CN117085116A - Glucose-responsive microneedle patch and preparation method and application thereof - Google Patents

Abstract

The invention discloses a glucose responsive microneedle patch, and a preparation method and application thereof. The invention also discloses a raw material composition of the glucose-responsive microneedle, which comprises an active ingredient and a matrix composition for forming a drug-carrying polymer, wherein the active ingredient is insulin and glucagon analogues, and the glucagon analogues are natural glucagon modified by positive charge and maintain the activity of the natural glucagon; the matrix composition includes a phenylboronic acid-based monomer having a glucose-responsive group and a liquid cationic compound. The novel microneedle patch with dual response to high and low blood sugar levels can be used for co-delivery of two blood sugar regulating hormone insulin and glucagon analogues, and can release correct medicaments in corresponding doses at corresponding blood sugar levels, namely, release correct medicaments at correct time; and the microneedle patch is simple to prepare, low in cost and simple and comfortable to use.

Description

Glucose-responsive microneedle patch and preparation method and application thereof

Technical Field

The invention relates to the field of biological materials, medical equipment and drug delivery related to diabetes treatment, in particular to a glucose-responsive microneedle patch and a preparation method and application thereof, and particularly relates to a raw material composition of a glucose-responsive microneedle, a drug composition containing the glucose-responsive microneedle, a preparation method of the microneedle, a microneedle patch, a preparation method of the microneedle patch, a double-closed-circuit delivery system for co-carrying insulin and glucagon and application thereof.

Background

Diabetes is a metabolic disease characterized by accumulation of glucose in blood, and has a large number of patients and huge medical pressure. With the development of society and the improvement of medical level, attention on how to treat diabetes comfortably is unprecedented. The traditional open-loop subcutaneous insulin injection administration mode has obvious pain and the insulin delivery modes such as an insulin pen which is subjected to wound and is developed later and an electronic closed-loop device insulin pump which is subjected to algorithm precision and sensor reliability cannot meet the requirements of the current patients on high-efficiency treatment and high life quality, so that the novel administration mode capable of intelligently, long-time, low-frequency and comfortably delivering diabetes therapeutic drugs is urgent.

Microneedle systems have been used for many years for transdermal drug delivery with technical and cost advantages of far beyond syringes, pumps, pens, etc. Although various modes of delivery of blood glucose regulating drugs have been developed at this stage, such as insulin microneedle patches, GLP-1 microneedle patches, insulin analogue microneedle patches, and glucagon microneedle patches. These responsive microneedle patches have typically produced tremendous effects, but still do not meet the need to completely avoid the risk of hypoglycemia from hypoglycemic agents. The ideal insulin and glucagon hybridization delivery microneedle patch also has the defects of complex preparation process, response capability to be improved and the like.

The present inventors disclosed in a previous patent application CN201880087087.7 a charge switchable polymer depot for glucose triggered insulin delivery with ultra fast response; a microneedle array patch with a glucose responsive matrix for closed loop insulin delivery is also disclosed in CN 201980068619.7. Both of the above microneedle patches are microneedle patches that deliver insulin at hyperglycaemia, and do not involve the delivery of glucagon. Furthermore, the inventors of the present invention disclosed in WO2022010698 a two-type microneedle shared co-polymeric matrix in a therapeutic hybrid microneedle patch that contains varying proportions of key monomers that have a "dual response" to hyperglycemic and hypoglycemic conditions. The hybrid microneedle patch includes a first plurality of microneedles (comprising a biocompatible polymer loaded with insulin) and a second plurality of microneedles (comprising a biocompatible polymer loaded with glucagon), i.e., insulin and glucagon are located in different microneedle bodies and function independently of each other. However, the conventional microneedle patch described above has the following drawbacks:

(1) The glucose response behavior in the body is to be improved (for all); (2) The simplified preparation process (WO 2022010698) is not achieved; (3) The method can not realize that two blood sugar regulating hormone medicines are loaded in the same microneedle body so as to completely avoid the risk (all) of hypoglycemia; (4) The duration of regulation of blood glucose in the body remains to be improved (in total).

Disclosure of Invention

In order to overcome the technical defects, the invention aims to provide a glucose-responsive microneedle patch, and a preparation method and application thereof. The invention designs the common microneedle drug delivery patch into the microneedle patch with double response and high and low blood sugar level for co-delivery of two blood sugar regulating hormones insulin and glucagon or analogues thereof, on one hand, the accurate and long-time drug delivery can be realized; on the other hand, the microneedle patch is simple and comfortable to use, simple to prepare, low in cost and can be used for treating abnormal blood sugar, particularly diabetes.

As known to those skilled in the art, the ideal design of a bi-directionally regulated blood glucose microneedle patch would meet the following requirements: (1) Rapid in vivo glucose response behavior with similar pharmacokinetics to islet α, β cells; (2) Sufficient drug loading capacity for daily use to reduce blood glucose and circumvent the risk of hypoglycemia; (3) Convenient and comfortable to use, miniaturized, ultrathin and waterproof; (4) The equipment is stable, the cost is low, the process is simple, and the large-scale production can be realized; (5) good biocompatibility, no acute and long-term toxicity.

However, in the research and development process, the inventor finds that the main technical difficulty to be overcome is to further control the drug release mode by screening the formulation and the preparation process of the drug composition on the microneedle patch so as to realize rapid release of insulin under hyperglycemia and glucagon under hypoglycemia, and simulate the physiological activities of the islet alpha and beta cells as far as possible. Through many researches, the inventor selects an electrostatic interaction mode from a plurality of different drug release modes of the insulin microneedle patch so as to achieve quick response; the selection of positive and negative charge components in the polymer loaded with two different active ingredients, including insulin or analogues thereof, glucagon or analogues thereof and positive and negative charge monomers in the drug-loaded polymer, and the adjustment of the molar ratio between the components are further studied, and the precise drug release is carried out through reasonable charge difference so as to achieve the regulation of the two diametrically opposite directions of high and low blood sugar. In this process, the inventors have surprisingly found that the desired effect can be achieved by adjusting a certain charge difference between glucagon and insulin, in particular by increasing the isoelectric point of glucagon, and that the preparation is simple, low cost and suitable for the later industrial production. However, it is a further technical difficulty of the present invention to adjust the isoelectric point of glucagon while retaining activity to maximize control by electrostatic interactions.

Specifically, a first aspect of the present invention provides a raw material composition of glucose-responsive microneedles, comprising an active ingredient that is insulin and a glucagon analog (GCA) that is native glucagon positively charge modified and retains the activity of native glucagon, and a matrix composition that forms a drug-loaded polymer; the matrix composition includes a phenylboronic acid-based monomer having a glucose-responsive group and a liquid cationic compound.

As known to those skilled in the art, insulin has an isoelectric point (pis) of about 5.4 and thus exhibits negative charge and high solubility at physiological pH, in contrast to native glucagon, which has a lower solubility in the pH range of 6-8 because of its isoelectric point of about 7.1. According to the present invention, the insulin may be conventional in the art, as long as various natural insulins or recombinant and modified insulins having isoelectric points less than 7.4 in physiological environments are applicable.

Preferably, the isoelectric point (uncharged pH) of the glucagon analog is increased by at least 2 compared to native glucagon, in which case GCA is capable of achieving a low glycemic release compared to native Glucagon (GC), and a nearly non-glycemic release profile of the drug; the isoelectric point is preferably increased by 2 to 7, more preferably by 4 to 6. For example, isoelectric points are improved by 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4 or 5.5, and the like, and according to researches, the efficacy of the GCA with the higher isoelectric points is the same as that of the GCA, the problem of biotoxicity is avoided, and the release speed of the hypoglycemia is faster.

In a preferred embodiment, the glucagon analog is a natural glucagon having two to ten arginines, preferably four to seven arginines, at the C-terminus; more preferably six arginines, at which point the isoelectric point is increased by 5.3.

As known to those skilled in the art, glucose responsive groups are commonly known as arylboronic acid or arylboronic ester groups, such as halophenylboronic acid groups, which are typically negatively charged. According to the present invention, the phenylboronic acid-based monomer may be selected from known phenylboronic acid group-containing monomers, preferably selected from (4- ((2-acrylamidoethyl) carbamoyl) -3-fluorophenyl) boronic acid (FPBA), 3- (acrylamido) phenylboronic acid (3 APBA) and 4- (bromoethyl) phenylboronic acid.

In the feed composition, the liquid cationic compound is used to bind negatively charged insulin, in the preferred embodiment of the present invention 2- (dimethylamino) ethyl acrylate (DMAEA).

The phenylboronic acid monomer and the liquid cationic compound primarily affect dissociation of the insulin from the microneedle in a hyperglycemic environment, while the glucagon analog dissociates from the microneedle in a hypoglycemic environment.

In the raw material composition, the matrix composition preferably further includes: a liquid monomer solvent. When the polymer is used for loading the drug on the micro-needle, on one hand, the phenylboronic acid monomer and the liquid cationic compound can be dissolved, and on the other hand, the polymer is also used for forming a skeleton matrix of the drug-loading polymer in the micro-needle patch. It may be selected from monomers containing at least one vinyl group commonly used in the microneedle patch field, such as N-vinyl pyrrolidone (NVP), and polyvinyl alcohol (PVA) or methyl acrylate PVA (m-PVA) may be used instead. In a preferred embodiment, when the drug-loaded polymer of the present invention is crosslinked, the matrix composition further comprises a crosslinking agent. The cross-linking polymerization reaction can better lead the polymer obtained by the whole polymerization to form a network structure, and the network structure can lead the polymer needle body not to be dissolved in the skin, and can be pulled out of the body after the use. The cross-linking agent can be selected from cross-linking agents known in the art, and is commonly used as Ethylene Glycol Dimethacrylate (EGDMA).

In another preferred embodiment, when the drug-loaded polymer of the present invention is subjected to photopolymerization, the matrix composition further comprises a photoinitiator; when ultraviolet light is selected, the photoinitiator is preferably 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropionacetone.

According to the invention, in the raw material composition, the release speed and dosage of the carried medicine active ingredient can be controlled by adjusting the mole ratio of the phenylboronic acid monomer to the liquid cationic compound, and the release speed and dosage are preferably 1: (1.1-1.3), more preferably, the molar ratio of the positive charge monomer to the negative charge monomer is 1:1.2, and 1:1.2, so that the microneedle patch can meet the requirements of high response performance and sufficient drug release amount, and achieve similar rapid in-vivo glucose response behaviors of islet alpha and beta cells under the condition of approaching physiological states.

In a preferred embodiment, the mass ratio of phenylboronic acid monomer to liquid cationic compound is (1.4-4.5): 1, more preferably 2.2:1;

in the raw material composition, the mass of the insulin and the liquid cationic compound is preferably (0.8 to 6.6): 1, preferably (1.3 to 3.3): 1 or (2.6-6.6): 1.

As known to those skilled in the art, under physiological conditions, the mass ratio of insulin to glucagon is 3:1, so that the two active ingredients of the present invention are calculated on the basis of the contained natural insulin and glucagon, and the above ratio is satisfied. In one embodiment of the invention, the mass ratio of the glucagon analog to the liquid cationic compound in the starting composition is preferably (0.29-2.2): 1, preferably (0.44-1.1): 1 or (0.8-0.2.2): 1, e.g., (0.44-0.56): 1.

In the raw material composition, the mass ratio of the liquid cationic compound to the liquid monomer solvent is (0.04-0.14): 1, for example, 0.09:1.

In the raw material composition, the mass ratio of the crosslinking agent to the liquid monomer solvent is (0.001 to 0.15): 1, for example, 0.01 to 0.015.

In the raw material composition, the mass ratio of the photoinitiator to the liquid monomer solvent is (0.01-0.15): 1, for example, 0.01-0.015.

In a preferred embodiment, the starting composition comprises 100 parts by weight of N-vinylpyrrolidone, (4- ((2-acrylamidoethyl) carbamoyl) -3-fluorophenyl) boric acid 20 parts by weight or 10 parts by weight, 2- (dimethylamino) ethyl acrylate 9 parts by weight or 4.5 parts by weight, insulin 12-30 parts by weight and 4-10 parts by weight of glucagon analog having six arginines at the C-terminus of natural glucagon.

In a preferred embodiment, the feedstock composition further comprises 0.1 to 1.5 parts by weight of ethylene glycol dimethacrylate and/or 1 to 1.5 parts by weight of 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropionate.

In another aspect, the present invention provides a pharmaceutical composition comprising an active ingredient and a drug-carrying polymer, the active ingredient being the active ingredient in the raw material composition according to the present invention, i.e. insulin and glucagon analogues; the drug-carrying polymer is polymerized by a matrix composition in the raw material composition, and the active ingredient is connected or dissociated with the drug-carrying polymer through electrostatic action.

In a preferred embodiment, the polymerization is a cross-linking polymerization, preferably an ultraviolet cross-linking polymerization, for example using a wavelength of 365nm, 10-130 mW/cm ² For 0.1 to 10 minutes) (pre-curing) such as 1 to 5 minutes, and/or the photo-crosslinking polymerization reaction is carried out at 3 to 25 c, for example 4 c.

Preferably, the drug-loaded polymer comprises or is poly (N-vinylpyrrolidone-co-2- (dimethylamino) ethyl acrylate-co- (4- ((2-acrylamidoethyl) carbamoyl) -3-fluorophenyl) boric acid, and may also comprise or be poly (N-vinylpyrrolidone-co-2- (dimethylamino) ethyl acrylate-co-3- (acrylamido) phenylboric acid.

In another aspect, the present invention provides a microneedle carrying the pharmaceutical composition of the present invention.

As known to those skilled in the art, microneedles are typically present in an array, also referred to herein as a microneedle array.

In a preferred embodiment of the present invention, the pharmaceutical composition is cured on the microneedles by a photo-e.g. uv-crosslinking curing method commonly used in the art.

According to the present invention, since the microneedles are loaded with insulin and glucagon/analogues, the biological properties of the different pharmaceutically active ingredients, particularly the proteinaceous molecules, are different; although the invention adopts the conventional in-situ photo-curing process in the field of microneedles, in order to obtain better drug effect, the conditions of the in-situ photo-curing process such as ultraviolet wavelength, intensity, duration and the like need to be further controlled, so that the activity loss of the drugs in the microneedles is avoided to the greatest extent. Accordingly, another aspect of the present invention provides a method for preparing microneedles, which vacuum-fills the raw material composition of the present invention into the microneedles and causes the raw material composition and the microneedles to undergo a crosslinking curing reaction together.

The crosslinking curing reaction is preferably an ultraviolet crosslinking curing reaction, for example, a wavelength of 365nm, 10 to 130mW/cm ² For 0.1 to 10 minutes (pre-curing) such as 1 to 5 minutes.

For example, the photo-crosslinking polymerization reaction is carried out at 3 to 25℃such as 4 ℃.

In another aspect, the present invention provides a microneedle patch (hereinafter referred to as GRD-MN) comprising the microneedle of the present invention, and a microneedle substrate and patch commonly used for the existing insulin microneedle patch.

Generally, the two sides of the microneedle substrate are respectively connected with the patch and the microneedles, and the microneedle substrate is made of an adhesive. The adhesive is preferably a Norland optical adhesive, such as Norland NOA86H ultraviolet light optical UV curable adhesive.

The invention also provides a preparation method of the microneedle patch, which is prepared by an in-situ photo-curing method.

In a preferred embodiment, the preparation method comprises the following steps:

(1) Preparing a microneedle by adopting the preparation method disclosed by the invention;

(2) And in-situ drip-strengthening the base material, and enabling the micro needle and the micro needle substrate, and the micro needle substrate and the patch to generate a crosslinking curing reaction.

Preferably, in the step (2), when the cured substrate is an ultraviolet light cured substrate, the crosslinking curing reaction is achieved by ultraviolet light irradiation.

In a preferred embodiment, the UV curable substrate is an adhesive, such as a Norland optical adhesive, and the conditions for the photo-curing crosslinking reaction are a wavelength of 365nm and an intensity of 50-200mW/cm ² For 7 to 20 minutes, such as 10 to 15 minutes.

Preferably, the photocuring crosslinking reaction is carried out at a temperature of 3-25 ℃, e.g. 4 ℃.

In another aspect, the invention provides a dual closed circuit delivery system for co-carrying insulin and glucagon comprising a feedstock composition of the invention, a pharmaceutical composition of the invention, a microneedle of the invention, or a microneedle patch of the invention.

The microneedle patches of the present invention may be used in a manner conventional in the art, such as by pre-disinfecting the skin surface prior to use.

In one aspect, the invention discloses methods of treating, reducing, inhibiting or preventing hyperglycemia or a disease comprising hyperglycemia as a symptom in a subject, including but not limited to diabetes (type I or type II), and treating, reducing, inhibiting or preventing hypoglycemia in a subject, particularly more damaging hypoglycemia produced during the course of treating hyperglycemia, comprising administering to a subject a microneedle patch or self-regulating insulin and glucagon analog delivery system of any of the foregoing aspects.

Therefore, the invention also provides the application of the raw material composition, the pharmaceutical composition, the microneedle or the microneedle patch in preparing medicines for treating diseases related to abnormal blood sugar.

The disorder associated with dysglycemia is preferably manifested as hyperglycemia or hypoglycemia, such as diabetes and/or hypoglycemia.

The present invention also provides a method of treating a disorder associated with a blood glucose abnormality by administering to a patient in need thereof a therapeutically effective amount of a feedstock composition of the present invention, a pharmaceutical composition of the present invention, a microneedle of the present invention, or a microneedle patch of the present invention. The size of the microneedle patch can be adjusted along with the weight of a patient (or mice and piglets), and the size of the microneedle patch is about 1cm when used on the mice ² 。

The CAS numbers for each of the compounds described in the present invention are shown in Table 1:

TABLE 1

The invention has the positive progress effects that:

the invention designs the common microneedle drug delivery patch into a novel microneedle patch with double response to high and low blood sugar levels for co-delivery of two blood sugar regulating hormone insulin and glucagon analogues, on one hand, accurate and long-time drug delivery can be realized, the accurate implementation of the microneedle patch can detect blood sugar concentrations at different levels, and the release mode and release speed of the two drugs can be regulated, namely, the correct drug with corresponding dosage (the corresponding dosage is accurately regulated) is released at the corresponding blood sugar level, and the correct drug is released at the correct time; on the other hand, the microneedle patch is simple and comfortable to use, simple to prepare and low in cost.

In the embodiment of the invention, the following aspects can be embodied specifically:

insulin in the GRD-MN patch shows high blood sugar rapid release and GCA shows low blood sugar rapid release, so that the GRD-MN realizes high and low glucose accurate responsiveness of two hormone medicines:

the GRD-MN patch has the capability of rapidly responding to the glucose concentration within half an hour to one hour, and simultaneously realizes the release of the correct medicament corresponding to the dosage under the corresponding glucose concentration, specifically, the GCA responds within half an hour to one hour under the condition of low blood sugar, the release quantity restores the blood sugar to the normal value within one hour, the insulin responds within half an hour under the condition of high blood sugar, and the release quantity restores the blood sugar to the normal value within half an hour:

the microneedle patch coated by the two medicaments has the same glucose response release mode as the medicament coated by the medicament alone, and the interaction between the two medicaments has little influence on the result;

insulin activity was preserved for at least three months;

the glucagon analogues and native glucagon have no significant differences in glycemic activity;

the GRD-MN patch has no obvious biotoxicity;

the GRD-MN patch effectively regulates and controls blood sugar of the diabetic mice for 8 hours, and no hypoglycemic event occurs;

The GRD-MN patch effectively regulates and controls blood sugar of the diabetic pig for 22 hours, and no hypoglycemia event occurs;

the GRD-MN patch has good glucose resistance;

the GRD-MN patch is effective in preventing and treating hypoglycemia.

Drawings

In the drawings in the specification of the present invention, reference lines are set at a blood glucose level of 50mg/dL, with the blood glucose level as the ordinate, and are indicated by hatching.

Fig. 1A and 1B show the composition of the inventive hyperglycemic and hypoglycemic dual-responsive insulin and glucagon analog microneedle patches. Fig. 1A shows that the microneedle patch consists of a transparent PU adhesive patch, a microneedle substrate, and a microneedle array. Fig. 1B shows a top view of the microneedle patch (with the transparent PU adhesive patch removed), showing a substrate with a number of pyramid-shaped microneedles attached.

Fig. 2A, 2B and 2C show schematic diagrams of glucose-responsive insulin and glucagon analog delivery systems having a microneedle array patch with a glucose-responsive matrix (phenylboronic acid derivative). Fig. 2A shows a schematic of a process for manufacturing a smart insulin and glucagon analog co-delivery patch (GRD-MN) from a silicone mold using an in situ photopolymerization strategy. Figure 2B shows the characteristics of a GRD-MN: (i) a representative photograph of a GRD-MN patch; (ii) a photograph of the flexibility of the GRD-MN patch; (iii) Representative Scanning Electron Microscope (SEM) images of microneedle arrays. Scale bar, 500 μm; (iv) Fluorescence microscopy images of FITC-labeled insulin (bright) in microneedle patches; (v) Fluorescence microscopy images of rhodamine B labeled glucagon analogs (dimmed) in microneedle patches; (vi) Combined fluorescence microscopy images of FITC-labeled insulin (bright) and rhodamine B-labeled glucagon analog (dim) in microneedle patches, scale bar: 500 μm. FIG. 2C shows the mechanism of glucose triggered release of insulin and glucagon analogs from GRD-MNs.

Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H and 3I show in vitro characteristics of the GRD-MN patch. FIG. 3A shows the mechanical properties of GRD-MNs. Figures 3B and 3C show the release of in vitro accumulated insulin (3B) and glucagon analog (3C) from a glucose responsive polymer matrix at 37 ℃, ph7.4, at different glucose concentrations, respectively (n=3). Figures 3D and 3E show the pulsatile release profiles of insulin (3D) and glucagon analog (3E), alternating between solutions of 50mg/dL and 400mg/dL glucose concentration (pH 7.4, 37 ℃) showing the insulin and glucagon analog release rate as a function of glucose concentration for a GRD-MN patch loaded with insulin and glucagon analog (n=3). Figures 3F and 3G show the glucose responsive release of insulin (3F) and glucagon analog (3G) from GRD-MN patches at the indicated glucose concentration (pH 7.4, 37 ℃) and quantification of the cumulative release (n=3) using fluorescence detection. Fig. 3H shows the hypoglycemic activity (n=5) of insulin extracted from an insulin-only microneedle patch stored at room temperature (25 ℃) in diabetic mice, indicating that insulin stored in the microneedle patch is able to maintain hypoglycemic activity for at least 3 months. Fig. 3I shows the glycemic activity of glucagon analogs in normal mice (n=5) extracted from a microneedle patch that is stored at room temperature (25 ℃) and loaded with only glucagon analogs, indicating that the glucagon analogs stored in the microneedle patch are capable of maintaining the glycemic activity for at least 3 months. In fig. 3B-I, the data are expressed as mean ± standard deviation.

Fig. 4 shows a comparison of glycemic activity of glucagon analogs and native glucagon in healthy mice (n=5). Mice were tested for blood glucose changes after each defined time point with the blood glucose level of the mice at zero point when the two drugs were injected subcutaneously separately. Indicating that there is no significant difference in glycemic activity between glucagon analogs and native glucagon.

FIG. 5 shows the glucose response performance of insulin and GCA in microneedles with varying molar ratios of DMAEA and FPBA. At various glucose concentrations (37 ℃, ph 7.4), the DMAEA and FPBA molar ratios were observed in vitro to be 1:1.2 (fig. 5A) and GCA release (fig. 5B). The DMAEA and FPBA ratio of insulin released by in vitro accumulation was 1:1.1 (fig. 5C) and 1:1.3 (FIG. 5D). Fig. 5A shows that insulin is released 2-4 times more at hyperglycemia than at hypoglycemia and glucagon of fig. 5B is released 4-8 times more at hyperglycemia, all with a significant difference in half an hour to one hour. The data result shows that the molar ratio of the positive charge monomer to the negative charge monomer of 1:1.2 can meet the requirements of optimal high response performance and sufficient drug release amount of the microneedle patch, and the GRD-MN achieves the rapid in-vivo glucose response behavior similar to pharmacokinetics of islet alpha and beta cells. In addition, figures 5E and 5F show drug release rates exhibited by equal proportions decreasing by a factor of two (figure 5E) or increasing by a factor of two (figure 5F) the dosing quality of DMAEA and FPBA at an equal 1:1.2 molar ratio. The results indicate that too high a dosing ratio or too low a dosing ratio may result in too slow or too fast a drug release rate. The results show that changing the dosing ratio can affect the drug release rate to achieve a more rapid or gradual hypoglycemic goal to address the hypoglycemic demands of patients of varying degrees. Data are expressed as mean ± standard deviation (n=3).

Fig. 6 shows cytotoxicity experiments of the microneedle patches just prepared. The cytotoxicity of GRD-MN and control PBS buffer was tested using CCK-8 kit, and the result demonstrated that the GRD-MN patch was not significantly biotoxic (n=4).

Fig. 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H, 7I, 7J and 7K show in vivo evaluation of GRD-MN patches in STZ-induced diabetic mouse models. FIG. 7A shows the treatment of the back skin (left) of a mouse with a microneedle patch; trace on the skin of the mice after uncovering the microneedle patch (right). Figures 7B and 7C show the blood glucose levels of STZ-induced diabetic mice (figure 7B) and plasma human insulin and glucagon analog concentrations (figure 7C) (n=5) after treatment with PBS solution, insulin solution (insulin dose: 0.05 mg), glucose-responsive insulin patch (hereinafter, GRI-MN) or GRD-MN patch (insulin dose: 0.729mg, glucagon analog dose: 0.243 mg), comparing the plasma insulin concentrations of both insulin delivery modes. Fig. 7D shows a comparison of in vivo intraperitoneal glucose tolerance test 3 hours after administration of GRD-MN or subcutaneous insulin in diabetic mice with healthy control mice (n=5), glucose dose: 1.5g/kg. Figure 7E shows the response calculated based on the area under the curve over 120 minutes, with the baseline set to 0 minutes of plasma glucose reading. FIG. 7F shows that intraperitoneal glucose challenge promotes glucose responsive insulin release in vivo 3 hours after administration of GRD-MN in diabetic mice, glucose dose: 3g/kg. Fig. 7G shows release of an in vivo hypoglycaemic responsive glucagon analog, subcutaneous insulin challenge 2 hours after administration of GRD-MN, resulting in an insulin-induced increase in blood glucose levels in hypoglycemic mice (n=5), insulin dose: 500. Mu.g/kg. Figures 7H and 7I show blood glucose levels (7H) and low glycemic index (7I) (n=5) fasted by diabetic mice after 6 hours of treatment with a gre-MN patch (insulin dose: 0.729 mg) and a GRD-MN patch (insulin dose: 0.729mg, GCA dose: 0.243 mg). FIGS. 7J and 7K show that diabetic mice (n=5) subcutaneously injected blood glucose levels (7J, left) and low glycemic index (7K) of 50 μg/kg insulin 2 hours (indicated by arrows) after administration of GRI-MN patch (insulin dose: 0.729 mg) and GRD-MN patch (insulin dose: 0.729mg, glucagon analog dose: 0.243 mg), and a partial enlarged view of blood glucose for 0.5-4.5 hours is presented on the right of FIG. 7J; the severe hypoglycemic region (< 50 mg/dL) of the mice is shown in the shaded area, and the black dashed line represents the upper limit of normal blood glucose levels (200 mg/dL) of the mice. In fig. 7B-K, the data are expressed as mean ± standard deviation. The statistical significance was determined by a two-tailed student t-test. * P <0.05, < P <0.01, < P <0.001, and < P <0.0001.

FIGS. 8A, 8B, 8C, 8D and 8E show in vivo evaluation of GRD-MN patches in STZ-induced diabetic mini-pig models. FIG. 8A shows a schematic representation of mini-pig leg treatment with GRD-MN and monitoring with CGMS (upper panel); a photograph of GRD-MN was attached to the leg of the mini pig (bottom panel). Fig. 8B shows normal blood glucose excursions in normal diabetic type I piglets. Black arrows indicate the time points of feeding. FIG. 8C shows the blood glucose excursion of a diabetic type I piglet after treatment with an appropriate amount of insulin by subcutaneous injection (insulin dose: 0.12 IU/kg). The light arrows and the black arrows indicate the time points of insulin subcutaneous injection and feeding, respectively. FIG. 8D shows blood glucose excursions of a diabetic type I piglet following GRD-MN patch treatment (insulin dose: 14.4 mg, glucagon analog dose: 4.8 mg). The light arrows and the black arrows indicate the time points of administration and feeding, respectively, of the GRD-MN patch. Figure 8E shows a pooled graph of STZ-induced blood glucose levels of diabetic mini-pigs (n=3) and normal blood glucose of diabetic type I piglets as controls after treatment with GRD-MN (insulin dose: 14.4 mg, glucagon analogue dose: 4.8 mg) and subcutaneous insulin (insulin dose: 0.12 IU/kg). The light up arrow, light down arrow and black arrow represent the time points of microneedle patch administration, insulin subcutaneous injection and feeding, respectively. Figure 8F shows an in vivo oral glucose tolerance test of diabetic piglets (n=3) 3 hours after administration of GRD-MN or subcutaneous insulin at 0.12 IU/kg. Glucose dose: 1g/kg. Figure 8G shows that the responsiveness of diabetic minipigs (n=3) was calculated from the area under the curve of 0-150 minutes, with a baseline set to 0 minutes of plasma glucose readings. OGTT, oral glucose tolerance test. FIGS. 8F-G illustrate that GRD-MN patches have good glucose tolerance. Figure 8H shows the in vivo efficacy of GRD-MN compared to glucose-responsive insulin patches, increasing blood glucose levels in insulin-induced hypoglycemic piglets (n=3) by the microneedle patch challenge given within 2 hours after insulin injection. Insulin dosage: 0.8IU/kg. Fig. 8I shows the hypoglycemic time of diabetic piglets in the hypoglycemic challenge. The results demonstrate that the hypoglycemic piglets treated by the GRD-MN patch can return to normal blood sugar in about half an hour, and the duration of the hypoglycemic treatment is far smaller than that of the GRI-MN patch containing only insulin. In fig. 8H-I, the data are expressed as mean ± standard deviation. The statistical significance was determined by a two-tailed student t-test. * P < 0.05, P < 0.01, P < 0.001, and P < 0.0001.

FIGS. 9A, 9B and 9C show the response release pattern of glucagon and its analog drugs under various modifications carried in GRD-MN patches. The results show that unmodified native glucagon (fig. 9A) did not show significantly different release kinetics of high/low glycemia in the GRD-MN patch; the 2R-glucagon modified by two arginines at the C end (shown in figure 9B) shows a certain blood sugar response difference, and accords with the ideal drug release behavior of the GRD-MN patch; while six arginine-modified 6R-glucagon (fig. 9C), GCA, had a distinct difference in release of the high/low glycemic response with ideal drug release kinetics regulated by electrostatic interactions.

FIG. 10 is an in vivo evaluation of GRD-MN patches in STZ-induced diabetic mouse models. The diabetic mice were pre-administered with excess insulin for 2 hours to induce a hypoglycemic mouse model, followed by transdermal administration of a glucose responsive insulin patch and a GRD-MN patch, respectively, to the hypoglycemic mice, which showed that the GRD-MN patch enabled the mice to break away from hypoglycemia within half an hour and return to the normoglycemic range within one hour, greatly reducing the duration of hypoglycemia.

Detailed Description

The subject matter of the present invention will now be described more fully hereinafter with reference to the accompanying examples and drawings, in which representative embodiments are shown. The inventive subject matter, however, may be embodied in different forms. And should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of these embodiments to those skilled in the art.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently described subject matter belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Throughout the specification and claims, a given formula or name shall encompass all active optical and stereoisomers, as well as racemic mixtures of such isomers and mixtures.

Interpretation of the terms

While the following terms are considered well understood by those of ordinary skill in the art, the following definitions are set forth to aid in the interpretation of the subject matter of the present disclosure.

The micro-needle is formed by combining the micro-needles into an array, so that the local delivery micro-needle of the mediated medicine can pierce the skin cuticle, generate reversible micro-channels on the skin, promote the medicine to smoothly reach the capillary vessel of the dermis layer through the micro-channels, and further achieve the effect of local or systemic treatment.

The microneedle is a stimulus-responsive drug delivery system, and the stimulus-responsive drug delivery system utilizes the biological signal (such as glucose concentration of diabetes patients) difference of skin and pathological process to realize on-demand drug administration of small-molecule drugs and biological macromolecule drugs loaded in the polymer microneedle.

As used herein, the term "microneedle" refers to a needle-like structure having at least one region with a dimension of less than about 1000 micrometers (μm). In some embodiments, the term "microneedle" refers to a structure having a size of about 1 micron to about 1,000 microns (e.g., about 1, 5, 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1,000 microns).

As used herein, the term "insulin" refers to insulin from a human or other mammal. In some embodiments, the term "insulin" refers to human insulin. In some embodiments, the term "insulin" refers to recombinant human insulin.

In some embodiments, the insulin may be human insulin, recombinant human insulin, insulin from a non-human animal source (e.g., bovine, porcine), or any other insulin, including insulin derivatives. In some embodiments, the insulin is of the same species as the intended recipient, i.e., human insulin for use in treating humans. Insulin or a biologically active derivative thereof may comprise a mixture of different insulins and/or derivatives thereof. Insulin or a biologically active derivative thereof may include a fast acting insulin, a fast acting insulin analogue and/or a long acting insulin. In some embodiments, the insulin or biologically active derivative thereof is a fast acting or rapid acting insulin. In some embodiments, the insulin or biologically active derivative thereof is recombinant human insulin.

Quick acting insulin begins to act within 1-20 minutes, peaking after about one hour for three to five hours. Quick acting insulin requires about two hours to be fully absorbed into the systemic circulation. Quick acting insulins include conventional recombinant human insulins (e.g., HUMULINM commercially available from Lilly and NOVOLINM commercially available from NovoNordisk). Bovine insulin and porcine insulin differ from human insulin in several amino acids, but are biologically active in humans and are also fast acting insulins.

Quick acting insulins include insulins that have been modified or have had their amino acid positions altered to increase their rate of absorption. Three types of fast acting commercial insulin analogues can be used: insulin lispro (lysine-proline insulin, sold by Eli Lilly as HUMALOGTM), insulin glycyrrhizinate (sold by Sanofi-Aventis as apifratm) and insulin aspart (sold by Novo Nordisk as NOVOLOGMH).

Long acting insulin includes Eli Lilly's humulin u (UltralenteM long acting human insulin (recombinant DNA source) zinc suspension); and insulin glargine (lanntus aventis). Insulin glargine is a recombinant human insulin analogue that can have a duration of up to 24 hours. It differs from human insulin in that it has glycine instead of asparagine at position 21 and two arginines are added at the carboxy terminus of the β chain. Lantus consists of insulin glargine (1001U, 3.6378mg insulin glargine, 30 micrograms zinc, 2.7mg m-cresol, 20mg glycerol 85% and to 1mL water) dissolved in a clear aqueous fluid.

As used herein, a glucagon analog refers to a glucagon in which one or more amino acid residues of the native glucagon are replaced or deleted by another amino acid residue, wherein the chain in the native glucagon is extended by the addition of one or more amino acid residues at the C-terminus, and/or wherein the native insulin is modified by the addition of one or more chemical substituents. Natural glucagon can functionally replace natural insulin and retain the biological activity of natural glucagon. Glucagon analogs may have a different pharmacokinetics than the pharmacokinetics of the endogenous peptide or protein. The dosage may be optimized based on the pharmacokinetics of the glucagon analog relative to native glucagon or the pharmacokinetics of native glucagon based on pharmacokinetics known to those of skill in the art.

As used herein, "monomer" refers to a molecule that can undergo polymerization, thereby contributing structural units, i.e., atoms or groups of atoms, to the basic structure of the macromolecule.

The terms "polymer" and "polymeric" refer to chemical structures having repeating building blocks (i.e., multiple copies of a given chemical substructure or "monomeric unit"). As used herein, a polymer may refer to a group having more than 10 repeat units and/or a group in which the repeat units are not methylene. The polymer may be formed from a polymerizable monomer. Polymerizable monomers are monomers that contain one or more reactive moieties (e.g., siloxy ethers, hydroxyl groups, amines, vinyl groups (i.e., carbon-carbon double bonds), halides (i.e., cl, br, F and I), carboxylic acids, esters, activated esters, etc.) that can react with other molecules to form bonds. Typically, each polymerizable monomer molecule can be bonded to two or more other molecules. In some cases, the polymerizable monomer bonds only with another molecule, forming the end of the polymeric material. Some polymers contain biodegradable linkages, such as esters or amides, such that they can degrade over time under biological conditions (e.g., at a particular pH present in the body or in the presence of an enzyme).

"copolymer" refers to a polymer derived from more than one monomer. Each monomer will provide a different type of monomer unit.

The term "crosslinker" refers to a compound that comprises at least two reactive functional groups (or groups that can be deblocked or deprotected to provide reactive functional groups) that can be the same or different. In some embodiments, the two reactive functional groups may be chemically different (e.g., two reactive functional groups are reactive (e.g., form a bond, such as a covalent bond) with different types of functional groups on other molecules or both), or one of the two reactive groups may tend to react faster than the other reactive functional group with a particular functional group on another molecule.

As used herein, the term "hyperglycemia" or "hyperglycemia" may refer to a condition in which a subject has an elevated amount of circulating glucose in the plasma relative to a healthy individual. Hyperglycemia may be diagnosed using methods known in the art, including measuring fasting blood glucose levels.

As used herein, the term "hypoglycemia" or "hypoglycemia" may refer to a condition in which the amount of circulating glucose in a subject's plasma is reduced. The decrease in glucose level predictive of hypoglycemia may vary depending on the age and health of the subject. For diabetic adults, a blood glucose level of 70mg/dL or less may be referred to as hypoglycemia. For non-diabetic adults, a blood glucose level of 50mg/dL or less may be referred to as hypoglycemia. The hypoglycemia may be diagnosed using methods known in the art, including using commercially available fingertip blood glucose monitors, continuous blood glucose monitors, venous blood glucose level measurements, and the like. Symptoms of hypoglycemia include, but are not limited to, tremors, blurred vision, sweating, pale complexion, changes in personality, headache, weakness, hunger, somnolence, nausea, dizziness, inattention, arrhythmia, confusion, seizures, and coma.

In some embodiments, the hypoglycemia may be associated with an elevated level of circulating insulin in the blood, i.e., hyperinsulinemic hypoglycemia. In some embodiments, hyperinsulinemic hypoglycemia may be the result of treating type 1 or type 2 diabetes with insulin replacement therapy (e.g., insulin injection) and/or with another diabetes therapeutic agent, such as sulfonylurea or meglitinide. Thus, in some embodiments, hypoglycemia may be caused by an excessive injection of insulin. In some embodiments, the hypoglycemia may be caused by excess endogenous insulin. In some embodiments, hyperinsulinemic hypoglycemia can be caused by, for example, congenital hyperinsulinemia, insulinomas (e.g., islet cell adenoma or carcinoma), gastric dumping syndrome, autoimmune insulin syndrome, reactive hypoglycemia, or non-insulinomatous pancreatic-derived hypoglycemia. In some embodiments, the use of certain drugs, such as, but not limited to, sulfonylureas, meglitinides, aspirin, valeramide, quinine, or propioamide (disoperamide), may result in hypoglycemia.

In some embodiments, the subject matter of the present invention relates to methods for delivering insulin (or a biologically active derivative thereof) or a glucagon analog to a subject in need thereof. It may also be used to deliver other negatively charged proteins and small molecule therapeutics, such as anti-cancer/anti-inflammatory drugs and/or other drugs (such as diabetes therapeutics disclosed herein) to treat diabetes and/or hyperglycemia and/or side effects thereof. In particular, in some embodiments, the compositions of the present disclosure may provide glucose-sensitive, "smart," closed-loop insulin or glucagon delivery to a subject in need thereof, thereby providing more cost-effective and easier diabetes, hypoglycemic control, to improve health, hypoglycemia, quality of life for diabetic patients, and to prevent the hypoglycemic complications of diabetes treatment.

More specifically, the subject matter of the present invention is based on an electrostatically interaction driven complex formed between a negatively charged therapeutic agent, such as insulin (or a biologically active derivative thereof), and a charge switchable polymer comprising a glucose sensing moiety and a positively charged moiety. In the presence of glucose, the glucose sensing moiety can rapidly or transiently bind to glucose and introduce a negative charge into the polymer, thereby reducing the amount of positive charge in the polymer, thereby releasing a negatively charged therapeutic agent, e.g., insulin (or a biologically active derivative thereof), from the complex. By reasonably adjusting the ratio between the positively charged moiety and the glucose sensing moiety of the polymer, the therapeutic agent can be released slowly from the complex at normal blood glucose, and can be released rapidly (e.g., instantaneously) at high blood glucose.

"composition" is intended to include a combination of an active agent and another compound or composition that is inert (e.g., a detectable agent or label) or active, such as an adjuvant.

An "effective amount" is an amount sufficient to produce a beneficial or desired result. The amount of an "effective" agent will vary from subject to subject, depending on the age and general condition of the subject, the particular agent or agents, and many other factors. Therefore, it is not always possible to specify a quantized "effective amount". However, an appropriate "effective amount" in any subject case can be determined by one of ordinary skill in the art using routine experimentation. Furthermore, as used herein, and unless otherwise specifically indicated, an "effective amount" of an agent may also be meant to encompass both a therapeutically effective amount and a prophylactically effective amount. The "effective amount" of the agent required to achieve a therapeutic effect may vary depending on factors such as the age, sex, and weight of the subject. The dosage regimen may be adjusted to provide the optimal therapeutic response. For example, several separate doses may be administered daily, or the dose may be proportionally reduced in accordance with the urgency of the treatment situation.

The following description of the preferred embodiments of the present invention is given with reference to the accompanying drawings, so as to explain the technical scheme of the present invention in detail.

The following is presented in terms of three aspects of structure, method of manufacture, and use of a high-low glycemic dual-responsive insulin and glucagon analog microneedle patch.

Example 1 Structure of the present high Low blood sugar Dual responsiveness microneedle Patch

As shown in fig. 1A, the high-low blood sugar dual-response microneedle patch comprises a soft skin-adhering single-sided adhesive patch 1 made of transparent PU (refer to the patch material of a common transparent adhesive bandage), a microneedle substrate 2 is arranged below the patch 1, the two are tightly attached, and the microneedle substrate 2 has the function of connecting a lower microneedle array 3 with an upper patch. The patch 1 can enable the micro needle to be attached to the skin and fixed on the surface, so that the micro needle is not easy to fall off, and the waterproof property can be applied to water environment. The microneedle substrate 2 has transparent and soft characteristics, and the close combination with the patch 1 is favorable for pressing drug delivery and uncovering drug withdrawal. Underneath the substrate is a microneedle array 3 containing a drug and a polymer, which can contact glucose in interstitial fluid to create a change in the charge of the polymer and thereby control the drug delivery pattern. The ultrathin patch 1 and the microneedle substrate 2 are designed, so that the thickness of the whole device can be reduced, the actual skin fitting is facilitated, and the comfort and the reliability are improved.

As shown in fig. 1B, which is a top view of a dual-responsiveness microneedle patch with high and low blood sugar, the dual-responsiveness microneedle patch mainly comprises a microneedle substrate 2 and a microneedle array 3, wherein the microneedle array 3 is a plurality of arrays, the specification of a single microneedle and the interval between two adjacent microneedles can be manufactured according to practical situations, and the microneedle substrate 2 is used for fixing the position of each microneedle and connecting with the patch so as to facilitate administration and stopping of drugs.

Example 2 method for producing the present high-Low blood sugar Dual-responsive microneedle patch

Fig. 2A shows a schematic of a process for manufacturing a smart insulin and glucagon analog co-delivery patch (GRD-MN) from a silicone mold using an in situ photopolymerization strategy. The silicone mold is prepared by 3D printing in the prior art, and the specification can be self-made. The microneedle size for the subsequent mouse test was 300 μm wide and 900 μm high; the width of the feed is 500 mu m and the height of the feed is 1200 mu m.

The high-low blood sugar dual-response microneedle patch is prepared by an in-situ photo-curing method. As shown in FIG. 2A, a microneedle needle solution was first prepared, 100 parts by weight of N-vinylpyrrolidone (NVP, available from Allatin), 4.5 or 9 parts by weight of 2- (dimethylamino) ethyl acrylate (DMAEA, available from Allatin), 10 to 20 parts by weight of (4- ((2-acrylamidoethyl) carbamoyl) -3-fluorophenyl) boric acid (FPBA, available from ambed) and 0.1 to 1.5 parts by weight of ethylene glycol dimethacrylate (EGDMA, available from Allatin), after ultrasonic dissolution, a photoinitiator (all usual in the art, used herein is 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylbenzophenone (Irgacure 2959)) was added, and then 12 to 30 parts by weight of insulin (recombinant human insulin, available from Beijing Seisaku technology Co., ltd.) and a glucagon analogue (glucose) were added to form a micro-scale preparation of a biochemical composition of 4 to glucose-sensitive invention. Covering the raw material composition with a hollow microneedle mould, and filling microneedles in vacuum; subsequently, the microneedle needle body solution (365 nm, 10-130 mW/cm) placed under low temperature-room temperature conditions (3-25 ℃, optimally 4 ℃) was cured using ultraviolet light ² The method comprises the steps of carrying out a first treatment on the surface of the Pre-curing for 0.1-10 minutes) to form a microneedle solid; next, a microneedle substrate (the substrate is made of Norland optical adhesive, here Norland NOA86H ultraviolet optical UV curable adhesive) was prepared, and an ultraviolet curable substrate (Norland optical adhesive) was applied dropwise in situ and covered with a transparent PU adhesive patch (the adhesive was pushed flat to be uniformly distributed over microperforated holes and the liquid level was made thin as much as possible), followed by curing crosslinking (365 nm, 50-200 mW/cm) under low temperature-room temperature conditions (3-25 ℃, optimal 4 ℃) and ultraviolet light ² The method comprises the steps of carrying out a first treatment on the surface of the 7-20 minutes) to crosslink and solidify the microneedle body with the substrate and the patch; finally, the patch is uncovered, and the high-low blood sugar dual-response microneedle patch is obtained, and the characteristics of the patch are shown in (i) to (iii) of figure 2. The whole manufacturing process is simple, the operability is strong, the patch is thin, the waterproof effect can be realized, and the use is comfortable, convenient and stable.

NVP is a liquid monomer which can be used as a solvent to dissolve three subsequent compounds; DMAEA is a liquid cationic compound that can be introducedThe over-protonated dimethylamino groups establish electrostatic attraction (B ^- -N ⁺ ) To stabilize FPBA, accelerate dissolution of FPBA in the preparation process, and utilize electrostatic interaction of FPBA to participate in regulating release behaviors of two drugs; the EGDMA liquid compound is used as a cross-linking agent to improve the cross-linking and hardness of the polymer grid matrix, and is a liquid compound which is beneficial to the dissolution of solid compounds; the FPBA (solid powder) compound monomer is more electronegative than phenylboronic acid, has a pKa lower than phenylboronic acid, has a more rapid glucose binding capacity in glucose solution, and thus acts as a glucose-sensitive stimulation module in the microneedle matrix, and uses its electrostatic interaction with protein drugs to regulate the release behavior of both drugs.

FIG. 2C shows the mechanism of glucose triggered release of insulin and glucagon analogs from GRD-MNs. Under the hyperglycemic state, negative charge formed by the glucose-boric acid ester compound is increased, so that the electrostatic action between negatively charged insulin and the polymer can be weakened, the volume of the polymer matrix is induced to be increased, and the rapid release of the insulin from the micro needle is promoted; the increased electrostatic interaction between the positively charged glucagon analog and the negatively charged complex may attract the glucagon analog to remain in the microneedle. In hypoglycemia, the negative charge of the complex is reduced, resulting in a smaller volume of the polymer matrix, inhibited insulin release, and a weakening of the electrostatic interaction between the positively charged glucagon analog and the complex, resulting in a rapid release of the glucagon analog. The release of insulin and glucagon analogs is regulated as a function of blood glucose levels.

Example 3 use of the present high Low blood glucose Dual responsive microneedle Patch

As shown in figure 1B, the adhesive plaster is used in a similar way to that of a band-aid, the package is unpacked to uncover the adhesive paper on the adhesive plaster, the skin surface is sterilized in advance, one side of the microneedle is pressed into the skin and fixed, and the microneedle is removed after a set time.

Example 4 in vitro Release and kinetic test experiments of the present high Low blood glucose Dual responsiveness microneedle Patch

In step one, the release of insulin and GCA molecules in GRD-MN patches is performed in a fluorescence labeling assay, wherein insulin is labeled with FITC (excitation wavelength: 490nm, emission wavelength: 520 nm), and GCA is labeled with rhodamine isothiocyanate B (excitation wavelength: 540nm, emission wavelength: 592 nm). Specifically, 150mg of GCA or insulin powder was weighed and dissolved in a 20ml glass bottle using deionized water and appropriate 0.1M hydrochloric acid; 3mg of rhodamine isothiocyanate B or FITC powder is weighed and respectively dissolved in the responsive medicine solutions, and evenly mixed. After stirring overnight at room temperature in the absence of light, dialyzing in pure water for 2 days by using a dialysis bag with a molecular weight cut-off of 1000Da, and freeze-drying to obtain FITC-labeled insulin and rhodamine B-labeled GCA powder.

Step two, GRD-MN patches were prepared as in examples 1 and 2 using the fluorescence-labeled drug powder obtained in step one, as shown in FIGS. 2B (iv) to (vi). Step three, the GRD-MN patch obtained in step two was incubated in PBS solution (pH 7.4, 37 ℃) containing 100mg/dL glucose for 30 minutes. After removal of the samples, incubation was continued for an additional 30 minutes in PBS containing 400mg/dL glucose. This cycle was repeated twice to obtain the pulse release content of the continuous GRD-MN patch bihormonal drug. At a predetermined 30 minute time point, 100. Mu.L of supernatant was collected and the amount of released INS/GCA was quantitatively measured on a 96-well plate using a fluorescence assay, respectively. As shown in fig. 3D and 3E, insulin in the GRD-MN patch showed high blood glucose rapid release and GCA showed low blood glucose rapid release, thereby proving that the GRD-MN achieved high and low glucose accurate responsiveness of both hormonal drugs.

Step four, the GRD-MN patch sample obtained in step two was placed in 1mL of PBS solution (pH 7.4) having different glucose concentrations (0, 50, 100 and 400 mg/dL) and gently shaken (400 rpm) at 37 ℃. At predetermined time points (0.5, 1, 2, 3, 4 h), 100 μl of supernatant was collected into 96-well plates and assayed for released insulin or GCA by fluorescence detection. As shown in fig. 3F and 3G, GRD-MN achieves high and low glucose responsiveness for both hormonal drugs.

FIGS. 3B and 3C represent drug release patterns of microneedle patches containing only insulin or GCA in pure glucose solutions of different concentrations (pH 7.4, 37 ℃) at different times. The results demonstrate that microneedle patches have the ability to rapidly respond to glucose concentrations in half an hour to one hour, and simultaneously achieve the release of the correct drug at the corresponding dose at the corresponding glucose concentration. Fig. 3F/3G is a conclusion that in order to investigate whether the effect between two drugs changes the one in fig. 3B/3C, which proves that the microneedle patch coated with the two drugs together has the same glucose response release pattern as the drug coated with the drug alone, the interaction between the two drugs has little effect on the result.

The mechanical strength of the microneedles was determined by pressing them against a stainless steel plate (fig. 3A). The initial gauge was set to 5.00mm between the microneedle tips and the stainless steel plate, and the capacity of the load cell was 50.00N. The upper stainless steel plate was moved toward the microneedles at a speed of 1.00 mm/min. The breaking force of the microneedle at the beginning of the needle bending, i.e. the force at which the microneedle breaks, was recorded. The results show that each microneedle had a mechanical strength of 0.64n±0.05N, which was sufficient for the microneedle to puncture the skin without damage.

Example 5 Room temperature shelf Life of the drug carried by the present high Low blood glucose Dual responsiveness microneedle Patch

Step one, a glucose-responsive patch containing only insulin or GCA was prepared as in example 1 and stored dry in batches at room temperature.

And step two, soaking the microneedle patch stored in the step one in 1M hydrochloric acid for 4 hours, extracting the solution, and adjusting the pH to about 7.4 by using NaOH. As shown in fig. 3H and 3I, the GCA solution obtained by subcutaneous injection into healthy mice (commercially available mice) was compared to the GC solution newly dissolved by subcutaneous injection, and it was found that GCA in the microneedle patch stored for three months still maintained the glycemic activity similar to GC. Similarly, insulin activity was preserved for at least three months in a solution obtained by subcutaneous injection into diabetic mice, as compared to a solution obtained by subcutaneous injection into the New sharp boundary of insulin.

Example 6 GCA Activity detection of the present high Low blood sugar Dual responsiveness microneedle Patch

Step one, a 1wt% glucagon analog solution and a 1wt% native glucagon solution are prepared. Accurately weighing 50.0mg of glucagon analog (GCA) or natural Glucagon (GC) in a 5mL centrifuge tube, adding 2mL of deionized water, uniformly mixing for 5s by a vortex instrument, dissolving the solid (if insoluble, adding 10 mu L of hydrochloric acid (1M), uniformly mixing, adding a proper amount of NaOH (1M) as soon as possible, adjusting the pH value of a solution system to be about 7.4), fixing the volume to 5.00mL by using deionized water, labeling, and uniformly mixing for later use (storing at 4 ℃).

Step two, in order to investigate whether glucagon with six arginine modified at the C end has the activity of increasing blood sugar, the method of subcutaneously injecting GCA into mice is adopted to compare with the activity of increasing blood sugar of natural glucagon. The activity test experiments were divided into two groups. Healthy mice (n=3) of the control group subcutaneously inject 1wt% of the formulated native glucagon, and blood glucose changes are observed and recorded over one hour; healthy mice (n=3) of the experimental group were subcutaneously injected with an equivalent of formulated glucagon analog at 1wt%, and the change in blood glucose was observed and recorded over one hour. As shown in fig. 4, comparing the two sets of results, it was concluded that GCA and GC have similar glycemic activity, and that GC can be completely replaced in the present microneedle patch for preventing the occurrence of hypoglycemia.

Example 7 experiments on the ratio of Positive to negative Charge monomer of the high Low blood sugar Dual-responsiveness microneedle patch

To evaluate glucose responsive release of insulin or GCA molecules, GRI-MN and GRG-MN patch samples obtained at different positive and negative charge monomer (molar or charge) ratios were placed in 1mL of PBS solution (pH 7.4) containing different glucose concentrations (0, 50, 100 and 400 mg/dL) and gently shaken (400 rpm) at 37℃as described in example 2. At predetermined time points, 10 μl of supernatant was collected into 96-well plates and assayed for released insulin or GCA using the Coomassie (Bradford) protein assay (Thermo Fisher Scientific). Absorbance was measured at 595 nm on an Infinite 200Pro multimode flat-panel reader (Tecan Group) and concentration was calculated using insulin (15-100 μg/mL) or glucagon (8-500 μg/mL) standard curves. As shown in fig. 5A, 5C and 5D, the glucose response releasing ability of insulin was better than 1:1.1 and 1:1.3 at a molar ratio of phenylboronic acid based monomer to liquid cationic monomer of 1:1.2. Meanwhile, as shown in fig. 5B, glucagon also showed superior glucose response release behavior at a molar ratio of 1:1.2 between the monomers. The release rate comparison of insulin at different dosing ratios between phenylboronic acid monomer and liquid cationic monomer is shown in fig. 5A, 5E and 5F. The results showed that the overall release rate of insulin was increased in magnitude with decreasing dosing ratio (30%: 13.5% <20%:9% <10%: 4.5%). A 20%:9% dosing ratio for the moderate release rate was selected as a representative protocol in example 2.

Example 8 cytotoxicity experiment of the present high Low blood sugar Dual responsiveness microneedle Patch

GRD-MN patches were performed in cytotoxicity assays according to the protocol of CCK-8 kit (available from Japan, tonic.). Before the start of the experiment, B16F10 cells (purchased from the national academy of sciences cell bank) were plated in 96-well plates at a density of 5000 cells per well. After the cells had adhered, the cell culture medium was removed from the 96-well plate. Three drug-free microneedles prepared as in example 2 were placed in a transwell dish to full swelling, medium was added to a 96-well plate, incubated in an incubator for 24 hours, the transwell dish was removed, CCK-8 solution was added to each well, and further incubated for 4 hours, and absorbance was read at 450nm using an Infinite 200Pro multi-mode plate reader (Tecan Group). As shown in fig. 6, the microneedles themselves are not significantly toxic to cells and have negligible damage to humans.

Example 9 in vivo release experiments in mice of the present hyperglycemic dual-responsive microneedle patches

The glucose responsive insulin patch (GRI-MN) differs from the GRD-MN patch in that the GRI-MN carries only insulin, and the preparation process and the like of the GRI-MN patch are identical. This group of controls is to demonstrate that the mere inclusion of insulin for the treatment of diabetics, without the constraint of glucagon, an hypoglycemic agent, is insufficient to completely avoid hypoglycemia during the treatment, especially at night.

The diabetic mice were subcutaneously injected with PBS solution, insulin solution, transdermally administered glucose-responsive insulin patch and GRD-MN patch, respectively, and the micropin patch-treated mice were photographed as shown in fig. 7A, and the blood glucose of the mice was continuously monitored using a blood glucose meter. At a predetermined time, 20. Mu.L of mouse plasma was collected by orbital bleeding, and plasma insulin content was measured using ELISA kit, and plasma GCA content was measured using fluorescent labeling method. As shown in fig. 7B and 7C, the GRD-MN patch was effective in regulating blood glucose in diabetic mice for up to 8 hours, and no hypoglycemic events occurred. Both subcutaneous insulin and GRD-MN patch treatments were able to develop adequate plasma insulin concentrations within 1 hour, indicating similar efficacy of GRD-MN and subcutaneous insulin during the first 2 hours. After the peak period of subcutaneous insulin injection (the first 2 hours), little insulin remained in the mice, whereby the mice exhibited a tendency to recover hyperglycemia; the GRD-MN treated mice still keep a certain dose of insulin stably released later for maintaining normal blood sugar, and glucagon is released along with the decrease of blood sugar, so that insulin is restricted and hypoglycemia is avoided. Furthermore, as can be seen from fig. 7B and 7C, in the first half hour in fig. 7C, the in vivo insulin content increases sharply, corresponding to the hyperglycemia level in fig. 7B, whereas in the next half hour in fig. 7C, the insulin content decreases because the blood glucose in fig. 7B returns to the normal range at the first hour, thereby deducing that insulin can be released in response to the release in half hour, and the hypoglycemic function is achieved in the next half hour, indicating that the GRD-MN patch of the present invention can reach hyperglycemia when insulin is responded in half hour, and the released amount returns the blood glucose to the normal value in half hour.

For the intraperitoneal glucose tolerance test (IPGTT) triggered insulin release test, diabetic mice (insulin dose: 0.729mg, GCA dose: 0.243 mg) were treated with GRD-MN patches. Abdominal glucose (0.3 g/mL in PBS) was administered 3 hours after treatment at a dose of 3g/kg or 1.5g/kg to peak an increase in blood glucose levels, which were measured by a blood glucose meter via tail vein blood (-3. Mu.L). For the quantification of plasma insulin, blood (50 μl) was collected over a predetermined time interval, the plasma was centrifuged off and stored at-20 ℃ until measured with ELISA kit. As shown in fig. 7D, 7E and 7F, the GRD-MN patch has good glucose tolerance.

For intra-abdominal insulin resistance test (IPITT) triggered diabetic mice GCA release, insulin (2.5 mg/kg) was administered 2 hours after GRD-MN patch (insulin dose: 0.729mg, GCA dose: 0.243 mg) administration to achieve the purpose of lowering blood glucose. Blood glucose levels were monitored with a blood glucose meter. Blood (50 μl) was collected at selected time points, plasma was isolated, and GCA analysis was performed using fluorescence detection. As shown in FIGS. 7G-K, the GRD-MN patch is effective in preventing and treating hypoglycemia.

Example 10 in vivo Release experiments in piglets of the present hyperglycemic Dual-responsive microneedle patches

The safety and effectiveness of GRD-MN patches were verified on STZ-induced type I diabetic piglets (Shanghai A Stem Biotechnology Co., ltd.). Type I diabetic piglets wear a continuous subcutaneous blood glucose meter (CGMS, freeStyle library, yapeia) for detecting blood glucose changes, and a GRD-MN patch was attached to the inner thigh of the piglets for subsequent experiments (fig. 8A). The daily hyperglycemic state of type I diabetic piglets (FIG. 8B), the glycemic state after subcutaneous injection of insulin solution (FIG. 8C) and the glycemic state of the patch for transdermal administration of GRD-MN (FIG. 8D) were examined, respectively. FIG. 8E shows the combined curves of FIGS. 8B-D, wherein the doses of GRD-MN patch were 14.4mg insulin and 4.8mg GCA. As shown in fig. 8C and 8D, the GRD-MN patch effectively regulated diabetic pig blood glucose for up to 22 hours, and no hypoglycemic events occurred, compared to the risk of hypoglycemia that occurred with subcutaneous insulin injection treatment. All treatment measures occurred before the piglets fed on the same day, two meals a day during each piglet treatment period.

For the Oral Glucose Tolerance Test (OGTT) triggered hypoglycemic test, diabetic piglets were treated with GRD-MN patch (insulin dose: 14.4mg, GCA dose: 4.8 mg). Oral glucose solution was administered 3 hours after treatment at a dose of 1g/kg to peak an increase in blood glucose levels, and the challenge of the abrupt high-glucose diet to the GRD-MN patch was observed, with CGMS measuring blood glucose levels. As shown in fig. 8F and 8G, the GRD-MN patch had a good glucose tolerance compared to the rapid recovery of hyperglycemia by subcutaneous insulin, and the blood glucose was re-lowered to the normal range within three hours.

For the subcutaneous insulin resistance test (SPITT) triggered anti-hypoglycemia capability test, subcutaneous insulin injections (2.5 mg/kg) were administered 2 hours after administration of GRD-MN patch (insulin dose: 14.4mg, GCA dose: 4.8 mg) and GRI-MN patch (insulin dose: 14.4 mg) to achieve the objective of inducing hypoglycemia in piglets. Blood glucose levels were monitored with CGMS. As shown in fig. 8H and 8I, the GRD-MN patch was effective in preventing and treating hypoglycemia, while the GRD-MN patch greatly reduced the duration of hypoglycemia, returning to normal blood glucose in half an hour to one hour, compared to the microneedle patch containing only insulin.

Example 11 glucagon analog screening experiments carried by the present high-low blood glucose dual-responsive microneedle patch

To preserve the glycemic activity of glucagon and to increase its isoelectric point to enhance electrostatic interaction with microneedles, glucagon analogs (commercially available products) with various modifications at the C-terminus of glucagon were screened as pharmaceuticals meeting the requirements of the present invention. Microneedle patches containing only glucagon and analogs thereof were prepared as described in example 2. The resulting patch samples were placed in 1mL of PBS solution (pH 7.4) with different glucose concentrations (0, 50, 100 and 400 mg/dL) and gently shaken (400 rpm) at 37 ℃. At predetermined time points (0.5, 1, 2, 3, 4 h), 10 μl of supernatant was collected into 96-well plates and the released protein concentration was measured using the Coomassie (Bradford) protein assay (Thermo Fisher Scientific). Absorbance was measured at 595 nm on an Infinite 200Pro multi-mode flat-panel reader (Tecan Group) and concentrations were calculated using corresponding glucagon and analogs (8-500 μg/mL) standard curves. Figures 9A, 9B and 9C show the responsive release of unmodified native glucagon, two arginine-modified glucagon and six arginine-modified glucagon, respectively. The results show that except for unmodified native glucagon which did not exhibit responsive release, glucagon with an elevated isoelectric point all exhibited some responsive release behavior, with six arginine modified glucagon exhibiting the best responsive release.

Example 12 in vivo response Rate and Release experiments of two drugs carried by the Dual-responsive high Low blood glucose microneedle patch

To determine whether the blood glucose response efficiency and release amount of insulin/GCA in vivo are sufficient to treat hyperglycemia/hypoglycemia, the present hyperglycemic dual response microneedle patch was applied to both a hyperglycemic mouse model and a hypoglycemic mouse model. Insulin patches and GRD-MN patches were prepared as described in example 2. Diabetes mice were pre-administered insulin for 2 hours to induce a hypoglycemic mouse model, followed by transdermal administration of a glucose responsive insulin patch and a GRD-MN patch, respectively, to the hypoglycemic mice, microneedle patch-treated mice photographs as shown in fig. 7A, with continuous monitoring of the mice blood glucose using a blood glucose detector. As shown in FIG. 10, the GRD-MN patch was able to break away from hypoglycemia within half an hour and return to the normal glycemic range within an hour, while the GRD-MN patch greatly reduced the duration of hypoglycemia, compared to the microneedle patch containing only insulin. In addition, figure 7G further illustrates that when diabetic mice reached hypoglycemia, the plasma GCA levels reached a maximum and returned to normal blood glucose within one hour.

Claims (14)

1. A feedstock composition of glucose-responsive microneedles comprising an active ingredient and a matrix composition forming a drug-carrying polymer, characterized in that the active ingredient is insulin and a glucagon analog that is a natural glucagon positively charge modified and retains the activity of the natural glucagon; the matrix composition includes a phenylboronic acid-based monomer having a glucose-responsive group and a liquid cationic compound.

2. The feedstock composition according to claim 1, characterized in that the isoelectric point of the glucagon analogue is increased by at least 2, preferably by 2-7, more preferably by 4-6, such as 5.3, compared to native glucagon.

3. The feedstock composition according to claim 1 or 2, characterized in that the glucagon analogue carries two to ten arginines at the C-terminus of native glucagon, preferably four to seven arginines, more preferably six arginines.

4. A feedstock composition according to any one of claims 1 to 3, wherein the phenylboronic acid monomer is selected from (4- ((2-acrylamidoethyl) carbamoyl) -3-fluorophenyl) boronic acid, 3- (acrylamido) phenylboronic acid and 4- (bromoethyl) phenylboronic acid, and/or the liquid cationic compound is 2- (dimethylamino) ethyl acrylate.

5. The feedstock composition according to any one of claims 1 to 4, wherein the matrix composition further comprises: a liquid monomer solvent, preferably further comprising a cross-linking agent and/or a photoinitiator;

more preferably, the matrix composition satisfies one or more of the following conditions:

the liquid monomer solvent is selected from N-vinyl pyrrolidone, polyvinyl alcohol and methyl acrylate;

The cross-linking agent is ethylene glycol dimethacrylate;

the photoinitiator is 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone.

6. The feedstock composition according to any one of claims 1 to 5, wherein the feedstock composition satisfies one or more of the following conditions:

the molar ratio of the phenylboronic acid monomer to the liquid cationic compound is 1 (1.1-1.3), preferably 1:1.2; preferably, the mass ratio of the phenylboronic acid monomer to the liquid cationic compound is (1.4-4.5): 1, for example, 2.2:1;

the mass ratio of the insulin to the liquid cationic compound is (0.8-6.6): 1, preferably (1.3-3.3): 1 or (2.6-6.6): 1;

the mass ratio of the glucagon analog to the liquid cationic compound is (0.29-2.2): 1, preferably (0.44-1.1): 1 or (0.8-0.2.2): 1;

the mass ratio of the liquid cationic compound to the liquid monomer solvent is (0.04-0.14): 1, preferably 0.09:1 or 0.045:1;

the mass ratio of the cross-linking agent to the liquid monomer solvent is (0.001-0.15) 1, preferably 0.01-0.015;

the mass ratio of the photoinitiator to the liquid monomer solvent is (0.01-0.15): 1, preferably 0.01-0.015;

Preferably, the raw material composition comprises 100 parts by weight of N-vinyl pyrrolidone, 20 parts by weight or 10 parts by weight of (4- ((2-acrylamidoethyl) carbamoyl) -3-fluorophenyl) boric acid, 9 parts by weight or 4.5 parts by weight of 2- (dimethylamino) ethyl acrylate, 12 to 30 parts by weight of insulin and 4 to 10 parts by weight of glucagon analogue with six arginines at the C-terminal of natural glucagon;

more preferably, it further comprises 0.1 to 1.5 parts by weight of ethylene glycol dimethacrylate and/or 1 to 1.5 parts by weight of 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropenone.

7. A pharmaceutical composition comprising an active ingredient and a drug-loaded polymer, wherein the active ingredient is an active ingredient in the raw material composition according to any one of claims 1 to 6, the drug-loaded polymer is polymerized from a matrix composition in the raw material composition according to any one of claims 1 to 6, and the active ingredient is connected or dissociated with the drug-loaded polymer by electrostatic action;

preferably, the drug-loaded polymer is poly (N-vinylpyrrolidone-co-2- (dimethylamino) acrylic acid ethyl ester-co- (4- ((2-acrylamidoethyl) carbamoyl) -3-fluorophenyl) boric acid, and/or the polymerization is a cross-linking polymerization, preferably a photo-cross-linking polymerization, more preferably a wavelength of 365nm, 10-130 mW/cm ² For 0.1 to 10 minutes, and/or the photo-crosslinking polymerization reaction is carried out at 3 to 25 ℃, for example 4 ℃.

8. A microneedle carrying the pharmaceutical composition of claim 7; preferably, the pharmaceutical composition is cured on the microneedles by an ultraviolet crosslinking curing method.

9. A method for producing a microneedle, comprising vacuum-filling a microneedle with the raw material composition according to any one of claims 1 to 6, and subjecting the raw material composition and the microneedle to a crosslinking curing reaction, preferably an ultraviolet crosslinking curing reaction, more preferably a wavelength of 365nm, 10 to 130mW/cm ² For 0.1 to 10 minutes, and/or the uv curing reaction is carried out at 3 to 25 c, for example 4 c.

10. A microneedle patch comprising the microneedle of claim 8, a microneedle substrate, and a patch.

11. The microneedle patch of claim 10, wherein the patch and the microneedles are connected on both sides of the microneedle substrate, and the microneedle substrate is made of an adhesive, preferably a Norland optical adhesive such as Norland NOA86H ultraviolet optical UV curable adhesive.

12. A method of preparing a microneedle patch according to claim 10 or 11, by in situ photocuring, preferably comprising the steps of:

(1) Preparing a microneedle using the preparation method of claim 9;

(2) In-situ drip-strengthening the base material, and enabling the micro needle and the micro needle substrate, and the micro needle substrate and the patch to generate a crosslinking curing reaction;

preferably, when the cured substrate is an ultraviolet light cured substrate, the crosslinking curing reaction is realized by ultraviolet light irradiation;

more preferably, the UV curable substrate is an adhesive such as Norland optical adhesive, and the curing and crosslinking reaction conditions are a wavelength of 365nm, an intensity of 50-200mW/cm ² The ultraviolet irradiation of (2) is preferably carried out at 3 to 25℃for 7 to 20 minutes, for example at 4 ℃.

13. A dual closed circuit delivery system for co-carrying insulin and glucagon comprising the raw material composition of any one of claims 1 to 6, the pharmaceutical composition of claim 7, the microneedle of claim 8 or the microneedle patch of claim 10 or 11.

14. Use of the raw material composition according to any one of claims 1 to 6, the pharmaceutical composition according to claim 7, the microneedle according to claim 8, or the microneedle patch according to claim 10 or 11 for the preparation of a medicament for treating diseases associated with abnormal blood glucose; preferably, the disorder associated with dysglycemia is manifested as a higher or lower glycemia, such as diabetes and/or hypoglycemia.