGA) 15 copolymer in a ratio of about 70% lactide: 30% glycolide. The amount of each active ingredient in a sustained release preparation depends on the required number of days for a controlled / delayed release (e.g., about 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or about 10 days). In some embodiments, the ratio of the copolymers and encapsulation of the active ingredient deliver up to about 1.0 μg per day (e.g., about 0.02; 0.04; 0.06; 0.07; 0.1; 0.2; 0.4 ; 0.5; 0.6; 0.8; or any other amount from about 0.02 μg to about 100.0 μg per day) of salmeterol 1 and / or up to about 0.5 μg (e.g., about 0.02 ; 0.04; 0.06; 0.07; 0.08; 0.09; 0.1; 0.2; 0.3; 0.4 or any other amount from about 0.02 μg to about 0, 5 mcg per day) of formoterol and up to 5 mcg per day (e.g., approximately 0.2; 0.4; 0.5; 0.7; 0.9; 1.0; 1.5; 2, 2.5 ; 3; 3,5; 4; 4,5 or any other quantity from at ERNO 0.2 to about 5 g per day) fluticasone and / or budesonide 6 to about 1 mg of copolymer for up to about 30 days. In another exemplary embodiment, the sustained release preparation to be administered comprises methylprednisolone acetate in the form of a suspension of crystalline microparticles with a beta-2 adrenergic agonist. A beta-2 adrenergic agonist may be prepared in the form of a solution or may also be prepared in the form of a suspension of crystalline microparticles. In some embodiments, the sustained release formulation also contains soluble methylprednisolone succinate to provide immediate effects (due to the quick release), in addition to the prolonged effect of the crystalline form of methylprednisolone acetate.

In some embodiments, one or more beta-2 agonists and methylprednisolone are mixed with each other before administration (e.g., before injection). In other embodiments, one or more beta-2 agonists and methylprednisolone are mixed during administration (e.g., injection).

In some embodiments, a selected sustained release corticosteroid preparation (e.g., a suspension of methylprednisolone crystals, a solution of methylprednisolone, or a combination of a suspension of crystals and solution) is delivered alone in about 7 days (e.g., at least 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or any other period of time from about 12 hours to about 7 days) to a beta-2 agonist to provide up-regulation of the beta receptor.

In some embodiments, the subject to be treated is given an immediate release preparation. In some embodiments, an immediate release formulation after a single dose provides the activity of one or more long-acting selective beta-2 agonists for a period of time from about four hours to about 24 hours, for example, for 6 hours, 8 hours, 10 hours , 12 hours, 16 hours, 18 hours, 21 hours, or during any other period of activity of the beta-2 agonist from about four hours to about 24 hours.

In other embodiments, the subject to be treated is given an immediate release preparation containing selective short-acting beta-2 agonists that have activities lasting less than about four hours (e.g., about 3.5 hours, 3 hours, 2, 5 hours, 2 hours, 1.5 hours, 1.3 hours, about 1 hour, 0.5 hours or any other period of time from less than about four hours to about 0.5 hours).

In an exemplary embodiment, the immediate release injectable preparations contain from about 100 μg to about 250 μg (e.g., 105, 110, 125, 150, 175, 190, 200, 210, 225, or any other amount from about 100 μg to about 250 μg) salmeterol xinafoate and from about 500 μg to about 1000 μg (e.g., 600, 650, 700, 730, 740, 800, 825, 875, 900, 930, 950 or any other amount from about 500 μg to about 1000 μg) of fluticasone propionate prepared in volumes up to about 10 ml (for example, 0.3; 0.5; 0.7; 1.1; 1.5; 2; 2.5; 3; 3.5; 4; 5; 6; 7; 8; 9 or any other volume from about 0 , 3 to about 10 ml) of an excipient compatible with administration into the orbit. The excipient concentration may be maintained at less than 1% (e.g., 0.05%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, or any other concentration from approximately 0.05% to less than 1%).

In some embodiments, the preparations described herein are delivered through the eye using any suitable method, for example, in the form of drops for local application, or using a reservoir placed under the eyelid.

In other embodiments, where the drug to be delivered contains long-acting beta-2 agonists such as formoterol 2, salmeterol 1 or bambuterol 3, and glucocorticosteroids, topical application is preferred. In some embodiments, sustained-release formulations delivered transdermally include a biodegradable biocompatible preparation of the active ingredient with a polymer or a liposome preparation, as discussed above.

In some embodiments, there is provided a method of treating thyroid ophthalmopathy by local delivery of a preparation containing one or more immunosuppressants (e.g. rapamycin, sirolimus or everolimus) to inhibit fibroblast proliferation and convert preadipocytes to adipocytes. In some embodiments, immunosuppressants are administered in a sustained release formulation (eg, a depot preparation) containing one or more immunosuppressants in a suspension of crystalline microparticles. In other embodiments, the sustained release preparation comprises polymer microparticles (as described herein) loaded with one or more immunosuppressants. The administration of a sustained release immunosuppressant drug may be administered by ocular, periorbital, or retrobulbar injection.

In some embodiments, one of the aforementioned preparations includes thyroid hormone to upregulate beta receptors, or to increase the number of beta receptors on the cell surface, or to downregulate alpha receptors, or to decrease the number of alpha receptors on the cell surface. For this purpose, you can use both thyroxine (T4) 18, and triiodothyronine (T3) 19, and stereoisomers of these hormones. Levothyroxine is a stereoisomer of thyroxine, which may have a longer half-life. These thyroid hormones can be combined with beta-agonists and administered by ocular, periorbital or retrobulbar injection. One or more thyroid hormones can also be combined with beta agonists in a sustained release preparation, such as a polymer or liposome, as described above, to reduce the frequency of administration. T3 and T4 can be combined with selective long-acting beta-2 agonists in polylactide or polylactide / glycolide polymer microspheres for injection and sustained release.

In some embodiments, the preparations described above additionally include one or more flavonoids from the group of flavones and flavinones (e.g., quercetin and fisetin) that act as inhibitors of cAMP phosphodiesterase and thus enhance the beta adrenergic signaling pathway.

EXAMPLES

The following specific examples should be construed only as illustrative and not limiting the rest of the description in any way. Without further elaboration, it is believed that one skilled in the art can use the present invention to its fullest extent on the basis of the description provided herein. All publications cited herein are hereby incorporated by reference in their entirety. Where a link is made to a URL or other such identifier or address, it is understood that such identifiers may change and specific information on the Internet may appear and disappear, but equivalent information can be found by searching the Internet. The link to them proves the availability and public distribution of such information.

Example 1: Analysis of lipolysis of rat adipocytes in vitro under the influence of beta-agonists and glucocorticosteroids

In an in vitro lipolysis assay, glycerol was determined in cell culture media by spectrophotometric measurement after chemical oxidation with hydrogen peroxide. Glycerin was measured for three hours. Lipolysis levels in cultured human adipocytes were tested after exposure to one beta agonist, one glucocorticosteroid, or a combination of two for one or more than one pre-incubation period, as described in more detail below.

Isolation of preadipocytes and differentiation into adipocytes:

In the analysis of lipolysis used human subcutaneous adipocytes. Adipose tissue was selected after liposuction or lipectomy, and preadipocytes were isolated as follows. Briefly, adipose tissue was minced and incubated at 37 ° C in Krebs-Ringer bicarbonate buffer containing 1% bovine serum albumin and 0.1% collagenase in an oxygen enriched shaking chamber (5% CO ₂ ; 75 shakes / min) in within 1 hour. The suspension was filtered through a 400 μm mesh network and centrifuged for 1 min at 100g. The preadipocytes in the supernatant were washed twice and then transferred to 96-well plates at a cell density / well. Preadipocytes were cultured in a support medium for seven days while they differentiated into adipocytes.

Reagents

Wash Buffer (Krebs-Ringer Buffer (KRB) without serum;

[Sigma, K4002-10X1L]) - stored at 4 ° C.

Assay buffer (KRB with 1% FBS (cow fetal serum); [FBS from Gibco, 26140-079]) - stored at 4 ° C.

The support medium was stored at 4 ° C.

Glycerin A reagent (Zen-Bio, RGTL-15 or RGTL-40) - after dissolution, stored at 4 ° C in a dark place.

The mother liquor of glycerol (1 M) obtained by diluting glycerol [Sigma G2025-500ML) in washing buffer (without serum) was stored at -20 ° C.

Lipolysis Analysis:

21 hours before lipolysis analysis, the medium was removed from each well and replaced with 75 μl of the support medium containing the appropriate concentrations of the drug or DMSO (dimethyl sulfoxide) (vehicle) (see the Experimental Scheme section below). Treatment with each test drug or control was performed in 8 wells / group (12 treatment groups per 96-well plate). 3 hours before lipolysis analysis, each well was washed twice with wash buffer (200 μl / wash), filled with test or control solutions prepared on analysis buffer (75 μl / well) and then incubated for three hours, i.e. before measuring the glycerol content in the assay buffer. For some groups, the drug was added only during the three-hour incubation period (see the Experimental and control groups section below). One hour before analysis, seven glycerol standards were prepared with concentrations ranging from 200 μM to 3.125 μM by serial dilution in assay buffer.

The glycerol content in the assay buffer from each well after incubation was used as a lipolysis index, where an increase in glycerol content indicated lipolysis. Glycerol levels were colorimetrically analyzed using a commercially available glycerol analysis kit (Randox Laboratory, United Kingdom) and quantified by comparison with a standard glycerol sequential dilution curve (3 μM-200 μM). Glycerol concentrations for each well were standardized to cell density.

Experiment Design:

In each of the following control or experimental groups, n = 8 corresponds to glycerol measurements for 8 wells from a 96-well cell culture plate.

Table 1 Generalization of the experimental scheme for the analysis of lipolysis in vitro Group (s) 18 hour incubation 3 hour incubation The results are shown in FIG. Experiment 1 1 (negative control) 0.1% DMSO 0.1% DMSO 2 * 2-11 0.1% DMSO Formoterol
(10 ^-13 M-10 ^-4 M) 2 12 (positive control) 0.1% DMSO Isoproterenol 10 ^-6 M 2 Experiment 2 13 (negative control) 0.1% DMSO 0.1% DMSO 3 * 14-18 0.1% DMSO Salmeterol (10 ^-8 M-10 ^-4 M) 3 19 (positive control) 0.1% DMSO Isoproterenol 10 ^-6 M 3 Experiment 3 20 (negative control) 0.1% DMSO 0.1% DMSO four* 21-22 0.1% DMSO Budesonide
10 ^-10 M ^vs 10 ^-7 M four 23-26 Budesonide
(10 ^-12 M-10 ^-6 M) Budesonide (10 ^-12 M-10 ^-6 M) four 27 (positive control) 0.1% DMSO Isoproterenol 10 ^-6 M four Experiment 4 28 (negative control) 0.1% DMSO 0.1% DMSO 5* 29th Salmeterol 10 ^-6 M Salmeterol 10 ^-6 M 5 thirty Salmeterol 10 ^-6 M + Budesonide 10 ^-6 M Salmeterol 10 ^-6 M + Budesonide 10 ^-6 M 5 31 Salmeterol 10 ^-8 M Salmeterol 10 ^-8 M 5 32 Salmeterol 10 ^-8 M + Budesonide 10 ^-6 M Salmeterol 10 ^-8 M + Budesonide 10 ^-6 M 5 33 (positive control) Isoproterenol 10 ^-6 M Isoproterenol 10 ^-6 M 5 ^* Data for all groups are presented on the graph as the ratio or% of differences relative to the data for the negative control group

As shown in FIG. 2, a long-acting beta-2 adrenergic receptor agonist induced a dose-dependent increase in lipolysis by more than six times after a three-hour incubation, which for a concentration of 10 ^-6 M and higher was greater than the increase observed for isoproterenol. Similarly, a long-acting beta-2 adrenergic receptor agonist also induced a dose-dependent increase in lipolysis after a three-hour incubation, although this effect (slightly more than a twofold increase in lipolysis) was not as strong as the effect observed for formoterol. Salmeterol-induced lipolysis was equal to or less than the lipolysis observed for isoproterenol.

As shown in FIG. 3, the budesonide glucocorticosteroid induced a slight increase (up to about 1.5-fold) of lipolysis after three hours, which was less than the increase observed for isoproterenol (about 2.5-fold). In contrast, incubation with budesonide alone for 18 hours actually caused a slight inhibition of lipolysis in vitro.

Incubation of adipocytes with salmeterol (10 ^-6 M) for 18 hours reduced lipolysis (Figure 4). Similarly, treatment with isoproterenol for 18 hours resulted in reduced lipolysis. However, when salmeterol was combined with budesonide during the 18-hour incubation period, an increase in lipolysis was observed (Figure 4).

Based on these data, the inventors concluded that formoterol and salmeterol effectively induce lipolysis in cultured adipocytes over a three-hour period of time. However, salmeterol actually reduces lipolysis when used within 18 hours, probably due to desensitization or downregulation of the receptor. In addition, in the presence of glucocorticosteroid budesonide, salmeterol is able to induce lipolysis even after 18 hours. Thus, budesonide can be used to maintain or restore the ability of a beta-2 adrenergic agonist to induce lipolysis over long periods of time, probably by preventing downregulation of beta-2 adrenergic receptors.

Example 2: Inhibition of Lipogenesis by Beta-Agonists and Glucocorticosteroids

A non-limiting example of such an inhibition of lipogenesis is as follows:

Cell culture:

The 3T3-L1 preadipocyte cell line (ATCC, Manassas, VA) was seeded with a density of 4 × 10 ⁵ cells per T75 ml vessel in Dulbecco's modified Eagle medium (DMEM) with 10% normal calf serum and 1% antibiotics (penicillin / streptomycin). Cells were incubated at 37 ° C, 5% CO ₂ . After three days, the cells were trypsin-free, counted and resuspended in 24-well plates at a density of 6 × 10 ⁵ cells per well in 2 ml of medium. After 1-2 days, the cells were close to confluence and ready for lipogenesis.

Substances for lipogenesis:

Lipogenesis initiation medium: DMEM / 10% bovine serum / 0.5 mM IBMX / 1 μM dexamethasone.

Medium for the development of lipogenesis: DMEM / 10% serum of cow fruit / 10 μg / ml insulin.

Lipogenesis Maintenance Medium: DMEM / 10% cow serum.

Negative control medium: DMEM / 10% normal calf serum.

Lipogenesis Protocol:

1.8 ml of medium is removed from the wells and 2 ml of medium is added to initiate lipogenesis per well. The plates are incubated for 48 hours at 37 ° C, 5% CO ₂ . 2 ml of medium is removed and 2 ml of medium per well is added to develop lipogenesis. The plates are incubated for 48 hours at 37 ° C, 5% CO ₂ . 2 ml of medium is removed and 2 ml of medium per well is added to maintain lipogenesis. The plates are incubated for at least 48 hours at 37 ° C, 5% CO ₂ . Intracellular lipid droplets accumulate in the cells for at least 5 days.

Experiment Design:

Before lipogenesis, 3T3-L1 preadipocyte cells are pretreated at different stages with a beta-2 agonist and / or glucocorticosteroid. 24 hours before adding the medium to initiate lipogenesis, the cells are treated as follows:

Group 1: no processing

Group 2: 10 ^-10 M salmeterol

Group 3: 10 ^-8 M salmeterol

Group 4: 10 ^-6 M salmeterol

Group 5: 10 ^-4 M salmeterol

Group 6: 10 ^-10 M salmeterol + 10 ^-6 M budesonide

Group 7: 10 ^-8 M salmeterol + 10 ^-6 M budesonide

Group 8: 10 ^-6 M salmeterol + 10 ^-6 M budesonide

Group 9: 10 ^-4 M salmeterol + 10 ^-6 M budesonide

Group 10: 10 ^-6 M budesonide

Group 11: 10 ^-10 M budesonide

Group 12: 10 ^-6 M capsaicin, a known inhibitor of lipogenesis.

Another batch of cells is treated with the above groups 24 hours before the addition of medium for the development of lipogenesis and another batch of cells is treated 24 hours before the addition of the medium to maintain lipogenesis. In a control batch, cells are treated with the above groups 24 hours before the addition of medium for negative control. In two other batches of 12 groups, salmeterol was replaced by a long-acting beta-2 agonist formoterol in one batch and a short-acting beta-2 agonist albuterol in another batch.

Visualization of intracellular lipids:

Cells are harvested 5 days after the addition of medium to maintain lipogenesis. The cell medium is removed and the plates washed twice with phosphate buffered saline (PBS). Add 0.5 ml of a solution of Oil Red O (0.36% Oil Red O in 60% isopropanol) per well and the plates are incubated for 15 minutes at room temperature. The staining solution was removed and the wells were washed three times with 60% isopropanol. The stained tablets are then photographed and / or scanned for visual analysis. Lipids are colored red.

Quantification of lipids:

0.25 ml of dye extraction solution (CHEMICON International) is added to stained wells. The tablets are mounted on an orbital shaker or shaker for 15-30 minutes. The solution with extracted dye was transferred to a cuvette and the absorbance was read on a spectrophotometer at 520 nm.

Example 3: Beta-2 agonists in combination with glucocorticosteroids reduce the mass of epididymal fat bodies

The inventors tried to determine whether a glucocorticosteroid can reduce the fat content in vivo in a manner consistent with the data of the inventors on in vitro lipolysis, as described in Example 1. For this, the inventors measured the mass of epididymal fat bodies in rats treated with beta-2-adrenergic long-acting agonist formoterol, alone or in combination with budesonide.

Male Sprague Dawley rats (~ 500 g) were anesthetized under 4% isoflurane using a Matrx 3000 vaporizer. Then, animals, as listed in Table 2 below, were injected with 5 ml of vehicle (2% PEG) 5 mm in front of the posterior end of the adipose body. ); formoterol (3.48 μg / ml; dose = 1.39 μg) in a carrier or formoterol (3.48 μg / ml) and budesonide (10 μg / ml; dose = 1.39 μg formoterol and 4 μg budesonide) in a carrier . Each animal received a drug treatment on one side and a carrier treatment (2% PEG) on the opposite side; for each treatment group, the right and left sides were balanced in relation to the drug and the carrier (see Table 2).

table 2
Experimental in vivo lipolysis assay Group Animal number Right Epididymal Fat Body Left Epididymal Fat Body one one One formoterol Carrier 2 One formoterol Carrier 3 Carrier One formoterol four Carrier One formoterol 2 5 Formoterol + Budesonide Carrier 6 Formoterol + Budesonide Carrier 7 Carrier Formoterol + Budesonide 8 Carrier Formoterol + Budesonide

The injections were repeated after 24 and 48 hours, a total of three injections. Twenty-four hours after the last injection, the animals were sacrificed by an intraperitoneal excess dose of pentobarbital (150 mg / kg), the left and right epididymal fat bodies from each animal were collected and weighed (the results are shown in Table 3 and Figure 6). A paired t-test and a standard t-test were used for statistical analysis.

Table 3 Change in body fat mass (carrier drug) Group one 2 Animal 1 and 5 -0.036 -0.599 Animal 2 and 6 -0.138 -0.115 Animal 3 and 7 0.118 -0.574 Animal 4 and 8 0.166 -0.124 Average 0,028 -0.353 SD 0.140 0.270

The data on the treatment with one formoterol and formoterol + budesonide, shown in Table 3, were analyzed using paired t-student test, the results of this analysis are shown in Table 4.

Table 4 Statistical analysis of changes in the mass of fat bodies (carrier drug) after treatment with one formoterol or formoterol + budesonide Groups The value of bilateral p Group 1 (formoterol) relative to the carrier 0.63 Group 2 (formoterol + budesonide) relative to the carrier 0,0792

As shown in Table 4, Group 1 animals (treated with formoterol alone) showed differences and variability consistent with naive untreated controls. The average treatment effect with formoterol alone was + 0.028 g ± 0.140 g. Statistical analysis yielded a p value of 0.63, which is consistent with the lack of a trend for the treatment effect. On the other hand, Group 2 animals (treated with formoterol + budesonide) showed a treatment effect. The average treatment effect was -0.353 ± 0.270 with a value of p = 0.079.

The authors of this invention also conducted a statistical analysis (Student's t-test) of significant differences between the effect of treatment with one drug and a combination of drugs, as well as significant differences between treatment with a combination of drugs relative to untreated control animals. The results of these statistical analyzes are shown in Table 5.

Table 5 Statistical analysis of differences in the effects of one formoterol relative to formoterol + budesonide and formoterol + budesonide relative to untreated Comparison (average weight difference of epididymal fat bodies) The value of bilateral p Group 2 (formoterol + budesonide) relative to Group 1 (one formoterol) 0.05 Group 2 relatively untreated 0.0131

As shown in Table 5 and FIG. 6, there was a statistical difference between Group 2 and Group 1 (p = 0.05). There was a significant difference between the average decrease in body fat mass in Group 2 relative to untreated animals with p = 0.013.

In a subsequent experiment, the inventors tested the ability of formoterol plus budesonide to reduce body fat in the assay described above. As shown in FIG. 7, the administration of a combination of doses of 1.4 μg / day of formoterol + 4.0 μg / day of budesonide led to a significant difference in the average epididymal mass in the treated group relative to the untreated group (p = 0.01). Similarly, even with a lower dose combination (0.7 μg / day formoterol + 2.0 μg / day budesonide, which was achieved by dosing every other day), the difference between the treated combination and the untreated control animals was significant (p = 0.04).

Based on these data and their analysis, the authors of the present invention concluded that the combination of a long-acting beta-2 agonist (e.g., formoterol) and a glucocorticosteroid (e.g., budesonide) appears to be effective for inducing lipolysis and reducing fat in vivo.

Example 4: Clinical Testing of the Treatment of Graves Ophthalmopathy with Compositions Containing Beta Agonist and Glucocorticosteroid

A non-limiting example of such clinical testing for the treatment of Graves' ophthalmopathy is the following:

Patient selection:

Patients should be 18 years of age or older and should not have hypersensitivity to the drugs administered. They showed symptoms of proptosis associated with Graves' ophthalmopathy using ultrasonography, exophthalmometry, MRI (magnetic resonance imaging) or computed tomography. In particular, patients with unilateral or bilateral proptosis of 0.5 mm or 3 mm or more, with or without other symptoms, were selected. Patients may also have double vision, restriction of eye movement in extreme positions and obvious restriction of movement, ulceration of the cornea, pain, cosmetic defects and poor quality of life. Patients may have undergone thyroidectomy for hyperthyroidism. Other steroid therapy should not be used to treat hyperthyroidism. All studies should be subject to approval by the institutional ethics committee and patient consent.

Study design:

Test 1: This is a multicenter study with an increase in the dose of combination therapy with a long-acting beta-2 agonist with salmeterol and glucocorticosteroid budesonide. Patients receive a daily injection of the parenteral drug composition. Patients who do not achieve improvement in proptosis or a partial or complete response, but who have a stable disease after 1 week of therapy, will receive an additional 1-week therapy with a higher dose than the one prescribed initially. Cohorts of 3-6 patients receive increasing doses of the combined drug until the maximum tolerated dose (MTD) is determined. MTD is defined as the dose preceding the dose at which 2 of 3 or 2 of 6 patients experience dose-limiting toxicity.

Test 2: This is a randomized, multicenter trial. The duration of this study is 60 days. Patients were randomized in 1 of 18 treatment groups. In group 1, patients are given daily one salmeterol or formoterol in MTD. In group 2, patients are given one budesonide per MTD daily. In group 3, patients are given simultaneously salmeterol and budesonide in MTD. In group 4, patients are given salmeterol or formoterol daily and budesonide every other day. In group 5, patients are given daily budesonide and salmeterol or formoterol every other day. In group 6, patients are given budesonide on every odd day and salmeterol on every even day. Groups 7-12 have the same dosage schedule as groups 1-6, except that the dosage is one-fourth MTD. Groups 13-18 also have the same dosage schedule as groups 1-6, except that the dosage is one tenth of the MTD. In addition to the treated groups, the control group was left untreated.

Assessment of Expected Results:

Patients are evaluated for a decrease in proptosis and a decrease in the amount of orbital fat and external muscles of the eye at the time of completion of the study. The improvement in eyelid closure and eye movement is also evaluated. A 20% decrease within 60 days is considered a positive result.

Example 5: Pharmaceutical Compositions

Non-limiting examples of such pharmaceutical compositions are as follows:

Parenteral compositions

Example 5A To obtain a parenteral pharmaceutical composition suitable for administration by injection, approximately 100 μg of a water-soluble salt of formoterol and approximately 3 mg of ketotifen are dissolved in DMSO and then mixed with 10 ml of 0.9% sterile saline. This mixture is included in a unit dosage form suitable for administration by injection.

Example 5B To obtain a parenteral pharmaceutical composition suitable for injection by injection, about 50 μg of a water-soluble salt of salmeterol and about 100 μg of fluticasone propionate are dissolved in DMSO and then mixed with 10 ml of 0.9% sterile saline containing about 20% v / about. PEG-400. Hyaluronidase is added to this mixture to a final concentration of 8 IU / ml. The resulting mixture is included in a unit dosage form suitable for administration by injection.

Example 5C. To obtain a parenteral pharmaceutical composition suitable for administration by injection, about 50 to 100 μg of a water-soluble salt of salmeterol is dissolved in DMSO and then mixed with 10 ml of 0.9% sterile physiological saline containing about 20% v / v. PEG-400. This mixture is included in a unit dosage form suitable for administration by injection.

Example 5D To obtain a parenteral pharmaceutical composition suitable for injection, approximately 50 μg of the water-soluble salt of fluticasone propionate is dissolved in DMSO and then mixed with 10 ml of 0.9% sterile saline. Hyaluronidase is added to this mixture to a final concentration of 10 IU / ml. The resulting mixture is included in a unit dosage form suitable for administration by injection.

Gel formulations for topical administration

Example 5E To obtain a pharmaceutical composition in the form of a gel for topical administration, approximately 100 mg of salmeterol and approximately 100 mg of prednisolone are mixed with 1.75 g of hydroxypropyl cellulose, 10 ml of propylene glycol, 10 ml of isopropyl myristate and 100 ml of purified USP alcohol (USP). The resulting gel mixture is then placed in containers, such as tubes, which are suitable for topical administration.

Example 5F. To obtain a topical gel pharmaceutical composition, approximately 100 mg of formoterol and approximately 100 mg of budesonide are mixed with approximately 10 mg of hyaluronidase, 1.75 g of hydroxypropyl cellulose, 10 ml of propylene glycol, 10 ml of isopropyl myristate and 100 ml of purified USP alcohol. The resulting gel mixture is then placed in containers, such as tubes, which are suitable for topical administration.

Example 5G. To obtain a topical gel pharmaceutical composition, approximately 100 mg of salmeterol is mixed with approximately 10 ml of PEG-400, 1.75 g of hydroxypropyl cellulose, 10 ml of isopropyl myristate and 100 ml of purified USP alcohol. The resulting gel mixture is then placed in containers, such as tubes, which are suitable for topical administration.

Example 5H To obtain a pharmaceutical composition in the form of a gel for topical administration, approximately 100 mg of prednisolone is mixed with approximately 10 ml of PEG-400, 1.75 g of hydroxypropyl cellulose, 10 ml of isopropyl myristate and 100 ml of purified USP alcohol. The resulting gel mixture is then placed in containers, such as tubes, which are suitable for topical administration.

Ophthalmic Solution Compositions

Example 5I To obtain a pharmaceutical composition in the form of an ophthalmic solution, approximately 100 mg of salmeterol compound and approximately 100 mg of budesonide are mixed with 0.9 g of NaCl in 100 ml of purified water and filtered using a 0.2 μm filter. The resulting isotonic solution is then placed in ophthalmic delivery devices, such as eye drop containers, which are suitable for ophthalmic administration.

Example 5J. To obtain a pharmaceutical composition in the form of an ophthalmic solution, about 100 mg of the formoterol compound and about 100 mg of budesonide are mixed with 0.9 g of NaCl in 100 ml of about 10% v / v. PEG-400 in purified water and filtered using a 0.2 μm filter. The resulting isotonic solution is then placed in ophthalmic delivery devices, such as eye drop containers, which are suitable for ophthalmic administration.

Example 5K. To obtain a pharmaceutical composition in the form of an ophthalmic solution, approximately 100 mg of the formoterol compound and approximately hyaluronidase (to a final concentration of 10 IU / ml) are mixed with 0.9 g of NaCl in 100 ml of about 10% v / v. PEG-400 in purified water and filtered using a 0.2 μm filter. The resulting isotonic solution is then placed in ophthalmic delivery devices, such as eye drop containers, which are suitable for ophthalmic administration.

Example 5L. To obtain a pharmaceutical composition in the form of an ophthalmic solution, about 100 mg of the ketotifen compound are mixed with 0.9 g of NaCl in 100 ml of about 20% v / v. PEG-400 in purified water and filtered using a 0.2 μm filter. The resulting isotonic solution is then placed in ophthalmic delivery devices, such as eye drop containers, which are suitable for ophthalmic administration.

Oral composition

Example 5M To obtain an oral pharmaceutical composition, approximately 100 mg of the prednisolone compound is mixed with 750 mg of starch. This mixture is included in an oral dosage form, such as a hard gelatin capsule, which is suitable for oral administration.

Example 5N. To obtain an oral pharmaceutical composition, approximately 50 mg of budesonide compound is mixed with 375 mg of gelatin. This mixture is included in an oral dosage form, such as a hard gelatin capsule, which is suitable for oral administration.

Example 5O. To obtain an oral pharmaceutical composition, approximately 200 mg of the ketotifen compound is mixed with 1,500 mg of hydroxypropyl methylcellulose. This mixture is included in an oral dosage form, such as a hard gelatin capsule, which is suitable for oral administration.

Example 5P To obtain an oral pharmaceutical composition, approximately 50 mg of fluticasone propionate compound is mixed with 600 mg of starch. This mixture is included in an oral dosage form, such as a hard gelatin capsule, which is suitable for oral administration.

Example 6: Schemes for the administration of beta-agonists and glucocorticosteroid

Non-limiting examples of such administration regimens are as follows:

Example 6A A patient suffering from Graves' ophthalmopathy is administered a therapeutically effective amount of the 5C composition per day 1 and every subsequent odd day of treatment. On an even day, a therapeutically effective amount of 5D composition is administered to a patient.

Example 6B A patient suffering from Graves' ophthalmopathy is administered a therapeutically effective amount of 5D composition per day 1 and on each subsequent odd day of treatment. On an even day, the patient is administered a therapeutically effective amount of composition 5C.

Example 6C A therapeutically effective amount of 5C composition is administered daily to a patient suffering from Graves ophthalmopathy. On an even day, a therapeutically effective amount of 5D composition is administered to a patient.

Example 6D A therapeutically effective amount of 5D composition is administered daily to a patient suffering from Graves ophthalmopathy. On an even day, the patient is administered a therapeutically effective amount of composition 5C.

Example 6E A patient suffering from Graves' ophthalmopathy is administered a therapeutically effective amount of the 5C composition per day 1, followed by a weekend for two days. On the second weekend, a therapeutically effective amount of 5D composition is administered to the patient. This introduction is then repeated.

Example 6F A patient suffering from Graves' ophthalmopathy is administered a therapeutically effective amount of the 5D composition per day 1, followed by a weekend for two days. On the second weekend, a therapeutically effective amount of composition 5C is administered to the patient. This introduction is then repeated.

Example 6G. A patient suffering from Graves' ophthalmopathy is administered a therapeutically effective amount of the 5M composition per day 1 and every subsequent odd day of treatment. On an even day, the patient is administered a therapeutically effective amount of composition 5C.

Example 6H A therapeutically effective amount of 5C composition is administered daily to a patient suffering from Graves ophthalmopathy. On an even day, a therapeutically effective amount of 5M composition is administered to a patient.

The inventor conducted experiments and obtained data that establish the industrial applicability of the claimed compositions. For example, two such experimental tests confirm that a beta adrenergic agonist and a compound to reduce desensitization of target tissue to a beta adrenergic agonist effectively treat a disease involving ocular fat accumulation, including Graves disease as an example.

1. Experiment 1: Injection of a beta adrenergic agonist and desensitizing compound into human periorbital tissue

The experiment was carried out by injection of salmeterol xinafoate (beta-adrenergic agonist) and budesonide (compounds to reduce desensitization to beta-adrenergic agonist) in tissue samples from the orbital fatty body of normal people and people with Graves' disease.

A. Protocol: Eye fat was obtained after informed consent from patients undergoing orbital decompression for the treatment of severe Graves ophthalmopathy, or from individuals without a known autoimmune disease or thyroid disease undergoing blepharoplasty with fat removal. Tissue samples of an initial volume of 1 ml (estimated by fluid displacement) were implanted in the right and left flanks of SCID NODγ ^{- / -} mice. After reconstitution after surgery (14 days), 0.1 ml of the carrier (3.5% PEG400 in 0.9% saline) or 0.1 ml of the carrier containing 10 μg of salmeterol xinafoate was injected subcutaneously, directly into the implanted fat, directly into the implanted fat and 4 mcg budesonide daily for 10 days, and then every 3-5 days for 6 weeks. Then the explants were collected and the volume of tissue was determined.

B. Results: No change in volume was observed in explants treated with vehicle, either from normal subjects or from subjects with Graves disease. In explants treated with 10 μg of salmeterol xinafoate and 4 μg of budesonide, the volume was reduced by 30% -80% in explants from subjects with Graves' disease; a change in volume was not observed in explants from normal subjects.

B. Table 1 The therapeutic effects of varying injection doses of a beta adrenergic agonist and compound to reduce the desensitization of target tissue compared to control doses of a carrier: Mouse No. / Implant Side Fabric source Treatment Initial Volume (ml) The final volume (ml) % Reduction 2 / L Graves disease Carrier 0.1 0.1 0 3 / P Graves disease Carrier 0.1 0.1 0 3 / L Normal Carrier 0.1 0.1 0 1 / P Graves disease 10 mcg SX + 4 mcg budesonide 0.1 0.02 80 1 / L Graves disease 10 mcg SX + 4 mcg budesonide 0.1 0,07 thirty 4 / P Graves disease 10 mcg SX + 4 mcg budesonide 0.1 0.05 fifty 4 / L Normal 10 mcg SX + 4 mcg budesonide 0.1 0.1 0 SX-Salmeterol Xinafoate

2. Experiment 2: Injection of a desensitizing compound alone and in combination with a beta adrenergic agonist into human periorbital tissue

The second experiment was performed by injection of salmeterol xinafoate (beta-adrenergic agonist) and fluticasone propionate (compounds to reduce desensitization to beta-adrenergic agonists) into tissue samples from the orbital fatty body of people with Graves' disease.

A. Protocol: Eye fat was obtained after informed consent from patients undergoing orbital decompression for the treatment of severe Graves ophthalmopathy, or from individuals without a known autoimmune disease or thyroid disease undergoing blepharoplasty with fat removal. Tissue samples of an initial volume of 1 ml (estimated by fluid displacement) were implanted in the right and left flanks of SCID NODγ ^{- / -} mice. After recovery from surgery (14 days), 0.1 ml of the carrier (3.5% PEG 400 in 0.9% saline) or 0.1 ml of the carrier containing 10 μg of salmeterol xinafoate was injected subcutaneously, directly into the implanted fat, into the explants and 4 μg of fluticasone propionate daily for 10 days, and then every 3-5 days for 6 weeks. Then the explants were collected and the volume of tissue was determined.

B. Results: In implants treated with the drug carrier, an increased volume in the range of 2.3- to 4-fold was manifested. The volumes of those implants that received fluticasone as the sole agent remained the same as their original volumes. The introduction of the combination resulted in a decrease in volume, which was in the range from 20% to 70%. This combination also led to a significant violation of the normal structure of the fat body and significant fibrosis.

B. Table 2 The therapeutic effects of varying injectable doses of a desensitizing compound, alone and in combination with a beta adrenergic agonist: Mouse number Fabric source Treatment Initial Volume (ml) The final volume (ml) % Changes one Graves disease Diluent 0.05 0.20 + 4x 2 Graves disease Diluent 0.1 0.23 + 2.3x 3 Graves disease 4 mcg FP 0.1 0.1 0 four Graves disease 4 mcg FP 0.1 0.1 0 5 Graves disease 10 mcg SX + 4 mcg FP 0.1 0.08 -twenty% 6 Graves disease 10 mcg SX + 4 mcg FP 0.1 0,03 -70% SX-Salmeterol Xinafoate, FP-Fluticasone Propionate

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes to them are within the essence and content of this application and the scope of the attached claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entireties for all purposes.

Claims (39)

1. A method of reducing the accumulation of orbital fat in a patient in need thereof, comprising administering to said patient
(a) a therapeutically effective amount of at least one beta adrenergic agonist; and
(b) a therapeutically effective amount of at least one compound to reduce desensitization of the beta adrenergic receptor. 2. The method according to claim 1, where the specified patient suffers from thyroid ophthalmopathy. 3. The method according to claim 1, where the introduction is parenteral, oral, intraocular, intraocular, ophthalmic, periorbital, retrobulbar, intrathoracic, local, intramuscular, transdermal, sublingual, intranasal or airway. 4. The method according to claim 1, where at least one compound is administered to at least one beta adrenergic agonist. 5. The method according to claim 4, where at least one compound is administered from about 3 days to about 7 days to at least one beta adrenergic agonist. 6. The method according to claim 5, where at least one compound is administered by the intraocular, intraocular, ocular, periorbital, retrobulbar or intrathoracic routes of administration. 7. The method according to claim 6, where at least one compound is administered in the form of a suspension of crystalline microparticles. 8. The method according to claim 4, where at least one compound is administered orally and at least one beta adrenergic agonist is administered to the eye. 9. The method of claim 8, where at least one beta adrenergic agonist is introduced into the preparation of a suspension of crystalline microparticles. 10. The method according to claim 1, where at least one beta adrenergic agonist is a long acting beta adrenergic agonist. 11. The method according to claim 1, where at least one beta-adrenergic agonist is a beta-adrenergic agonist that is selective for the beta-2-adrenergic receptor. 12. The method according to claim 1, where at least one beta-adrenergic agonist is salmeterol, formoterol, or any combination thereof. 13. The method of claim 12, wherein the at least one beta adrenergic agonist is salmeterol and the therapeutically effective amount of salmeterol is from about 0.01 μg / day to about 100 μg / day. 14. The method according to item 13, where at least one beta-adrenergic agonist is formoterol, and a therapeutically effective amount of formoterol is from about 0.001 μg / day to about 50 μg / day. 15. The method according to claim 1, where at least one compound is a glucocorticosteroid, antihistamine, or any combination thereof. 16. The method according to claim 1, where at least one compound is dexamethasone, prednisone, methylprednisolone, fluticasone propionate, budesonide, ketotifen, or any combination thereof. 17. The method according to claim 6, where at least one compound is a glucocorticosteroid, and at least one long-acting beta-adrenergic agonist is salmeterol, formoterol, or a combination thereof. 18. The method according to claim 1, further comprising administering a therapeutically effective amount of an immunosuppressant by intraocular, intraocular, ophthalmic, periorbital, retrobulbar or intrathoracic route prior to administration of at least one compound. 19. The method of claim 18, wherein a therapeutically effective amount of an immunosuppressant is administered in the form of a suspension of crystalline microparticles. 20. A method of treating proptosis, comprising administering to a patient in need thereof a composition comprising a therapeutically effective amount of at least one beta adrenergic agonist. 21. The method according to claim 20, where at least one beta adrenergic agonist is a long acting beta adrenergic agonist. 22. The method of claim 20, wherein the at least one beta adrenergic agonist is a beta adrenergic agonist that is selective for the beta 2 adrenergic receptor. 23. The method according to claim 20, where at least one beta-adrenergic agonist is salmeterol, formoterol, bambuterol ,formoterol, isoproterenol, albuterol or fenoterol. 24. The method according to claim 20, where the composition comprises a mixture of at least one long-acting beta-adrenergic agonist and at least one short-acting beta-adrenergic agonist. 25. The method according to item 23, where the composition contains salmeterol, and a therapeutically effective amount of salmeterol is from about 0.01 μg / day to about 100 μg / day. 26. The method according to item 23, where the composition contains formoterol, and a therapeutically effective amount of formoterol is from about 0.001 μg / day to about 50 μg / day. 27. The method according to item 23, where the introduction is parenteral, oral, intraocular, intraocular, intrathoracic, ophthalmic, retrobulbar, local, intramuscular, transdermal, sublingual, intranasal or airway administration. 28. The method according to claim 20, where the composition further comprises a therapeutically effective amount of hyaluronidase. 29. A method of reducing the accumulation of orbital fat in a patient in need thereof, comprising administering to said patient
(a) a therapeutically effective amount of one or more than one compound, stimulating the pathway associated with the adrenergic receptor; and
(b) a therapeutically effective amount of at least one compound to reduce desensitization of the beta adrenergic receptor. 30. The method according to clause 29, where one or more than one compound that stimulates the pathway associated with the adrenergic receptor is a catecholamine, alpha-adrenergic antagonist, forskolin, aminophylline or their analogues. 31. An ophthalmic pharmaceutical composition comprising an ophthalmologically acceptable excipient and a therapeutically effective amount of methylprednisolone acetate or fluticasone propionate in the form of a suspension of crystalline microparticles. 32. The ophthalmic pharmaceutical composition of claim 31, further comprising solubilizing methylprednisolone acetate or solubilizing fluticasone propionate. 33. The ophthalmic pharmaceutical composition of claim 32, further comprising a therapeutically effective amount of at least one long-acting beta-2 agonist in the form of a suspension of crystalline microparticles. 34. An ophthalmic pharmaceutical composition comprising an ophthalmologically acceptable excipient and a therapeutically effective amount of at least one long-acting beta-2 agonist in the form of a suspension of crystalline microparticles. 35. The ophthalmic pharmaceutical composition according to claim 34, wherein the at least one long-acting beta-2 agonist is salmeterol or formoterol. 36. The ophthalmic pharmaceutical composition of claim 34, further comprising a therapeutically effective amount of at least one solubilized, long-acting beta-2 agonist. 37. The ophthalmic pharmaceutical composition according to claim 34, further comprising a therapeutically effective amount of at least one compound to reduce desensitization of the beta adrenergic receptor in the form of a suspension of crystalline microparticles. 38. The use of at least one beta adrenergic agonist and at least one compound to reduce desensitization of target tissue to a beta adrenergic agonist for the manufacture of a medicament for the treatment of a disease including accumulation of orbital fat. 39. The use of at least one beta adrenergic agonist and at least one compound to reduce desensitization of target tissue to a beta adrenergic agonist for use in a method of treating a disease comprising accumulation of orbital fat.

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