CN111632054A

CN111632054A - A pharmaceutical preparation for treating rheumatoid arthritis by transdermal administration, and its preparation method

Info

Publication number: CN111632054A
Application number: CN202010655272.0A
Authority: CN
Inventors: 储晓琴; 吴文青; 蔡叶; 桂双英
Original assignee: Anhui University of Traditional Chinese Medicine AHUTCM
Current assignee: Anhui University of Traditional Chinese Medicine AHUTCM
Priority date: 2020-07-09
Filing date: 2020-07-09
Publication date: 2020-09-08

Abstract

A medicinal preparation for treating rheumatoid arthritis by percutaneous administration and a preparation method thereof, belonging to the technical field of medicaments for treating rheumatoid arthritis. The pharmaceutical composition consisting of sinomenine hydrochloride and cinnamaldehyde in a weight ratio of 1: 0.1-10, one or more pharmaceutically acceptable carriers and excipients are prepared into clinically acceptable external preparations, including carbomer gel, liquid crystal gel, ointment and transdermal patches. The composition of sinomenine hydrochloride and cinnamaldehyde can obviously improve the treatment effect of rheumatoid arthritis, and the cinnamaldehyde not only has the function of anti-inflammatory immunity, but also has the function of promoting the transdermal absorption of sinomenine hydrochloride. Therefore, after the combined medication, the respective administration dosage can be reduced, the treatment effect can be improved, the occurrence of adverse reaction can be reduced, and the compliance of patients can be improved.

Description

A pharmaceutical preparation for treating rheumatoid arthritis by transdermal administration, and its preparation method

Technical Field

The invention belongs to the technical field of rheumatic arthritis treatment medicines, and particularly relates to a pharmaceutical preparation for treating rheumatoid arthritis through transdermal administration and a preparation method thereof.

Background

Rheumatoid arthritis is a common chronic, systemic autoimmune disease. The clinical manifestations are mainly erosive and symmetric polyarthritis. The affected joints are mainly facet joints, the pathological manifestation of main target organs is synovitis accompanied with various extraarticular manifestations, and diseases erode the joints to cause joint damage and joint dysfunction, which are the main causes of disability. At present, the main measures taken by western medicine for treating rheumatoid arthritis are non-steroids, antibiotics, hormones, immunosuppressants or surgical treatment. Although the medicine inhibits the deterioration of the disease to a certain extent, the side effect is large, and the operation treatment cannot completely solve the cause of the disease. Through long-term practice, the advantages of the traditional Chinese medicine in treating the disease are gradually highlighted, the treatment effect is better than that of western medicine, the side effects are small, the implementation is simple, and the Chinese medicine is approved by many patients.

Sinomenine is isoquinoline alkaloid monomer extracted from caulis Sinomenii. The hydrochloride of the compound, namely the sinomenine hydrochloride, is commonly used clinically. Sinomenine hydrochloride has analgesic, tranquilizing, local anesthetic, and anti-inflammatory effects, and can be used for reducing hypertension, and treating rheumatic and rheumatoid arthritis. At present, sinomenine hydrochloride is mainly used for treating rheumatoid arthritis, and the treatment effect is more obvious. However, sinomenine hydrochloride still has a plurality of defects as clinical medication, such as instability to light and heat and easy decomposition, and the defects can cause the curative effect of sinomenine to be reduced. The biological half-life of the sinomenine hydrochloride is short, the sinomenine hydrochloride is mostly taken orally, and the dosage is large; sinomenine hydrochloride is used as a strong histamine releasing agent, and clinical adverse reactions such as rash, gastrointestinal irritation and the like can be caused by the release of histamine.

Cinnamaldehyde is the main effective component of ramulus Cinnamomi, and has antiinflammatory and immunity enhancing effects. The cinnamic aldehyde can also be used as a transdermal absorption enhancer to improve the absorption of skin to drugs, and the combined use of sinomenine and cinnamic aldehyde can significantly reduce the dosage of each drug, increase the curative effect, reduce adverse reaction and improve the compliance of patients.

The self-assembly liquid crystal medicine carrying system has high medicine carrying and medicine releasing performance, and thus has attracted much attention in recent years. The drug delivery system is formed by self-assembly of amphiphilic substances consisting of hydrophilic head groups and hydrophobic long hydrocarbon chains in a solvent, and generally comprises lamellar phases, hexagonal phases and cubic phases. The cubic liquid crystal has a unique double-channel structure which is not communicated with each other, one channel is connected with the outside, the other channel is completely closed, the hexagonal phase consists of densely filled and infinitely long straight water columns or is radially oriented outwards from the center of the water columns through direct interaction in the lipid hydrophobic part, and the specificity of the liquid crystal structure can accommodate drug molecules with various polarities.

In recent years, cubic phase and hexagonal phase can entrap drugs with different properties, and become new hot spots in drug delivery system research. The liquid crystal material has good biocompatibility, controllable drug release, simple preparation, different drug loading behaviors, large cubic phase drug loading, simple preparation and targeting effect, so the liquid crystal material is widely applied to transdermal drug delivery preparations.

Disclosure of Invention

The invention mainly solves the technical problem of providing a composition of sinomenine hydrochloride and cinnamaldehyde which can treat rheumatoid arthritis and has a synergistic effect, the combination of the two medicines can obviously improve the treatment effect of the rheumatoid arthritis, and the cinnamaldehyde not only has the function of anti-inflammatory immunity, but also has the function of promoting the transdermal absorption of the sinomenine hydrochloride. Therefore, after the combined medication, the respective administration dosage can be reduced, the treatment effect can be improved, and the occurrence of adverse reaction can be reduced.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a pharmaceutical composition for treating rheumatoid arthritis by percutaneous administration comprises Sinomenine Hydrochloride (SH) and Cinnamaldehyde (CA) in a weight ratio of 1: 0.1-10.

The pharmaceutical composition is mixed with one or more pharmaceutically acceptable carriers and excipients to prepare clinically acceptable external preparations, including carbomer gel, liquid crystal gel, ointment and transdermal patch.

According to a preferable technical scheme, the liquid crystal gel is prepared by emulsifying a water phase and an oil phase, the water phase is prepared by dissolving sinomenine hydrochloride in distilled water, and the oil phase is prepared by a lipid material and cinnamaldehyde; the phase composition comprises, by weight, 0.5-2 parts of sinomenine hydrochloride, 0.25-1 part of cinnamaldehyde, 65-75 parts of lipid material and 25-35 parts of distilled water; the method comprises the following specific steps:

(1) precisely weighing sinomenine hydrochloride and distilled water, and performing vortex oscillation to prepare a water phase;

(2) precisely weighing lipid materials, placing the lipid materials in a water bath with the melting temperature corresponding to the melting temperature of the lipid materials, adding cinnamaldehyde after the lipid materials are completely melted, carrying out vortex oscillation, and uniformly mixing to form an oil phase;

(3) mixing the water phase and the oil phase while the solution is hot, vortex and shake the mixture, and standing the mixture for 48 hours at normal temperature to obtain the cubic-phase sinomenine hydrochloride cinnamaldehyde liquid crystal gel.

According to a preferable technical scheme, the liquid crystal gel is prepared by emulsifying a water phase and an oil phase, the water phase is prepared by dissolving sinomenine hydrochloride in distilled water, and the oil phase is prepared by a lipid material, a hexagonal phase inducer and cinnamaldehyde; the phase component comprises, by weight, 0.5-2 parts of sinomenine hydrochloride, 0.5-1 part of cinnamaldehyde, 65-77 parts of lipid material, 3-5 parts of hexagonal phase inducer and 20-30 parts of distilled water; the method comprises the following specific steps:

(2) precisely weighing a lipid material and a hexagonal phase inducer, placing the lipid material and the hexagonal phase inducer in a water bath at the corresponding melting temperature of the lipid material, adding cinnamaldehyde after the lipid material is completely melted, carrying out vortex oscillation, and uniformly mixing to form an oil phase;

(3) mixing the water phase and the oil phase while the solution is hot, vortex and shake the mixture, and standing the mixture for 72 hours at normal temperature to obtain the hexagonal phase sinomenine hydrochloride cinnamaldehyde liquid crystal gel.

Further preferably, the hexagonal phase inducer employs one or more of Tricaprylin (TAG), vitamin E acetate (VitEA), and Paraffin Oil (PO).

Further preferably, the lipid material employs one or more of Phytantriol (PHYT), Glycerol Monooleate (GMO), Monoglyceride (MO) and Sorbitol Monooleate (SMO).

As a preferred technical scheme, according to the weight ratio of carbomer 940, Sinomenine Hydrochloride (SH) and Cinnamaldehyde (CA) in sinomenine hydrochloride and cinnamaldehyde carbomer gel, the gel sequentially comprises the following components in percentage by weight: 1.5-2.5%, 0.5-2%, 0.25-1% to prepare carbomer gel, the specific steps are as follows:

uniformly spreading carbomer 940 in a proper amount of distilled water, standing, and then placing in a water bath for warming for later use; weighing Sinomenine Hydrochloride (SH), adding a proper amount of distilled water to completely dissolve the Sinomenine Hydrochloride (SH), pouring the Sinomenine Hydrochloride (SH) into warm carbomer, and continuously stirring to dissolve the Sinomenine Hydrochloride (SH); adding Cinnamaldehyde (CA) dissolved in a small amount of ethanol, then dropwise adding triethanolamine while stirring to control the pH value to 6.5-7.0, adding distilled water to the required mass, continuously stirring, cooling to room temperature to obtain sinomenine hydrochloride cinnamaldehyde carbomer gel, and storing in a shade container for later use.

As a preferred technical scheme, the ointment comprises the following components in sequence according to the weight ratio of Sinomenine Hydrochloride (SH) and Cinnamaldehyde (CA): 0.5-2% and 0.25-1%, stearic acid, white vaseline, liquid paraffin, glyceryl monostearate and lanolin are used as oil phase components, sodium lauryl sulfate, ethylparaben, glycerol and distilled water are used as water phase components to prepare an ointment, and the preparation method specifically comprises the following steps:

weighing oil phase components in a small beaker, placing the small beaker on a water bath, heating the small beaker to 70-80 ℃ to melt the oil phase components, adding cinnamaldehyde, and stirring uniformly; putting the water phase components into a small beaker, putting the beaker on a water bath, heating the beaker to 70-80 ℃ to dissolve the water phase components, adding sinomenine hydrochloride, and uniformly stirring the sinomenine hydrochloride; adding the water phase component into the oil phase component in a trickle shape under the constant temperature, continuously heating and stirring for 5-10 min on a water bath, and then stirring at room temperature until the mixture is condensed to obtain the sinomenine hydrochloride cinnamaldehyde ointment.

As a preferred technical scheme, the transdermal patch is prepared according to the weight ratio of Sinomenine Hydrochloride (SH) to Cinnamaldehyde (CA) of 0.5-2: 0.25-1, and comprises the following specific steps:

adding sinomenine hydrochloride and a proper amount of polyacrylate pressure-sensitive adhesive into a beaker, adding a proper amount of absolute ethyl alcohol and cinnamaldehyde, stirring to completely dissolve and uniformly mix the medicines, removing bubbles by ultrasonic waves, uniformly coating the medicines on an anti-sticking layer, standing at room temperature, then putting into an oven to be dried, taking out, cooling, and then pressing and coating a backing material to obtain the sinomenine hydrochloride cinnamaldehyde transdermal patch.

Aiming at the defects of short half-life period, low bioavailability, large dosage, gastrointestinal adverse reactions and the like of sinomenine after oral administration in the prior art of rheumatoid arthritis, the invention provides a pharmaceutical composition capable of treating rheumatoid arthritis by transdermal administration, which is prepared from sinomenine hydrochloride and cinnamaldehyde, and also provides an external preparation containing the pharmaceutical composition, including carbomer gel, liquid crystal gel, ointment and transdermal patch. Compared with the prior art, the invention has the beneficial effects that:

(1) after the two medicines are combined, based on the synergistic effect, the dosage of each medicine can be obviously reduced, the curative effect is increased, the adverse reaction is reduced, and the compliance of patients is improved.

(2) The cinnamaldehyde has anti-inflammatory and immunity enhancing effects, can promote percutaneous absorption of sinomenine hydrochloride, can further improve bioavailability of sinomenine hydrochloride, and can reduce administration dosage.

(3) The liquid crystal gel has a microstructure similar to that of a biological membrane, and can increase skin permeation of the medicine and improve bioavailability of the medicine.

(4) The liquid crystal gel preparation for treating rheumatoid arthritis is safe, nontoxic and good in biocompatibility, and the adopted lipid material is comfortable, safe and non-irritant.

(5) The invention has simple manufacturing process and high long-term stability.

Drawings

FIG. 1 is a photograph taken by a polarizing microscope (magnification 100 times) of a cubic liquid crystal gel of sinomenine hydrochloride prepared in example 4.

FIG. 2 is a polarization microscope photograph (magnification 100 times) of sinomenine hydrochloride cinnamaldehyde hexagonal phase liquid crystal gel prepared in example 5.

FIG. 3 is a SAXS detection spectrum of the sinomenine hydrochloride cinnamaldehyde cubic phase liquid crystal gel prepared in example 4.

FIG. 4 is a SAXS detection spectrum of sinomenine hydrochloride cinnamaldehyde hexagonal phase liquid crystal gel prepared in example 5.

FIG. 5 is a graph of the in vitro cumulative release of sinomenine hydrochloride versus time.

Figure 6 is an in vitro cumulative cinnamaldehyde release versus time curve.

FIG. 7 is a graph of cumulative permeation of sinomenine hydrochloride versus time.

FIG. 8 is a cumulative permeation of cinnamaldehyde versus time curve.

FIG. 9 is a graph showing the effect of gel on paw volume in various groups of rats.

FIG. 10 is a graph showing the effect of gels on IL-1 β, IL-6, IL-10, TNF- α production in various groups of rats.

FIG. 11 is a graph showing the effect of gel on histopathological changes in groups of rats. A: a saline control group; b: an AA model group; c: group of Zhengqing Fengtongning tablets (Sin); d: gel 2: 1; e: gel 4: 1; f: gel 6: 1. (HE staining, magnification 40 times; 500 scale. mu.m).

Detailed Description

The pharmaceutical preparation for treating rheumatoid arthritis by transdermal administration and the preparation method thereof according to the present invention will be described in detail with reference to the accompanying drawings and examples.

Example 1

The preparation of the sinomenine hydrochloride cinnamaldehyde ointment comprises the following steps:

the mixing ratio is as follows:

cinnamic aldehyde (0.5%, w/w, the same below).

Sinomenine hydrochloride (1%).

Oil phase components: stearic acid (6.5%), white vaseline (8%), liquid paraffin (10%), glyceryl monostearate (4%), lanolin (4%).

Water phase composition: sodium lauryl sulfate (0.5%), ethylparaben (0.1%), glycerol (7.4%), distilled water (58%).

Accurately weighing the oil phase components according to the mixture ratio, putting the oil phase components into a small beaker, heating the beaker on a water bath to 70-80 ℃ to melt the oil phase components, adding cinnamaldehyde, and stirring uniformly. Putting the water phase component into a small beaker, heating the small beaker on a water bath to 70-80 ℃ to dissolve the water phase component, adding sinomenine hydrochloride, stirring uniformly, adding the water phase component into the oil phase component in a trickle shape under an isothermal condition, continuously heating and stirring on the water bath for 5min, and then stirring at room temperature until the water phase component is condensed to obtain sinomenine hydrochloride (1%) cinnamaldehyde (0.5%) ointment.

Example 2

The preparation of the sinomenine hydrochloride cinnamaldehyde transdermal patch comprises the following steps:

adding 0.1g sinomenine hydrochloride and 1g polyacrylate pressure-sensitive adhesive (Duro-Tak 872677) into a 50mL beaker, adding 10mL absolute ethyl alcohol and 0.05g cinnamaldehyde, stirring for 2h to completely dissolve the medicine and uniformly mix, removing bubbles by ultrasonic wave, uniformly coating on an anti-sticking layer, standing at room temperature for 20min to volatilize the solvent, then placing in a 40 ℃ oven to dry for 30min, taking out, cooling, and then covering a backing material by pressing to obtain the sinomenine hydrochloride cinnamaldehyde transdermal patch.

Example 3

Preparation of sinomenine hydrochloride cinnamaldehyde carbomer gel:

200mg (2%, w/w) of carbomer 940 is precisely weighed and uniformly sprinkled into a small beaker filled with a proper amount of distilled water (about 2.5mL), kept stand for 12 hours, and placed in a water bath to be warmed for later use. Accurately weighing 100mg (1%, w/w) of sinomenine hydrochloride, adding a proper amount of distilled water to completely dissolve the sinomenine hydrochloride, pouring the sinomenine hydrochloride into warm carbomer, and continuously stirring to dissolve the sinomenine hydrochloride into a gel matrix. And then adding 50mg (0.5%, w/w) of cinnamaldehyde dissolved in a small amount of ethanol, dropwise adding triethanolamine while stirring to control the pH value to be about 6.5-7.0, adding distilled water to 10g, continuously stirring, cooling to room temperature to obtain sinomenine hydrochloride (1%) cinnamaldehyde (0.5%) carbomer gel, and storing in a shady and cool place and a lightproof container for later use.

For comparison, in the above method, no cinnamaldehyde was added to prepare carbomer gel-1% SH. ② carbomer gel-0.5 percent CA is prepared without adding sinomenine hydrochloride. ③ without adding sinomenine hydrochloride, adjusting the addition of cinnamaldehyde to 100mg (1 percent) to prepare carbomer gel-1 percent CA.

Example 4

The preparation of the sinomenine hydrochloride cinnamaldehyde cubic phase liquid crystal gel comprises the following steps:

the mixing ratio is as follows: sinomenine hydrochloride 100mg, cinnamaldehyde 50mg, phytantriol 7000mg and distilled water 3000 mg.

The preparation method comprises the following steps:

(1) precisely weighing sinomenine hydrochloride and distilled water in proportion, and performing vortex oscillation for 10min to prepare a water phase.

(2) Accurately weighing phytantriol in proportion, placing in water bath at 60 ℃, adding cinnamaldehyde in proportion after phytantriol is completely melted, vortex vibrating, and mixing to form oil phase.

(3) Mixing the water phase and oil phase while hot, vortex and shaking for 10min, standing at room temperature for 48 hr to obtain sinomenine hydrochloride cinnamaldehyde cubic phase liquid crystal gel (marked as cubic liquid crystal-1% SH + 0.5% CA).

Example 5

Preparing sinomenine hydrochloride cinnamaldehyde hexagonal liquid crystal gel:

the mixing ratio is as follows: 100mg of sinomenine hydrochloride, 100mg of cinnamaldehyde, 7200mg of phytantriol, 400mg of tricaprylin and 2400mg of distilled water. The preparation method comprises the following steps:

(2) Weighing phytantriol and tricaprylin in proportion, putting the phytantriol and tricaprylin in a water bath at 60 ℃, adding cinnamaldehyde in proportion after the phytantriol is completely melted, and performing vortex oscillation and uniform mixing to form an oil phase.

(3) Mixing the water phase and the oil phase while hot, vortexing and shaking for 10min, and standing at room temperature for 72h to obtain sinomenine hydrochloride cinnamaldehyde hexagonal liquid crystal gel (marked as hexagonal liquid crystal-1% SH + 1% CA).

Sinomenine hydrochloride cinnamaldehyde liquid crystal gels prepared in examples 4 and 5 were taken and observed by a polarization microscope, and in order to examine the internal morphology of the gel, a small amount of a sample was placed on a glass slide and covered with a glass cover, and then observed by a polarization microscope at room temperature at a magnification of 100 times. As shown, the cubic phase liquid crystal prepared in example 4 was dark-field (fig. 1), and the hexagonal phase liquid crystal prepared in example 5 was fan-shaped texture-like (fig. 2).

When the sinomenine hydrochloride cinnamaldehyde liquid crystal gels prepared in example 4 and example 5 are subjected to SAXS detection, the SAXS spectrum of the cubic phase liquid crystal prepared in example 4 is shown in FIG. 3, and the ratio of Bragg peaks from left to right can be seen in the spectrum

Shown as cubic phases. The SAXS spectrum of the hexagonal phase liquid crystal prepared in example 6 is shown in FIG. 4, wherein the ratio of Bragg peaks from left to right is shown as

Showing a hexagonal phase. The results were consistent with those observed with a polarizing microscope.

Comparative example 1

The preparation method is substantially the same as that of example 4, and is characterized in that:

firstly, cubic liquid crystal-1% SH is prepared without adding cinnamaldehyde.

② preparing cubic liquid crystal-0.5 percent CA without adding sinomenine hydrochloride.

Comparative example 2

The preparation method is substantially the same as that of example 5, except that:

firstly, preparing hexagonal liquid crystal-1% SH without adding cinnamaldehyde.

② preparing hexagonal liquid crystal-1 percent CA without adding sinomenine hydrochloride.

Example 6

Sinomenine hydrochloride cinnamaldehyde liquid crystal gel and carbomer gel in-vitro release experiment

Firstly, dynamic dialysis bag method test: the liquid crystal gels prepared in examples 4 and 5 and comparative examples 1 and 2, respectively, 0.1g of carbomer gel prepared in example 3, are precisely weighed, placed in a 6 cm-long pretreated dialysis bag, both ends are tied, placed in a centrifuge tube containing 8mL of physiological saline, then placed in an air bath constant temperature oscillator, the temperature is set at 32 ℃, the rotating speed is set at 100r/min, samples are taken according to

time points

1, 2, 4, 6, 12, 24, 36 and 48h (taking hexagonal liquid crystal to 60h), 1mL of release medium with the same volume is supplemented at each time, the samples are filtered through a 0.45 mu m microporous filter membrane, filtrate is taken, HPLC determination is carried out, carbomer gel is used as a control group, and 3 parts of carbomer gel are parallelly distributed in each group.

Secondly, data processing and analysis: cinnamic aldehyde can be oxidized to cinnamic acid, so after measuring CA in release medium and cinnamic acid concentration by established HPLC method, cinnamic acid is converted into cinnamic aldehyde, and the cumulative release amount (Q) of sinomenine hydrochloride and cinnamic aldehyde is calculated_n) And cumulative release rate (Q)_n%) the cumulative release rate of the drug was plotted as a function of time (t).

Wherein C is_nRepresents the concentration of drug in the release medium at each sampling time point, C_iIs the drug concentration of the ith sample, V₀And V_iRepresenting the volume of release medium and sample taken, respectively.

Thirdly, analyzing results:

1. cumulative percent release curve

The cumulative release percentage curves of sinomenine hydrochloride and cinnamaldehyde at different time points are shown in fig. 5 and fig. 6, respectively. The in vitro release results show that:

as can be seen from fig. 5: the sinomenine hydrochloride in the carbomer gel is released fastest within 1-6 h, and most of the medicine is released (82.61%) and is in an obvious burst release phenomenon. The release curves of the cubic liquid crystal and the hexagonal liquid crystal are obviously gentle, and the release amount of the hexagonal liquid crystal is obviously reduced compared with the corresponding cubic liquid crystal.

As can be seen from fig. 6: when the time is 48 hours, the cumulative release amount of the cinnamaldehyde in the cubic liquid crystal containing the cinnamaldehyde is larger than that of the hexagonal phase, the cinnamaldehyde in the cubic liquid crystal and the hexagonal liquid crystal is released quickly in the first 12 hours, the cumulative release amount reaches the peak value from 12 hours to 24 hours, the release amount is reduced, and the cumulative release amount is gradually increased after 36 hours; in carbomer cinnamaldehyde was released faster in the first 6h and most of the drug was released (83.69%), with cinnamaldehyde release entering a plateau after 6 h.

2. Release Curve fitting

The release kinetics fitting result (table 1) of the sinomenine hydrochloride shows that the release of the sinomenine hydrochloride in each preparation conforms to the Higuchi equation, so that each cubic liquid crystal and hexagonal liquid crystal of the sinomenine hydrochloride have a certain slow release effect; with reference to fig. 5, the release amount of the cubic liquid crystal is much higher than that of the hexagonal liquid crystal, and the release percentage of the cubic liquid crystal is 70-90% at the end of 48h, so that the slow release effect of the hexagonal liquid crystal is better than that of the cubic liquid crystal. The carbomer gel is hydrophilic gel, the Higuchi equation can only roughly simulate the release behavior of the carbomer gel, the burst effect exists in the first 6h, the release amount after 6h does not change greatly, and therefore the slow release performance of the cubic liquid crystal is superior to that of the carbomer gel. According to the fitting result of the Ritger-Peppas equation, the release of the sinomenine hydrochloride in the cubic liquid crystal, the hexagonal liquid crystal and the carbomer gel (Cb) meets n <0.45, so that the in-vitro release of the sinomenine hydrochloride in each preparation meets the Fick's diffusion mechanism.

TABLE 1 sinomenine hydrochloride Release kinetics fitting results

②, the cinnamic aldehyde release kinetics fitting results (tables 2 and 3) show that the release of cinnamic aldehyde in cubic liquid crystal basically conforms to the Higuchi equation, so the cubic liquid crystal containing cinnamic aldehyde has a certain slow release function; the release of cinnamaldehyde in the hexagonal liquid crystal basically conforms to a first-order kinetic equation, so the hexagonal liquid crystal containing cinnamaldehyde also has a certain slow release effect. Carbomer gel is hydrophilic gel, and Higuchi can only slightly simulate the release behavior; according to the fitting result of the Ritger-Peppas equation, the cubic liquid crystal gel and the hexagonal liquid crystal gel both meet the condition that n is more than 0.45 and less than 0.89, so that the in-vitro release of the cinnamaldehyde in each preparation conforms to a composite mechanism with the coexistence of diffusion and erosion. The mechanism of releasing the water-soluble substance from the two-component liquid crystal gel is consistent with that of the single-component gel, mainly based on Ficks diffusion, namely greatly influenced by the concentration, the water content and the water channel of the medicine. The release mechanism of the lipophilic substance in the two-component liquid crystal gel is a composite mechanism of diffusion and erosion, and is influenced by various factors such as drug concentration, system bulk density, viscosity and the like.

TABLE 2 cinnamic aldehyde Release kinetics fitting results

TABLE 3 cinnamic aldehyde Release kinetics fitting results-

Example 7

Sinomenine hydrochloride cinnamaldehyde liquid crystal gel and carbomer gel in-vitro transdermal experiment

Firstly, preparing in-vitro skin: taking healthy male rats with weight of 200 +/-20 g, firstly anesthetizing with pentobarbital, then removing abdominal hair with a shaver and depilatory cream, then washing abdominal skin with a large amount of physiological saline, surgically cutting off the skin at the middle part of the abdomen, removing subcutaneous fat and blood vessels, then cleaning with physiological saline, spreading in a preservative film, and freezing and refrigerating for later use.

II, an in-vitro transdermal experimental method: in vitro transdermal experiments were performed using a modified Franz diffusion cell, and about 400mg of the liquid crystal gels prepared in examples 4 and 5 and comparative examples 1 and 2, respectively, were applied to the skin surface, and a carbomer gel was prepared in example 3, and magnetically stirred at 200r/min in a water bath at a temperature of 32 ℃. Sampling 1mL in 0.5, 1, 2, 6, 8, 12, 24, 36 and 48 hours respectively, simultaneously supplementing blank receiving solution with the same temperature and the same volume, filtering the sample by a 0.45 mu m microporous membrane, taking the subsequent filtrate, measuring the concentrations of sinomenine hydrochloride and cinnamaldehyde by HPLC, taking self-made carbomer gel as a control group, and calculating the cumulative permeation quantity Qn (the total permeation quantity Qn of the medicine in the skin of different time points and unit areas), (the total permeation quantity Qn is calculated by taking the self-made carbomer gel as the control groupμg/cm²)

Wherein Q is_nRepresents the amount of the drug per unit area accumulated per transdermal area (. mu.g/cm) at the nth sampling site²) (ii) a V denotes the receiving well volume (24 mL); c_n、C_iRespectively representing the mass concentration (mu g/mL) of the medicine in the receiving liquid of the nth sampling point and the i sampling point; v₀Sample volume (1 mL); a is the area of the diffusion cell (3.14 cm)²)。

Thirdly, analyzing results:

1. cumulative permeation versus time curve

The cumulative permeation of sinomenine hydrochloride versus time at different time points is shown in FIG. 7. The graph shows that the cumulative permeation amount of sinomenine hydrochloride in the cubic phase is larger at 48h, and the next is carbomer gel, which is the lowest in hexagonal liquid crystal. The cumulative permeation of cinnamaldehyde versus time for the different time points is shown in FIG. 8. As can be seen from the figure, the cumulative permeation amount of the carbomer gel is larger at 48h, and then the hexagonal liquid crystal is arranged, and the cubic liquid crystal is the lowest.

2. And (3) fitting a transdermal curve:

fitting a transdermal curve of the drug according to a first-order equation, a Higuchi equation, a zero-order equation and a Ritger-Peppas equation respectively to determine the release property of the drug in each preparation. The permeation kinetics fitting result (table 4) of Sinomenine Hydrochloride (SH) shows that the release of sinomenine hydrochloride in cubic liquid crystal and hexagonal liquid crystal can be fit with a zero-order equation better basically, so that sinomenine hydrochloride cubic phase and hexagonal phase liquid crystal gel have a certain slow release effect. The fitting result of the Ritger-Peppas equation shows that the cubic phase liquid crystal gel has 0.45< n <0.89, and is a non-Fick diffusion mechanism, namely a Fick diffusion mechanism and a gel skeleton corrosion mechanism. N in hexagonal phase is greater than 0.89, which is skeleton corrosion mechanism.

TABLE 4 Sinomenine Hydrochloride (SH) osmotic kinetics fitting results

②, the permeation kinetics fitting result (table 5) of the Cinnamaldehyde (CA) shows that the permeation of the cinnamaldehyde in the cubic phase liquid crystal gel and the hexagonal phase liquid crystal gel can better fit the Higuchi equation, so that the cubic phase liquid crystal gel and the hexagonal phase liquid crystal gel have certain slow release effect. The fitting result of the Ritger-Peppas equation shows that both the cubic phase liquid crystal gel and the hexagonal phase liquid crystal gel meet the condition that n is more than 0.45 and less than 0.89, and the release mechanism of the cinnamaldehyde follows a non-Fick diffusion mechanism, namely a Fick diffusion mechanism and a gel framework corrosion mechanism.

TABLE 5 Cinnamic Aldehyde (CA) osmotic kinetics fitting results

Example 8

Pharmacodynamic experiment of sinomenine hydrochloride cinnamaldehyde carbomer gel

Firstly, preparing carbomer liquid crystal gel:

accurately weighing 200mg of carbomer 940, uniformly scattering the carbomer 940 in a small beaker filled with a proper amount of distilled water, standing for 12 hours, and putting the beaker in a water bath for warming for later use. 120mg (1.2%, w/w) of sinomenine hydrochloride is precisely weighed, a proper amount of distilled water is added to completely dissolve the sinomenine hydrochloride, and the sinomenine hydrochloride is poured into warm carbomer and is continuously stirred to be dissolved in the gel matrix. Then adding 20mg, 30mg and 60mg of cinnamaldehyde dissolved in a small amount of ethanol respectively, then dropwise adding triethanolamine while stirring to control the pH value to be about 6.5-7.0, adding distilled water to 10g, continuously stirring, and cooling to room temperature to obtain the following 3 compound gels with different proportions:

1.2％SH+0.2％CA(6∶1)，1.2％SH+0.3％CA(4∶1)，1.2％SH+0.6％CA(2∶1)。

storing each compound gel in a shady and cool place in a lightproof container for later use.

Secondly, establishing an adjuvant arthritis rat model:

SD rats 60 were acclimatized for one week. The rats were then modelled with Freund's Complete Adjuvant (FCA) subcutaneously and randomized on day 17 according to the degree of paw volume swelling.

The normal rats injected with normal saline are normal control group (control group), the rats injected with FCA are divided into model group (AA group), Zhengqing Fengtongning group (Sin group), compound gel (2: 1) group, compound gel (4: 1) group and compound gel (6: 1) group, and each group contains 10 rats.

Thirdly, the administration method comprises the following steps:

the skin dose is approximately 5mg/kg, referred to the daily oral dose in rats. The above groups are applied to abdominal skin of rats according to dosage at 18d, 400mg each time, 2 × 2cm of application area, 1 time per day, and 7d continuously, and blank carbomer gel is administered to abdominal skin of normal group and model group, and Zhengqing Fengtongning tablet is administered by intragastric administration.

Fourthly, evaluation indexes:

(1) foot swelling assessment

The degree of foot swelling is determined by measuring foot volume. The volumes of the inflamed side feet of the rats were measured by toe volume measuring instruments at the day (0d) before the inflammation and at the 11 th, 14 th, 17 th, 20 th and 24d after the inflammation, respectively, so that the changes in the volumes of the feet before and after the inflammation were used as evaluation indexes.

(2) Inflammatory cytokine level detection

At the end of the experiment, all animals were anesthetized with a dose of 30mg/kg sodium pentobarbital. The abdominal aorta was bled and centrifuged at 3000rpm for 15min, the supernatant was collected and stored at-80 ℃ for testing. The ELISA kit method is used for measuring the concentrations of IL-1 beta, IL-6, IL-10 and TNF-alpha.

(3) Histopathological examination

At 2h after the last administration, the inflammation-causing ankle joint of a rat is taken and placed in 10% formaldehyde solution for fixation, then decalcification treatment is carried out, decalcification is carried out by using 5% nitric acid solution, and the decalcification solution is replaced every day until the tissue is not subjected to resistance feeling by using a needle. After decalcification, the tissue was rinsed with running water overnight. Dehydrating with gradient alcohol at room temperature, making xylene transparent, performing longitudinal incision on tibia and joint center of epiphysis, embedding in paraffin, slicing, attaching tissue to slide glass treated with anticreep agent, oven drying at 60 + -0.5 deg.C for 12 hr, and performing HE staining.

HE staining method: paraffin sections are subjected to xylene dewaxing → hydration → hematoxylin staining for 10min (staining cell nucleus) → hydrochloric acid alcohol differentiation → eosin staining (staining cytoplasm) → gradient alcohol dehydration → xylene transparency → resin glue sealing piece → observation of pathological manifestations of rat ankle under an optical microscope.

Fifthly, statistical analysis:

all results are expressed as mean values

Standard Deviation (SD), and analyzed by Graphpad Prism version 6.0. One-way ANOVA analyzed significant differences between the different experimental groups. The minimum level of statistical significance (P) was considered at a P value of less than 0.0001<0.0001)。

Sixthly, result analysis:

the swelling of the foot, inflammatory cytokine levels and histopathology were as follows:

figure 9 shows that after successful modeling, there was a significant reduction in left hind paw swelling (P <0.05) in all treatment groups.

FIG. 10 shows that IL-1 β (23.9pg/mL), IL-6(151.7pg/mL), TNF- α (263.9pg/mL) were significantly increased (P <0.0001) and IL-10 levels were significantly decreased (25.5pg/mL) (P <0.0001) in the AA model group compared to the normal control group. Compared with AA group, the Sin group, the compound gel (2: 1) group and the compound gel (4: 1) group all significantly reduce the IL-1 beta, IL-6 and TNF-alpha levels in serum and significantly improve the IL-10 level (P < 0.0001). Compound gel (6: 1) had no significant effect on IL-6 and TNF- α.

FIG. 11 shows that pathological sections of the ankle joint of model rats showed significant infiltration of inflammatory cells, causing synovial inflammatory reaction and vascular proliferation, with microscopic evidence of synovial congestion, hypertrophy, villous formation and pannus formation, and some synovial intimal hyperplasia and interstitial cell proliferation and fibrosis; compared with the model group, the ankle joint of the normal group of rats has no obvious inflammatory reaction symptom; the positive control group of rats showed a light inflammatory reaction with only a small amount of inflammatory cell infiltration and less vascular proliferation, indicating that the positive control drug has a treatment effect on RA; compared with the model group, the compound gel (2: 1) and (4: 1) groups show that inflammatory cell infiltration is obviously reduced greatly, pannus formation is obviously relieved, and fibrosis of interstitial cells is not seen, which indicates that the compound gel has excellent treatment effect on RA; the pathological section of group (6: 1) was observed to have a small amount of inflammatory cell infiltration, pannus formation and vascular hyperplasia were also relieved and restored to some extent, indicating that the therapeutic effect on RA was similar to that of the positive drug.

Example 9

the mixing ratio is as follows: sinomenine hydrochloride 200mg, cinnamaldehyde 25mg, sorbitol monooleate 6500mg and distilled water 3500 mg. The preparation method comprises the following steps:

(2) Precisely weighing sorbitol monooleate in proportion, placing at room temperature, adding cinnamaldehyde in proportion, vortex vibrating, and mixing to form oil phase.

(3) Mixing the water phase and the oil phase, vortex and shaking for 10min, standing at room temperature for 48h to obtain sinomenine hydrochloride cinnamaldehyde cubic phase liquid crystal gel.

Example 10

Preparing sinomenine hydrochloride cinnamaldehyde hexagonal liquid crystal gel:

the mixing ratio is as follows: sinomenine hydrochloride 50mg, cinnamaldehyde 50mg, glyceryl monooleate 7700mg, paraffin oil 500mg and distilled water 2000 mg. The preparation method comprises the following steps:

(2) Weighing glycerol monooleate and paraffin oil in proportion, placing in a water bath at 40 ℃, adding cinnamic aldehyde in proportion after the glycerol monooleate is completely melted, vortex shaking, and mixing to form an oil phase.

(3) Mixing the water phase and the oil phase while hot, vortex and shake for 10min, and standing at room temperature for 72h to obtain sinomenine hydrochloride cinnamaldehyde hexagonal liquid crystal gel.

The foregoing is merely exemplary and illustrative of the principles of the present invention and various modifications, additions and substitutions of the specific embodiments described herein may be made by those skilled in the art without departing from the principles of the present invention or exceeding the scope of the claims set forth herein.

Claims

1. A pharmaceutical composition for treating rheumatoid arthritis by percutaneous administration comprises sinomenine hydrochloride and cinnamaldehyde in a weight ratio of 1: 0.1-10.

2. A pharmaceutical preparation for treating rheumatoid arthritis by transdermal administration is prepared from the pharmaceutical composition of claim 1, one or more pharmaceutically acceptable carriers and excipients, and is prepared into clinically acceptable external preparations, including carbomer gel, liquid crystal gel, ointment and transdermal patch.

3. The method for preparing the liquid crystal gel according to claim 2, wherein the liquid crystal gel is prepared by emulsifying an aqueous phase and an oil phase, the aqueous phase is prepared by dissolving sinomenine hydrochloride in distilled water, and the oil phase is prepared from a lipid material and cinnamaldehyde; the phase composition comprises, by weight, 0.5-2 parts of sinomenine hydrochloride, 0.25-1 part of cinnamaldehyde, 65-75 parts of lipid material and 25-35 parts of distilled water; the method comprises the following specific steps:

4. The method for preparing the liquid crystal gel according to claim 2, wherein the liquid crystal gel is prepared by emulsifying an aqueous phase and an oil phase, the aqueous phase is prepared by dissolving sinomenine hydrochloride in distilled water, and the oil phase is prepared by using a lipid material, a hexagonal phase inducer and cinnamaldehyde; the phase component comprises, by weight, 0.5-2 parts of sinomenine hydrochloride, 0.5-1 part of cinnamaldehyde, 65-77 parts of lipid material, 3-5 parts of hexagonal phase inducer and 20-30 parts of distilled water; the method comprises the following specific steps:

5. The method for preparing the liquid crystal gel according to claim 4, wherein the hexagonal phase inducer is one or more of tricaprylin, vitamin E acetate and paraffin oil.

6. The method of producing a liquid crystal gel according to any one of claims 3 to 5, wherein the lipid material is one or more of phytantriol, glycerol monooleate, monoglyceride and sorbitol monooleate.

7. The method of preparing the carbomer gel of claim 2, wherein the weight ratio of carbomer 940, sinomenine hydrochloride, and cinnamaldehyde to sinomenine cinnamaldehyde carbomer gel is, in order: 1.5-2.5%, 0.5-2%, 0.25-1% to prepare carbomer gel, the specific steps are as follows:

uniformly spreading carbomer 940 in a proper amount of distilled water, standing, and then placing in a water bath for warming for later use; weighing sinomenine hydrochloride, adding appropriate amount of distilled water to dissolve completely, pouring into warm carbomer, and stirring to dissolve; and adding cinnamaldehyde dissolved in a small amount of ethanol, then dropwise adding triethanolamine while stirring to control the pH value to be 6.5-7.0, adding distilled water to the required mass, continuously stirring, cooling to room temperature to obtain sinomenine hydrochloride cinnamaldehyde carbomer gel, and storing in a shady and cool container for later use.

8. The method for preparing the ointment of claim 2, wherein the ointment comprises the following components in the weight ratio of sinomenine hydrochloride and cinnamaldehyde: 0.5-2% and 0.25-1%, stearic acid, white vaseline, liquid paraffin, glyceryl monostearate and lanolin are used as oil phase components, sodium lauryl sulfate, ethylparaben, glycerol and distilled water are used as water phase components to prepare an ointment, and the preparation method specifically comprises the following steps:

9. The method for preparing the transdermal patch according to claim 2, wherein the transdermal patch is prepared according to the weight ratio of sinomenine hydrochloride to cinnamaldehyde of 0.5-2: 0.25-1, and comprises the following steps:

10. Use of a pharmaceutical composition according to claim 1 for transdermal administration in the treatment of rheumatoid arthritis.