CN115887464A - Tofacitinib citrate calcium carbonate lipid nano spray as well as preparation method and application thereof - Google Patents
Tofacitinib citrate calcium carbonate lipid nano spray as well as preparation method and application thereof Download PDFInfo
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- CN115887464A CN115887464A CN202211313390.9A CN202211313390A CN115887464A CN 115887464 A CN115887464 A CN 115887464A CN 202211313390 A CN202211313390 A CN 202211313390A CN 115887464 A CN115887464 A CN 115887464A
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- calcium carbonate
- tofacitinib citrate
- lipid
- spray
- amorphous calcium
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- 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 125
- 229910000019 calcium carbonate Inorganic materials 0.000 title claims abstract description 105
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- 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
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 pharmaceutical salts thereof in preparation of medicines for protecting hair follicles and/or preventing alopecia areata and/or promoting hair regeneration. The present invention also provides a pharmaceutical formulation comprising: 1-4 wt% of tofacitinib citrate; 2-8 wt% of calcium carbonate nanoparticles; 1-5 wt% of phospholipid; 0.5-4 wt% of a lipid material; 2 to 6 weight percent of surfactant; the balance being water. The tofacitinib citrate-containing amorphous calcium carbonate lipid nano-spray has good biocompatibility and self pH sensitivity and can control the release of the medicine; the lipid material in the lipid nanoparticle has affinity to hair follicles, can improve the targeting effect and permeability of tofacitinib citrate on the hair follicles, and promotes the growth and development of the hairs.
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 which is clinically common in dermatology and is generally characterized by rapid and complete hair loss in one or more well-defined circular or oval areas. The prevalence rate of alopecia areata is about 1-2%, the alopecia is more common in young and strong years, and the psychological, working and social activities of patients are greatly influenced. Current treatment options for alopecia areata include drug therapy, photochemotherapy and bioimmunotherapy, but the above treatment options also have some drawbacks in the treatment of alopecia areata. For example, oral or topical administration of the sebaceous steroid clobetasol propionate may cause epifolliculitis and rare adverse reactions such as skin atrophy, and the epifolliculitis and the rare adverse reactions are easy to relapse after stopping administration; photochemotherapy, such as psoralen in combination with ultraviolet a (PUVA) therapy, has significantly promoted hair regrowth in bald areas in some studies but with high recurrence rates, and long-term use results in unacceptably high uv accumulation; immunotherapy such as topical application of antrocin can lead to irritant dermatitis, require long-term maintenance of the therapy, have limited efficacy, and the like.
The etiology of alopecia areata is currently not completely understood, and may involve a combination of genetic susceptibility and environmental cues, with evidence supporting the etiology as an autoimmune mechanism predominating, and it is believed that disruption of the immune privileged function of the hair follicle is one of the important pathogenesis. In alopecia areata patients, the inflammatory factor IFN- γ plays a major role in this disease process, and this pathway is mediated by JAK kinases, and therefore JAK inhibitor therapy represents a new treatment option for alopecia areata, and only barretinib (JAK inhibitor) is currently approved for alopecia areata indications. Tofacitinib (TFB) belongs to one of JAK inhibitors, and was approved by the U.S. Food and Drug Administration (FDA) for use in the treatment of moderate-severe rheumatoid arthritis with methotrexate intolerance or poor response in 2012.
Therefore, there is a need for a medicament and a preparation thereof for effectively treating alopecia areata.
Disclosure of Invention
The inventor finds that the tofacitinib citrate has a small molecular structure and is easy to permeate into the skin in the research process, and local administration may be a beneficial means for treating alopecia areata and can avoid the side effect of oral administration; however, the penetration effect of the drug is not satisfactory due to the barrier function of the skin, and there are problems that the individual difference of hair growth promotion is large and the effect is not satisfactory.
The technical development of hair loss prevention and hair fixation from chemical treatment to surgical hair transplantation has gone for nearly 60 years. With the rapid rise of nanotechnology nowadays, the inventors try to solve core problems such as scalp hair follicle arousal and repair by delivering active ingredients through a nano-targeting technology. The amorphous calcium carbonate lipid nanoparticles are selected as active ingredient carriers, so that the active ingredient carriers have good hair follicle targeting property and transdermal efficiency, and the drug subcutaneous and hair follicle accumulation concentration is increased through hair follicle targeted delivery; the structure of the hair follicle determines that the hair follicle can be used as a weakly acidic warehouse for storing the medicine, and the medicine is controlled to release and activate a signal path related to hair papilla cells under an acidic condition, so that the growth of the hair in a alopecia areata area is promoted, the curative effect of the medicine is improved, and the adverse reaction of the medicine to the whole body is reduced.
Aiming at the problem in the aspect of application of tofacitinib citrate in locally treating alopecia areata part hair regeneration, the invention provides an amorphous calcium carbonate lipid nano-spray containing tofacitinib citrate for promoting alopecia areata hair regeneration, the lipid nano-particles have hair follicle targeting and pH-sensitive drug release control capability, locally-delivered drugs have higher intradermal retention and deeper hair follicle diffusion depth, and compared with a tofacitinib citrate solution, the lipid nano-particles have a better effect of promoting alopecia areata part hair growth.
The present invention aims to solve at least to some extent one of the technical problems of the prior art, and therefore, in a first aspect of the invention, the invention provides the use of tofacitinib citrate and/or a pharmaceutically acceptable salt thereof for the preparation 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) 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) calcium carbonate nanoparticles;
2-2.5% (w/v) of phospholipid;
1% (w/v) of a lipid material;
1.5% (w/v) of a 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 lauryl sulfate;
the balance being pure water.
In one or more embodiments of the present invention, the pharmaceutical formulation is in the form of a spray. Preferably, the dosage form of the pharmaceutical preparation is 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 nanoparticles are 200nm, and the tofacitinib citrate is loaded on the amorphous calcium carbonate nanoparticles.
In one or more embodiments of the present invention, the phospholipid is selected from at least one of distearoyl lecithin, didecyl lecithin, egg yolk lecithin, and soybean lecithin; preferably, the lipid material is selected from at least one of glyceryl monostearate, stearic acid, and polyglyceryl fatty acid ester.
In one or more embodiments of the invention, the mass ratio of phospholipid to lipid material is 0.5 to 4; preferably 2 to 2.5.
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 lauryl sulfate.
In a third aspect of the invention, there is provided the use of a pharmaceutical formulation as described in the second aspect of the invention in 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 invention, there is provided a process for the preparation of a pharmaceutical formulation according to the second aspect of the invention, comprising the steps of:
1) Weighing tofacitinib citrate, amorphous calcium carbonate nanoparticles, phospholipid, a lipid material and a 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 the tofacitinib citrate solution is cooled, and stirring to obtain a solution of amorphous calcium carbonate nanoparticles loaded with tofacitinib citrate;
4) Slowly adding the lipid mixture obtained in the step 2) into the amorphous calcium carbonate nanoparticle solution loaded with tofacitinib citrate obtained in the step 3);
5) And (3) carrying out rotary evaporation treatment on the reaction liquid obtained in the step 4), preparing a surfactant into a 1-4 wt% aqueous solution, adding the aqueous solution into the mixed liquid after the rotary evaporation treatment, fixing the volume, and carrying out ultrasonic treatment to obtain the medicinal preparation.
In one or more embodiments of the present invention, in the step 2), the ultrasonic treatment is ultrasonic treatment at a power of 800 to 1600W for 10 to 30 minutes;
preferably, in the step 3), the heating, condensing and refluxing is carried out at the temperature of 50-65 ℃ for 0.5-2 h; the stirring is carried out for 2 to 4 hours at the temperature of between 30 and 50 ℃;
preferably, in the step 4), the reaction is carried out for 2 to 4 hours at the temperature of between 35 and 50 ℃ with stirring;
preferably, in the step 5), the rotary steaming treatment is rotary steaming at 38-50 ℃ for 30-60 minutes; the ultrasonic treatment is carried out for 10-30 minutes under the condition of 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 a medicinal salt thereof in preparing a medicament 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 carriers, 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 the targeting effect and permeability of tofacitinib citrate on the hair follicle, and increase the retention of tofacitinib citrate in skin and hair follicle. Meanwhile, the hair follicle similar to a funnel-shaped structure can be used as a weakly acidic storage drug warehouse, the release and activation of tofacitinib citrate are controlled under an acidic condition, hair papilla cells are stimulated, JAK1/3 channels are inhibited, the expression of IFN-gamma is reduced, the hair follicle epithelial cells are prevented from being identified and attacked by killer T cells, the symptom of alopecia areata is relieved, and the growth and development of hair are promoted.
3. The invention also provides a preparation method of the tofacitinib citrate-containing amorphous calcium carbonate lipid nano spray, and the preparation method is simple to operate and easy to industrialize.
Drawings
FIG. 1 is a graph showing particle size diagrams of tofacitinib citrate amorphous calcium carbonate lipid nanospray samples 3A,3B and 3C prepared in example 3, in order from left to right;
fig. 2 is a transmission electron microscope image of tofacitinib citrate amorphous calcium carbonate lipid nano-spray 3B prepared in example 3;
fig. 3 is a graph of the in vitro stability results of tofacitinib citrate amorphous calcium carbonate lipid nano-spray 3B prepared in example 3;
fig. 4 is a graph showing the in vitro release behavior results of tofacitinib citrate amorphous calcium carbonate lipid nano-spray 3B prepared in example 3 at different pH values;
fig. 5 is a schematic diagram of the permeation behavior of the amorphous calcium carbonate lipid nano-carrier in vitro pig ear skin hair follicles, wherein fig. 5a is a transverse cutting diagram, and fig. 5b is a longitudinal cutting diagram;
FIG. 6 is a graph showing the protection and repair experiment of human hair follicle organs;
fig. 7 is an evaluation graph of promoting the hair regeneration of a alopecia areata model mouse, wherein fig. 7a is a comparison of the hair regeneration of the alopecia areata model mouse for each treatment group; figure 7b is the alopecia areata model mouse hair regrowth score for each treatment group;
in the figures, where asterisks are indicated in the figures, i.e., values for significance differences P were calculated by Graphpad Prism 8, where P < 0.1, P < 0.01, P < 0.001, P < 0.0001, and ns were no significance differences.
Detailed Description
The present invention is further illustrated by the following examples and figures, but the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The following examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer, by using conventional methods known in the art without specific descriptions, and by using consumables and reagents which were commercially available without specific descriptions. Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
Example 1
The embodiment provides an amorphous calcium carbonate lipid nano-spray containing tofacitinib citrate for promoting the regeneration of alopecia areata hair, which comprises 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) of a polyglycerin fatty acid ester;
5) 1.5% (w/v) sodium lauroylaminoethylsulphate;
6) The balance of pure water;
the embodiment also provides a preparation method of an amorphous calcium carbonate lipid nano-spray containing tofacitinib citrate for promoting the regeneration of alopecia areata hair, which comprises the following steps:
1) Weighing 450mg of egg yolk lecithin and 200mg of polyglycerol fatty acid ester, dissolving in 100mL of chloroform, and carrying out 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 2h at 60 ℃, adding 400mg of amorphous calcium carbonate nanoparticles with the particle size of 200nm after the tofacitinib citrate solution is cooled, and stirring for 2h at 40 ℃ to obtain a solution containing calcium carbonate nanoparticles loaded with tofacitinib citrate;
3) Transferring the lipid mixture obtained in the step 1) into a constant-pressure funnel, slowly adding the lipid mixture into the solution containing the tofacitinib citrate-loaded calcium carbonate nanoparticles obtained in the step 2), stirring while adding, and reacting at 40 ℃ for 2 hours to obtain a mixed solution;
4) And (3) carrying out 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)% of lauroyl aminoethyl sodium sulfate solution for hydration, carrying out ultrasonic treatment at the power of 800W for 30min, 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 embodiment provides an amorphous calcium carbonate lipid nano-spray containing tofacitinib citrate for promoting the regeneration of alopecia areata hair, which comprises the following components:
1) 2% (w/v) tofacitinib citrate;
2) 2% (w/v) amorphous calcium carbonate nanoparticles;
3) 2% (w/v) distearoyl lecithin;
4) 1% (w/v) stearic acid;
5) 1.5% (w/v) of 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 the regeneration of alopecia areata hair, which comprises the following steps:
1) Weighing 400mg of distearoyl lecithin and 100mg of stearic acid, dissolving in 100mL of chloroform, and performing ultrasonic treatment for 30 minutes under the condition of ultrasonic wave with the power of 800W to obtain a lipid mixture;
2) Adding 400mg of tofacitinib citrate into 100mL of absolute ethyl alcohol, condensing and refluxing for 2h at 60 ℃, adding 400mg of amorphous calcium carbonate nanoparticles with the particle size of 200nm after the tofacitinib citrate solution is cooled, and stirring for 2h at 40 ℃ to obtain a solution of calcium carbonate nanoparticles loaded with tofacitinib citrate;
3) Transferring the lipid mixture obtained in the step 1) into a constant-pressure funnel, slowly adding the lipid mixture into the solution of the tofacitinib citrate-loaded calcium carbonate nanoparticles obtained in the step 2), stirring while adding, and reacting for 2 hours at 40 ℃ to obtain a mixed solution;
4) And 3) carrying out 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 sodium fatty acid solution to hydrate, carrying out ultrasonic dispersion for 30min under the ultrasonic wave with the power of 800W, and fixing the 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 being pure water
Comprises the following steps
1) Weighing three parts of 400mg of soybean lecithin and 200mg of glyceryl monostearate respectively, dissolving in 100mL of chloroform respectively, and performing ultrasonic treatment for 30 minutes under the condition of the power of 800W to obtain a lipid mixture;
2) Adding three 200mg tofacitinib citrate into 100mL of absolute ethanol, condensing and refluxing for 2h at 60 ℃, preparing three same batches of tofacitinib citrate ethanol solutions after the tofacitinib citrate solution is cooled, adding 400mg of amorphous calcium carbonate nanoparticles with the particle sizes of 100nm, 200nm and 400nm into the ethanol solutions, and stirring for 2h 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 and slowly adding the solution 1,2 and 3 of the tofacitinib citrate-loaded calcium carbonate nanoparticles obtained in the step 2), stirring while adding, and reacting for 2 hours at 40 ℃ to obtain a mixture 1,2 and 3;
4) And (3) carrying out 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, carrying out ultrasonic dispersion for 30min under the condition of 800W ultrasonic wave power, and fixing the 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. Characterization of particle size and morphology of tofacitinib citrate amorphous calcium carbonate lipid nano spray
The tofacitinib citrate amorphous calcium carbonate lipid nanospray samples 3a,3b and 3C obtained in example 3 above were diluted 100-fold respectively, and the particle sizes of the tofacitinib citrate amorphous calcium carbonate lipid nanospray samples 3a,3b and 3C were 145.9nm,286nm and 459nm, respectively, and the polydispersity numbers (PDIs) were 0.121,0.254 and 0.312, respectively, as determined by using a laser particle sizer of marvens, usa, and the results are shown in fig. 1; FIG. 1 is a particle size diagram of samples 3A,3B and 3C of tofacitinib citrate amorphous calcium carbonate lipid nanospray prepared in example 3, in order from left to right.
The tofacitinib citrate amorphous calcium carbonate lipid nano-spray sample 3B obtained in the above example 3 was diluted by 10 times, mixed and dyed with 2% phosphotungstic acid at a volume ratio of 1.
B. Stability investigation of tofacitinib citrate amorphous calcium carbonate lipid nano-spray
The method for preparing 3 batches of tofacitinib citrate amorphous calcium carbonate lipid nano-spray sample 3B is as described in 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 are measured in 1 day, 3 days, 5 days, 7 days, 15 days and 30 days, and the result is shown in fig. 3.
Example 5: in-vitro drug release evaluation of tofacitinib citrate amorphous calcium carbonate lipid nano-spray
And (3) evaluating the in-vitro drug release behavior of the tofacitinib citrate amorphous calcium carbonate lipid nano spray in release media with different pH values by a dialysis method. Taking 3 parts of tofacitinib citrate amorphous calcium carbonate lipid nano-spray sample 3B (2 mL) obtained in the example 3 into a dialysis bag (molecular weight is 3500 Da), respectively placing the dialysis bag into 150mL of PBS buffer solution (pH 5.5/6.5/7.4), vibrating at constant temperature under the conditions that the temperature is 32 ℃ and the rotating speed is 100rpm, taking 2mL of release medium at the time points of 0.5h, 1h, 2h, 4h, 8h, 12h and 24h, supplementing the same amount of release medium in time, and calculating the cumulative release amount of tofacitinib citrate by using an ultraviolet spectrophotometry. As can be seen from fig. 4, the cumulative release amount of tofacitinib citrate is obviously increased with the decrease of pH, and the cumulative release amount of tofacitinib citrate in the release medium in the acidic environment (pH = 5.5) is 2 times of that in the release medium in the neutral environment (pH = 7.4), so that the release behavior is well controlled in the pH sensitivity.
Example 6: in-vitro transdermal evaluation of tofacitinib citrate amorphous calcium carbonate lipid nano-spray
Preparing a tofacitinib citrate solution: adding 200mg of tofacitinib citrate into 20mL of 1.5% (W/v) SDS solution, and performing ultrasonic dispersion for 30 minutes under the ultrasonic wave with the power of 800W to obtain 1% (W/v) tofacitinib citrate solution.
Using fresh C57BL/6 mouse dorsal skin, treating and fixing between a supply pool and a receiving pool of a Franz diffusion pool, using PBS buffer solution (0.1m, ph = 6.8) as a receiving solution, respectively taking 1mL of tofacitinib citrate amorphous calcium carbonate lipid nanospray samples 1,2, and 3a,3b, and 3c obtained in example 1, example 2, and example 3 to act on the skin cutin layer of the supply pool, comparing with the same volume of 1% (w/v) of tofacitinib citrate solution, performing transdermal experiment under magnetic stirring (800 rpm) at 32 ℃, and respectively taking 2mL of release medium and supplementing the same volume of fresh blank medium at set time points of 0.5h, 1h, 2h, 4h, 8h, 12h, and 24 h; and respectively measuring the osmotic quantity of the tofacitinib citrate in the release medium of each sample at different time points and the retention quantity of the tofacitinib citrate in the skin after 24 hours of release by adopting a high performance liquid chromatography.
The results are shown in table 1, comparing tofacitinib citrate amorphous calcium carbonate lipid nanospray samples 1,2 and 3B prepared with different lipid nanospray formulations, and show that: the steady transdermal speed and the skin retention amount of the tofacitinib citrate amorphous calcium carbonate lipid nano-spray prepared by the prescription in the example 3 are obviously higher than those of the samples prepared by the prescriptions in the examples 1 and 2 in 24 hours, but the tofacitinib citrate amorphous calcium carbonate lipid nano-spray (carrier group) is obviously higherHigher than the control tofacitinib solution group, indicating that the amorphous calcium carbonate lipid nano has better transdermal and subcutaneous target site accumulation capacity, the amorphous calcium carbonate lipid nano spray formula of embodiment 3 is preferred; compared with a control group tofacitinib citrate solution, the tofacitinib citrate amorphous calcium carbonate lipid nano-spray (carrier group) can obviously enhance the steady transdermal rate and intradermal retention of tofacitinib citrate, and the steady transdermal rate of the tofacitinib citrate lipid nano-spray 3B is 25.13 mu g cm -2 ·h -1 The retention time of the tofacitinib citrate in the solution group is 1.8 times that of the tofacitinib citrate in the solution group, and the retention time of the tofacitinib citrate in the solution group is 339.0 mu g/g and 3.9 times that of the tofacitinib citrate in the solution group within 24 hours. In addition, single factor investigation shows that the tofacitinib citrate lipid nano-spray prepared from amorphous calcium carbonate with different particle sizes has significant difference on two transdermal evaluation indexes of steady transdermal rate and 24h skin retention, and the lipid nano-spray of tofacitinib citrate prepared from amorphous calcium carbonate with 200nm has 1.31 times and 1.56 times of those prepared from amorphous calcium carbonate with 100nm and 400nm respectively on the skin retention of 24h, which indicates that the lipid nano-spray of tofacitinib citrate prepared from amorphous calcium carbonate with 200nm stores more drugs at a subcutaneous target site, and the formula of the embodiment 3B is preferred.
Table 1 tofacitinib solution and amorphous calcium carbonate lipid nanospray in vitro transdermal results (n = 3)
Example 7: permeation behavior of amorphous calcium carbonate lipid nano-carrier in-vitro pig ear skin hair follicle
The preparation method of sample 3B in example 3 was followed except that 20mg of rhodamine B was used in place of tofacitinib citrate to prepare rhodamine B amorphous calcium carbonate lipid nanoparticles.
Selecting fresh pig ear skin (taken from a local slaughterhouse), cleaning with cold water, wiping with a paper towel, trimming pig ear hair to 1-2 mm, marking 3 areas of 2cm multiplied by 3cm, respectively coating 100 mu L PBS (a control group), 0.1% (w/v) rhodamine B solution (a rhodamine B solution group) and 0.1% (w/v) rhodamine B amorphous calcium carbonate lipid nanoparticles (a rhodamine B lipid nanocarrier group) on different areas of 1,2 and 3, then incubating at a constant temperature of 32 ℃ for 6h, cleaning redundant rhodamine B on the skin surface by using a phosphate buffer solution, separating skin tissues in the medicine application area from the lower cartilage by using a scalpel, preparing transversely and longitudinally cut skin frozen sections by using a freezing microtome, and observing the depth of fluorescence diffusion to hair follicles by using a fluorescence microscope. The results are shown in fig. 5, wherein fig. 5a is a cross-sectional view of different treatment groups, and fig. 5B is a longitudinal-sectional view of different treatment groups, and it can be seen from the cross-sectional view of fig. 5a that 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 nanoparticles can reach the sebaceous gland part (700 μm) of hair follicle, indicating that the penetration effect of the amorphous calcium carbonate lipid nanocarrier group is good, the drug can be delivered to deeper part of hair follicle, and good hair follicle permeability and hair follicle targeting are shown; fig. 5b shows deeper penetration ability and stronger fluorescence intensity of the fluorescently labeled amorphous calcium carbonate lipid nanocarrier in the longitudinal section of hair follicles of different treatment groups.
Example 8: test for protection and repair of citric acid tofacitinib 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) classes I and II can be detected only under the interference of immune collapse of the hair follicle; in the experiment, 24 hair follicles of the complete growth type IV of the scalp of a healthy male person are taken and divided into 8 groups, and each group comprises 3 hair follicles. Namely four repair groups: a blank group, an interferon-gamma treatment group, a tofacitinib citrate solution group and a tofacitinib citrate amorphous calcium carbonate lipid nano-spray group; four protection groups: a blank group, an interferon-gamma treatment group, a tofacitinib citrate solution group and a tofacitinib citrate amorphous calcium carbonate lipid nano-spray group; adding a blank culture medium into a blank group in the repair group for incubation for 6 days, treating the hair follicles by using interferon-gamma for 4 days to induce immune collapse, and then respectively adding the blank culture medium, the tofacitinib citrate solution and the amorphous calcium carbonate lipid nano-spray sample B prepared in the example for incubation for 2 days; the treatment modes of the hollow white group and the repairing group in the protective group are the same, the hair follicles of the other three groups are treated by using a blank culture medium, a tofacitinib citrate solution group and a tofacitinib citrate lipid nano-spray for 2 days respectively, and then interferon-gamma is added into each group for 4 days. After the treatment of the protection group and the repair group is finished, the hair follicles are taken out to be embedded and frozen into slices, and the immunohistochemical fluorescent staining is carried out to detect the expression of MHC-I molecules. The results are shown in fig. 6, and it can be seen from the graph that the tofacitinib citrate amorphous calcium carbonate lipid nano-spray group or tofacitinib citrate solution group does not repair damaged hair follicles, but can protect the hair follicles from IFN-gamma damage, which is of great significance for preventing alopecia areata.
Example 9: influence of tofacitinib citrate amorphous calcium carbonate lipid nano-spray on cyclophosphamide-induced alopecia areata model mouse hair regeneration
The amorphous calcium carbonate lipid nano-spray sample 3B containing tofacitinib citrate obtained in the step 4 of the example 3 is taken as a sample to examine the influence of the sample on the hair regeneration of mice of a cyclophosphamide-induced alopecia areata model.
20C 57BL/6 mice (7 weeks old, female, body mass 18 + -2 g, provided by Wuhan disease and control center), were anesthetized by intraperitoneal injection of phenobarbital sodium, shaved with a shaver, and the depilated area was 2cm × 3cm, followed by applying depilatory cream to the depilated area, and after waiting for 2min, washed with PBS. After shaving, when the skin on the back of the mouse begins to turn black, the normal control group (4 mice) is not treated, the mice in the other groups are injected with 150mg/kg of cyclophosphamide in a single dose abdominal cavity, and the hair on the head and the back of the mice begin to fall after 4-5 days, so that the serious hair fall is successfully induced, and the animal model for long-term baldness at birth is prepared. The model mice are randomly divided into 4 groups, each group comprises 4 mice, namely a model group, a tofacitinib citrate solution group (solution group), a commercially available tofacitinib citrate oral group and a tofacitinib citrate amorphous calcium carbonate lipid nano-spray group (lipid nano-carrier group). The model group, the tofacitinib citrate solution group and the tofacitinib citrate amorphous calcium carbonate lipid nano-spray group are respectively and locally administered with physiological saline, 1% (w/v) tofacitinib citrate solution group, 0.1mL of each of 1% (w/v) tofacitinib citrate amorphous calcium carbonate lipid nano-spray sample B, and the commercially available oral group is intragastrically filled with 3% (w/v) tofacitinib suspension 1mL (the tofacitinib tablet is prepared into 3% (w/v) suspension by using 0.9% NaCl as a solvent), and the change of the back hair of the mouse is observed by photographing for 21 days after continuous administration. Finally, analysis was performed using Image J, and data were quantified for black hair regrowth on day 21 of dosing and scored. The scoring standard is grade 0: no new black hair regeneration; stage 1: the new hair black accounts for 0-20% of the total area of the alopecia; and 2, stage: regeneration of 20-40% of black hair; and 3, level: 40-60% of new black hair regeneration; 4, level: 60-80% black hair regeneration: and 5, stage: 80-100% of black hair regeneration.
The results are shown in FIG. 7a, where the rates of blackening of hair growth were as follows: the normal group > tofacitinib citrate amorphous calcium carbonate lipid nano-spray group > commercial oral group > model group, which shows that the tofacitinib citrate amorphous calcium carbonate lipid nano-spray group after molding has better hair regeneration effect in a mouse alopecia areata area than the tofacitinib citrate amorphous calcium carbonate lipid nano-spray group and the commercial oral group, and proves the superiority of the amorphous calcium carbonate lipid nano-carrier; after 21 days of administration, the result of the black hair regeneration score of the mice is shown in figure 7b, the normal group is more than or equal to the tofacitinib citrate amorphous calcium carbonate lipid nano-spray group, the commercially available oral group, the tofacitinib citrate solution group and the model group, after treatment is finished, the black hair regeneration score of the mice with the 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 commercially available oral group, which shows that the tofacitinib citrate amorphous calcium carbonate lipid nano-spray has better hair regeneration induction effect on cyclophosphamide induced alopecia areata model mice.
Although the embodiments of the present invention have been shown and described, it is understood that the above embodiments are illustrative and not restrictive, and that those skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention and that they should be included in the protection scope of the present invention.
Claims (10)
1. Application of tofacitinib citrate and/or pharmaceutically acceptable salts thereof in preparing medicines for protecting hair follicles and/or preventing alopecia areata and/or promoting hair regeneration.
2. A pharmaceutical formulation, comprising:
1-4% (w/v) tofacitinib citrate;
2-8% (w/v) 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.
3. The pharmaceutical formulation of claim 2, wherein the pharmaceutical formulation is in the form of a spray; preferably, the dosage form of the pharmaceutical preparation is a lipid nanoparticle spray.
4. The pharmaceutical formulation according to claim 2, wherein the calcium carbonate nanoparticles have a particle size of 100 to 400nm; preferably, the amorphous calcium carbonate nanoparticles are 200nm, and the tofacitinib citrate is loaded on the calcium carbonate nanoparticles.
5. The pharmaceutical formulation of claim 2, wherein the phospholipid is selected from at least one of distearoyl lecithin, didecyl lecithin, egg yolk lecithin, and soybean lecithin; preferably, the lipid material is selected from at least one of glyceryl monostearate, stearic acid, and polyglyceryl fatty acid ester.
6. The pharmaceutical formulation according to claim 2, wherein the mass ratio of phospholipid to lipid material is 0.5 to 4.
7. The pharmaceutical formulation of claim 2, wherein the surfactant is selected from at least one of sodium lauroyl aminoethyl sulfate, sodium sulfated fatty acid, and sodium lauryl sulfate.
8. Use of a pharmaceutical formulation according to any one of claims 2 to 7 in the manufacture of a medicament for protecting hair follicles and/or preventing alopecia areata and/or promoting hair regrowth.
9. A process for the preparation of a pharmaceutical formulation according to any one of claims 2 to 7, comprising the steps of:
1) Weighing tofacitinib citrate, calcium carbonate nanoparticles, phospholipid, a lipid material and a 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 the tofacitinib citrate solution is cooled, and stirring to obtain a solution of calcium carbonate nanoparticles loaded with tofacitinib citrate;
4) Slowly adding the lipid mixture obtained in the step 2) into the solution of the calcium carbonate nanoparticles loaded with tofacitinib citrate obtained in the step 3);
5) And (3) carrying out rotary evaporation treatment on the reaction liquid obtained in the step 4), preparing a surfactant into a 1-4 wt% aqueous solution, adding the aqueous solution into the mixed liquid after the rotary evaporation treatment, fixing the volume, and carrying out ultrasonic treatment to obtain the medicinal preparation.
10. The method for preparing a pharmaceutical preparation according to claim 9, wherein the sonication in step 2) is carried out for 10 to 30 minutes under ultrasonic waves with a power of 800 to 1600W;
preferably, in the step 3), the heating, condensing and refluxing is carried out at the temperature of 50-65 ℃ for 0.5-2 h; the stirring is carried out for 2 to 4 hours at the temperature of between 30 and 50 ℃;
preferably, in the step 4), the reaction is carried out for 2 to 4 hours at the temperature of between 35 and 50 ℃ with stirring;
preferably, in the step 5), the rotary steaming treatment is rotary steaming at 38-50 ℃ for 30-60 minutes; the ultrasonic treatment is carried out for 10-30 minutes under the condition of ultrasonic wave with the power of 800-1600W.
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| 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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