CN114272511B - Semiglutide soluble microneedle patch and preparation method thereof - Google Patents
Semiglutide soluble microneedle patch and preparation method thereof Download PDFInfo
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- CN114272511B CN114272511B CN202210028787.7A CN202210028787A CN114272511B CN 114272511 B CN114272511 B CN 114272511B CN 202210028787 A CN202210028787 A CN 202210028787A CN 114272511 B CN114272511 B CN 114272511B
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
Abstract
The invention relates to a semaglutin soluble microneedle patch and a preparation method thereof. The soluble microneedle patch comprises a substrate and a needle body on the substrate; the needle body comprises the following components: high molecular polymer skeleton material, semaglutin and penetration promoter; the substrate comprises a high molecular polymer framework material; the penetration enhancer is at least one of amino acids. The microneedle patch is used for transdermal administration of the semaglutin, obviously improves the release and permeation speed of the semaglutin in skin, obviously increases the concentration of a drug entering blood circulation, and improves the bioavailability of the semaglutin in vivo after delivery and administration of the semaglutin by a microneedle.
Description
Technical Field
The invention relates to the field of medicines, in particular to a semaglutin soluble microneedle patch and a preparation method thereof.
Background
Data published by the international diabetes union in 2019 shows that about 4.63 million adults worldwide have diabetes, and it is expected that 2045 years will increase to 7 million. In 2019, diabetics in China have about 1.16 hundred million, and the trend of increasing year by year is estimated to reach 1.43 hundred million in 2030. China has become the most diverse country for diabetics in the world, of which about 95% are type 2 diabetics. The long-term effective control of blood sugar is significant for preventing and reducing diabetic complications. Glucagon-like peptide 1receptor agonists (Glucagon-like peptide 1receptor agonists,GLP-1 RA) can agonize the GLP-1 receptor, stimulate insulin secretion and inhibit Glucagon release.
The semaglutin (original name of Ma Lutai) has 94% homology with natural GLP-1, the amino acid skeleton of the semaglutin is connected with a fatty acid side chain, the half life is prolonged to 165h, and the semaglutin is administrated by subcutaneous injection 1 time per week. Compared with other several agonists, the semaglutin has greater advantages in the aspects of reducing blood sugar, reducing weight, benefiting the cardiovascular system, safety and the like. On day 20, 9 in 2019, the FDA approved 1-day oral semaglutin (Rybelsus) to be marketed, and the formulation adopts 2-hydroxy benzamide as an absorption enhancer, thereby improving bioavailability and simultaneously getting rid of inconvenience, pain and psychological affliction caused by injection. However, the oral administration still has certain defects that the specific polypeptide structure is easy to be degraded by gastric acid after oral administration and has poor stability under normal temperature environment, so that the bioavailability of the oral tablet is lower, the required administration dosage is far higher than that of a subcutaneous injection, adverse reactions of gastrointestinal tracts are easy to occur after oral administration, and symptoms comprise nausea, vomiting, diarrhea, abdominal pain, constipation and the like; and the tablet is orally taken once daily, so that patients can take the tablet easily without taking the tablet, and the curative effect is unstable.
The soluble microneedle technology is used as a novel transdermal delivery technology, can improve infection and pain caused by injection administration, can avoid the first pass effect of the gastrointestinal tract, and can reduce adverse reactions of the gastrointestinal tract. The principle of operation is to increase the permeability of the drug by creating tiny holes in the sebum layer, which is a mode of administration between transdermal patches and subcutaneous injections. However, since the stratum corneum of the skin can obstruct the transdermal absorption of the drug, even if penetrant is added, the small molecular drug can not penetrate the skin to achieve effective drug concentration, and especially for the semaglutin macromolecular drug, the small molecular drug is more easily trapped and retained in the skin epidermis layer and is difficult to enter the blood circulation. If the absorption rate is slow due to permeation, the corresponding therapeutic effect is still not satisfied.
Disclosure of Invention
Based on the above, the invention aims to provide a soluble microneedle patch combining semaglutin and amino acid permeation enhancer and a preparation method thereof, which obviously improve the release and permeation speed of semaglutin in skin, obviously increase the concentration of semaglutin entering blood circulation and improve the bioavailability of polypeptide or protein drugs in vivo after delivery and administration by a microneedle.
In a first aspect of the invention, there is provided a semaglutin soluble microneedle patch comprising a substrate and a needle on the substrate; the substrate is prepared from a high molecular polymer framework material;
the needle body is prepared from the following components in percentage by weight:
the penetration enhancer is at least one of amino acids.
In a second aspect of the present invention, a method for preparing the soluble microneedle patch described above is provided, comprising the steps of:
dissolving the permeation enhancer in a solvent, adding the high molecular framework material, then dissolving, adjusting the pH value to 7.0-9.0, adding the semaglutin, stirring, and uniformly mixing to obtain a needlepoint solution;
then dissolving the high molecular polymer framework material in a solvent to obtain a substrate solution;
and sequentially placing the needle point solution and the substrate solution into a microneedle mould to prepare the soluble microneedle patch.
Compared with the prior art, the invention has the following beneficial effects:
in order to solve the technical problems that the binding force between the semaglutin and subcutaneous tissue is strong after the semaglutin is administrated by a microneedle, so that the medicament is prevented from entering blood circulation and the bioavailability is low, the invention provides the soluble microneedle patch combining the semaglutin and the permeation enhancer of proper amino acids for the first time, and the soluble microneedle patch has good bioavailability.
In the semaglutin soluble microneedle patch of the present invention, a suitable amino acid is adopted as a penetration enhancer for the first time, which is different from the conventional accelerator which acts on the superficial stratum corneum, but is selected to act subcutaneously: after the amino acid in the microneedle is combined with the dometacin, the biological transdermal of the simetacin can be increased, so that the subcutaneous release and permeation speed of the simetacin are obviously improved, the concentration of a drug entering blood circulation is increased, and the bioavailability of the simetacin in a body after being delivered and administrated by the microneedle is improved.
The semaglutin soluble microneedle patch provided by the invention has the advantages of complete and good morphology, uniform needle tip length and regular arrangement. The mechanical strength is high, and the strength required by puncturing the stratum corneum of human skin is satisfied. And after contacting body fluid, the needle body can be rapidly dissolved and separated from the basal layer, so that the needle is convenient to use, short in wearing time and good in patient compliance.
Drawings
Fig. 1 is a morphology diagram of the semaglutinin soluble microneedle patch of example 1 under electron microscopy.
FIG. 2 is a graph showing the in vivo pharmacokinetic results of the variable amounts of the permeation enhancer, semaglutin, soluble microneedle patches of example 1; wherein A represents a permeation enhancer histidine which accounts for 40% of the mass ratio of the semaglutin soluble microneedle patch; b represents a permeation promoter histidine which accounts for 50% of the weight of the semaglutin soluble microneedle patch; c represents a permeation enhancer histidine which accounts for 60% of the mass ratio of the semaglutin soluble microneedle patch.
FIG. 3 is a graph of the in vivo pharmacokinetic results of the semaglutin soluble microneedle patch of example 2; wherein A represents a commercially available injection of semaglutin; b represents a semaglutin soluble microneedle patch without a penetration enhancer; c represents a semaglutin soluble microneedle patch containing arginine permeation enhancer; d represents a semaglutin soluble microneedle patch containing glycine penetration enhancer; e represents a semaglutin soluble microneedle patch containing a lysine permeation enhancer.
FIG. 4 is the results of an in vitro transdermal diffusion test of a semaglutin soluble microneedle patch, wherein A represents a commercially available injection of semaglutin; b represents a semaglutin soluble microneedle patch without a penetration enhancer; c represents a semaglutin soluble microneedle patch containing arginine permeation enhancer; d represents a semaglutin soluble microneedle patch containing glycine penetration enhancer; e represents a semaglutin soluble microneedle patch containing a lysine permeation enhancer.
Detailed Description
The experimental methods of the present invention, in which specific conditions are not specified in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
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 this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having" and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.
In the present invention, the term "plurality" means two or more. "and/or", describes an association relationship of an association object, and indicates that there may be three relationships, for example, a and/or B, and may indicate: a exists alone, A and B exist together, and B exists alone. The character "/" generally indicates that the context-dependent object is an "or" relationship.
The inventor of the invention discovers that when the delivered medicine is polypeptide or protein macromolecular medicine in the long-term research process of the microneedle patch preparation, the medicine release effect of the conventional soluble microneedle patch is obviously deteriorated, and the release speed of the conventional permeation enhancer cannot be obviously improved after the conventional permeation enhancer is added. In order to solve the problem, the inventor of the present invention has conducted long-term research and finally found that the main reason for the unsatisfactory subcutaneous drug release of the microneedle patch of polypeptide or protein drugs (such as semeglutide) is that the subcutaneous drug release effect is poor: (1) The polypeptide or protein drugs usually have a certain charge, are easy to combine with epidermis or subcutaneous tissue, are easy to be trapped in the epidermis or subcutaneous tissue after microneedle administration, and are difficult to diffuse into blood, so that the blood concentration is low; (2) The polypeptide or protein medicine has large molecular structure, amphipathy and easy aggregation and adsorption in subcutaneous fat-soluble environment. On the basis, the invention provides a semaglutin soluble microneedle patch for the first time, amino acid is selected as a permeation promoter, the permeation promoter of amino acid and the charge of polypeptide or protein medicines can be effectively combined to form a neutral charge compound on the premise of not changing the drug loading capacity and the mechanical strength of the microneedles, the interception effect of epidermis or subcutaneous tissue on the polypeptide or protein medicines is effectively relieved, and finally, the invention realizes the quick drug release of the semaglutin medicine microneedle patch by selecting at least one of the amino acid as the permeation promoter of the semaglutin medicine microneedle patch for the first time.
In some embodiments of the invention, a semaglutin soluble microneedle patch is provided comprising a substrate and a needle on the substrate; the substrate is a high molecular polymer framework material;
the material for preparing the needle body can be prepared from the following components in percentage by weight except the solvent:
the penetration enhancer is at least one of amino acids.
The proportions of the four components described above, in some embodiments, add up to 100%, which is the core material from which the needle is made,
in the preparation, the core material is added into the solvent, and other auxiliary materials can be added according to the requirement.
The preparation method of the semaglutin soluble microneedle patch comprises the following steps:
dissolving the permeation enhancer in a solvent, adding the high molecular framework material, then dissolving, adjusting the pH value to 7.0-9.0, adding the semaglutin, stirring, and uniformly mixing to obtain a needlepoint solution;
dissolving the high molecular polymer framework material in a solvent to obtain a substrate solution;
and then placing the needle tip solution and the substrate solution in a microneedle mould in sequence, and preparing the soluble microneedle patch according to the prior art mastered by the person skilled in the art.
In some embodiments, the needle body of the semaglutin microneedle patch is prepared from the following components in percentage by weight:
in some embodiments, the amino acid is at least one of glutamic acid, glycine, lysine, histidine, aspartic acid, arginine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, selenocysteine, and pyrrolysine.
In some embodiments, the amino acid is at least one of lysine, gan Ji acid, arginine, histidine. More preferably, the amino acid is Gan Ji acid.
In some of these embodiments, the semaglutin in the microneedle patch is present in the microneedle in solid form. The biological macromolecular medicament is stored in a solid state, so that the activity of the medicament is effectively protected, and meanwhile, the harsh cold chain storage and transportation of injection are not required.
In some embodiments, the high molecular polymer matrix material in the needle or the substrate is each independently selected from at least one of polyvinyl alcohol, dextran, hyaluronic acid, mannitol, chitosan, gelatin, silk fibroin, carboxymethyl cellulose, polyvinylpyrrolidone, and vinyl propionate copolymer.
In some embodiments, the stabilizer is at least one of trehalose, sucrose, glucose, decanoyl sucrose, and mannitol.
In some preferred embodiments, the compositions are further optimized for suitable amino acids to improve the release and penetration rate of semaglutin in skin, resulting in better results.
In some embodiments, the soluble microneedle patch is prepared from the following components in weight percent:
in some embodiments, the needle is prepared from the following components in percentage by weight:
semaglutin: 7-15% of a high molecular framework material: 35-40%, arginine: 35-40%, mannitol: 5-10%;
still more preferably: semaglutin: 10-15% of a high molecular framework material: 35-40%, arginine: 35-40%, mannitol: 5-8%.
In some embodiments, the needle is prepared from the following components in percentage by weight: semaglutin: 10-12% of a high molecular framework material: 40-45%, lysine: 40-45%, sucrose: 5-10%; more preferably: semaglutin: 10-12% of a high molecular framework material: 42-45%, lysine: 40-43%, sucrose: 5-8%.
In some embodiments, the needle is prepared from the following components in percentage by weight: semaglutin: 15-18.6% of high molecular framework material: 30-40%, glycine: 45-50%, trehalose: 3-7%.
In some preferred embodiments, the needle is prepared from the following components in percentage by weight: semaglutin: 16.5-17.5%, high molecular framework material: 33-37.5%, glycine: 45-48%, trehalose: 3-5%.
In some embodiments of the aforementioned semaglutin microneedle patch, the high molecular polymer matrix material in the needle body is a vinyl propionate copolymer.
In some of these embodiments, the polymeric matrix material in the needle is dextran, preferably the dextran has a molecular weight of (30-60) kDa, further (30-50) kDa, further (40+ -5) kDa.
In some of these embodiments, the polymeric backbone material in the substrate is polyvinylpyrrolidone, preferably polyvinylpyrrolidone-K90.
In some of these embodiments, the solvent from which the needle tip solution is prepared is at least one of deionized water, acetic acid solution, phosphoric acid solution.
In some of these embodiments, the concentration of the high molecular polymer matrix material in the tip solution is 0.07 to 0.50g/mL, more preferably 0.07 to 0.2g/mL, and still more preferably 0.07 to 0.15g/mL.
In some of these embodiments, the solvent from which the base solution is prepared is absolute ethanol.
In some of these embodiments, the concentration of the high molecular polymer backbone material in the base solution is 0.2 to 0.4g/mL.
The raw materials used in the embodiment of the invention are as follows:
the purity of the semaglutin is more than 98%, and the semaglutin is a commercial product and is purchased from Zhejiang surge peptide biotechnology Co;
PVP/VA (vinyl propionate copolymer): purchased from Shanghai basf technologies Co., ltd;
histidine: purchased from merck chemical technology (Shanghai);
glycine: purchased from sienna n pharmaceutical excipients limited;
lysine: purchased from san-ri pharmaceutical technologies, inc. Of shanxi;
l-arginine: purchased from Pfanstiehl, inc;
trehalose: purchased from rocuronium medicine technologies, inc;
dextran: molecular weight 40kDa, available from Shanghai Ala Biochemical technologies Co., ltd;
mannitol: purchased from Shenzhen Utility medicine Co., ltd;
sucrose: purchased from the pharmaceutical industry, inc. of Tian le, guangxi.
The present invention will be described in further detail with reference to specific examples.
Example 1
The implementation of the drug of the semaglutin soluble microneedle patch comprises the following steps: the high molecular polymer skeleton material is selected from dextran, the penetration enhancer is histidine, and the stabilizer is selected from mannitol. The high molecular polymer framework material in the substrate is selected from PVP K90, and the concentration in the substrate solution is 0.3g/mL. The mass of the semaglutin, dextran, histidine and mannitol is shown in the following groups (1) to (3).
(1) The weight ratio of the penetration enhancer histidine to the semaglutin (5%), the dextran (50%), the histidine (40%) and the mannitol (5%) is 0.5:5:4:0.5.
(2) The weight ratio of the penetration enhancer histidine to the semaglutin to the dextran, the histidine and the mannitol is 0.5:4:5:0.5.
(3) The weight ratio of the penetration enhancer histidine to the semaglutin to the dextran, the histidine and the mannitol is 0.5:3:6:0.5.
wherein the concentration of the high molecular polymer framework material (dextran) in the needle tip solution is 0.075-0.1g/mL.
The solvent for preparing the substrate solution is absolute ethyl alcohol, and the solvent for preparing the needle point solution is deionized water.
Dissolving the corresponding penetration enhancer in a solvent (ionized water), adding a high molecular framework material, then dissolving, adjusting the pH value to 7.0-9.0, adding the semaglutin, stirring, and uniformly mixing to obtain a plurality of needle tip solutions. Dripping the prepared drug solution into a polydimethylsiloxane mould by using a pipetting gun, and spreading the drug solution over holes of the mould; putting the die in the center of a hanging basket of a centrifugal machine, setting the parameters of the centrifugal machine to be 0-10 ℃, centrifuging for 10min at 4000rpm, turning a rotary cup for 180 degrees, and centrifuging for 10min under the same parameter condition, so that the needle point solution is fully filled in the micro cavity of the female die; then, the residual excessive solution on the upper layer is scraped off and recovered; dissolving a polymer framework material of a substrate in absolute ethyl alcohol (PVP K90, the concentration is 0.30 g/mL), adding the solution into a female mold, and centrifuging for 3min under the same centrifugation parameter condition; after centrifugation, the mixture is placed in a dryer to be dried for 36 hours at room temperature, and the microneedle patch is gently demolded by forceps and taken out, thus obtaining the soluble microneedle.
Example 2
The implementation of the drug of the semaglutin soluble microneedle patch comprises the following steps: the high polymer skeleton material is selected from vinyl propionate copolymer, the permeation promoter is one of arginine, glycine or lysine, and the stabilizer is selected from mannitol, sucrose or trehalose. The high polymer skeleton material in the substrate is selected from PVP K90, the concentration in the substrate solution is 0.3g/mL, the solvent for preparing the substrate solution is absolute ethyl alcohol, and the solvent for preparing the needle point solution is deionized water.
The mass of the semaglutin, the vinyl propionate copolymer, the permeation enhancer, and the mannitol are shown in the following groups (1) to (3).
(1) When the permeation promoter is arginine, the mass ratio is as follows:
semaglutin (15%): macromolecular framework material (40%): arginine (40%): mannitol (5%) 1.5:4:4:0.5.
(2) When the penetration enhancer is lysine, the mass ratio is as follows:
semaglutin (10%): macromolecular framework material (45%): lysine (40%): sucrose (5%) was 1:4.5:4:0.5.
(3) When the permeation promoter is glycine, the mass ratio is as follows:
semaglutin (17%): macromolecular framework material (35%): glycine (45%): trehalose (3%) was 1.7:3.5:4.5:0.3.
wherein the high molecular polymer framework material is PVP/VA (vinyl propionate copolymer) and the concentration of the PVP/VA (vinyl propionate copolymer) in the needle point solution is 0.1-0.15g/mL.
The preparation method of the semaglutin soluble microneedle patch provided by the embodiment is as follows:
1. the preparation method of the semaglutin soluble microneedle with arginine as the permeation enhancer comprises the following steps:
(1) Preparing a needle tip solution:
accurately weighing 120mg of arginine, 120mg of PVP/VA and 15mg of mannitol in a 5mL centrifuge tube, adding 1mL of ultrapure water, stirring and dissolving to obtain an auxiliary material solution. A certain amount of 0.25mol/L NaOH solution is added into the auxiliary material solution dropwise, and the pH is regulated to about 7.0. 45mg of semaglutin is precisely weighed and added into the auxiliary material solution for regulating the pH value, and the mixture is stirred and dissolved to obtain the pinpoint solution containing arginine.
(2) Preparing a substrate solution:
10g PVP K90 was weighed into a 50mL centrifuge tube, 30mL absolute ethanol was added, and the mixture was stirred and swelled overnight to give a base solution.
(3) Preparation of soluble microneedles:
sucking 200 mu L of the needle tip solution into a microneedle female die, and centrifuging at 4000rpm for 10min at 0-10 ℃; after the centrifugation is finished, the die is turned over for 180 degrees, and the die is further centrifuged at 4000rpm for 10min at 0-10 ℃. Taking out the mould, scraping residual needlepoint solution on the upper layer, and recycling; adding a certain amount of the substrate solution into a female die filled with pinholes, and centrifuging at 4000rpm for 5min at 0-10 ℃; the mixture was dried in a desiccator for 36 hours, and the mold was carefully peeled off. Obtaining the semaglutin soluble microneedle with arginine as a permeation enhancer.
2. The preparation method of the semaglutin soluble microneedle with lysine as the penetration enhancer comprises the following steps:
(1) Preparing a needle tip solution:
120mg of lysine, 135mg of PVP/VA and 15mg of sucrose are precisely weighed in a 5mL centrifuge tube, 1mL of ultrapure water is added, and stirring and dissolution are carried out, so as to obtain an auxiliary material solution. A certain amount of 0.25mol/L NaOH solution is added into the auxiliary material solution dropwise, and the pH is regulated to about 7.0. Precisely weighing 30mg of semaglutin, adding the semaglutin into the auxiliary material solution with the pH adjusted, and stirring for dissolution to obtain the needle tip solution containing lysine.
(2) Preparing a substrate solution: as above.
(3) Preparation of soluble microneedles: as above.
Obtaining the semaglutin soluble microneedle with lysine as a penetration enhancer.
3. The preparation method of the semaglutin soluble microneedle with glycine as the permeation enhancer comprises the following steps:
(1) Preparing a needle tip solution:
precisely weighing 135mg glycine, 105mg PVP/VA and 9mg trehalose in a 5mL centrifuge tube, adding 1mL ultrapure water, stirring and dissolving to obtain an auxiliary material solution. A certain amount of 0.25mol/L NaOH solution is added into the auxiliary material solution dropwise, and the pH is regulated to about 7.0. Precisely weighing 51mg of semaglutin, adding the semaglutin into the auxiliary material solution with the pH adjusted, and stirring for dissolution to obtain a needle tip solution containing glycine.
(2) Preparing a substrate solution: as above.
(3) Preparation of soluble microneedles: as above.
Obtaining the semaglutin soluble microneedle with glycine as a permeation enhancer.
Test example 1 semaglutin soluble microneedle patch scanning electron microscope experiment
The experimental method comprises the following steps: the prepared soluble microneedle carrying the semeglutide is fixed on a V-shaped copper sheet by using a conductive adhesive tape, after metal spraying, the working voltage is set to be 15kV by using an electron scanning microscope (Scanning Electron Microscope, SEM), the shape of the microneedle patch is observed, and an image is acquired.
Taking the drug-loaded soluble microneedle patch taking the permeation enhancer as glycine in the embodiment 2 as an example, the experimental result is shown in a scanning electron microscope image as shown in fig. 1, and the result shows that the semaglutin soluble microneedle patch is consistent with the design of a main mould, the polymer solution has good fluidity in the preparation process and can be fully filled in micropores of a female mould, the array shape of the microneedles is maintained in the drying process, and the combination of a quadrangular prism and a quadrangular pyramid is uniform in length and regular in appearance form.
Experimental example 2 in vivo pharmacokinetic experiments on the Semiglutide microneedle patch described in example 1
The semaglutin microneedle patch experimental method described in example 1: 9 SD rats of 200-250 g were randomly divided into 3 groups of 3 rats each. The rats were fasted for 12 hours before dosing, excess hair was removed from the rat neck and back area using depilatory cream, and the skin surface was wiped clean of moisture. The 3 microneedles (n=3) prepared dried in example 1 were pressed vertically against the neck and back of the rat for 3min, fixed on the skin using a medical tape, and administration time was recorded. Blood was taken from the retroorbital veins of the rats at the time of dosing, respectively, at about 0.5mL. Placing the blood samples of each group in a centrifuge tube, centrifuging the blood samples in a low-temperature centrifuge at 4 ℃ and rpm for min, collecting supernatant blood samples, detecting the content of each drug in the supernatant, calculating the concentration of the drug, and drawing a pharmacokinetic curve of the drug.
The results of pharmacokinetic experiments with histidine added in different proportions in this test example are shown in FIG. 2. The results show that as the proportion of histidine in the formulation increases, the greater the amount of semaglutin that permeates from the skin into the blood circulation, the better the permeation promoting effect of histidine. Wherein when the amount of histidine is 60%, the area under the curve of the microneedle administration is 1.94 times that of the case where the amount of histidine is 50%, and when the amount of histidine is 60%, the area under the curve of the microneedle administration is 3.78 times that of the case where the amount of histidine is 40%; the amino acid can be combined with the semaglutin to form a neutral charge compound, so that the percutaneous permeation of the semaglutin is promoted, and the greater the dosage of the amino acid, the greater the percutaneous permeation amount of the semaglutin. However, the amount of amino acid used will also have an effect on the mechanical strength of the microneedle, so the preferred histidine amount of the present invention is 60%.
Experimental example 3 in vivo pharmacokinetic experiments on the Semiglutide microneedle patch described in example 2
The experimental method comprises the following steps: 200-250 g SD rats were randomly divided into 5 groups of 3 rats. Removing longer hair on the skin of the back by using a special hair razor for pets, completely covering the back area with the depilating paste for 5min, scraping down on the surface of the skin, cleaning the residual depilating paste, wiping the skin watchband with qualitative filter paper, standing for 10min, and recording the initial weight of the rat for later use. The microneedle patch is fixed at the probe of the tension meter by using double faced adhesive tape and medical adhesive tape, the back skin area of the mouse is fixed on the receiving plate, the parameters of the tension meter are set to be 100N, the speed is 60mm/min for 5min, then the medical adhesive tape is used for fixing, and the microneedle is removed after being kept for 2 h.
And blood is collected by 0.5mL at the orbits of 15min, 30min, 1h, 2h, 4h, 8h, 12h, 24h, 48h and 72h after administration, and the orbits are placed in a centrifuge tube with the inner wall coated with heparin sodium. Centrifuging at 5000rpm for 10min, collecting supernatant blood sample, detecting each drug content in supernatant, calculating drug concentration, and drawing pharmacokinetic curve of drug.
TABLE 3.1 in vivo drug metabolism detection results for Semiglutide soluble microneedle patches
Referring to table 3.1 and fig. 3, the results show that the blood concentration of the soluble microneedle patch without the permeation enhancer group after administration is low, while the soluble microneedle patch breaks the barrier of the skin stratum corneum, the molecular weight of semaglutinin is large, about 4113.58, and the microneedles are easily trapped at the epidermis or are difficult to diffuse into the blood plasma in subcutaneous tissues after transdermal administration, resulting in a low blood concentration and a relative bioavailability of about 14.48%. After glycine, lysine or arginine is added, the blood concentration and the relative bioavailability of the medicine in the body are improved. Wherein, the relative bioavailability of the microneedle group added with lysine after administration is 24.80%; the relative bioavailability of arginine-added microneedle groups following administration was 36.90%; the relative bioavailability of the microneedle group added with glycine after administration is 53.54% as high as possible; the optimal relative bioavailability can be achieved after the combination of the semaglutin microneedle patch and glycine, and the infiltration of the drug into blood is promoted.
Test example 4 external transdermal drug delivery experiment of semaglutinin soluble microneedle patch body
Experimental method of transdermal diffusion experiment: after the rats are anesthetized, long hair on the back is shaved by a special pet shaver, the residual short hair is removed by using the depilatory cream, and the residual depilatory cream is washed clean and killed. The skin on the back is cut off, the subcutaneous fat is carefully scraped off, and the skin is washed and soaked by normal saline, and the skin is preserved at-20 ℃. When in use, the filter paper is soaked in normal saline for thawing, sheared into small pieces, and the filter paper completely absorbs water. The microneedle patch is fixed at the probe of a tension meter by using double faced adhesive tape and medical adhesive tape, the parameters of the tension meter are set to be 100N, the speed is 60mm/min, the tension meter acts on the mouse skin for 5min, and then the tension meter is fixed on a Franz diffusion cell. Wherein the supply tank is sealed with a sealing film, and the receiving tank is filled with ultrapure water as a receiving liquid. Franz diffusion cells were maintained at 37℃and experiments were performed on a transdermal diffusion device at 100rpm, and 0.5mL of the receiving solution was withdrawn at a specified time (0.5, 1, 2, 4, 8, 12, 24 h) while an equal amount of fresh receiving solution was replenished. And measuring the dosage in the receiving liquid at each time point by high performance liquid chromatography, calculating the release percentage, and preparing a transdermal release curve by taking time as an abscissa and the release percentage as an ordinate.
TABLE 4.1 in vitro transdermal Release test results of Semiglutide soluble microneedle patches
Referring to table 4.1 and fig. 4, the results show that the addition of the permeation enhancer to the formulation improved the release and permeation rate of semaglutinin in the skin to varying degrees, significantly increasing the concentration of the drug into the blood circulation. The original ground injection group has almost no transdermal process, and the subcutaneous medicine injected 8h after administration basically completely penetrates the skin, and the accumulated permeability reaches more than 95%. Whereas the transdermal release rate and release amount of the permeation enhancer-free microneedle (group B) were the lowest in each group, and the cumulative permeation rate for 24 hours was only about 12%. The reason may be that semaglutin has a relatively large molecular weight and has a relatively high retention in the skin. After the penetration enhancer is added, the cumulative penetration rate is significantly increased. The glycine (group D) is added to have the best permeation promotion effect on the semaglutin, and the cumulative permeation rate reaches about 50%. The reason is probably that glycine has small molecular weight and simple structure, is amphoteric amino acid, is easier to combine with the semaglutin to form a neutral charge complex, and promotes the percutaneous permeation of the semaglutin. Secondly, arginine and lysine are added, and the cumulative permeability of the semaglutin is about 36% and 28% respectively after the arginine and the lysine are added, and the two are relatively close. Arginine (group C) and lysine (group E) are both basic amino acids and have similar penetration enhancing effects. In summary, glycine added to the formulation more promotes permeation of semaglutin.
The experiment shows that the semaglutin soluble microneedle patch improves the release and permeation speed of semaglutin in skin and obviously increases the concentration of drugs entering blood circulation.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (14)
1. The semaglutin soluble microneedle patch comprises a substrate and a needle body on the substrate, and is characterized in that the substrate is prepared from a high-molecular polymer framework material;
the needle body is prepared from the following components in percentage by weight:
3-20% of semaglutin;
25-50% of high molecular polymer framework material;
35-65% of penetration enhancer;
3-15% of stabilizer;
the total weight of the semaglutin, the high molecular polymer framework material, the penetration enhancer and the stabilizer is 100 percent;
the penetration enhancer is glycine.
2. The semaglutin soluble microneedle patch of claim 1, wherein the needle is prepared from the following components in percentage by weight:
3.5-18.6% of semaglutin;
25-45% of high molecular polymer framework material;
35-54% of penetration enhancer;
5-10% of stabilizer.
3. The semaglutin soluble microneedle patch of claim 1, wherein the high molecular polymer matrix material in the needle body or the substrate is each independently selected from at least one of polyvinyl alcohol, dextran, hyaluronic acid, mannitol, chitosan, gelatin, silk fibroin, carboxymethyl cellulose, polyvinylpyrrolidone, and vinyl propionate copolymer.
4. The semaglutin soluble microneedle patch of claim 1, wherein the high molecular polymer matrix material in the needle body is a vinyl propionate copolymer; and/or the high molecular polymer framework material in the substrate is polyvinylpyrrolidone.
5. The semaglutin soluble microneedle patch of claim 1, wherein the stabilizer is at least one of trehalose, sucrose, glucose, decanoyl sucrose, and mannitol.
6. The semaglutin soluble microneedle patch of claim 1, wherein the needle is prepared from the following components in percentage by weight:
semaglutin: 15-18.6%
High molecular framework material: 30-40%
Glycine: 45-50%
Trehalose: 3-7%.
7. The semaglutin soluble microneedle patch of claim 6, wherein the high molecular polymer scaffold material is a vinyl propionate copolymer.
8. The semaglutin soluble microneedle patch of claim 7, wherein the needle is prepared from the following components in percentage by weight:
semaglutin: 17 percent,
high molecular framework material: 35%,
glycine: 45 percent,
trehalose: 3%.
9. A method for preparing the semaglutin soluble microneedle patch according to any one of claims 1 to 8, comprising the steps of:
dissolving the permeation enhancer in a solvent, adding the high-molecular polymer framework material, then dissolving, adjusting the pH value to 7.0-9.0, adding the semaglutin, stirring, and uniformly mixing to obtain a needlepoint solution;
dissolving the high molecular polymer framework material in a solvent to obtain a substrate solution;
and sequentially placing the needle point solution and the substrate solution in a microneedle mould to prepare the soluble microneedle patch.
10. The method of claim 9, wherein the solvent for preparing the needle tip solution is at least one of deionized water, acetic acid solution, and phosphoric acid solution.
11. The method of claim 9, wherein the solvent for preparing the base solution is absolute ethanol.
12. The method of claim 9, wherein the concentration of the high molecular polymer matrix material in the tip solution is 0.07-0.50 g/mL.
13. The method of claim 12, wherein the concentration of the high molecular polymer matrix material in the tip solution is 0.07-0.20 g/mL.
14. The method of claim 9, wherein the concentration of the high molecular polymer backbone material in the base solution is 0.2-0.4 g/mL.
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| CN116440058A (en) * | 2022-10-09 | 2023-07-18 | 浙江大学 | Glucagon-carrying microneedle patch |
| KR102553293B1 (en) * | 2022-12-23 | 2023-07-07 | 대원제약주식회사 | Microneedle comprising semaglutide and manufacturing method thereof |
| CN116966274A (en) * | 2023-04-14 | 2023-10-31 | 广州新济生物医药研究院有限公司 | Abaparib soluble microneedle patch and preparation method thereof |
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| JP2004277352A (en) * | 2003-03-17 | 2004-10-07 | Sansho Seiyaku Co Ltd | External preparation for skin |
| US7442682B2 (en) * | 2004-10-19 | 2008-10-28 | Nitto Denko Corporation | Transepithelial delivery of peptides with incretin hormone activities |
| CA2607777A1 (en) * | 2005-04-28 | 2006-11-09 | Japan Science And Technology Agency | Transdermal absorption enhancer |
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| JP5879126B2 (en) * | 2008-10-07 | 2016-03-08 | ジン・トゥオＪＩＮ Ｔｕｏ | Phase change polymer microneedle |
| US20130323293A1 (en) * | 2011-02-24 | 2013-12-05 | Takuya Umemoto | Glp-1 analogue composition for microneedle devices |
| CN102805896A (en) * | 2011-05-30 | 2012-12-05 | 惠觅宙 | Transdermal administration method, preparation and system |
| CN104080506B (en) * | 2011-11-30 | 2017-03-08 | 3M创新有限公司 | The microneedle devices comprising peptide therapeutics and aminoacid and preparation and use its method |
| WO2014060472A1 (en) * | 2012-10-17 | 2014-04-24 | Novo Nordisk A/S | Fatty acid acylated amino acids for oral peptide delivery |
| CN103705494A (en) * | 2013-12-26 | 2014-04-09 | 湖北医药学院 | Insulin nano transdermal patch and preparation method thereof |
| WO2017060500A1 (en) * | 2015-10-07 | 2017-04-13 | Cyprumed Gmbh | Pharmaceutical formulations for the oral delivery of peptide drugs |
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