CN103735501B - Methotrexate chitosan thermosensitive hydrogel and application thereof - Google Patents

Abstract

The invention provides a kind of MTX subcutaneous depot injection-methotrexate chitosan thermosensitive hydrogel (CS/GP/MTX), containing following composition in the aqueous solution of often liter of methotrexate chitosan thermosensitive hydrogel: chitosan 16-22g, acetic acid 0.05-0.1mol, sodium β-glycerophosphate 70-150g, methotrexate 0.1-0.12mol, solubilizing agent HP-β-CD0.1-0.12mol, isoosmotic adjusting agent 5-50g and pH adjusting agent.The stable in properties of described CS/GP/MTX, quality controllable, appearance character, t _gel, η, pH value, content, primary stability all meet thermosensitive hydrogel requirement.Under 37 DEG C of conditions, CS/GP/MTX is at PBS(pH7.4) in slow release 70% medicine in 5 days.Compare with MTX injection, CS/GP/MTX after rabbit back subcutaneous injection, C _maxreduce by 82%, AUC _0-tincrease 2.37 times, t _{0.02 μM}extend 12.93 times; CS/GP/MTX can reduce untoward reaction, increases curative effect.

Description

Methotrexate chitosan thermosensitive hydrogel and application thereof

Technical field

The invention belongs to field of pharmaceutical preparations, be specifically related to a kind of methotrexate chitosan thermosensitive hydrogel and uses thereof.

Background technology

Rheumatoid arthritis (RheumatoidArthritis, RA) is a kind of agnogenic chronic auto-immune disease, and its disability rate is high, has a strong impact on patient work's ability and daily life, causes white elephant to patient and society.

Methotrexate (methotrexate, MTX, structural formula is shown in formula 1) is the grappling medicine (anchordrug) for the treatment of RA, when namely patient makes a definite diagnosis and needs medication, as without contraindication, then should consider to take MTX; When conditions of patients controls, and when starting to subtract medicine gradually, as there is not the relevant side effect of MTX, then first should subtract and remove other medicine, finally just subtract and remove MTX.At present, MTX is still not clear as the mechanism for the treatment of RA, it is generally acknowledged, low dose of MTX by number of mechanisms onset, and connects each other between each mechanism of action.The onset first step: MTX suppresses the chemotaxis of neutrophilic granulocyte, cell death inducing, directly suppresses synovial cell and immune inflammation hyperplasia; Maintain long-term efficacy: many glutamate methotrexate (Methotrexatepol _yglutamate, MTX-PG) promote that adenosine discharges, be combined with adenosine receptor and produce antiinflammatory action, or regulate generation and the activity of inflammatory cytokine.

The chemical structural formula of formula 1MTX and pKa thereof

MTX treats the route of administration of RA and is mainly oral, intravenous injection, intramuscular injection and subcutaneous injection, European wind resistance damp disease alliance (EuropeanLeagueAgainstRheumatism, EULAR) in 2010 determines that subcutaneous injection treats the new way of RA goldstandard as MTX.Compared with oral, subcutaneous injection MTX bioavailability is high, absorption is complete, evident in efficacy, better tolerance, and can reduce biological preparation use, reduces the treatment cost of patient.Compared with intravenous injection, subcutaneous injection MTX peak concentration (maximumplasmadrugconcentration, C _max) low, lower area of blood concentration-time curve (areaundertheplasmaconcentrationtimecurve, AUC) is large, thus safety is effective more to make subcutaneous injection.Compared with intramuscular injection, subcutaneous injection toleration is better, can reduce patient pain.Existing self injecting type MTX subcutaneous injection agent at present, as German MedacGmbH the VIBEX of U.S. AntarespharmaInc ^tMmTX.Subcutaneous injection has above-mentioned multiple advantage, but subcutaneous injection still has 66% patient to have the untoward reaction such as dizziness, vomiting, abdominal discomfort, sense of taste disorder, at least there is a kind of untoward reaction in 41% patient, the adverse reactions of patients of 53% is relevant with medicine, 5.7% patient has serious adverse reaction, and the patient of 9.3% abandons the treatment of MTX because standing untoward reaction.

MTX is oral tablet and the injection of conventional use dosage form at present.Although it determines it is one of various tumor and the active drug for the treatment of rheumatoid arthritis, and some limitation cannot solve all the time.The pharmacokinetic properties of MTX is unsatisfactory, and clinical therapeutic efficacy may be caused not good.The pharmacokinetic parameter of bibliographical information, comprises peak concentration, peak time, and lower area of blood concentration-time curve (AUC), individual variation is between 30 and 90%.A large amount of MTX that gives is discharged by kidney at short notice, causes 5 – 8h plasma half-lifes short, and lower in target tissue concentration.Such as, tumor, inflamed joint etc.MTX is transported to cell by two kinds of different processes, is respectively folacin receptor mediated Energy Dependence transhipment and Passive diffusion.Once MTX molecule enters cell, if do not carry out outer row transhipment, it can be polymerized glutamate side chain and generate a kind of effective DHFR inhibitor, i.e. MTX polyglutamic acid.The dosage increasing MTX may have better curative effect, but this result also in the risk of larger side effect.Generally, the main cause of clinical inactive MTX is not lack effectiveness but toxicity.Gastrointestinal toxicity, if nauseating, abdominal discomfort, oral inflammation etc. are common sympton.Although the precise mechanism of toxicity is still unclear, a part of side effect is directly related with the metabolic pathway of MTX.Gastrointestinal upset and bone marrow seemingly directly related with suppression folic acid antagonist because these tissue cell proliferation updating decision, need a large amount of purine, thymine.7-OH-MTX is the main metabolites of MTX, and it is water insoluble, may cause nephrotoxicity after giving high dose MTX.The clinical MTX that limits of these side effect uses maximal dose.

Pharmacokinetics-Pharmacodynamics (thepharmacodynamicsandpharmacokinetics, PK/PD) correlation analysis shows, MTX treats C after the untoward reaction of RA and MTX administration _maxclosely related, reduce C _maxthe generation of untoward reaction can be reduced to a certain extent.And MTX treats curative effect and the AUC and 0.02 of RA _μthe M time, (namely in one week, MTX Drug-time curve maintained the time of blood drug level more than 0.02 μM, t _{0.02 μM}) closely related, increase AUC, extend t _{0.02 μM}obviously can improve the state of an illness of RA patient, increase curative effect; And do not find curative effect and C _maxrelevant.Therefore build a kind of MTX subcutaneous depot injection meaning very great, this system can reduce C _maxand extend t _{0.02 μM}and (or) increase effective AUC, thus reach the object reducing untoward reaction, increase (or maintenance) curative effect.

Chitosan thermosensitive hydrogel (thermosensitivechitosan-basedhydrogel, CS/GP) be the syringeability sustained-release drug carrier that a kind of degradable biocompatibility is good, its pH value is in physiological pH range, be low viscosity solution under room temperature, semi-solid gel can be transformed under body temperature (37 DEG C) condition.

In the present invention, many places use english abbreviation, and concrete meaning is as shown in the table:

English abbreviation vocabulary

Summary of the invention

For the deficiencies in the prior art, the present invention is intended to the untoward reaction of reduction MTX and increases its curative effect, in conjunction with MTX character and CS/GP characteristic, build a kind of novel MTX subcutaneous depot injection-methotrexate chitosan thermosensitive hydrogel (CS/GP/MTX), and carry out galenic pharmacy property representation and the evaluation of rabbit Internal pharmacokinetics.For the research and development of MTX subcutaneous injection sustained release drug delivery systems provide experimental basis, and provide reference for the research of the injection slow release formulation of similar drugs.

For achieving the above object, technical scheme of the present invention is:

A kind of methotrexate chitosan thermosensitive hydrogel, containing following composition in the aqueous solution of often liter of methotrexate chitosan thermosensitive hydrogel:

Containing following composition in the aqueous solution of preferred often liter of methotrexate chitosan thermosensitive hydrogel:

Containing following composition in the aqueous solution of more preferably often liter of methotrexate chitosan thermosensitive hydrogel:

Described chitosan is the chitosan that deacetylation is preferably greater than 95%.

Described isoosmotic adjusting agent is preferably sodium chloride or glucose, is more preferably sodium chloride.

Described pH adjusting agent is preferably sodium hydroxide.

The mole dosage of described solubilizing agent HP-β-CD is preferably equal with the mole dosage of methotrexate.

The preparation method of described methotrexate chitosan thermosensitive hydrogel is:

(1) preparation of solution:

By described proportioning, take chitosan, add water appropriate, after magnetic agitation makes into chitosan suspension, then add acetic acid, continue stirring and chitosan is dissolved completely, obtain chitosan solution; Take sodium β-glycerophosphate, add water appropriate, ultrasonic dissolution, obtains sodium β-glycerophosphate solution; Take methotrexate and pH adjusting agent, solubilizing agent, isoosmotic adjusting agent, add water appropriate, ultrasonic dissolution, obtains methotrexate solution; Add water described in obtain solution process and be as the criterion to make added material dissolve completely in right amount;

(2) methotrexate chitosan thermosensitive hydrogel solution is prepared:

Under ice bath stirs, by sodium β-glycerophosphate dropwise instillation chitosan solution, after dripping off, continue ice bath and stir 10-20min, obtain chitosan sodium β-glycerophosphate solution; Equally under ice bath stirs, methotrexate dropwise is instilled in described chitosan sodium β-glycerophosphate solution, after dripping off, continue ice bath and stir 10-20min, obtain methotrexate chitosan thermosensitive hydrogel solution.

Methotrexate chitosan thermosensitive hydrogel of the present invention is as the application in drugs for rheumatoid arthritis.

The present invention will be further explained below:

MTX chitosan thermosensitive hydrogel is a kind of novel MTX preparation, at home and abroad there is no relevant report.The MTX chitosan thermosensitive hydrogel of the present invention's research is a kind of slow releasing preparation of injectable.

MTX chitosan thermosensitive hydrogel of the present invention solves following technical barrier:

1. study discovery through the present invention, invention increases the dissolubility of MTX in chitosan thermosensitive hydrogel system (not adding sodium HP-β-CD, isoosmotic adjusting agent and pH adjusting agent).MTX is the insoluble and water-insoluble medicine of fat, and drug loading is 5%(50mg/ml) time, MTX not exclusively dissolves in chitosan thermosensitive hydrogel system, and this system is also precipitation shape.MTX at the dissolubility of chitosan thermosensitive hydrogel system, exploitative experiment result shows that the chances are 1mg/ml, but can 50mg/ml be increased in methotrexate chitosan thermosensitive hydrogel of the present invention.

2. study discovery through the present invention, invention increases the Thermo-sensitive of system.Chitosan thermosensitive hydrogel system (not adding sodium HP-β-CD, isoosmotic adjusting agent and pH adjusting agent) is loaded into drug loading when being the MTX of 5%, and have impact on the Thermo-sensitive of this system greatly, namely 37 DEG C time, not gelling is still solution shape.But methotrexate chitosan thermosensitive hydrogel of the present invention can in minutes gelling.

That is, component in methotrexate chitosan thermosensitive hydrogel of the present invention and amounts of components very important, create interaction between component, have impact on dissolubility and Thermo-sensitive, the performance of described adjuvant to product has a great impact, and even directly determines whether final products can use.The performance of preparation method to product also has a certain impact.

Compared with prior art, advantage of the present invention is:

The stable in properties of the CS/GP/MTX 1, prepared by the present invention, quality controllable, appearance character, t _gel, η, pH value, content, primary stability all meet thermosensitive hydrogel requirement.Under 37 DEG C of conditions, CS/GP/MTXgel is at PBS(pH7.4) in slow release 70% medicine in 5 days.

2, compare with MTX injection, CS/GP/MTX after rabbit back subcutaneous injection, C _maxreduce 82%, AUC _0-tincrease 2.37 times, t _{0.02 μM}extend 12.93 times; Prompting CS/GP/MTX can reduce untoward reaction, increases curative effect.

3, the present invention can be the developmental research of MTX subcutaneous injection sustained release drug delivery systems and provides experimental basis, and for similar drugs injection slow releasing preparation research reference is provided.

Accompanying drawing explanation

Fig. 1 is CS/GP/MTXgel microstructure;

Fig. 2 is the FTIR figure of CS (a), GP (b), MTX (c), HP-β-CD (d), physical mixture (e) and CS/GP/MTXgel (f);

Fig. 3 is the tablets in vitro curve (n=6) of CS/GP/MTX;

Fig. 4 is Korsmeyer-Peppas models fitting curve (n=6);

Fig. 5 is average Drug-time curve (n=6) after New Zealand white rabbit subcutaneous injection R preparation and T preparation.

Detailed description of the invention

Below in conjunction with embodiment, the present invention is described further.

The structure of embodiment 1 methotrexate chitosan thermosensitive hydrogel

1 method and result

1.1 solution preparation

Raw material and consumption:

1.1.1 chitosan solution

Take recipe quantity CS, add water appropriate, after magnetic agitation makes into CS suspension, more dropwise instill recipe quantity acid flux material, continue stirring and CS is dissolved completely, obtain CS solution.

1.1.2 phosphoglycerol two sodium solution

Take recipe quantity GP, add water appropriate, ultrasonic dissolution, obtains GP solution.

1.1.3 methotrexate solution

Take recipe quantity MTX and other adjuvants (NaOH, solubilizing agent, isoosmotic adjusting agent), add water appropriate, ultrasonic dissolution, obtains MTX solution.

Preserve under the solution of above-mentioned preparation is placed on 4 DEG C of environment, for subsequent use.

1.2CS/GP/MTXsol preparation technology

Under ice bath stirs, in GP dropwise instillation CS solution, after dripping off, continue ice bath and stir 10min, obtain CS/GPsol.Under ice bath stirs, in MTX dropwise instillation CS/GPsol, after dripping off, continue ice bath and stir 10min, obtain CS/GP/MTXsol.

1.3 evaluation index

For CS/GP, t _gelbe evaluate its Thermo-sensitive common counter, rheological characteristic often adopts viscosity (viscosity, η) to evaluate, and pH value is its important galenic pharmacy parameter.Herein with t _gel, η and pH value be CS/GP/MTX primary evaluation index.

1.3.1 gelling time

Adopt test tube anastrophe ^[28]measure colloidal sol t _gel.CS/GPsol(or CS/GP/MTXsol will be housed) cillin bottle put in 37 DEG C of thermostat water baths, every 1min take out, observe and record t _gel.Determine that the method for solution gel is inverted by cillin bottle, solution keeps 10S not flow, then think its gelling.T _gelshorter, illustrate that Thermo-sensitive is better.

1.3.2 viscosity

Under normal temperature condition, measure colloidal sol η with Digital Viscometer.η is less, and illustrate that mobility is better, injectable performance is better ^[30].

1.3.3pH value

Under normal temperature condition, measure the pH value of colloidal sol with pH meter.PH value is the important galenic pharmacy parameter of CS/GP, and pH value is relevant to CS/GP Thermo-sensitive and rheological characteristic to a certain extent, by various factors in prescription.

1.4 single factor exploration

CS/GP delivery system has pH and temperature dual sensitivity, and Thermo-sensitive and pH value have and necessarily contact, and pH value could form CS/GP in 6.8-7.3 scope, and pH value is higher or lower all can not build CS/GP.Except pH value, the temperature sensitive of this system is also subject to the various factors such as CS type and consumption, dissolving CS acid flux material, GP consumption.MTX is a kind of Multiple Weak Acid medicine (pKa≤5.7), and MTX is loaded in the pH value that can affect CS/GP to a certain extent, needs to add suitable pH adjusting agent.MTX is the insoluble medicine of a kind of water not fat melting, for building a kind of CS/GP/MTX of more satisfactory high drug level, need screen appropriate solubilising agent.Screen suitable CS type, acid flux material, pH adjusting agent, solubilizing agent and isotonic adjustment by single factor exploration and determine CS and GP amount ranges.

1.4.1 chitosan model is selected

Model (deacetylation, molecular weight or the viscosity) t to CS/GP of CS _gel, η and pH value all have impact.Select the CS (A: deacetylation 90.26%, viscosity 130.0m.Pas of three kinds of different models; B: deacetylation 95.26%, viscosity 147.0m.Pas; C: deacetylation 95.49%, molecular weight 510KDa), CS model is screened.

Table 1-1CS model is to the t of CS/GP _gel, η and pH value impact (n=3)

Fixation of C S consumption 2%(W/V), HAc concentration 0.100molL ^-1, GP consumption 7%(W/V), prepare CS/GPsol as stated above with the CS of three kinds of models respectively, measure t _gel, η and pH value, the results are shown in Table 1-1.Three kinds of model C S are to the t of CS/GP _gel, η and pH value impact as follows: t _gel, A>B>C; η, A<B ≈ C; PH, A<B<C.Along with the increase of CS deacetylation, t _gelshorten, pH value increases.Result shows, CS/GP Thermo-sensitive prepared by C model C S is better, therefore selects this model C S to carry out follow-up study.

1.4.2 acid flux material is selected

Select HCl and HAc herein, acid flux material is screened.

Fixation of C S consumption 2%(W/V), acid flux material concentration 0.100molL ^-1, GP consumption 7%(W/V), prepare CS/GPsol with acid flux material HCl or HAc respectively, measure t _gel, η and pH value, the results are shown in Table 1-2.The η preparing CS/GPsol relative to acid flux material HCl, HAc is slightly large, pH value is slightly low, t _gelobviously shorter.Result shows, CS/GP Thermo-sensitive prepared by HAc is better, therefore selects acid flux material HAc to carry out follow-up study.

Table 1-2 acid flux material is on t, η of CS/GP and pH value impact (n=3)

1.4.3 the determination of drug loading

MTX is a kind of Multiple Weak Acid medicine (pKa≤5.7), and MTX is loaded in the pH value that can affect CS/GP to a certain extent, and then affects CS/GP Thermo-sensitive.For reducing CS/GP/MTX Thermo-sensitive influence factor, suitable MTX concentration can be selected to carry out follow-up study.

The survey showed that for curative effect satisfaction, and 93% patient is more ready to select 50mgmL ^-1subcutaneous injection agent, and 50mgmL ^-1subcutaneous injection agent toleration better.The MTX subcutaneous injection agent concentration of the current clinical practice of patient mostly is 50mgmL ^-1, the research such as Striesow confirms further, 50mgmL ^-1it is more convenient that pre-filled MTX subcutaneous injection agent uses, and patient tolerability is better.Therefore determine that drug loading is 50mgmL ^-1(i.e. 0.110molL ^-1).

1.4.4pH regulator is selected

MTX is weak acidic drug (pKa≤5.71), and CS/GP Thermo-sensitive is relevant to pH value, and pH value is within the scope of 6.8-7.3, and pH value is higher, the t of CS/GP _gelshorter.MTX can reduce the pH value of CS/GPsol, makes the t of CS/GP _gelextend or without temperature sensitive.Preliminary experiment finds, MTX makes the pH value of CS/GPsol lower t _gelextend.Select NaOH to be pH adjusting agent, make MTX solution ph within the scope of 6.8-7.3.

Press n respectively _naOH: n _mTXbe 1.6,1.7,1.8,1.9 preparation MTX solution (50mgmL ^-1), survey pH value, the results are shown in Table 1-3.N _naOH: n _mTXwithin the scope of 1.7-1.8, MTX solution ph is within the scope of 6.8-7.3.N _naOH: n _mTXwithin the scope of 1.7:1-1.8:1, further by investigation NaOH to the t of CS/GP/MTX _gel, η and pH value impact, determine n _naOH: n _mTX.

Table 1-3NaOH is on MTX solution ph impact (n=3)

Fixation of C S consumption 2%(W/V), HAc concentration 0.100molL ^-1, GP consumption 7%(W/V), MTX concentration 0.110molL ^-1, HP-β-CD concentration 0.110molL- ¹, respectively with n _naOH: n _mTXfor 1.7:1 or 1.8:1 prepares CS/GP/MTXsol, measure t _gel, η and pH value, the results are shown in Table 1-4.η and pH value are with n _naOH: n _mTXincrease and have increased slightly, but t _gelincrease with NaOH and obviously shorten.Result shows, n _naOH: n _mTXduring=1.8:1, CS/GP/MTX Thermo-sensitive is better, therefore determines n _naOH: n _mTX=1.8:1, NaOH concentration is 0.198molL ^-1.

Table 1-4NaOH is to the t of CS/GP/MTX _gel, η and pH value impact (n=3)

1.4.5 solubilizing agent is selected

MTX is water insoluble, is soluble in weak acid and weak base, at HEPES(25mmolL ^-1, pH7.4) in dissolubility be 4.0mgmL ^-1.Preliminary result shows, MTX can not dissolve completely in CS/GPsol.Fixation of C S consumption 2%(W/V), HAc concentration 0.100molL ^-1, GP consumption 7(W/V), MTX concentration 0.110molL ^-1, NaOH concentration 0.198molL ^-1, preparation CS/GP/MTX, CS/GP/MTXsol are yellow suspension.For making MTX be dissolved completely in CS/GPsol, need to screen suitable solubilizing agent.

Preliminary result shows, HP-β-CD and SBE-β-CD solubilizing effect better.Therefore select HP-β-CD and SBE-β-CD, solubilizing agent is screened.

Fixation of C S consumption 2%(W/V), HAc concentration 0.100molL ^-1, GP consumption 7%(W/V), MTX concentration 0.110molL ^-1, NaOH concentration 0.198molL ^-1, prepare CS/GP/MTXsol with solubilizing agent HP-β-CD, SBE-β-CD respectively, observe CS/GP/MTXsol whether clear, if its clear, then measure t _gel, η and pH value, the results are shown in Table 1-5.N _cD: n _mTXduring for 1:1, CS/GP/MTXsol is clear yellow solution.Compare with SBE-β-CD, the t of CS/GP/MTX prepared by solubilizing agent HP-β-CD _gelobviously shorter, η is less.Result shows, when HP-β-CD is solubilizing agent, CS/GP/MTX Thermo-sensitive is better, therefore determines that HP-β-CD is solubilizing agent, and n _hP-β-CD: n _mTX=1:1, HP-β-CD concentration is 0.110molL ^-1.

Table 1-5 solubilizing agent is to clarity, the t of CS/GP/MTX _gel, η and pH value impact (n=3)

1.4.6 isoosmotic adjusting agent is selected

Select 0.9%NaCl and 5%Glu, isoosmotic adjusting agent is screened.

Fixation of C S consumption 2%(W/V), HAc concentration 0.100molL ^-1, GP consumption 7%(W/V), MTX concentration 0.110molL ^-1, NaOH concentration 0.198molL ^-1, HP-β-CD concentration is 0.110molL ^-1, use H respectively ₂o, 0.9%NaCl or 5%Glu are solvent, and preparation CS/GP/MTXsol, measures t _gel, η and pH value, the results are shown in Table 1-6.Isoosmotic adjusting agent is on the η of CS/GP/MTX and pH value substantially without impact, and isotonic adjustment 0.9%NaCl is to the t of CS/GP/MTX _gelwithout impact, and isotonic adjustment 5%G extends the t of CS/GP/MTX _gel.Therefore, 0.9%NaCl is selected to be isoosmotic adjusting agent.

Table 1-6 isoosmotic adjusting agent kind is to the t of CS/GP/MTX _gel, η and pH value impact (n=3)

1.4.7 chitosan dosage screening

CS consumption is to the t of CS/GP/MTX _gel, η and pH value etc. have a certain impact, and when CS consumption is 1.5%, can not form thermosensitive hydrogel, but due to the restriction of CS dissolubility in an acidic solution, when CS consumption is greater than 2.2%, CS can not dissolve completely, therefore research CS consumption is 1.6%-2.2%(W/V) time, to the t of CS/GP/MTX _gel, η and pH value impact.

Table 1-7CS consumption is to the t of CS/GP/MTX _gel, η and pH value impact (n=3)

Fixing HAc concentration 0.100molL ^-1, GP consumption 7%(W/V), MTX concentration 0.110molL ^-1, NaOH concentration 0.198molL ^-1, HP-β-CD concentration is 0.110molL ^-1, isotonic adjustment is 0.9%NaCl, prepare the CS/GP/MTXsol of different CS consumption (1.6%, 1.8%, 2.0%, 2.2%), and measure t _gel, η and pH value, the results are shown in Table 1-7.Along with the increase of CS consumption, the t of CS/GP/MTX _gelshorten, η increases, and pH value is without significant change and in physiologic pH values.Desirable CS/GP/MTX should possess following three kinds of conditions substantially: (1) t _gelshort, (2) η is little, and (3) pH value is in physiological pH range.Therefore, tentatively determine that CS consumption is within the scope of 1.6%-2.2%, follow-up employing Star point design test optimizes CS consumption further.

1.4.8 phosphoglycerol disodium consumption screening

According to document ^[26]and preliminary result, research GP consumption is at 5.0%-15.0%(W/V) in scope, it is to the t of CS/GP/MTX _gel, η and pH value impact.

Fixation of C S consumption 1.8%(W/V), HAc concentration 0.1molL ^-1, MTX concentration 0.110molL ^-1, NaOH concentration 0.198molL ^-1, HP-β-CD concentration is 0.110molL ^-1, isotonic adjustment is 0.9%NaCl, prepares the CS/GP/MTXsol of different GP consumption (5.0%, 7.5%, 10.0%, 15.0%), measures t _gel, η and pH value, the results are shown in Table 1-8.With the increase of GP consumption, the t of CS/GP/MTX _gelshorten, pH value has increased slightly, and η is without significant change.

Preliminary experiment finds, increases with CS concentration, preparation t _gelduring identical CS/GP/MTX, required GP consumption reduces.Therefore tentatively determine that GP amount ranges is 7.0%-15.0%, follow-up employing Star point design test optimizes GP consumption further.

Table 1-8GP consumption is on t, η of CS/GPsol and pH value impact (n=3)

1.5 Star point design-effect surface method optimization experiment

Each experimental point of CCD-RSM is equal apart from the distance of central point, the level of independent variable testing in the scope allowed, an optimization experiment optimal value.CCD-RSM should complete following work: (1) sets up suitable mathematical model equation, the correct relation describing each investigation factor and index or result.(2) preferably formulation and technology is chosen according to mathematical model equation.The key of dealing with problems sets up mathematical model exactly, except wanting accurate Control release condition, also needs to select suitable mathematical model.Single index optimization can directly experimentally result select, but when index more (>=2), often influence each other between each index, the condition favourable to a certain index may be unfavorable to another index, then need to be optimized according to the comprehensive effect of each index.Overall desirability (overalldesirability, OD) is by mathematical method by each index comprehensive, represents whole effect with OD, sets up suitable mathematical model and carries out forecast and promote ^[42].Each index is all standardized as the normalizing value (desirability, d) between 0-1, and each index d asks calculation geometric mean by formula (1-1), obtains OD.The index that the index the smaller the better for value and value are the bigger the better, adopts Hassan method to carry out mathematical transformation by formula (1-2) and formula (1-3) respectively, asks d _minand d _max.

k is index number formula (1-1)

D _min=(y _max-y _i)/(y _max-y _min) formula (1-2)

D _max=(y _i-y _max)/(y _max-y _min) formula (1-3)

1.5.1 Star point design-preferred result of effect surface method is:

The preferred proportioning raw materials of 1-9, described W/V

Remarks: described 1.69%(W/V) refer to that 2%, 13.03%, 0.9% is all these implications containing 1.69gCS in 100 milliliters of finished products.

1.5.2 analyze:

The Thermo-sensitive impact of molecular weight (or η) on CS/GP of CS is less, and this experiment finds that the Thermo-sensitive of CS/GP/MTX can affect the η of CS/GP/MTXsol to a certain extent, but its affecting laws needs to be studied further.

PH value is within the scope of 6.8-7.3, and pH value obviously affects the Thermo-sensitive of CS/GP, along with pH value increases, and the t of CS/GP _gelshorten, before and after CS/GP gelling, pH value does not change.Single factor exploration finds, GP consumption and acid flux material type affect larger on the pH value of CS/GP/MTXsol.With the increase of GP consumption, the pH value of CS/GP/MTX increases, t _gelshorten.Infer that possible cause is: GP consumption increases, with-the NH of GP electrostatical binding ₃ ⁺more, under heating condition, more-NH ₃ ⁺proton captured by GP, make hydrogen bond and hydrophobic interaction comparatively strong, thus make the t of CS/GP/MTX _gelshorter.Compare with acid flux material HCl, the pH value of CS/GP/MTXsol prepared by HAc increases, t _gelshorter.Because HAc is acid weak compared with HCl, may be unprotonated-NH in CS solution on the one hand ₂increase, add in CS molecule and intermolecular hydrogen bond and hydrophobic interaction, under heating condition, the hydrogen bond of CS/GP/MTX and hydrophobic interaction are comparatively strong, make its t _gelshorter; On the other hand, may be H free in CS solution ⁺reduce, make-the NH with GP electrostatical binding ₃ ⁺more, under heating condition, in CS/GP/MTX, there is more-NH ₃ ⁺proton is captured by GP, makes hydrogen bond and hydrophobic interaction comparatively strong, makes its t _gelshorter.As pH>7.4, CS/GP is separated, and CS separates out, and CS/GPsol is without Thermo-sensitive.May be alkalescence stronger time, with-the NH of GP electrostatical binding ₃ ⁺increase severely or-NH ₃ ⁺proton is captured ,-NH ₃ ⁺between electrostatic repulsion obviously weaken or disappear, in CS molecule and intermolecular hydrogen bond and hydrophobic interaction occupy an leading position in system, when not heating, CS/GP forms gel fast, or is separated CS is precipitated.

2 brief summaries

With t _gelfor primary evaluation index, CS model and consumption, GP consumption, acid flux material type, drug loading, pH adjusting agent, solubilizing agent, isoosmotic adjusting agent etc. are comprised on the formulation factors affecting CS/GP/MTX Thermo-sensitive and carries out single factor exploration, and adopt CCD-RSM to carry out formulation optimization to CS/GP/MTX, finally determine CS/GP/MTX prescription: CS model is C type CS, its consumption is 1.69%(W/V); GP is 13.03%(W/V); Acid flux material is HAc, and its concentration is 0.100molL ^-1; MTX concentration is 0.110molL ^-1; PH adjusting agent is NaOH, and its concentration is 0.198molL ^-1; Solubilizing agent is HP-β-CD, and its concentration is for being MTX concentration; Isoosmotic adjusting agent is 0.9%NaCl.The viscosity of CS/GP/MTX, t _gel(184.0 ± 12.5) m.Pas, (6 ± 2) min and 7.01 ± 0.05 is respectively with pH value.Result shows, the prescription of CS/GP/MTX is comparatively reasonable, and good process repeatability, CS/GP/MTXsol is the low viscosity solution of pH value in physiological pH range, can fast gelation under 37 DEG C of conditions.

The research of methotrexate chitosan thermosensitive hydrogel galenic pharmacy character.

The research of embodiment 2 methotrexate chitosan thermosensitive hydrogel galenic pharmacy character

1 method and result

1.1 outward appearance

CS/GP/MTXsol is light yellow transparent solution, and perusal has no insoluble composition or block aggregate.CS/GP/MTXsol obtains CS/GP/MTXgel after 37 DEG C of water-bath gellings, in faint yellow semi-solid.

The microstructure of 1.2 gels

CS/GP/MTXgel sample, after lyophilization 24h, adopts that liquid nitrogen is sudden coldly to be broken disconnected, to its section vacuum metal spraying process, characterizes the internal structure of CS/GP/MTXgel with SEM.As shown in Figure 1, the internal structure of CS/GP/MTXgel is irregular cellular.

1.3 viscosity, gelling time and pH value

Prepare 3 crowdes of CS/GP/MTXsol by optimization formulation, measure its t _gel, η and pH value, in Table 2-1.Result shows, CS/GP/MTX prescription is reasonable, and process repeatability is better, the CS/GP/MTXsol of preparation is the pH value low viscosity solution that Thermo-sensitive is good in physiological pH range, it gelling can become semi-solid at about 6min, and η is about 188.0m.Pas, and pH value is about 7.01.

The t of table 2-1CS/GP/MTX _gel, η and pH value (n=3)

1.4 infrared analysis

By the INFRARED ABSORPTION of FTIR spectrum (FourierTransformInfraredSpectroscopy, FTIR) characterizing sample, the chemical property of research CS/GP/MTX, inquires between MTX-adjuvant and adjuvant-adjuvant whether there is interaction.

Sample: (a) CS powder, (b) GP powder, (c) MTX crude drug, (d) HP-β-CD powder (e) physical mixture thing, (f) CS/GP/MTXgel.

Sample pretreatment: get appropriate amount of sample (a) and (b), (c), (d) powder respectively in tared dish, 40 DEG C of vacuum drying 24h.First take MTX, CS, GP, HP-β-CD of optimization formulation amount, fully mixing makes even sample (e), gets appropriate amount of sample (e) in tared dish, 40 DEG C of vacuum drying 24h.Prepare CS/GP/MTXsol by optimization formulation, get the addition of C S/GP/MTXsol in 37 DEG C of water-baths, after its gelling, obtain sample (f), sample (f) is in-80 DEG C of pre-cooling 2h, lyophilization 24h.Infrared figure as shown in Figure 2, as can be seen from Figure 2, has interaction between sample.

1.5 tablets in vitro character

1.5.1 the foundation of MTX method for measurement of concentration in release medium

HPLC-UV method is adopted to measure the concentration of the MTX in release medium.

1.5.1.1 chromatographic condition: chromatographic column: lP-C ₁₈(2 μm, 4.6 × 250mm); Mobile phase:

PBS(pH6.5,0.01molL ^-1)-acetonitrile (89:11, V/V); Determined wavelength: 302nm; Flow velocity:

1.0mLmin ^-1; Column temperature: 40 DEG C; Sample size: 20 μ L.

1.5.1.2 the stability of methotrexate in release medium

With PBS(pH7.4,0.05molL ^-1) the basic, normal, high concentration of solution preparation (2.5,25.0,350.0 μ gmL ^-1) each 5 parts of MTX solution, in constant temperature oscillation instrument ((37 ± 0.5) DEG C, (100 ± 5) rpm), respectively at 0,5 day sampling, 0.45 μm of microporous filter membrane crossed by sample, gets the analysis of subsequent filtrate sample introduction, record peak area, by standard curve calculation sample concentration, compare with 0h sample, in Table 2-2.It is stable in 120h under release conditions in vitro that the basic, normal, high concentration samples RSD of result MTX is all less than 0.88%, MTX.

Table 2-2MTX stability (n=5) of 5 days in release medium

1.5.1.3 absolute recovery

Get a certain amount of blank CS/GPsol suspension, add not commensurability MTX storing solution, obtain basic, normal, high concentration (2.5,25.0,350.0 μ gmL ^-1) blank each 5 parts of the CS/GPsol suspension of dosing, 0.45 μm of microporous filter membrane crossed by sample, and get the analysis of subsequent filtrate above-mentioned chromatographic condition sample introduction, record peak area is (A ₁).The basic, normal, high concentration of another preparation (2.5,25.0,350.0 μ gmL ^-1) each 5 parts of MTX solution, analyze by above-mentioned chromatographic condition sample introduction, with peak area (A ₀), with A ₁/ A ₀calculate absolute recovery, in Table 2-3.Result shows, and the MTX absolute recovery of basic, normal, high concentration is respectively 99.69%, 100.16%, 100.04%, RSD and is all less than 1.41%, meets the concentration determination requirement of release medium Chinese medicine.

Table 2-3HPLC-UV method measures the absolute recovery (n=5) of MTX release in vitro sample

1.5.2 vitro release experiment

Document often adopts without film leaching research thermosensitive hydrogel release in vitro, namely first by the gelling in vitro of temperature sensitive colloidal sol, then add release medium 100rpm vibration under 37 DEG C of conditions, sample at setting-up time and supplement equal-volume equality of temperature release medium, sample feeding is analyzed, and draws tablets in vitro curve.The present invention adopts the tablets in vitro behavior investigating CS/GP/MTX without film leaching.

Release medium commonly used by MTX preparation is PBS(pH7.4), MTX is stable in properties in the medium, the higher (4.0mgmL of dissolubility ^-1) ^[55], substantially meet sink conditions and can physiological condition be simulated, therefore selecting PBS(pH7.4) and be release medium.

Take 6 parts of 1.000gCS/GP/MTXsol(respectively about containing MTX0.036g) in 50mL tool plug teat glass, 37 DEG C of waters bath with thermostatic control, make its complete gelation, add 20mLPBS(pH7.4,0.05molL in test tube ^-1), in (37 ± 0.5) DEG C, isothermal vibration under (100 ± 5) rpm condition, 0.5,1,2,4,8,12,24,36,48,72,96,120h time, release medium is all taken out, and supplements the release medium of the fresh isothermal of 20mL.Sample is after 0.25 μm of filtering with microporous membrane, and HPLC-UV sample introduction is analyzed, and record peak area, by standard curve calculation sample concentration.

Accumulation dissolution (the Q of different time points is calculated by formula 2-1 _t), with Q _tfor vertical coordinate, t are that abscissa draws tablets in vitro curve, see Fig. 3.From tablets in vitro curve, the release in vitro of CS/GP/MTX is two-phase release, and namely the initial stage dashes forward and releases and later stage slow release.4h cumulative release about 20%, MTX can rapid-onset, after 36h cumulative release about 50%, 96h, MTX release slowly, only release 0.2% between 96-120h.

$Q\%=\frac{\underset{i-1}{\overset{n-i}{\mathrm{\&Sigma;}}}{C}_{i}V}{m_0}\&times;100\%$ Formula

（2-1）

Q%-Accumulation dissolution; C _i-the i-th sample point drug level; V-release medium volume; m ₀-medicine total amount

1.5.3 tablets in vitro mechanism

For slow releasing preparation, conventional Zero order release model (Q=k ₀t), first-order release model (Q=100 × [1-Exp (-k ₁t)), Higuchi model (Q=k _ht ^0.5), Korsmeyer-Peppas model (Q=k _kPt ⁿ), Hixson-Crowell model (Q=100 × [1-(1-k _hCt) ³]) etc. model carry out tablets in vitro equation model, Fig. 4 is shown in matched curve, by selecting suitable releasing theory research slow releasing preparation tablets in vitro mechanism.

By Q _tcarry out release in vitro models fitting, the correlation coefficient of more each equation, in Table 2-4.Result show, tablets in vitro curve more meets Korsmeyer-Peppas model (n=0.292), prompting CS/GP/MTX at PBS(pH7.4) in release Mechanisms be that Fick ' s spreads.

The release in vitro models fitting of table 2-4CS/GP/MTX

1.6 primary stability experiments

With t _gel, η and pH value be inspection target, investigates CS/GP/MTXsol at 25 DEG C, 4 DEG C, freezing and lyophilization condition stability inferior, for the condition of storage of CS/GP/MTX and application provide experimental basis.

Under CS/GP/MTXsol is stored in 25 DEG C or 4 DEG C of conditions, respectively at 0,6,12,24h sampling, measure t by embodiment 1 _gel, η and pH value, the results are shown in Table 2-13.Under 25 DEG C of conditions, the t of the CS/GP/MTXsol of 0h and 12h _gelbe respectively (6 ± 1) min and (4 ± 0) min, η and be respectively (187.1 ± 8.8) m.Pas and (194.7 ± 11.6) m.Pas, pH value is respectively 7.04 ± 0.07 and 6.98 ± 0.08.Under 4 DEG C of conditions, the t of the CS/GP/MTXsol of 0h and 12h _gelbe respectively (4 ± 1) min and (4 ± 1) min, η and be respectively (190.0 ± 9.5) m.Pas and (198.6 ± 12.5) m.Pas, pH value is respectively 7.01 ± 0.02 and 7.00 ± 0.04.Result shows, under 25 DEG C and 4 DEG C of conditions, and the t of CS/GP/MTXsol in 12h _gel, η and pH value change less, more stable in its 12h.

Table 2-5CS/GP/MTXsol places 24h stability (Mean ± SD, n=3) under 25 DEG C and 4 DEG C of conditions

CS/GP/MTXsol is in-20 DEG C of stored frozen after-80 DEG C of pre-cooling 2h, and in 0 day and sampling in 30 days, ice bath thawed, and thawing solution is pressed " 1.3 " item in example 1 and measured t _gel, η and pH value, the results are shown in Table 2-6.Under freezing conditions, the t of the CS/GP/MTXsol of 0 day and 30 days _gelbe respectively (6 ± 2) min and (5 ± 1) min, η and be respectively (179.3 ± 10.6) m.Pas and (190.4 ± 10.7) m.Pas, pH value is respectively 6.97 ± 0.02 and 6.99 ± 0.02.Result shows, under freezing conditions, and the t of CS/GP/MTXsol in 30 days _gel, η and pH value change less, more stable in its 30 days.

Table 2-6CS/GP/MTXsol under freezing conditions places 30 days stability (Mean ± SD, n=3)

CS/GP/MTXsol, adds freeze drying protectant 10% mannitol ,-80 DEG C of pre-cooling 2h, then uses freezer dryer dry, and lyophilized powder adds prescription water gaging (4 DEG C), stirring and dissolving under condition of ice bath ^[30], measure t by chapter 1 " 2.3 " item _gel, η and pH value, the results are shown in Table 2-15.Before and after lyophilization, the t of CS/GP/MTXsol _gelbe respectively (6 ± 2) min and (8 ± 2) min, η and be respectively (189.8 ± 13.8) m.Pas and (192.6 ± 3.8) m.Pas, pH value is respectively 7.02 ± 0.03 and 6.99 ± 0.04.Result shows, before and after CS/GP/MTXsol lyophilization, and its t _gel, η and pH value change less, more stable after CS/GP/MTXsol lyophilization process, prompting CS/GP/MTXsol can the long term storage of lyophilized powder form.

η, t before and after table 2-7CS/GP/MTXsol lyophilization process _geland pH value change (Mean ± SD, n=3)

To sum up, CS/GP/MTXsol can store under freezing or lyophilization condition; Under 25 DEG C of conditions, its stability meets the requirement of injection thermosensitive hydrogel substantially.

2 brief summaries

The present embodiment has prepared CS/GP/MTX by optimization formulation, to its appearance character, microstructure, pH value, viscosity, t _gel, infrared suction, content, the galenic pharmacy character such as tablets in vitro and primary stability is studied.Result shows, CS/GP/MTX stable in properties, quality controllable, appearance character, t _gel, η, pH value, content, primary stability all meet thermosensitive hydrogel requirement, under 37 DEG C of conditions, CS/GP/MTXgel slow release 70% medicine in 5 days in PBS solution.

Pharmacokinetic studies in embodiment 3 methotrexate chitosan thermosensitive hydrogel rabbit

1.1 laboratory animal

Healthy male New Zealand rabbit (1.8-2.5Kg), credit number: SCXK(Hunan) 2009-0012, Changsha Tian Qin Bioisystech Co., Ltd.

2 methods and result

2.1 reagent

2.1.1CS/GP/MTXsol preparation

With embodiment 1.

2.1.2 the preparation of methotrexate inj

Taking NaOH appropriate, is 0.198molL by 0.9%NaCl solution preparation concentration ^-1naOH normal saline solution.Be 50mgmL with NaOH normal saline solution compound concentration ^-1mTX injection.

2.2 experimental program

12 New Zealand white rabbit are divided into 2 groups (n=6) at random, i.e. experimental group and matched group.By 5mgkg ^-1dorsal sc injection administration, experimental group injection CS/GP/MTXsol, matched group injection MTX injection.Fasting 10h before administration, can't help water (5% glucose solution).Experimental group, before administration and after administration 5,15,30,45,60min, 2,3,4,5,6,8,10,12,24,48,72,96,120h auricular vein gets about 0.7mL blood in heparinization EP pipe; Matched group, before administration and after administration 5,15,30,45,60min, 2,3,4,5,6,8,10,12h auricular vein gets about 0.7mL blood in heparinization EP pipe; The centrifugal 10min separated plasma of 4000rpm ,-20 DEG C of preservations are to be measured.

In 2.3 blood plasma, Concentration of Methotrexate assay method is set up

The present embodiment adopts novel LC/LC-UV method to measure MTX concentration in blood plasma.Novel LC/LC-UV method overlaps independently HPLC use in conjunction by ten-way valve system by two, wherein one-level liquid chromatograph (LC _l) be responsible for example enrichment and be separated, secondary liquid chromatograph (LC ₂) be responsible for separation detection.This system has in-line purification and enriched sample, the automatically function such as control deviation and target substance transfer, and sample treatment is simple, without the need to interior mark, accuracy and precision are high and automaticity is high.

2.3.1 chromatographic condition

LC _l: extraction-chromatography post is ASTON-RGC ₁₈post (4.6mm × 50mm, 5 μm, ANAX), mobile phase (pump 1) is 70% acetonitrile-20% methanol solution: 16mmolL ^-1ammonium dihydrogen phosphate (20% ethylene glycol ammonium is adjusted to pH6.26) (13:87, V/V); Focusing on mobile phase (pump 2) is 0.02% phosphoric acid solution; LC ₂: chromatographic column is ASTON-RGSCX post (4.6mm × 250mm, 5 μm, ANAX), and mobile phase (pump 3) is ammonium dihydrogen phosphate (90mmolL ^-1): methanol (70:30, V/V, pH5.05).Chromatographic column temperature 45 DEG C, determined wavelength 306nm, sample size 250 μ L.System runs program is in Table 3-1.

Table 3-1LC/LC-UV method chromatographic condition setting-temporal sequence

2.3.2 storing solution preparation

Take MTX reference substance 10.0mg, be placed in the brown volumetric flask of 10mL, with PBS(pH7.4,0.05molL ^-1) dissolve and standardize solution, obtain MTX storing solution (1.0mgmL ^-1).

2.3.3 plasma sample process

Precision gets blood plasma 300 μ L, puts in 1.5mLEP pipe, Plus acidic protein precipitant (6% perchloric acid) 600 μ L, the centrifugal 8min of vortex 1min, 12000rpm, gets supernatant 700 μ L, add saturated ammonium sulfate solution 70 μ L, vortex 30S, analyze by " 2.3.1 " item chromatographic condition sample introduction.

2.3.4 sample room temperature shelf-stability

With blank plasma preparation be respectively 10 containing MTX concentration, 250,4000ngmL ^-1sample, room temperature is placed after 6h, by after the process of " 2.3.3 " item, analyzes by " 2.3.1 " item chromatographic condition sample introduction, and record peak area, by standard curve calculation sample concentration, compares with 0h sample, in Table 3-5.Result MTX is basic, normal, high, and concentration samples RSD is all less than 1.54%, and it is basicly stable that sample room temperature places 6h.

Table 3-5MTX plasma sample room temperature places 6h stability (n=5)

2.3.5 sample treatment liquid room temperature shelf-stability

With blank plasma preparation MTX concentration be respectively 10,250,4000ngmL ^-1sample, by after the process of " 2.3.3 " item, after treatment fluid and ambient temperatare are put 12h, analyze by " 2.3.1 " item chromatographic condition sample introduction, record peak area, by standard curve calculation sample concentration, compare with 0h sample, in Table 3-6.Result MTX is basic, normal, high, and concentration samples RSD is all less than 3.08%, and it is basicly stable that sample treatment liquid room temperature places 12h.

After the process of table 3-6MTX plasma sample, room temperature places 12h stability (n=5)

2.3.6 freeze-thaw stability

With blank plasma preparation MTX basic, normal, high concentration be respectively 10,100,4000ngmL ^-1sample, put-20 DEG C of refrigerators, take out thaw after put into refrigerator again, through three circulation after take out sample, by after the process of " 2.3.3 " item, analyze by " 2.3.1 " item chromatographic condition sample introduction, record peak area, by standard curve calculation sample concentration, compare with before freeze thawing, in Table 3-7.Result MTX is basic, normal, high, and concentration samples RSD is all less than 2.58%, and sample freeze thawing 3 times is basicly stable.

Table 3-7MTX plasma sample freeze-thaw stability (n=5)

2.4 determination of plasma concentration results

Get plasma sample by by after the process of " 2.3.3 " item, analyze by " 2.3.1 " item chromatographic condition sample introduction, record peak area, by standard curve calculation sample concentration, as exceeded the range of linearity, then after dilution, sample introduction measures.MTX normal injection (T) and plasma drug concentration data CS/GP/MTX(R) are in Table 3-8,3-9, and average Drug-time curve is shown in Fig. 5.

Table 3-8 New Zealand white rabbit subcutaneous injection R preparation (5mgkg ^-1) after blood drug level (ngmL ^-1)

Table 3-9 New Zealand white rabbit subcutaneous injection T preparation (5mgkg ^-1) after blood drug level (ngmL ^-1)

2.5 pharmacokinetic parameters

Adopt DASVer2.1 to calculate the pharmacokinetic parameters of MTX, adopt statistical moment non-compartment model fitting process to calculate the pharmacokinetic parameters t of MTX in each animal blood slurry _max, C _max, AUC _0-t, AUC _0-∞, MRT _0-t.T _{0.02 μM}(concentration maintains 9ngmL ^-1the above time) be measured value, if during last blood sampling point, its blood drug level is still greater than 9ngmL ^-1, then t _{0.02 μM}count this time.Self-control CS/GP/MTX(T) and the pharmacokinetic parameters of MTX normal injection agent (R) respectively in Table 3-10,3-11.

Main pharmacokinetic parameters after table 3-10 subcutaneous injection R preparation

Main pharmacokinetic parameters after table 3-11 subcutaneous injection T preparation

2.6 statistical analysis

Calculate R and T preparation main pharmacokinetic parameters (C _max, t _max, AUC _0-t, AUC _0-∞, MRT _0-t, t _{0.02 μM}) ratio.By the pharmacokinetic parameters AUC of CS/GP/MTX and MTX injection _0-t, AUC _0-∞, C _maxone factor analysis of variance is carried out after Logarithm conversion; The pharmacokinetic parameters MRT of CS/GP/MTX and MTX normal injection agent _0-t, t _{0.02 μM}adopt one factor analysis of variance; t _maxadopt nonparametric rank test.The results are shown in Table 3-12,3-13 and 3-14.

Table 3-12T and R preparation main pharmacokinetic parameters ratio

Main pharmacokinetic parameters the results of analysis of variance after the administration of table 3-13T and R preparation

The C of CS/GP/MTX _max, t _max, AUC _0-t, AUC _0-∞, MRT _0-tand t _{0.02 μM}0.18,4.49,3.37,3.52,38.47 and 13.93 times of MTX injection respectively, main pharmacokinetic parameters (C _max, t _max, AUC _0-t, AUC _0-∞, MRT _0-t, t _{0.02 μM}) between all have significant difference (P<0.05).Result shows, CS/GP/MTX can reduce the C of MTX _max, increase AUC and t _{0.02 μM}, there is certain slow release effect.

Main pharmacokinetic parameters non parametric tests result after the administration of table 3-14T and R preparation

2.7 relative bioavailability

AUC is calculated according to statistical moment parameter _0-tcalculate the relative bioavailability F of CS/GP/MTX and MTX injection _rel(%), computing formula is shown in formula (3-1).

$F_{\mathrm{rel}}\%=\frac{{\mathrm{AUC}}_{0-t}\left(T\right)}{{\mathrm{AUC}}_{0-t}\left(R\right)}\&times;100\%$ Formula (3-1)

The relative bioavailability F of MTX can be obtained through above formulae discovery _relfor (341.08 ± 61.49) %.

3 discuss

3.1 laboratory animals and dosage are selected

New Zealand white rabbit is RA animal model conventional animal.Release in vitro result shows, cumulative release about 70% in CS/GP/MTX5 days, slow release effect is good.For more complete Drug-time curve after acquisition CS/GP/MTX subcutaneous administrations, its sampling time may be longer.After MTX injection subcutaneous injection, medicine is eliminated fast in 12h, and blood drug level is lower than lower limit of quantitation (4.5ngmL ^-1).Because plasma sample drug concentration is lower, and still there is curative effect when 0.02 μM, reaching 5ngmL for making the lower limit of quantitation of LC/LC-UV method ^-1, the volume of plasma sample at least needs 300 μ L.To sum up, this experiment selects New Zealand white rabbit as laboratory animal.

MTX treats RA and adopts pulsatile administration method (namely Per-Hop behavior once), clinical administration dosage 30mgweek ^-1calculate by the direct Commutation Law of dose,equivalent, in MTX rabbit body, unit dosage form is about 5mgkg ^-1.Therefore determine that rabbit back subcutaneous doses is 5mgkg ^-1.

3.2 plasma sample processing methods are selected

In blood plasma, MTX concentration adopts novel LC/LC-UV system measurement, and this system has in-line purification and enriched sample, the automatically function such as control deviation and target substance transfer, therefore plasma sample only needs simple albumen precipitation process.MTX plasma sample commonly uses perchloric acid precipitation of protein, but sample is highly acid after perchloric acid albumen precipitation.When treatment fluid acidity is stronger, easily affect the chromatographic behavior of MTX and destroy chromatographic column, and MTX is unstable under acidic condition, more stable under neutrality or meta-alkalescence condition.For increasing plasma sample treatment fluid stability, protection chromatographic column, MTX plasma sample is after perchloric acid albumen precipitation, and the appropriate saturated ammonium sulfate of supernatant is adjusted to pH7.0.

3.3 pharmacokinetic parameters

Compare with MTX injection, CS/GP/MTX after rabbit back subcutaneous injection, AUC _0-tincrease 2.37 times.Trapani etc., research finds, CS/CD system can increase the absorption of model drug glutathion, infers that CS/CD system can increase the absorption of medicine.Relative to MTX injection, subcutaneous injection slow releasing preparation MVL-CD-MTX or MTXPLGA microsphere, can increase the AUC of MTX, infers subcutaneous injection MTX slow releasing preparation, and MTX can not tachymetabolism and increase AUC in body.Containing CS and CD in subcutaneous depot formulation C S/GP/MTX, then it can increase the absorption of medicine by CS/CD system and reduce tachymetabolism medication amount in body, its AUC is increased, but the AUC of CS/GP/MTX is increased mechanism and increase MTX treatment RA curative effect and need be confirmed further by the zoopery of RA pharmacodynamics model.

3.4 laboratory animal local skin reactions

After New Zealand white rabbit subcutaneous injection MTX injection or CS/GP/MTXsol, there is not abnormal response in laboratory animal, injection site has no red swelling of the skin reaction, and prompting MTX injection or CS/GP/MTXsol biocompatibility better, but also need to be proved further by subcutaneous injection irritant experiment.CS/GP good biocompatibility, at healthy adult SD rat back subcutaneous injection CS/GPsol, it can fast and subcutaneous fibrotic cell membrane fusion, and inflammatory reaction is not observed in injection site.

4 brief summaries

The present embodiment establishes blood sample LC/LC-UV system measurement method in MTX New Zealand white rabbit body, adopt perchloric acid precipitation of protein processing sample, in blood plasma, impurity does not disturb MTX to measure and separating degree is better, and linear relationship is good, accuracy, precision and absolute recovery are high, and lower limit of quantitation is 5ngmL ^-1.Data, through DAS2.0 process, adopt SPSS17.0 to carry out statistical analysis to main pharmacokinetic, found that, relative MTX injection, the C of CS/GP/MTX _maxreduce 82%, AUC _0-tincrease 2.37 times, t _{0.02 μM}extend 12.93 times.Result shows, CS/GP/MTX, after rabbit back subcutaneous injection, can reduce the C of medicine _max, increase AUC, extend t _{0.02 μM}; Prompting CS/GP/MTX can reduce untoward reaction, increases curative effect.

Claims (7)

1. a methotrexate chitosan thermosensitive hydrogel, is characterized in that, containing following composition in the aqueous solution of often liter of methotrexate chitosan thermosensitive hydrogel:

The mole dosage of described HP-β-CD is equal with the mole dosage of methotrexate.

2. methotrexate chitosan thermosensitive hydrogel according to claim 1, is characterized in that, containing following composition in the aqueous solution of often liter of methotrexate chitosan thermosensitive hydrogel:

3. methotrexate chitosan thermosensitive hydrogel according to claim 1 or 2, is characterized in that, containing following composition in the aqueous solution of often liter of methotrexate chitosan thermosensitive hydrogel:

4. methotrexate chitosan thermosensitive hydrogel according to claim 1 or 2, is characterized in that, described chitosan is the chitosan that deacetylation is greater than 95%.

5. methotrexate chitosan thermosensitive hydrogel according to claim 1 or 2, is characterized in that, described isoosmotic adjusting agent is sodium chloride or glucose.

6. methotrexate chitosan thermosensitive hydrogel according to claim 1 or 2, is characterized in that, described pH adjusting agent is sodium hydroxide.

7. methotrexate chitosan thermosensitive hydrogel according to claim 1 or 2, is characterized in that, the preparation method of described methotrexate chitosan thermosensitive hydrogel is:

(1) preparation of solution:

By the described proportioning of one of claim 1-2, take chitosan, add water appropriate, after magnetic agitation makes into chitosan suspension, then add acetic acid, continue stirring and chitosan is dissolved completely, obtain chitosan solution; Take sodium β-glycerophosphate, add water appropriate, ultrasonic dissolution, obtains sodium β-glycerophosphate solution; Take methotrexate and pH adjusting agent, HP-β-CD, isoosmotic adjusting agent, add water appropriate, ultrasonic dissolution, obtains methotrexate solution; Add water described in obtain solution process and be as the criterion to make added material dissolve completely in right amount;

(2) methotrexate chitosan thermosensitive hydrogel solution is prepared:

Under ice bath stirs, by sodium β-glycerophosphate dropwise instillation chitosan solution, after dripping off, continue ice bath and stir 10-20min, obtain chitosan sodium β-glycerophosphate solution; Equally under ice bath stirs, methotrexate dropwise is instilled in described chitosan sodium β-glycerophosphate solution, after dripping off, continue ice bath and stir 10-20min, obtain methotrexate chitosan thermosensitive hydrogel solution.