CN101905022B - Stable medicinal composition containing artemisia pollen allergen and preparation method thereof - Google Patents
Stable medicinal composition containing artemisia pollen allergen and preparation method thereof Download PDFInfo
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Abstract
The invention provides a stable liquid medicinal composition. The liquid medicinal composition contains therapeutically effective amount or diagnostically effective amount of artemisia pollen allergen, buffering effective amount of buffer solution with pH of 5.0 to 7.0, and pharmaceutically acceptable carrier. The form of the liquid medicinal composition is preferable oral liquid, injection, sublingual buccal tablets, aerosol, nasal agent or skin pricking agent. The invention also provides a method for preparing the liquid medicinal composition. The liquid medicinal composition provided by the invention can be used for treating or diagnosing various allergic diseases caused by artemisia pollen, and has broad application prospect and clinical value in the field of allergic disease diagnosis and treatment.
Description
Technical field
The invention belongs to the bio-pharmaceutical technical field, be specifically related to a kind of stable composition of liquid medicine that contains Artemisia pollen allergen and preparation method thereof and application.
Background technology
Anaphylactic disease is one of illness common, the most obstinate in the world today, is a kind of disease of serious threat human health.The sickness rate of anaphylactic disease accounts for greatly 20% of population, all might occur to the middle-aged and elderly people each age group from neonate, does not have obvious gender tendency, but obvious heritability tendency is arranged.
Cause the irritated original dirt demodicid mite of main allergic effect, pollen, room dirt, mycete, feather etc., wherein pollen is the important anaphylactogen of a class.Pollen is the male sex-cell of higher plant, under the effect of wind or worm, propagates in air, and some people just can produce anaphylaxis inhale people's pollen in respiratory after.Flying away of pollen has obvious seasonality.The anemophilous pollen multi-source in spring is in trees; The anemophilous pollen multi-source of late spring and early summer is in herbage; Anemophilous pollen multi-source at the beginning of late summer and autumn is in weeds.
The at present known pollen of human allergy that can cause mainly contains artimesia (Artemisia), Ambrosia pollen (Ambrosia), Ricinus pollen (Ricinus), Humulus pollen (Hummlus), Chenopodium pollen (Chenlpldum), Amaranthus pollen (Amaranthus), Casuarina pollen (Casuarina), Betula pollen (Betula), Pinus pollen (Pinus), Picea pollen (Picea), Cryptomeria pollen (Cryptomeria), plane pollen (Platanus) etc.The pollen of these plants is strong because of its pathogenicity, and the sensitization rate is high, and quantity is large, and the scope of disseminating is wide; And the scope that plant self distributes is wide, and quantity is also abundant, and is large on the impact of pollen contamination, thereby these plants become more important pollen contamination source plant.Because the impact of the factors such as distribution climate, soil, biology and landform of pollen contamination source plant, thereby different regions, its main pollen contamination source plant is also not identical.At America ﹠ Canada, the pollen contamination source is the most important with artemisiifolia; In Britain, Czech, Denmark, France, Italy, Spain and Switzerland, take grass as main; In South Africa, Palestine, Australia and New Zealand, divided by grass be main outside, trees class plant is outbalance also; And in Japan, what cause pollinosis mainly is Cortex Cryptomeriae Fortunei Radicis and cedar wood.In China, because the main pollen in most areas (such as Beijing, Xinjiang, Shanxi, Shandong, Wuhan, Shenyang, Guangzhou, Ningxia and other places) is Artemisia Plant Pollen, so the pollen contamination source plant of China is the most serious with sagebruss.
Treatment about anaphylactic disease all can't solve both at home and abroad fully, and at present main prevention comprises with the treatment measure avoids contacting allergen, symptomatic drugs treatment and specific active immunotherapy.Specific active immunotherapy is a kind of method of etiological treatment, it is with main allergenic substance unescapable and that confirm or suspect through skin pricking method test or additive method, make the allergen preparation of series concentration, carry out administration (being generally subcutaneous injection or sublingual administration) with the method for ascending-dose and concentration gradually, impel to produce corresponding antibody in the body.This antibody-like belongs to the IgG4 type, and after these specific antibodies improved in body fluid, when accepting external specific allergen once again, the at first with it combination of this antibody-like was with original sIgE antibody competition in the body.SIgE in the serum, can prevent anaphylactoid generation, thereby reach the purpose of desensitization when following to the irritated threshold of reaction in continuously later on gradually decline of desensitization treatment.
Because composition and the complex structure of Artemisia pollen allergen material itself, be subject to the impact of physics or chemical factor and lose activity, reduce and tire, even sometimes in low temperature environment, also can't guarantee the long-term stability of its allergenic activity, this makes such medicine " shelf life " all shorter, is unfavorable for long preservation.
Summary of the invention
Technical problem to be solved by this invention namely overcomes defects, and a kind of composition of liquid medicine of Artemisia pollen allergen that can be steady in a long-term is provided.
For achieving the above object, first aspect present invention provides a kind of stable composition of liquid medicine, it is characterized in that it comprises the treatment effective dose or diagnoses buffer and the pharmaceutically acceptable carrier of the pH 5.0～7.0 of the Artemisia pollen allergen of effective dose, buffering effective dose.In a better embodiment, described composition of liquid medicine is comprised of the buffer of pH 5.0～7.0 of the Artemisia pollen allergen for the treatment of effective dose or diagnosis effective dose, buffering effective dose and pharmaceutically acceptable carrier.
In another better embodiment, the Artemisia pollen allergen lixiviating solution of described Artemisia pollen allergen for making in order to the below method: take artimesia as raw material, through pollen defat, drying, lixiviate, concentrated and obtain.
In a preferred embodiment, the dosage form of described composition of liquid medicine is selected from the liquid dosage forms such as oral agents, injection, aerosol, nasal cavity agent, skin prick agent or sublingual administration agent.
In another preferred embodiment, described Artemisia pollen allergen is selected from north Chinese mugwort pollen allergen, Herba Artemisiae annuae pollen allergen, Artemisia or its combination.
In also having a preferred embodiment, the buffer of described pH 5.0～7.0 is selected from sodium hydrogen phosphate-citrate buffer solution, citric acid-sodium citrate buffer, sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution or acetic acid-sodium acetate buffer.In a preferred embodiment, the buffer of described pH 5.0～7.0 is selected from pH 5.0 sodium hydrogen phosphates-citrate buffer solution, pH 6.0 sodium hydrogen phosphates-citrate buffer solution, pH 6.4 sodium hydrogen phosphates-citrate buffer solution, pH 5.0 citric acid-sodium citrate buffer, pH 6.0 citric acid-sodium citrate buffer, pH 6.4 sodium hydrogen phosphates-phosphate sodium dihydrogen buffer solution, pH 7.0 sodium hydrogen phosphates-phosphate sodium dihydrogen buffer solution, pH 5.0 acetic acid-sodium acetate buffer or pH 5.4 acetic acid-sodium acetate buffer.
The present invention provides a kind of method for preparing described stable composition of liquid medicine on the other hand, and its buffer and pharmaceutically acceptable carrier that comprises the steps: the pH 5.0～7.0 of Artemisia pollen allergen, buffering effective dose that will the treatment effective dose mixes.
In a preferred embodiment, described method comprises the steps:
A) take artimesia as raw material, with pollen defat, lixiviate, concentrated, make the Artemisia pollen allergen lixiviating solution;
B) with step a) the Artemisia pollen allergen lixiviating solution of gained be diluted to preparation in 1: 1 to 1: 100000000 scope of volume ratio with the buffer of pH 5.0～7.0 of buffering effective dose; Add again pharmaceutically acceptable carrier, filtration sterilization.
In a preferred embodiment, described step a) in, using pH value in the described leaching process is 7.5～8.9 buffer lixiviate; Use the buffer of the pH 5.0～7.0 of buffering effective dose to replace the lixiviate buffer in the described concentration process.
Third aspect present invention also relates to the described stable application of composition of liquid medicine in the anaphylactic disease medicine that preparation is treated or diagnosis is caused by artimesia.
In a better embodiment, described anaphylactic disease is selected from the pollinosis that is caused by artimesia.In another better embodiment, described anaphylactic disease is selected from the I metallergy diseases such as allergic asthma, allergic rhinitis, anaphylaxis conjunctivitis or atopic dermatitis that caused by artimesia.
Can learn that from long-term stable experiment Artemisia pollen allergen composition of liquid medicine of the present invention has good stability, under 2～8 ℃ of conditions, can deposit 36 months.
Specific embodiments
Particularly, the present invention at first provides a kind of stable composition of liquid medicine, it is characterized in that it comprises the treatment effective dose or diagnoses buffer and the pharmaceutically acceptable carrier of the pH 5.0～7.0 of the Artemisia pollen allergen of effective dose, buffering effective dose.Term herein " comprising ... " refers to can also contain any other component in this medicine, these components can exist with any content, as long as this component that exists with this content is that human body is acceptable, and does not have substantial impact to get final product for the biological activity of active component in the pharmaceutical composition of the present invention.
In a preferred embodiment, described composition of liquid medicine is basically to be comprised of the Artemisia pollen allergen for the treatment of effective dose or diagnosis effective dose, buffer and the pharmaceutically acceptable carrier of pH 5.0～7.0 of buffering effective dose.In a preferred embodiment, described composition of liquid medicine is comprised of the buffer of pH 5.0～7.0 of the Artemisia pollen allergen for the treatment of effective dose or diagnosis effective dose, buffering effective dose and pharmaceutically acceptable carrier.
The preferred north of artemisia of the present invention (Artemisia) pollen allergen Chinese mugwort (Artemisia vulgaris Linn.) pollen allergen, Herba Artemisiae annuae (Artemisia annua Linn.) pollen allergen, Artemisia sieversiana Willd. (Artemisia sieversianaEhrhart ex Willd.) pollen allergen, Chinese mugwort (Artemisia argyi L é vl.et Van.) pollen allergen or its combination.Artemisia pollen allergen of the present invention can be liquid form or solid form; Can be to buy from commercial channels, the method that also can use the mode that do not make allergen degraded or inactivation in any prior art to extract from natural artimesia obtains (being the form of Artemisia pollen allergen lixiviating solution); It can be the Artemisia pollen allergen (for example north Chinese mugwort pollen Art v 1 recombinant allergen) of single component, it also can be the allergen mixture (for example Herba Artemisiae annuae pollen allergen mixture) of the artimesia in source identical (single source), under the prerequisite that is not affecting Artemisia pollen allergen self activity and tiring, also can be the multiple Artemisia pollen allergen Artemisia pollen allergen compositions source, that mix by any way (for example compositions of Herba Artemisiae annuae pollen allergen and northern Chinese mugwort pollen allergen).
In a better embodiment, the Artemisia pollen allergen lixiviating solution of described Artemisia pollen allergen for making in order to the below method: as raw material, pass through pollen defat, drying, lixiviate, concentrate and obtain with artimesia (should use dry artimesia).
Concentration (dosage) when term " treatment effective dose " refers to that composition of liquid medicine of the present invention is applied to specific active immunotherapy (desensitization treatment).Specific active immunotherapy is a kind of method of etiological treatment, it is with main allergenic substance unescapable and that confirm or suspect through skin pricking method test or additive method, make the allergen preparation of series concentration, carry out administration (being generally subcutaneous injection or sublingual administration) with the method for ascending-dose and concentration gradually, by repeatedly making the contact patients specific allergen, prevent anaphylactoid generation, thereby reach the purpose of desensitization.Generally speaking, Artemisia pollen allergen (lixiviating solution) total protein concentration of " treatment effective dose " is preferably 0.01 μ g/ml～10mg/ml, more preferably 0.1 μ g/ml～1mg/ml.According to the difference of route of administration, the concentration (dosage) of general sublingual administration is hypodermic 10 times～800 times.
Term " diagnosis effective dose " refers to that composition of liquid medicine of the present invention is applied to the skin prick concentration (dosage) in when diagnosis.Generally speaking, Artemisia pollen allergen (lixiviating solution) total protein concentration of " diagnosis effective dose " is preferably 1 μ g/ml～10mg/ml, more preferably 100 μ tg/ml～1mg/ml.
Composition of liquid medicine of the present invention can be made various medically acceptable dosage forms, and can by the doctor according to patient age, body weight and roughly the factor such as disease condition determine the useful dosage of patient is used.The dosage form of described composition of liquid medicine is selected from the liquid dosage forms such as oral agents, (subcutaneous) injection, sublingual administration agent, aerosol, nasal cavity agent or skin prick agent; Preferably (subcutaneous) injection, sublingual administration agent or skin prick agent.Wherein, (subcutaneous) injection, sublingual administration agent generally are dosage forms commonly used during as specific active immunotherapy, and the skin prick agent is dosage form commonly used when detecting as allergen in the body.
" buffer " of the present invention is well-known to those skilled in the art, and can be used safely in the pharmaceutical formulations, include but not limited to: glycine-hydrochloride buffer, phthalic acid-hydrochloride buffer, sodium hydrogen phosphate-citrate buffer solution, citric acid-sodium hydroxide-hydrochloride buffer, the citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer, sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution, sodium hydrogen phosphate-potassium phosphate buffer, potassium dihydrogen phosphate-sodium hydrate buffer solution, barbital sodium-hydrochloride buffer, Tris-hydrochloride buffer, boric acid-borate buffer solution, glycine-sodium hydrate buffer solution, Borax-sodium hydrate buffer solution, sodium carbonate-sodium bicarbonate buffer liquid, PBS buffer (sodium hydrogen phosphate-sodium dihydrogen phosphate-sodium chloride buffer) etc.Those skilled in the art can by the above buffer of commercially available acquisition, also can prepare the buffer of certain pH value as required.Unless have in addition describedly, " pH value " described in the present invention all is the pH value of surveying under 25 ℃ of conditions.
In composition of liquid medicine of the present invention, what preferably use is the buffer of pH 5.0～7.0.In a preferred embodiment, the buffer of described pH 5.0～7.0 is selected from sodium hydrogen phosphate-citrate buffer solution, citric acid-sodium citrate buffer, sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution or acetic acid-sodium acetate buffer.Also want in the preferred embodiment at one, the buffer of described pH 5.0～7.0 is selected from pH 5.0 sodium hydrogen phosphates-citrate buffer solution, pH 6.0 sodium hydrogen phosphates-citrate buffer solution, pH 6.4 sodium hydrogen phosphates-citrate buffer solution, pH 5.0 citric acid-sodium citrate buffer, pH 6.0 citric acid-sodium citrate buffer, pH 6.4 sodium hydrogen phosphates-phosphate sodium dihydrogen buffer solution, pH 7.0 sodium hydrogen phosphates-phosphate sodium dihydrogen buffer solution, pH 5.0 acetic acid-sodium acetate buffer or pH 5.4 acetic acid-sodium acetate buffer.
Term " buffering effective dose " refers to that the concentration of described buffer is enough to make the pH value of described composition of liquid medicine to maintain a specific pH value (preferred pH 5.0～7.0) ± 0.5 (better ± 0.3, good ± 0.2 also, best ± 0.1) in the individual pH unit.Such as but not limited to, pH6.0 ± 0.2 (being pH5.8～6.2), pH5.0 ± 0.3 (being pH4.7～5.3) etc.The concentration of concrete described buffer is relevant with the factors such as concrete composition of the kind of buffer, pH value, whole pharmaceutical composition, and it can be determined according to the normal experiment of limited number of time easily by those skilled in the art.Generally, the concentration of described acidic buffer is that (preferred 10mM～400mM, more preferably 30mM～300mM also want preferred 50mM～200mM) to 5mM～500mM.
Term " pharmaceutically acceptable carrier " should with medicine of the present invention in allergen compatible, can be with its blend the biological activity of decrease medicine (allergenic activity) under normal conditions not.Can be used as pharmaceutically acceptable carrier includes but not limited to: saccharide, such as lactose, glucose, sucrose, trehalose; Starch is such as corn starch, potato starch; The cellulose or derivatives thereof is such as sodium carboxymethyl cellulose, ethyl cellulose, methylcellulose; The tragakanta powder; Gelatin; Talcum; Kollag is such as stearic acid, magnesium stearate; Calcium sulfate; Vegetable oil is such as Oleum Arachidis hypogaeae semen, Oleum Gossypii semen, Oleum sesami, olive oil, Semen Maydis oil, cupu oil; Polyhydric alcohol is such as propylene glycol, glycerol, Sorbitol, mannitol, Polyethylene Glycol; Aminocaproic acid, alginic acid; Emulsifying agent is such as tween; Wetting agent is such as sodium lauryl sulfate; Coloring agent; Flavoring agent; Antioxidant; Pharmaceutical preservative etc., or its combination.The preferred pharmaceutically acceptable carrier of the present invention is the combination of polyhydric alcohol (being preferably glycerol) and pharmaceutical preservative (being preferably phenol or sorbate).
The effect of polyhydric alcohol is to give the allergen medicine suitable infiltration tension force.Polyhydric alcohol includes but not limited to, propylene glycol, glycerol, Sorbitol, mannitol, Polyethylene Glycol etc.In whole composition of liquid medicine, preferred 20%～80% (v/v) of the concentration of polyhydric alcohol, more preferably 30%～70% (v/v) also wants preferred 40%～60% (v/v), most preferably 45%～55% (v/v).
Pharmaceutical preservative is well-known to those skilled in the art, and its effect is the growth that prevents or suppress pathogenic microorganism.Pharmaceutical preservative includes, but not limited to phenol, thimerosal, benzoic acid, sorbic acid or its esters, parabens (parabens), benzyl alcohol, phenethanol etc., or its combination.The kind of the general and antiseptic of the consumption of pharmaceutical preservative, the many factors such as the kind of medicine, dosage form, pH value are relevant.In whole composition of liquid medicine, the concentration of preferred pharmaceutical preservative is 0.01%～3.0% (w/v).
Therefore, in a preferred embodiment, pharmaceutical composition of the present invention comprises the Artemisia pollen allergen for the treatment of effective dose or diagnosis effective dose, buffer, 0.01%～3.0% (w/v) pharmaceutical preservative and 20%～80% (v/v) polyhydric alcohol that cushions the pH 5.0～7.0 of effective dose.In a preferred embodiment, described pharmaceutical composition is basically to be comprised of the Artemisia pollen allergen for the treatment of effective dose or diagnosis effective dose, buffer, 0.01%～3.0% (w/v) pharmaceutical preservative and 20%～80% (v/v) polyhydric alcohol of pH 5.0～7.0 of buffering effective dose.Also want in the preferred embodiment at one, described pharmaceutical composition is comprised of buffer, 0.01%～3.0% (w/v) pharmaceutical preservative and 20%～80% (v/v) polyhydric alcohol of the pH 5.0～7.0 of the Artemisia pollen allergen for the treatment of effective dose or diagnosis effective dose, buffering effective dose.
In such scheme, preferred 0.2%～0.3% (w/v) phenol of described pharmaceutical preservative or 0.1%～0.2% (w/v) potassium sorbate; Described polyhydric alcohol preferred 45%～55% (v/v) glycerol.
In specific embodiments of the present invention, long-term stable experiment mainly is that the indexs such as total allergenic activity of Artemisia pollen allergen and main allergen content have been done investigation.Suppose that the initial total allergenic activity of Artemisia pollen allergen preparation is 100%, under certain condition through after a while, use same detection means, its total allergenic activity is changed into more than 70% of initial activity (preferred more than 80%, more preferably more than 90%); And main allergen content is under certain condition through after a while, and its content changes when also being more than 70% of initial amount (preferred more than 80%, more preferably more than 90%); Be " stablizing " with regard to the biological activity of assert this Artemisia pollen allergen, it is used for the allergen specific immunization therapy or the diagnosis of allergen skin prick also is reliable.
The present invention provides a kind of method for preparing described stable composition of liquid medicine on the other hand, and its buffer and pharmaceutically acceptable carrier that comprises the steps: the pH 5.0～7.0 of Artemisia pollen allergen, buffering effective dose that will the treatment effective dose mixes.
In a preferred embodiment, described method comprises the steps:
A) take artimesia as raw material, with pollen defat, lixiviate, concentrated, make the Artemisia pollen allergen lixiviating solution;
B) with step a) the Artemisia pollen allergen lixiviating solution of gained be diluted to the preparation of a plurality of concentration in 1: 1 to 1: 100000000 scope of volume ratio with the buffer of pH 5.0～7.0 of buffering effective dose; Add again pharmaceutically acceptable carrier, filtration sterilization.
Wherein, the preferred north of described Artemisia pollen allergen Chinese mugwort (Artemisia vulgaris Linn.) pollen allergen, Herba Artemisiae annuae (Artemisia annua Linn.) pollen allergen, Artemisia sieversiana Willd. (Artemisia sieversiana Ehrhart exWilld.) pollen allergen, Chinese mugwort (Artemisia argyi L é vl.et Van.) pollen allergen or its combination.
In such scheme, employed degreasing agent is conventional during described pollen defat, and degreasing agent includes but not limited to, ether, acetone and other organic solvent.
When preparation Artemisia pollen allergen lixiviating solution, the inventor has attempted multiple lixiviating solution, and has compared their extracting efficiency.Employed lixiviating solution comprises: coca ' the s liquid of sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of sodium hydrogen phosphate-citrate buffer solution of normal saline, pH5.0, sodium hydrogen phosphate-citrate buffer solution of pH6.0, pH7.5, sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of pH8.0, pH8.2, the Tris-hydrochloride buffer of pH8.9 etc.Experimental result confirms that the extracting efficiency of the buffer of use pH 7.5～8.9 is higher, and this may be the relevant of slant acidity with pollen class material.In a better embodiment, the sodium hydrogen phosphate of use pH 8.0-phosphate sodium dihydrogen buffer solution lixiviate Artemisia pollen allergen.With traditional coca ' s liquid phase ratio, although the extracting efficiency of sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of pH8.0 does not significantly improve, but the pH value that can make whole lixiviating solution keeps stable, and this lixiviating solution long storage time also can not produce precipitation, has overcome some defective of coca ' s liquid.
In another preferred scheme, described step a) in, use the buffer of the pH 5.0～7.0 of buffering effective dose to replace the lixiviate buffer in the described concentration process.
It is pointed out that it is well-known to those skilled in the art that above-mentioned concentrated allergen leachate is also replaced the method for buffer simultaneously, includes but not limited to the methods such as ultrafiltration, dialysis.The aperture of damming of selected film can be determined as required, requires to remove under the prerequisite that keeps as far as possible allergenic activity the impurity such as micromolecule pigment of non-activity, and the molecular weight that preferably dams is 3～10kD.In one embodiment of the invention, use the ultrafilter membrane of the molecular weight 3kD that dams to carry out the concentrated allergen leachate of method of ultrafiltration and replace simultaneously buffer.
The buffer of employed pH 5.0～7.0 when a) replacement lixiviate buffer of described step, with at described step b) dilution in the buffer of employed pH 5.0～7.0 can be identical, also can be different, as long as both can melt mutually, and mixed buffer system has stable buffer capacity, make the pH value of whole medicine maintain a specific pH value (preferred pH 5.0～7.0) ± 0.5 (better ± 0.3, good ± 0.2 also, best ± 0.1) gets final product in the individual pH unit.The buffer of the pH 5.0～7.0 of the buffering effective dose that general preferred use is identical, for example, when a) replacement lixiviate buffer of described step, use pH 6.0 sodium hydrogen phosphates of buffering effective dose-citrate buffer solution, at described step b) dilution in use pH 6.0 sodium hydrogen phosphates of buffering effective dose-citrate buffer solution.
In a better embodiment, the buffer of described pH 5.0～7.0 is selected from sodium hydrogen phosphate-citrate buffer solution, citric acid-sodium citrate buffer, sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution or acetic acid-sodium acetate buffer.In a better embodiment, the buffer of described pH 5.0～7.0 is selected from pH 5.0 sodium hydrogen phosphates-citrate buffer solution, pH 6.0 sodium hydrogen phosphates-citrate buffer solution, pH 6.4 sodium hydrogen phosphates-citrate buffer solution, pH 5.0 citric acid-sodium citrate buffer, pH 6.0 citric acid-sodium citrate buffer, pH 6.4 sodium hydrogen phosphates-phosphate sodium dihydrogen buffer solution, pH 7.0 sodium hydrogen phosphates-phosphate sodium dihydrogen buffer solution, pH 5.0 acetic acid-sodium acetate buffer or pH 5.4 acetic acid-sodium acetate buffer.
Therefore, in an embodiment that is more preferably, the method for preparing stable composition of liquid medicine of the present invention comprises the steps:
Step 1: the preparation of Artemisia pollen allergen lixiviating solution.
(1) defat: can use continuously organic solvent (for example acetone) to soak dry artimesia (press mass volume ratio at every turn and added acetone in 1: 2～1: 10), defat 2～6 times, each 0.1～24 hour (preferred 1～12 hour), acetone to the defat be colourless after, with the solids natural drying after the defat to without weighing behind the acetone flavor.Laboratory operation should be finished in fume hood, and the vacuum concentration extraction pot is adopted in pilot scale.
(2) lixiviate: the two is take 1: 10～1 to make buffer through the artimesia behind the degreaser drying and pH 7.5～8.9 (sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of preferred pH 8.0): (better is 1: 20～1 to 50W/V (be behind per 1 gram degreaser drying artimesia with the buffer lixiviate of 2～50 milliliters of pH 7.5～8.9): 40W/V, better is 1: ratio 30W/V) is extracted, fully the contact lixiviate (was preferably carried out 1～60 hour in 0.1～72 hour, more preferably 12～48 hours), centrifugal or/and filter, collect crude extract.Wherein, described lixiviate operation should be carried out under low temperature (preferred 2～8 ℃); In order to prevent or suppress the growth of pathogenic microorganism, should carry out in advance sterilization treatment to the lixiviate buffer.
(3) concentrated: it is that the ultrafilter membrane of 3～10kD carries out ultrafiltration that crude extract obtained above is used the molecular weight that dams, the buffer that repeatedly adds pH 5.0～7.0 is replaced original lixiviate buffer (preferred ultrafiltration 2～5 times), remove the impurity such as micromolecule pigment, finally make concentrated 2～10 times of crude extract volume, obtain the Artemisia pollen allergen lixiviating solution.
Step 2: the preparation of composition of liquid medicine.
(1) dilution: the Artemisia pollen allergen lixiviating solution that step 1 is made is diluted to 1: 1 to 1: 100000000 interior preparation of scope of volume ratio with the buffer of the pH 5.0～7.0 of buffering effective dose, add pharmaceutically acceptable carrier (preferably add polyhydric alcohol and pharmaceutical preservative, the final concentration that makes polyhydric alcohol is that the final concentration of 20%～80% (v/v), pharmaceutical preservative is 0.01%～3.0% (w/v)).
(2) degerming: the aseptic microporous filter membrane that uses the aperture to be not more than 1 μ m (preferred 0.22 μ m) carries out filtration sterilization, and packing, embedding make described composition of liquid medicine finished product.
It should be noted that the present invention when describing the described composition of liquid medicine method of preparation, the sentence formula of having used " comprising the steps: ... ".That is to say, those skilled in the art can also be as required, when the described composition of liquid medicine of preparation, increase other technical step, such as but not limited to, use existing routine techniques that described Artemisia pollen allergen lixiviating solution is carried out lyophilizing, when formulated (medicine), re-use suitable buffer (buffer of preferred pH 5.0～7.0) dissolving; Perhaps after lixiviate, first crude extract is regulated pH value, carry out again ultrafiltration and concentration; Can also carry out quality testing (as measuring protein content etc.), convenient follow-up dilution step for described Artemisia pollen allergen lixiviating solution.Certainly, the technical step of increase must guarantee basically can not affect the biological activity (allergenic activity) of described composition of liquid medicine.
Third aspect present invention also relates to the described stable application of composition of liquid medicine in the anaphylactic disease medicine that preparation is treated or diagnosis is caused by artimesia.
In a better embodiment, described anaphylactic disease is selected from the pollinosis that is caused by artimesia.In another better embodiment, described anaphylactic disease is selected from the I metallergy diseases such as allergic asthma, allergic rhinitis, anaphylaxis conjunctivitis or atopic dermatitis that caused by artimesia.
In another better embodiment, described artimesia is selected from north Chinese mugwort (Artemisia vulgaris Linn.) pollen, Herba Artemisiae annuae (Artemisia annua Linn.) pollen, Artemisia sieversiana Willd. (Artemisia sieversiana Ehrhart exWilld.) pollen, Chinese mugwort (Artemisia argyi L é vl.et Van.) pollen or its combination.
When composition of liquid medicine of the present invention is applied to diagnose the anaphylactic disease that is caused by artimesia (allergen skin prick diagnostic test), except composition of liquid medicine of the present invention, generally also should comprise negative controls, positive control solution and pricking needles.Negative controls is generally the non-allergenie liquid of human body (such as mixture of glycerol and saline solution etc.), and positive control solution is generally the histamine phosphate of 1.0～5.0mg/ml/histamine hydrochloride solution (for example 1.7mg/ml histamine phosphate solution).
In addition, composition of liquid medicine of the present invention also can be applied in the patch test.Its principle is: suspicious sensitizer (allergen) is applied on the patient skin, enters behind the body by antigen-presenting cell antigen presentation to the T lymphocyte by skin or mucosa, make the T lymphocyte specific activation, bring out inflammatory reaction.
Below in conjunction with embodiment the present invention is described in further detail.Yet should be appreciated that and enumerate these embodiment just in order to play illustration, and be not to limit the scope of the invention.
The preparation of embodiment 1 Herba Artemisiae annuae pollen allergen sublingual administration agent
(1) defat: accurately take by weighing dry Herba Artemisiae annuae pollen, press mass volume ratio and add acetone at 1: 4, continuous stirring defat 2 hours was left standstill 30 minutes again, removed upper strata acetone, repeated above-mentioned skimming processes 4 times; Solid after the defat is paved, dry in ventilating kitchen, to being powdered, and there is not the acetone abnormal smells from the patient.
(2) lixiviate: the defat pollen that step (1) is obtained and sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of 50mM pH8.0 the two with 1: 30 (W/V) ratio, 4 ℃ of continuous stirring lixiviates 48 hours; 4 ℃, centrifugal 30 minutes of 10000r.p.m. collects supernatant; Successively use common filter paper, 0.45 μ m microporous filter membrane to filter successively, remove finely ground particle substance, obtain crude extract.
(3) concentrated: the crude extract that step (2) is obtained is that the ultrafilter membrane of 3kD carries out ultrafiltration with holding back the aperture, repeatedly add the sodium hydrogen phosphate of 50mM pH6.0-citrate buffer solution and replace lixiviate buffer (ultrafiltration 3 times), remove the impurity such as micromolecule pigment, finally make concentrated 6 times of crude extract volume, obtain Herba Artemisiae annuae pollen allergen lixiviating solution.
(4) determination of protein concentration: the Herba Artemisiae annuae pollen allergen lixiviating solution that step (3) is obtained carries out total protein concentration mensuration (the BCA protein determination kit with Pierce company is measured protein content), when if total protein concentration is not less than 3mg/ml, namely meet the requirements.
(5) dilution: the Herba Artemisiae annuae pollen allergen lixiviating solution that step (4) is made is diluted to the preparation of the interior a plurality of concentration of 1: 1 to 1: 100000000 scope of volume ratio with sodium hydrogen phosphate-citrate buffer solution of 50mM pH6.0, add 1: 1 glycerol of volume ratio (making its final concentration is 50% (v/v)), add again phenol (making its final concentration is 0.25% (w/v)).
(6) degerming: use the aseptic microporous filter membrane of 0.22 μ m to carry out filtration sterilization, packing, embedding make Herba Artemisiae annuae pollen allergen sublingual administration agent finished product.Respectively name: Herba Artemisiae annuae pollen allergen sublingual administration agent No. 6～No. 1, total protein concentration are followed successively by 500 μ g/ml, 100 μ g/ml, 20 μ g/ml, 4 μ g/ml, 0.8 μ g/ml, 0.16 μ g/ml.
The preparation of embodiment 2 Herba Artemisiae annuae pollen allergen skin prick liquid
Step (1)-(4) are with embodiment 1, but the sodium hydrogen phosphate of use 150mM pH7.0-phosphate sodium dihydrogen buffer solution is replaced the lixiviate buffer when ultrafiltration and concentration.
(5) dilution: it is 1200 μ g/ml that the Herba Artemisiae annuae pollen allergen lixiviating solution that step (4) is made is diluted to total protein concentration with sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of 150mM pH7.0, add 1: 1 glycerol of volume ratio (making its final concentration is 50% (v/v)), add again phenol (making its final concentration is 0.2% (w/v)).
(6) degerming: use the aseptic microporous filter membrane of 0.22 μ m to carry out filtration sterilization, packing, embedding make Herba Artemisiae annuae pollen allergen skin prick liquid finished product, and the total protein concentration of pricking method liquid finished product is 600 μ g/ml.
The preparation of embodiment 3 Artemisia subcutaneous injection agent
(1) defat: accurately take by weighing dry Artemisia sieversiana Willd. pollen, press mass volume ratio and add acetone at 1: 4, continuous stirring defat 2 hours was left standstill 30 minutes again, removed upper strata acetone, repeated above-mentioned skimming processes 4 times; Solid after the defat is paved, dry in ventilating kitchen, to being powdered, and there is not the acetone abnormal smells from the patient.
(2) lixiviate: the defat pollen that step (1) is obtained and sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of 50mM pH8.0 the two with 1: 30 (W/V) ratio, 4 ℃ of continuous stirring lixiviates 48 hours; 4 ℃, centrifugal 30 minutes of 10000r.p.m. collects supernatant; Successively use common filter paper, 0.45 μ m microporous filter membrane to filter successively, remove finely ground particle substance, obtain crude extract.
(3) concentrated: the crude extract that step (2) is obtained is that the ultrafilter membrane of 3kD carries out ultrafiltration with holding back the aperture, the citric acid-sodium citrate buffer that repeatedly adds 100mM pH5.0 is replaced lixiviate buffer (ultrafiltration 3 times), remove the impurity such as micromolecule pigment, finally make concentrated 6 times of crude extract volume, obtain the Artemisia lixiviating solution.
(4) determination of protein concentration: the Artemisia lixiviating solution that step (3) is obtained carries out total protein concentration mensuration (the BCA protein determination kit with Pierce company is measured protein content), when if total protein concentration is not less than 3mg/ml, namely meet the requirements.
(5) dilution: the Artemisia lixiviating solution that step (4) is made is diluted to the preparation of the interior a plurality of concentration of 1: 1 to 1: 100000000 scope of volume ratio with the citric acid-sodium citrate buffer of 100mM pH5.0, add glycerol (making its final concentration is 45% (v/v)), add again phenol (making its final concentration is 0.25% (w/v)).
(6) degerming: use the aseptic microporous filter membrane of 0.22 μ m to carry out filtration sterilization, packing, embedding make Artemisia subcutaneous injection agent finished product.Respectively name: Artemisia subcutaneous injection agent No. 4～No. 1, total protein concentration is followed successively by 1000ng/ml, 100ng/ml, 10ng/ml, 1ng/ml.
The preparation of embodiment 4 north Chinese mugwort pollen allergen sublingual administration agent
(1) defat: accurately take by weighing dry north Chinese mugwort pollen, press mass volume ratio and add acetone at 1: 4, continuous stirring defat 2 hours was left standstill 30 minutes again, removed upper strata acetone, repeated above-mentioned skimming processes 4 times; Solid after the defat is paved, dry in ventilating kitchen, to being powdered, and there is not the acetone abnormal smells from the patient.
(2) lixiviate: the defat pollen that step (1) is obtained and sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of 50mM pH8.0 the two with 1: 30 (W/V) ratio, 4 ℃ of continuous stirring lixiviates 48 hours; 4 ℃, centrifugal 30 minutes of 10000r.p.m. collects supernatant; Successively use common filter paper, 0.45 μ m microporous filter membrane to filter successively, remove finely ground particle substance, obtain crude extract.
(3) concentrated: the crude extract that step (2) is obtained is that the ultrafilter membrane of 3kD carries out ultrafiltration with holding back the aperture, repeatedly add the sodium hydrogen phosphate of 200mM pH6.4-phosphate sodium dihydrogen buffer solution and replace lixiviate buffer (ultrafiltration 3 times), remove the impurity such as micromolecule pigment, finally make concentrated 6 times of crude extract volume, obtain north Chinese mugwort pollen allergen lixiviating solution.
(4) determination of protein concentration: the north Chinese mugwort pollen allergen lixiviating solution that step (3) is obtained carries out total protein concentration mensuration (the BCA protein determination kit with Pierce company is measured protein content), when if total protein concentration is not less than 3mg/ml, namely meet the requirements.
(5) dilution: the north Chinese mugwort pollen allergen lixiviating solution that step (4) is made is diluted to the preparation of the interior a plurality of concentration of 1: 1 to 1: 100000000 scope of volume ratio with sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of 200mM pH6.4, add glycerol (making its final concentration is 55% (v/v)), add again potassium sorbate (making its final concentration is 0.15% (w/v)).
(6) degerming: use the aseptic microporous filter membrane of 0.22 μ m to carry out filtration sterilization, packing, embedding make north Chinese mugwort pollen allergen sublingual administration agent finished product.Respectively name: north Chinese mugwort pollen allergen sublingual administration agent No. 5～No. 1, total protein concentration is followed successively by 300 μ g/ml, 100 μ g/ml, 10 μ g/ml, 1 μ g/ml, 0.1 μ g/ml.
The preparation of embodiment 5 north Chinese mugwort pollen allergen skin prick liquid
Step (1)-(4) are with embodiment 4, but the acetic acid of use 200mM pH5.4-sodium acetate buffer is replaced the lixiviate buffer when ultrafiltration and concentration.
(5) dilution: it is 800 μ g/ml that the north Chinese mugwort pollen allergen lixiviating solution that step (4) is made is diluted to total protein concentration with acetic acid-sodium acetate buffer of 200mM pH5.4, add 1: 1 glycerol of volume ratio (making its final concentration is 50% (v/v)), add again phenol (making its final concentration is 0.3% (w/v)).
(6) degerming: use the aseptic microporous filter membrane of 0.22 μ m to carry out filtration sterilization, packing, embedding make north Chinese mugwort pollen allergen skin prick liquid finished product, and the total protein concentration of pricking method liquid finished product is 400 μ g/ml.
The bioactive mensuration of embodiment 6 Artemisia pollen allergen
(1) mensuration of total allergenic activity
Artemisia to be measured (such as north Chinese mugwort) pollen allergen preparation is mixed (100 μ l+100 μ l) with serum equal-volume in the artimesia standard serum storehouse (Zhejiang Wolwo Biotech Co., Ltd. provides), use simultaneously in this standard serum storehouse serum in contrast, 37 ℃ of incubator incubations 1 hour, then take out and be statically placed in 4 ℃ of refrigerator overnight (9～12 hours).Sample after 4 ℃ spent the night is transferred in the sterilized teat glass, measure the relative amount (according to the Immuno CAP diagnostic system of Sweden Pharmacia Corp, the operation of the automatic vitro detection anaphylactogen of Unicap system specification) of its sIgE with Unicap100 instrument (Sweden Pharmacia Corp).The sIgE relative amount that serum records in the artimesia standard serum storehouse is 78.29KUA/L.
Serum in the standard serum storehouse contains for allergenic specific IgE (sIgE).Allergenic activity in the product to be tested becomes the sIgE of branch in serum to be combined to generate complex, sIgE concentration free in the serum is reduced, detect the content of sIgE in the serum with the Unicap system again.Allergenic activity is higher in the product to be tested, and after serum and the product to be tested effect, its sIgE concentration will be fallen lowlyer.Detect with the Unicap system and can find that sIgE free in the serum can corresponding minimizing this moment, and the variation by sIgE concentration in the serum before and after relatively product to be tested adds just can obtain the total allergenic activity in the product to be tested.SIgE is by in the situation of the complete combination of allergen in the product to be tested in serum, in this process in total allergenic activity and the serum reduction degree of sIgE concentration be positively related.Can obtain total allergenic activity in the product to be tested according to the suppression ratio of sIgE, the suppression ratio of sIgE is higher, and total allergenic activity is higher in the product to be tested.
(2) mensuration of main allergen content
Use the main allergen protein 1 assay test kit of artimesia (being produced by middle money Chinese arteries and veins (Beijing) Bioisystech Co., Ltd), according to its subsidiary description operation.Need to prove, capture antibody in the main allergen protein 1 assay test kit of artimesia is for the artimesia monoclonal antibody that (comprising Herba Artemisiae annuae, Artemisia sieversiana Willd., north Chinese mugwort etc.), main allergen protein 1 common epitope produced, therefore, this test kit can detect the not content of the main allergen protein 1 of pollen of the same race of artemisia.
Embodiment 7 long-term stable experiments are investigated
Investigate sample:
Sample 1-6), (be denoted as in the following table: sample 1-1) for No. 1 No. 6, the Herba Artemisiae annuae pollen allergen sublingual administration agent of embodiment 1 preparation (is denoted as in the following table:;
The Herba Artemisiae annuae pollen allergen skin prick liquid of embodiment 2 preparations (is denoted as in the following table: sample 2);
The Artemisia subcutaneous injection agent of embodiment 3 preparations (is denoted as in the following table: sample 3-4) for No. 4;
Sample 4-5), (be denoted as in the following table: sample 4-1) for No. 1 No. 5, the north Chinese mugwort pollen allergen sublingual administration agent of embodiment 4 preparation (is denoted as in the following table:;
The north Chinese mugwort pollen allergen skin prick liquid of embodiment 5 preparations (is denoted as in the following table: sample 5);
Contrast 1: use coca ' s liquid (5.0g sodium chloride, 2.75g sodium bicarbonate, add purified water to 1000ml) obtain Herba Artemisiae annuae pollen allergen lixiviating solution after the lixiviate, add 1: 1 glycerol of volume ratio (making its final concentration is 50% (v/v)), add again phenol (making its final concentration is 0.25% (w/v)), and carry out filtration sterilization with the aseptic microporous filter membrane of 0.22 μ m.The total protein concentration of said preparation is 500 μ g/ml;
Contrast 2: step (1)-(4) are with embodiment 4, but the use normal saline is replaced the lixiviate buffer when ultrafiltration and concentration.Again with normal saline dilution north obtained above Chinese mugwort pollen allergen lixiviating solution, add glycerol (making its final concentration is 55% (v/v)), add again potassium sorbate (making its final concentration is 0.15% (w/v)), and carry out filtration sterilization with the aseptic microporous filter membrane of 0.22 μ m.The total protein concentration of said preparation is 300 μ g/ml;
Experimental condition: 2 ℃～8 ℃;
Test sampling time point: detect in test sampling in the 3rd, 6,9,12,18,24,36 month;
The investigation project: pH value (the results are shown in Table 1), total allergenic activity (the results are shown in Table 2), main allergen protein 1 content (the results are shown in Table 3), wherein, "-" representative does not detect significant numerical value.
The testing result of table 1pH value
| 0 day | March | June | JIUYUE | December | 18 months | 24 months | 36 months | |
| Sample 1-6 | 6.02 | 6.04 | 6.05 | 6.10 | 6.11 | 6.09 | 6.03 | 6.06 |
| Sample 1-1 | 6.08 | 6.11 | 6.08 | 6.07 | 6.02 | 6.09 | 6.04 | 6.03 |
| Sample 2 | 7.06 | 7.05 | 7.08 | 7.03 | 7.02 | 7.04 | 7.00 | 7.01 |
| Sample 3-4 | 5.11 | 5.05 | 5.07 | 5.10 | 5.04 | 5.02 | 5.05 | 5.09 |
| Sample 4-5 | 6.42 | 6.41 | 6.44 | 6.50 | 6.48 | 6.40 | 6.43 | 6.45 |
| Sample 4-1 | 6.43 | 6.45 | 6.45 | 6.42 | 6.46 | 6.47 | 6.42 | 6.46 |
| Sample 5 | 5.42 | 5.44 | 5.40 | 5.41 | 5.44 | 5.43 | 5.48 | 5.47 |
| Contrast 1 | 8.14 | 9.00 | 9.26 | 9.45 | 9.88 | 10.21 | 10.40 | 10.67 |
| Contrast 2 | 5.84 | 6.15 | 6.48 | 6.80 | 6.97 | 7.04 | 7.19 | 7.27 |
The testing result of the total allergenic activity of table 2
| 0 day | March | June | JIUYUE | December | 18 months | 24 months | 36 months | |
| Sample 1-6 | 100％ | 98.87％ | 99.04％ | 98.27％ | 96.55％ | 93.70％ | 91.66％ | 89.47％ |
| Sample 1-1 | 100％ | 99.25％ | 97.50％ | 98.20％ | 97.76％ | 94.56％ | 92.41％ | 88.81％ |
| Sample 2 | 100％ | 98.55％ | 96.26％ | 96.89％ | 95.54％ | 94.30％ | 92.09％ | 88.90％ |
| Sample 3-4 | 100％ | 97.38％ | 98.14％ | 96.60％ | 95.68％ | 93.57％ | 91.68％ | 89.40％ |
| Sample 4-5 | 100％ | 98.60％ | 97.53％ | 96.74％ | 95.30％ | 93.07％ | 91.91％ | 88.64％ |
| Sample 4-1 | 100％ | 97.68％ | 97.89％ | 95.67％ | 96.27％ | 94.20％ | 92.57％ | 89.03％ |
| Sample 5 | 100％ | 97.57％ | 96.99％ | 95.21％ | 95.52％ | 93.67％ | 91.50％ | 88.79％ |
| Contrast 1 | 100％ | 94.75％ | 68.57％ | 56.20％ | 41.67％ | 23.89％ | 10.88％ | 1.64％ |
| Contrast 2 | 100％ | 95.64％ | 70.63％ | 61.63％ | 49.36％ | 30.47％ | 15.31％ | 3.79％ |
Annotate: the total allergenic activity during take 0 day is as 100%, and the numerical value contrast of the numerical value of measuring At All Other Times during with 0 day carried out ratio Analysis (percent), the variation of the total allergenic activity of detection different time points.
The main allergen protein 1 assay result of table 3 artimesia (μ g/ml)
| 0 day | March | June | JIUYUE | December | 18 months | 24 months | 36 months | |
| Sample 1-6 | 101.26 | 99.87 | 96.53 | 91.27 | 86.27 | 81.22 | 76.27 | 72.87 |
| Sample 2 | 124.67 | 121.25 | 118.20 | 114.29 | 100.05 | 99.85 | 93.62 | 87.53 |
| Sample 4-5 | 58.16 | 57.64 | 56.07 | 54.98 | 50.84 | 47.07 | 44.60 | 41.66 |
| Sample 5 | 81.59 | 78.71 | 75.53 | 71.62 | 68.09 | 65.70 | 62.23 | 58.05 |
| Contrast 1 | 102.62 | 82.41 | 49.85 | 20.52 | 8.24 | 1.66 | - | - |
| Contrast 2 | 60.21 | 48.53 | 30.28 | 16.76 | 6.99 | 1.36 | - | - |
The clinical efficacy of embodiment 8 Herba Artemisiae annuae pollen allergen sublingual administration agent
(1) Herba Artemisiae annuae pollen allergen skin prick test
1. the composition of pricking method test kit:
1) Herba Artemisiae annuae pollen allergen skin prick liquid (embodiment 2 prepares);
2) positive control (the histamine phosphate solution of 1.7mg/ml, solvent are the equal-volume mixed liquor of sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of glycerol and 150mM pH7.0, and add the phenol of final concentration 0.2% (w/v));
3) negative control (the equal-volume mixed liquor of sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of glycerol and 150mM pH7.0, and the phenol of adding final concentration 0.2% (w/v)).
2. pricking method operating procedure:
1) cleans the forearm palmar skin with ethanol first before the diagnosis;
2) with Herba Artemisiae annuae pollen allergen skin prick liquid, negative control, positive control from top to bottom each one in the cleaning after skin on, between two drops the distance 3～5cm, to prevent that reacting blush merges mutually;
3) during pricking method, taught skin sees through drop with pricking needles, vertically thrusts skin, to every kind of test solution with a new pricking needles;
4) wipe debris on the skin away with cotton swab after 2～3 minutes, note not mixing adjacent drops;
5) after the pricking method 10～20 minutes, viewing test result.The skin of positive reaction can present welt and these 2 kinds of phenomenons of blush, describes sideline around welt and the blush with the oil pen.Welt refers to the flat protuberance that is higher than skin that occurs behind the contact skin anaphylactogen blush is arranged on every side.If the positive findings welt is obvious, just compare the welt area, if welt is not obvious, can compare the blush area;
6) relatively the easiest method of welt area is range estimation, do correction with negative control welt or blush area, the area that compares Herba Artemisiae annuae pollen allergen skin prick liquid and positive control welt or blush, if Herba Artemisiae annuae pollen allergen skin prick liquid welt and more than 25% of the positive contrast area of blush area, can judge that this patient Herba Artemisiae annuae pollen allergen pricking method test is positive, can consider to use specific active immunotherapy (desensitization treatment);
7) the welt area more accurate such as needs, the circle that available adhesive tape is described the oil pen is transferred on millimeter electrocardiograph paper, determine this patient to Herba Artemisiae annuae allergen anaphylaxis intensity by the shared grid number of welt that compares Herba Artemisiae annuae pollen allergen skin prick liquid and positive control, at last testing result is recorded in the pricking method test data sheet originally.
3. the result judges:
If pricking method position skin presents welt or blush, be positive reaction; Judge the reaction rank according to welt Area Ratio due to Herba Artemisiae annuae pollen allergen skin prick liquid and the positive control:
● the positive contrast welt 0～25% of ratio or identical person with negative control are (one);
● the positive contrast welt 26%～50% of ratio is (+);
● the positive contrast welt 51%～100% of ratio is (++);
● the positive contrast welt 101～200% of ratio is (+++);
● the positive contrast welt 200% above person of ratio is (++ ++).
(2) clinical efficacy of Herba Artemisiae annuae pollen allergen sublingual administration agent
1. case:
Select pollinosis patient totally 83 examples: the course of disease is more than 3 years, and proves Herba Artemisiae annuae pollen hypersensitivity (++ more than) through above-mentioned dermatological puncture test.Wherein, male 39 examples, women 44 examples, the oldest 54 years old, minimum 8 years old, 35 years old mean age.
2. drug usage and consumption:
Medicine: the concrete total protein concentration of Herba Artemisiae annuae pollen allergen sublingual administration agent (embodiment 1 prepares) is followed successively by: No. 1 (0.16 μ g/ml), No. 2 (0.8 μ g/ml) of Herba Artemisiae annuae pollen allergen sublingual administration agent, No. 3 (4 μ g/ml), No. 4 (20 μ g/ml), No. 5 (100 μ g/ml), No. 6 (500 μ g/ml).
Usage (sublingual administration): drip in the Sublingual, swallow every day 1 time after containing about 1～2 minute.
Consumption: 1 of No. 1, the 1st day sublingual administration Herba Artemisiae annuae pollen allergen sublingual administration agent, the 2nd day 1, the 3rd day 2, the 4th day 2, the 5th day 3, the 6th day 3, the 7th day 4, the 8th day 4, the 9th day 5, the 10th day 5; No. 2, the 11st day～the 20th day sublingual administration Herba Artemisiae annuae pollen allergen sublingual administration agent, No. 1, the dosage of every day and the agent of Herba Artemisiae annuae pollen allergen sublingual administration are identical; No. 3, the 21st day～the 30th day sublingual administration Herba Artemisiae annuae pollen allergen sublingual administration agent, No. 1, the dosage of every day and the agent of Herba Artemisiae annuae pollen allergen sublingual administration are identical; No. 4, the 31st day～the 40th day sublingual administration Herba Artemisiae annuae pollen allergen sublingual administration agent, No. 1, the dosage of every day and the agent of Herba Artemisiae annuae pollen allergen sublingual administration are identical; No. 5, the 41st day～the 50th day sublingual administration Herba Artemisiae annuae pollen allergen sublingual administration agent, No. 1, the dosage of every day and the agent of Herba Artemisiae annuae pollen allergen sublingual administration are identical; The agent of the 51st day beginning sublingual administration Herba Artemisiae annuae pollen allergen sublingual administration is maintained until treatment for No. 6 and finishes, and every day, sublingual administration was 2.
Points for attention: general begin treatment when allergic symptom (pollinosis symptom) is slight.No. 1～No. 5 is ascending-dose, and No. 6 is maintenance dose.If take the sx↑ reaction is arranged in rear 24 hours, inferior daily dose should reduce 3 grades and (as just using the person No. 6, use instead No. 3; If still in initial stage for the treatment of, then time daily dose is reduced to minimum dose, increases progressively gradually after the tolerance again.)
The course for the treatment of: treatment full half a year of person is totally 32 examples; Full 1 year person for the treatment of is totally 27 examples; Full 2 years persons for the treatment of are totally 24 examples.
3. curative effect is judged:
Clinic control: with ratio before the treatment, nose, eye symptom or the pollen hypersensitivity asthma of pollen period (8～October) pollinosis obviously alleviate and reach 2 more than the pollen season.Produce effects: with ratio before the treatment, nose, eye symptom or the pollen hypersensitivity asthma of pollen period (8～October) pollinosis alleviate and reach 2 more than the pollen season; Perhaps obviously alleviate but 2 pollen season of elapsed-time standards less than.Take a turn for the better: with ratio before the treatment, pollen period (8～October) pollinosis symptom or pollen hypersensitivity asthma slightly alleviate.Invalid: with ratio before the treatment, pollen period (8～October) pollinosis symptom or pollen hypersensitivity asthma are unchanged or increase the weight of.
4. result:
Clinic control 43 examples (accounting for 51.8%) behind the 83 routine patient treatments, produce effects 26 examples (accounting for 31.3%), 11 examples that take a turn for the better (accounting for 13.3%), invalid 3 examples (accounting for 3.6%), total effective rate (clinic control+produce effects) is 83.1%.And the result shows that also the desensitization treatment time is longer, and effective percentage is higher.
Although the invention describes concrete example, having a bit is significantly to those skilled in the art, namely can make various changes and change the present invention under the prerequisite that does not break away from aim of the present invention, main points and scope.Therefore, claims have covered all these changes within the scope of the present invention.
Claims (41)
1. a stable composition of liquid medicine is characterized in that, this composition of liquid medicine comprises buffer and the pharmaceutically acceptable carrier of the Artemisia pollen allergen for the treatment of effective dose or diagnosis effective dose, the pH5.0 of buffering effective dose～7.0,
Wherein, the Artemisia pollen allergen lixiviating solution of described Artemisia pollen allergen for making in order to the below method: take artimesia as raw material, through pollen defat, drying, lixiviate, concentrated and obtain,
The use pH value is 7.5～8.9 buffer lixiviate in the described leaching process, and the buffer of the pH5.0 of the described buffering effective dose of use～7.0 is replaced the lixiviate buffer in the described concentration process.
2. composition of liquid medicine according to claim 1, it is characterized in that described composition of liquid medicine is comprised of the buffer of the pH5.0～7.0 of the Artemisia pollen allergen for the treatment of effective dose or diagnosis effective dose, buffering effective dose and pharmaceutically acceptable carrier.
3. composition of liquid medicine according to claim 1 and 2, it is characterized in that the pH value that uses in the described leaching process is that 7.5～8.9 buffer is selected from sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of pH7.5, sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of pH8.0, coca ' the s liquid of pH8.2 or the Tris-hydrochloride buffer of pH8.9.
4. composition of liquid medicine according to claim 1 and 2 is characterized in that, the dosage form of described composition of liquid medicine is selected from oral agents, injection, sublingual administration agent, aerosol, nasal cavity agent or skin prick agent.
5. composition of liquid medicine according to claim 1 and 2 is characterized in that, described Artemisia pollen allergen is selected from north Chinese mugwort pollen allergen, Herba Artemisiae annuae pollen allergen, Artemisia or its combination.
6. composition of liquid medicine according to claim 1 and 2, it is characterized in that the buffer of described pH5.0～7.0 is selected from sodium hydrogen phosphate-citrate buffer solution, citric acid-sodium citrate buffer, sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution or acetic acid-sodium acetate buffer.
7. composition of liquid medicine according to claim 1 and 2, it is characterized in that the buffer of described pH5.0～7.0 is selected from pH5.0 sodium hydrogen phosphate-citrate buffer solution, pH6.0 sodium hydrogen phosphate-citrate buffer solution, pH6.4 sodium hydrogen phosphate-citrate buffer solution, pH5.0 citric acid-sodium citrate buffer, pH6.0 citric acid-sodium citrate buffer, pH6.4 sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution, pH7.0 sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution, pH5.0 acetic acid-sodium acetate buffer or pH5.4 acetic acid-sodium acetate buffer.
8. composition of liquid medicine according to claim 1 and 2 is characterized in that, described pharmaceutically acceptable carrier is the combination of polyhydric alcohol and pharmaceutical preservative.
9. composition of liquid medicine according to claim 8 is characterized in that, described pharmaceutical preservative is selected from phenol, thimerosal, benzoic acid, sorbic acid, sorbic acid salt, parabens, benzyl alcohol, phenethanol or its combination.
10. composition of liquid medicine according to claim 8 is characterized in that, described pharmaceutical preservative concentration is 0.01%～3.0%w/v.
11. composition of liquid medicine according to claim 8 is characterized in that, described pharmaceutical preservative is the phenol of 0.2%～0.3%w/v.
12. composition of liquid medicine according to claim 8 is characterized in that, described pharmaceutical preservative is the potassium sorbate of 0.1%～0.2%w/v.
13. composition of liquid medicine according to claim 8 is characterized in that, described polyhydric alcohol is selected from propylene glycol, glycerol, Sorbitol, mannitol, Polyethylene Glycol.
14. composition of liquid medicine according to claim 8 is characterized in that, the concentration of described polyhydric alcohol is 20%～80%v/v.
15. composition of liquid medicine according to claim 14 is characterized in that, the concentration of described polyhydric alcohol is 30%～70%v/v.
16. composition of liquid medicine according to claim 15 is characterized in that, the concentration of described polyhydric alcohol is 40%～60%v/v.
17. composition of liquid medicine according to claim 16 is characterized in that, the concentration of described polyhydric alcohol is 45%～55%v/v.
18. composition of liquid medicine according to claim 8 is characterized in that, described polyhydric alcohol is the glycerol of 45%～55%v/v.
19. method for preparing claim 1 or 2 described stable composition of liquid medicine, it is characterized in that, comprise the steps: buffer and the pharmaceutically acceptable carrier of the Artemisia pollen allergen for the treatment of effective dose or diagnosis effective dose, the pH5.0 of buffering effective dose～7.0 are mixed
Wherein, the Artemisia pollen allergen lixiviating solution of described Artemisia pollen allergen for making in order to the below method: take artimesia as raw material, through pollen defat, drying, lixiviate, concentrated and obtain,
The use pH value is 7.5～8.9 buffer lixiviate in the described leaching process, and the buffer of the pH5.0 of the described buffering effective dose of use～7.0 is replaced the lixiviate buffer in the described concentration process.
20. method according to claim 19 is characterized in that, described Artemisia pollen allergen is selected from north Chinese mugwort pollen allergen, Herba Artemisiae annuae pollen allergen, Artemisia or its combination.
21. method according to claim 19, it is characterized in that the buffer of described pH5.0～7.0 is selected from sodium hydrogen phosphate-citrate buffer solution, citric acid-sodium citrate buffer, sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution or acetic acid-sodium acetate buffer.
22. method according to claim 19, it is characterized in that the buffer of described pH5.0～7.0 is selected from pH5.0 sodium hydrogen phosphate-citrate buffer solution, pH6.0 sodium hydrogen phosphate-citrate buffer solution, pH6.4 sodium hydrogen phosphate-citrate buffer solution, pH5.0 citric acid-sodium citrate buffer, pH6.0 citric acid-sodium citrate buffer, pH6.4 sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution, pH7.0 sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution, pH5.0 acetic acid-sodium acetate buffer or pH5.4 acetic acid-sodium acetate buffer.
23. method according to claim 19 is characterized in that the method comprises the steps:
A) take artimesia as raw material, with pollen defat, lixiviate, concentrated, make the Artemisia pollen allergen lixiviating solution;
B) with step a) the Artemisia pollen allergen lixiviating solution of gained be diluted to preparation in 1: 1 to 1: 100000000 scope of volume ratio with the buffer of the pH5.0～7.0 of buffering effective dose; Add again pharmaceutically acceptable carrier, filtration sterilization,
Wherein, described step a) in, using pH value in the described leaching process is 7.5～8.9 buffer lixiviate; The buffer of the pH5.0 of use buffering effective dose～7.0 is replaced the lixiviate buffer in the described concentration process.
24. method according to claim 19, it is characterized in that the pH value that uses in the described leaching process is that 7.5～8.9 buffer is selected from sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of pH7.5, sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of pH8.0, coca ' the s liquid of pH8.2 or the Tris-hydrochloride buffer of pH8.9.
25. method according to claim 23 is characterized in that, the buffer of employed pH5.0～7.0 when a) replacement lixiviate buffer of described step is and at described step b) dilution in the buffer of employed pH5.0～7.0 identical.
26. method according to claim 19 is characterized in that, described concentrated method is ultrafiltration or dialysis.
27. method according to claim 19 is characterized in that, described pharmaceutically acceptable carrier is the combination of polyhydric alcohol and pharmaceutical preservative.
28. method according to claim 27 is characterized in that, described pharmaceutical preservative is selected from phenol, thimerosal, benzoic acid, sorbic acid, sorbic acid salt, parabens, benzyl alcohol, phenethanol or its combination.
29. method according to claim 27 is characterized in that, described pharmaceutical preservative concentration is 0.01%～3.0%w/v.
30. method according to claim 27 is characterized in that, described pharmaceutical preservative is the phenol of 0.2%～0.3%w/v.
31. method according to claim 27 is characterized in that, described pharmaceutical preservative is the potassium sorbate of 0.1%～0.2%w/v.
32. method according to claim 27 is characterized in that, described polyhydric alcohol is selected from propylene glycol, glycerol, Sorbitol, mannitol, Polyethylene Glycol.
33. method according to claim 27 is characterized in that, the concentration of described polyhydric alcohol is 20%～80%v/v.
34. method according to claim 33 is characterized in that, the concentration of described polyhydric alcohol is 30%～70%v/v.
35. method according to claim 34 is characterized in that, the concentration of described polyhydric alcohol is 40%～60%v/v.
36. method according to claim 35 is characterized in that, the concentration of described polyhydric alcohol is 45%～55%v/v.
37. method according to claim 27 is characterized in that, described polyhydric alcohol is the glycerol of 45%～55%v/v.
38. method according to claim 19 is characterized in that, the dosage form of described composition of liquid medicine is selected from oral agents, injection, sublingual administration agent, aerosol, nasal cavity agent or skin prick agent.
39. each described stable composition of liquid medicine application in the anaphylactic disease medicine that preparation is treated or diagnosis is caused by artimesia of claim 1-18.
40. described application is characterized in that according to claim 39, the pollinosis of described anaphylactic disease for being caused by artimesia.
41. described application is characterized in that according to claim 39, described anaphylactic disease is selected from allergic asthma, allergic rhinitis, anaphylaxis conjunctivitis or the atopic dermatitis that is caused by artimesia.
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| CN102512673B (en) * | 2011-12-21 | 2014-09-10 | 北京新华联协和药业有限责任公司 | Injectable artemisia pollen allergen vaccine and preparation method thereof |
| CN102512672B (en) * | 2011-12-21 | 2014-09-10 | 北京新华联协和药业有限责任公司 | Artemisia pollen allergen vaccine and preparation method thereof |
| CN102552900A (en) * | 2011-12-21 | 2012-07-11 | 北京新华联协和药业有限责任公司 | Artemisia pollen allergen vaccine, and preparation method and application thereof |
| JP5931449B2 (en) * | 2012-01-11 | 2016-06-08 | 日東電工株式会社 | Pharmaceutical composition and method for producing the same |
| CN102988979B (en) * | 2012-12-13 | 2014-05-07 | 北京新华联协和药业有限责任公司 | Artemisia pollen allergen vaccine lozenge and preparation method thereof |
| CN104491858B (en) * | 2014-11-21 | 2018-04-13 | 浙江我武生物科技股份有限公司 | A kind of composition of liquid medicine containing animal wool scurf allergen of stabilization |
| CN104447943A (en) * | 2014-11-21 | 2015-03-25 | 浙江我武生物科技股份有限公司 | Method for extracting dog hair dander allergens |
| CN108265065B (en) * | 2016-12-31 | 2021-04-27 | 江苏众红生物工程创药研究院有限公司 | Recombinant artemisia annua 1-class allergen protein and application thereof |
| CN107308120A (en) * | 2017-08-06 | 2017-11-03 | 周海云 | A kind of preparation technology of Chinese medicinal granule |
| CN109939226B (en) * | 2018-03-23 | 2022-10-21 | 中国医学科学院北京协和医院 | Artemisia sieboldii pollen allergen extract, extract thereof and preparation method thereof |
| CN110064052B (en) * | 2018-03-23 | 2022-11-15 | 中国医学科学院北京协和医院 | England phoenix tree pollen allergen extract, its extract and preparation method thereof |
| CN110124028A (en) * | 2018-03-23 | 2019-08-16 | 中国医学科学院北京协和医院 | A kind of pollen humuli scandentis allergen extract, immersion liquid and preparation method thereof |
| CN108743935A (en) * | 2018-06-27 | 2018-11-06 | 杨永锋 | Contact carries the method and product of allergy raw material rehabilitation seasonal allergic rhinitis |
| CN111175512A (en) * | 2018-11-09 | 2020-05-19 | 北京新华联协和药业有限责任公司 | Preparation and test of artemisia pollen specific IgE antibody standard serum |
| CN113018445A (en) * | 2020-10-28 | 2021-06-25 | 杨永锋 | Composition for preventing and relieving allergic rhinitis and preparation method and application thereof |
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