CN115212197A - Pharmaceutical preparation containing engelizin and preparation method thereof - Google Patents

Pharmaceutical preparation containing engelizin and preparation method thereof Download PDF

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Publication number
CN115212197A
CN115212197A CN202110712920.6A CN202110712920A CN115212197A CN 115212197 A CN115212197 A CN 115212197A CN 202110712920 A CN202110712920 A CN 202110712920A CN 115212197 A CN115212197 A CN 115212197A
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China
Prior art keywords
preparation
emp
empagliflozin
pharmaceutical
water
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CN202110712920.6A
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Chinese (zh)
Inventor
魏彦君
李琦琦
刘希望
邢艳平
徐青景
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Shandong Weizhi Zhongke Pharmaceutical Co ltd
Weizhi Pharmaceutical Co ltd
Shandong Viwit Baike Pharmaceutical Co ltd
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Shandong Weizhi Zhongke Pharmaceutical Co ltd
Weizhi Pharmaceutical Co ltd
Shandong Viwit Baike Pharmaceutical Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/10—Antioedematous agents; Diuretics

Abstract

The invention discloses a pharmaceutical preparation containing empagliflozin and a preparation method thereof. The medicinal preparation has good curative effect on treating and/or preventing pancreatitis and other inflammations and/or complications thereof. In vivo test results show that the empagliflozin has a good treatment effect on inflammation (or pancreatic diseases) such as pancreatitis and/or complications thereof, can improve the levels of proinflammatory cytokines such as interleukin-1 beta, interleukin-6, nuclear factor-kappa B (NF-kappa B), transforming growth factor beta (TGF-beta), intercellular adhesion molecule 1 (ICAM-1), tumor necrosis factor alpha (TNF-alpha) and the like, enables the levels to approach normal levels, relieves symptoms such as tissue edema, inflammatory cell infiltration and cell necrosis, reduces the death rate caused by the diseases, and prolongs the survival life of the diseases; meanwhile, the effect of synergy of the empagliflozin and the stevioside is further shown.

Description

Pharmaceutical preparation containing engelizin and preparation method thereof
Technical Field
The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a pharmaceutical preparation containing empagliflozin and a preparation method thereof.
Background
Pancreatic disease, which is a generic term for all diseases of the pancreas, includes inflammatory diseases of the pancreas, damaged diseases of the pancreas, cystic lesions of the pancreas, and secretory tumors of the pancreas.
Pancreatitis is an inflammation caused by the pancreatin digestion of the pancreas and its surrounding tissues, and is mainly manifested by edema, hyperemia or hemorrhage, and necrosis of the pancreas. Clinically, symptoms such as abdominal pain, abdominal distension, nausea, vomiting, fever and the like may occur, and the content of amylase in laboratory blood and urine is increased and the like. Generally, the fatality rate of acute pancreatitis is about 10%, and the fatality rate of severe necrotizing pancreatitis is 50% or higher, which seriously threatens the life health of people.
Empagliflozin (Empagliflozin) is white to yellowish powder and is hardly soluble in water at 25 ℃. The product IS developed by the German Boringer-Yiger-Han company and the American Gift company, IS a novel oral hypoglycemic drug, and effectively reduces the blood sugar and improves the Insulin Sensitivity (IS) and the function of islet beta cells by a mechanism independent of insulin secretion and insulin action (see Chinese patent applications CN 111214450A, CN 112618495A, CN 110655511A and the like).
Currently, the formulation of the engelazin marketed product is mainly tablets, with the trade name Jardiance, approved for marketing in the european union and the us, respectively, in 2014. Steviol glycoside, which is readily soluble in water, is a high-sweetness, low-calorie sweetener extracted from the leaves of stevia rebaudiana, which has a sweetness far higher than that of sucrose, and is considered as a safe natural plant extract by the european union and the united states at the same time.
Until now, no relevant literature report on the use of empagliflozin or a complex thereof for treating and/or preventing inflammation such as pancreatitis and/or complications thereof has been searched.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
In view of the problems and/or disadvantages of the prior art, it is an object of the present invention to provide a pharmaceutical preparation comprising empagliflozin and a method for preparing the same. The medicinal preparation has good curative effect on treating and/or preventing inflammation and/or complications thereof such as pancreatitis and the like.
In particular, the method comprises the following steps of,
the invention provides a pharmaceutical preparation, which comprises engagliflozin and a water-soluble carrier.
Further, in the above-mentioned case,
in any of the above technical solutions (pharmaceutical preparations), the water-soluble carrier is steviol glycoside; preferably, the steviol glycoside is a rebaudioside (e.g., rebaudioside A, B, C, D, E, F, etc. disclosed in CN 112512337A); more preferably, the rebaudioside is rebaudioside a.
Further, in the above-mentioned case,
in any of the above technical solutions (pharmaceutical preparations), the weight ratio of the engagliflozin to the water-soluble carrier is 1; preferably, the weight ratio of the empagliflozin to the water-soluble carrier is 1; more preferably, the weight ratio of the empagliflozin to the water-soluble carrier is 1.
Further, in the above-mentioned case,
in any of the above embodiments (pharmaceutical formulations), the encapsulation efficiency of the empagliflozin in the pharmaceutical formulation is at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.); preferably, the encapsidation rate of the empagliflozin is more than or equal to 90 percent.
Further, in the above-mentioned case,
in any of the above technical solutions (pharmaceutical preparations), the pharmaceutical preparation is a pharmaceutical preparation for treating and/or preventing inflammation and/or its complications.
Further, in the above-mentioned case,
in any of the above technical solutions (pharmaceutical preparations), the inflammation is an inflammatory disease of the pancreas; preferably, the pancreatic inflammatory disease is acute pancreatitis or chronic pancreatitis.
In a further aspect of the present invention,
in any of the above technical means (pharmaceutical preparations), the inflammation is an inflammatory disease caused by abnormality of L-arginine.
Further, in the above-mentioned case,
in any of the above embodiments (pharmaceutical preparations), the inflammation is an inflammation associated with a proinflammatory cytokine; the proinflammatory cytokine comprises one or more than two of interleukin-1 beta, interleukin-6, nuclear factor-kB (NF-kB), transforming growth factor beta (TGF-beta), intercellular adhesion molecule 1 (ICAM-1) and tumor necrosis factor alpha (TNF-alpha).
Further, in the above-mentioned case,
in any of the above technical solutions (pharmaceutical preparations), the pharmaceutical preparation is a pharmaceutical preparation for treating and/or preventing pancreatic diseases and/or complications thereof.
Further, in the above-mentioned case,
in any of the above technical solutions (pharmaceutical preparations), the pancreatic disease is an inflammatory disease of the pancreas; preferably, the pancreatic inflammatory disease is acute pancreatitis or chronic pancreatitis.
In a further aspect of the present invention,
in any of the above technical means (pharmaceutical preparations), the pancreatic disease is an inflammatory disease caused by abnormality of L-arginine.
Further, in the above-mentioned case,
in any of the above technical solutions (pharmaceutical preparations), the pharmaceutical preparation further comprises pharmaceutically acceptable adjuvants (for example, stevioside can be used as a carrier, and carboxymethyl cellulose or a salt thereof can be used as a viscosity-increasing agent) and/or auxiliary components.
Further, in the above-mentioned case,
in any of the above technical solutions (pharmaceutical preparations), the pharmaceutical preparation is an oral administration preparation, an injection preparation, a sublingual administration preparation, a buccal administration preparation or a transdermal absorption preparation.
Further, in the above-mentioned case,
in any of the above technical schemes (pharmaceutical preparations), the pharmaceutical preparation is a decoction, a pill, a powder, a paste, a film, a patch, a pellet, an injection, a medicated wine, a granule, an oral liquid, a tablet, an injection, a capsule, a granule, a suppository, a spray, a gel, a syrup, a liniment, a film, a solution, an emulsion, a suspension, an implant, a lotion, a rinse, an enema, a mixture, a sustained release preparation, a controlled release preparation, a delayed release preparation, a targeted preparation or a prodrug preparation.
In a further aspect of the present invention,
in any of the above technical solutions (pharmaceutical preparations), the pharmaceutical preparation consists of engagliflozin and the water-soluble carrier.
In any of the above technical solutions (pharmaceutical preparations), the pharmaceutical preparation contains only engagliflozin and/or steviol glycosides as active ingredients.
In a further aspect of the present invention,
in any of the above technical solutions (pharmaceutical preparations), the amount of the empagliflozin and/or steviol glycoside is therapeutically effective.
In a further aspect of the present invention,
in any of the above technical solutions (pharmaceutical preparations), the administration subject of the engagliflozin and/or steviol glycoside is a mammal, such as a cow, a horse, a sheep, a pig, a cat, a dog, a mouse, a rat, a rabbit, a guinea pig, a monkey, a human.
Further, in the above-mentioned case,
in any of the above technical solutions (pharmaceutical preparations), the pharmaceutical preparation is a nano-micelle preparation comprising engagliflozin and the water-soluble carrier, and preferably is a self-assembled nano-micelle preparation comprising engagliflozin and the water-soluble carrier.
Further, in the above-mentioned case,
in any of the above technical solutions (pharmaceutical preparations), the pharmaceutical preparation is a nano-micelle preparation formed by engagliflozin and the water-soluble carrier, and is preferably a self-assembled nano-micelle preparation formed by engagliflozin and the water-soluble carrier.
Further, in the above-mentioned case,
in any of the above technical solutions (pharmaceutical preparations), the pharmaceutical preparation is a solution, and the solvent thereof is selected from water, PBS buffer solution, or 0.5wt% sodium carboxymethyl cellulose aqueous solution.
In a further aspect of the present invention,
in any of the above technical solutions (pharmaceutical preparations), when the concentration of empagliflozin in the solution is 1.0mg/mL,
the micelle size of the solution is 1-25 nm; preferably 2 to 8nm;
and/or the polydispersity of the solution is less than or equal to 0.7; preferably ≤ 0.2 or 0.1;
and/or the zeta potential of the solution is-15-0 mV; preferably from-5 to 0mV.
Further, in the above-mentioned case,
in any of the above technical solutions (pharmaceutical preparations), the pharmaceutical preparation is a pharmaceutical preparation comprising any of the following solutions:
the first scheme comprises the following steps:
the preparation method comprises the following steps of (1) preparing a stevioside-engagliflozin nano micelle preparation solution, wherein water is used as a solvent, the drug loading ratio is 1;
scheme II:
the preparation method comprises the following steps of (1) taking stevioside-engelizin nano micelle preparation solution and 0.5wt% sodium carboxymethylcellulose aqueous solution as solvents, wherein the traditional Chinese medicine carrying ratio is 1;
the third scheme is as follows:
the stevioside-engeletin nano-micelle preparation solution takes PBS buffer solution as a solvent, the traditional Chinese medicine carrying ratio is 1.
Further, in the above-mentioned case,
in any of the above technical solutions (pharmaceutical preparations), the pharmaceutical preparation is prepared by a method comprising the steps of: dissolving the engeletin and the water-soluble carrier in a solvent together, uniformly mixing, and then removing the solvent to obtain the product;
preferably, the preparation method satisfies one or more of the following conditions:
(1) the solvent is ethanol;
(2) after the solvent is removed, sieving is also included, and the sieving is preferably carried out by a sieve of 80 meshes;
(3) the solvent removal was by evaporation at 40 ℃ under reduced pressure.
In addition to this, the present invention is,
the invention also provides a preparation method of any one of the pharmaceutical preparations, which comprises the following steps: dissolving the engeletin and the water-soluble carrier in a solvent together, uniformly mixing, and then removing the solvent to obtain the product; preferably, the solvent is ethanol.
In any of the above technical solutions (preparation methods), the preparation method satisfies one or more of the following conditions:
(1) the solvent is ethanol;
(2) after the solvent is removed, sieving is also included, and the sieving is preferably carried out by a sieve of 80 meshes;
(3) the solvent removal was by evaporation at 40 ℃ under reduced pressure.
Another object of the present invention is to provide a new use of engeletin: can be used for preparing medicine for treating and/or preventing inflammation and/or its complications such as pancreatitis.
In particular to application of empagliflozin and/or stevioside in preparation of medicines for treating and/or preventing inflammation and/or complications thereof.
Further, in the above-mentioned case,
in any of the above embodiments (uses), the inflammation is an inflammatory disease of the pancreas; preferably, the pancreatic inflammatory disease is acute pancreatitis or chronic pancreatitis.
Further, in the above-mentioned case,
in any of the above technical means (uses), the inflammation is an inflammatory disease caused by abnormality of L-arginine.
The invention also provides: use of engagliflozin and/or stevioside for the preparation of a medicament for the treatment and/or prevention of pancreatic diseases and/or complications thereof.
In a further aspect of the present invention,
in any of the above technical solutions (uses), the pancreatic disease is an inflammatory disease of the pancreas; preferably, the pancreatic inflammatory disease is acute pancreatitis or chronic pancreatitis.
In a further aspect of the present invention,
in any of the above technical means (uses), the pancreatic disease is an inflammatory disease caused by abnormality of L-arginine.
In a further aspect of the present invention,
in any of the above technical solutions (uses), the inflammation is an inflammation associated with a proinflammatory cytokine; the proinflammatory cytokine comprises one or more than two of interleukin-1 beta, interleukin-6, nuclear factor-kB (NF-kB), transforming growth factor beta (TGF-beta), intercellular adhesion molecule 1 (ICAM-1) and tumor necrosis factor alpha (TNF-alpha).
In a further aspect of the present invention,
in any of the above technical solutions (uses), the steviol glycoside is a rebaudioside (e.g. rebaudioside A, B, C, D, E, F, etc. disclosed in CN 112512337A); preferably, the rebaudioside is rebaudioside a.
Further, in the above-mentioned case,
in any of the above technical solutions (uses), the empagliflozin and/or steviol glycoside is in a therapeutically effective amount.
Further, in the above-mentioned case,
in any of the above technical schemes (uses), the medicament only takes the empagliflozin and/or stevioside as active ingredients.
Further, in the above-mentioned case,
in any of the above technical solutions (uses), the medicament consists of engagliflozin and steviol glycosides.
Further, in the above-mentioned case,
in any of the above embodiments (uses), the drug has an envelopment ratio of at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.); preferably, the encapsulation efficiency of the empagliflozin is more than or equal to 90 percent.
Further, in the above-mentioned case,
in any of the above technical solutions (uses), the weight ratio of entecazin to steviol glycoside is 1; preferably, the weight ratio of the empagliflozin to the stevioside is 1; more preferably, the weight ratio of the empagliflozin to the steviol glycoside is 1.
Further, in the above-mentioned case,
in any of the above technical solutions (uses), the drug is a nano-micelle preparation containing engeletin and stevioside, and preferably a self-assembled nano-micelle preparation containing engeletin and stevioside.
In a further aspect of the present invention,
in any of the above technical solutions (uses), the drug is a nano-micelle preparation formed by engagliflozin and stevioside, and is preferably a self-assembled nano-micelle preparation formed by engagliflozin and stevioside.
In a further aspect of the present invention,
in any of the above technical solutions (uses), the medicament further includes a pharmaceutically acceptable adjuvant (for example, stevioside may be used as a carrier, carboxymethyl cellulose or a salt thereof may be used as a viscosity-increasing agent), and/or an auxiliary component.
In a further aspect of the present invention,
in any technical scheme (application), in the medicine, the empagliflozin is a main active ingredient, and/or the stevioside is a carrier.
In any of the above technical solutions (uses), the medicament further comprises a pharmaceutically acceptable adjuvant and/or an auxiliary component, and the pharmaceutically acceptable adjuvant or auxiliary component is preferably carboxymethyl cellulose or a salt thereof.
Further, in the above-mentioned case,
in any of the above technical schemes (uses), the medicament is an oral administration preparation, an injection preparation, a sublingual administration preparation, a buccal administration preparation or a transdermal absorption preparation.
Further, in the above-mentioned case,
in any technical scheme (application), the medicament is decoction, pill, powder, paste, membrane, patch, emplastrum, pellet, injection, medicinal liquor, electuary, oral liquid, tablet, injection, capsule, granule, suppository, spray, gel, syrup, liniment, plastics, solution, emulsion, suspension, implant, lotion, rinse, enema, mixture, sustained-release preparation, controlled-release preparation, delayed-release preparation, targeted preparation or prodrug preparation.
Further, in the above-mentioned case,
in any technical scheme (application), in the medicines, the medicines are engelizin and stevioside, and are prepared by a solvent evaporation method; preferably, the solvent is ethanol;
specifically, the medicine is prepared by the method comprising the following steps: dissolving the engeletin and the water-soluble carrier in a solvent together, uniformly mixing, and then removing the solvent to obtain the product; preferably, the solvent is ethanol.
With respect to definitions of terms used herein, the initial definitions provided for the terms herein apply to the terms throughout, unless otherwise specified; for terms not specifically defined herein, the meanings that would be afforded to them by a person skilled in the art, in light of the disclosure and/or the context, should be given.
The term "steviol glycoside" refers to a glycoside of steviol; including but not limited to: naturally occurring steviol glycosides, for example: <xnotran> , A, , D, , A, B, B, , G, A, B, C, A, E, E2, E4, E6, E3, D, I, AM, D7, M, M4, 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, 1i, 1j, 1k, 1l, 1m, 1n, 1o, 1p, 1q, 1r, 1s, 1t, 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, 2i, 2j, 2k, 2l, 2m, 2n, 2o, 2p, 2q, 2r, 2s; </xnotran> And synthetic steviol glycosides, e.g.: enzymatically glycosylated steviol glycosides and combinations thereof (see: chinese patent application CN 112512337A, applicant: spectrally seideae, U.S. gmbh).
The term "pharmaceutically acceptable" means that the carrier, cargo, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients comprising a pharmaceutical dosage form and physiologically compatible with the recipient.
The mode of administration of the drug of the present invention is not particularly limited, and representative modes of administration include, but are not limited to: oral, parenteral (intravenous, intramuscular, or subcutaneous), and topical administration.
Solid dosage forms for oral administration include capsules, tablets, pills, powders, granules and the like. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), or with the following ingredients: (ii) (a) a filler or compatibilizer; (b) tackifiers such as: carboxymethyl cellulose and salts thereof; (c) a humectant; (d) a disintegrant; (e) a slow solvent; (f) an absorption accelerator; (g) a wetting agent; (h) an adsorbent; and (i) lubricants, and the like. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.
Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds from such dosage forms may be delayed in release in a certain part of the digestive tract. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration include: a pharmaceutically acceptable emulsion, solution, suspension, syrup, or tincture. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art, such as water or other solvents, as well as solubilizing agents and emulsifiers and the like. In addition to these inert diluents, the medicaments may also comprise auxiliaries, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents. Suspensions, in addition to the active compounds, may contain suspending agents.
Medicaments for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients, and the like.
Pharmaceutical dosage forms for topical administration include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if desired.
The pharmaceutically acceptable auxiliary material of the invention refers to a substance contained in a dosage form except for an active ingredient.
The pharmaceutically acceptable auxiliary components have certain physiological activity, but the addition of the components does not change the dominant position of the medicine in the disease treatment process, but only plays auxiliary effects, and the auxiliary effects are only the utilization of the known activity of the components, and are auxiliary treatment modes which are commonly used in the field of medicine. If the auxiliary components are used together with the medicament of the invention, the auxiliary components still belong to the protection scope of the invention.
The positive progress effects of the invention are as follows: the in vivo test result shows that the pharmaceutical preparation containing the empagliflozin has better treatment effect on inflammation (or pancreatic diseases) such as pancreatitis and the like and/or complications thereof, can improve the levels of proinflammatory cytokines such as interleukin-1 beta, interleukin-6, nuclear factor-kappa B (NF-kappa B), transforming growth factor beta (TGF-beta), intercellular adhesion molecule 1 (ICAM-1), tumor necrosis factor alpha (TNF-alpha) and the like, enables the levels to approach normal levels, relieves the symptoms such as tissue edema, inflammatory cell infiltration and cell necrosis, reduces the death rate caused by the disease, and prolongs the survival life of the disease; meanwhile, the effect of synergy of the empagliflozin and the stevioside is further shown.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
Drawings
FIG. 1 is an appearance diagram of RA-EMP powder of the present invention.
FIG. 2 is an SEM image of EMP (B-1), RA (B-2), RA-EMP (B-4), and a physical blend of RA and EMP (B-3).
FIG. 3 is an IR plot of EMP (I-1), RA (I-2), RA and a physical blend of EMP (I-3), RA-EMP (I-4).
FIG. 4 is an XRD pattern of EMP (X-1), RA (X-2), the physical blend of RA and EMP (X-3), RA-EMP (X-4).
FIG. 5 is a DSC of EMP (D-1), RA (D-2), physical blend of RA and EMP (D-3), RA-EMP (D-4).
FIG. 6 shows the PBS solution of EMP on the left and the aqueous solution of RA-EMP on the right.
FIG. 7 is a TEM image of an RA-EMP aqueous solution.
FIG. 8 is a hydrodynamic size distribution plot of an aqueous RA-EMP solution.
FIG. 9 is a zeta potential diagram of an aqueous RA-EMP solution.
FIG. 10 is a graph of micelle size for different loading ratios of RA-EMP.
FIG. 11 is the polydispersity index for different loading ratios RA-EMP.
FIG. 12 shows zeta potentials for different loading ratios RA-EMP.
FIG. 13 shows the encapsulation efficiency for different drug loading ratios RA-EMP.
FIG. 14 shows micelle sizes for 1-12 weeks of storage of RA-EMP.
FIG. 15 shows zeta potentials of RA-EMP stored for 1-12 weeks.
FIG. 16 is an in vitro release profile of EMP in RA-EMP.
FIG. 17 is a hemolysis observation pattern of RA-EMP.
FIG. 18 shows the hemolysis rate of RA-EMP.
Figure 19 is a graph of EMP concentration versus time in plasma following oral administration of EMP and RA-EMP.
Figure 20 is a graph of EMP concentration in the gastrointestinal segment following oral administration of EMP and RA-EMP.
FIG. 21 is a graph showing EMP concentrations in organ tissues after oral administration of EMP and RA-EMP.
FIG. 22 is a fluorescence visualization of RA-EMP and EMP in the duodenum, jejunum, ileum.
FIG. 23 is a graph showing the fluorescence visualization of RA-EMP and EMP in kidney and pancreas.
Fig. 24 shows survival rates of mice from different experimental groups tested in vivo for acute pancreatitis.
FIG. 25 shows the pancreas of mice tested in vivo for acute pancreatitis in different experimental groups.
FIG. 26 is the ratio of moisture content, pancreas weight/body weight for different experimental groups in vivo experiments for acute pancreatitis.
FIG. 27 is the serum amylase and lipase levels in different experimental groups in vivo for acute pancreatitis.
Fig. 28 shows ELISA results of different experimental groups in vivo experiments on acute pancreatitis.
FIG. 29 shows the results of evaluation of tissue oxidative stress in different experimental groups in vivo for acute pancreatitis.
Fig. 30 is the histopathological examination results of different experimental groups in the acute pancreatitis in vivo experiment.
FIG. 31 is a graph showing the evaluation of apoptosis in different experimental groups in an in vivo acute pancreatitis test.
Detailed Description
While the present invention will be described more fully hereinafter with reference to the accompanying specific embodiments, it is to be understood by those skilled in the art that the following descriptions are provided for purposes of illustration only and are not intended to limit the scope of the present invention.
In the present invention, those who do not specify specific conditions are performed according to conventional conditions or conditions recommended by the manufacturer, and those who do not specify the reagents or instruments used are conventional products commercially available.
For example:
engeletin (Empagliflozin, abbreviated as EMP) and L-arginine, available from shanghai alatin biochem-related technologies, inc;
with Rebaudioside A (RA, CAS: 58543-16-1) with purity of 98% or more, purchased from Jining stevia products GmbH;
male SD rats (. About.200 g) and male BALB/c mice (8 weeks old) were purchased from Jinanpunyue laboratory animal breeders Co., ltd.
Example 1
1 preparation of RA-EMP
RA-EMP was prepared by a solvent evaporation technique using ethanol as a solvent (ref: 1, hou, Y., et al, novel self-stabilizing dispersed base on reagent A: a porous nano platform for an organic delivery of curcumin. Int J Nanomedicine,2019.14, p.557-571 2, wang H, cui Y, fu Q, et al. A phosphor complex to organic bio availability of flavanoids. Drug Dev. 11 (10): 16993-1703.).
Ethanol evaporation method: taking 50mg of Empagliflozin (EMP), mixing the empagliflozin with the Rebaudioside A (RA) in different drug carrier ratios of 1; then, ethanol was completely removed by evaporation under reduced pressure at 40 ℃ using a rotary evaporator to obtain a white powdery product, which was passed through a 80-mesh sieve, i.e., RA-EMP (EMP self-assembled nano-micellization formulation with RA as a carrier), as shown in fig. 1 (drug loading ratio of 1 to 15), which was fine and showed high uniformity and fluidity.
1.1SEM test
The morphology of the RA-EMP powder was evaluated using a scanning electron microscope (SEM, hitachi S-4800, japan). SEM images of EMP, RA and physical blend of EMP (weight ratio of EMP to RA 1.
The results show that the boundaries of the rod-like crystals are somewhat more blurred in the SEM image of RA-EMP (B-4 of FIG. 2) than the physical blend of RA and EMP (B-3 of FIG. 2).
1.2 Infrared testing
The molecular interaction of RA and EMP in RA-EMP (FTIR, nicolet iS10, thermoFisher, madison, wis., USA) was studied by Fourier transform infrared spectroscopy. IR patterns of EMP, RA and physical blends of EMP (weight ratio of EMP to RA 1.
The results show that no new absorption peak (I-4 of FIG. 3) is found in the IR profile of RA-EMP compared to the physical blend of RA and EMP (I-3 of FIG. 3), indicating that no chemical reaction has occurred during the preparation of RA-EMP.
1.3XRD testing
Changes in the crystalline characteristics of EMP in RA-EMP were investigated by X-ray diffraction (XRD, D/max-2400, rigaku, japan). XRD patterns of EMP, RA and physical blend of EMP (weight ratio of EMP to RA 1.
The results showed that characteristic peaks of EMP (X-1 of fig. 4) were present at 2 θ =14.52, 18.68, 20.2, 25.04 degrees, whereas these characteristic peaks of EMP (X-4 of fig. 4) were not present in the spectrum of RA-EMP, preliminarily indicating that EMP in RA-EMP is amorphous.
1.4DSC test
Differential scanning calorimetry (DSC, Q20, TA, USA) was used to analyse the characteristics of EMP in RA-EMP. DSC profiles of the physical blends of EMP, RA and EMP (weight ratio of EMP to RA 1.
The results showed that the DSC curve of EMP has a distinct endothermic peak at 154.03 deg.C (D-1 in FIG. 5), while the DSC curve of RA-EMP has a corresponding endothermic peak of EMP which disappears (D-4 in FIG. 5), and the melting point range is expanded, further indicating that EMP exists in RA-EMP in an amorphous state.
2 self-micellization characteristic of RA-EMP in aqueous solution
2.1 solubility
Solubility of RA-EMP: readily soluble in water, as a pale yellow transparent solution (very pale in color), see fig. 6: the left side is a PBS solution of EMP and the right side is an aqueous solution of RA-EMP (loading ratio 1. The apparent solubility of EMP in RA-EMP (loading ratio 1, 15) was 14.721 + -1.401 mg/mL, while the apparent solubility of EMP was 0.051 + -0.001 mg/mL, and the apparent solubility of EMP in a physical blend of RA and EMP (weight ratio of EMP to RA 1.
2.2 micelle size, polydispersity index and zeta potential
The EMP concentration in the aqueous RA-EMP solution was adjusted to about 1.0mg/mL and the following analytical tests were performed: the micelles were observed microscopically with a transmission electron microscope (TEM, JEM-1200EX, JEOL, tokyo, japan) and the sample was stained with 1.5% phosphotungstic acid before observation, and the results are shown in FIG. 7; the hydrodynamic size distribution (see fig. 8) and zeta potential (see fig. 9) of the micelles were determined using the Dynamic Light Scattering (DLS) method of Zetasizer Nano ZS90 (Malvern Instruments, worcester county, uk).
The micelle size (micell size), polydispersity index (PDI for short) and zeta potential of RA-EMPs obtained at different drug loading ratios (1.
The results show that RA-EMP has an ultra-small micelle size, homogeneity and an almost neutral zeta potential, contributing to an increase in its water solubility.
2.3 encapsulation efficiency
RA-EMP (e.g., a drug loading ratio of 1. The unsupported EMP was separated by filtration through a 0.22 μm filter. The pre-and post-filtration solution of 100. Mu.l RA-EMP was diluted appropriately with acetonitrile to disrupt micelles. The EMP concentration of the RA-EMP aqueous solution is determined by high performance liquid chromatography. The encapsulation efficiency is the ratio of the amount of EMP detected in the RA-EMP aqueous solution after filtration to the amount of EMP detected in the RA-EMP aqueous solution before filtration.
High Performance Liquid Chromatography (HPLC) for determining EMP concentration (samples include micelle solution, in vitro release solution, plasma sample, tissue sample, etc.): agilent 1100 chromatography system using a 250 x 4.6mm column packed with 5 μm reverse phase C18 (agilent ZORBAX SB-C18, usa) at 26 ℃, mobile phase acetonitrile-water (volume ratio =45, 55), flow rate 1.0mL/min, uv detector (detection at 225 nm), retention time of EMP around 5 minutes. The encapsulation efficiency of the resulting RA-EMPs was shown in fig. 13 for different drug loading ratios (1.
The results showed that the encapsulation efficiency of RA-EMP was 45.89% at a drug loading ratio of 1.
Example 2
Storage stability: RA-EMP (loading ratio 1. At predetermined time points, 3 vials were randomly drawn, RA-EMP was dissolved with water, and micelle size (see FIG. 14), zeta potential (see FIG. 15), and encapsulation efficiency were determined.
The result shows that no obvious agglomeration phenomenon appears after the RA-EMP stored for 1-12 weeks is dissolved in water, the micelle size and the zeta potential of the aqueous solution are basically kept stable, and the encapsulation efficiency is more than or equal to 99 percent.
Example 3
EMP in vitro release evaluation: the in vitro release profile of RA-EMP was analyzed by dialysis.
Test solutions:
(1) 1mL of an RA-EMP (drug loading ratio 1;
(2) 1mL of EMP suspension: EMP was dispersed in 0.5% aqueous sodium carboxymethylcellulose at 5mg/mL.
Test solutions were placed in dialysis membrane bags (molecular weight cut-off [ MWCO ]]=3500 Da). Over the first 2 hours, 100mL of simulated gastric fluid (SGF, 0.15M HCl,0.05M KCl, pH =1.2,0.5% (w/v) tween 80) was incubated with shaking at 100rpm at 37 ℃; then transferred to 100mL simulated intestinal fluid (SIF, 8g NaCl, 0.2g KCl, 1.44g Na) 2 HPO 4 、0.24g KH 2 PO 4 1000mL of water, pH =6.8,0.5% (w/v) tween 80) for 22h. Samples of culture broth (SGF or SIF) were collected at predetermined time points (1 mL) and rapidly supplemented with an equal volume of culture broth to keep the volume constant (100 mL). The sample was filtered through a 0.22 μm filter and the amount of EMP released into the culture was measured by HPLC. The in vitro release profile of EMP in RA-EMP was determined by dialysis and is shown in FIG. 16.
The results show that there is a significant difference in the amount of RA-EMP released compared to EMP (P < 0.05): in the first 2 hours, 11.16 + -1.07% of EMP was released from the EMP suspension into SGF, while 22.03 + -1.87% of EMP was released from the RA-EMP aqueous solution; over the next 22 hours, the EMP suspension released 27.91. + -. 0.82% of EMP, while the aqueous RA-EMP solution released 56.28. + -. 7.18% of EMP.
Example 4
Blood compatibility test: freshly collected rat blood was used.
And (3) suspending the red blood cells into physiological saline to obtain red blood cell suspension. 200 μ L of the erythrocyte suspension was added to 800 μ L of the test solution, mixed gently and incubated at 37 ℃ for 4h. After the incubation, 1mL of the test sample solution containing red blood cells was centrifuged at 2500rpm for 5min, and the absorbance of the supernatant of each sample was measured using a microplate reader (Infinite 200 Pro) having a wavelength of 540 nm. Deionized water, physiological saline solution were used as positive control (100% lysis) and negative control (0% lysis), respectively, as shown in fig. 17.
The hemolysis rate was calculated using the following formula and the results are shown in fig. 18:
hemolysis rate (%) = (OD) Sample (I) -OD Negative control )/(OD Positive control -OD Negative control )×100
The results show that 5, 15 and 25 μ g/mL of RA-EMP (drug loading ratio 1.
Example 5
1 in vivo pharmacokinetic evaluation
Rats (male SD rats, about 200 g) were randomly divided into two groups, fasted for 12h before the experiment, and freely fed and drunk water after oral administration. 35mg/kg of EMP suspension (0.5 wt% sodium carboxymethylcellulose in water as dispersion solvent, 5 mg/mL) was administered to 6 rats; another 6 rats were administered 35mg/kg of RA-EMP solution (drug loading ratio 1. At 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 hours post-dose, 0.25mL of blood sample was drawn from the tail vein, collected in a 1.5mL anticoagulation tube, immediately centrifuged at 4 ℃ at 4000g for 10min, and the plasma stored at-80 ℃ until HPLC detection.
To determine the plasma drug concentration, 100. Mu.L of plasma was mixed with 100. Mu.L of oxcarbazepine in acetonitrile (1.0. Mu.g/mL, internal standard), centrifuged at 10000rpm for 10min, and the supernatant was filtered through a 0.22 μm filter and injected into the HPLC system. Data were analyzed using pharmacokinetic programs Drug and staticiscs 2.0 and the results are shown in table 1. The plasma EMP concentration-time curves after oral administration of EMP and RA-EMP are shown in FIG. 19, with significant differences in the release concentration of RA-EMP compared to EMP (P < 0.05).
TABLE 1 pharmacokinetic parameters of EMP after oral administration of EMP and RA-EMP (n = 6)
Figure BDA0003134364740000111
Figure BDA0003134364740000121
Wherein the content of the first and second substances,
peak concentration of drug (C) max ): maximum plasma concentration after administration; this parameter is an important index reflecting the rate and extent of absorption of the drug in vivo.
Area under the curve (AUC) at time of drug: area enclosed by the blood concentration curve versus time axis; the parameter is an important index for evaluating the absorption degree of the drug and reflects the exposure characteristic of the drug in vivo.
Time to peak (T) max ): the time required to reach the peak concentration of the drug after administration; the parameter reflects the speed of the drug entering the body, and the peak reaching time is short when the absorption speed is high.
Mean Residence Time (MRT): the mean value of the residence time of the drug molecules in the body represents the time required to eliminate 63.2% of the drug from the body.
The results showed that both RA-EMP and EMP reached peak concentration (T) at 30min max ) But maximum plasma concentration (C) of RA-EMP max ) 93.38% higher than EMP (P)<0.05). The area under the curve (AUC 0-t) for RA-EMP was significantly increased, 2.19-fold greater than for EMP suspensions. Mean Residence Time (MRT) for RA-EMP 0-t ) The elongation is 0.89 times than that of EMP group. These all indicate that RA-EMP has a better effect than EMPThe oral bioavailability of (a).
2 evaluation of tissue distribution in vivo
After oral administration to rats, the biodistribution of EMP in the intestinal tract and major organs was measured, and biodistribution was measured at the pharmacokinetic Tmax time point (30 min) and the elimination period time point (4 h).
The rats were randomly divided into two groups, fasted for 12h and allowed free access to water. 35mg/kg of EMP suspension (0.5 wt% sodium carboxymethylcellulose in water as dispersion solvent, 5 mg/mL) was administered to 6 rats; another 6 rats were administered 35mg/kg of RA-EMP solution (drug loading ratio 1. At 30min and 4h, 3 rats per group were sacrificed at random, and the heart, liver, spleen, lung, kidney, brain, pancreas, and other organ tissues, as well as the stomach, duodenum, jejunum, ileum, cecum, colon, rectum, and other gastrointestinal segments were collected and washed. The collected tissue was divided into two parts, one part was used for quantitative measurement by high performance liquid chromatography (in the measurement, water in the tissue was blotted with filter paper, 1.0. Mu.g/mL of acetonitrile solution of oxcarbazepine was added after weighing, and detection was performed by high performance liquid chromatography), and the other part was used for fluorescence visualization analysis (EMP showed fluorescence excitation).
For fluorescence visualization, samples were prepared by frozen sections, nuclei were stained blue with 4,' 6-diamidino-2-phenylindole Dihydrochloride (DAPI), and F-actin was stained red with rhodamine-labeled phalloidin (rhodamine-phaseolin); the stained tissue sections were observed with a fluorescence microscope (BX 53F2, olympus, tokyo, japan).
The EMP concentration of stomach, duodenum, jejunum, ileum, caecum, colon, rectum, etc. is shown in figure 20; the EMP concentration of heart, liver, spleen, lung, kidney, brain, pancreas and other organs is shown in FIG. 21.
The results showed that the EMP concentration in the gastrointestinal tract of stomach, duodenum, jejunum, ileum, etc. at 30 min: the RA-EMP administration group is significantly higher than the EMP administration group, and is 1.4-10 times (P < 0.05) higher than the EMP administration group. RA-EMP administration group: EMP concentrations in tissues were greatest in the ileum, duodenum, jejunum, stomach, etc. EMP administration group: EMP concentrations in tissues were greatest in the duodenum, stomach, jejunum, ileum, etc. EMP concentration in organ tissues such as pancreas at 30 min: the group administered with RA-EMP was significantly higher than the group administered with EMP (P < 0.05). At 240min, the situation is similar (P < 0.05).
EMP can excite green-yellow fluorescence, so that permeability and biodistribution of RA-EMP and EMP in different intestinal segments (such as duodenum, jejunum and ileum) and organs (such as pancreas and kidney) can be observed by fluorescence. The fluorescence visualization results of duodenum, jejunum and ileum are shown in fig. 22; the fluorescence visualization of kidney and pancreas is shown in FIG. 23.
The results showed that the EMP fluorescence signals of the group administered with RA-EMP were significantly stronger in the duodenum, jejunum and ileum than those of the group administered with EMP, and that the serosal layers of all three sections of intestine also had strong EMP fluorescence signals, indicating that RA-EMP could more easily pass through the intestinal wall and rapidly enter the systemic circulation. Also, the EMP fluorescence signal was significantly stronger in the pancreas and kidney in the RA-EMP-administered group than in the EMP-administered group. These data and data further demonstrate that RA-EMP has better oral bioavailability than EMP.
Example 6
In vivo therapeutic effect test for acute pancreatitis
Experiment design:
mice (male BALB/c mice, 8 weeks old) received two intraperitoneal injections of L-arginine (dissolved in physiological saline at pH 7.0) at a dose of 4g/kg body weight, with 1h interval between injections. After the first injection, all rats had free access to water and food. The time for the second injection of L-arginine was recorded as 0h. After the second injection of L-arginine, a mouse model of Acute Pancreatitis (AP) was successfully established. Mice injected with L-arginine twice were divided into seven groups:
(1) AP group: respectively taking 7mL/kg of physiological saline orally 2, 4 and 6 hours after the injection of the L-arginine for the second time by 22 to 24 mice;
(2) EMP group: respectively orally taking EMP suspension (0.5 wt% sodium carboxymethylcellulose aqueous solution as a dispersion solvent) 35mg/kg of body weight for 2, 4 and 6 hours after the 22-24 mice are injected with the L-arginine for the second time;
(3) group RA: respectively taking an oral RA water solution of 525mg/kg of body weight for 2, 4 and 6 hours after the injection of the L-arginine for the second time by 22 to 24 mice;
(4) RA & EMP physical cocktail group: respectively orally taking mixed solution of 525mg/kg body weight of RA aqueous solution and 35mg/kg body weight of EMP suspension (0.5 wt% sodium carboxymethylcellulose aqueous solution as a dispersing solvent) 2, 4 and 6 hours after the 22-24 mice are injected with the L-arginine for the second time;
(5) RA-EMP low dose group: 20-22 mice were orally administered with RA-EMP solution (loading ratio 1;
(6) dose groups in RA-EMP: 20-22 mice were orally administered with RA-EMP solution (loading ratio 1;
(7) RA-EMP high dose group: 18 to 20 mice were orally administered with RA-EMP solution ( loading ratio 1, 15, PBS buffer as solvent) at 35mg/ kg body weight 2, 4, and 6 hours after the second injection of L-arginine, respectively.
Normal control group: another 18 normal mice not injected with L-arginine were orally administered with physiological saline 7mL/kg body weight for 2, 4, and 6 hours, respectively.
The difference between groups was determined using multiple comparisons in analysis of variance. And analyzing the difference of the blood concentration of EMP and RA-EMP at different time points by adopting independent sample t test. All analyses were performed using the sps 11.5 software (spsinc., chicago), with P <0.05 indicating significance.
The survival rates of the above groups of mice after 24 hours are shown in FIG. 24.
The results show that the mouse mortality rate is up to 40 percent (AP group in figure 24) within 24 hours after two injections of L-arginine, and EMP, RA & EMP and RA-EMP can effectively reduce the death of the mouse and improve the survival rate of the mouse, especially the mouse can completely survive after the administration of RA-EMP under medium and high doses, and the survival life of the mouse after the pancreatitis is infected by the RA-EMP is prolonged. This indicates that RA-EMP has a good therapeutic effect for the prevention and/or treatment of pancreatitis.
About 0.8mL of blood was collected from all the mice by tail-cutting after 24 hours from the second injection of L-arginine, and serum was obtained by coagulation and stored at-80 ℃ for biochemical analysis of serum amylase, lipase level, etc.
Each group of mice was sacrificed, pancreatic tissue was collected and weighed (wet weight), and the pancreatic weight/body weight ratio was determined. 3 pancreatic tissues were randomly taken from each group and the pancreatic water content was determined. 6 pancreatic tissues were randomly stored at-80 ℃ for ELISA (enzyme linked immunosorbent assay) and MDA (malondialdehyde) detection. Randomly 3 pancreatic tissues were stored at-80 ℃ for SOD and GSH assays. 2 pancreatic tissues were randomly selected, fixed in 10% formaldehyde for 24 hours, and examined histologically. Other organs, including heart, liver, spleen, lung, kidney and brain, and sections of the gastrointestinal tract, including stomach, duodenum, jejunum, ileum, caecum, colon and rectum were collected and fixed with 10% formaldehyde for histology/tunnel examination.
(1) Evaluation of edema
The pancreas separated from the fat and blood samples (see FIG. 25) was weighed (wet weight), and then dried at 60 ℃ for 12 hours to obtain its dry weight; moisture content = (wet-dry weight)/wet weight. The degree of pancreatic edema is evaluated by using indices such as moisture content, pancreas weight/body weight ratio, and the like.
The water content, pancreas weight/body weight ratio of the different experimental groups are shown in fig. 26.
Compared to normal pancreatic tissue, the pancreas of the AP group exhibits the following characteristics: translucency and edema of pancreatic tissue, saponification spots in the tissue, bleeding in the tissue (see the AP group in FIG. 25) were visually observed; pancreatic water content and pancreatic weight/body weight were significantly increased, indicating severe pancreatic edema (see AP group of fig. 26).
The results show that EMP, RA & EMP, RA-EMP all reduce the edema symptoms of the pancreas to some extent: compared with the AP group, the moisture content and pancreas weight/body weight ratio of EMP, RA & EMP and RA-EMP (including three doses) show significant difference (P < 0.05), and especially the curative effect of RA-EMP is best: the moisture content, pancreas weight/body weight ratio of RA-EMP (including: medium and high dose) showed significant differences compared to the RA & EMP group.
(2) Biochemical analysis
Serum amylase and lipase levels were performed according to kit instructions (Nanjing institute of bioengineering); the results are shown in FIG. 27.
The results showed that serum amylase levels in the AP group were significantly elevated compared to the normal control group (P <0.05 compared to the normal control group). Serum amylase levels were not improved in both EMP and RA groups (P >0.05 compared to AP group), whereas serum amylase levels were improved to different degrees in both RA & EMP and RA-EMP groups (P < 0.05).
Serum lipase aspect: serum lipase levels in the AP group were significantly elevated compared to the normal control group (P < 0.05). The serum lipase levels were not improved in the EMP group, RA & EMP group (P >0.05 compared to AP group), while the serum lipase levels were significantly improved in the RA-EMP group (P < 0.05). In particular, serum lipase levels at a dose of 35mg/kg for RA-EMP were restored to normal (P >0.05 compared to normal control).
(3) Pancreatic enzyme-linked immunosorbent assay
Proinflammatory cytokines including Interleukin (IL) -1 beta, interleukin-6, nuclear factor-kappa B (NF-kappa B), transforming growth factor beta (TGF-beta), intercellular adhesion molecule 1 (ICAM-1), and tumor necrosis factor alpha (TNF-alpha) were detected using a mouse ELISA kit (Shanghai enzyme-linked Biotechnology Co., ltd.). Combining two pancreases into one sample, and repeatedly testing three samples; the results are shown in FIG. 28.
The results show that all these proinflammatory cytokine levels in the AP group were significantly higher than the normal control group. EMP, RA & EMP and RA-EMP can improve the level of proinflammatory cytokines to a certain extent, so that the content of the proinflammatory cytokines tends to a normal level; compared with the AP group, the levels of IL-1 beta, IL-6, TGF-beta and TNF-alpha of EMP, RA & EMP and RA-EMP (including three doses) show significant difference (P < 0.05), the levels of NF-kappa B of EMP, RA & EMP and RA-EMP show significant difference (P < 0.05), but the level of NF-kappa B of RA does not show significant difference, and the levels of ICAM-1 of RA, RA & EMP and RA-EMP show significant difference, but the level of ICAM-1 of EMP does not show significant difference, especially the curative effect of RA-EMP is optimal: the TNF-alpha levels of RA-EMP showed significant differences compared to the RA & EMP group.
(4) Evaluation of tissue oxidative stress
The level of oxidative stress in pancreatic tissue is assessed by measuring the levels of Glutathione (GSH), superoxide dismutase (SOD), and Malondialdehyde (MDA) in the pancreatic tissue. Each pancreatic tissue is divided into two parts: one part is used for SOD detection, and the other part is used for GSH detection; the solution provided in the kit was homogenized and its SOD and GSH activities were determined according to the kit instructions (Biyuntian biotechnology, jiangsu, china). The test sample (pancreas) of enzyme-linked immunosorbent assay (ELISA) was used to determine the Malondialdehyde (MDA) content in the tissue and tested according to the kit instructions (bi yun tian biotechnology, china jiangsu).
SOD and GSH activity are expressed as U/mg (wet weight of pancreatic tissue). MDA content is expressed in nmol/mg (wet weight of pancreatic tissue). The results are shown in FIG. 29.
The results show that the GSH level, MDA level and SOD activity of the mice in the AP group are reduced and increased compared with the normal control group (P < 0.05). GSH levels in the EMP, RA & EMP groups showed no improvement compared to the AP group (P > 0.05). Different doses of RA-EMP have positive effects on improving GSH level of mouse pancreatic tissue (P is less than 0.05 compared with AP group), and are dose-dependent. In particular, levels of GSH at 35mg/kg dose of RA-EMP returned to normal (P >0.05 compared to normal controls). MDA aspect: EMP, RA & EMP, and RA-EMP all reduced the MDA level to some extent (P <0.05 compared with AP group), making its content approach normal level.
(5) Histopathological evaluation
The 10% formaldehyde-fixed tissue and organ sections were stained with hematoxylin-eosin and examined histopathologically. The results are shown in FIG. 30.
The results show that the pathological manifestations of the AP group compared to healthy pancreatic tissue of the normal control group: pancreatic tissue structural disorders such as: pancreatic edema, inflammatory cell infiltration, acinar cell necrosis, vacuolization, lobular space expansion, etc. The improvement of the pancreatic injury by the EMP, RA & EMP groups was weak, and the destruction of the pancreatic tissue structure could still be easily observed. The pancreatic injury of the RA-EMP group is obviously reduced, which shows that the RA-EMP has better treatment effect on acute pancreatitis. Moreover, the effect is dose-dependent, and especially the curative effect of RA-EMP is very obvious under 35mg/kg dose.
Meanwhile, histopathological examination proves that the organs such as heart, liver, spleen, lung, kidney, intestine and the like of the AP group mice also have edema, hyperemia and necrosis and are accompanied with serious inflammatory cell infiltration; RA-EMP also has protective effect on these organs/tissues, and the damage condition is obviously reduced.
(6) Evaluation of apoptosis
The pancreatic and renal tissues were evaluated for apoptosis using immunohistochemical apoptosis staining. The results are shown in FIG. 31 (apoptosis positive cells are brown).
The results show that the apoptosis positive cells of pancreas and kidney of the AP group are obviously increased compared with the normal control group. The apoptosis positive cells of pancreas and kidney of RA-EMP group are relatively less, and are closer to the normal control group. This suggests that RA-EMP is effective in protecting pancreatic acinar cells, renal cells, and the like.
The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it is therefore intended that all such changes and/or modifications be considered as within the spirit or scope of the present invention as defined by the appended claims.

Claims (14)

1. A pharmaceutical formulation characterized by: the pharmaceutical preparation comprises the empagliflozin and a water-soluble carrier.
2. The pharmaceutical formulation of claim 1, wherein: the water-soluble carrier is stevioside; preferably, the steviol glycoside is rebaudioside; more preferably, the rebaudioside is rebaudioside a.
3. The pharmaceutical formulation according to claim 1 or 2, characterized in that: in the pharmaceutical preparation, the weight ratio of the empagliflozin to the water-soluble carrier is 1; preferably, the weight ratio of the empagliflozin and the water-soluble carrier is 1; more preferably, the weight ratio of the empagliflozin to the water-soluble carrier is 1.
4. The pharmaceutical formulation according to any one of claims 1 to 3, characterized in that: in the pharmaceutical preparation, the encapsidation rate of the empagliflozin is at least 80 percent; preferably, the encapsulation efficiency of the empagliflozin is more than or equal to 90 percent.
5. The pharmaceutical formulation according to any one of claims 1 to 3, characterized in that: the pharmaceutical preparation is a pharmaceutical preparation for treating and/or preventing inflammation and/or complications thereof;
preferably, the inflammation is an inflammatory disease of the pancreas; more preferably, the pancreatic inflammatory disease is acute pancreatitis or chronic pancreatitis;
alternatively, the inflammation is an inflammatory disease caused by an abnormality of L-arginine.
6. The pharmaceutical formulation of claim 5, wherein: the inflammation is inflammation associated with proinflammatory cytokines; the proinflammatory cytokine comprises one or more than two of interleukin-1 beta, interleukin-6, nuclear factor-kB, transforming growth factor beta, intercellular adhesion molecule 1 and tumor necrosis factor alpha.
7. The pharmaceutical formulation according to any one of claims 1 to 3, characterized in that: the pharmaceutical preparation is a pharmaceutical preparation for treating and/or preventing pancreatic diseases and/or complications thereof;
in a preferred embodiment of the method of the invention,
the pancreatic diseases are pancreatic inflammatory diseases; more preferably, the pancreatic inflammatory disease is acute pancreatitis or chronic pancreatitis;
alternatively, the pancreatic disorder is an inflammatory disorder caused by abnormality of L-arginine.
8. The pharmaceutical formulation according to any one of claims 1 to 3, characterized in that: the pharmaceutical preparation also comprises pharmaceutically acceptable auxiliary materials and/or auxiliary components.
9. The pharmaceutical formulation according to any one of claims 1 to 3, characterized in that: the medicinal preparation is an oral administration preparation, an injection preparation, a sublingual administration preparation, a buccal administration preparation or a transdermal absorption preparation.
10. The pharmaceutical formulation according to any one of claims 1 to 3, characterized in that: the medicinal preparation is decoction, pills, powder, ointment, cataplasm, film agent, patch, emplastrum, pellet, injection, medicinal liquor, medicinal granules, oral liquid, tablets, injection, capsules, granules, suppository, spray, gel, syrup, liniment, solution, emulsion, suspension, implant, lotion, flushing agent, enema, mixture, sustained release preparation, controlled release preparation, delayed release preparation, targeted preparation or prodrug preparation.
11. The pharmaceutical formulation according to any one of claims 1 to 3, characterized in that: the pharmaceutical preparation is a nano-micelle preparation formed by the empagliflozin and the water-soluble carrier, and preferably is a self-assembled nano-micelle preparation formed by the empagliflozin and the water-soluble carrier;
preferably, the pharmaceutical preparation is a nano-micelle preparation formed by the empagliflozin and the water-soluble carrier, and is preferably a self-assembled nano-micelle preparation formed by the empagliflozin and the water-soluble carrier.
12. The pharmaceutical formulation according to any one of claims 1 to 3, characterized in that: the medicinal preparation is a solution, and the solvent is selected from water, PBS buffer solution or 0.5wt% sodium carboxymethylcellulose aqueous solution.
13. The pharmaceutical formulation of claim 12, wherein: when the concentration of the empagliflozin in the solution is 1.0mg/mL,
the micelle size of the solution is 1-25 nm; preferably 2 to 8nm;
and/or the polydispersity of the solution is less than or equal to 0.7; preferably ≤ 0.2 or 0.1;
and/or the zeta potential of the solution is-15-0 mV; preferably from-5 to 0mV.
14. A process for the preparation of a pharmaceutical formulation according to any one of claims 1 to 11, characterized in that it comprises the following steps: dissolving the engeletin and the water-soluble carrier in a solvent together, uniformly mixing, and then removing the solvent to obtain the product; preferably, the solvent is ethanol.
CN202110712920.6A 2021-04-16 2021-06-25 Pharmaceutical preparation containing engelizin and preparation method thereof Pending CN115212197A (en)

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