WO2012031342A2

WO2012031342A2 - Polymer system and method for encapsulating human amylin and agonist analogues, and use thereof; released amylin functional evaluation method

Info

Publication number: WO2012031342A2
Application number: PCT/BR2011/000316
Authority: WO
Inventors: Luis Mauríco TRAMBAIOLI DA ROCHA E LIMA; Luiz Henrique Guerreiro Rosado; Camile Moreira Mascarenhas; Eduardo RICCI JÚNIOR
Original assignee: Universidade Federal Do Rio De Janeiro
Priority date: 2010-09-08
Filing date: 2011-09-08
Publication date: 2012-03-15
Also published as: WO2012031342A3; BRPI1003424A2

Abstract

Diabetes mellitus is a disease which, according to the World Health Organisation, is responsible for a significant proportion of deaths throughout the world. In spite of the large number of glycemic control products aimed at diabetes, there still exist pharmacological and pharmacokinetic gaps to be filled. The present invention aims at describing a method for developing a polymer system for encapsulating amylin and agonist analogues in the form of nanoparticles and microparticles. Another object of the invention is a method of functional in vitro evaluation of amylin released from polymer encapsulation systems. The invention further describes the use of the system for producing a medicament for amylin replenishment therapy in patients, which can be used for treating or preventing metabolic diseases.

Description

DESCRIPTIVE REPORT

POLYMERIC SYSTEM OF CONFINEMENT OF HUMAN AMYLINE AND AGONIST ANALOGS, PROCESS AND USE; EVALUATION PROCESS

AMILINE RELEASE

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 2030, with a growth from 171 million cases in 2000 to 366 million in 2030. The highest prevalence of this group of diseases is 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. Its absence or deficient action incur the clinical condition 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 check antibodies that identify an autoimmune process against β cells. This autoimmune response culminates in failure of insulin production and therefore patients with DTIM must replenish this hormone by administering exogenous insulin.

DMTII corresponds to 90-95% of diagnosed cases 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 mentioned above. .

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 has 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 condition. 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 the cysteine 2 and 7 side chain thiols, having the sequence: H ₂ N-Lys-Cys-Asn-A Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Asn-Asn-Phe-Gly-Ala- Ile-Leu-Ser- Ser-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-CONH ₂

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 characteristics of amylin is its ability to form amyloid fibers under physiological / pathophysiological conditions, which gives it a prominent role in the pathogenesis of DMTII, as over 90% of patients with DMTII have amyloid deposits. In their pancreas, cases of DMTII are reported in other mammalian species that express amylins with a propensity to form 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 analog 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 Clinical Evaluation for Clinical Evaluation. : Diabetes 45 (2), 235A, 1996).

A better understanding of the stability of amylin and its agonist analogs enabled us to arrive at the amino acid sequence that constitutes the pramlintide biopharmaceutical, which was originally called AC137. 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 lower weight gain when compared with the group that received insulin only. Clinical trials also revealed that pramlintide, in combination with insulin, was able to reduce postprandial blood glucose by lowering the intake of by reducing gastric emptying rate and inhibiting glucagon secretion (KLEPPINGER, EL; VVIAN, EM 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 insulin-treated type 2 diabetes patients. In: Obes Res. 12 (4), 661 -8, 2004; ARONNE, L. Progressive reduction in body weight after treatment with the amylin analog pramlintide in obese subjects: a phase 2, randomized, placebo-controlled, dose-escalation study In: J Clin Endocrinol Metab 92 (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 of insulin doses required for proper maintenance of blood glucose (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. such as insulin from other organisms and mutants.

Although the literature lacks information on long-term adverse effects (YOUNG, A. Alin: Physiology and Pharmacology. 2005. 341 P. Elsevier Academic Press, London, 2005; SINGH-FRANCO, D., ROBLES, G., GAZZE, D. Pramlintide acetate injection for 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 pramlintide therapy (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; PULLMA, J.; DARSOW, T.; FRIAS, JP Pramlintide in the management of insulin-using patients with type 2 and type 1 diabetes. Health Risk Manag 2 (3), 203-12, 2006; SINGH-FRANCO, D., ROBLES, G., GAZZE, D. Pramlintide acetate injection for the tre treatment 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 insulin-treated type 2 diabetes patients. In: Obes Res. 12 (4), 661-8, 2004; EDELMA, SV Does addition of pramlintide to basal insulin improve glycemic control in type 2 diabetes mellitus? In: Nature Reviews Endocrinology 4, 194-195, 2008). Since pramlintide is a peptide, its administration by subcutaneous, intramuscular or intravenous injection is preferred in order to prevent digestive tract degradation. The bioavailability of subcutaneous pramlintide injections is 30 to 40% (compared to the bioavailability of intravenous applications) and the commonly used dosages are 30 to 120 pg before meals. The maximum plasma concentration, Cmax, occurs approximately 20 minutes after subcutaneous administration (EDELMAN, SV.): Nature Reviews Endocrinology 4, 194-195, 2008). The low availability coupled with the high incidence of hypoglycemic events arising from the need for careful administration to avoid dosage variations point 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 development of new release systems that can keep human amylin stable.

Thus, confined release systems would allow meeting these explicit demands of maintaining the stability of human amylin and agonist analogs and achieving sustained release, which They aim to prolong the permanence of drugs in their therapeutic ranges in the 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 the chances of underdose.

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.

THE . Amylin is known for several other pharmacological actions, such as: secretion of glucagon, insulin, amylin and other pancreatic hormones, bone metabolism, anorexia, gastric emptying, stomach acid secretion, insulin degradation enzyme control, glucagon, peptide A- beta and amylin, promotion of pancreatic β cell regeneration, lipid, glycidic, lactic, glycogen metabolism, calcitonin receptor regulation, and peptides related to the calcitonin, cardiovascular and heraodynamic effects, central amino acid transport, body temperature, activation of the dopaminergic system, hippocampal modulation and memory, regulation of motor activity, anti-inflammatory action, dyslipidemia, coagulopathies, renal metabolism, immunological disorders, among others (YOUNG, A. Amyliri: 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.

WO2010017215-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 peptide. amyloid, insoluble, unstable, and whose system, proposed in 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 for no aggregation to occur, as its concentration is very low and transit by biological means where it would be associated with cofactors that would prevent 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. The third object refers to an in vitro functional evaluation process 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 X, 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, familial Creobutyl amyloid disease , 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-therapy or beta-cell transplantation, restoration of glucose sensitivity response in tissues composed of pancreatic cells, beta cells, adipose cells, central nervous system , 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 In the present invention, a polymeric confinement system of amylin and agonist analogues, in the form of nanoparticles, between 100 nm and 800 nm in diameter, and microparticles, between 5 μπ \ and 100 μτη, was developed using as matrix polymer is a compound comprised of the group consisting of polycaprolactone, poly-ε-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, preventing 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 for the production of nanoparticles and microparticles, respectively, containing amylin and agonist analogs.

The process of preparing the 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 c) Vacuum extraction of organic solvents

d) Washing by centrifugation or filtration

e) Removal of water Initially, in step (a), the two 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), with an average molecular weight of 10 to 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 μq / vL of amylin (human or animal) or agonist analogues, preferably 150 to 250 μq being used in organic solvent such as dichloromethane, acetone, trifluoroethane, chloroform, ethanol, methanol, ethyl acetate and 1, 1, 3, 3, 3, -hexafluoroisopropanol, in the range 100 to 10000 μΐ, at 4 to 35 ° C with the aid of an ultrasound bath (10 to 400 watts). Amylin agonist analogues should be comprised of the group consisting of pramlintide, chemically synthesized in solution, chemically synthesized solid phase, biosynthetic, amidated and unamidated, pegylated and not plegated, with intramolecular oxidation between tanks ( forming cystine) and non-oxidized, incubated with small aggregation inhibitors or not, such as resveratrol, thioflavin, insulin and insulinomimetics. The organic solvent used should belong to the group consisting of dichloromethane (DCM), acetone and trifluoroethanol (TFE), chloroform, ethanol, methanol, ethyl acetate and hexafluoroisopropanol (HFIP). Then, in step (b), an amount between 2.5 and 7.5 mL of FA with 0 to 2.0% PVA concentration was stirred and dispersed using a mechanical system. Stirring can be performed using one of the high-speed turbine tip (sonicator), homogenizer, mixer and diffuser equipment, and preferably the sonicator adjusted from 10 to 200 watts in continuous cycles for a time from 2 to 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 containment systems and analogs Agonists for the production of microparticles consist of the same steps already 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 isotonized buffered aqueous medium containing surfactant or emulsifying lipid agent (lauryl ether, sodium lauryl sulfate, polysorbate, or a nonionic surfactant) or phospholipids (dodecylphosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, phosphatidylcholine) was evaluated. 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 comprised between 100 and 300 μΐ. of an amyline-containing solution, released in an isotonized, buffered aqueous medium, containing a surfactant or emulsifier lipid agent (lauryl ether, sodium lauryl sulphate, polysorbate, or a nonionic surfactant) or phospholipids (dodecylphosphatidylcholine, phosphatidylcholine, phosphatidylglycerine, phosphatidylglycerine, ) from 0.001 to 2% w / v, from containment systems, were mixed in a buffered, isotonized aqueous medium containing a surfactant or emulsifier lipid agent (lauryl ether, sodium lauryl sulfate, polysorbate, or a nonionic surfactant) or phospholipids (dodecylphosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol) 0.001 to 2% w / v, with a volume in the range 100 to 300 μ]. derivatizing agent (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, being preferably 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 pancreatic amyloidosis cardiomyopathy, Familial pancreatic amyloid disease , myocardial infarction, coagulopathies, inflammatory diseases, dyspepsia, gastric ulcers, food intake, modulation of the bone metabolism, regulation of glycemic metabolism, insulin secretion, amylin secretion, glucagon secretion, proteostasis, decreased beta cell apoptosis, increased beta cell function and mass, adjuvant cell therapy or beta cell transplantation, restoration of glucose sensitivity response in tissues composed of pancreatic, beta, adipose, central nervous system, hepatic, cardiac and pulmonary cells.

This medicine can be found in solid, liquid suspension, gel, or semi-solid dosage forms such as resuspension powder, inhalable powder, tablets, coated tablets, capsules, lozenges, suppositories, ointments, injectables and transdermal patches for oral, sublingual use. , topical, implant, intramuscular, intravenous, or by inhalation, preferably being injected.

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: H ₂ N-Lys-Cys-Asn- Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser- Ser-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-CONH ₂ .

The following examples are for illustrative purposes only and are intended to substantiate the embodiments of the inventors. The data contained in the examples should therefore not serve to delimit the depositor's rights. EXAMPLES

Example 1: Nanoparticles production process 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 sonicator with a cycle of 1 and amplitude of 70% was used to perform the agitation. FO was homogenized in the 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 15 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 mass of 65 kDa, was dissolved in 1mL of DCM, 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. The FO was homogenized in the AF and, afterwards, a 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 pm were obtained (Figure 2).

Example 3: In vitro release method of amylin. The method using fluorescamine, when used for detection and quantification of amylin, showed good linearity, expressed by the R ² value of 0.999 in the concentration range of 0.1 mg to 50 μg / mL (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 could not be analyzed by light microscopy as they had very small diameters.

Nanoparticle suspensions were diluted with water and kept in sealed acrylic cuvettes. The flasks were placed in the light scattering equipment analysis chamber so that the laser beam passed through the suspension without any internal filter effect.

The sample containing human amylin had a diameter of 153.3 nm and polydispersity of 0.116, as shown in Figure 6.

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

156.210.101

148.20 108

154.50 139

Average 153.3 0, 116

Standard Error 2.1 0.011

Example 4.2: Scanning Electron Microscopy (SEM) morphological analysis

Using a micropipette, the nanoparticle 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 μΐι of the nanoparticle suspensions, after 30 seconds another 5 pL of uranyl (supersaturated) supersaturated 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. 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: In Vitro Amylin Nanoparticles Release Profile

After washes, samples were diluted in 10 mL of the release buffer (PBS pH 7.4 with 0.02% NaN ₃ and 0.1% polysorbate-80). After dilution, the material was divided into ten 1.5 mL microtubes, each tube receiving 1 mL of sample and all transferred to the greenhouse and maintained at 37 ° 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 shows a release profile of 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. Example 5.2: In vivo Pharmacological Profile of Amyline 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 (Νρ-hIAPPwt group, n = 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 L of 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 formulation containing nanoconfinished human amylin was 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 It is also a potential use of the formulation to prepare a medicament for treating diabetes mellitus and other metabolic disorders. The animal experiments were performed in order to minimize their discomfort or suffering and these experiments were previously submitted to analysis and approved by the animal experimentation ethics committee of the animal.

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

Claims

1. Polymeric system for confinement of amylin and agonist analogues characterized by consisting of a polymeric matrix, in the form of nanoparticles, with an average size between 20 and 600 nm, and microparticles, with an average size between 1 and 100 m, of release controlled and sustained use of amylin and agonist analogues directly in the body.

2. Polymeric system, according to claim 1, characterized by using as a polymeric matrix a compound comprised in the group consisting of polyglycolic acid, polylactic acid, polycaprolactone, poly-ε-caprolactone (PCL), poly (DL-lactide-co-caprolactone), poly(lactic-glycolic) acid (PLGA), phospholipids, liposomes, chitosan and alginates.

3. Process for preparing formulations based on polymeric amylin confinement systems and agonist analogues characterized by consisting of a simple emulsification methodology, followed by solvent extraction, to form nanoparticles and microparticles.

4. Process, according to claim 3, characterized in that it is used for the production of nanoparticles containing amylin and agonist analogues, using the following procedure:

a) Preparation of the emulsion phases

b) Formation of the emulsion by stirring or sonication

c) Extraction of organic solvents by vacuum

d) Washing by centrifugation or filtration

e) Water removal

5. Process, according to claims 3 and 4, characterized by the fact that the aqueous phase, in step (a) is prepared by adding emulsifying agent to the water ultra-pure heated in a range between 20 and 90°C.

6. Process, according to claim 5, characterized by the fact that the emulsifying agent is included in the group consisting of polyvinyl alcohol and polaxamer, both in concentrations between 0 and 5%.

7. Process, according to claim 6, characterized by the fact that the polyvinyl alcohol has an average molecular mass between 10 and 90 kDa.

8. Process, according to claims 3 and 4, characterized by the fact that the organic phase, in step (a) is obtained using amylin or analogues, in a range between 10-1000 g/mL dissolved in an organic solvent , totaling a volume in the range between 10-5000 yL.

9. Process, according to claim 8, characterized by the fact that the mass of amylin and agonist analogues used in the dissolution is comprised, preferably, in a range between 100-500 μς.

10. Process, according to claims 3 to 9, characterized by the fact that the amylin analogue is included in the group consisting of pramlintide, synthetic chemically in solution, synthetic chemically in solid phase, biosynthetic, amidated and non-amidated , pegged and non-peglated, with intramolecular oxidation between cysteines and non-oxidized, incubated with small molecules that inhibit aggregation and non-incubated.

11. 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.

12. Process, according to claims 3 and 4, characterized by the fact that the dissolution of the polymer, in step (a) is carried out, using a mass ranging from 1 to 300 mg, in a volume of 1 to 10000 µm. organic solvent.

13. Process, according to claim 12, characterized by the fact that the mass of the polymer used in the dissolution is comprised, preferably, in a range between 10 and 30 mg.

14. Process, according to claim 13, characterized by the fact that the polymer has an average molecular mass of 5,000 to 100,000 Da.

15. Process, according to claims 3 to 8, characterized by the fact 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.

16. Process, according to claims 3 and 4, characterized by the fact that the nanoemulsion, in step (b), contains the organic phase and an amount between 2.5 and 7.5 mL of PVA aqueous solution with a concentration of 0 .5 to 2.0%.

17. Process, according to claim 16, characterized by the fact that the nanoemulsion is obtained using one of the equipment comprising a sonicator, homogenizer, mixer and high-speed turbine diffuser.

18. Process, according to claim 17, characterized by the fact that the nanoemulsion is obtained, preferably, with the aid of a sonicator for a time between 2 and 15 minutes.

19. Process, according to claim 18, characterized by the fact that the nanoemulsion is obtained with the aid of a sonicator, preferably for a period of between 5 and 10 minutes.

0. Process, according to claims 17 to 19, characterized by the fact that the formation of the nanoemulsion can occur with the sonicator adjusted between 10 and 200 watts of power in a continuous cycle.

1. Process, according to claims 3 and 4, characterized in that the vacuum, in step (c) is used for 30 to 60 minutes under cooling.

2. Process, according to claims 3 and 4, characterized by the fact that the system is resuspended, in step (e) in a cryoprotectant solution for subsequent drying by methods such as freeze-drying and fluidized bed ("spray-dry").

3. 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 silicone dioxide, polyethylene glycol and polaxamer.

4. Process, according to claims 3 to 23, characterized in that it is used for the production of microparticles containing amylin and agonist analogues.

5. Process, according to claim 24, characterized by the fact that the formation of the microemulsion, in step (b), occurs by agitation using a high-speed turbine diffuser.

6. Process according to claim 25, characterized in that the high-speed turbine diffuser is operated at a speed between 10,000 and 20,000 RPM.

27. Process of functional evaluation of in vitro release of amylin and agonist analogues from polymeric confinement systems dispersed in a 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) between 0.001 and 2% w/v, characterized by consisting of the dosage of amylin and agonist analogues by spectrofluorimetry with fluorescamine.

28. Process, according to claim 27, characterized by generating a conjugate between amylin and agonist analogues with the fluorescent substance as product.

29. Use of the polymeric amylin confinement system and agonist analogues characterized by being used for the production of medication for amylin replacement therapy in individuals.

30. Use of the polymeric confinement system, according to claim 29, characterized in that the medicine is used in solid, liquid suspension, gel or semi-solid pharmaceutical forms, such as powder for resuspension, inhalable powder, tablets, coated tablets, capsules, lozenges , suppositories, ointments, injectables and transdermal patches, for oral, sublingual, topical, implant, intramuscular, intravenous, or inhalation use, allowing the sustained release of amylin and agonist analogues.

31. Use, according to claim 30, characterized in that it is used, preferably, by injectable route.

32. Use of the polymeric amylin confinement system and agonist analogues characterized by being used for 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, amyloidoses such as Alzheimer's , Parkinson's, cerebral amyloid angiopathy, leptomeningeal amyloidosis, familial visceral amyloidosis, primary cutaneous amyloidosis, senile systemic amyloidosis, familial amyloid polyneuropathy, familial amyloid cardiomyopathy, Creutzfeldt-Jakob disease, pancreatic amyloidosis, coagulopathies, other vascular diseases, inflammatory diseases, dyspepsia, gastric ulcers, decreased food intake, modulation of bone metabolism, proteostasis, decreased apoptosis of beta cells, increased function and mass of beta, adipose, central nervous system, hepatic, cardiac and pulmonary cells, adjuvant in cellular or beta cell transplantation, restoration of the glucose sensitivity response in tissues composed of pancreatic, adipose, central nervous system, liver, heart and lung cells.

33. Amylin agonist analogue characterized by 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, Phe, Pro, Ser, Thr, Trp, Tyr, Val, which can be introduced at one or more points in the human amylin chain.