BRPI1103164A2 - insulin confinement polymeric system, process and use of said system - Google Patents

Abstract

POLYMERIC INSULIN CONFINEMENT SYSTEM, PROCESS AND USE OF SUCH SYSTEM. The present invention discloses a polymeric insulin confinement system consisting of a polymeric matrix, preferably in the form of nanoparticles and microparticles, of sustained controlled release of insulin directly into the body, as well as its use for the preparation of a medicament for diabetes treatment. The process for preparing this system consists of one. double emulsification method and solvent evaporation.

Description

POLYMERIC INSULIN CONFINEMENT SYSTEM, PROCESS AND USE OF SUCH SYSTEM

Field of the Invention

The present invention relates to a polymeric insulin confinement system developed for use, among others, as a medicament in the replacement therapy of this pancreatic hormone in diabetes. The present invention also discloses the process of preparing said polymeric system. Background of the Invention

Insulin is a pancreatic protein with hormonal activity involved in the regulation of blood glucose in postprandial and basal situations. Insulin replacement therapy has been in place for decades, especially with the advent of recombinant human insulin.

In Brazil there is a prevalence of 5.2% of diabetes mellitus patients in the adult population, which generates high expenses with the purchase and distribution of medicines by the Ministry of Health (SCHMIDT, MI et al. Prevalence of diabetes and hypertension based on self -reported morbidity survey, Brazil, 2006. Rev Saúde Pública 2009; 43 (Suppl 2): 74-82).

Human insulin therapy is currently performed by subcutaneous administration of formulations of this hormone obtained by biosynthetic or semi-biosynthetic route, aiming at the control of glycemia at various levels and time scales.

Regular human insulin has a range of action around 2 to 5 hours. In order for this insulin to be used, it must be given about 30 minutes before the meal. This way, it is not able to take immediate action, nor can it maintain circulating insulin levels for a long time.

Nowadays only human insulin analogues

maintains circulating insulin levels for this extended time. These analogs are based on point mutations (eg B28LysB29Pro, B28Arg, B24Asp) or chemically derivatized (with fatty acid or PEG attached to the polypeptide chain). These innovations bring immunogenic reactions as inconvenient, such as past use of bovine insulin that differs from human in only one amino acid.

There are several commercially available insulin-based products, both native and chemically modified (derivatization or point mutations) reaching a wide pharmacokinetic range of action.

Replacement of basal insulin levels in diabetic subjects with prolonged coverage of

Baseline of insulin in the fasting state, does not have

native human insulin (not chemically modified). There are some of these products in the pharmaceutical market, but they have the characteristics that they are not homologous to insulin or in the presence of protamine, a protein heterologous to the human organism. This raises the problem of immunogenicity, a barrier that was overcome with the introduction of recombinant human insulin and semi-synthetic human insulin in the 1980s.

There are several prior art documents which

30 disclose controlled release formulations containing insulin. However, no reference was found that anticipated the object of the present invention.

Patent application WO 2010/028257, for example, discloses the use of non-aqueous vehicles such as di- and triglycerides or fatty acids for formulations containing microparticles without, however, describing the preparation of the microparticles. This document addresses the resuspension of protein lyophilisate in an oil as a carrier without the use of nano technology and / or polymeric microencapsulation. WO 96/31231, in turn, discloses the preparation

of polycanoacrylate nanoparticles containing insulin. In comparing this reference with the present invention it is noted that different polymers are used for the formation of nano and microparticles. From a process point of view, the differences are also striking, since in WO 96/31231, matrix polymerization occurs in situ and is concurrent with insulin nanoencapsulation and nanoparticle formation. On the other hand, in the present invention the polymer used is already formed and it is only dissolved in a compatible solvent which, after solubilization of the polymer, is incorporated into the formulation by employing kinetic energy for microparticle formation and insulin encapsulation. . Also, in this prior art document WO 96/31231, the authors make use of a different class of polymeric material, which requires the use of a completely different technology of the present invention. The difference in the duration of the pharmacological effect is another point to be emphasized, since in the present invention there was an effect ensured for at least 24 hours, whereas the data presented in WO 96/31231 point to effects lasting up to 6 hours.

Finally, WO 2006/088473 describes chitosan micro and nanoparticles containing peptides or proteins, and such particles may or may not be coated by lipophilic polymers such as polycaprolactone. In the present invention, poly-ε-caprolactones are the matrix forming agents and there is no particle coating. In the technology now revealed, the system is totally chitosan independent for its functionality. It is further noted that the processes for obtaining the particles also differ, as the present formulations are obtained by double emulsion followed by extraction of volatile solvents, whereas in the cited application, WO 2006/088473, insulin is incorporated directly into the particles. Chitosan

Summary of the Invention

Surprisingly, the present invention discloses a polymeric insulin confinement system developed for use, among others, as a medicament in the replacement therapy of this pancreatic hormone in diabetes. The present invention also discloses the process of preparing said polymeric system.

In this context, in order to obtain a formulation capable of being administered as a drug capable of prolonged human insulin-based glycemic control, a polymeric particulate system was developed, with size distribution particularly in the micro and nanometric ranges, with a two-phase release profile. of insulin, able to act on blood glucose both in the fast phase (1 to 4 30 hours after subcutaneous injection) and in the slow phase, with in vitro release for at least 10 days.

Brief Description of the Figures Figure 1: Size distribution of insulin-containing microparticles (data obtained by dynamic light scattering).

Figure 2: Image of particles containing human insulin obtained by scanning electron microscopy (SEM) (magnification and size bar indicated at the bottom of the microscopy).

Figure 3: In vitro release kinetics (saline phosphate buffer - PBS pH 7.4 at 370C) of microencapsulated insulin in poly-8-caprolactone - PCL particles. A) linear scale. Inset: Detail showing initial times. Continuous lines are adjustments with equation Cobs = C0 + A1 * e (_kl * t) + A2 * and ("k2 * t) double-order first kinetics.

Figure 4: Pharmacological effect of insulin-containing microparticles in diabetic mice (streptozotocin induction, blood glucose greater than 300 mg / dL). Figure 5: Release of human insulin from 50:50 poly D7L-laticoglycolic acid polymer microparticles, MW ~ 43,000 to 65,000.

Description of the Invention

The polymeric insulin confinement system of the present invention preferably contains particles of polycaprolactone and poly D 1 -loglycolic acid. Its use as a medicine for insulin replacement therapy occurs in the slow and rapid phases. The process of preparing said polymeric system uses double emulsion followed by extraction of volatile solvents. The problem that the present invention lends itself to solving is to provide a mixed-action polymeric insulin confinement system with mixed actions. A system based on polymer particles selected from the group consisting of polycaprolactone and poly D, L-laticoglycolic acid capable of sustaining fast and slow insulin release in two phases was developed, as evidenced by pharmacological measures in vivo.

The system is preferably injectable, even more preferably subcutaneous, biocompatible, bioerosible, capable of decomposing in vivo to non-toxic products, capable of controlled release in vitro for at least 240 hours. In vivo pharmacological trials in fasting diabetic mice demonstrated the efficacy of controlled release, as observed by their ability to significantly reduce basal glucose (compared to control) for at least 48 h compared to controls administered with regular soluble human insulin. These data together establish a proof of concept of the ability to overcome the technological challenge of a biocompatible human insulin system for sustained / prolonged release, to date not available worldwide.

The immediate needs to be met by the present invention are the provision of:

- a system for use in human insulin therapy and

25 analogues;

- a slow controlled release system for use in human insulin replacement therapy and analogues;

- a slow and controlled release system in which the preparation is a form of insulin confined in a matrix

30 polymeric for use in human insulin replacement therapy and analogs;

- a slow, controlled release system in which the preparation is a form of insulin confined in a polymeric matrix for injection (subcutaneous) administration.

or other), oral, transdermal or other for use in human insulin replacement therapy and the like;

- a slow, controlled release system in which the preparation is a form of insulin confined in a polymeric matrix for injection (subcutaneous) administration.

or other), orally, transdermally or otherwise, wherein the polymeric matrix is selected from the group consisting of polycaprolactone, poly D, L-laticoglycolic acid (as in Figure 5) and others for use in human insulin replacement therapy and the like. The use of these polymers is

desirable for biocompatibility, its atoxicity to the polymeric form and its decomposition products. Their use is determined by the desired parameters of release rate, in vivo degradation rate and absorption, compatibility with encapsulated drug;

- a process for the preparation of the system containing

a matrix for molecular confinement of human insulin and analogs;

- the use of the system for the preparation of a diabetes medicine;

- a system containing a lyophilised powder containing

encapsulated, particularly microencapsulated or nanoencapsulated insulin and an aqueous carrier; and

a product for extemporaneous use, which after reconstitution with a suitable vehicle may be applied with

3 0 syringe aid. The examples below are illustrative only and should not limit the scope of the present invention. Preparation of insulin containing particles

The production process consists of a method of emulsifying and evaporating the solvent as follows:

- Firstly, 10 to 4,000 μ] ^, preferably 400 μ] 1 l of regular human insulin (0.5 to 20 mg / mL human insulin, preferably 3.7 mg / mL stabilized with 0 to 30 mg / mL of metacresol or phenol, preferably 3 mg / mL

metacresol; 0 to 1 mg / mL ZnCl2 preferably at 7 μg / mL, and 0 to 200 mg / mL deglycerol, preferably 16 mg / mL) and 0 to 1,000 μL of 0.01 to% w / v polyvinyl alcohol (PVA) , preferably 100 μL at 5% w / v (internal aqueous phase - FAi) are homogenized with 0 to 2,000 mg of

PCL, preferably 100 mg, dissolved in 0.01 to 20 mL dichloromethane - DCM (oil phase - FO), preferably 5 mL, using a S10N-5G shear mixer at 1,000 at 50,000 rpm preferably 18,000 to 22,000

rpm, forming the primary water-in-oil (A / O) emulsion.

- Next, this A / O emulsion is dripped dispersed under flow from 0.1 to 50 mL / min, preferably at 5 mL / min, over 0.1 to 250 mL of PVA, preferably 25 mL, from 0 to PVA, 01 to 20% w / v, preferably 1.5% w / v, under

gentle stirring at 50 to 1,000 rpm, preferably at 500 rpm with magnetic stirring, forming the water-in-water (W / O / W) microemulsion.

- This microemulsion is then subjected to vacuum (200 to 70 mm Hg, preferably 60 mm Hg) at a temperature of

10 to 60 ° C, preferably 25 ° C, for removal of DCM.

The particulate suspension is washed three times with PVA aqueous solution at a concentration between 0 and 20% w / v, preferably 1.5% w / v, and then preferably dried by lyophilization for storage, or used immediately for testing. release

Process yield is calculated based on the mass recovered after lyophilization and the encapsulation efficiency obtained by dosing insulin in the supernatants after washing.

Importantly, several formulations were tested using single emulsion (with insulin dissolved directly in organic solvent) instead of double emulsion, or double emulsion, but using insulin without stabilization. None of these attempts resulted in an adequate formulation, and unsatisfactory results were obtained, possibly due to the structural degradation of insulin.

The efficiency calculated from the wash supernatants was 64.5% with a standard deviation of 6.8%. The yield was obtained from weighing the material after lyophilization which was 88.4% ± 4.2%. Quantitative insulin spectrophotometric analysis

Insulin-containing samples were quantified by the modified Bradford method. For the quantification of the present insulin, 500 μΐ; from each sample were mixed with 500 μΐι of the twice concentrated Bradford reagent (200 mg Coomassie G-250, 100 mL 95% ethanol, 200 mL 85% H3PO4 and ultrapure water qsp 1 L) and after mixing the samples, Using a vortex, the absorbances at 595 nm were measured using a spectrophotometer. The insulin calibration curve in the release buffer (PBS pH 7.4, 0.02% azide and 0.1% polysorbate-80) was performed to verify the adequacy of the method to the expected concentrations during the release experiments. and to obtain the linearity of the method. The specificity of this methodology was verified using samples containing all components of the formulation except insulin itself.

Particle Size Analysis

Particle suspensions, particularly nanoparticles and microparticles, were diluted with water and kept in sealed acrylic cuvettes. The vials were placed in the analysis chamber of the

light scattering so that the laser beam passes through the suspension to its full extent without any internal filtering effect. The average diameter value and polydispersity index were provided by Shimadzu- SALD-2201 and Brookhaven equipment.

2 0 Instruments Corporation - ZetaPlus Zeta Potential Analyzer.

The particles resulting from the double-emulsion method employing magnetic stirrer presented varying sizes. The analysis of the samples by dynamic light scattering showed that they had diameter values

mean and standard deviation equal to 9.82 μτη and 0.56 μιτι respectively (as shown in Figure 1).

Morphological analysis by scanning electron microscopy (SEM)

The particle suspension was spread over slides

3 0 glass and cooled in liquid nitrogen. After lyophilization, the samples received treatment (gold coating) and were observed in the scanning electron microscope, as shown in Figure 2.

In vitro particle insulin release kinetic profile

After washes, samples were diluted in 15 mL of the release buffer (PBS pH 7.4 and 0.02% azide). After dilution, the material was divided into fifteen 1.5 mL microtubes, with each tube receiving 1 mL of sample, which

It was then transferred to the greenhouse and kept at 37 ° C during the experiment. At each time a microtube was removed from the oven and centrifuged at 20,000 g for 30 minutes at 120 ° C. Insulin present in supernatants was measured by the previously described Bradford method.

Several experiments performed on different days

indicated that this system has a biphasic release kinetics, with amplitudes of 50% for each one, the first lasting about 2h and the second until 48h. Figure 3 shows the release profile.

20 obtained from insulin-containing particle preparations.

Data were fitted with a first-order double kinetics function as follows:

Cobs = C0 + A2 ewt '+ A2 * and (k2 * t)

where Cobs is the insulin concentration of time t, C0 is

the insulin concentration at time 0, A is the total effect, k is the kinetic constant and 1 and 2 are the kinetic phases. Considering that the glycemic effect is biphasic, with restoration of the original blood glucose levels, in vivo pharmacological data were adjusted using Al = A2.

The table below shows in vitro release kinetic data and in vivo pharmacological effects of human amylin from PCL particles:

In vitro release In vivo release (blood glucose) MP: Insulin MP: Humulin® Ai 43.9 + 9.9 --- --- ki 0.76 + 0.39 h "1 2.58 h" 1 2.59 h "1 A2 54.7 + 9.8 --- --- k2 0.023 + 0.0095 h "1 0.047 h" 1 0.18 h "1 r2 0. 916 0. 92 0. 93

Pharmacological evaluation of insulin-containing particle preparation

8-week-old male Swiss mice were divided into two large groups: control (n = 4) and diabetic (n = 13). The animals were housed in a temperature controlled room with a 12h light-dark cycle and had free access to water and feed. Type 1 diabetes was induced by an intraperitoneal (Ip.) Injection of streptozotocin (STZ, 200 mg / kg) dissolved in fresh citrate buffer (100 mM, pH 4.5). The control group received only the vehicle (0.1 ml). Five days after STZ administration, mouse tail blood was collected, and glucose levels were measured by a glucometer (Accu-Chek® Active - Roche). The animals with glucose levels above 300 mg / dl were considered diabetic.

Then the diabetic group was subdivided into three new groups. The first group (placebo group, n = 3) received 100 μΐ; of insulin-free particle formulation produced according to the same protocol as the preparation of insulin-containing particle formulation; the second group (Regular Human Insulin group 100 U / mL, η = 5) was treated with 5 U / kg commercial regular human insulin (Humulin®); and the third group (Test group, η = 5) received 10 0 μΙ <from the microencapsulated insulin formulation. On the sixth day, the animals' blood glucose was determined at time zero, and 100 μΙ; each sample was injected subcutaneously into each animal. All mice were in fed state. Thereafter, blood glucose concentration was monitored at times: 15, 30, 60, 120, 360, 1440 and 2880 minutes. This protocol was approved by the Animal Experimentation Bioethics Committee of the Health Sciences Center (CCS) - UFRJ, under the registration number DFBCICB 038.

Animals that received empty particles (without insulin) did not show significant variations in their glycemia. Comparison between mean blood glucose levels of the group receiving commercial regular human insulin (100 U / mL) and the group receiving insulin-containing particles showed that these glycemia differed statistically significantly at 6h and 24h, indicating that the insulin released by the particles was able to reduce the

2 5 blood glucose levels of these animals for a period longer than

commercial pharmaceutical.

In Figure 4 the first hours of the experiment are highlighted. The initial phase of blood glucose decline was similar to that observed for soluble human insulin.

In turn, it was noteworthy that the preparation tested kept the animals' blood glucose lower for a longer period of time, with a return of blood glucose, but still maintaining for a lower level than the control group administered with commercial formulation (longer pharmacological evaluation was not possible in this experimental protocol due to the need to return the animals' food). The kinetic constants of the two phases of glycemic effect, decline and recovery, were equivalent to those observed for in vitro release (Table above) suggesting a strong in vitro χ correlation in vivo for this formulation.

Claims (10)

1. Polymeric insulin confinement system characterized by the fact that it consists of a polymeric matrix, controlled and sustained release of insulin directly into the body. Polymeric insulin confinement system according to claim 1, characterized in that the polymeric matrix is in the form of nanoparticles and microparticles. Polymeric insulin confinement system according to claim 1 or 2, characterized in that the polymeric matrix consists of a compound selected from the group consisting of poly-e-caprolactone and poly D, L-laticoglycolic acid. Polymeric insulin confinement system according to claim 1, 2 or 3, characterized in that it has a biphasic insulin release profile. Process for the preparation of the polymeric insulin confinement system as defined in claim 1, 2, 3 or 4, characterized in that it consists of a method of emulsifying and evaporating the solvent in which: - first, 10 to 4,000 μΐϋ regular human insulin and 0 to 1,000 μΐ; 0.01 to 10% w / v polyvinyl alcohol (PVA) (internal aqueous phase - FAi) are homogenized with 0 to 2,000 mg poly-caprolactone (PCL) dissolved in 0.01 to 20 mL dichloromethane - DCM (oil phase - FO), using a shear mixer at 1,000 to 50,000 rpm, forming the primary water-in-oil (A / O) emulsion; - this A / 0 emulsion is then dripped dispersed in a flow rate of from 0.1 to 50 mL / min, over 0.1 to 250 mL of PVA, from 0.01 to 20% w / v PVA, under gentle agitation. 50 to 1,000 rpm with magnetic stirring, forming the microemulsion water in oil in water (A / O / A); - this microemulsion is then vacuumed (200 to 70 mmHg) at a temperature of 10 to 60 ° C for the removal of DCM; The particulate suspension is washed three times with aqueous PVA solution at a concentration between 0 and 20% w / v, and then dried or used immediately in the release assays. Process according to Claim 5, characterized in that the regular human insulin consists of 0.5 to 20 mg / mL human insulin, stabilized with 0 to 30 mg / mL of metacresol or phenol, Oal mg / mL of ZnCl2. , and 0 to 200 mg / ml glycerol. Process according to Claim 5, characterized in that the mixing speed in the first process step is 18,000 to 22,000 rpm. Use of a system as defined in claim 1, 2, 3 or 4, characterized in that it is for the preparation of a medicament for human isulin replacement therapy. Use according to claim 8, characterized in that the system is capable of biphasic release of insulin, being a fast phase and a slow phase. Use according to claim 9, characterized in that the rapid phase occurs 1 to 4 hours after subcutaneous injection and the slow phase has a pharmacological effect for at least 48 hours.