BRPI1003424A2 - human amylin containment polymeric system and agonist analogs, process and use; functional assessment process of released amylin - Google Patents

Abstract

HUMAN AMYLINE CONFINEMENT POLYMERIC SYSTEM AND AGONIST ANALOGUE, PROCESS AND USE; Released amylin functional assessment process Diabetes Meilitus is a disease that according to the World Health Organization is responsible for a significant proportion of deaths worldwide. Despite the large repertoire of glycemic control products aimed at diabetes, there are still pharmacological and pharmacokinetic gaps to be filled. The present invention aims to describe the process of developing a polymeric amylin confinement system and agonist analogs in the form of nanoparticles and microparticles. Another object of the invention relates to a process of functional in vitro evaluation of amylin released from polymeric containment systems. In addition, the invention describes the use of the drug producing system for amylin replacement therapy in individuals that can be used for treatment or prevention of metabolic diseases.

Description

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DESCRIPTIVE REPORT

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HUMAN AMYLINE CONFINEMENT POLYMER SYSTEM AGONIST ANALOGS, PROCESS AND USE; RELEASED AMILINE FUNCTIONAL EVALUATION PROCESS

FIELD OF INVENTION

The present invention describes a polymeric amylin confinement system and agonist analogs. The invention also relates to the process of preparing formulations based on polymeric human amylin confinement systems and agonist analogs. Another object of the invention relates to a process of functional in vitro evaluation of amylin released from polymeric containment systems. In addition, the invention describes the use of a human amylin polymeric confinement system and agonist analogs for the manufacture of medicaments for amylin replacement therapy in individuals. Another object relates to the use of the polymeric amylin confinement system and agonist analogues for treating or preventing metabolic diseases. Finally, the sixth object of this invention corresponds to human amylin agonist analogs.

STATE OF ART

Currently, diabetes mellitus (DM) is responsible for 5% of reported deaths worldwide, and in 2005, 1.1 million people died as a result of the complications of this disease. The World Health Organization considers that the incidence of DM cases has reached such a high level that it estimates a pandemic by 2 030, with a 2/24 ο · OJ ·

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increase of 171 million cases in 2000 to.

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resides in poor and developing countries (80% of cases). The age distribution also varies according to the level of development and in the poorest countries the highest risk group is made up of adults aged 45-64, while in the rich countries the highest prevalence group is the elderly.

DM is a group of chronic diseases that occur when the pancreas does not produce enough insulin or when there is resistance to the action of this hormone and, in some cases, deficient insulin production and action are observed. Insulin is a peptide hormone secreted by pancreatic islet β cells and has effects on the regulation of plasma glucose (glucose) concentration. Your

absence or deficient action incur the clinical picture known as hyperglycemia.

Although there are numerous subtypes of DM, types I and II together account for over 95% of diagnosed cases. The incidence of type II is 90-95% of cases. The correlation between type II diabetes and amylin deposit amyloidosis has been shown.

DMTI occurs due to an autoimmune dysfunction that leads to the destruction of pancreatic β cells. The main symptoms used for the initial diagnosis are: polyuria, intense thirst, weight loss and tiredness. Laboratory diagnosis of immunomediated MTID is performed by immunoassays performed to detect antibodies that identify an autoimmune process against β cells. This autoimmune response culminates in insulin production failure and therefore patients with DTIM must replace this hormone by administering exogenous ins.

DMTII corresponds to 90-95% of diagnosed cases

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and was called non-insulin dependent diabetes and covers individuals who have insulin resistance and usually have relative (not absolute) insulin deficiency. It is characterized by peripheral and hepatic resistance to insulin action, and sometimes this hormone is present at normal levels with no hypoglycemic effect. Another important feature of DMTII is the state of glucose tolerance, which corresponds to an increase in insulin secretion followed by β cell failure. Therefore, in patients with DMTII, elevated insulin levels may be observed along with hyperglycemia. An important feature of this condition is that hormone replacement is not always necessary. In fact, often in its early stages this condition can be treated only by dietary re-education in combination with aerobic exercise. There are probably many different causes of this form of diabetes, although the specific etiology is not known, autoimmune destruction of β cells is not present, and patients do not have the other causes of diabetes that fit them into the other groups previously mentioned. .

Despite its great importance in the pathogenesis of DMTII, insulin is not the only hormone involved in this disease. In this regard, other hormones (glucagon, GLP1, amylin, etc.) have been associated with this disorder. Amylin plays a prominent role in the pathogenesis of DMTII, since most patients with this disease have amyloid deposits, mostly amylin, in their pancreas. From the above, it is evident that glycemic control is a highly complex process, and replacement with exogenous insulin may not be sufficient to ensure the health of patients with DM, as this is not the only altered hormone. in this pathology. In addition, currently marketed drugs are unable to restore the physiological patterns of secretion of such hormones.

Despite the large repertoire of glycemic control products aimed at diabetes, there are still pharmacological and pharmacokinetic gaps to be filled.

Currently two hormones and agonist analogues are employed in the treatment of diabetes, namely insulin and amylin (pancreatic amyloid polypeptide, IAPP).

Amylin is a 37 amino acid pancreatic protein amidated at the carboxy-terminal end of the intramolecular disulfide bridged chain comprised of cysteine side chain thiols 2 and 7, with sequence: H2N-Lys-Cys-Asn-Thr -Ala-Thr-Cys-Ala-Thr-Gln-Arg- Leu-Ala-Asn-Phe-Leu-Val-His-Being-Asn-Asn-Phe-Gly-Ala- Ile-Leu-Ser-Ser Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-CONH2

Amylin has hormonal activity, involved in the regulation of a series of metabolic events such as gluconeogenesis, glycogen synthesis, glucose uptake, gastric emptying, anorexia, among others. Human amylin replacement therapy has not yet been possible due to the limited solubility of IAPP in aqueous medium.

Apparently the amylin gene is present in both mammals and birds, located on chromosome 12 in humans. One of the most evident features of amylin 30 is its ability to form amyloid fibers under physiological / pathophysiological conditions, which gives it a prominent role in the pathogenesis of DMTII, as more than 90% of patients with DMTII have amyloid deposits. . ·· Λ? T f. ..- Cv- Vf-,

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in their pancreas and cases of AMD in M

mammalian species expressing amylins prone to formation of amyloid structures.

As amylin is a hormone acting on blood glucose, its use for therapeutic purposes was proposed along with its discovery. However, the use of amylin as a biopharmaceutical was hampered due to its low aqueous solubility and tendency to form amyloid aggregates in aqueous media.

Amlintide, a synthetic analogue of amylin, was the

first prototype to be produced. However, amlintide also formed amyloid aggregates, as did amylin and was therefore unsuitable for therapeutic applications (USAN council. List No. 392. New names Amlintide. In: Clin Pharmacol Ther. 61 (4), 500, 1997). Several amylin agonist analogs were produced and tested until it was concluded that proline substitutions in regions 25, 28 and 29 are determinant for peptide stability (JANES, S. et al. The Selection of Pramlintide for Clinical Evaluation. Diabetes 45 (2), 235A, 1996).

A better understanding of the stability of amylin and its agonist analogs has enabled us to arrive at the amino acid sequence that constitutes the pramlintide biopharmaceutical, which was originally called ACl37. Clinical studies have shown that in the groups of patients taking pramlintide (given together with insulin) there was better glycemic control (evidenced by reduced glycated hemoglobin A - HbAlc levels) and these patients also had 30 lower weight gain, compared to the insulin-only group. Clinical tests also revealed that pramlintide, in combination with insulin, was able to reduce postprandial blood glucose by lower intake of 6/24 pf ■ ν

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inhibition of glucagon secretion (KLEPPINGER, E.L .; VIVIAN, E.M. Pramlintide for the treatment of diabetes mellitus. In: Ann Pharmacother 37 (7-8): 1082-1089, 2003). In summary, the main results found were: weight loss and appetite (HOLLANDER, P. et al. Effect of pramlintide on weight in overweight and obese insulin-treated type 2 diabetes patients. In: Obes Res. 12 (4), 661-8, 2004; ARONNE, L. Progressive reduction in body weight after treatment with amylin analog pramlintide in obese subjects: a phase 2, randomized, placebo-controlled, dose-escalation study In: J Clin Endocrinol Metab. 8), 2977-83, 2007), better glycemic control (HAYDEN, MR; TYAGI, SC "A" for Amylin and Amyloid in type 2 Diabetes Mellitus. In: J Pancreas 2 (4), 124-139, 2001) and decreased insulin doses required for proper blood glucose maintenance (HOLLANDER, P. et al. Effect of pramlintide on weight in overweight and obese insulin-treated type 2 diabetes patients. In: Obes Res. 12 (4), 661-8 , 2004; YOUNG, A. Amylin: Physiology and Pharmacology, 2005. 341 P. Elsevier Academic Press, London, 2005).

The lack of formulations capable of preventing human amylin aggregation has created the need to develop a more stable biopharmaceutical (pramlintide). However, pramlintide is a human amylin analogue, with four different amino acids, three of which are prolines (amino acids known to induce the formation of random secondary structures). Thus, it can be assumed that pramlintide may have significant differences in metabolic and immunological responses compared to endogenous amylin and, therefore, several adverse effects, as historically observed for other biological products 7/24 φ 1?

such as insulin from other mutant organisms.

Although the literature lacks information on long-term adverse effects (YOUNG, A. Amylin: physiology and pharmacology. 2005. 341 P. Elsevier Academic Press, London, 2005; SINGH-FRANCO, D., ROBLES, G., GAZZE , D. Pramlintide acetate injection for the treatment of type 1 and type 2 diabetes mellitus In: Clinical Therapeutics 29 (4), 535-562, 2007), some effects such as nausea, vomiting episodes and anorexia have often been associated with therapy. with pramlintide (HOLLANDER, P. et al. Effect of pramlintide on weight in overweight and obese insulin-treated type 2 diabetes patients. In: Obes Res. 12 (4), 661-8, 2004; YOUNG, A. Amylin: physiology and pharmacology, 2005. 341 P. Elsevier Academic Press, London, 2005; PULLMAN, J .; DARSOW, T.; FRIAS, JP Pramlintide in the management of insulin-using patients with type 2 and type 1. diabetes. Risk Manag 2 (3), 203-12, 2006; SINGH-FRANCO, D., ROBLES, G., GAZZE, D. Pramlintide 20 acetate injection for the tr food of type 1 and type 2 diabetes mellitus. In: Clinical Therapeutics 29 (4), 535-562, 2007), which may be justified by point mutations in the native amylin sequence.

Another issue to consider when evaluating pramlintide therapy is drug release rate and bioavailability. Pramlintide use has been associated with a high risk of insulin hypoglycaemia, especially in type I diabetic patients (WHITEHOUSE, F. et al. A randomized study and open-label extension evaluating the long-term efficacy of pramlintide as an adjunct to insulin therapy in type 1 diabetes In: Diabetes Care 25 (4), 724-30, 2002; HOLLANDER, P. et al Effect of pramlintide on weight in overweight and obese 8/24 «β <

insulin-treated type 2 diabetes patients. In: Obes Re "12 (4), 661-8, 2 004; EDELMAN, SV Does addition of ^ A baseline insulin improve glycemic control in type 2 diabetes mellitus? In: Nature Reviews Endocrinology 4, 194-195 Since pramlintide is a peptide, its administration by subcutaneous, intramuscular or intravenous injections is preferred to avoid degradation in the digestive tract.The bioavailability of subcutaneous injections of pramlintide is 30 to 40% ( compared to the bioavailability of intravenous applications) and the commonly used dosages are 30 to 120 μς before meals.The maximum plasma concentration, Cmax, occurs approximately 20 minutes after subcutaneous administration (EDELMAN, SV). improve glycemic control in type 2 diabetes mellitus? In: Nature Reviews Endocrinology 4, 194-195, 2008) The low availability coupled with the high incidence of hypoglycemic events arising from the need The need for careful administration to avoid dosage variations points to the need for the development of new human amylin release systems and agonist analogs. The inability to restore basal fasting amylin levels points to the need for development of new sustained release systems of human amylin and agonist analogs. The need to use human amylin with its native and amidated sequence also points to the need for the development of new release systems that can keep human amylin stable.

Thus, the confined release systems would allow to meet these explicit demands of maintaining the stability of human amylin and agonist analogs and the accomplishment of sustained release, which aim to prolong the permanence of drugs, in their therapeutic ranges, in body. These * "" << 'systems increase drug safety because the risk of overdose is low, and improve efficacy both by improving patients' adherence to treatment (who do not have to take multiple doses of medication) and by reducing their chances of underdosing.

Despite the aforementioned advantages and disadvantages of using pramlintide in combination with insulin, both in terms of safety and efficacy, as well as the treatment regimen, we emphasize that replacement of baseline amylin levels in diabetic subjects is not covered with pramlitide, nor is there any other drug available with based on human amylin or sustained release agonist analogues capable of establishing this hormone replacement of importance for diabetes and other pathophysiological conditions, as set forth below. Therefore, fasting glucose control for diabetic individuals remains dependent on current therapies, based on oral hypoglycemic agents and slow-release insulin, whose efficacy varies greatly depending on the individual. The product object of the invention may therefore be employed as a supplement in the treatment of diabetes patients.

Amylin is known for several other pharmacological actions, such as: glucagon secretion, insulin, amylin and other pancreatic hormones, bone metabolism, anorexia, gastric emptying, stomach acid secretion, insulin degradation enzyme control, glucagon, peptide Α -beta and amylin, promotion of pancreatic β-cell regeneration, lipid, glycidic, lactic, glycogen metabolism, calcitonin receptor regulation and calcitonin gene-related peptides, cardiovascular and over-the-counter effects. ^ hemodynamics, central arainoacid transport, body temperature, dopaminergic system activation, hippocampal modulation and memory, motor activity regulation, anti-inflammatory action, dyslipidemia, coagulopathies, renal metabolism, immunological disorders, among others (YOUNG, A. Amylin: physiology and pharmacology, 2005. Elsevier Academic Press, London, 2005). Thus, the product object of the invention, consisting of a human amylin release system and amylinimimetic agonist analogues, may be employed in amylin replacement and amylin agonists for intervention on these pathophysiological conditions.

The specialized technical literature presents some examples of containment systems for slow and controlled release of bioactive agents.

W02010017215-A2 discloses the development of a microstructured and coated system for releasing a hydrogel based agent. The system was developed and tested with atropine, a nonpeptide and nonprotein small organic molecule for transocular operation. The present invention relates to a polymeric system employed in the form of nanocapsules and microcapsules dispersed in liquid phase and which does not require coating for its in vitro and in vivo operation. Furthermore, the present invention describes a hydrophobic substance confinement system which has a method of preparation distinct from systems using hydrophilic substances. Another differential and additional advantage of the present invention over said patent is the confinement of protein / peptide material, which has been tested in vitro and in vivo for a γ'ii peptide.

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insoluble, unstable, and whose system, proposed by the present invention, generates stability thereof.

Protein / peptide aggregation is a phenomenon dependent on several factors, including protein / peptide concentration and the condition of the medium in which it is found. Circulating amylin in individuals, as well as several other proteins, would be in a favorable condition to avoid aggregation, since its concentration is very low and transit by biological means where it would be associated with cofactors that would avoid the aggregation phenomenon. With this hypothesis, the present invention describes a polymeric amylin confinement system capable of slow release directly into the body, avoiding a high momentary concentration and also allowing it to associate with cofactors, and together preventing it from occurring. protein aggregation and favoring its release into the body. This system could be employed by various routes of administration in the body, allowing sustained release for days to replace basal amylin levels in individuals. In addition, a method for functional evaluation of particles and peptide in in vitro systems has also been developed.

SUMMARY OF THE INVENTION

The present invention describes a polymeric amylin confinement system and agonist analogs.

The second object of this invention relates to the process of preparing formulations based on polymeric amylin confinement systems and agonist analogs. tf% 12/24 <7 „\ <k%

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The third object relates to a process for in vitro functional assessment of amylin released from polymeric containment systems.

The fourth object of the invention describes the use of a polymeric amylin confinement system and agonist analogues for the manufacture of medicaments for amylin replacement therapy in individuals.

The fifth object of the invention describes the use of a polymeric amylin confinement system and agonist analogues for the manufacture of medicaments for the prevention and treatment of diabetes mellitus, glucose tolerance, glycemic control, obesity, hypertension, syndrome X7 dyslipidemia, cognitive disorders. atherosclerosis, amyloidoses such as Alzheimer's, Parkinson's, Huntington's, cerebral amyloid angiopathy, leptomeningeal amyloidosis, visceral familial amyloidosis, primary cutaneous amyloidosis, senile systemic amyloidosis, familial amyloid polyneuropathy, familial amyloidosis cardiomyopathy, Jakut's disease, Jakob's disease

2 0 myocardial infarction, coagulopathies, inflammatory diseases,

dyspepsia, gastric ulcers, decreased food intake, modulation of bone metabolism, regulation of glycemic metabolism, insulin secretion, amylin secretion, glucagon secretion, proteostasis, decreased beta cell apoptosis, increased beta cell function and mass, adjuvant in cell or beta-cell transplantation therapies, restoration of glucose sensitivity response in tissues composed of pancreatic cells, beta cells, adipose cells, nervous system

30 central, hepatic, cardiac and pulmonary.

The sixth object of this invention corresponds to a human amylin agonist analog. BRIEF DESCRIPTION OF DRAWINGS

Figure 1 illustrates the size distribution of amylin-containing nanoparticles.

Figure 2 illustrates the size distribution of amylin-containing microparticles.

Figure 3 shows the dosage of human amylin by fluorimetry using fluorescamine as a derivatizer.

Figure 4 shows representative spectra of various fluorescamine derivatized human amylin concentrations.

Figure 5 shows fluorescence spectra of empty PCL nanoparticles and human amylin.

Figure 6 illustrates the size distribution of sonicator-produced human amylin-containing nanoparticles obtained by dynamic light scattering.

Figure 7 shows the image of the human amylin-containing nanoparticles obtained by SEM. The magnification and size bars are indicated at the bottom of the microscopy.

Figure 8 shows the images of human amylin-containing nanoparticles taken by MET at 25,000X magnification.

Figure 9 illustrates the release profile of the nanoconfinished human amylin-containing system.

Figure 10 shows fluorescence spectra of samples containing nanoconfinished amylin formulations collected at different times.

Figure 11 shows the in vivo pharmacological action of human amylin from the nanoconfinished human amylin-containing preparation by monitoring and evaluating the glycemic curve. The assays were performed with fasting normal (non-diabetic) mice.

DETAILED DESCRIPTION OF THE INVENTION ■ I

In the present invention, a polymeric confinement system for arailin and nanoparticulate agonist analogs having a diameter of between 100 nm and 800 nm and microparticles with a diameter of between 5 μτη and 100 μπι has been developed. using as a polymeric matrix a compound comprised in the group consisting of poly-caprolactone, poly-e-caprolactone (PCL), poly (DL-

lactide-co-caprolactone), poly (lactic glycolic acid) (PLGA), phospholipids, liposomes, chitosan and alginates. This polymeric confinement system is capable of controlled and sustained release of amylin directly into the body, avoiding a momentary concentration of amylin and also allowing the possible association of amylin with natural cofactors, resulting in the prevention of protein aggregation and thus favoring of its release into the body.

The second object of this invention relates to the process of preparing formulations based on polymeric amylin confinement systems and agonist analogs. A new simple emulsification methodology followed by solvent extraction was used to produce nanoparticles and microparticles, respectively, containing amylin and agonist analogs.

The process of preparing formulations based on polymeric amylin confinement systems and agonist analogs for the production of nanoparticles consists of the following steps:

a) Preparation of emulsion phases

b) Emulsion formation by stirring or sonification 30 c) Vacuum extraction of organic solvents

d) Washing by centrifugation or filtration

e) Removal of water Initially, in step (a), the emulsion phases are prepared separately. The aqueous phase (FA) is prepared by adding emulsifying agent to the ultra pure water heated to a range between 20 and 90 ° C. The emulsifying agent should be comprised of the group consisting of polyvinyl alcohol (PVA) having an average molecular weight of 90 kDa and polaxamer, both in a concentration of 0 to 5%. The system was kept under magnetic stirring until the emulsifying agent was completely dissolved and then cooled in an ice bath. The organic phase (FO) is obtained from the dissolution of the matrix forming polymer, with an average size of 5000 to 100,000 kDa, with a mass between 1-300 mg, preferably 10-30 mg, together with 10 to 1000 μς / ηιΐ: amine (human or animal) or agonist analogues, preferably 150 to 250 μς, in organic solvent such as dichloromethane, acetone, trifluoroethane, chloroform, ethanol, methanol, ethyl acetate and 1,1,1,3,3, 3 -hexafluoroisopropanol in the range 100 to 10000 μ! At 4 to 35 ° C with the aid of an ultrasound bath (10 to 400 watts). Amylin agonist analogues shall be comprised of the group consisting of pramlintide, chemically synthesized in solution, chemically solid, biosynthetic, amidated and non-amidated, pegylated and non-plegated, with intramolecular oxidation between cysteines ( forming cystine) and non-oxidized, incubated with small aggregation inhibitors or not, such as resveratrol, thioflavin, insulin and insulinomimetics. The organic solvent used should be 30 belonging to the group consisting of dichloromethane (DCM), acetone and trifluoroethanol (TFE), chloroform, ethanol, methanol, ethyl acetate and hexafluoroisopropanol (HFIP). Λ'ί '*

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Then, in step (b), an amount of 2.5 to 7 mL of FA with a concentration of 0 to 2.0% PVA was stirred and dispersed using a mechanical system. Agitation can be performed by employing one of the ultrasound nozzle (sonicator), homogenizer, mixer, and high-speed turbine diffuser equipment, with the sonicator set at 10 to 200 watts in continuous cycles for a period of 2 to 2 hours. 15 minutes. The FO is then slowly added to the AF under mechanical stirring until emulsion formation. The emulsion is then added, if necessary, with 100% silicone oil to reach a final concentration of up to 10%.

In step (c), the emulsion is then vacuumed for 30-60 minutes under cooling for complete removal of organic solvents, which has led to the formation of a nanosuspension.

In step (d), the system is then separated for 10 to 20 minutes by centrifugation or filtration and washed several times using 0 to 5% aqueous PVA in the washes.

Finally, in step (e), the system is resuspended in a cryoprotectant solution for further drying by methods such as lyophilization or spray-dry bed. The cryoprotectant solution contains varying amounts of up to 20% w / w of one of the polyhydric compounds such as mannitol, sorbitol, inositol, propylene glycol, ethylene glycol, mannose, ribose, lactose, sucrose, fructose, galactose, arabinose, glycerol, polyvinyl alcohol, trehalose, colloidal silicon dioxide, polyethylene glycol and polaxamer.

The process of preparing formulations based on polymeric amylin confinement systems and agonist analogs for the production of microparticles consists of the same steps as described for the production of nanoparticles, except for step (b). In step (b), microemulsion formation occurs by agitation employing high-rotation turbine diffuser (ultra-disperser) at a speed of from 10,000 to 20,000 RPM.

The third object of the present invention relates to an in vitro functional evaluation process of amylin and agonist analogs released from polymeric containment systems. For this purpose, the in vitro release method in an isotonized buffered aqueous medium containing surfactant or emulsifier lipid agent (lauryl ether, sodium lauryl sulfate, polysorbate, or a nonionic surfactant) or phospholipids (dodecylphosphatidylcholine, phosphatidylcholine) was evaluated.

phosphatidylglycerol, phosphatidylserine, phosphatidylinositol) at 0.001 to 2% w / v by the dosage of amylin and agonist analogs by fluorescamine spectrofluorimetry generating a fluorescent product.

Initially, a volume of from 100 to 300 μL of an amylin-containing solution released in an isotonized buffered aqueous medium containing a surfactant or emulsifying lipid agent (lauryl ether, sodium lauryl sulfate, polysorbate, or a nonionic surfactant) or phospholipids (dodecylphosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol) from 0.001 to 2% w / v, from containment systems, were mixed in buffered, isotonized aqueous medium containing surfactant or emulsifying lipid lauryl sulfate sodium lauryl ether , polysorbate, or a nonionic surfactant) or phospholipids (dodecylphosphatidylcholine, phosphatidylcholine,

phosphatidylglycerol, phosphatidylserine, phosphatidylinositol).

0.001 to 2% w / v, with a volume in the range of 100 to 300 µL of the derivatizer (at a concentration of from 0.2 to 1.0 mg / ml of fluoresamine in DMSO). After this, sample homogenization was performed using a shaker. The sample was analyzed in a fluorimeter with parameters defined by a technician in the subject.

The fourth object of the invention describes the use of a polymeric amylin confinement system and agonist analogs for producing a sustained release drug for days to replace basal amylin levels in individuals, which may be found in solid dosage forms. , suspended liquids, gel, or semisolid, such as resuspension powder, inhalable powder, tablets, coated tablets, capsules, lozenges, suppositories, ointments, injectables and transdermal patches, for oral, sublingual, topical, implant, intramuscular, intravenous, or by inhalation, preferably being used in injectable form.

The fifth object of the invention describes the use of a polymeric amylin confinement system and agonist analogues for the manufacture of medicaments for the prevention and treatment of diabetes mellitus, glucose tolerance, glycemic control, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders. , atherosclerosis, myocardial infarction, amyloidoses such as Alzheimer's, Parkinson's, Huntington's, cerebral amyloid angiopathy, leptomeningeal amyloidosis, visceral familial amyloidosis, primary cutaneous systemic amyloidosis, familial amyloid polyneuropathy, familial amyloidosis cardiomyopathy, Familial pancreatic amyloid disease , myocardial infarction, coagulopathies, inflammatory diseases, dyspepsia, gastric ulcers, food intake, 4JI modulation

bone metabolism, glycemic metabolism regulation ^^ ""

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insulin secretion, amylin secretion, secretion

glucagon, proteostasis, decreased beta-cell apoptosis, increased beta-cell function and mass, adjunct to cell or beta-cell transplantation therapies, restoration of glucose sensitivity response in tissues composed of pancreatic cells, beta-cells, adipose, of the central nervous system, liver, heart and lung.

This medicine can be found in the forms

solid pharmaceuticals, liquids in suspension, gel, or semi-solid, such as resuspension powder, inhalable powder, tablets, coated tablets, capsules, lozenges, suppositories, ointments, injectables and transdermal patches, for oral, sublingual, topical, implant, intramuscular, intravenously, or by inhalation, and is preferably used in injectable form.

The human amylin agonist analog corresponds to all alpha L-amino acids, alpha-D-amino acids and beta-L-amino acids such as Ala, Arg, Asn, Asp, Cys, Glu, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val and others known in the art which may be introduced at one or more points of the following human amylin chain: H2N-Lys-Cys-Asn-Thr- Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Being-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser- Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-CONH2.

The following examples are illustrative only and are intended to substantiate the embodiments 30 made by the inventors. The data contained in the examples should therefore not serve to delimit the depositor's rights. Example 1: Production Process EXAMPLES

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using sonicator.

FA was prepared using 5 mL of 1.5% PVA aqueous solution. For the preparation of FO, 20 mg of PCL was dissolved in 0.6 mL of DCM using an ultrasound bath for 10 min. 0.4 mL TFE containing 0.2 mg human amylin was added to the FO. 5mL of FA was transferred to an ice bath cooled tube. A cycle sonicator equal to 70% amplitude was employed to perform agitation. FO was homogenized in AF and then vacuum (700 mmHg) was used in an ice bath for 30 to 40 min. Washing was performed 3 times with 1.5% PVA by centrifugation for min.

At the end of the process, particles ranging in diameter from 100 to 300 nm were obtained (Figure 1). Example 2: Microparticle production process.

FA was prepared using 5mL of 1% PVA aqueous solution. For the preparation of FO, 30mg of PCL, with an average molecular weight of 65 kDa, was dissolved in DCM ImL, using an ultrasound bath for 10 min. 0.1 mL TFE containing 0.2 mg human amylin was added to FO. 5mL FA were transferred to a 50mL flask of nominal capacity. An ultra-disperser at 14,000 RPM was employed to perform agitation. FO was homogenized in the AF and, afterwards, vacuum (700 mmHg) was used in an ice bath for 15min. Washing was performed 3 times with 1% PVA by centrifugation for 15min.

At the end of the process, particles of diameter from 1.0 to 2.5 μπι were obtained (Figure 2). Example 3: In vitro release method of amylin. ^ Fis-

The method using fluorescamine, when employed from amylin detection and quantification, showed good linearity, expressed by the value of R2 equal to 0.999 in the concentration range of 0.1 mg to 50 μς / πΛ (Figures 3 and 4).

In addition, Figure 4 indicates that increasing amylin concentration leads to increased flowering intensity.

This method also presents adequate selectivity, since the components of the empty formulation do not interfere with the obtained signal (Figure 5).

Example 4: Characterization of particle size.

Example 4.1: Determination of Dynamic Light Scattering (DLS) Size

Human amylin-containing nanoparticles were prepared using a sonicator. These samples are not

could be analyzed by light microscopy because they had very small diameters.

Nanoparticle suspensions were diluted with water and kept in sealed acrylic cuvettes. The vials were placed in the analysis chamber of the

20 light scattering so that the laser light beam

cross the suspension to its full extent without any internal filtering effect.

The sample containing human amylin had a diameter of 153.3 nm and polydispersity of 0.116, as shown

Figure 6

Table 1 shows the size distribution of human amylin nanoparticles produced using sonicator (data obtained by dynamic light scattering).

3 0 Table 1. Size distribution of nanoparticles with

amylin produced with sonicator Diameter (nm) Polydispersity 156, 2 0, 101 148, 2 0, 108 154, 5 0, 139 Average 153.3 0, 116 Standard error 2.1.0

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Example 4.2: Scanning Electron Microscopy (SEM) morphological analysis

Using a micropipette, the nanoparticles suspension was spread over glass slides and cooled in liquid nitrogen. After lyophilization, the samples received treatment (gold coating) and were observed under the scanning electron microscope.

The images obtained by SEM are in agreement with the DLS results, indicating nanometric diameters for most of the particle population present in the sample. In addition to the nanometric diameters, it is possible to observe that the particles have spheroidal morphology compatible with the expected. Figure 7 shows an image of the human amylin-containing nanoparticles obtained by SEM.

Example 4.3: Transmission Electron Microscopy (MET) morphological analysis

The samples used for this technique were prepared as follows: each MET grid received 5 μ] 1 of the 20 nanoparticle suspensions, 30 seconds elapsed and another 5 μΐϋ of supersaturated (contrasting) uranyl acetate solution was added to each grid. . A further 30 seconds were allowed until excess liquid could be removed using absorbent paper. The samples were then left for two hours in a desiccator until they could be viewed under the transmission electron microscope. 23/24;

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The result indicated a nanometric particle population. However, Figure 8 shows that the particle morphology obtained by MET points to more elliptical shapes. Another important feature of MET images is the absence of amyloid fibers, indicating that the process prevented human amylin from forming amyloid aggregates. Example 5: Functional assessment of amylin released from polymeric containment systems.

Example 5.1: Release Profile of Amyline Nanoparticles in vitro

After washes, samples were diluted in 10 mL of the release buffer (PBS pH 7.4 with 0.02% NaN3 and 0.1% polysorbate-80). After dilution, the material was divided into ten 1.5 mL microtubes, with each tube receiving 1 mL of sample and all transferred to the greenhouse and maintained at 370 ° C during the experiment. At each time a microtube was removed from the oven and centrifuged at 20,000g for 30 minutes at 12 ° C. The amylin present in the supernatants was dosed by

spectrofluorimetry.

Several experiments performed on different days with independently prepared formulations indicated that this system has a release kinetics greater than four days.

Figure 9 presents a release profile of the

nanoconfinished amylin formulations, with release kinetics of more than three days.

Some of the fluorescence spectra obtained during both releases are shown in Figure 10, where it is possible to observe the increase in fluorescence intensity over time, indicating that amylin is being released. 24/24 j? Example 5.2: In vivo Pharmacological Profile

i

amylin nanoparticles

8-week-old male Swiss mice were divided into two large groups: Control (n = 6) and NpHIAPPwt (n = 6). The animals were housed in a temperature controlled room with a 12 h light-dark cycle and had free access to water and feed, which was suspended 12 h before the start of the experiment and was made available again after the end of the experiment. The first group (Control group, n = 6) received 100 µl of the non-amylin formulation (vehicle), the second group (Np-hIAPPwt group, η = 6) was treated with 100 µl of the formulation with nanoconfinished amylin. Blood glucose was determined at time zero by means of a glucometer and 100 pL from each sample was injected subcutaneously into each animal. All mice were fasting during the experiment. After that, blood glucose concentration was monitored at times: 6 h, 10 h and 32 h.

The nanoconfinished human amylin-containing formulation was 20 able to promote a hypoglycemic effect in fasting mice as shown in Figure 11. This hypoglycemic effect lasted until the end of the experiment (32 h) indicating the systemic pharmacological effectiveness of the amylin released by the product. , and also a potential use of

2 5 formulation for preparing a medicament for treating the

diabetes mellitus and other metabolic diseases. Animal experiments were performed in order to minimize their discomfort or suffering and such experiments were previously submitted to analysis and

30 approved by the animal experimentation ethics committee of the

Health Sciences Center of the Federal University of Rio de Janeiro.

Claims (33)

1. Polymeric confinement system of amylma and agonist analogs, consisting of a nanoparticulate polymeric matrix with an average size between 20 and 600 nm and microparticles with an average size between 1 and 100 μπι controlled and sustained amylin and agonist analogues directly in the body. Polymeric system according to Claim 1, characterized in that the polymeric matrix comprises a compound comprised in the group consisting of polyglycolic acid, poly-lactic acid, polycaprolactone, poly-s-caprolactone (PCL), poly (DL-lactide-caprolactone), poly (lactic-glycolic) acid (PLGA), phospholipids, liposomes, chitosan and alginates. Process for the preparation of formulations based on polymeric amylin confinement systems and agonist analogs characterized by a simple emulsification methodology followed by solvent extraction to form nanoparticles and microparticles. Process according to Claim 3, characterized in that it is used for the production of amylin-containing nanoparticles and agonist analogs using the following procedure: a) Preparation of emulsion phases b) Emulsion formation by agitation or sonication c) Extraction organic solvents by vacuum d) Washing by centrifugation or filtration e) Water removal Process according to Claims 3 and 4, characterized in that the aqueous phase in step (a) is prepared by adding emulsifying agent to the ultra-pure water heated in the range 2CT7 to 90 ° C. Process according to Claim 5, characterized in that the emulsifying agent is comprised of the group consisting of polyvinyl alcohol and polaxamer, both in a concentration between 0 and 5%. Process according to Claim 6, characterized in that the polyvinyl alcohol has an average molecular weight of between 10 and 90 kDa. Process according to Claims 3 and 4, characterized in that the organic phase in step (a) is obtained by using the amylin or the like in a range of 10-1000 pg / ml dissolved in an organic solvent. totaling a volume in the range 10-5000 pL. Process according to Claim 8, characterized in that the mass of amylin and agonist analogues employed in the dissolution is preferably in the range 100-500 pg. Process according to Claims 3 to 9, characterized in that the amylin analog is comprised of the group consisting of pramlintide, chemically synthesized in solution, chemically solid, biosynthetic, amidated and unamidated. , pegylated and non-pegylated, with intramolecular oxidation between cysteines and non-oxidized, incubated with small aggregation inhibitor molecules and not incubated. Process according to Claim 10, characterized in that the amylin analog incubated with small aggregation inhibiting molecules belongs to the group consisting of resveratrol, thioflavin, insulin and insulinomimetics. Process according to Claims 3 and -4, characterized in that the dissolution of the polymer in step (a) is carried out employing a mass in the range of 1 to 300 mg in a volume. 1 to 10000 μϋ of organic solvent. Process according to Claim 12, characterized in that the mass of the polymer employed in the dissolution is preferably in a range of 10 to 30 mg. Process according to Claim 13, characterized in that the polymer has an average molecular weight of 5,000 to 100,000 Da. Process according to Claims 3 to 8, characterized in that the organic solvent used in step (a) belongs to the group consisting of dichloromethane, acetone and trifluoroethanol, chloroform, ethanol, methanol, ethyl acetate and 1.1. , 1,3,3,3-hexafluoroisopropanol. Process according to Claims 3 and 4, characterized in that the nanoemulsion in step (b) contains the organic phase and an amount of between 2.5 and 7.5 ml of aqueous PVA solution with a concentration of 0 0.5 to 2.0%. Process according to Claim 16, characterized in that the nanoemulsion is obtained by employing one of the equipment comprised of sonicator, homogenizer, mixer and high speed turbine diffuser. Process according to Claim 17, characterized in that the nanoemulsion is preferably obtained with the aid of a sonicator for a time of 2 to 15 minutes. Process according to Claim 18, characterized in that the nanoemulsion is obtained with the aid of a sonicator, preferably for a period of 5 to 10 minutes. Process according to Claims 17 to 19, characterized in that the formation of nanoemulsion may occur with the sonicator set at 10 to 200 watts of continuous cycle power. Process according to Claims 3 and 4, characterized in that the vacuum in step (c) is employed for 30 to 60 minutes under cooling. Process according to Claims 3 and 4, characterized in that the system is resuspended in step (e) in a cryoprotectant solution for further drying by methods such as lyophilization and spray-dry bed. Process according to Claim 22, characterized in that the cryoprotectant solution contains varying amounts of up to 20% w / w of one of the polyhydric compounds such as mannitol, sorbitol, inositol, propylene glycol, ethylene glycol, mannose, ribose. , lactose, sucrose, fructose, galactose, arabinose, glycerol, polyvinyl alcohol, trehalose, colloidal silicon dioxide, polyethylene glycol and polaxamer. Process according to Claims 3 to 23, characterized in that it is used for the production of amylin-containing microparticles and agonist analogs. Process according to Claim 24, characterized in that the formation of the microemulsion in step (b) occurs by agitation employing a high rotation turbine diffuser. Process according to Claim 25, characterized in that the high-speed turbine diffuser is operated at a speed of from 10000 to 20000 RPM. 27. Process for functional evaluation of in vitro release of amylin and agonist analogues from confinement polymeric sister dispersed in medium containing surfactant or emulsifying lipid agent (lauryl ether, sodium lauryl sulfate, polysorbate, or a nonionic surfactant) or phospholipids (dodecylphosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol) between 0.001 to 2% w / v, characterized in that it consists of amylin dosage and agonist analogues by fluorescamine spectrofluorometry. Process according to Claim 27, characterized in that it generates a conjugate between amylin and agonist analogues with the fluorescent substance as a product. 29. Use of the polymeric amylin confinement system and agonist analogs characterized for being used for the production of medicaments for amylin replacement therapy in individuals. Use of the polymeric containment system according to claim 29, characterized in that the medicament is employed in solid, liquid suspension, gel or semisolid pharmaceutical forms, such as resuspension powder, inhalable powder, tablets, coated tablets, capsules, lozenges. , suppositories, ointments, injectables and transdermal patches, for oral, sublingual, topical, implant, intramuscular, intravenous, or inhalation use, allowing sustained release of amylin and agonist analogs. Use according to claim 30, characterized in that it is preferably employed by injection. 32. Use of the polymeric amylin confinement system and agonist analogues for use in the treatment or prevention of hyperglycemia, diabetes, type I diabetes, type II diabetes, glucose tolerance, regulation of glycemic metabolism, insulin secretion, amylin secretion. , glucagon secretion, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atherosclerosis, myocardial infarction, Alzheimer's amyloidosis, Parkinson's disease, cerebral amyloid angiopathy, visceral amyloidosis, primary cutaneous amyloidosis, primary cutaneous amyloidosis, systemic polysilumosis familial, familial amyloidotic cardiomyopathy, Creutzfeldt-Jakob disease, pancreatic amyloidosis, coagulopathies, other vascular diseases, inflammatory diseases, dyspepsia, gastric ulcers, decreased bone metabolism, proteostasis, decreased beta cell apoptosis, increased function and mass of beta, adipose, central nervous system, hepatic, cardiac and pulmonary cells, adjuvant in cell therapy or beta cell transplantation, restoration of glucose sensitivity response in tissues composed of pancreatic, adipose, central nervous system, hepatic, cardiac, pulmonary. Amylin agonist analog having one or more alpha L-amino acids, Alpha-D-amino acids and beta-L-amino acids such as Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu , Lys, Met, Phe7 Pro, Ser, Thr, Trp, Tyr, Val, which may be introduced at one or more points in the human amylin chain.