CN115141136B - Co-crystals of tecan Wei Ruiyao and method for preparing same - Google Patents
Co-crystals of tecan Wei Ruiyao and method for preparing same Download PDFInfo
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Abstract
The invention relates to a cocrystal of tecovirime and a preparation method thereof, a pharmaceutical composition containing the cocrystal, and application of the cocrystal in preparing medicines for treating smallpox. The co-crystal comprises tecovirime and a co-crystal ligand, wherein the co-crystal ligand is pharmaceutically acceptable organic acid or organic base. The eutectic crystal has the advantages of high solubility, high in vitro dissolution rate, high in vivo relative bioavailability and the like.
Description
Technical Field
The invention belongs to the technical field of medicines, relates to a co-crystal of tecovirime and a preparation method thereof, also relates to a pharmaceutical composition containing the co-crystal, and further relates to application of the co-crystal in preparation of medicines for treating smallpox.
Background
Tecovirimat (Tecovirimat, code ST-246, chemical name 4-trifluoromethyl-N- (3, 3a, 4a, 5a,6 a-octahydro-1, 3-dioxo-4, 6-vinylidenecyclopropano [ f ] isoindol-2 (1H) -yl) -benzamide, formula 1) is a highly active small molecule viral inhibitor for anti-smallpox therapy, which acts by binding to viral genes to prevent release of the virus in the cell. In addition, the combined application of the tecovirimide and the smallpox vaccine can also prevent and treat adverse reactions caused by the adverse reactions, reduce the injury level and accelerate the wound healing. However, the poor water solubility of tecovirime, less than 3 μg/ml, and the low solubility creates bioavailability problems that limit its clinical application.
Pharmaceutical co-crystals are multicomponent systems composed of pharmaceutically active ingredients (API) and co-crystal ligands (cocrystals former, CCF) in fixed stoichiometric ratios by non-covalent bonding such as hydrogen bonding, van der waals forces, pi-pi conjugation, and halogen bonding. The pharmaceutical co-crystal can improve the physicochemical properties of the medicine, such as melting point, stability, solubility, bioavailability and the like, on the basis of not changing the chemical structure of the medicine. In recent years, pharmaceutical co-crystals have become a new strategy for drug development, and have shown attractive application prospects in the field of pharmaceutical biology.
Disclosure of Invention
The inventors have unexpectedly obtained a co-crystal of tecoviral that has improved solubility, dissolution and relative bioavailability in vivo of poorly soluble tecoviral compared to the prior art.
The invention provides a eutectic, which comprises tecovirime and a eutectic ligand, wherein the eutectic ligand is pharmaceutically acceptable organic acid or organic base.
The invention also provides a pharmaceutical composition comprising the co-crystal, and a pharmaceutically acceptable carrier or excipient.
The invention also provides a method for preparing the eutectic, which comprises the following steps:
a) Dissolving tecovirimide and a co-crystal ligand in a solvent;
b) Cooling until crystals are separated out;
c) Optionally, the crystals are isolated and optionally dried.
The invention also provides another method for preparing the eutectic, which comprises the following steps:
a) Dissolving tecovirimide and a eutectic ligand in a solvent, and stirring;
b) Separating the solid sample;
c) Optionally, the solid sample is dried.
The invention also provides application of the eutectic crystal in preparing medicines for treating smallpox.
The invention also provides application of the pharmaceutical composition in preparing medicines or vaccines for treating and/or preventing smallpox.
Detailed Description
In certain embodiments, the organic acid described herein is selected from the group consisting of p-hydroxybenzoic acid, benzoic acid.
In certain embodiments, the organic acid described herein is p-hydroxybenzoic acid.
In certain embodiments, the organic base of the present invention is selected from the group consisting of isonicotins, nicotinamide, acetamides, benzamides, piperazines, monoethanolamines.
In certain embodiments, the organic base of the present invention is isonicotins, nicotinamide, acetamides, or benzamides.
In certain embodiments, the organic base of the present invention is isonicotinic acid or nicotinamide.
In certain embodiments, the organic base of the present invention is isonicotinic.
In certain embodiments, the molar ratio of tecovirime to co-crystal ligand in the co-crystals of the present invention is about 1:0.5 to 50, preferably about 1:0.5 to 25, more preferably about 1:0.5 to 10, even more preferably about 1:0.5 to 5.
In certain embodiments, the co-crystal ligand described herein is p-hydroxybenzoic acid, isonicotins, benzoic acid, or nicotinamide. In certain embodiments, the molar ratio of tecovirimir to co-crystal ligand is about 1:0.5 to 5, about 1:0.8 to 4, about 1:0.8 to 3, about 1:0.8 to 2.5, about 1:0.8 to 1.5, about 1:0.8 to 1.2, e.g., about 1:1.
In certain embodiments, the co-crystals described herein are co-crystals of tecovirimide with parahydroxybenzoic acid, isonicotinal, benzoic acid or nicotinamide, and the molar ratio of tecovirimide to co-crystal ligand is about 1:1.
In certain embodiments, the co-crystal of the present invention wherein the co-crystal ligand is parahydroxybenzoic acid and the molar ratio of tecovirimide to co-crystal ligand is about 1:1.
In certain embodiments, the co-crystals described herein are co-crystals of tecovirimide and parahydroxybenzoic acid, and the molar ratio of tecovirimide to co-crystal ligand is about 1:1.
In certain embodiments, the co-crystals of tecovirime and parahydroxybenzoic acid described herein have characteristic peaks at 14.16 ° ± 0.2 °, 11.49 ° ± 0.2 ° in an X-ray powder diffraction pattern expressed in degrees 2θ obtained using Cu-ka radiation.
In certain embodiments, the co-crystals of tecovirime and parahydroxybenzoic acid described herein also have characteristic peaks at 6.49 ° ± 0.2 °, 19.88 ° ± 0.2 ° in an X-ray powder diffraction pattern expressed in degrees 2θ obtained using Cu-ka radiation.
In certain embodiments, the co-crystals of tecovirimide and parahydroxybenzoic acid described herein also have characteristic peaks at 18.85 ° ± 0.2 °, 21.16 ° ± 0.2 °, 20.73 ° ± 0.2 °, 14.65 ° ± 0.2 ° in an X-ray powder diffraction pattern expressed in degrees 2θ obtained using Cu-ka radiation.
In certain embodiments, the co-crystals of tecovirimide and parahydroxybenzoic acid described herein also have characteristic peaks at 25.79 ° ± 0.2 °, 16.69 ° ± 0.2 °, 27.26 ° ± 0.2 °, 30.48 ° ± 0.2 ° in an X-ray powder diffraction pattern expressed in degrees 2θ obtained using Cu-ka radiation.
In certain embodiments, the co-crystals of tecovirime and parahydroxybenzoic acid described herein also have characteristic peaks at 12.87 ° ± 0.2 °, 15.93 ° ± 0.2 °, 9.60 ° ± 0.2 ° in an X-ray powder diffraction pattern expressed in degrees 2θ obtained using Cu-ka radiation.
In certain embodiments, the co-crystals of tecovirimide and parahydroxybenzoic acid described herein have characteristic peaks at 14.16 ° ± 0.2 °, 11.49 ° ± 0.2 °, 6.49 ° ± 0.2 °, 19.88 ° ± 0.2 °, 18.85 ° ± 0.2 °, 21.16 ° ± 0.2 °, 20.73 ° ± 0.2 °, 14.65 ° ± 0.2 °, 25.79 ° ± 0.2 °, 16.69 ° ± 0.2 °, 27.26 ° ± 0.2 °, 30.48 ° ± 0.2 °, 12.87 ° ± 0.2 °, 15.93 ° ± 0.2 °, 9.60 ° ± 0.2 ° in an X-ray powder diffraction pattern expressed in 2θ degrees obtained using Cu-ka radiation.
In certain embodiments, the co-crystal of tecovirime and parahydroxybenzoic acid described herein has an X-ray powder diffraction pattern substantially the same as shown in figure 5 using Cu-ka radiation.
In certain embodiments, the co-crystals of tecovirimide and parahydroxybenzoic acid described herein have no characteristic peak at 13.77 ° ± 0.2 °,42.13 ° ± 0.2 °, and characteristic peaks at 14.16 ° ± 0.2 °, 11.49 ° ± 0.2 ° in an X-ray powder diffraction pattern expressed in 2θ degrees obtained using Cu-ka radiation, as compared to a tecovirimide drug substance.
In certain embodiments, the co-crystals of tecovirimide and parahydroxybenzoic acid described herein exhibit melting endotherm in the range of 160 to 170 ℃ as measured by differential scanning calorimetry.
In certain embodiments, the co-crystals of tecovirimide and parahydroxybenzoic acid described herein have substantially the same Differential Scanning Calorimeter (DSC) profile as shown in figure 7 as measured by differential scanning calorimetry.
In certain embodiments, the co-crystals are present in the pharmaceutical compositions of the invention in an amount effective to treat smallpox.
In certain embodiments, the pharmaceutical compositions of the invention further comprise a smallpox vaccine. In certain embodiments, the pharmaceutical composition of the invention, the smallpox vaccine is present in an amount effective to prevent smallpox.
In certain embodiments, the solvent according to the present invention is at least one selected from the group consisting of water, alcohol solvents, ester solvents, ketone solvents, ether solvents, nitrile solvents, alkane solvents, haloalkane solvents.
In certain embodiments, the alcoholic solvent of the present invention is at least one selected from the group consisting of methanol, ethanol, and isopropanol.
In certain embodiments, the ester solvent of the present invention is at least one selected from the group consisting of methyl acetate, ethyl acetate, and propyl acetate.
In certain embodiments, the ketone solvent of the present invention is at least one selected from the group consisting of acetone and methyl isobutyl ketone.
In certain embodiments, the ether-based solvent of the present invention is at least one selected from the group consisting of methyl t-butyl ether, cyclopentyl methyl ether, and tetrahydrofuran.
In certain embodiments, the nitrile solvent of the present invention is acetonitrile.
In certain embodiments, the alkane solvent of the present invention is n-hexane.
In certain embodiments, the halogenated hydrocarbon solvent of the present invention is methylene chloride.
In certain embodiments, the solvent of the present invention is at least one selected from the group consisting of methanol, ethanol, t-butyl methyl ether, n-hexane, and water.
In certain embodiments, the solvent of the present invention is a mixed solvent of methanol and ethanol. In certain embodiments, the volume ratio of methanol to ethanol in the mixed solvent is 15:0.1-1, e.g., 15:0.2-0.8, 15:0.3-0.7, 15:0.4-0.6, 15:0.5.
In certain embodiments, the solvent of the present invention is n-hexane.
Definition of terms
As used herein, the term "substantially identical" as used to define the figures is intended to mean that the figures are considered identical to the reference figures by those skilled in the art in view of the deviations that are acceptable in this field. Such deviations may be caused by factors known in the art that relate to instrumentation, operating conditions, and artifacts, among others. For example, those skilled in the art will appreciate that the onset and peak temperatures of endotherms, as measured by Differential Scanning Calorimetry (DSC), can vary significantly from experiment to experiment. In some embodiments, two plots are considered substantially identical when the positions of the characteristic peaks of the plots do not vary by more than ±5%, ±4%, ±3%, ±2% or ±1%. For example, one skilled in the art can readily identify whether two X-ray diffraction patterns or two DSC patterns are substantially the same. In some embodiments, the X-ray diffraction patterns are considered substantially the same when the 2 theta angle of the characteristic peaks of the two X-ray diffraction patterns does not vary by more than ± 0.3 °, ± 0.2 ° or ± 0.1 °.
As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired therapeutic or prophylactic effect, e.g., an amount effective to reduce symptoms associated with a disease to be treated (e.g., smallpox), or an amount effective to avoid, reduce, prevent, or delay the occurrence of a disease (e.g., smallpox). Determination of such effective amounts is within the ability of those skilled in the art. Generally, the co-crystals of the present invention may be used in a therapeutic daily dose of about 1 to 1000 milligrams.
As used herein, the term "treatment" is intended to alleviate, mitigate, ameliorate or eliminate a disease state or disorder for which it is intended. A subject is successfully "treated" if the subject has received a therapeutic amount of the co-crystal or pharmaceutical composition according to the methods described herein, and the subject's one or more indications and symptoms exhibit an observable and/or detectable decrease or improvement. It is also to be understood that the treatment of the disease state or condition includes not only complete treatment, but also less than complete treatment, but achieves some biologically or medically relevant result.
As used herein, the term "preventing" is intended to avoid, reduce, prevent or delay the appearance of a disease or disease-related symptoms, and the absence of such disease or disease-related symptoms prior to administration of the relevant drug. "preventing" is not required to completely prevent the occurrence of a disease or disease-related symptom, e.g., a subject may be reduced in risk of developing a particular disease or disease-related symptom after administration of a related agent, or may be considered "preventing" the occurrence or progression of the disease by reducing the severity of the related symptom that later develops.
As used herein, the term "about" is understood to be within normal tolerances in the art, such as, for example, within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1%, ±0.05% or ±0.01% of the stated value. Unless otherwise apparent from the context, all numbers provided herein are modified by the term "about".
As used herein, the term "pharmaceutically acceptable carrier or excipient" refers to a diluent, supplement, or vehicle with which a therapeutic agent is administered, and which is suitable for contacting the tissues of humans and/or other animals without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio, within the scope of sound medical judgment.
Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of the present invention include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, water is an exemplary carrier. Physiological saline and aqueous solutions of glucose and glycerol can also be used as liquid carriers, in particular for injections. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition may also optionally contain minor amounts of wetting agents, emulsifying agents, pH buffering agents, preservatives, antioxidants, flavoring agents, perfuming agents, co-solvents, solubilizing agents, tonicity adjusting agents, coloring agents and the like. Oral formulations may contain standard carriers such as binders, fillers, disintegrants, lubricants and the like.
The pharmaceutical compositions of the present invention may be administered by methods well known in the art, such as, but not limited to, any of the following: oral, spray inhalation, rectal, nasal, buccal, topical, parenteral, e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or by means of an explanted reservoir. Among them, oral, intramuscular or intravenous injection administration is preferable.
For these routes of administration, the pharmaceutical compositions of the present invention may be administered in suitable dosage forms.
The dosage form may be a solid, semi-solid, liquid, or gaseous formulation including, but not limited to, tablets, capsules, powders, granules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, suspensions, elixirs, syrups.
The pharmaceutical compositions of the present invention may be prepared by any method well known in the art, for example by mixing, dissolving, granulating, dragee-coating, levigating, emulsifying, lyophilizing, and the like.
Advantageous effects
Compared with the raw material medicines of the tecovirimide, the eutectic crystal of the tecovirimide provided by the invention has one or more of the following advantages:
1) The solubility is high;
2) The in vitro dissolution rate is high;
3) Has high in vivo relative bioavailability.
In addition, the invention utilizes the principle of crystal engineering and combines the characteristic of amide groups in the Tecowry structure, and increases the contact opportunity between molecules by using a cooling crystallization method or a mechanical stirring method, thereby accelerating the eutectic generation rate.
The method for preparing the eutectic provided by the invention has one or more of the following advantages:
1) The operation is simple;
2) The preparation time is short;
3) The obtained eutectic has stable quality;
4) The method has strong controllability and good reproducibility;
5) The cost is low.
Drawings
FIG. 1 shows the equilibrium solubility results of the product obtained in example 1 of the present invention for Japanese patent application No. Wei Ruiji;
FIG. 2 shows the equilibrium solubility results of the product obtained in example 2 of the present invention for specific reference Wei Ruiji;
FIG. 3 shows the results of the in vitro dissolution profile of the product obtained in example 1 of the present invention, specific reference Wei Ruiji;
FIG. 4 shows the results of the in vitro dissolution profile of the product obtained in example 2 of the present invention, specific reference Wei Ruiji;
FIG. 5 shows a PXRD pattern of a tecovirimide/parahydroxybenzoic acid co-crystal prepared in example 1 of the present invention;
FIG. 6 shows a PXRD contrast pattern of a tecavir co-crystal/parahydroxybenzoic acid and a physical mixture of tecavir, parahydroxybenzoic acid, and tecavir/parahydroxybenzoic acid prepared in example 1 of the present invention;
FIG. 7 shows DSC spectra of tecovirimide, parahydroxybenzoic acid, a tecovirimide/parahydroxybenzoic acid co-crystal prepared in example 1 of the present invention;
FIG. 8 shows the FIIR spectrum of tecovirime;
FIG. 9 shows the FIIR spectrum of p-hydroxybenzoic acid;
FIG. 10 shows the FIIR spectrum of the tecovirimide/parahydroxybenzoic acid co-crystal prepared in example 1 of the present invention;
FIG. 11 shows an HPLC chart of the tecovirimide/parahydroxybenzoic acid co-crystal prepared in example 1 of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail with reference to the following examples, but it will be understood by those skilled in the art that the following examples and experimental examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions in examples and test examples were not specified, and the reagents or equipment used were conventional products commercially available, either under conventional conditions or under conditions recommended by the manufacturer.
The tecovirimide used in the examples of the present invention and commercially available ones may also be prepared by reference to the prior art. The co-crystal ligands used are all commercially available, for example urea, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, p-hydroxybenzoic acid, benzoic acid, nicotinamide, acetamide and benzamide are commercially available from the national pharmaceutical chemicals company, inc.; isonicotinic is available from alas Ding Shiji limited.
Example 1: tecowry Rui eutectic prepared by cooling crystallization method
The tecovirimide and 12 eutectic ligands (respectively urea, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, p-hydroxybenzoic acid, isonicotinyl amine, benzoic acid, nicotinamide, acetamide and benzamide) are mixed according to a molar ratio of 1:2, then dissolved in 15.5ml of methanol/ethanol (15:0.5, v/v) mixed solvent, stirred for 6 hours at 350rpm, cooled at a speed of 10 ℃/h until crystals are separated out, suction filtration is carried out after 3 hours, a sample is obtained, and the sample is dried under reduced pressure, thus obtaining the product.
Example 2: preparation of tecovirens eutectic by mechanical stirring method
The tecovirimide and 12 eutectic ligands (respectively urea, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, p-hydroxybenzoic acid, isonicotinic acid, benzoic acid, nicotinamide, acetamide and benzamide) are mixed according to a molar ratio of 1:2, then are dissolved in 0.5ml of normal hexane solvent, stirred for 3 days at 1000rpm, a solid sample is centrifugally separated, and the sample is dried under reduced pressure to obtain the product.
Test example 1: equilibrium solubility test
The test method comprises the following steps: adding excessive tecoviril and the products obtained in example 1 and example 2 respectively by taking 1.0% Sodium Dodecyl Sulfate (SDS) aqueous solution as a solvent, adding a small amount of small glass beads, sealing, oscillating at constant temperature of 37 ℃, centrifuging, taking the supernatant, and filtering with a 0.45 μm microporous filter membrane to obtain filtrate as a sample solution; in addition, the tecovirimide crude drug is precisely weighed as a reference substance to be about 5mg, and diluted to proper concentration to be a reference substance solution. And measuring peak areas of the sample solution and the reference solution at 224nm according to high performance liquid chromatography, and calculating the content and equilibrium solubility by an external standard method.
Chromatographic conditions:
detection wavelength: 240nm; chromatographic column: venusil MP C18 column (specification: 4.6 mm. Times.250 mm,5 μm); mobile phase: acetonitrile: 50mM sodium dihydrogen phosphate in water=60:40 (v/v); flow rate: 1ml/min; sample injection amount: 20 μl; column temperature: 30 ℃.
The equilibrium solubility results of the tecovirimide and the products obtained in example 1 are shown in fig. 1, and in 12 eutectic ligands, compared with the tecovirimide bulk drugs, the equilibrium solubility of the products with the p-hydroxybenzoic acid, the isonicotin, the benzoic acid and the nicotinamide as the ligands is greatly improved, which indicates that the eutectic is formed and the effect of improving the solubility is achieved; while the remaining 8 ligands were not significantly improved, indicating that no co-crystals were formed.
The equilibrium solubility results for each of the products obtained in example 2 are shown in FIG. 2 and are substantially the same as those obtained in example 1, indicating that the products obtained in both methods are substantially the same.
Test example 2: in vitro dissolution test
The test method comprises the following steps: taking 500ml of aqueous solution containing 1.0% SDS as a dissolution medium, adding a proper amount of tecovirime and each product obtained in the above example 1 and example 2, taking 5ml of solution according to a dissolution rate measurement method (2020 edition of Chinese pharmacopoeia general rule 0931 second method), taking 5ml of solution according to the method, and filtering with 0.45 mu m microporous filter membrane at 37 ℃ and rotating speed of 100rpm according to the method operation, wherein the 5ml of solution is 5, 10, 30, 60, 90, 120, 150, 180, 240, 300 and 360min respectively, and filtering to obtain a sample solution; in addition, the tecovirimide crude drug is precisely weighed as about 5mg of a reference substance, and diluted to proper concentration by a dissolution medium to obtain a reference substance solution. According to the chromatographic conditions in test example 1, the peak areas of the sample solution and the reference solution were measured at 224nm by high performance liquid chromatography, the elution amounts at different time points were calculated by external standard method, and the cumulative elution profile was drawn.
The in vitro dissolution curves of the products obtained in the example 1 are shown in fig. 2, and the results show that the products with the p-hydroxybenzoic acid, the isonicotinyl, the benzoic acid and the nicotinamide as ligands can be completely dissolved in 2 hours (> 80%), which shows that the 4 ligands and the tecovirimide form eutectic crystals, and the effect of improving the in vitro dissolution rate is achieved; the in vitro dissolution rates of the tecovirimide drug substance and the products of the remaining 9 ligands are all less than <20%, indicating that no co-crystals are formed.
The in vitro dissolution profile of each product obtained in example 2 is shown in FIG. 4 and is substantially the same as that obtained in example 1.
Test example 3: co-crystal characterization of tecovirimide/p-hydroxybenzoic acid
The tecovirimide/parahydroxybenzoic acid co-crystals prepared in example 1 or example 2 were characterized by X-ray powder diffraction (XRPD), differential scanning calorimetric analysis (DSC), fourier transform infrared spectroscopy (FTIR), HPLC, and the like.
a. The powder diffraction pattern of the pharmaceutical co-crystals obtained in example 1 was determined using a Bruker D8 Advance diffractometer (Bruker, germany) under the following test conditions: cu, K alpha, 40kV and 40mV are used as light sources, the step length is 0.0128 DEG, the scanning range is 3-45 ℃ and the room temperature is realized.
The tecovirimide/parahydroxybenzoic acid co-crystal prepared in example 1 had characteristic peaks at 2θ (°) of 6.49 ° ± 0.2 °, 19.88 ° ± 0.2 ° using Cu-ka radiation; characteristic peaks at 18.85 ° ± 0.2 °, 21.16 ° ± 0.2 °, 20.73 ° ± 0.2 °, 14.65 ° ± 0.2 °; characteristic peaks at 25.79 ° ± 0.2 °, 16.69 ° ± 0.2 °, 27.26 ° ± 0.2 °, 30.48 ° ± 0.2 °; characteristic peaks at 12.87°±0.2°, 15.93°±0.2°, 9.60°±0.2°, 14.16°±0.2°, 11.49°±0.2°.
Specifically, the XRPD pattern of the tecovirimide/parahydroxybenzoic acid co-crystal prepared in example 1 is shown in fig. 5 or fig. 6.
Compared with the tecovirimide bulk drug, the X-ray powder diffraction of the tecovirimide/parahydroxybenzoic acid eutectic provided by the invention has the characteristic peaks disappeared at the diffraction angles 2 theta (error + -0.2 degrees) of 13.77 DEG + -0.2 DEG and 42.13 DEG + -0.2 DEG, and has the novel characteristic peaks appearing at the diffraction angles 2 theta (error + -0.2 DEG) of 14.16 DEG + -0.2 DEG and 11.49 DEG + -0.2 deg.
The XRPD pattern of the tecovirimide/parahydroxybenzoic acid co-crystal prepared in example 2 is substantially the same as in figure 5.
b. The pharmaceutical co-crystals obtained in example 1 were measured using a TA Q2000 differential scanning calorimeter (TA Instruments) under the following test conditions: about 5mg of sample aluminum tray package was heated to 25-300 deg.C at a rate of 10.0 deg.C/min with a purge of 50ml/min nitrogen and temperature calibration was performed using NIST indium metal.
The DSC spectrum of the tecovirimide/parahydroxybenzoic acid eutectic prepared in example 1 is shown in figure 7, and the melting points of the tecovirimide, parahydroxybenzoic acid and tecovirimide/parahydroxybenzoic acid eutectic are 196 ℃, 216 ℃ and 166 ℃ respectively. A change in melting point indicates the formation of a new phase.
The DSC profile of the tecovirimide/parahydroxybenzoic acid co-crystal prepared in example 2 is substantially the same as figure 7.
c. The pharmaceutical co-crystal obtained in example 1 was measured using a Nicolet 6700 fourier transform infrared spectrometer (Thermo Fisher Scientific) under the following test conditions: KBr dry tabletting is adopted, and the scanning range is 4000-400 cm -1 。
As shown in FIG. 8, FIG. 9 and FIG. 10, the N-H absorption peak in the FTIR spectrum of the tecovirimide/parahydroxybenzoic acid co-crystal prepared in example 1 was determined to be 3471.49cm -1 Displacement to 3398.46cm -1 The C=O absorption peak is defined by 1676.38cm -1 Displacement to 1519.33cm -1 It is illustrated that the N-H bond in tecovirime and the c=o bond in parahydroxybenzoic acid may form hydrogen bonding.
The FTIR spectrum of the tecovirimir/parahydroxybenzoic acid co-crystal prepared in example 2 was substantially the same as that of fig. 10.
d. The co-crystal of the tecovirimide drug/parahydroxybenzoic acid prepared in example 1 or example 2 was checked by HPLC, and the molar ratio of the tecovirimide and the ligand parahydroxybenzoic acid in the co-crystal was determined to be about 1:1, and the chromatogram is shown in fig. 11.
Test example 4: test method for in vivo pharmacokinetic test of rat: male SD rats (purchased from Beijing Vitrendy laboratory animal technologies Co., ltd.) were taken at 200+ -10 g weights and randomly divided into three groups: test formulation group 1 (tecovirimide/parahydroxybenzoic acid physical mixture group), test formulation group 2 (tecovirimide/parahydroxybenzoic acid co-crystal group), and reference formulation group (tecovirimide drug substance group), 4 each. Fasted and free drinking water is taken the day before the experiment. The rats of group 3 were each dry powder intragastrically dosed with a dose of tecovirimide equivalent to 25mg/kg using a dry powder dosing device. Orbital bleeding was performed at 5min, 15min, 30min, 45min, 1h, 2h, 3h, 4h, 6h, 8h, 12h, 24h, respectively, after administration. Centrifuging in heparinized EP tube for 10min, precisely measuring 100 μl of supernatant, adding into 1.5mL EP tube, adding 10 μl of glibenclamide internal standard solution (500 ng/mL), placing on vortex machine, swirling for 30s, adding 0.8mL of methyl tert-butyl ether, swirling for 5min, and centrifuging for 5min 14000 r/min; the upper organic phase was transferred to a clean 2.0ml EP tube and the solvent was evaporated at 40 ℃ on a centrifugal concentrator. The residue was dissolved in 200. Mu.L of mobile phase, vortexed for 2min and centrifuged at 14000r/min for 5min, and the supernatant was taken into an autosampler sample tube, and the plasma concentration was determined and calculated by LC-MS/MS, and pharmacokinetic parameters were calculated by DASS2.0 software.
a. Chromatographic conditions
Chromatographic column: venusil MP C18 column (gauge: 2.1 mm. Times.50 mm,3 μm); mobile phase: acetonitrile: water (0.1% formic acid) =56:44; flow rate: 0.3ml/min; sample injection amount: 5 μl; column temperature: 30 ℃.
b. Mass spectrometry conditions
Ion polarity: a positive ion; ionization mode: pneumatically assisted electrospray ionization (ESI); ion detection mode: multiple Reaction Monitoring (MRM); detecting an object: ST246[ M+H ] +, M/z 377.0.fwdarw. 173.0, internal standard [ M+H ] +, M/z 494.1.fwdarw.369.1; the fragmentation voltages were respectively: 100V and 80V; collision energy: ST246 20eV, internal standard 10eV, drying gas flow rate: 10L/min; atomization chamber pressure: 30psi; drying gas temperature: capillary voltage at 350 ℃): 4000V.
c. Pharmacokinetic data processing
The resulting blood concentration data were analyzed by DAS 2.0 analysis software.
d. Measurement results
After rats respectively take the tecovirimide raw material drug, the tecovirimide/parahydroxybenzoic acid physical mixture and the tecovirimide/parahydroxybenzoic acid eutectic prepared in the example 1, main pharmacokinetics parameters and relative bioavailability are calculated according to average blood concentration (mug/mL) at different time points, and the results are shown in tables 1 and 2.
Table 1: the main pharmacokinetic parameters of each preparation are n=4
Note that: AUC (AUC) 0→Tn 、AUC 0→∞ Areas under the blood concentration curves of 0 to Tn and 0 to infinity, respectively, C max And T max Peak concentration and peak arrival time, respectively.
Table 2: relative bioavailability results for the tested formulations, n=4
Note that: relative bioavailability = AUC Test formulations /AUC Reference formulation 。
The above experimental results show that the relative bioavailability of the test formulation 1 (the physical mixture of tecovirimide/parahydroxybenzoic acid for the formulation in example 1) and the reference formulation (the crude drug of tecovirimide) is 90.33%, comparable to the reference formulation; whereas the relative bioavailability of the test formulation 2 (tecovirimide/parahydroxybenzoic acid co-crystal prepared in example 1) was 196.88% from the reference formulation. Therefore, the tecovirimide/parahydroxybenzoic acid pharmaceutical co-crystal provided by the invention can obviously improve the bioavailability of the tecovirimide, but the physical mixture with the same prescription proportion has no effect of improving the bioavailability, which indicates that certain interaction exists between active ingredients and co-crystal ligands in the pharmaceutical co-crystal, thereby promoting the in vivo absorption of the pharmaceutical.
In conclusion, compared with the prior art, the tecovirimide pharmaceutical co-crystal provided by the invention has a simple prescription, and the physicochemical property of the active ingredient tecovirimide Wei Ruixin is endowed on the basis of not changing the molecular structure of the medicine. The in vitro dissolution curve and the in vivo pharmacokinetics experiment result of the rat show that the pharmaceutical co-crystal provided by the invention is superior to the tecovirimide bulk drug and the physical mixture with the same proportion, and has the advantages of simple preparation process, low cost, easy operation, stable quality, strong controllability, good reproducibility, high bioavailability and the like.
Claims (29)
1. A co-crystal comprising tecovirimide and a co-crystal ligand, wherein the co-crystal ligand is p-hydroxybenzoic acid, the molar ratio of the tecovirimide to the co-crystal ligand is 1:0.8-2.5, and
in contrast to the tecovirimide drug substance, the co-crystal had no characteristic peak at 13.77 ° ± 0.2 °,42.13 ° ± 0.2 ° and characteristic peaks at 14.16 ° ± 0.2 °, 11.49 ° ± 0.2 ° in an X-ray powder diffraction pattern expressed in 2θ degrees obtained using Cu-ka radiation.
2. The co-crystal of claim 1, wherein the molar ratio of tecovirime to co-crystal ligand is 1:0.8-1.5.
3. The co-crystal of claim 2, wherein the molar ratio of tecovirime to co-crystal ligand is 1:0.8-1.2.
4. The co-crystal of claim 3, wherein the molar ratio of tecovirime to co-crystal ligand is about 1:1.
5. The co-crystal of claim 1, wherein the co-crystal further has characteristic peaks at 6.49 ° ± 0.2 °, 19.88 ° ± 0.2 ° in an X-ray powder diffraction pattern expressed in terms of 2Θ angles obtained using Cu-ka radiation.
6. The co-crystal of claim 5, wherein the co-crystal further has characteristic peaks at 18.85 ° ± 0.2 °, 21.16 ° ± 0.2 °, 20.73 ° ± 0.2 °, 14.65 ° ± 0.2 ° in an X-ray powder diffraction pattern expressed in 2Θ angles obtained using Cu-ka radiation.
7. The co-crystal of claim 6, wherein the co-crystal further has characteristic peaks at 25.79 ° ± 0.2 °, 16.69 ° ± 0.2 °, 27.26 ° ± 0.2 °, 30.48 ° ± 0.2 ° in an X-ray powder diffraction pattern expressed in 2Θ angles obtained using Cu-ka radiation.
8. The co-crystal of claim 7, wherein the co-crystal further has characteristic peaks at 12.87 ° ± 0.2 °, 15.93 ° ± 0.2 °, 9.60 ° ± 0.2 ° in an X-ray powder diffraction pattern expressed in terms of 2Θ angles obtained using Cu-ka radiation.
9. The co-crystal of claim 8, wherein Cu-ka radiation is used, the co-crystal having an X-ray powder diffraction pattern substantially the same as shown in figure 5.
10. The co-crystal of any one of claims 1 to 9, wherein the co-crystal exhibits a melting endotherm in the range of 160 to 170 ℃ as measured by a differential scanning calorimeter.
11. The co-crystal of claim 10, wherein the co-crystal has a differential scanning calorimetry curve substantially the same as shown in figure 7.
12. A pharmaceutical composition comprising the co-crystal of any one of claims 1 to 11, and a pharmaceutically acceptable carrier or excipient.
13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition further comprises a smallpox vaccine.
14. A method of preparing the co-crystal of any one of claims 1 to 11, comprising:
a) Dissolving tecovirimide and a co-crystal ligand in a solvent;
b) Cooling to precipitate crystals.
15. The method of claim 14, further comprising:
c) The crystals are isolated and optionally dried.
16. The method according to claim 14, wherein the solvent is at least one selected from the group consisting of water, alcohol solvents, ester solvents, ketone solvents, ether solvents, nitrile solvents, alkane solvents, haloalkane solvents,
the alcohol solvent is at least one selected from the group consisting of methanol, ethanol and isopropanol;
the ester solvent is at least one selected from the group consisting of methyl acetate, ethyl acetate and propyl acetate;
the ketone solvent is at least one selected from the group consisting of acetone and methyl isobutyl ketone;
the ether solvent is at least one selected from the group consisting of methyl tertiary butyl ether, cyclopentyl methyl ether and tetrahydrofuran;
the nitrile solvent is acetonitrile;
the alkane solvent is n-hexane;
the halogenated hydrocarbon solvent is dichloromethane.
17. The method of claim 16, wherein the solvent is at least one selected from the group consisting of methanol, ethanol, t-butyl methyl ether, n-hexane, and water.
18. The method of claim 17, wherein the solvent is a mixed solvent of methanol and ethanol.
19. The method of claim 18, wherein the volume ratio of methanol to ethanol in the mixed solvent is 15:0.1-1.
20. The method of claim 19, wherein the volume ratio of methanol to ethanol in the mixed solvent is 15:0.2-0.8.
21. The method of claim 20, wherein the volume ratio of methanol to ethanol in the mixed solvent is 15:0.3-0.7.
22. The method of claim 21, wherein the volume ratio of methanol to ethanol in the mixed solvent is 15:0.4-0.6.
23. The method of claim 22, wherein the volume ratio of methanol to ethanol in the mixed solvent is 15:0.5.
24. A method of preparing the co-crystal of any one of claims 1 to 11, comprising:
a) Dissolving tecovirimide and a eutectic ligand in a solvent, and stirring;
b) The solid sample was isolated.
25. The method of claim 24, further comprising:
c) The solid sample was dried.
26. The method of claim 24, wherein the solvent is at least one selected from the group consisting of water, alcohol solvents, ester solvents, ketone solvents, ether solvents, nitrile solvents, alkane solvents, haloalkane solvents,
the alcohol solvent is at least one selected from the group consisting of methanol, ethanol and isopropanol;
the ester solvent is at least one selected from the group consisting of methyl acetate, ethyl acetate and propyl acetate;
the ketone solvent is at least one selected from the group consisting of acetone and methyl isobutyl ketone;
the ether solvent is at least one selected from the group consisting of methyl tertiary butyl ether, cyclopentyl methyl ether and tetrahydrofuran;
the nitrile solvent is acetonitrile;
the alkane solvent is n-hexane;
the halogenated hydrocarbon solvent is dichloromethane.
27. The method of claim 26, wherein the solvent is n-hexane.
28. Use of the co-crystal of any one of claims 1 to 11 in the manufacture of a medicament for the treatment of smallpox.
29. Use of a pharmaceutical composition according to claim 12 or 13 for the preparation of a medicament or vaccine for the treatment and/or prevention of smallpox.
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