CN1842325A - Dosage form - Google Patents
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- CN1842325A CN1842325A CNA2004800243309A CN200480024330A CN1842325A CN 1842325 A CN1842325 A CN 1842325A CN A2004800243309 A CNA2004800243309 A CN A2004800243309A CN 200480024330 A CN200480024330 A CN 200480024330A CN 1842325 A CN1842325 A CN 1842325A
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- sporopollenin
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Abstract
A pharmaceutical or dietetic dosage form comprising of effective quantity of an active substance chemically or physically bound to a support comprising, sporopollenin, or other similar exine coating of spores, of a plant of fungus, optionally with further excipients.
Description
The present invention is about the dosage form on a kind of pharmacopedics or the threpsology, a kind of manufacture method of this kind dosage form and utilize the Therapeutic Method of this kind dosage form.Provide human and dosage form for animals.
Spore powder Quality is the exine of each kind of plant, liverwort, mushroom and algae.Remove by continuous processing and may be attached on the exine or lipid, carbohydrate, protein and nucleic acid that exine is contained, Bao Fen Quality can be separated from spore with solvent, alkali and acid.Also used the method for enzyme.Bao Fen Quality physically is steady statue chemically reaching, and it is described to be similar to carotenoid and has hydrophobicity.Other can have similar chemistry and physical stability by the exine that glucosan, mannan and chitin form.Yet the interior membrane portions of some spores is made of cellulose; This inner membrance can be decomposed (F.Zetzsche and K.Huggler Annalen, 1928,461,89) by these chemical treatments to a great extent.
DE-A-19902724 has disclosed a kind of dosage form, and wherein microcapsule is made by the Bao Fen Quality sporangium of having filled active substance.These dosage forms have a shortcoming, and the release of this kind or these active substances depends on the integrity of sporangium and decides exactly.Different with this announcement, the present invention utilizes the existence of ethanol and the filling that flexible nature is assisted Bao Fen Quality capsule thereof, particularly is used for improving processing speed, and utilizes specific exine size to determine to carry target.
US-A-5013552 has disclosed being used for induction system by getting fully loaded cellulose shell in the pollen particle.This kind shell and inner membrance are similar, and promptly its leaching process that can be used to obtain sporopollenin destroys.
According to a first aspect of the invention, dosage form on a kind of pharmacopedics or the threpsology comprises the active substance of effective dose, this active substance and a carrier chemical bond, select in the exine of this carrier by plant, liverwort, mushroom, algae or its scrappy part, optionally further have excipient.
According to a second aspect of the invention, dosage form on a kind of pharmacopedics or the threpsology comprises the active substance of effective dose, this active substance is incorporated in the carrier physically, select in the exine of this carrier by plant, liverwort, mushroom, algae or its scrappy part, can select further to have excipient.
When this active substance was combined physically, it can be adsorbed on the carrier.
As another selection and often more preferably keep this active substance in the hollow exine for example the sporopollenin wall of sporopollenin be linked as in the holistic cavity, or remain in the central cavity.This three aspect all makes the said preparation form adventitia be decomposed carry out dispenser with the method for release of active agent subsequently by its absorption is entered blood flow.
According to a third aspect of the invention we, the manufacture method of a kind of pharmacopedics or threpsology's dosage form comprises following steps:
Contact an exine with an infiltration auxiliary liquid
With an active substance contact this exine and this material can be infiltrated in the Bao Fen Quality wall cavity and/or
Contact exine and allow this material infiltrate exine inside with an active substance, and remove this infiltration auxiliary liquid subsequently and allow this exine drying so that this material is retained in the adventitia.
A kind of preferred infiltration auxiliary liquid can be for by a solvent of selecting among the group of gang or solubilising medium, and this group comprises C
1To C
4Ethanol, more preferably ethanol and aqueous C
1To C
4Ethanol is preferably aqueous ethanol.
The exine of Bao Fen Quality can be dipped in a kind of solution that is dissolved with active substance.Can be chosen in addition and adventitia is dipped in solvent before contact with active substance or other permeate in the auxiliary liquid.
The exine of spore powder Quality can quilt: (i) under atmospheric pressure or be pressed into tablet in a vacuum, then it is contacted with active substance (or only for having or do not have the liquid active substance of infiltration auxiliary liquid) solution; The vacuum that (ii) places active substance to exist, wherein this active substance has or does not have the infiltration auxiliary liquid.These processes can be operated under the temperature of room temperature or high 250 degree extremely Celsius.This active substance can comprise the mixture of a kind of medicine, a kind of medicinal mixture, a kind of nutrient substance, a kind of mixture of nutrients or a kind of medicine and nutrient substance.The example of nutrient substance comprises mineral and essential oil.The cholesterol reducing dosage form can be provided.Vitamin, mineral, food flavouring agent and other nutritional supplementation active substances can utilize dosage form according to the present invention to use.
Can be with in this respect dosage form be incorporated in the food according to the present invention, cholesterol reducing food for example as the grain strip.Also provide nutrition product for animals.
Can carry active substance in a large number, for example reach several times of exine weight.The ability that this kind can hold other quite a large amount of materials promoted interpolation nutritional supplementation material or other compositions or additive for example spice, antiseptic, antioxidant or mineral at other products for example food and beverage.Spore powder Quality adventitia can provide protection against the tide, acid-proof, alkali resistant oxidation or anti-photolytic protection for active substance before product is taken.Some material, for example copper sulfate is not easy to discharge in the aqueous solution, and other (those only comprise physically) then discharge slowly.Scanning electron microscope (SEM) X-ray shows that down the copper sulfate of whole Bao Fen Quality shell is absorbed equably.When being transferred through intestinal, active component can slowly be discharged.There is above-mentioned situation then can on Bao Fen Quality adventitia, increase another adventitia as not wishing to postpone release.Exine can for example be used a low viscosity resin, as Radix Acaciae senegalis by derivation to reduce its semipermeability.Do not enter in the blood flow if do not wish to be absorbed, can use Bao Fen Quality (greater than 100 microns) than macroparticle.
In preferred dosage form, exine comprises Bao Fen Quality, glucosan, mannan or chitin.These exines have chemically reaching and physically are steady statue, use and use convenient and prepare simple, cheap advantage.It does not contain usually and can leach impurity and can be designed to provide the function of high activity material bearing capacity, and it has protein-free advantage, can prevent allergy or other physiological effects that is caused by protein or denatured protein.The latter may be present in the strict inadequately leaching process, for example described in the US-A-5013552 like that.
In a preferred embodiment of the invention, this carrier is made up of exine basically, and this exine comprises Bao Fen Quality, chitin, glucosan or mannan, and is substantially free of protein.
Be preferably protein content and be no more than 0.5%, preferably be no more than 0.1%.
The protein content of preferred adventitia is low to moderate to be enough to 6%
w/
vThe aqueous potassium hydroxide in do not observe further protein again after refluxing 2 hours and run off.
According to dosage form of the present invention, it has in acid or alkaline medium and is steady statue, but especially can biolytic apace advantage in blood under certain environment.This catabolite can be nontoxic and the NIP reaction.The time of staying in gastrointestinal tract is short.Decomposition can be in blood flow takes place rapidly, and the degree in gastrointestinal tract may be less, and it makes using effectively of medicine or active substance and rapidly, for example a few minutes as 20 minutes time in.
The said preparation form can comprise a conjugated biomolecule closes, and promptly by the macromolecular complex of chemical bond with the synthetic method acquisition, for example the drug molecule covalent bond is connected to one and comprises on the carrier or culture medium of sporopollenin or other exines.Although it is comparatively desirable that the covalency keyed jointing is used for same application, but also can use ion keyed jointing, hydrogen bond keyed jointing, Van der Waals force or in exine the method for envelope capsule, but especially in the time can not needing the powerful keyed jointing of medicine or other active substances and carrier application examples as sucking in the agent of writing out a prescription.Active substance or medicine can be with the sporopollenin direct reaction or are adhered to it physically to produce a conjugated biomolecule and close.Yet sporopollenin or other exines are functionalized in an embodiment of the present invention, thereby medicine can adhere to by covalency keyed jointing or other chemical bondings with suitable stability.For example, under the situation of carrying, can be chosen in the keyed jointing that is steady statue in the acid solution, thereby active substance and carrier can enter intestinal by stomach by oral area.The protection that maybe can select to utilize exine to provide makes the medicine of capsule envelope be steady statue.For example use Radix Acaciae senegalis or starch can reach Additional Protection by an extra adventitia to the active substance of physical attachment or chemical attachment.Can also use traditional thin film, for example the cellulose of hydroxypropyl cellulose or other improvement.
Dosage form according to these data has application-specific aspect the dispenser, promptly its in liquid or especially acid medium for example beta-lactam (be labile state in β-lactams), cephalosporin (cephalosporins) or the sweet acid of two deoxidation glands (dideoxyadenosine).Can reduce and discharge into the preceding decomposition of blood.Also can promote that for example ciclosporin (cyclosporins), paclitaxel (taxanes) or other macrolides (macrolides) are exerted in the blood flow for example ciclosporin (ciclosporin), mebendazole (mebendazole), nystatin (nystatin), propofol (propofol), Paclitaxel (paclitaxel), miconazole anthracyclinones or ciprofloracin (ciprofloxacin) with insoluble or soluble hardly medicine.
It is that if carrier is made up of sporopollenin or other exines or comprise sporopollenin or other exines, because its persorption ability of entering blood and tachymetabolism (20 minutes to 2 hours) being arranged fast after oral, the polymer that medicine or other active substances are transmitted on sale being used to compared to the market has apparent advantage.It is easy to by derivation to adhere to multiple medicine with different dissolubilities and stability in addition.This kind carrier can be protected acid instability molecule and nontoxic according to its biological origin keeping chemically reaching unanimity on the form.Moreover also not having anaphylaxis, partly cause when oral sporopollenin or other exines or when sucking it in lung is that the protein that closes with these reacting phases and carbohydrate are by removing in the plant spore raw material.Sporopollenin or exine decompose rapidly in blood discharges active component rapidly.This kind adventitia can keep its size and form in dispenser and absorption process.The size of specific species and the exine that comes and the unification of form make the dosage form can be according to the pattern of the bearing capacity of medicine and conveying and optimised.Bigger particle can comprise the active substance of higher proportion, for example medicine or functional food composition.We have found that 25 microns particle can hold the active substance more than equivalent weight in central cavity.Proposed in the past, this kind macroparticle can not enter blood flow (ML Wierner, FdChem.Toxic., 1988,26 (10) 867-880) by the intestinal wall persorption.
There are the paper, comment and the books conjugated biomolecule that much relate to conjugated biomolecule and close this problem to close that (two famous quoting comprise: Bioconjugate Techniques, Greg T Hermanson, 1996, AcademicPress Inc and Bioconjugation in Pharmaceutical Chemistry, Il Farmaco, 1999,54,497-526).A lot of coupling agents are revealed.Carbodiimides such as DCC (dicyclohexylcarbodiimide dicyclohexylcarbodiimide) or EDC{1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride} are very effective.Can directly combine with primary amine or with more bonded medicines of carboxylate group (carboxylate groups) and probe with non-derivation sporopollenin.
Can use separately according to dosage form of the present invention,, but select at least a suitable pharmaceutical excipient, the diluent or carrier relevant usually with drug delivery route especially for the suction dispenser.
The said preparation form can be manufactured to and be suitable for oral area, buccal or Sublingual with tablet, capsule, comprises the form administration of soft gel capsule, ovule, elixir solution or suspension.The said preparation form can be manufactured to and be applicable to immediately, postpone, discharge through improvement or under control and carry.The effect of the exine in the agent of compression prescription is comparatively favourable sometimes, because it has elasticity, can make tablet or be kept perfectly when needs dwindle tablet sizes.
The said preparation form also can be manufactured to and be applicable to quick dissipation or dissolve administration fast.
Can use compressed tablets or other compressed preparation forms.Can applying waterborne and non-aqueous skin.The prescription agent maybe can select to use a disintegrant or foaming agent combination so that dissipate in liquid.
Can comprise excipient according to tablet of the present invention, microcrystalline Cellulose for example, lactose, sodium citrate, calcium carbonate, Bibasic Calcium Phosphate, glycine and starch, be preferably frumentum, Rhizoma Solani tuber osi or tapioca, disintegrant, carboxymethylstach sodium (sodium starch glycollate) for example, cross-linked carboxymethyl cellulose sodium (croscarmelose sodium) and granulation adhesive, for example polyvinylpyrrolidone (polyvinylpyrollidone), hydroxypropyl emthylcellulose (hydroxypropylmethylcellulose), hydroxypropyl cellulose (hydroxypropylcellulose), methylcellulose, sucrose and gel.Also can make with lubricator, for example magnesium stearate and fluidizer, for example Talcum or silica gel.
Can be according to dosage form of the present invention as the implant of gel capsule.Preferred excipient comprises lactose, mixed crystal sugar, starch, cellulose and Polyethylene Glycol.Maybe can select to use the freeze-dried preparation form, the Zydis that reaches various and RP Scherer company that for example in GB1548022, is disclosed
TMThe patent that agent is relevant.
Can comprise aqueous suspension or dryness particle or other compositions that are used to replenish as suspension according to dosage form of the present invention.
The release of improvement or pulsation delivery formulations form can comprise that discharging the frequency modifying agent comprises hydroxypropyl emthylcellulose, methylcellulose, sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate, poly(ethylene oxide), Xanthan gum (xanthan gum), carbomer (Carbomer), oil, wax and methacrylate (methacrylate) copolymer.
Also can use through the skin administering mode, especially use the scrappy part of little sporopollenin.
Dosage form of the present invention is manufactured to and is applicable to pulmonary administration in particularly preferred embodiment.Sporopollenin or other exines can be selected from a liverwort, fungus or plant species with the particle that selected size is provided and make dosage form can be penetrated into required administration position, for example descend pulmonary.A preferred especially size is about the particle of 1-100 micron, preferably is about the 1-30 micron, more preferably about 1-10 micron, especially 1-5 micron.
In this respect dosage form has the unified advantage of particle size according to the present invention.In addition, with acid, alkali or organic solution spore is extracted after the size and the shape of particle roughly constant.The exine that is extracted can keep to its from spore in size and keep similar in shape.Relatively, micronized medicine for example is used for the sort of for the treatment of asthma traditionally, and it has many kinds of particle sizes, comprises the young fine particle of a part, and bigger grumeleuse is also arranged.Each is from the spore of specific species, and the differentiation on its size is all very little and have consistent shape, and can be selected to provide one to be suitable at a selected position or the dosage form that carries out administration with a selected pattern.Can disclose like that according to following publication according to useful spore of the present invention and to obtain from gymnosperm, angiosperm, pteridosperms, mushroom and algae, it is incorporated into this description as a reference:
G.Shaw, Sporopollenin in Phytochemical Phylogeny, J.B.Harborne (Ed), Academic press, London and New York, the 3rd chapter, (1997), 31-5.
P.D.Moore, J.A.Webb and M.E.Collinson, Pollen analysis, second edition, Blackwell Scientific Publications, (1999).
J.Brooks, Some Chemical and Geochemical Studies onSporopollenin in:Sporopollenin, J.Brooks, M.Muir, P.Van Gijzel and G.Shaw, (Eds) Academic press, London and New York, (1971), 305-348.
The spore size of typical case's species is as follows:
Spore size example
1.2 microns of bacillus subtilises (Bacillus subtilis)
Myosotis (Myosotis sylvatica) (Myosotis (Forgetmenot)) 2.4-5 micron
4 microns of aspergillus nigers (Aspergillus niger)
Penicillium (Penicillium) 3-5 micron
Little chanterelle (Cantharellus minor) 4-6 micron
Ganoderma (Ganomerma) 5-6.5 micron
The edge of a field mushroom (Agrocybe) 10-14 micron
Urtica dioica (Urtica dioica) 10-12 micron
Black anthracene is spent mould (Periconia) 16-18 micron
20 microns in attached coccus (Epicoccum)
25 microns of Herba Lycopodiis (Lycopodium clavatum)
125 microns of firs (Abies)
200 microns in short Fructus Cucurbitae moschatae (Cucurbitapapo)
250 microns in Fructus Cucurbitae moschatae (Cuburbita)
Why the spore that causes disease such as farmer's lung for example can cause that disease is because it lodges in an ad-hoc location of pulmonary.Contain protein and other related substanceses to make exine by removing from spore, remaining harmless body can transfer to useful medium these identical positions in the pulmonary.
Can be according to a dosage form of the present invention by the mode administration that pulmonary sucks or nasal cavity sucks, and can be by using the spraying of a dry powder inhalant or a certain amount of inhalation aerosol or by a pressurizing vessel, pump, spraying or the suitable propellant of nebulizer utilization, for example 1,1,1,2-tetrafluoroethane (HFA 134a) or 1,1,1,2,3,3,3-heptafluoro-propane (HFA 227) transmits easily.
According to traditional method, the metered dose inhalation aerosol composition can be in conjunction with a surfactant or cosolvent.
Can be according to dosage form of the present invention with the form administration of suppository or vaginal suppository.Dosage form especially in conjunction with the scrappy part of sporopollenin or spore, can be used with the form of gel, moisture coagulant liquid, solution, Emulsion, ointment or powder in external application.This prescription agent also can be on skin or through the skin administration, for example by using skin to paste.
Dosage form of the present invention can offer human or for animals.
The present invention's method by way of example further describes, but it does not mean any restriction.
Sporopollenin or other exines can be separated by using organic solution and strong acid and alkaline compositions that spore is carried out the strictness processing.
Example 1: from Herba Lycopodii (Lycopodium clavatum), separate sporopollenin
Make Herba Lycopodii (250 grams, Fluka is on sale) be suspended in the acetone (700 milliliters) and under refluxing and stir 4 hours.Leach solid residue, with clean acetone rinsing, (850 milliliters is 6% in the concentration of water to reinstall in the reaction bulb and make once more it to be suspended in potassium hydroxide solution
w/
v) in.Then this mixture was stirred 6 hours under refluxing.Leach residue, fully wash, reinstall in the reaction bulb, then the repeated hydrogen oxidation processes with hot water.After the filtration solid matter is washed once more with hot water, hot ethanol and water.Residue was stirred 2 hours under ethanol (750 milliliters) refluxes, filter, then successively with clean ethanol and dichloromethane rinse.Make the gained solid suspension again in clean dichloromethane (750 milliliters), under refluxing, stirred 2 hours, leach again air-dry 24 hours.
Make the particle suspension that leaches then in orthophosphoric acid (85%, 800 milliliter), under the backflow of gentleness, stirred 5 days and filter.Blot again with capacity hot water injection residue.Repeat orthophosphoric acid processing and dry again.Use hot water, ethanol and dichloromethane rinse particle then.At last this solid is refluxed at ethanol (800 milliliters) and stirred 2 hours down, filter and also use dichloromethane rinse, air-dry then and vacuum drying is with production sporopollenin (50 restrain).
Example 2: thyroxinic physical attachment and biological assessment
Sporopollenin (0.5 gram) be compressed in 10 tonnes following 2 minutes.The gained tablet is added in the solution, and this solution is for comprising the thyroxinic 0.3 milliliter of DMSO of 300 micrograms (dimethyl sulfoxide) and 1.5 milliliters of ethanol.This tablet just absorbed solution less than one minute, then this sample is placed under the vacuum of phosphorus pentoxide and be dried to constant weight under 5 degree Celsius.Give volunteer oral the thyroxinic sporopollenin of physical attachment.Less than the 1 Nai Mimo/litre (nano mole/litre) that risen of thyroxine level in 15 minutes patient bloods.In this rising, can be observed the sporopollenin particle of significant amounts or the particle that part is decomposed, as viewed in the example 18.Therefore, supposing has 5 liters of blood in the volunteer body, finds that 4 microgram thyroxine then were equivalent to 0.66% of medicine transmission amount after oral 15 minutes.Its amount (0.6%) with the sporopollenin particle of transmission is consistent.The rapid rising of this thyroxine level should just take place after 2 hours in 1, absorbed than the jejunum (jeujenum) of upper/lower positions because it at first can be positioned at digestive tract.
Example 3: Recombinant Somatropin .'s Physical Absorption
Sporopollenin (0.5 gram) formed tablet (taking advantage of 3 millimeters for 16 millimeters) in 2 minutes in 10 tonnes of lower compression.This tablet is added one with [5.5 milligrams of human body recombinant human growth hormone solid prescription agent, it comprises the mixture of this hormone (1 milligram) and a mannitol, glycine, sodium hydrogen phosphate, sodium hydroxide and/or phosphoric acid] be dissolved in the solution of a mixture, this mixture comprises (0.5 milliliter of a diluent; Comprise glycerol, metacresol water (m-cresol water) and sodium hydroxide and/or hydrochloric acid) and ethanol (2.0 milliliters).This sporopollenin tablet promptly (15 seconds) increases to almost 4 times of its original volume, absorbs all solution simultaneously.The gained powder taking the photograph under the third constellations 5 degree and the vacuum dry 48 hours, is reached constant weight at this section in the period.
Example 4: the Physical Absorption of solubilized insulin prescription agent
The solution of one solubilized insulin (comprising one 2 cubic centimetres prescription agent of 0.18 gram insulin, rDNA (rDNA), zinc chloride, glycerol, metacresol, sodium hydroxide, hydrochloric acid and water) and ethanol (1 milliliter) is added in the sporopollenin (1 gram), and the sporopollenin of this 1 gram is compressed under 10 tonnes in advance and formed tablet in 2 minutes.Rise to almost in 4 times of its original volume at the sporopollenin tablet, this solution was absorbed in 20 seconds.With this gained powder under 5 degree Celsius and vacuum dry 48 hours, during this period of time reach constant weight.
Example 5: the Physical Absorption of Oleum helianthi
By in one 16 millimeters moulds, preparing sporopollenin (0.5 gram) tablet in 2 minutes with 10 tonnes of pressure compressions.These tablets are at room temperature added an Oleum helianthi (1 milliliter) and auxiliary an absorption in the mixtures of liquids (table 1).The gained powder there is being P in an exsiccator
2O
5(phosphorus pentoxide) and take the photograph under the degree of the third constellations 50 and dry to constant weight.
This tablet will keep no change to surpass 3 hours in fact when without solvent.
Table 1
| The auxiliary liquid that absorbs | Absorb the used time fully |
| Ethanol | 20 seconds |
| Diethyl ether | 1 minute 5 seconds |
| Petroleum ether | 3 hours |
| Dichloromethane | 35 seconds |
| Hexane | 3 hours |
| Ethyl acetate | 50 seconds |
| Acetonitrile | 20 seconds |
| Toluene | 2 minutes 15 seconds |
Repeat the physical attachment of ethanol and Oleum helianthi under 40 degree Celsius, it has shortened the used time of this sample that absorbs fully; Promptly finish absorption after 12 seconds.The time that is used for absorbing fully relies on employed auxiliary absorption liquid and temperature very much.
Example 6: the Physical Absorption of glycine
One sporopollenin tablet (0.5 gram) is added in the 1M glycine solution (0.5 milliliter) with ethanol (0.5 milliliter) dilution.Solution is absorbed rapidly, again the gained powder is dried to constant weight having under the vacuum of phosphorus pentoxide.The bearing capacity of sporopollenin is found to be 1.9 millis and rubs/restrain.Repeat this experiment, but needn't compress sporopollenin.The bearing capacity of sporopollenin is found to be 0.38 milli and rubs/restrain.This demonstrates when sporopollenin is compressed, and its absorption to glycine obviously increases.
Sporopollenin was found its high degree of agitation in water can be kept 15% water soluble amino acetic acid after 30 minute.Same sample is found in high degree of agitation after skin is covered with starch can keep 35% glycine after 30 minutes.
Example 7: the Physical Absorption of copper sulfate
One sporopollenin tablet (0.5 gram) is added in the 1M copper-bath (0.5 milliliter) with ethanol (0.5 milliliter) dilution.This solution is absorbed rapidly, then the gained powder is dried to constant weight having under the vacuum of phosphorus pentoxide.The bearing capacity of sporopollenin is found to be 2.5 millis and rubs/restrain.The copper sulfate that shows whole sporopollenin shell under scanning electron microscope (SEM) X-ray is absorbed equably.
Sporopollenin stirs to be found after 30 minutes in water and has kept 75% copper sulfate.
Example 8:LR White Resin
Physical Absorption
One sporopollenin tablet (0.1 gram) is added 50%LR White Resin
(2 milliliters: wherein contain 80% polyhydroxy and replace bisphenol a dimethacrylate resin (polyhydroxysubstituted bisphenol A dimethacrylate resin) of alcoholic solution, 19.6%C12 methacrylate (C12 methacrylate ester), 0.9% dimethyl-p-toluidine (dimethyl paratoluidine)) in.This mixture was leniently stirred 2 hours.The gained particle is also further used LR White Resin by the centrifuging separation
(2 milliliters) were handled 12 hours again.Then this mixture is placed a capsule under 60 degree Celsius, to heat 8 hours.Resulting polymers fills up sporopollenin particle and wall inner chamber body thereof, as (Figure 15) shown under the scanning electron microscope.
Example 9: the derivation of sporopollenin (Fig. 1)
Can be by the sporopollenin of separating in the Herba Lycopodii in the one about 5 level halogenation (F.Zetsche and K.Huggler, Liebigs Ann.Chem., 1928,461,89) that rubs/restrain in the least by direct bromination.It can be by with dichlormetbylether and butter of tin generation effect and by chloromethylation, with obtain bearing capacity be about the chlorine that 1 milli rubs/restrain (G.Mackenzie and G.Shaw, Int.J.PeptideProtein Res., 1980,15,298-300).Use traditional Merrifield method to synthesize single three peptides with the chloromethane group.Maybe can select with 1, the 3-diaminopropanes makes the sporopollenin ammonification so that its obtain bearing capacity be 1.6 the milli rub/restrain alkali (R Adamson, S.Gregson and G.Shaw, Int.J.Peptide Protein Res., 1983,22,560-564).This 1,3-diaminopropanes sporopollenin can produce reaction with monochloroacetic acid anhydride or with chloracetyl chloride, links the agent sodium salt with the 4-salicylic alcohol subsequently and handles.Available subsequently benzyl stops interleaving agent and synthesizes one or four peptides with traditional method.The diaminoethanes derivative of sporopollenin can produce amberlite lipid material, and this material and bromoacetate/alkaline hydrolysis (bearing capacity is that 1.4 millis rub/restrain) reaction can obtain acid.Reaction by sporopollenin and chlorosulfonic acid (bearing capacity is that 1.6 millis rub/restrain) can obtain an acid product (G.Shaw, M.Sykes, R.W.Humble, G.Mackenzie, D.Marsdan, E.Phelivan, Reactive Polymers, 1988,9,211-217).
Sporopollenin has compared to many markets polymer on sale and reaches chemically firm physically but relatively be easy to simultaneously the advantage of derivation.The most frequently used conjugated group that closes as conjugated biomolecule is BNH in the polymer on sale of market
2,-OH, SH and CO
2H.Sporopollenin has and is easy to this kind conjugated group is guided to its surperficial advantage, and with directly or produce a covalent bond by an interleaving agent or link agent group and a medicine and connect, it forms a short chain between anchoring group and medicine.Link agent and conjugated group on the sporopollenin that we have improved in the document to be disclosed, and summed up in the part in front.In the application of sporopollenin as drug delivery material with these derivation, below described new link agent/conjugated group in document, put down in writing those have convenient attachment, bearing capacity improves, and adheres to the advantage of stablizing and toxic by-products being reduced to minimum.The sporopollenin particle that should be noted that same source is in its form and chemically intimate consistent.This concordance employed man-made polymer in controlling the medicine field may not have.According to a forth aspect of the invention, provide the sporopollenin of a primary amine functionalization.
Several markets polymer on sale that is used for drug conveying, for example poly (L-lysine) and poly (L-aspartic acid), it has one and is used for the primary amine group that medicine adheres to, and is used for the multiduty especially link agent/combined function group that multiple drug media is used because its effective nucleophilicity can become it.Generally, with carbodiimides reagent with amine groups with contain the carboxyl medicine and combine.Primary amine also can react to produce isourea (Bioconjugate Techniques with two succinimidyl carbonates (disuccinimidyl carbonate) and triethylamine (triethylamine), Greg T Hermanson, 1996, Academic Press Inc and Bioconjugation in pharmaceutical chemistry, IlFarmaco, 1999,54,497-526 and wherein reference), it can adhere to the medicine of carrying amine, for example insulin.
The present invention one preferred aspect provides the method for a novelty to come the derivation sporopollenin with a primary amine functional group with minimum chemical action, and avoids the use of nontoxic interleaving agent group, for example former employed diamidogen.The method comprises with ammonia (0.880) at room temperature handles sporopollenin to produce the amination shape of sporopollenin, uses LiAlH subsequently
4(lithium aluminium hydride reduction) reduction is to produce the primary amine groups (NH of polymer
2) shape.Can obtain the bearing capacity that about 1 milli rubs/restrains.
Example 10: the preparation of the sporopollenin of primary amine functionalization (Fig. 2)
Sporopollenin (2 gram) was stirred 4 days under room temperature in 0.880 ammonia.Collect sporopollenin by filtering, then water (10 * 100 cubic centimetres), EtOH (ethanol) (2 * 30 cubic centimetres) and DCM (dichloromethane) (2 * 30 cubic centimetres) flushing.Then sporopollenin is dried to constant weight in a vacuum.N in dioxane (dioxane) (100 cubic centimetres)
2(nitrogen) stirs LiAlH down
4(lithium aluminium hydride reduction) (3.6 gram).Add aminating sporopollenin (2 gram) and in N
2Refluxed 4 days in (nitrogen).Cool off this mixture.Add ethyl acetate (100 cubic centimetres) and cooling carefully.With Glass rod insoluble bulk is blended.Add entry (100 cubic centimetres) lentamente and add 2M sulphuric acid (200 cubic centimetres) then.Water (2 * 250 cubic centimetres), EtOH (2 * 250 cubic centimetres), DCM (2 * 250 cubic centimetres) flushing primary amine sporopollenin are dried to constant weight (1.8 gram) then in a vacuum then.
According to a fifth aspect of the present invention, provide the exine of a poly-amino functional.Also provide a dosage form that comprises chemically in conjunction with the poly-amino functional sporopollenin of effective dose active substance.
Some polyamino compounds for example are present in intravital spermine and spermidine naturally.They are nontoxic, and therefore can be used as the application of interleaving agent group.In a preferred embodiment, can in an atent solvent, make a polyamino compounds and sporopollenin reaction to form covalent bond by heating or backflow.
With respect to diamidogen and primary amine, use polyamine to have an advantage, promptly it has and is used for the extra nucleophilic amine groups that medicine adheres to, so that the derivation sporopollenin and the similar exine of the medicine delivered payload capability that these derivation have raising to be provided.Also available similar methods is used aromatic polyamine, and for example 1,3,5-o-phenylenediamine and relevant heterocycle, for example tripolycyanamide.Use phosgene or thiophosgene together with the medicine that has hydroxy respectively, for example nucleoside (for example stavudine (D4T), acyclovir (acyclovir) and Qi Tafuding (AZT)) can easily activate into aromatic amines respectively isocyanates or isothiocyanate hydrogen-oxygen group.Example 11 has disclosed the method that these combinations or interleaving agent group are adhered to.
Example 11: the preparation of the sporopollenin of amine functional polysiloxaneization (Fig. 3)
Spermidine (0.7 milli rubs), 1,3,5-o-phenylenediamine (0.7 milli rubs) and tripolycyanamide, respectively with sporopollenin (0.1 gram) at all kinds of solvents toluene (10 milliliters) for example, refluxed 24 hours in dimethyl sulfoxide (DMSOdimethyl sulphoxide) and the dimethyl formamide (DMF dimethyl formamide) so that it is after filtration, use toluene (2 * 10 cubic centimetres) then, 2M HCl (hydrochloric acid) (2 * 10 cubic centimetres), water (3 * 10 cubic centimetres), EtOH (2 * 10 cubic centimetres) and DCM (2 * 10 cubic centimetres) flushing, and the bearing capacity that is dried in a vacuum after the constant weight is respectively 1.6,0.95 and 0.54 milli rubs/restrains.
According to a sixth aspect of the invention, provide the exine of monocarboxylic acid functionalization.Also provide the dosage form that comprises chemically in conjunction with the carboxylic acid functional exine of effective dose active substance.
One is used to form the method for drug-polymer conjugate, comprises by N-maloyl imines ester utilizing N-maloyl imines and carbodiimides activation monocarboxylic acid salt functional.These Acibenzolars can be effectively and the medicine that has primary amine peptide for example, and the medicine that contains hydroxy for example nucleoside hydroxy unity close.Several markets carbodiimides on sale dissolves in aqueous or organic solvent.The carboxylic acid link agent that is attached to sporopollenin revealed (G.Shaw, M.Sykes, R.W.Humble, G.Mackenzie, D.Marsdan, E.Phelivan, Reactive Polymers, 1988,9,211-217).This effect has comprised 1, and 3-diaminopropanes derivation sporopollenin and bromoacetate produce the reaction after saponification.For example, carry out saponification subsequently again, can improve bearing capacity by making above-mentioned poly-amino derivation sporopollenin and for example succinic anhydrides or bromoacetate reaction.Another method that can guide the carboxylic acid function is monoamino-acid or the adhering to of cripetura propylhomoserin chain of Gly (glycine)-Phe (phenylalanine)-Ala (alanine)-Leu (leucine) or Gly (glycine)-Phe (phenylalanine)-Leu (leucine)-Gly (glycine) for example.One attached to more noticeable as the aminoacid of link agent on the exine because its by in an appropriate solvent with a unshielded aminoacid or or amino acid whose ethyl ester or other Arrcostabs and exine heat or reflux and can obtain easily.The further advantage of these link agent is its avirulence.Example 12 has disclosed the method that a non-derivation aminoacid is attached to sporopollenin.
Example 12: preparation aminoacid functional sporopollenin is as a kind of method (Fig. 4) of adhering to the carboxylic acid function
A kind of glycine (0.1 gram) refluxed in DMSO 24 hours with the mixture of sporopollenin (0.1 gram).By filter collecting sporopollenin, and with toluene (2 * 10 cubic centimetres), EtOH (2 * 10 cubic centimetres), 2M HCl (2 * 10 cubic centimetres), water (3 * 10 cubic centimetres), EtOH (2 * 10 cubic centimetres) and DCM (2 * 10 cubic centimetres) flushing so that its bearing capacity is 3.6 to rub/restrain in the least.
Example 13 demonstrates one and is used for amino-acid ester is attached to the method that sporopollenin also provides carboxyl function subsequently.
Example 13: preparation amino-acid ester functionalization sporopollenin is as a kind of method (Fig. 5) of adhering to the monocarboxylic acid function
Glycine ethyl ester hydrochloride (0.1 gram) is stirred in toluene (20 cubic centimetres) and triethylamine (2 milliliters).Adding sporopollenin (0.1 gram) also also stirred this mixture backflow in 24 hours.Collect refrigerative derivation spore by filtering, with toluene (2 * 10 cubic centimetres), EtOH (2 * 10 cubic centimetres), 2M HCl (2 * 10 cubic centimetres), water (3 * 10 cubic centimetres), EtOH (2 * 10 cubic centimetres) and DCM (2 * 10 cubic centimetres) flushing.Then that sporopollenin is dry in a vacuum, its shown bearing capacity is that 1.7 millis rub/restrain.By refluxing 2 hours, can make the sodium salt of corresponding carboxylic acid carry out the hydrolysis of ethyl ester function at 2M NaOH (20 milliliters).At room temperature neutralize with 2M HCl (40 milliliters), water flushing subsequently is also dry under vacuum, promptly gets needed acid (bearing capacity is that 2.8 millis rub/restrain).
It is the ethyl ester hydrochloride that 1.0 to the 2.5 for example β-alanines that rub/restrain in the least, L-rely amino acid, α-L-alanine, aspartic acid, glutamic acid and amidomalonic acid that above process also can be used for the bearing capacity scope.The advantage of adhering to amidomalonic acid, aspartic acid or glutamic acid has extra medicament-carried ability for it has two carboxylate functional groups.
Provide the exine of a polyhydroxy functionalization according to a seventh aspect of the present invention.Also provide a kind of dosage form that comprises chemically in conjunction with the polyhydroxy functionalization exine of effective dose active substance.
Most of by the polyhydroxy link agent that obtains in the carbohydrate, owing to have the hydrogen-oxygen group that a plurality of attributes according to sugar are attached to exine, it has and is easy to adhere to, and is nontoxic, the advantage that bearing capacity is high.Have a large amount of hydrogen-oxygen groups on the exine, have the advantage that to adhere to multiple medicine.The stability of polyhydroxy derivation exine and chemically with morphologic concordance, for example have advantage starch, cellulose and the non-spore derivation chitin with respect to the Polysaccharides drug media.Polymer described later is undiscovered have with sporopollenin or the exine that equates with it same chemically and/or modal concordance, or same soda acid resistance and water absorption.
Hydrogen-oxygen group on these conjugate materials can be converted into the active specy that much is suitable for synthetic drug-polymer conjugates.For example the hydrogen-oxygen group can activate into mesylate and toluene fulfonate with succinimido carbonate and diaza cyclopentadienyl carbonate and p-nitrophenyl formates (p-nitrophenylformates).All these is easy to and the medicine that contains primary amine, and for example peptide produces reaction.The hydrogen-oxygen group also can be oxidized to aldehydes or ketones.
The medicine that comprises primary amine can adhere to by reduction amination.The also available CNBr of hydrogen-oxygen group on the polymer (Bromine cyanide .) activation is to form cyanate, and it can produce reaction with the medicine that comprises amine.Therefore a kind of can the combination with an exine to produce one from a carbohydrate-derived drug-polymer conjugate has the conjugate of a large amount of reactive hydrogen-oxygen groups and its and has concordance chemically reaching form.Example 14 demonstrates a kind of method that polyhydroxy link agent can be attached to sporopollenin.
Example 14: the preparation (Fig. 6) of polyhydroxy functionalization sporopollenin
In DMSO (10 cubic centimetres), Pyrusussuriensis hydramine (Sorbitolamine) (0.13 gram, 0.69 milli rubs) is stirred.Add sporopollenin (0.1 gram, 0.23 milli rubs), this mixture was refluxed 24 hours.Collect refrigerative spore by filtering, and wash with DMSO (2 * 10 cubic centimetres), water (100 cubic centimetres), EtOH (2 * 10 cubic centimetres) and DCM (2 * 10 cubic centimetres).Sporopollenin is dried to constant weight under vacuum, making its bearing capacity is that 1.7 millis rub/restrain.
Example 15: the preparation (Fig. 7) of trihydroxymethylaminomethane (tris (hydroxymethy1) methylamine)-sporopollenin
One program similar to example 14 uses trihydroxymethylaminomethane (tris (hydroxymethyl) methylamine) can reach the bearing capacity that 0.83 milli rubs/restrains.
Example 16: the preparation that fluorescein and thyroxine covalent bond adhere to sporopollenin
Fluorescein (0.5 gram) refluxes in DMSO (20 milliliters) with sporopollenin (0.1 gram) respectively with thyroxine (0.5 gram).Collect refrigerative sporopollenin and water (100 cubic centimetres), EtOH (2 * 10 cubic centimetres) and DCM (2 * 10 cubic centimetres) flushing by filtering.Sporopollenin is dried to constant weight in a vacuum so that its bearing capacity is respectively 1.0 and 0.37 milli rubs/restrain.
If medicine or other active substances are enough stable to keep out reflow step, then this adhering mode is very effective.This direct adherence method is relatively chemically not as having apparent advantage the polymer on sale of the stable many markets of sporopollenin.
More unsettled medicine, for example insulin can and use DCC and HOBt (I-hydroxybenzotriazole) to be attached to amino sporopollenin as coupling agent by the amino interleaving agent of a for example succinyl.Polymer on sale has chemical conforming advantage to sporopollenin in this kind combination compared to the market, thereby makes the expection bearing capacity of the medicine that is bonded to every group of sporopollenin very consistent.One medicine, for example the adherence method of insulin may further comprise the steps:
Example 17: the preparation of the sporopollenin of bound insulin
The butanedioic anhydride (0.57 gram, 5.7 millis rub) that will be dissolved in the dried dimethyl formamide (DMF, 15 milliliters) is incorporated in amino sporopollenin (1 gram) suspension of doing DMF (30 milliliters).This reactant mixture is at room temperature stirred a whole night in nitrogen.By its product of isolated by filtration, with acetone rinsing and P is being arranged
2O
5Drying is 48 hours under the vacuum that exists.The DCC (3.80 grams, 18.4 millis rub) and the HOBt (2.27 grams, 1.84 millis rub) that will do among the DMF add in the amino sporopollenin suspension of the succinyl of doing among the DMF (20 milliliters) continuously again.This mixture at room temperature stirred then cryodesiccated insulin (0.2 gram) to be added in 3 hours in nitrogen do among the DMF (20 milliliters) as a solution.This mixture was at room temperature stirred 48 hours.Stop this reaction by adding entry (10 milliliters) at 0 degree Celsius.This mixture was at room temperature leniently stirred 2 hours, and filtering and washing to produce bearing capacity with acetone and ether is the amino sporopollenin (insulin-succinylamido sporopollenin) of insulin-succinyl that 0.03 milli rubs/restrains.
Above example shows how protein and enzyme are attached to sporopollenin and similar exine.Oligonucleotide, for example antisense oligonucleotide can adhere to by similar chemical reaction so that this medicine can be oral.In the past these chemical compounds often as PEG (Polyethylene Glycol)-3 '-oligonucleotide or PEG-5 '-oligonucleotide conjugates by injection delivery.Can be by utilizing above-mentioned hydroxylation in conjunction with sporopollenin, carry out those and be used for step like the synthesis of coupling reacting phase of synthetic PEG-3 '-oligonucleotide or PEG-5 '-oligonucleotide conjugates.
Application has water miscible medicine hardly, and for example the sporopollenin of paclitaxel and ciclosporin-drug type conjugate has special benefit.These medicines can be attached to sporopollenin under many circumstances, for example, at efficient solvent, for example use coupling agent among the DMSO or in the buffering aqueous solvent.Highly dissipation mode of medicine discharges when oral sporopollenin-medicine type conjugate arrives blood flow, therefore can be better by the serum solvation.Therefore sporopollenin and similar exine also can be used as and use Polyethylene Glycol (PEGs) a kind of selection in addition, and it often is used to derivation insoluble,practically medicine in aqueous systems.The PEG-drug conjugates that need inject during with respect to use, it is that it can be oral that this kind conjugate has an advantage.
Example 18: biological assessment
Take sporopollenin (1 gram for two clinical trial persons; 25 microns; Derive from Herba Lycopodii), extracted blood sample every 30 minutes.
Blood sample (20) separated eight minutes with 3000 rev/mins speed with centrifuge.Move apart serum then, and the residual substance water is washed (totally 10 milliliters) several times, and be transferred to bigger test tube.Then sample fully being mixed the reuse centrifuge separates once.Remove the supernatant, granule and residual liquid (0.5 milliliter) are suspended in 0.5 milliliter of glycerol once more.Get the about 0.1 milliliter five equilibrium of per minute and use light microscopy.Entire portion below the inspection cover plate.Calculate the sporopollenin number of particles in order to following method.By calculating complete particle and collecting together the sum that calculates particle in the quantity of one scrappy part.These scrappy parts are taken as and derive from same sporopollenin particle, but it may comprise the composition of several particles.
The microscope test result is as follows:
Sample (feed back) from the first human body experimenter
1) use sporopollenin before:
Do not find particle.
2a) use 30 minutes after:
80 particles wherein have 12 for complete, and all the other are 10-20 a small bundle of straw, etc. for silkworms to spin cocoons on that scrappy part forms.
2b) use 30 minutes after:
Find 45 particles, wherein 7 is complete, and remaining is scrappy part.
3a) use 60 minutes after:
Find the scrappy part of 2 complete particles and 27 groups little.
3b) use 60 minutes after:
Find 20 groups little scrappy part, do not have complete particle.
4) use 90 minutes after:
Observe the scrappy part of 1 complete particle and 12 groups very little.
5) use 120 minutes after:
Observe a small amount of little scrappy part.
Sample (before the feed) from the second human trial person:
1) use sporopollenin before:
Do not find particle.
2a) use back 30 minutes:
Find 3 complete particles and 48 very little scrappy parts.
2b) use 30 minutes after:
Find 4 complete particles and 35 scrappy parts.
3a) use 60 minutes after:
Observe 1 complete particle and 17 minimum scrappy parts.
3b) use 60 minutes after:
As seen 2 particles and 15 scrappy parts.
4) use 90 minutes after:
Do not find particle or scrappy part.
The minimum scale that is entered in the blood flow by 1 gram sporopollenin is estimated as 0.60%.
The dosage of estimating medicable required medicine-sporopollenin conjugate is:
Suppose that average bearing capacity is that 1 milli rubs/restrains, then the medicine total amount that is entered in the blood flow by 1 gram sporopollenin will be rubbed for 0.006 milli.
For 0.03 sporopollenin that rubs/restrain in the least that 1 gram physically or covalently is attached with insulin (M.W.6000), the insulin of 11 milligrams of (0.018 millimeter) 30 ius (IU) is entered in the blood flow.The normal person produces the 24u insulin every day, and common diabetics needs 60u every day, supposes that the subcutaneous injection agent is identical with oral result, and then 2 of maximum gram sporopollenins can be equivalent to 1 day insulin supply.
Is for the 0.37 milli sporopollenin that rubs/restrain for 1 gram with thyroxine (M.Wt.776.9) bearing capacity, and it can provide 1.7 milligrams thyroxine, [being about 17 doses (mean dose is 100 micrograms)].Physical attachment is that the thyroxine of the sporopollenin of 600 microgram/grams can expectedly carry 3.6 micrograms to enter blood flow in bearing capacity.This value obtains proof by the thyroxine of finding in the volunteer blood is analyzed, and the thyroxine amount after wherein oral 15 minutes is 4 micrograms/5 liter blood (promptly having transmitted 0.66%, consistent with the amount of transmission sporopollenin particle).
Sporopollenin or other similar exines can be by multiple derivation.Can be used in a dosage, transmit by this mode sporopollenin more than a kind of medicine.In addition, a medicine also can be incorporated in the same sporopollenin particle with an active medium.In addition; this kind multi-functional can be used to the guiding function group; a fatty acid chain for example; to form a kind of lipid conjugates; or one Polyethylene Glycol (PEG) to form a PEG-lipid conjugates; in case the particle that adheres to arrives in the blood flow, can and medicine be discharged more slowly by the endosome space obstacle and protect the medicine that adheres to.For example, the residual double bonds of sporopollenin and hydroxy-acid group can be by derivation respectively.Therefore this pair key at first produces to react with bromine and forms the bromo sporopollenin, makes itself and sodium azide reaction form the repeatedly nitrilo sporopollenin of a novelty, with lithium aluminium hydride reduction it is reduced to form a primary amine groups sporopollenin.Make primary amine in buffer solution and with EDC as coupling agent and a medicine, for example anti-virus medicine AZT monophosphate reaction is to produce AZT list phosphoric acid sporopollenin.The hydroxy-acid group and the cetyl ammonia that will remain in subsequently on the sporopollenin concentrate with DDC.Medicine then ought adhere in cetyl half family provides extra protection for it in the blink of stomach.One example of the adherence method of this type following (Fig. 8).
According to an eighth aspect of the present invention, provide a halogen functionalization exine.Also provide a dosage form that comprises chemically in conjunction with the halogen functionalization exine of effective dose active substance.
Example 19: by bromination, repeatedly nitrogenize and reduction be to the guiding of primary amine
Sporopollenin (1 gram) was stirred 24 hours in the acetic acid that 30% bromine is arranged (10 cubic centimetres).Regain sporopollenin by filtration, and with the flushing of methanol (10 * 5 cubic centimetres) and ether (5 cubic centimetres), and under vacuum dry with produce the bromo sporopollenin (A, Fig. 8).Add sodium azide (1.25 grams, 19.2 millis a rub) solution that is dissolved among the DMSO (30 cubic centimetres) and also this mixture is spent heating 48 hours with Celsius 60.By filtration, flushing, the dry (N that 1.8 millis rub/restrain
3) and refluxed 1 hour with THF (15 a cubic centimetres) mixture that contains lithium aluminium hydride reduction (0.2 gram), with collect the repeatedly nitrilo sporopollenin that forms (B, Fig. 8).This mixture is cooled to room temperature, and flushing is also dry to obtain amido sporopollenin (C, Fig. 8) (NH that 1.4 millis rub/restrain
2).
Adhering to of example 20:AZT monophosphate
AZT monophosphate (2.1 milli rub) is added one at 0.1M MES (MES), and pH value is amido sporopollenin (0.3 gram in the buffer solution (15 cubic centimetres) of 4.7-6.0; 1.4 the NH that milli rubs/restrains
2) the stirring suspension.Add EDC (2.1 millis rub), and mixture was at room temperature stirred 18 hours.Leach particle, the water flushing is also dry to draw AZT monophosphate amidatioon sporopollenin (D, Fig. 8) (1 milli rubs/restrains) under vacuum.
Example 21: the adhering to of hexadecylamine
With an AZT monophosphate sporopollenin (0.3 gram; 1 milli rubs/restrain) and the mixture of DCC (2.1 rub in the least) in DMSO (10 cubic centimetres), stirred 18 hours.By filter and under vacuum dry cetyl amino (1 milli rubs/restrain)/AZT monophosphate amidated (1 rubs/the restrains in the least) sporopollenin that removes gained (E, Fig. 8).
Bearing capacity is about 1 sporopollenin that rubs/restrain in the least and has the remaining hydrogen oxygen groups, gained (route A among Fig. 9) in analyzing as Fmoc (fluorenes methoxy carbonyl acyl group).These functional groups can be used for directly or by the interleaving agent group adhering to medicine.
Example 22: the alkanisation of hydrogen-oxygen group
Contain sporopollenin (1 gram with one; 1 milli rubs/restrain) and 6M NaOH (50 cubic centimetres) suspension of monoxone (6 milli rub) stirred 18 hours, obtaining bearing capacity after flushing and the drying is the 0.8 sporopollenin-acetic acid (route B among Fig. 9) that rubs/restrain in the least.
Example 23: the acidylate of hydrogen-oxygen group
I) use acid anhydride and acid chloride:
Residual hydrogen-oxygen group is easy to that for example chloroacetic chloride or acetic anhydride are acetylation by reagent
2,3,4, and by benzoyl chloride by benzoylation, with under standard state to obtain the sporopollenin (route C among Fig. 9) of acidylate.Benzoyl chloride (5 milli rub) is added one that mix and contain sporopollenin (0.1 gram; The 1 milli OH that rubs/restrain) DCM (10 cubic centimetres), DMAP (4-dimethylamino naphthyridine) and in ice bath in the suspension of refrigerative pyrimidine (3 rub in the least).The sporopollenin of benzoylation (0.6 milli rub/restrain benzoylation) is filtered, wash drying.
Ii) use a N-protected (N-protected) aminoacid and coupling agent:
With N-protected (N-protected) aminoacid for example Fmoc glycine (route D among Fig. 8) and coupling agent for example the catalytic DCC of DMAP be not disclosed as yet with direct acidylate hydrogen-oxygen group.Therefore an example of the method is as follows: will contain the DCM (20 cubic centimetres) of DCC (0.5 milli rubs) and Fmoc glycine (1 milli rubs) and DMF (1 cubic centimetre) solution stirring 20 minutes.Then the DCM evaporation is removed, residue is dissolved in the formed solution adding one of DMF (10 cubic centimetres) in DMF (10 cubic centimetres), contains in the suspension of sporopollenin (0.1 milli rubs/restrains).Adding one is stirred the gained mixture 24 hours then in the solution that DMF (2 cubic centimetres) contains DMAP (0.1 milli rubs).Leach Fmoc glycine sporopollenin, with DMF, DCM and MeOH (methanol) flushing and dry (0.41 milli rub/restrain Fmoc glycine).
Can use a similar adherence method to adhere to peptide and pharmaceutical grade protein.
The (iii) carbamic acidization of hydrogen-oxygen group:
Remaining hydrogen-oxygen group can react with isocyanates (route E among Fig. 9).The method can be used for forming the difunctional link agent of out-phase, comes in conjunction with the hydrogen-oxygen group to use p-maleimide isocyanates (p-maleimidophenyl isocyanate).Step with carbanil derivation sporopollenin is as follows: with sporopollenin (1 gram; 1 milli rubs/restrains) with the suspension stirring of carbanil and with 80 degree heating Celsius 18 hours, to obtain sporopollenin carbaminate (0.9 milli rubs/restrains), it is leached, with DMSO and washed with methanol and drying.
The (iv) halogenation of hydrogen-oxygen group:
The remaining hydrogen-oxygen group of sporopollenin SOCl
2, POCl
3Or PCl
5Can be easily by halogenation (route F among Fig. 9).This halogenated form of sporopollenin can be used for by a link agent and uses the further derivation polymer of a medicine subsequently.The halogenation of hydrogen-oxygen group is described below: with Anhydrous potassium carbonate (6.37 millis rub) and sporopollenin (1 gram; The 1 milli OH that rubs/restrain) in DCM, contains PCl in 0 degree adding one Celsius
5Solution in.This mixture was stirred 15 minutes, leach sporopollenin then, with DCM and alcohol flushing and dry (1 milli rub/restrain C1).
Reference:
1.S.Kettley，PhD?University?of?Hull，2001。
2.G.Shaw, The Chemistry of Sporopollenin, in: Sporopollenin, J.Brooks, M.Muir, P.Van Gijzel and G.Shaw, (Eds) Academic press, London and New York, 1971,305-348.
3.F.Zetche，P.Kalt，J.Liechti，E.Ziegler，J.Prakt.Chem，，1937，148，267。
4.P.Fawcett, D.Gree, R.Holleyhead and G.Shaw, Grana, 1970,10,246.
5.M.E.Annunziato, U.S.Patel, M.Ranade and P.S.PalumboBiocon jugate Chem., 1993,4,212.
Some conjugated biomolecules close and use mercaptan conjugated group (Biocojugate Techniques, Greg THermanson, 1996, Academic Press Inc and Bioconjugation inpharmaceutical chemistry, Il Farmaco 1999,54,497-526).Sporopollenin is easy to by derivation, with by for example, earlier the two keys of sporopollenin is carried out bromination, uses thiourea (Figure 10) subsequently or guides thiol group with the NaSH processing method.
According to a ninth aspect of the present invention, provide the exine of a thiol functionization.Also provide a dosage form that comprises chemically in conjunction with the thiol function exine of effective dose active substance.
Example 24: thiol group is to adhere to (Figure 10) of sporopollenin
(1 restrains, and 4.5 millis rub/restrain to contain the bromo sporopollenin with one in DMSO (10 cubic centimetres); Method obtains by the bromine reaction in sporopollenin and the acetic acid as previously described) refluxed 24 hours with the suspension of thiourea (60 millis rub).Collect gains by filtering, and wash with DMSO, water, 2M HCl, water, EtOH and DCM.These particles are being refluxed and stirring 6 hours in 25%KOH.After the cooling with this particulate filter and water, 2M HCl, water and washed with methanol and dry sporopollenin (5.2 millis rub/restrain) to obtain mercaptanization.
Example 25:p-Nitrobenzol acyl-oxygen phosphinylidyne (p-nitrobenzoyloxycarbonyl) group is to adhere to (Figure 11) of sporopollenin
With the acidylate (Figure 11) of p-Nitrobenzol acyl-oxygen carbonic acyl radical chloride (p-nitrobenzoyloxycarbonyl chloride) to thiol group:
Mercaptan sporopollenin (5.2 millis rub/restrain) is at N
2Following to DCM (25 cubic centimetres) stirring under refluxing that contains p-Nitrobenzol acyl-oxygen carbonic acyl radical chloride (p-nitrobenzoyloxycarbonyl chloride) (30 millis rub) and triethylamine (30 millis rub).Particle is leached, with DCM and washed with methanol and dry to obtain p-Nitrobenzol acyl-oxygen carbonic acyl radical (p-nitrobenzoylthiooxycarbonylated) sporopollenin (2.45 millis rub/restrain).
Have and manyly be used to make carbon-to-carbon to be attached to the method for sporopollenin.This structure quite complicated but that reach the polymeric material that chemically is steady statue is not physically known as yet fully; Therefore be difficult to know and find to have assorted one reaction to produce in the future on its surface.Yet, one simple examples of carbon-carbon bond structure comprises diethylmalonate and the reaction of bromo sporopollenin in the methylamino ethoxy sodium solution, with with the hydrolysis of KOH solution and with a mineral acid in and the back on sporopollenin, produce one or two acid functions (structure C, Figure 12) and increase CO thus
2The bearing capacity of H group.One example of the method is as follows:
Provide an exine by the carbon-carbon bond functionalization according to a tenth aspect of the present invention.Also provide one and comprise an exine, a carbon-carbon bond, with the dosage form of bonded other functional groups of effective dose active substance.
Example 26: the functional group carbon-carbon bond adheres to sporopollenin
Diethylmalonate (25 millis rub) is slowly added (30 cubic centimetres of sodium methoxides under 50 degree Celsius; The 25 milli sodium that rub) in the solution.Gained solution is slowly added one in ethanol (50 cubic centimetres), contain bromo sporopollenin (1 gram; Mixing in the suspension 5 milli bromines that rub/restrain).Adding back (15 minutes) refluxes this mixture 18 hours.Leach diethyl malonyl sporopollenin (A, Figure 12) and use alcohol flushing.To add to again in the ethanol (10 cubic centimetres) in the potassium hydroxide (15 millis rub) water-soluble (2 cubic centimetres).This solution is added to one in ethanol (40 cubic centimetres), contain diethyl sporopollenin diethylmalonate (A, mixing in the suspension and refluxed 18 hours Figure 12).Leach particle and water and alcohol flushing dry again with obtain potassium salt (B, Figure 12).With the suspension that places the B of frozen water dilute sulphuric acid acidify, after water and alcohol flushing and drying, to obtain sporopollenin malonic acid (C, Figure 12) (1 milli rubs/restrains).
Metal be attached to be attached to sporopollenin the interleaving agent group to produce a medium and to have nothing to do in medicine transmission { G.Shaw, M.Sykes, R.W.Humble, G.Mackenzie, D.Marsdan, E.Phelivan, Reactive Polymers, 9, (1988), 211-217}.Yet do not disclose medicine so far as yet with metal complex.Ensuing example discloses a kind of method, wherein sporopollenin as the dentate of platinum (D, Figure 12).Itself and known antitumor drug be cisplatin (cisplatin), carboplatin (CBDCA) and JM-40 similar (Figure 13) for example.Among Figure 12 simple displaying go out the synthesizing new derivative (its details is as follows for D, method Figure 12):
Example 27: cisplatin (cisplatin) adhering to sporopollenin
Comprise cis-[PtCl with one
2(NH
3)
2] DMF (20 cubic centimetres) solution of (suitable-two hydrazine dichloride platinum) (1.33 milli rub) is added to one and contains sporopollenin malonic acid (C, mixing in suspension Figure 12) in DMF (20 cubic centimetres).Add 0.1M aqueous KOH (27 cubic centimetres) then and the gained mixture was stirred 48 hours under 60 degree Celsius.Leach platinum spore (Sporoplatin) (D, Figure 12 also shows with this title) and water, ethanol and ether (0.9 milli rubs/restrains) flushing in Figure 13.
Some gold (I) mercaptides (gold (I) thiolate) complex shows that rheumatoid arthritis is had effective active.Allochrysine and early oral Ruide (Ridaura) (R.Bau, J.Am.Chem.Soc., 1998,120,9380).Ruide (Ridaura) is one thiosugar/hydrogen phosphide and golden complex, and can be decomposed in digestive system as most sugars.The complex of relevant sporopollenin/hydrogen phosphide and gold should be more stable and be easier to gold (I) mercaptides (gold (I) thiolate) is delivered in the blood flow to treat rheumatoid arthritis in intestinal portion.Also can use platinum, ruthenium, gadolinium and technetium complex.
Example 28: the gold of sporopollenin (I) mercaptides (gold (I) thiolate) complex (Figure 14)
The alcohol-water (1: 4 that will comprise potassium carbonate (1 milli rub); 50 cubic centimetres) (1 restrains to be added to a mercaptan sporopollenin suspension; 1 milli rubs/restrains) in, at room temperature stirred 5 hours and be cooled to 0 degree Celsius.Slowly add an alcohol-water that comprises (triethylphosphine) auric chloride (I) (1.1 milli rub) in 0 degree Celsius.Can be with this solution elevating to room temperature, and further stirred again 12 hours.Should (triethylphosphine) (sporopollenin-S) gold (I) derivative (0.8 milli rubs/restrains) water, ethanol and ether flushing and dry.
Claims (40)
1. the dosage form on pharmacopedics or the threpsology, it comprises the active substance of effective dose, chemically or physically be incorporated into or be packaged in the carrier, select in the exine of this carrier by a plant, liverwort, mushroom or algae or its scrappy part, optionally further have excipient.
2. dosage form as claimed in claim 1, wherein this carrier comprises sporopollenin, chitin or glucosan.
3. dosage form as claimed in claim 1, wherein this adventitia comprises sporopollenin, chitin, glucosan or the mannan that derives from plant, liverwort, mushroom or algae spore.
4. the described dosage form of any one claim as described above, it is suitable for oral, buccal, Sublingual or through the skin administration.
5. dosage form as claimed in claim 4 is by selecting in tablet, capsule, ovule, elixir, solution and the outstanding agent.
6. as any one described dosage form in the claim 1 to 3, it is used for pulmonary administration.
7. dosage form as claimed in claim 6, wherein the particle size of carrier is in 1 to 100 micron the scope.
8. dosage form as claimed in claim 7, wherein the particle size of carrier is in 1 to 10 micron the scope.
9. dosage form as claimed in claim 8, wherein the particle size of carrier is in 1 to 5 micron the scope.
10. the described dosage form of any one claim as described above, wherein active substance comprises peptide or hereditary material.
11. the described dosage form of any one claim, wherein sporopollenin or other exines or sporocyst functionalization as described above.
12. the described dosage form of any one claim as described above, wherein active substance is unstable or unstable to acid to alkali.
13. as any one described dosage form in the claim 1 to 11, wherein active substance has low-solubility in water.
14. dosage form as claimed in claim 13, wherein active substance is metal or metal derivative.
15. dosage form that is used for pulmonary administration and comprises the effective dose active substance, wherein active substance is contained in the seal, select in its complete exine by a strain plant, liverwort, mushroom or algae, or be contained in the intraparietal cavity of sealing, and optionally further have excipient.
16. dosage form as claimed in claim 15, wherein the sealing body comprises sporopollenin, chitin or glucosan.
17. dosage form that is used for pulmonary administration and comprises the effective dose active substance, wherein this active substance is absorbed in the surface of carrier, select in its complete exine, optionally further have excipient by a strain plant, liverwort, mushroom or algae.
18. as claim 16 and 17 described dosage forms, wherein complete exine has a scope and is in about 1 to 10 micron average diameter.
19. as claim 16 and 17 described dosage forms, wherein complete exine has a scope and is in about 1 to 5 micron average diameter.
20. the sporopollenin of a primary amine functionalization or other exines.
21. the sporopollenin of a thiol functionization or other exines.
22. a method that forms sporopollenin or other exines of primary amine functionalization comprises with ammonia to sporopollenin or other adventitias are handled and carry out step of reducing with for example lithium aluminium hydride reduction.
23. the sporopollenin of a poly-amino functional or other exines.
24. one kind forms the sporopollenin of poly-amino functional or the method for other exines, comprises the step with sporopollenin or other exines and polyamino compounds heating.
25. the sporopollenin of a carboxylic acid functional or other exines.
26. the sporopollenin of an aminoacid functionalization or other exines.
27. a method of making sporopollenin or other exines of aminoacid functionalization comprises the step with its amino-acid ester and sporopollenin or the heating of other exines.
28. the sporopollenin of a poly-hydroxy functionalization or other exines.
29. the sporopollenin of a halogen functionalization or other exines.
30. the sporopollenin of a functionalization or other exines wherein have a carbon-carbon bond between adventitia and active component.
31. one kind includes covalent bond and is connected to the sporopollenin of insulin or the dosage form of other exines, optionally further has one or more excipient.
32. threpsology or flavouring agent dosage form, it comprises a kind of threpsology or functionalization material that is contained in physically in the exine.
33. threpsology or flavouring agent dosage form, it comprises physically a kind of or chemically is attached to the threpsology or the functionalization material of exine.
34. threpsology or flavouring agent dosage form, its comprise a kind of be included in the exine the threpsology's or the functionalization material.
35. one kind comprises C with being combined in of pressure or vacuum
1To C
4There is the method for filling exine down in the auxiliary permeation liquid of ethanol.
36. there is the method for filling exine down in the aqueous ethanol that is combined in pressure or vacuum.
37. as any one described threpsology or flavouring agent dosage form in the claim 32 to 34, wherein the exine skin further is covered with an Edible material with auxiliary retentive activity composition.
38. as any one described dosage form in the claim 1 to 9, wherein the exine skin further is covered with a kind of material with auxiliary retentive activity composition.
39. as the dosage form described in the claim 1 to 18 any one, with the medicine of its preparation treatment disease.
40. one kind is utilized an insoluble medium to carry an active component to enter method in the blood flow through oral area, wherein active component promptly entered in the blood flow less than 20 minutes and subsequently medium in 3 hours, decompose.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0315019A GB0315019D0 (en) | 2003-06-27 | 2003-06-27 | Dosage form |
| GB0315019.0 | 2003-06-27 | ||
| GB0411047A GB0411047D0 (en) | 2004-05-18 | 2004-05-18 | Dosage form |
| GB0411047.4 | 2004-05-18 | ||
| PCT/GB2004/002775 WO2005000280A2 (en) | 2003-06-27 | 2004-06-28 | Dosage form comprising an exine coating of sporopollenin or derivatized sporopollenin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1842325A true CN1842325A (en) | 2006-10-04 |
| CN1842325B CN1842325B (en) | 2012-02-29 |
Family
ID=27637467
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2004800243309A Expired - Fee Related CN1842325B (en) | 2003-06-27 | 2004-06-28 | Dosage form |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN1842325B (en) |
| GB (1) | GB0315019D0 (en) |
| ZA (1) | ZA200510179B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114831111A (en) * | 2022-06-17 | 2022-08-02 | 中国农业科学院蔬菜花卉研究所 | Pesticide carrier based on plant spore powder and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5275819A (en) * | 1989-02-06 | 1994-01-04 | Amer Particle Technologies Inc. | Drug loaded pollen grains with an outer coating for pulsed delivery |
| WO1998051711A1 (en) * | 1997-05-14 | 1998-11-19 | Japan As Represented By Director General Of National Institute Of Sericultural And Entomological Science Ministry Of Agriculture, Forestry And Fisherries | Chitin beads, chitosan beads, process for preparing these beads, carrier comprising said beads, and process for preparing microsporidian spore |
| DE19902724A1 (en) * | 1999-01-19 | 2000-07-27 | Dietmar Lerche | Microcapsules, e.g. useful as filtration and chromatography media, comprise intact macroporous pollen exine or sporoderm |
-
2003
- 2003-06-27 GB GB0315019A patent/GB0315019D0/en not_active Ceased
-
2004
- 2004-06-28 CN CN2004800243309A patent/CN1842325B/en not_active Expired - Fee Related
-
2005
- 2005-12-14 ZA ZA200510179A patent/ZA200510179B/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114831111A (en) * | 2022-06-17 | 2022-08-02 | 中国农业科学院蔬菜花卉研究所 | Pesticide carrier based on plant spore powder and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| GB0315019D0 (en) | 2003-07-30 |
| CN1842325B (en) | 2012-02-29 |
| ZA200510179B (en) | 2006-08-30 |
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