CN1842325B - Dosage form - Google Patents
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- CN1842325B CN1842325B CN2004800243309A CN200480024330A CN1842325B CN 1842325 B CN1842325 B CN 1842325B CN 2004800243309 A CN2004800243309 A CN 2004800243309A CN 200480024330 A CN200480024330 A CN 200480024330A CN 1842325 B CN1842325 B CN 1842325B
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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
Technical field
The present invention is about the dosage form on a kind of pharmacopedics or the threpsology, a kind of manufacturing approach of this kind dosage form and utilize the Therapeutic Method of this kind dosage form.Provide human and dosage form for animals.
Background technology
Sporopollenin is the exine of each kind of plant, liverwort, mushroom and algae.Remove through continuous processing and possibly be attached on the exine or lipid, carbohydrate, protein and nucleic acid that exine is contained, can sporopollenin be separated from spore with solvent, alkali and acid.Also used the method for enzyme.Sporopollenin 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 up 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 processed by the sporopollenin 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 the sporopollenin capsule thereof, particularly is used for improving processing speed, and utilizes specific exine size to confirm 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.
Summary of the invention
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; This carrier optionally further has excipient by selecting in plant, liverwort, mushroom, algae or its segmental exine.
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 in physically being incorporated into a carrier; This carrier can select further to have excipient by selecting in plant, liverwort, mushroom, algae or its segmental exine.
When this active substance when physically being combined, it can be adsorbed on the carrier.
As another selection and often more desirable be 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 get into blood flow through it is absorbed, and adventitia is decomposed carry out dispenser with the method for release of active agent subsequently.
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 this exine of active substance contact and this material can be infiltrated in the sporopollenin wall cavity and/or
Contact exine and let this material infiltrate exine inside with an active substance, and remove this infiltration auxiliary liquid subsequently and let 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 crowd 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 sporopollenin 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 or other permeate in the auxiliary liquid before contact with active substance.
The exine of sporopollenin can quilt: (i) under atmospheric pressure or be pressed into tablet in a vacuum, then it is contacted with active substance (or being merely the liquid active substance that has or do not have the 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 property 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.The sporopollenin 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 physically comprise) then discharge slowly.Scanning electron microscope (SEM) X-ray shows that down the copper sulfate of whole sporopollenin shell is absorbed equably.Can be when active component is transferred through intestinal by slow release.As do not hope to have above-mentioned situation then can on the sporopollenin adventitia, increase another adventitia to postpone release.Exine can for example be used a low viscosity resin, like Radix Acaciae senegalis by derivation to reduce its semipermeability.Do not get in the blood flow if do not hope to be absorbed, can use sporopollenin (greater than 100 microns) than macroparticle.
In preferred dosage form, exine comprises sporopollenin, 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 that kind described in the US-A-5013552.
In a preferred embodiment of the invention, this carrier is made up of exine basically, and this exine comprises sporopollenin, 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 maybe be less, and it makes using effectively of medicine or active substance and rapidly, for example a few minutes like 20 minutes time in.
The said preparation form can comprise a conjugated biomolecule closes, and promptly through 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 adhere to it to produce a conjugated biomolecule with the sporopollenin direct reaction or physically and close.Yet sporopollenin or other exines be by functionalization in an embodiment of the present invention, thereby medicine can adhere to through 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 get into intestinal through stomach through 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 through 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), miconazoleanthracyclinones 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 entering blood and the ability of tachymetabolism (20 minutes to 2 hours) is arranged fast oral after, 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 get into blood flow (ML Wierner, Fd Chem.Toxic., 1988,26 (10) 867-880) through 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; I1Farmaco; 1999,54,497-526).A lot of coupling agents are disclosed.Carbodiimides such as DCC (dicyclohexylcarbodiimide dicyclohexylcarbodiimide) or EDC{1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride} is 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, diluent or the 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 process tablet or when needs dwindle tablet sizes, be kept perfectly.
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 to combine so that in liquid, dissipate.
Can comprise excipient according to tablet of the present invention; For example microcrystalline Cellulose, lactose, sodium citrate, calcium carbonate, Bibasic Calcium Phosphate, glycine and starch; Be preferably frumentum, Rhizoma Solani tuber osi or tapioca; Disintegrant; For example carboxymethylstach sodium (sodium starch glycollate), 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, PEO, Xanthan gum (xanthan gum), carbomer (Carbomer), oil, wax and methacrylate (methacrylate) copolymer.
Also can use through the skin administering mode, especially use little sporopollenin fragment.
Dosage form of the present invention is manufactured to and is applicable to pulmonary administration in special 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, the size and the shape of particle were roughly constant after usefulness acid, alkali or organic solution were extracted spore.The exine that is extracted can keep with its from spore in size and keep similar in shape.Relatively, micronized medicine for example is used for the sort of of treating 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 that kind according to following publication according to useful spore of the present invention and from gymnosperm, angiosperm, pteridosperms, mushroom and algae, obtain, 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 of typical case's species is big or small 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
Attaches 20 microns in 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 process exine through removing from spore, remaining harmless body can transfer to these identical positions in the pulmonary with useful medium.
Can be according to a dosage form of the present invention, and can be through 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 through the mode administration that pulmonary sucks or nasal cavity sucks, 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 combine 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 combines the segmental of sporopollenin or spore, can external application with the administered of gel, moisture coagulant liquid, solution, Emulsion, ointment or powder.This prescription agent also can be on skin or through the skin administration, for example through using patch.
Dosage form of the present invention can offer human or for animals.
Description of drawings
Fig. 1 is extremely shown in Figure 14 to be the reaction equation of sporopollenin derivation/functional method of each embodiment according to the present invention;
Figure 15 is the electron micrograph image of the cross section after the sporopollenin of embedding acrylic resin is cut into slices.
The specific embodiment
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 through 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 with hot water, hot ethanol and water once more.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 has been less than just absorbing solution in one minute, this sample placed under the vacuum of phosphorus pentoxide under 5 degree Celsius, to be dried to constant weight then.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, after oral 15 minutes, finds that 4 microgram thyroxine then are equivalent to 0.66% of medicine transmission amount.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
Through 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 |
| Toluene | |
| 2 minutes 15 seconds |
The physical attachment of repetition ethanol and Oleum helianthi under 40 degree Celsius, it has shortened the used time of this sample that absorbs fully; Promptly accomplish after 12 seconds and absorb.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 came to light 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 under scanning electron microscope (SEM) X-ray, shows whole sporopollenin shell is absorbed equably.
Sporopollenin stirs to come to light after 30 minutes in water and has kept 75% copper sulfate.
Example 8:LR White
Physical Absorption
(2 milliliters: wherein contain 80% polyhydroxy and replace bisphenol a dimethacrylate resin (polyhydroxysubstituted bisphenol A dimethacrylate resin) of alcoholic solution that one sporopollenin tablet (0.1 gram) added 50%LR White
; 19.6%C12 methacrylate (C12 methacrylate ester), 0.9% dimethyl-p-toluidine (dimethyl paratoluidine)) in.This mixture was leniently stirred 2 hours.Also further use LR White
(2 milliliters) to handle again 12 hours through the centrifuging separation on the gained particle.Place a capsule under 60 degree Celsius, to heat 8 hours in this mixture then.Resulting polymers fills up sporopollenin particle and wall inner chamber body thereof, like (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 about 5 milli level halogenations (F.Zetsche and K.Huggler, Liebigs Ann.Chem., 1928,461,89) of rubbing/restraining through direct bromination.It can be through 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 M errifield 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 through 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).
Polymer on sale has and is physically reaching chemically firm but relatively be easy to the advantage of derivation simultaneously sporopollenin compared to many markets.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 through 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 stable and toxic by-products is reduced to minimum advantage.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 kinds of 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, Greg T Hermanson, 1996 with two succinimidyl carbonates (disuccinimidyl carbonate) and triethylamine (triethylamine); 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 a novel method 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 through filtering, then water (10x100 cubic centimetre), EtOH (ethanol) (2x30 cubic centimetre) and DCM (dichloromethane) (2x30 cubic centimetre) flushing.Then with the sporopollenin dried in vacuum to constant weight.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 (2x250 cubic centimetre), EtOH (2x250 cubic centimetre), DCM (2x250 cubic centimetre) wash the primary amine sporopollenin then, then dried in vacuum to constant weight (1.8 gram).
According to a fifth aspect of the present invention, provide one gather amino functional exine.Also provide one and comprise the dosage form that gathers the amino functional sporopollenin that chemically combines the 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 through 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; Nucleoside (for example stavudine (D4T), acyclovir (acyclovir) and Qi Tafuding (AZT)) for example, can be easily with aromatic amines respectively activation become 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; For example refluxed 24 hours so that it filters at warp in toluene (10 milliliters), dimethyl sulfoxide (DMSOdimethyl sulphoxide) and the dimethyl formamide (DMF dimethyl formamide) at all kinds of solvents with sporopollenin (0.1 gram) respectively; Use toluene (2x10 cubic centimetre), 2M HCl (hydrochloric acid) (2x10 cubic centimetre), water (3x10 cubic centimetre), EtOH (2x10 cubic centimetre) and DCM (2x10 cubic centimetre) flushing then, and the bearing capacity after dried in vacuum to the constant weight is respectively 1.6,0.95 and 0.54 milli and rubs/restrain.
According to a sixth aspect of the invention, provide the exine of monocarboxylic acid functionalization.Also provide the dosage form that comprises the carboxylic acid functional exine that chemically combines the effective dose active substance.
One is used to form the method for drug-polymer conjugate, comprises through N-maloyl imines ester utilizing a N-maloyl imines and a 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 disclosed (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 through making above-mentioned gather 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 through 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 of adhering to the carboxylic acid function (figure 4)
A kind of glycine (0.1 gram) refluxed in DMSO 24 hours with the mixture of sporopollenin (0.1 gram).Through filter collecting sporopollenin, and with toluene (2x10 cubic centimetre), EtOH (2x10 cubic centimetre), 2M HCl (2x10 cubic centimetre), water (3x10 cubic centimetre), EtOH (2x10 cubic centimetre) and DCM (2x10 cubic centimetre) 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 of adhering to the monocarboxylic acid function
(Fig. 5)
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 through filtering, with toluene (2x10 cubic centimetre), EtOH (2x10 cubic centimetre), 2M HCl (2x10 cubic centimetre), water (3x10 cubic centimetre), EtOH (2x10 cubic centimetre) and DCM (2x10 cubic centimetre) flushing.With the sporopollenin dried in vacuum, the bearing capacity that it showed is that 1.7 millis rub/restrain then.Through 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).
Above process also can be used for the ethyl ester hydrochloride that the bearing capacity scope is 1.0 to 2.5 the millis bad amino acid of for example β-alanine, L-, α-L-alanine, aspartic acid, glutamic acid and the amidomalonic acid that rub/restrain.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 the polyhydroxy functionalization exine that chemically combines the 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.After the polymer stated 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 use succinimido carbonate and diaza cyclopentadienyl carbonate and p-nitrophenyl formates (p-nitrophenylformates) activation to become mesylate and toluene fulfonate.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 through 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 from a carbohydrate-derived drug-polymer conjugate can combine with an exine with produce one have a large amount of reactive hydrogen-oxygen groups conjugate and its have concordance chemically reaching form.Example 14 demonstrates a kind of method that can polyhydroxy link agent 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 through filtering, and with DMSO (2x10 cubic centimetre), water (100 cubic centimetres), EtOH (2x10 cubic centimetre) and DCM (2x10 cubic centimetre) flushing.Sporopollenin is dried to constant weight under vacuum, making its bearing capacity is that 1.7 millis rub/restrain.
Example 15: trihydroxymethylaminomethane (tris (hydroxymethyl) methylamine)-sporopollenin
Preparation (Fig. 7)
One programs similar with routine 14 use trihydroxymethylaminomethanes (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 (2x10 cubic centimetre) and DCM (2x10 cubic centimetre) flushing through filtering.With sporopollenin dried in vacuum to constant weight so that its bearing capacity be respectively 1.0 and 0.37 the milli rub/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 through 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.Through 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 through adding entry (10 milliliters) at 0 degree Celsius.This mixture was at room temperature leniently stirred 2 hours, and filtering and use acetone and ether flushing is the amino sporopollenin (insulin-succinylamido sporopollenin) of insulin-succinyl that 0.03 milli rubs/restrains to produce bearing capacity.
Above example shows how protein and enzyme are attached to sporopollenin and similar exine.Oligonucleotide, for example antisense oligonucleotide can adhere to through 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 combine sporopollenin through utilizing above-mentioned hydroxylation, 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 its orally-ingestible 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), whenever extracted blood sample at a distance from 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.Through calculating complete particle and collecting together the sum that calculates particle one number of fragments.These fragments are taken as and derive from same sporopollenin particle, but it possibly comprise the composition of several particles.
The microscope test result is following:
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 a small bundle of straw, etc. for silkworms to spin cocoons on that 10-20 fragment forms.
2b) use 30 minutes after:
Find 45 particles, wherein 7 is complete, and remaining is a fragment.
3a) use 60 minutes after:
3b) use 60 minutes after:
Find 20 groups of little fragments, do not have complete particle.
4) use 90 minutes after:
Observe 1 complete particle and 12 groups of very little fragments.
5) use 120 minutes after:
Observe a small amount of little fragment.
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 fragments.
2b) use 30 minutes after:
Find 4 complete particles and 35 fragments.
3a) use 60 minutes after:
Observe 1 complete particle and 17 minimum fragments.
3b) use 60 minutes after:
It is thus clear that 2 particles and 15 fragments.
4) use 90 minutes after:
Do not find particle or fragment.
The minimum scale that is got 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, the medicine total amount that is then got in the blood flow by 1 gram sporopollenin will be rubbed for 0.006 milli.
For the 0.03 milli sporopollenin that rubs/restrain that 1 gram physically or covalently is attached with insulin (M.W.6000), can make in the insulin entering blood flow of 11 milligrams of (0.018 millimeter) 30 ius (IU).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 get into blood flow in bearing capacity.This value obtains proof through 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 through 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, forming a kind of lipid conjugates, or a Polyethylene Glycol (PEG) is to form a PEG-lipid conjugates; In case the particle that adheres to arrives in the blood flow, can and medicine discharged more slowly through 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 reacts formation bromo sporopollenin with the bromine generation, makes itself and sodium azide reaction form the nitrilo sporopollenin that changes 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 is (Fig. 8) as follows.
According to an eighth aspect of the present invention, provide a halogen functionalization exine.Also provide one comprise the halogen functionalization exine that chemically combines the effective dose active substance dosage form.
Example 19: through 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 through filtration, and with the flushing of methanol (10x5 cubic centimetre) and ether (5 cubic centimetres), and under vacuum dry with produce the bromo sporopollenin (A,
Figure8).Sodium azide (1.25 grams, the 19.2 millis rub) solution that adding one is dissolved among the DMSO (30 cubic centimetres) also heats this mixture 48 hours with 60 degree Celsius.Through filtration, flushing, the dry (N that 1.8 millis rub/restrain
3) and with one contain lithium aluminium hydride reduction (0.2 gram) THF (15 cubic centimetres) mixture refluxed 1 hour, 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.Through filter and under vacuum dry amino (1 milli rubs/restrain)/AZT monophosphate amidated of cetyl (1 rubs/the restrains in the least) sporopollenin that removes gained (E, Fig. 8).
Bearing capacity is about the 1 milli sporopollenin that rubs/restrain and has the remaining hydrogen oxygen groups, like gained (route A among Fig. 9) in Fmoc (the fluorenes methoxy carbonyl acyl group) analysis.These functional groups can be used for directly or through 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, obtain bearing capacity after flushing and the drying and be 0.8 sporopollenin-acetic acid (route B among Fig. 9) of rubbing/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 through reagent
2,3,4, and through 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 following: DCM (20 cubic centimetres) and DMF (1 cubic centimetre) solution stirring 20 minutes that will contain DCC (0.5 milli rubs) and Fmoc glycine (1 milli rubs).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 in the solution that DMF (2 cubic centimetres) contains DMAP (0.1 milli rubs) then.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, combines the hydrogen-oxygen group to use p-maleimide isocyanates (p-maleimidophenyl isocyanate).Step with carbanil derivation sporopollenin is following: 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 is used SOCl
2, POCl
3Or PCl
5Can be easily by halogenation (route F among Fig. 9).This halogenated form of sporopollenin can be used for through a link agent and uses the further derivation polymer of a medicine subsequently.The halogenation of hydrogen-oxygen group is set forth as follows: 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 Cl).
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.PalumboBioconjugate 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 through 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 one comprise the thiol function exine that chemically combines the effective dose active substance dosage form.
Example 24: the adhering to of thiol group to sporopollenin (Figure 10)
(1 restrains, and 4.5 millis rub/restrain in DMSO (10 cubic centimetres), to contain the bromo sporopollenin with one; Obtain through the bromine reaction in sporopollenin and the acetic acid like method described above) refluxed 24 hours with the suspension of thiourea (60 millis rub).Collect gains through filtering, and with DMSO, water, 2M HCl, water, EtOH and DCM flushing.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 cryptogam
The adhering to of matter (Figure 11)
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 is quite complicated but do not know fully as yet in the structure that physically reaches the polymeric material that chemically is steady statue; 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 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 following:
Provide an exine through 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 adhering to of functional group carbon-carbon bond 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.With adding 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).The suspension of the B that places frozen water is used the 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 following 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 in DMF (20 cubic centimetres), contains sporopollenin malonic acid (C, mixing in suspension Figure 12).Add 0.1M aqueous KOH (27 cubic centimetres) then and with the gained mixture in down stirring 48 hours of 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 in digestive system, be decomposed like most sugars.Relevant sporopollenin/hydrogen phosphide should more be stablized and be easier to gold (I) mercaptides (gold (I) thiolate) is delivered in the blood flow with the treatment rheumatoid arthritis in intestinal portion with the complex of gold.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.In 0 degree Celsius slowly add one comprise (triethylphosphine) auric chloride (I) (1.1 milli rub) alcohol-water.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 (19)
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, this carrier is by selecting in the sporopollenin exine of plant, liverwort, mushroom or algae or its fragment; Optionally further have excipient; Be that the dosage form of form is selected from tablet, soft gel capsule, ovule, elixir, solution, outstanding agent with food or medicine wherein, the said preparation form is applicable to pulmonary administration or with the form administration of suppository, vaginal suppository, gel, hydrogel lotion, Emulsion, ointment, powder and patch; And wherein the sporopollenin exine comprises and is no more than 0.5% protein.
2. dosage form as claimed in claim 1, wherein the sporopollenin exine comprises and is no more than 0.1% protein.
3. dosage form as claimed in claim 1, its miospore is selected from the spore of following material: Herba Lycopodii (Lycopodium clavatum), bacillus subtilis (Bacillus subtilis), myosotis (Myosotis), aspergillus niger (Aspergillus niger), penicillium (Penicillium), little chanterelle (Cantharellus minor), Ganoderma (Ganomerma), the edge of a field mushroom (Agrocybe), Urtica dioica (Urtica dioica), black anthracene are spent mould (Periconia), are attached coccus (Epicoccum), fir (Abies), short Fructus Cucurbitae moschatae (Cucurbitapapo), Fructus Cucurbitae moschatae (Cuburbita).
4. dosage form as claimed in claim 3, its miospore are the spore of Herba Lycopodii (Lycopodium clavatum).
5. as the described dosage form of aforementioned any one claim, it is suitable for oral or through the skin administration.
6. dosage form as claimed in claim 5, wherein the said preparation form is suitable for buccal or sublingual administration.
7. dosage form as claimed in claim 1; Wherein dosage form is applicable to pulmonary administration and comprises the active substance of the effective dose that is used for pulmonary administration; And wherein active substance is contained in the seal; Select in its complete sporopollenin exine, or be contained in the intraparietal cavity of sealing by plant, liverwort, mushroom or algae; And optionally further have an excipient.
8. dosage form as claimed in claim 1, wherein dosage form is applicable to pulmonary administration, and wherein the particle size of carrier is in 1 to 100 micron the scope.
9. dosage form as claimed in claim 8, wherein the particle size of carrier is in 1 to 10 micron the scope.
10. dosage form as claimed in claim 9, wherein the particle size of carrier is in 1 to 5 micron the scope.
11. like the described dosage form of aforementioned claim 1, wherein active substance is selected from peptide or hereditary material.
12. like the described dosage form of aforementioned claim 1, wherein the sporopollenin exine is by functionalization.
13. like the described dosage form of aforementioned claim 1, wherein active substance is unstable or unstable to acid to alkali.
14. dosage form as claimed in claim 1, wherein active substance has low-solubility in water.
15. dosage form as claimed in claim 14, wherein active substance is metal or metallic compound.
16. dosage form as claimed in claim 1, wherein sporopollenin exine skin further is covered with a kind of material with auxiliary retentive activity composition.
17. the dosage form on pharmacopedics or the threpsology is used to treat the purposes of the medicine of disease in preparation; Wherein dosage form comprises the active substance of effective dose; Chemically or physically be incorporated into or be packaged in the carrier, this carrier is selected by sporopollenin exine or its fragment of plant, liverwort, mushroom or algae; Optionally further have excipient; And wherein the sporopollenin exine comprises and is no more than 0.5% protein.
18. purposes as claimed in claim 17; Be that the dosage form of form is selected from tablet, soft gel capsule, ovule, elixir with food or medicine wherein, the said preparation form is applicable to pulmonary administration or with the form administration of suppository, vaginal suppository, gel, hydrogel lotion, Emulsion, ointment, powder and patch.
19. purposes as claimed in claim 17, wherein utilize this by exine form can not be water-soluble medium carry this active component to get in blood flow through oral area, wherein this active component promptly got 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 | ||
| GB0411047.4 | 2004-05-18 | ||
| GB0411047A GB0411047D0 (en) | 2004-05-18 | 2004-05-18 | Dosage form |
| 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 CN1842325A (en) | 2006-10-04 |
| CN1842325B true 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) |
Families Citing this family (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 |
Citations (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 |
| CN1226900A (en) * | 1997-05-14 | 1999-08-25 | 农林水产省蚕丝、昆虫农业技术研究所长代表的日本国 | 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
Patent Citations (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 |
| CN1226900A (en) * | 1997-05-14 | 1999-08-25 | 农林水产省蚕丝、昆虫农业技术研究所长代表的日本国 | 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 |
Non-Patent Citations (2)
| Title |
|---|
| G.SHAW et. al..THE USE OF MODIFIED SPOROPOLLIENIN FROM Lycopodium clavatum AS A NOVEL IONOR LIGAND-EXCHANGE MEDIUM.REACTIVE POLYMERS.ELSEVIER SCIENCE PUBLISHERS,1988,第9卷(第2期),211-218. * |
| R. ADAMSON et al..New applications of sporopollenin as a solid phase support for peptide synthesis and the use of sonic agitaion.Int. J. Peptide Protein Res..1983,第22卷(第5期),560-564. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1842325A (en) | 2006-10-04 |
| ZA200510179B (en) | 2006-08-30 |
| GB0315019D0 (en) | 2003-07-30 |
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