CN115887464B - Tofacitinib citrate calcium carbonate lipid nano spray and preparation method and application thereof - Google Patents
Tofacitinib citrate calcium carbonate lipid nano spray and preparation method and application thereof Download PDFInfo
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- CN115887464B CN115887464B CN202211313390.9A CN202211313390A CN115887464B CN 115887464 B CN115887464 B CN 115887464B CN 202211313390 A CN202211313390 A CN 202211313390A CN 115887464 B CN115887464 B CN 115887464B
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- calcium carbonate
- tofacitinib citrate
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- 229960004247 tofacitinib citrate Drugs 0.000 title claims abstract description 134
- SYIKUFDOYJFGBQ-YLAFAASESA-N tofacitinib citrate Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O.C[C@@H]1CCN(C(=O)CC#N)C[C@@H]1N(C)C1=NC=NC2=C1C=CN2 SYIKUFDOYJFGBQ-YLAFAASESA-N 0.000 title claims abstract description 131
- 229910000019 calcium carbonate Inorganic materials 0.000 title claims abstract description 113
- 239000007921 spray Substances 0.000 title claims abstract description 53
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Cosmetics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides tofacitinib citrate calcium carbonate lipid nano spray and a preparation method and application thereof, and particularly provides application of tofacitinib citrate and/or medicinal salt thereof in preparing medicines for protecting hair follicles and/or preventing alopecia areata and/or promoting hair regeneration. The present invention also provides a pharmaceutical formulation comprising: 1-4wt% tofacitinib citrate; 2-8 wt% of calcium carbonate nanoparticles; 1 to 5wt% of a phospholipid; 0.5 to 4wt% of a lipid material; 2-6wt% of surfactant; the balance being water. The tofacitinib citrate-containing amorphous calcium carbonate lipid nano spray has good biocompatibility and pH sensitivity, and can control drug release; the lipid material in the lipid nanoparticle has affinity to hair follicle, can improve targeting effect and permeability of tofacitinib citrate to hair follicle, and promote hair growth and development.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a calcium carbonate lipid nano spray containing tofacitinib citrate, and a preparation method and application thereof.
Background
Alopecia areata is a sudden, non-scarring inflammatory alopecia disease that is clinically common to dermatology and is generally characterized by the rapid and complete shedding of hair in one or more well-defined circular or oval areas. The prevalence of alopecia areata is about 1-2%, and the incidence is more common in young and old, which has a great influence on the psychological, work and social activities of patients. Current treatments for alopecia areata include drug therapy, photochemotherapy and biological immunotherapy, but these treatments have some drawbacks in the treatment of alopecia areata. For example, oral administration or topical use of the corticosteroid clobetasol propionate may cause folliculitis and rare skin atrophy, and may be prone to recurrence after drug withdrawal; photochemotherapy, such as the use of psoralen in combination with ultraviolet a (PUVA) therapy, has in some studies been shown to significantly promote hair regrowth in the bald areas but at a high rate of recurrence and long-term use can result in unacceptably high uv accumulation; immunotherapy such as topical application of aztrelin can lead to irritant dermatitis, requires prolonged maintenance of therapy, has limited efficacy, etc.
The etiology of alopecia areata is currently not completely understood, and may involve a combination of genetic susceptibility and environmental predisposition, where evidence supporting the pathogenesis of autoimmune mechanisms predominates, and the destruction of the immune privilege function of hair follicles is considered to be one of the important pathogenesis. In alopecia areata patients, the inflammatory factor IFN- γ plays an important role in this disease process, and this pathway is mediated by JAK kinase, so JAK inhibitor therapy represents a new treatment option for alopecia areata, and only barytanib (JAK inhibitor) is currently approved for alopecia areata indications. Tofacitinib (TFB) belongs to one of the JAK inhibitors and was approved by the united states Food and Drug Administration (FDA) in 2012 for use in the treatment of moderate to severe rheumatoid arthritis where methotrexate is intolerant or poorly responsive.
Therefore, it is necessary to provide a drug and a preparation thereof which can effectively treat alopecia areata.
Disclosure of Invention
The inventor finds that the tofacitinib citrate has small molecular structure and is easy to permeate in skin in the research process, and local administration is a beneficial means for treating alopecia areata, so that the side effect of oral administration can be avoided; however, the barrier function of the skin is unsatisfactory in the permeation effect of the drug, and there are problems that the individual difference in hair growth promotion is large and the effect is unsatisfactory.
From chemical drug therapy to surgical hair-planting therapy, the technical development of hair loss prevention and hair fixation has been carried out for nearly 60 years. With the rapid rise of nanotechnology nowadays, the inventor tries to deliver active ingredients through a nanotargeting technology to solve core problems such as scalp hair follicle arousal, repair and the like. The amorphous calcium carbonate lipid nanoparticle is selected as an active ingredient carrier, has better hair follicle targeting and transdermal efficiency, and increases the subcutaneous and hair follicle accumulation concentration of the drug through hair follicle targeting delivery; the hair follicle itself has a structure which determines that the hair follicle can be used as a weak acid warehouse for storing medicines, controls the medicine to release and activate the relevant signal paths of hair papilla cells under the acid condition, promotes the hair growth in the alopecia areata area, improves the medicine treatment effect and reduces the systemic adverse reaction of the medicines.
Aiming at the problem of application of tofacitinib citrate in the aspect of locally treating hair regeneration at the alopecia areata, the invention provides an amorphous calcium carbonate lipid nano spray containing tofacitinib citrate for promoting hair regeneration at the alopecia areata, the lipid nano particles have hair follicle targeting and pH sensitive controlled drug release capabilities, and the locally delivered drugs have higher intradermal retention and deeper hair follicle diffusion depth and have better effect of promoting hair growth at the alopecia areata compared with a tofacitinib citrate solution.
The present invention aims to solve, at least to some extent, one of the technical problems existing in the prior art, whereby, in a first aspect of the invention, the present invention provides the use of tofacitinib citrate and/or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for protecting hair follicles and/or preventing alopecia areata and/or promoting hair regrowth.
In a second aspect of the invention, the invention provides a pharmaceutical formulation comprising:
1-4% (w/v) tofacitinib citrate;
2-8% (w/v) of calcium carbonate nanoparticles;
1-5% (w/v) of a phospholipid;
0.5-4% (w/v) of a lipid material;
1-6% (w/v) of a surfactant;
the balance being pure water.
Preferably, the pharmaceutical formulation comprises:
1-2% (w/v) tofacitinib citrate;
2% (w/v) of calcium carbonate nanoparticles;
2-2.5% (w/v) of a phospholipid;
1% (w/v) of a lipid material;
1.5% (w/v) surfactant;
the balance being pure water.
More preferably, the pharmaceutical formulation comprises:
1% (w/v) tofacitinib citrate;
2% (w/v) amorphous calcium carbonate nanoparticles;
2% (w/v) soybean lecithin;
1% (w/v) glyceryl monostearate;
1.5% (w/v) sodium dodecyl sulfate;
the balance being pure water.
In one or more embodiments of the invention, the pharmaceutical formulation is in the form of a spray. Preferably, the dosage form of the pharmaceutical formulation is a lipid nanospray.
In one or more embodiments of the present invention, the calcium carbonate nanoparticles are amorphous calcium carbonate nanoparticles having a particle size of 100 to 400nm; preferably, the amorphous calcium carbonate nanoparticle is 200nm, and the tofacitinib citrate is loaded on the amorphous calcium carbonate nanoparticle.
In one or more embodiments of the present invention, the phospholipid is selected from at least one of distearoyl lecithin, didecanoyl lecithin, egg yolk lecithin, and soybean lecithin; preferably, the lipid material is selected from at least one of glyceryl monostearate, stearic acid, polyglyceryl fatty acid ester.
In one or more embodiments of the invention, the phospholipid and lipid materials are present in a mass ratio of 0.5 to 4:1; preferably 2 to 2.5:1.
In one or more embodiments of the present invention, the surfactant is selected from at least one of sodium lauroyl aminoethyl sulfate, sodium sulfated fatty acid, and sodium dodecyl sulfate.
In a third aspect of the invention, the present invention provides the use of a pharmaceutical formulation according to the second aspect of the invention for the manufacture of a medicament for protecting hair follicles and/or preventing alopecia areata and/or promoting hair regrowth.
In a fourth aspect of the present invention, there is provided a method of preparing a pharmaceutical formulation as described in the second aspect of the present invention, comprising the steps of:
1) Weighing tofacitinib citrate, amorphous calcium carbonate nanoparticles, phospholipid, lipid material and surfactant;
2) Dissolving phospholipid and lipid material in chloroform, and performing ultrasonic treatment to obtain lipid mixture;
3) Adding tofacitinib citrate into ethanol, heating, condensing and refluxing, adding amorphous calcium carbonate nanoparticles after cooling the tofacitinib citrate solution, and stirring to obtain a solution of amorphous calcium carbonate nanoparticles loaded with tofacitinib citrate;
4) Slowly adding the lipid mixture in the step 2) into the solution of the amorphous calcium carbonate nanoparticle loaded with tofacitinib citrate in the step 3);
5) Performing rotary steaming treatment on the reaction solution obtained in the step 4), preparing a surfactant into a 1-4wt% aqueous solution, adding the aqueous solution into the mixed solution after the rotary steaming treatment, fixing the volume, and performing ultrasonic treatment to obtain the pharmaceutical preparation.
In one or more embodiments of the present invention, in the step 2), the ultrasonic treatment is ultrasonic at a power of 800 to 1600W for 10 to 30 minutes;
preferably, in the step 3), the heating, condensing and refluxing is performed for 0.5 to 2 hours at a temperature of between 50 and 65 ℃; stirring for 2-4 h at 30-50 ℃;
preferably, in the step 4), the reaction is carried out for 2-4 hours under stirring at 35-50 ℃;
preferably, in the step 5), the spin-steaming treatment is spin-steaming for 30 to 60 minutes at 38 to 50 ℃; the ultrasonic treatment is carried out for 10-30 minutes under the ultrasonic wave with the power of 800-1600W.
The invention has the beneficial effects that:
1. the invention provides application of tofacitinib citrate and/or medicinal salt thereof in preparing medicines for protecting hair follicles and/or preventing alopecia areata and/or promoting hair regeneration.
2. The invention provides an amorphous calcium carbonate lipid nano spray containing tofacitinib citrate, which adopts amorphous calcium carbonate nanoparticles as a carrier, has good biocompatibility, is easy to synthesize and has pH sensitivity; the lipid material in the lipid nanoparticle has affinity to hair follicle, can improve targeting effect and permeability of tofacitinib citrate to hair follicle, and increase retention of tofacitinib citrate in skin and hair follicle. Meanwhile, the hair follicle itself can be used as a storage of weak acid stored medicines, and the release activation of tofacitinib citrate is controlled under the acid condition to stimulate hair papilla cells, inhibit JAK1/3 channels, reduce IFN-gamma expression, prevent hair follicle epithelial cells from being identified and attacked by killer T cells, relieve the symptoms of alopecia areata and promote hair growth and development.
3. The invention also provides a preparation method of the tofacitinib citrate-containing amorphous calcium carbonate lipid nano spray, which is simple to operate and easy to industrialize.
Drawings
Fig. 1 shows particle size diagrams of tofacitinib citrate amorphous calcium carbonate lipid nano-spray samples 3a,3b and 3C prepared in example 3 from left to right;
fig. 2 is a transmission electron microscope image of tofacitinib citrate amorphous calcium carbonate lipid nanospray 3B prepared in example 3;
FIG. 3 is a graph showing the in vitro stability results of tofacitinib citrate amorphous calcium carbonate lipid nanospray 3B prepared in example 3;
fig. 4 is a graph showing the results of in vitro drug release behavior of tofacitinib citrate amorphous calcium carbonate lipid nanospheres 3B prepared in example 3 at different pH;
FIG. 5 is a schematic diagram showing permeation behavior of an amorphous calcium carbonate lipid nanocarrier in an isolated porcine ear skin hair follicle, wherein FIG. 5a is a transverse drawing and FIG. 5b is a longitudinal drawing;
FIG. 6 is a protection and repair experiment of human follicular organs;
FIG. 7 is a graph showing evaluation of hair regeneration in a model alopecia areata mouse, wherein FIG. 7a is a comparison of hair regeneration in a model alopecia areata mouse for each treatment group; fig. 7b is hair regeneration scores for model mice with alopecia areata for each treatment group;
in the figures, the asterisks are obtained by calculating the significance difference P value by Graphpad Prism 8, wherein P is less than 0.1, P is less than 0.01, P is less than 0.001, P is less than 0.0001, and ns is no significance difference.
Detailed Description
The invention is further described below in conjunction with the specific examples and figures, which are provided to illustrate the invention and should not be construed to limit the scope of the invention. The following examples are conducted under conventional conditions or conditions recommended by the manufacturer, and the methods used are conventional methods known in the art, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any method or material similar or equivalent to those described may be used in the present invention.
Example 1
The present example provides an amorphous calcium carbonate lipid nanospray containing tofacitinib citrate for promoting hair regrowth of alopecia areata, comprising the following components:
1) 1% (w/v) tofacitinib citrate;
2) 2% (w/v) amorphous calcium carbonate nanoparticles;
3) 2.5% (w/v) egg yolk lecithin;
4) 1% (w/v) polyglycerol fatty acid ester;
5) 1.5% (w/v) sodium lauroyl aminoethyl sulfate;
6) The balance being pure water;
the embodiment also provides a preparation method of the amorphous calcium carbonate lipid nano spray containing tofacitinib citrate for promoting alopecia areata hair regeneration, which comprises the following steps:
1) Weighing 450mg of egg yolk lecithin, dissolving 200mg of polyglycerol fatty acid ester in 100mL of chloroform, and performing ultrasonic treatment for 30 minutes under the ultrasonic wave with the power of 800W to obtain a lipid mixture;
2) Adding 200mg of tofacitinib citrate into 100mL of absolute ethyl alcohol, condensing and refluxing for 2 hours at 60 ℃, adding 400mg of amorphous calcium carbonate nanoparticles with the particle size of 200nm after cooling the tofacitinib citrate solution, and stirring for 2 hours at 40 ℃ to obtain a solution containing the calcium carbonate nanoparticles loaded with tofacitinib citrate;
3) Transferring the lipid mixture in the step 1) into a constant pressure funnel, slowly adding the lipid mixture into the solution containing the calcium carbonate nanoparticles loaded with tofacitinib citrate obtained in the step 2), stirring while adding, and reacting for 2 hours at 40 ℃ to obtain a mixed solution;
4) And (3) performing rotary evaporation on the mixed solution obtained in the step (3) at 40 ℃ for 30min to remove an organic phase (ethanol and chloroform) to form a film, adding 20mL of 1.5 (W/v)% lauroyl aminoethyl sodium sulfate solution for hydration, performing ultrasonic treatment for 30min under 800W ultrasonic wave, dispersing, and fixing the volume to 20mL to obtain the amorphous calcium carbonate lipid nano spray sample 1 loaded with tofacitinib citrate.
Example 2
The present example provides an amorphous calcium carbonate lipid nanospray containing tofacitinib citrate for promoting hair regrowth of alopecia areata, comprising the following components:
1) 2% (w/v) tofacitinib citrate;
2) 2% (w/v) amorphous calcium carbonate nanoparticles;
3) 2% (w/v) distearyl lecithin;
4) 1% (w/v) stearic acid;
5) 1.5% (w/v) sodium sulfated fatty acid;
6) The balance being pure water;
the embodiment also provides a preparation method of the amorphous calcium carbonate lipid nano spray containing tofacitinib citrate for promoting alopecia areata hair regeneration, which comprises the following steps:
1) 400mg of distearyl lecithin and 100mg of stearic acid are weighed and dissolved in 100mL of chloroform, and the lipid mixture is obtained by ultrasonic treatment for 30 minutes under the ultrasonic wave with the power of 800W;
2) Adding 400mg of tofacitinib citrate into 100mL of absolute ethyl alcohol, condensing and refluxing for 2 hours at 60 ℃, adding 400mg of amorphous calcium carbonate nanoparticles with the particle size of 200nm after cooling the tofacitinib citrate solution, and stirring for 2 hours at 40 ℃ to obtain a solution of calcium carbonate nanoparticles loaded with tofacitinib citrate;
3) Transferring the lipid mixture in the step 1) into a constant pressure funnel, slowly adding the lipid mixture into the solution of the tofacitinib citrate-loaded calcium carbonate nanoparticle in the step 2), stirring while adding, and reacting for 2 hours at 40 ℃ to obtain a mixed solution;
4) And (3) performing rotary evaporation on the mixed solution obtained in the step (3) at 40 ℃ for 30min to remove an organic phase (ethanol and chloroform) to form a film, adding 20mL of 1.5 (W/v)% sulfated fatty acid sodium solution for hydration, performing ultrasonic dispersion for 30min under the ultrasonic wave with the power of 800W, and performing constant volume to 20mL to obtain the amorphous calcium carbonate lipid nano spray sample 2 loaded with tofacitinib citrate.
Example 3
The embodiment provides a preparation method of an amorphous calcium carbonate lipid nano spray containing tofacitinib citrate for promoting alopecia areata hair regeneration, which comprises the following components:
1) 1% (w/v) tofacitinib citrate
2) 2% (w/v) amorphous calcium carbonate nanoparticles
3) 2% (w/v) soybean lecithin
4) 1% (w/v) glyceryl monostearate
5) 1.5% (w/v) sodium lauryl sulfate
6) The balance of pure water
Comprises the following steps
1) Respectively weighing three parts of 400mg soybean lecithin and 200mg glyceryl monostearate, respectively dissolving in 100mL chloroform, and performing ultrasonic treatment under 800W ultrasonic wave for 30min to obtain lipid mixture;
2) Adding three 200mg of tofacitinib citrate into 100mL of absolute ethyl alcohol, condensing and refluxing for 2 hours at 60 ℃, preparing the same three batches of tofacitinib citrate ethanol solution after the tofacitinib citrate solution is cooled, adding 400mg of amorphous calcium carbonate nanoparticles with the particle diameters of 100, 200 and 400nm into the ethanol solution, and stirring for 2 hours at 40 ℃ to obtain solutions 1,2 and 3 of calcium carbonate nanoparticles loaded with tofacitinib citrate;
3) Transferring the lipid mixture obtained in the step 1) into a constant pressure funnel, respectively slowly adding the lipid mixture into the solution 1,2 and 3 of the tofacitinib citrate-loaded calcium carbonate nanoparticle obtained in the step 2), stirring while adding, and reacting for 2 hours at 40 ℃ to obtain the mixture 1,2 and 3;
4) And (3) performing rotary evaporation on the mixtures 1,2 and 3 obtained in the step (3) at 40 ℃ for 30min to remove organic phases (ethanol and chloroform), adding 20mL of 1.5 (W/v)% sodium dodecyl sulfate solution for hydration, performing ultrasonic dispersion for 30min under 800W ultrasonic wave, and performing constant volume to 20mL to obtain amorphous calcium carbonate lipid nano spray samples 3A,3B and 3C loaded with tofacitinib citrate.
Example 4: evaluation of characteristics of tofacitinib citrate amorphous calcium carbonate lipid nano spray
A. Particle size and morphology characterization of tofacitinib citrate amorphous calcium carbonate lipid nano spray
The tofacitinib citrate amorphous calcium carbonate lipid nanospray spray samples 3a,3b and 3C obtained in the above example 3 were diluted 100 times respectively, and the particle diameters were measured by using a laser particle sizer of malva company in the united states, and the particle diameters of the tofacitinib citrate amorphous calcium carbonate lipid nanospray spray samples 3a,3b and 3C were 145.9nm, 284 nm and 459nm, respectively, and the polydispersity index (PDI) was 0.121,0.254 and 0.312, respectively, and the results are shown in fig. 1; fig. 1 shows particle size diagrams of tofacitinib citrate amorphous calcium carbonate lipid nano-spray samples 3a,3b and 3C prepared in example 3 from left to right.
The tofacitinib citrate amorphous calcium carbonate lipid nano-spray sample 3B obtained in the above example 3 is diluted 10 times, mixed and dyed with 2% phosphotungstic acid according to a volume ratio of 1:1, and then dripped on a copper mesh to observe the morphology by a transmission electron microscope, and the result is shown in fig. 2, the tofacitinib citrate amorphous calcium carbonate lipid nano-particles are in a similar sphere shape with uniform size.
B. Stability investigation of tofacitinib citrate amorphous calcium carbonate lipid nano spray
3 batches of tofacitinib citrate amorphous calcium carbonate lipid nano-spray samples 3B are prepared according to the method described in the example 3, three batches of tofacitinib citrate amorphous calcium carbonate lipid nano-spray are placed in a refrigerator at 4 ℃ and the particle size and the dispersion coefficient of the tofacitinib citrate amorphous calcium carbonate lipid nano-spray samples are measured for 1 day, 3 days, 5 days, 7 days, 15 days and 30 days, the result is shown in figure 3, and the particle size and the dispersion of the tofacitinib citrate amorphous calcium carbonate lipid nano-spray samples are unchanged within one month, so that the tofacitinib citrate amorphous calcium carbonate lipid nano-spray samples have better stability.
Example 5: in vitro drug release evaluation of tofacitinib citrate amorphous calcium carbonate lipid nano spray
And evaluating the in vitro drug release behavior of the tofacitinib citrate amorphous calcium carbonate lipid nano spray in different pH release media by adopting a dialysis method. 3 parts of the tofacitinib citrate amorphous calcium carbonate lipid nano spray sample 3B (2 mL) obtained in the example 3 are taken and placed in dialysis bags (molecular weight is 3500 Da), respectively placed in 150mL of PBS buffer solution (pH 5.5/6.5/7.4), and are vibrated at constant temperature under the conditions that the temperature is 32 ℃ and the rotating speed is 100rpm, and the release mediums are taken at the time points of 0.5h, 1h, 2h, 4h, 8h, 12h and 24h respectively, 2mL of the release mediums are timely supplemented with the same release mediums, and the accumulated release amount of tofacitinib citrate is calculated by using an ultraviolet spectrophotometry. As can be seen from fig. 4, the accumulated release amount of tofacitinib citrate increases significantly with decreasing pH, and the accumulated release amount of tofacitinib citrate in the release medium in an acidic environment (ph=5.5) is 2 times that in the neutral release medium (ph=7.4), and the pH-sensitive controlled release behavior is good.
Example 6: in vitro transdermal evaluation of tofacitinib citrate amorphous calcium carbonate lipid nanospray
Preparing tofacitinib citrate solution: 200mg of tofacitinib citrate is added into 20mL of 1.5% (W/v) SDS solution, and the solution is dispersed for 30 minutes under the ultrasonic wave with the power of 800W, so as to obtain 1% (W/v) tofacitinib citrate solution.
Using fresh C57BL/6 mouse back skin, fixing between a supply tank and a receiving tank of a Franz diffusion tank after treatment, taking 1mL of PBS buffer (0.1 m, ph=6.8) as a receiving solution, taking 2mL of release medium respectively from 1mL, 2mL and 3a,3b,3C of tofacitinib citrate amorphous calcium carbonate lipid nanospray samples obtained in example 1, example 2 and example 3, acting on a skin cutin layer of the supply tank, comparing to 1% (w/v) tofacitinib citrate solution with the same volume, performing transdermal experiments under magnetic stirring (800 rpm) at 32 ℃, and taking 2mL of release medium respectively at set time points of 0.5h, 1h, 2h, 4h, 8h, 12h and 24h while supplementing the same volume of fresh blank medium; and (3) respectively measuring the permeation quantity of tofacitinib citrate in the release medium at different time points of each sample and the retention quantity of tofacitinib citrate in the skin after 24 hours of release by adopting a high performance liquid chromatography.
The results are shown in table 1, and compared with different lipid nanospray formulations to prepare tofacitinib citrate amorphous calcium carbonate lipid nanospray samples 1,2 and 3B, the results show that: the tofacitinib citrate amorphous calcium carbonate lipid nanospray spray prepared by adopting the prescription of the example 3 has significantly higher steady-state transdermal rate and 24 hours skin retention than the samples prepared by the prescriptions of the examples 1 and 2, but the tofacitinib citrate amorphous calcium carbonate lipid nanospray spray (carrier group) is significantly higher than the control tofacitinib solution group, which shows that the amorphous calcium carbonate lipid nanospray has better transdermal and subcutaneous target site accumulation capacity, and the amorphous calcium carbonate lipid nanospray spray prescription of the example 3 is preferred; compared with a control group tofacitinib citrate solution, the tofacitinib citrate amorphous calcium carbonate lipid nano-spray (carrier group) can significantly enhance the steady-state transdermal rate and intradermal retention of tofacitinib citrate, and the steady-state transdermal rate of tofacitinib citrate lipid nano-spray 3B is 25.13 mug cm -2 ·h -1 1.8 times of the tofacitinib citrate solution group (solution group), and the 24-hour intradermal retention is 339.0 mug/g, which is 3.9 times of the tofacitinib citrate solution group. In addition, single factor investigation shows that the tofacitinib citrate lipid nano spray prepared by amorphous calcium carbonate with different particle sizes has obvious difference in two transdermal evaluation indexes of steady-state transdermal rate and 24h skin retention, and the tofacitinib citrate prepared by amorphous calcium carbonate with 200nm is also preparedThe lipid nanospray of tinib was 1.31 times and 1.56 times that of 100nm and 400nm respectively at 24h skin hold up, indicating that tofacitinib citrate amorphous calcium carbonate lipid nanospray prepared with 200nm amorphous calcium carbonate accumulated more drug at subcutaneous target sites, preferred example 3B formulation.
Table 1 in vitro transdermal results of tofacitinib solution and amorphous calcium carbonate lipid nanosprays (n=3)
Example 7: osmotic behavior of amorphous calcium carbonate lipid nanocarriers in vitro in porcine ear skin hair follicles
The preparation method of sample 3B in example 3 was followed, except that 20mg of rhodamine B was used instead of tofacitinib citrate to prepare rhodamine B amorphous calcium carbonate lipid nanoparticles.
Fresh pig ear skin (from a local slaughterhouse) was selected, washed with cold water, wiped dry with paper towel, pig ear hair was trimmed to 1-2 mm, and 2cm x 3cm area 3 pieces were marked, 100 μl PBS (control group) was applied to each of the different areas 1,2,3, 0.1% (w/v) rhodamine B solution (rhodamine B solution group) and 0.1% (w/v) rhodamine B amorphous calcium carbonate lipid nanoparticle (rhodamine B lipid nanocarrier group), then incubated at constant temperature of 32 ℃ for 6 hours, excess rhodamine B on the skin surface was washed with phosphate buffer, and skin tissue in the drug application area was carefully separated from underlying cartilage with a scalpel, transected and longitudinally cut skin frozen sections were prepared using a frozen microtome, and fluorescence diffusion was observed to the depth of hair follicles using a fluorescence microscope. The results are shown in fig. 5, wherein fig. 5a is a cross-sectional view of the different treatment groups, fig. 5B is a longitudinal cutting view of the different treatment groups, and as can be seen from the cross-sectional view of fig. 5a, rhodamine B in the skin of the rhodamine B solution group is mainly distributed on the skin surface and stratum corneum (100 μm), while rhodamine B in the rhodamine B amorphous calcium carbonate lipid nanoparticle can reach sebaceous gland sites (700 μm) of hair follicles, which indicates that the amorphous calcium carbonate lipid nanocarrier group has good permeation effect, can deliver drugs deeper into hair follicles, and shows good hair follicle permeability and hair follicle targeting; fig. 5b shows a deeper penetration and a stronger fluorescence intensity of the fluorescently labeled amorphous calcium carbonate lipid nanocarriers in the longitudinal cut of hair follicles of different treatment groups.
Example 8: protection and repair test of tofacitinib citrate amorphous calcium carbonate lipid nano spray on human hair follicle organs
In the human scalp hair follicle, the expression of a large number of Major Histocompatibility Complex (MHC) class I and II can only be detected under the interference of the immune breakdown of the hair follicle; the experiment takes 24 hair follicles of which the scalp grows completely in normal male healthy people IV, and the hair follicles are divided into 8 groups of 3 hair follicles. I.e. repair group four: blank group, interferon-gamma treated group, tofacitinib citrate solution group and tofacitinib citrate amorphous calcium carbonate lipid nano-spray group; protection group four: blank group, interferon-gamma treated group, tofacitinib citrate solution group and tofacitinib citrate amorphous calcium carbonate lipid nano-spray group; adding a blank culture medium into a repairing group for incubation for 6 days, treating hair follicles with interferon-gamma for 4 days to induce immune collapse, and then respectively adding a blank culture medium, tofacitinib citrate solution and tofacitinib citrate amorphous calcium carbonate lipid nano-spray sample B prepared in the example for incubation for 2 days; the hollow white group in the protective group and the repair group are treated in the same way, the rest three groups are treated with blank culture medium, tofacitinib citrate solution group and tofacitinib citrate lipid nanometer spray for 2 days respectively, and then interferon-gamma is added into each group to interfere for 4 days. After the treatment of the protective group and the repair group is finished, hair follicles are taken out for embedding and frozen sections, and immunohistochemical fluorescent staining is carried out for detecting the expression of MHC-I molecules. As shown in fig. 6, the amorphous calcium carbonate lipid nano-spray or tofacitinib citrate solution group does not repair damaged hair follicles, but can protect hair follicles from IFN- γ, which is important for preventing alopecia areata.
Example 9: influence of tofacitinib citrate amorphous calcium carbonate lipid nano spray on hair regeneration of cyclophosphamide-induced alopecia areata model mice
The effect of the amorphous calcium carbonate lipid nanospray sample 3B containing tofacitinib citrate obtained in step 4 of example 3 above on hair regeneration of cyclophosphamide-induced alopecia areata model mice was examined as a sample.
C57BL/6 mice 20 (7 weeks old, female, quality 18+ -2 g, martial arts disease and control center supply), after the mice were anesthetized with phenobarbital sodium, the depilation area was 2cm×3cm using shaving Mao Qiti wool, then the depilation area was coated with depilation cream, and after waiting for 2min, washed with PBS. After shaving, when the skin at the back of the mice starts to be blackened, the normal control group (4 mice) does not carry out any treatment, the other groups of mice are subjected to single-dose intraperitoneal injection of cyclophosphamide of 150mg/kg, and after 4-5 days, the head and back hair of the mice start to fall off, and serious hair fall off is successfully induced, so that the growth-period baldness animal model is prepared. The model mice were randomly divided into 4 groups of 4, i.e., model group, tofacitinib citrate solution group (solution group), commercially available tofacitinib citrate oral group and tofacitinib citrate amorphous calcium carbonate lipid nanospray group (lipid nanocarrier group). In the model group, physiological saline is locally administrated to the skin of a back dehairing area every day in a tofacitinib citrate solution group and a tofacitinib citrate amorphous calcium carbonate lipid nanometer spray agent group respectively, 0.1mL of each tofacitinib citrate amorphous calcium carbonate lipid nanometer spray sample B is 1% (w/v), 1mL of tofacitinib suspension (3% (w/v) tofacitinib tablet is prepared into a suspension of 3% (w/v) by using 0.9% NaCl as a solvent) is administrated continuously for 21 days, and the change of the hair on the back of a mouse is observed by photographing. Finally, image J was used for analysis, and black hair regrowth at day 21 of dosing was quantified and scored. The scoring standard is grade 0: no new black hair regeneration; stage 1: the new-born black hair accounts for 0-20% of the total area of alopecia; 2 stages: regenerating 20-40% of black hair; 3 stages: regenerating black hair by 40-60%;4 stages: regeneration of black hair of 60-80 percent: 5 stages: 80-100% of black hair is regenerated.
As a result, as shown in fig. 7a, the rate of blackening hair growth was: the normal group > tofacitinib citrate amorphous calcium carbonate lipid nano spray group > tofacitinib citrate solution group > commercial oral group > model group, which shows that the tofacitinib citrate amorphous calcium carbonate lipid nano spray group has better hair regeneration effect in the mouse alopecia areata area compared with the tofacitinib citrate solution group and the commercial oral group after modeling, and proves the superiority of preparing the amorphous calcium carbonate lipid nano carrier; the black hair regeneration scoring result of the mice after 21 days of administration is shown in fig. 7b, the normal group is more than or equal to tofacitinib citrate amorphous calcium carbonate lipid nano spray group > commercial oral group > tofacitinib citrate solution group > model group, after treatment is finished, the black hair regeneration scoring of the mice in tofacitinib citrate amorphous calcium carbonate lipid nano spray group is close to that of the normal group, and the effect of the amorphous calcium carbonate lipid nano carrier group is better than that of the commercial oral group, which indicates that the tofacitinib citrate amorphous calcium carbonate lipid nano spray has better hair regeneration induction effect on cyclophosphamide-induced alopecia areata model mice.
While embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are illustrative and not to be construed as limiting the present invention, and that variations, modifications, alternatives and variations of the above embodiments may be made by those skilled in the art within the scope of the present invention and are intended to be included within the scope of the present invention.
Claims (11)
1. A pharmaceutical formulation, the pharmaceutical formulation comprising:
1-4% (w/v) tofacitinib citrate;
2-8% (w/v) of calcium carbonate nanoparticles;
1-5% (w/v) of a phospholipid;
0.5-4% (w/v) of a lipid material;
1-6% (w/v) of a surfactant;
the balance being water;
the calcium carbonate nanoparticles are amorphous calcium carbonate nanoparticles, and the tofacitinib citrate is loaded on the calcium carbonate nanoparticles;
the phospholipid is at least one of distearoyl lecithin, didecanoyl lecithin, egg yolk lecithin and soybean lecithin; the lipid material is at least one of glyceryl monostearate, stearic acid and polyglyceryl fatty acid ester;
the mass ratio of the phospholipid to the lipid material is 0.5-4:1;
the surfactant is at least one selected from lauroyl aminoethyl sodium sulfate, sulfated fatty acid sodium and sodium dodecyl sulfate;
the preparation method of the pharmaceutical preparation comprises the following steps:
1) Weighing tofacitinib citrate, calcium carbonate nanoparticles, phospholipid, lipid material and surfactant;
2) Dissolving phospholipid and lipid material in chloroform, and performing ultrasonic treatment to obtain lipid mixture;
3) Adding tofacitinib citrate into ethanol, heating, condensing and refluxing, adding calcium carbonate nanoparticles after cooling the tofacitinib citrate solution, and stirring to obtain a solution of the calcium carbonate nanoparticles loaded with tofacitinib citrate;
4) Slowly adding the lipid mixture in the step 2) into the solution of the tofacitinib citrate-loaded calcium carbonate nanoparticles in the step 3);
5) Performing rotary steaming treatment on the reaction solution obtained in the step 4), preparing a surfactant into a 1-4wt% aqueous solution, adding the aqueous solution into the mixed solution after the rotary steaming treatment, fixing the volume, and performing ultrasonic treatment to obtain the pharmaceutical preparation.
2. The pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation is in the form of a spray.
3. The pharmaceutical formulation of claim 1, wherein the formulation of the pharmaceutical formulation is a lipid nanoparticle spray.
4. The pharmaceutical formulation according to claim 1, wherein the mean particle size of the calcium carbonate nanoparticles is 100-400 nm.
5. The pharmaceutical formulation according to claim 4, wherein the average particle size of the calcium carbonate nanoparticles is 200nm.
6. Use of a pharmaceutical formulation according to any one of claims 1 to 5 for the preparation of a medicament for protecting hair follicles and/or preventing alopecia areata and/or promoting hair regrowth.
7. A method of preparing a pharmaceutical formulation according to any one of claims 1 to 5, comprising the steps of:
1) Weighing tofacitinib citrate, calcium carbonate nanoparticles, phospholipid, lipid material and surfactant;
2) Dissolving phospholipid and lipid material in chloroform, and performing ultrasonic treatment to obtain lipid mixture;
3) Adding tofacitinib citrate into ethanol, heating, condensing and refluxing, adding calcium carbonate nanoparticles after cooling the tofacitinib citrate solution, and stirring to obtain a solution of the calcium carbonate nanoparticles loaded with tofacitinib citrate;
4) Slowly adding the lipid mixture in the step 2) into the solution of the tofacitinib citrate-loaded calcium carbonate nanoparticles in the step 3);
5) Performing rotary steaming treatment on the reaction solution obtained in the step 4), preparing a surfactant into a 1-4wt% aqueous solution, adding the aqueous solution into the mixed solution after the rotary steaming treatment, fixing the volume, and performing ultrasonic treatment to obtain the pharmaceutical preparation.
8. The method of preparing a pharmaceutical formulation according to claim 7, wherein in step 2), the ultrasonic treatment is ultrasonic treatment at a power of 800 to 1600W for 10 to 30 minutes.
9. The method of preparing a pharmaceutical formulation according to claim 7, wherein in step 3), the heating, condensing and refluxing is performed at 50-65 ℃ for 0.5-2 hours; the stirring is carried out for 2-4 h at 30-50 ℃.
10. The method of preparing a pharmaceutical preparation according to claim 7, wherein in the step 4), the reaction is carried out at 35 to 50 ℃ with stirring for 2 to 4 hours.
11. The method of preparing a pharmaceutical formulation according to claim 7, wherein in step 5), the spin-steaming treatment is spin-steaming at 38 to 50 ℃ for 30 to 60 minutes; the ultrasonic treatment is carried out for 10-30 minutes under the ultrasonic wave with the power of 800-1600W.
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| CN113712919A (en) * | 2020-05-25 | 2021-11-30 | 南京帝昌医药科技有限公司 | Preparation of tofacitinib citrate spray for treating alopecia |
| CN113975280A (en) * | 2020-07-27 | 2022-01-28 | 杭州和正医药有限公司 | Pharmaceutical composition containing pharmaceutically acceptable salt of tofacitinib, preparation and application |
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| US10639327B1 (en) * | 2015-11-20 | 2020-05-05 | Washington University | Nano-calcium carbonate |
| CN106138011A (en) * | 2016-08-05 | 2016-11-23 | 浙江理工大学 | Calcium carbonate/hyaluronic acid combination drug carrier and preparation method thereof |
| CN111035768A (en) * | 2018-10-15 | 2020-04-21 | 北京大学 | Small intestine transporter targeted nano calcium carbonate composition for improving oral absorption of insoluble drugs |
| CN113712919A (en) * | 2020-05-25 | 2021-11-30 | 南京帝昌医药科技有限公司 | Preparation of tofacitinib citrate spray for treating alopecia |
| CN113975280A (en) * | 2020-07-27 | 2022-01-28 | 杭州和正医药有限公司 | Pharmaceutical composition containing pharmaceutically acceptable salt of tofacitinib, preparation and application |
| CN111773181A (en) * | 2020-08-03 | 2020-10-16 | 浙江大学 | Simvastatin-loaded bone-targeting composite lipid nanoparticle and application thereof |
| CN112402615A (en) * | 2020-11-11 | 2021-02-26 | 长沙晶易医药科技有限公司 | Compound external preparation for treating alopecia areata and preparation method thereof |
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