KR20160042819A - Non-agglomerating bioconjugates of amylin-mimetic compounds and polyethylene glycol - Google Patents

Abstract

The present invention relates generally to the novel use of amylin-mimetic compounds and novel non-aggregated bioadhesions of polyethylene glycols and their use in the treatment of diseases associated with extracellular amyloid deposition or accumulation that contribute to dysfunction or impairment of systemic organs such as the pancreas .

Description

[0001] NON-AGGLOMERATING BIOCONJUGATES OF AMYLIN-MIMETIC COMPOUNDS AND POLYETHYLENE GLYCOLS OF AMYLINE-MODIFIED COMPOUNDS AND POLYETHYLENE GLYCOL [0002]

The present invention relates generally to amylin-mimetic compounds and novel non-aggregated bioadhesives of polyethylene glycols, diseases associated with the deposition or accumulation of extracellular amyloid contributing to impaired or impaired systemic organs such as the pancreas And their use in therapy.

Certain embodiments of the present invention are directed to the use of the conjugates of natural or synthetic human amylin and polyethylene glycols and their use in the treatment of diabetes and the use of amylin s on the surface of the pancreatic beta- To the toxic effects caused by the accumulation of the polymer. This accumulation contributes to the pathogenesis of diabetes. It is therefore believed to be advantageous to preserve such cells that have already become debilitated in diabetics in the field.

Amylin, the amyloid polypeptide of Langerhans islands, identified as CAS RN: 106602-62-4, is a hormone in pancreatic beta-cells that is co-secreted with insulin in response to feeding, and the effect of insulin on postprandial glucose control Supplement. It is a 37-amino acid polypeptide that is co-secreted with insulin at a ratio of 100: 1 insulin: amylin.

The beneficial effects of amylin in diabetes and obesity are well known: increased satiety, resulting in reduced food intake and consequent weight loss; Slower gastric emptying, improved glucose metabolism profile with postprandial peak, and reduced glucagon levels in diabetic patients.

Patients with type 1 diabetes have virtually no natural amylin products, while patients with long-lasting type 2 diabetes have lower levels than healthy individuals.

Due to its physiochemical properties, it is not possible to use structurally unmodified amylin as a pharmaceutical active. Its limited solubility in aqueous solutions has led to the development of a more soluble amylin-mimetic compound, pramlintide, in which the three amino acids of the murine amylin are replaced by three-proline. There is a corresponding pramindide product named Symlin ^® .

Although the solubility of pramine tide is greater than the solubility of human amylin, Symlin ^® products are offered as acid solutions, since pramlintide is more similar to murine and amylin than to humans and does not have excellent stability at neutral pH. Rat and amylin exhibit reduced amyloid aggregation propensity compared to human amylin, suggesting a general propensity for amylin and amylin-mimetic materials. These products should be administered as subcutaneous injections immediately before meals to increase postprandial amylin levels. Due to its short half-life of 10-15 min, such pramine-tide injections increase the concentration of amylin in the bloodstream as a series of peaks and can not restore the basal level of amylin during fasting. In the scientific literature, pramlineide is associated with increased risk of severe insulin-induced hypoglycemia and other side effects such as nausea, vomiting, anorexia and fatigue.

The technical literature does not appear to focus on the agglomeration tendency of human amylin, or on the solution to the harmful effects of amylin deposition and its pancreatic beta-cell membrane; There is no available solution.

Without being bound by theory, there are indications that cholesterol in the plasma membrane of beta-cells is a major factor in the polymerization of so-called amylin, and its deposition control on the membrane. The low level or absence of cholesterol in the plasma membrane, which is a common aspect of diabetes, inhibits the internalization of the amylin oligomer and promotes extracellular accumulation, thereby potentiating amylin cytotoxicity in that state, Apoptosis < / RTI > or destruction of < RTI ID = 0.0 > On the other hand, the cellular internalization of the monomer amylin is independent of the cholesterol content of the plasma membrane.

Briefly, a series of studies correlate amylin polymerization and deposition on the plasma membrane surface of beta-cells with the absence or presence of less cholesterol and / or increased severity of type 2 diabetes mellitus, with corresponding toxicity.

To date, there is no known drug or pharmacological active substance known to control the glycoprotein, which also inhibits aggregation of human amylin or amylin-mimetic compounds.

The present invention aims at its use for the conjugation of human amylin to polyethylene glycol and for the inhibition of extracellular aggregation of amylin, generally without toxicity induction associated with the formation of amylin oligomers and clusters.

The following text should be understood as follows: (1) Reference to the bioadhesion of amylin and polyethylene glycol also includes bioadhesion of an amylin-mimetic compound and polyethylene glycol; (2) The expression "amylin-mimetic compound" also includes natural or synthetic, human amylin for ease of surface. (3) Reference to diabetes refers to the direct association of amylin- Or other diseases and dysfunctions that are indirectly related.

The use of the bio-conjugates of the invention, in particular human amylin bio-conjugates, exhibits the most favored physiological benefits, in particular the high stability of the diabetic organism, as a required feature for pharmaceuticals of this nature:

- Increase in plasma half-life compared to amylin mimetics such as amylin and pramineide, thus allowing longer duration in the bloodstream;

Lower doses or fewer injections to mimic the effects of natural amylin;

- excellent control of diabetes;

- reduction of nausea;

- high satiety associated with feeding;

- reduced gastric emptying rate;

- higher solubility than human amylin;

- Decreased glucagon levels, thus improving glucose control.

- Figure 1 - chromatogram for monopegylated amylin obtained by reaction of human amylin and mPEG;
Figure 2 - Mass spectrometry characterizing monopegylated human amylin;
- testing of RAMP2 and RAMP3 coceptor binding interactions for glass and tablet monopegylated human amylin in Figures 3 and 4;
FIG. 5 - Test for amyloid aggregates;
Figure 6 - Comparison of plasma stability between free amylin and monopegylated human amylin.

The present invention relates generally to the reduction or avoidance of aggregation (also referred to as polymerization in the literature), deposition and fibrillation of pancreatic beta-cells and thus the apoptosis or destruction of the pancreatic beta-cells, ≪ / RTI > and to avoid the toxic effects typical of human amylin and amylin-mimetic compounds.

In a first aspect, the present invention relates to a novel non-aggregated bioassay of amylin or amylin-mimetic compounds and polyethylene glycols, said bioassay being characterized by the presence of alpha and epsilon amine moes of lysine 1 residue of the amylin polypeptide chain Characterized in that it comprises at least one polyethylene glycol unit covalently bonded to two nitrogen atoms originating from the T (side chain) (therefore a single conjugate or multiple conjugate compound can be obtained).

It is therefore an object of the present invention to provide amylin-mimetic compounds of formula < RTI ID = 0.0 >

(R1-COX) m-R2

here

R1 represents a methoxypolyethylene glycol (mPEG) moiety and a functional spacer having various mean molar masses,

R2 represents an amylin or amylin-mimetic compound,

X represents NH or O,

m is an amidine-mimetic compound comprising an amylin-mimetic lacking Lysl (or: des-Lysl) via an amide or ester linkage by reaction of a primary amine or hydroxyl functional moiety and mPEG- Represents the number of units of the mPEG polymer (Rl) conjugated to the amylin-mimetic compound (R2), obtained from the conjugation of Dl;

And a non-agglomerated bioassay of a compound of formula (II)

(R1-X) m-R2

here

R1 represents a methoxypolyethylene glycol (mPEG) moiety and a functional spacer having various average molar masses,

R2 represents an amylin or amylin-mimetic compound,

X represents NH or O,

m represents the number of units of the mPEG polymer (R1) conjugated to the amylin-mimetic compound (R2) obtained from the conjugation of the amylin-mimetic compound and the mPEG-aldehyde.

Reference to an amylin-mimetic compound in a bioconjugate of the present invention includes active derivatives of the amylin-mimetic compound such as salts, isomers, hydrates, solvates, prodrugs, metabolites, polymorphs and copper co-crystals.

It is important to remember that references to amylin-mimetic compounds in the text include natural, synthetic or biologically synthetic human amylin itself.

In another aspect, the present invention provides a method for the treatment or prophylaxis of dysfunction or impairment of systemic organs (i.e., external organs or tissues external to the central nervous system), such as hyperglycemia, diabetes, low resistance to glucose or insufficient glucose metabolism, Disorders caused by elevated blood pressure, and vascular diseases such as atherosclerosis, myocardial infarction, stroke, coronary heart disease, cardiac disease. In general, Alzheimer's disease is caused by amyloid deposition or accumulation, And to the use of the novel non-aggregated bioassay of amylin-mimetic compounds and polyethylene glycols in the prophylaxis or treatment of diseases or problems that are or are promoted.

In another embodiment, the present invention relates to amylin-adenosine dehydrogenase inhibitors for the preparation of low toxic products, pharmaceuticals, compositions and associates useful in the prevention or treatment of diseases caused or facilitated by amyloid deposition or accumulation causing dysfunction or impaired systemic organs, Lt; RTI ID = 0.0 > polyethyleneglycol < / RTI >

In another aspect, the invention relates to a low toxic pharmaceutical composition comprising a therapeutically effective amount of an amylin-mimetic compound and polyethylene glycol, at least one novel non-aggregating bioadhesion product and at least one pharmaceutically acceptable excipient. Such compositions are suitable for all various dosage forms such as oral, enteral, non-intestinal, dermal, sublingual, nasal, dermal, epidermal, transdermal, mucosal, vaginal, rectal, ocular and the like.

The composition of the present invention may itself be administered in any necessary or appropriate dosage form such as a solution, suspension, emulsion, microemulsion, foam, paste, cream, tablet, capsule (hard or soft, suppository), bolus, gel, , Aerosols, sprays, and the like.

Pharmaceutically acceptable excipients used in the compositions of the present invention are known to those skilled in the art, for example, as described in "Remington's Pharmaceutical Sciences", 15th edition, Mack Publishing Co., New Jersey (1991). As is known, certain excipients are selected according to the route of administration required in the pharmacological arts practice.

The pharmaceutical composition of the present invention may contain one or more active principles separate from human amylin such as insulin (which does not exclude any other), ions (such as zinc and sodium), antidiabetic agents, antibiotics, antihypertensive agents , Antiretroviral agents, and the like. Such a composition may be of immediate, delayed or slow release, including the possibility of administration of a novel bioconjugate of human amylin either concurrently with or contiguous with the other active ingredient.

Another aspect of the invention is the use of a non-aggregated bioassay of human amylin and polyethylene glycol as adjuvant in the prevention or treatment of diseases caused or promoted by amyloid deposition or accumulation causing dysfunction or impaired systemic organs .

Another aspect of the invention relates to a medicament characterized in that it comprises a therapeutically effective amount of at least one bioconjugate of human amylin and polyethylene glycol.

Another aspect of the present invention relates to a bioadhesion product of human amylin and polyethylene glycol, as well as to products, pharmaceuticals, compositions and associations comprising them, characterized by their use in medicament therapy.

Another aspect of the invention is a method of treating a patient suffering from amyloid deposition or accumulation by amyloid deposition or accumulation, characterized in that it comprises administering to the patient a therapeutically effective amount of at least one biologic conjugate of an amylin-mimetic compound, particularly human amylin, and polyethylene glycol. Or to a method for the treatment or prevention of diseases which are promoted or promoted.

Example

The following examples are directed to specific embodiments of the invention and are not intended to limit the scope of the appended claims in any way.

1. Preparation of the conjugate according to the invention

Reaction of 5 mg / mL human amylin solution at 25 캜 for 2 h in the presence of molar excess of 5 mPEG / 1 human amylin and 10 mM PBS (phosphate buffer solution) pH 7.4.

The reaction is quenched by addition of an equal volume of 30% acetonitrile / 0.1% trifluoroacetic acid (in water) and then chromatographed on a C18 Kromasil reversed phase column at 4 mL / min on a detector set to 220 nm. (Kromasil is a product line commercialized by the Separation Products group of AkzoNobel company in Sweden). This purification process is shown in Fig. The chromatogram shows the peak of the monoPEGylated amylin at 12 min.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) was performed to characterize monopegylated human amylin. Figure 2 shows the peak of monopegylated human amylin at 9 Kda.

2. Human Amylin  And Monopegylation  human Amylin  Receptor binding assay

Purified monopegylated human amylin was tested for RAMP2 and RAMP3 coreceptor binding interactions.

RAMP (Receptor Activated Crystal Protein) is labeled with fluorescein isothiocyanate at 4 ° C for 1 h in PBS (fetal bovine serum) pH 7.4 and incubated with Sepharose G25 Lt; / RTI > (Ross-based chromatography medium). The label was confirmed by measurement of UV absorbance at A280 and A490 to evaluate coupling efficiency depending on about 0.5 fluorene / RAMP molecules. Binding was determined by measuring the fluorescence anisotropy of RAMP-FITC (fluorescein isothiocyanate) as a function of free murine and amylin, human amylin-pegylation and as a control for non-specific binding, One protein egg white lysozyme (HEWL) was used.

According to Figures 3 and 4, the binding assays are performed in duplicate (monopegylated human amylin C # 1, C # 2; and free amylin # 1 and # 2) on glass and purified monopep And exhibits a similar apparent affinity of affinity for both the agonized human amylin suggesting that the PEG moiety does not interfere with the receptor affinity. A control test with HEWL indicates non-specific binding to the coceptor.

Effect of Pegylation on the Cohesion Profiles of 3 - Human Amylin.

The purified monopegylated human amylin of Example 1 was resuspended in DMSO and diluted in PBS pH 7.4 and incubated at 37 [deg.] C. Samples were evaluated for amyloid aggregates using the amyloid chromogenic probe Thiopurebin T (ThT).

Figure 5 shows the results. Free human amylin aggregates very quickly. After 3 days, almost free human amylin aggregated in amyloid form. On day 7, monopegylated human amylin still does not show signs of aggregation. This test demonstrates that the pegylation process can inhibit aggregation of human amylin.

Pharmacokinetics of 4-Pegylated Human Amylin Products

The pharmacokinetics of the monopegylated human amylin product were characterized in vivo in Swiss mice. The mice were housed in a temperature-control room with a light-dark cycle of 12 h. Water and food were available at will. Two groups of controls (free human amylin, not pegylated) and pegylated amylin were formed. The animals received either 100 μL saline containing 10 μg human amylin peptide, free human amylin or purified monopegylated human amylin. Plasma was subjected to ELISA for human amylin as supplied by the manufacturer (Millipore, Cat Number EZHA-52K-http: //www.millipore.com/catalogue/item/ezha-52k).

Figure 6 shows rapid plasma decay of free amylin, whereas monopegylated human amylin exhibits a longer duration of stability in plasma. This test demonstrates that the pegylation process of human amylin can increase the half-life.

Skilled artisans will appreciate that, even though not explicitly described, the skilled artisan will appreciate that, in order to perform the same or an equivalent function to accomplish the same or similar results, and to perform the present invention in equivalent embodiments that fall within the scope of the appended claims, Descriptions, and teachings of the embodiments may be used.

Claims (25)

A non-aggregated bioassay of amylin-mimetic compounds and polyethylene glycol characterized by their use in medical therapy. 2. A compound according to claim 1, which is a compound of formula I (R1-COX) m-R2
Wherein R1 represents a methoxypolyethylene glycol (mPEG) moiety and a functional spacer having various average molar masses,
R2 represents an amylin or amylin-mimetic compound,
X represents NH or O,
m is amylin-mimetic compound comprising amylin-mimetic compound lacking Lysl (des-Lysl) through amide or ester linkage by reaction of primary amine or hydroxyl functional moiety with mPEG-succinimidyl Represents the number of units of the mPEG polymer (R1) conjugated to the amylin-mimetic compound (R2), obtained from the conjugation; or
A non-agglomerated bioconjugate of an amylin-mimetic compound and polyethylene glycol characterized by having a compound of formula II (R1X) m-R2
here
R1 represents a methoxypolyethylene glycol (mPEG) moiety and a functional spacer having various average molar masses,
R2 represents an amylin or amylin-mimetic compound,
X represents NH or O,
m represents the number of units of the mPEG polymer (R1) conjugated to the amylin-mimetic compound (R2), obtained from the conjugation of the amylin-mimetic compound and the mPEG-aldehyde. The non-aggregated bioassay of claim 1 or 2, wherein the amylin-mimetic compound is a natural or synthetic human amylin, and an amylin-mimetic compound and a polyethylene glycol. The amylin-mimetic compound of claim 1 or 2, wherein the amylin-mimetic compound is in the form of a salt, isomer, hydrate, solvate, prodrug, metabolite, polymorph or co- Non-aggregated bioassay of glycol. A non-aggregated bioadhesion product of an amylin-mimetic compound and a polyethylene glycol, wherein the amylin-mimetic compound is human amylin, and the pharmaceutical product, pharmaceutical product, composition, ≪ RTI ID = 0.0 > amylin-mimetic < / RTI > compound and polyethylene glycol. Non-aggregated bioassay of amylin-mimetic compounds and polyethylene glycols for use in the prevention or treatment of diseases and dysfunctions caused or promoted by amyloid deposition or accumulation. 7. The method of claim 6, wherein the disease or dysfunction is selected from the group consisting of hyperglycemia, diabetes, low or no resistance to glucose, insufficient glucose metabolism, obesity, metabolic syndrome, eating disorders, atherosclerosis, myocardial infarction, stroke, coronary heart disease, Disease In general, the non-aggregated bioassay of an amylin-mimetic compound and polyethylene glycol characterized by being at least one of Alzheimer's disease. 7. The non-aggregated bioassay of an amylin-mimetic compound and polyethylene glycol according to claim 6, characterized in that it is used for the prevention or treatment of diabetes mellitus. Non-aggregated bioassay of amylin-mimetic compounds and polyethylene glycol for use in the preparation of low toxicity products useful for the treatment or prevention of diseases caused or promoted by amyloid deposition or accumulation. 10. The non-aggregated bioassay of claim 9, wherein the amylin-mimetic is a natural or synthetic human amylin, and an amylin-mimetic compound and a polyethylene glycol. 10. The non-aggregated bioassay of an amylin-mimetic compound and polyethylene glycol according to claim 9, wherein said biojunction is as set forth in claim 2. Claims 1. A low toxic pharmaceutical composition comprising one or more amylin-mimetic compounds and a therapeutically effective amount of at least one non-aggregated bioactive conjugate of polyethylene glycol and one or more pharmaceutically acceptable excipients. 13. The composition of claim 12, wherein the amylin-mimetic compound is a natural or synthetic human amylin. 13. The composition of claim 12, wherein said biojunction is as set forth in claim 2. 15. A composition according to any of claims 12 to 14, further comprising at least one active ingredient which is distinct from the amylin-mimetic compound. 15. The pharmaceutical composition according to any one of claims 12 to 14, which is selected from ions such as insulin, zinc or sodium, antidiabetic agents, antibiotics, antihypertensive agents, antiretroviral agents, ≪ / RTI > Adjuvant for the prophylaxis or treatment of a disease, characterized in that it comprises a non-aggregated bioadhesive of an amylin-mimetic compound and polyethylene glycol. 18. The adjuvant according to claim 17, wherein the amylin-mimetic compound is a natural, synthetic or biosynthetic human amylin. 18. The adjuvant for the prevention or treatment of diseases according to claim 17, wherein the biodegradable conjugate is the biodegradable polymer. Claims 1. A medicament comprising a therapeutically effective amount of at least one non-aggregated bioadhesive of an amylin-mimetic agent and polyethylene glycol. 21. A medicament according to claim 20, wherein the amylin-mimetic compound is a natural or synthetic human amylin. 21. The medicament according to claim 20, wherein said biodegradable material is as defined in claim 2. A method for treating or preventing a disease caused or promoted by amyloid deposition or accumulation characterized by administering to a patient a therapeutically effective amount of at least one non-aggregating bioadhesive or amylin-mimetic compound and polyethylene glycol. 24. The method of claim 23, wherein the amylin-mimetic compound is a natural or synthetic human amylin. 24. The method of claim 23, wherein the non-aggregated bioconjugate is the same as set forth in claim 2.

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