CN101905022A - 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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- CN101905022A CN101905022A CN200910147470XA CN200910147470A CN101905022A CN 101905022 A CN101905022 A CN 101905022A CN 200910147470X A CN200910147470X A CN 200910147470XA CN 200910147470 A CN200910147470 A CN 200910147470A CN 101905022 A CN101905022 A CN 101905022A
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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 20% of population greatly, all might take place to the middle-aged and elderly people each age group from neonate, does not have tangible gender tendency, but the obvious genetic sexual orientation is arranged.
Cause the original dirt demodicid mite of main allergic effect hypersensitive, 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 seasonal.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.
At present knownly can cause that human pollen hypersensitive 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, sensitization rate height, and quantity is big, and the scope of disseminating is wide; And the scope that plant self distributes is wide, and quantity is also abundant, and is big to the influence of pollen contamination, thereby these plants become more important pollen contamination source plant.Because the distribution of pollen contamination source plant is subjected to the influence of factors such as weather, soil, biology and landform, thereby different regions, its main pollen contamination source plant is also inequality.In the U.S. and Canada, the pollen contamination source is the most important with artemisiifolia; In Britain, Czech, Denmark, France, Italy, Spain and Switzerland, based on grass; 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 of the main pollen in most areas (as Beijing, Xinjiang, Shanxi, Shandong, Wuhan, Shenyang, Guangzhou, Ningxia and other places) is sagebruss 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 combination at first with it of this antibody-like was with original sIgE antibody competition in the body.SIgE in the serum descends later on gradually at continuous desensitization treatment,, can prevent anaphylactoid generation, thereby reach the purpose of desensitization when following to the irritated threshold of reaction.
Because the composition and the complex structure of Artemisia pollen allergen material itself, be subject to the influence of physics or chemical factor and lose activity, reduce and tire, even in low temperature environment, also can't guarantee the long-term stability of its allergenic activity sometimes, 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 promptly overcomes above-mentioned defective, 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 preferable embodiment, described composition of liquid medicine is made up of the buffer of pH 5.0～7.0 of the Artemisia pollen allergen of treatment effective dose or diagnosis effective dose, buffering effective dose and pharmaceutically acceptable carrier.
In another preferable embodiment, the Artemisia pollen allergen lixiviating solution of described Artemisia pollen allergen for making in order to the below method: with the artimesia is raw material, through pollen defat, drying, lixiviate, concentrate and obtain.
In a preferred embodiment, the dosage form of described composition of liquid medicine is selected from 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 sieversiana Willd. pollen allergen or its combination.
In also having an embodiment preferred, 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) with the artimesia be raw material,, make the Artemisia pollen allergen lixiviating solution pollen defat, lixiviate, concentrated;
B) the Artemisia pollen allergen lixiviating solution of step a) gained is diluted to preparation in 1: 1 to 1: 100000000 scope of volume ratio with the buffer of the pH 5.0～7.0 of buffering effective dose; Add pharmaceutically acceptable carrier again, filtration sterilization.
In a preferred embodiment, in described step a), the use pH value is 7.5～8.9 buffer lixiviate in the described leaching process; 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 preferable embodiment, described anaphylactic disease is selected from the pollinosis that is caused by artimesia.In another preferable 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 influence 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 to be made up of the Artemisia pollen allergen of treatment effective dose or diagnosis effective dose, the buffer and the pharmaceutically acceptable carrier of pH 5.0～7.0 of buffering effective dose basically.In a preferred embodiment, described composition of liquid medicine is made up of the buffer of pH 5.0～7.0 of the Artemisia pollen allergen of treatment 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 sieversiana Ehrhart 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), do not influencing under the active and prerequisite of tiring of Artemisia pollen allergen self, also can be multiple Artemisia pollen allergen source, blended Artemisia pollen allergen compositions (for example compositions of Herba Artemisiae annuae pollen allergen and north Chinese mugwort pollen allergen) by any way.
In a preferable embodiment, the Artemisia pollen allergen lixiviating solution of described Artemisia pollen allergen for making in order to the below method: be raw material with artimesia (should use exsiccant artimesia), pass through pollen defat, drying, lixiviate, concentrated and obtain.
Term " treatment effective dose " is meant the concentration (dosage) when 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 making the contact patients specific allergen repeatedly, 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 " is meant the concentration (dosage) when composition of liquid medicine of the present invention is applied to the skin prick 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 g/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 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 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 a 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 also can prepare the buffer of certain pH value as required by the above buffer of commercially available acquisition.Unless have describedly in addition, " pH value " described in the present invention all is the pH value of being surveyed 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 embodiment preferred 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 " is meant that the concentration of described buffer is enough to make the pH value of described composition of liquid medicine to maintain in the individual pH unit of a specific pH value (preferred pH 5.0～7.0) ± 0.5 (better ± 0.3, also good ± 0.2, best ± 0.1).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 come to determine easily according to the normal experiment of limited number of time 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 be compatible with the allergen in the medicine of the present invention, can not reduce the biological activity (allergenic activity) of medicine with its blend under normal conditions significantly.Can be used as pharmaceutically acceptable carrier includes but not limited to: saccharide, as lactose, glucose, sucrose, trehalose; Starch is as corn starch, potato starch; The cellulose or derivatives thereof is as sodium carboxymethyl cellulose, ethyl cellulose, methylcellulose; The tragakanta powder; Gelatin; Talcum; Kollag is as stearic acid, magnesium stearate; Calcium sulfate; Vegetable oil is as Oleum Arachidis hypogaeae semen, Oleum Gossypii semen, Oleum sesami, olive oil, Semen Maydis oil, cupu oil; Polyhydric alcohol is as propylene glycol, glycerol, Sorbitol, mannitol, Polyethylene Glycol; Aminocaproic acid, alginic acid; Emulsifying agent is as tween; Wetting agent is as sodium lauryl sulfate; Coloring agent; Flavoring agent; Antioxidant; Pharmaceutical preservative etc., or its combination.The carrier of preferred pharmaceutical compositions 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 the whole liquid pharmaceutical composition, 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, multiple factors such as the kind of medicine, dosage form, pH value are relevant.In the whole liquid pharmaceutical composition, 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 to be made up of the Artemisia pollen allergen of treatment 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 basically.Also want in the embodiment preferred at one, described pharmaceutical composition is made up 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 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 the similar 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%); With regard to the biological activity of assert this Artemisia pollen allergen is " stablizing ", and 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) with the artimesia be raw material,, make the Artemisia pollen allergen lixiviating solution pollen defat, lixiviate, concentrated;
B) the Artemisia pollen allergen lixiviating solution of step a) gained is diluted to the preparation of a plurality of concentration in 1: 1 to 1: 100000000 scope of volume ratio with the buffer of the pH 5.0～7.0 of buffering effective dose; Add pharmaceutically acceptable carrier again, 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 ex Willd.) 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 preferable embodiment, use sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution lixiviate Artemisia pollen allergen of pH 8.0.With traditional coca ' s liquid phase ratio, though 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, in described step a), 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 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 allergenic activity as far as possible 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 buffer simultaneously.
The buffer of employed pH 5.0～7.0 when the replacement lixiviate buffer of described step a), with the buffer of employed pH 5.0～7.0 in the dilution of described step b) can be identical, also can be different, as long as both can melt mutually, and mixed buffer system has stable buffer capacity, the pH value of whole medicine is maintained in the individual pH unit of a specific pH value (preferred pH 5.0～7.0) ± 0.5 (better ± 0.3, also good ± 0.2, best ± 0.1) to get final product.The buffer of the pH 5.0～7.0 of the buffering effective dose that general preferred use is identical, for example, when the replacement lixiviate buffer of described step a), use the pH 6.0 sodium hydrogen phosphates-citrate buffer solution of buffering effective dose, in the dilution of described step b), use the pH 6.0 sodium hydrogen phosphates-citrate buffer solution of buffering effective dose.
In a preferable 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 soak exsiccant artimesia (press mass volume ratio at every turn and added acetone in 1: 2～1: 10) with organic solvent (for example acetone) continuously, defat 2～6 times, each 0.1～24 hour (preferred 1～12 hour), acetone to the defat be colourless after, the solids natural drying after the defat is not weighed after having the acetone flavor.Laboratory operation should be finished in fume hood, and the vacuum concentration extraction pot is adopted in pilot scale.
(2) lixiviate: make buffer through artimesia behind the degreaser drying and pH 7.5～8.9 (sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of preferred pH 8.0) the two (that preferable is 1: 20～1: 40 W/V with 1: 10～1: 50 W/V (be behind per 1 gram degreaser drying artimesia with the buffer lixiviate of 2～50 milliliters of pH 7.5～8.9), that better is 1: 30 W/V) ratio extract, 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 sterilization treatment in advance to the lixiviate buffer.
(3) concentrate: the crude extract use that will above-mentionedly the obtain molecular weight that dams is that the ultrafilter membrane of 3～10kD carries out ultrafiltration, the buffer that adds pH 5.0～7.0 is repeatedly replaced original lixiviate buffer (preferred ultrafiltration 2～5 times), remove impurity such as micromolecule pigment, finally make the crude extract volume concentrate 2～10 times, 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 the existing conventional technology 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, earlier crude extract is regulated pH value, carry out ultrafiltration and concentration again; 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 can not influence basically 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 preferable embodiment, described anaphylactic disease is selected from the pollinosis that is caused by artimesia.In another preferable 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 preferable 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 ex Willd.) 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 some pricker.Negative controls is generally the non-allergenie liquid of human body (for example mixture of glycerol and saline solution etc.), and positive control solution is generally histamine phosphate/histamine hydrochloride solution (for example 1.7mg/ml histamine phosphate solution) of 1.0～5.0mg/ml.
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 skin or mucosa and give the T lymphocyte by antigen-presenting cell with antigen presentation, 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, and be not to be used for limiting the scope of the invention just in order to play illustration.
The preparation of embodiment 1 Herba Artemisiae annuae pollen allergen sublingual administration agent
(1) defat: accurately take by weighing exsiccant 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) concentrate: the crude extract that step (2) is obtained is that the ultrafilter membrane of 3kD carries out ultrafiltration with holding back the aperture, sodium hydrogen phosphate-the citrate buffer solution that adds 50mM pH6.0 is repeatedly replaced lixiviate buffer (ultrafiltration 3 times), remove impurity such as micromolecule pigment, finally make the crude extract volume concentrate 6 times, 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 being not less than 3mg/ml as if total protein concentration, promptly meets 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 phenol (making its final concentration is 0.25% (w/v)) again.
(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.Name respectively: 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 sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of use 150mM pH7.0 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 phenol (making its final concentration is 0.2% (w/v)) again.
(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 sieversiana Willd. pollen allergen subcutaneous injection agent
(1) defat: accurately take by weighing exsiccant 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) concentrate: 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 adds 100mM pH5.0 is repeatedly replaced lixiviate buffer (ultrafiltration 3 times), remove impurity such as micromolecule pigment, finally make the crude extract volume concentrate 6 times, obtain Artemisia sieversiana Willd. pollen allergen lixiviating solution.
(4) determination of protein concentration: the Artemisia sieversiana Willd. 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 being not less than 3mg/ml as if total protein concentration, promptly meets the requirements.
(5) dilution: the Artemisia sieversiana Willd. 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 the citric acid-sodium citrate buffer of 100mM pH5.0, add glycerol (making its final concentration is 45% (v/v)), add phenol (making its final concentration is 0.25% (w/v)) again.
(6) degerming: use the aseptic microporous filter membrane of 0.22 μ m to carry out filtration sterilization, packing, embedding make Artemisia sieversiana Willd. pollen allergen subcutaneous injection agent finished product.Name respectively: Artemisia sieversiana Willd. pollen allergen 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 exsiccant 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) concentrate: the crude extract that step (2) is obtained is that the ultrafilter membrane of 3kD carries out ultrafiltration with holding back the aperture, sodium hydrogen phosphate-the phosphate sodium dihydrogen buffer solution that adds 200mM pH6.4 is repeatedly replaced lixiviate buffer (ultrafiltration 3 times), remove impurity such as micromolecule pigment, finally make the crude extract volume concentrate 6 times, 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 being not less than 3mg/ml as if total protein concentration, promptly meets 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 potassium sorbate (making its final concentration is 0.15% (w/v)) again.
(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.Name respectively: 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 acetic acid-sodium acetate buffer of use 200mM pH5.4 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 phenol (making its final concentration is 0.3% (w/v)) again.
(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 (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, take out then and be statically placed in 4 ℃ of refrigerator overnight (9～12 hours).Sample transfer after 4 ℃ spent the night is to 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 78.29 KUA/L.
Serum in the standard serum storehouse contains at allergenic specific IgE (sIgE).Allergenic activity in the product to be tested becomes branch to combine with sIgE in the serum to generate complex, makes that free sIgE concentration reduces in the serum, and reuse Unicap system detects the content of sIgE in the serum.Allergenic activity is high more in the product to be tested, and after serum and the product to be tested effect, its sIgE concentration will be fallen lowly more.Detect with the Unicap system and can find in the serum that free sIgE 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 under the complete bonded situation 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 high more, and total allergenic activity is high more in the product to be tested.
(2) main allergen Determination on content
Use the main allergen protein 1 assay test kit of artimesia (producing), according to its incidental description operation by middle money Chinese arteries and veins (Beijing) Bioisystech Co., Ltd.Need to prove, capture antibody in the main allergen protein 1 assay test kit of artimesia is at 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), No. 1 (note is done in the following table: sample 1-1) (note is done in the following table: for No. 6, the Herba Artemisiae annuae pollen allergen sublingual administration agent of embodiment 1 preparation;
The Herba Artemisiae annuae pollen allergen skin prick liquid of embodiment 2 preparations (do in the following table: sample 2) by note;
No. 4 (note is done in the following table: sample 3-4) in the Artemisia sieversiana Willd. pollen allergen subcutaneous injection agent of embodiment 3 preparations;
Sample 4-5), No. 1 (note is done in the following table: sample 4-1) (note is done in the following table: for No. 5, the north Chinese mugwort pollen allergen sublingual administration agent of embodiment 4 preparation;
The north Chinese mugwort pollen allergen skin prick liquid of embodiment 5 preparations (do in the following table: sample 5) by note;
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 phenol (making its final concentration is 0.25% (w/v)) again, 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.The above-mentioned north Chinese mugwort pollen allergen lixiviating solution that obtains of reuse normal saline dilution, add glycerol (making its final concentration is 55% (v/v)), add potassium sorbate (making its final concentration is 0.15% (w/v)) again, 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 1 pH 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 with 0 day is 100%, and the numerical value the when numerical value of Ce Dinging was with 0 day At All Other Times contrasts, and carries 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 earlier 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 will be put the pricker permeate and drip, and vertically thrust skin, to every kind of test solution with a new some pricker;
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 is meant the flat protuberance that is higher than skin that occurs behind the contact skin anaphylactogen, and 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 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; React rank according to Herba Artemisiae annuae pollen allergen skin prick liquid and welt area due to the positive control than judging:
● 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 is to Herba Artemisiae annuae pollen hypersensitivity (++ more than) through the evidence of above-mentioned anaphylactogen skin prick.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 back 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 of treatment, then time daily dose is reduced to minimum dose, increases progressively gradually after the tolerance again.)
The course of treatment: treatment full half a year of person is totally 32 examples; Full 1 year person of treatment is totally 27 examples; Full 2 years persons of treatment 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 alleviate slightly.Invalid: with ratio before the treatment, pollen period (8～October) pollinosis symptom or pollen hypersensitivity asthma no change 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 long more, and effective percentage is high more.
Although the invention describes concrete example, having a bit is significantly to those skilled in the art, promptly can do various variations and change to 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 (10)
1. a stable composition of liquid medicine is characterized in that, this composition of liquid medicine 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.
2. composition of liquid medicine according to claim 1, it is characterized in that described composition of liquid medicine is made up of the buffer of pH 5.0～7.0 of the Artemisia pollen allergen of treatment effective dose or diagnosis effective dose, buffering effective dose and pharmaceutically acceptable carrier.
3. 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.
4. 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 sieversiana Willd. pollen allergen or its combination.
5. composition of liquid medicine according to claim 1 and 2, it is characterized in that 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.
6. composition of liquid medicine according to claim 5, it is characterized in that 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.
7. method for preparing the described stable composition of liquid medicine of claim 1, it is characterized in that, comprise the steps: that Artemisia pollen allergen that will the treatment effective dose, the buffer of pH 5.0～7.0 and the pharmaceutically acceptable carrier of buffering effective dose mix.
8. method according to claim 7 is characterized in that this method comprises the steps:
A) with the artimesia be raw material,, make the Artemisia pollen allergen lixiviating solution pollen defat, lixiviate, concentrated;
B) the Artemisia pollen allergen lixiviating solution of step a) gained is diluted to preparation in 1: 1 to 1: 100000000 scope of volume ratio with the buffer of the pH 5.0～7.0 of buffering effective dose; Add pharmaceutically acceptable carrier again, filtration sterilization.
9. method according to claim 8 is characterized in that, in described step a), the use pH value is 7.5～8.9 buffer lixiviate in the described leaching process; Use the buffer of pH5.0～7.0 of buffering effective dose to replace the lixiviate buffer in the described concentration process.
10. the described stable application of composition of liquid medicine in the anaphylactic disease medicine that preparation is treated or diagnosis is caused by artimesia of claim 1.
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102512673A (en) * | 2011-12-21 | 2012-06-27 | 北京新华联协和药业有限责任公司 | Injectable artemisia pollen allergen vaccine and preparation method thereof |
| CN102512672A (en) * | 2011-12-21 | 2012-06-27 | 北京新华联协和药业有限责任公司 | Artemisia pollen allergen vaccine and preparation method thereof |
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| 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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