JP2648392B2 - Modified pollen grains for delivering biologically active substances to plants and animals - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to a new and improved carrier for delivering biologically active substances of various molecular sizes to plants and animals (typically humans). Or regarding shaped.
The present invention also relates to the long term release of these substances to appropriate target organs and bodies.

Background of the Invention Historically, many drugs have been developed to improve the development of drugs to their target organs with greater organ specificity, to release drugs over longer periods of time, and to provide specialized release characteristics. Methods have been developed. These controlled drug release methods are:
It usually requires the use of a carrier system to deliver the drug in a predetermined manner after it has been delivered near its target organ. These prior art carrier systems used a large number of substances, including a wide variety of lipids and phospholipids (liposomes), biodegradable and non-biodegradable polymers, mechanical devices, magnets, ultrasound, osmotic systems, and the like. . However, these systems have several disadvantages.

For example, none of these systems have the intrinsic ability to adhere to the target organ for a period of time sufficient to locally release the drug over an extended period of time. These systems do not promote the absorption of the drug or drug mixture contained therein into the bloodstream. Also, these systems load and release high molecular weight proteins and other macromolecules on these systems or devices, so that they can deliver the molecular size of biologically active substances that they can deliver. Is limited. Such systems also typically involve problems of incompatibility or reactivity of the carrier or excipient with the drug, potential environment and / or target organ or body.

OBJECTIVES OF THE INVENTION Accordingly, it is an object of the present invention to deliver biologically active substances such as compounds, drugs and other pharmacologically active materials that are less susceptible to such defects to target organs and bodies. To provide a system that can Another object is a delivery system that can deliver biologically active substances consisting of molecules from small to large (ie, proteins and other macromolecules) over time. It is to provide. Yet another object is to prepare and formulate a carrier or excipient in such a delivery system so that they can be released properly and, if desired, over an extended period of time. Is to provide a way. Other objects and advantages will become apparent from the description below.

SUMMARY OF THE INVENTION One or more of these objectives is to provide a pollen that incorporates a biologically active substance that is capable of being released to a plant or animal and that is foreign to naturally occurring pollen grains. This can be achieved by the present invention including compositions comprising granular porous cellulose grains. The present invention is directed to extracting at least a portion of a substance that can be extracted from naturally occurring pollen grains and releasing at least a portion of the extracted material from a biologically active pollen grain in a plant or animal. And a method for preparing the composition described above, which comprises The present invention partially modifies pollen grains by incorporating them with various biologically active substances, such as drugs, chemicals and other pharmacologically active substances. And then deliver it to a target organ, surface or site of a plant or animal to release the substances contained in the pollen grains. Molecules, especially large macromolecules, are usually absorbed into the blood stream of animals (e.g., humans) or reach the circulatory system without using pollen particles with biologically active substances as delivery vehicles. Can not do
The use of pollen grains is particularly useful for transferring molecules, particularly large macromolecules, into the blood stream of animals (eg, humans). Furthermore, when such pollen grains are to be administered to an animal, they are first defatted, then treated to remove antigenic substances (eg, proteins), and then mixed with a biologically active substance. Is preferred.

Thus, the present invention is directed to delivery vehicles for introducing biologically active substances, such as drugs, chemicals and other pharmacologically active substances, in or on plants and in the human or animal body. The invention relates to the use of modified pollen grains suitable for use as agents. Such pollen grains are suitable for delivering small and large (high molecular) molecules. Preferred pollen grains are those which have a rough or uneven surface which makes them more likely to adhere to tissue surfaces, especially mucous membranes. Pollen grains having barbed or irregular or fragmented surfaces are particularly preferred. A method of pretreating pollen grains to remove antigenic substances, a method of incorporating a biologically active substance into pollen grains, and a method of placing pollen grains thus pretreated in or on a plant or animal body Methods for formulating a formulation or dosage form suitable for introduction are also disclosed.

In general, the invention relates to the incorporation of a biologically active substance into the porous cellulose shell of pollen grains, wherein the substance is foreign to naturally occurring pollen grains,
A modified pollen grain is provided, which is characterized by being incorporated into the pollen grain, and in particular, being released later in a suitable environment.

The present invention uses such modified pollen grains to deliver biologically active substances to target flower beds or organs of plants and animals.

The invention also optimally provides for the controlled release of these substances over and over a period of time in or on plants and animals.

The present invention also provides a method of incorporating a biologically active substance into pollen grains, as described above.

DETAILED DESCRIPTION OF THE INVENTION Pollen grains have long been acting as delivery vehicles for naturally occurring genetic material and allergic proteins. They are in fact natural delivery devices for macromolecules as well as small molecules of the size of proteins and nucleic acids. The cross section of the pollen grains resembles a sponge and has a very large surface area. Also, most pollen grains are characterized by having a rough surface and adhering to the surface of mucous membranes and other target organs, optimally producing micro-surgical cuts. Utilizing these properties, various biologically active substances, such as chemicals, drugs and pharmacologically active substances, are incorporated into pollen grains, which are then planted (this term in its broadest sense) Used, trees, shrubs,
(Including grasses, flowers, fruit and vegetable plants, weeds, etc.) or animal target organs, where the substances contained in the pollen grains can be released. The bumpy surface of each pollen grain attaches to the tissue surface, especially the mucous membrane,
These pollen grains have been in contact with the target organ for a long time. Also, optimally, on the mucous membrane, the microsurgical cuts created by the bumpy and / or spiny surfaces of the pollen grains cause the substances contained therein to enter the bloodstream or circulatory system. Migration is enhanced. Therefore, the use of these pollen grains as delivery vehicles is limited to molecules, especially large macromolecules, since they cannot be absorbed into the bloodstream or reach the circulatory system without the use of pollen grains. In particular, it is particularly useful in transferring large macromolecules into the bloodstream. It is well known that molecules such as large allergic proteins can be released from pollen grains into the bloodstream, and naturally allergic proteins contained therein are released into the bloodstream, making it a sensitive solid. It is evident from causing an allergic reaction to.

Pollen grains have a specialized structure that houses sperm or male gametes of flowering plants. Pollen grains usually consist of two or three cells combined as a unit. Typically, each cell contains about 20% protein, 37% carbohydrate, 4%
% Lipids or natural oils and 3% minerals. The cellulose wall of pollen grains, which account for the remaining 36%, is extremely tough, due to hot concentrated acids and alkalis, prolonged destruction (as evidenced by fossilized pollen grains) and milling in a mixer. Is not decomposed. However, they are edible.

The Greer Laboratories Corporation of Lenoir N.
Pollen grains commercially available from C. are classified by origin: grass, weed, tree, shrub, flower (wild and cultivated strain) and cultivated plant. They are available as dried cereals with whole natural oils or as defatted cereals. As described above, pollen grains have a cross-section resembling a sponge and an extremely large surface area. Many are characterized by an irregular outer surface or shell, some of which are spiny. They are stable to temperature and chemical treatments within reasonable limits.

Ragweeds, Kaginoki (Broussonetia papyrifera)
And corn (Zea mays) are particularly preferred for the practice of the present invention. For example, ragweed has a barbed surface, which facilitates adhesion to mucous membranes and other target surfaces, creating a microsurgical cut there. It is spherical and is about 17-21 μm in size. However, many other pollen grains having suitable surface properties can also be used. Also, these properties are related to the shell, most preferably of a spiny or uneven nature (which can be determined by fractal geometry), whereby the pollen grains are transferred to the tissue surface. Especially on mucous membranes, causing prolonged contact and microsurgical cuts, more effectively releasing the biologically active substances contained therein and absorbing them into the bloodstream or target organs Is done. Thus, the following pollen grains with spiny shells are other preferred substances: Cocklebur (Xanthium commune), Goldenrod (Solidago species), Poverty weed (lva axillaris) Desert Ragweed (Ambrosia dumosa), False Ragweed (Ambrosia acanthicarpa), Giant Regweed (Ambrosia trifida), Short Regweed (Ambrosia artemsioli)
a), Slender Ragweed (Ambros
ia tenuifolia), Southern Ragwee (Southern Ragwee)
d) (Ambrosia bidentata), Western Ragweed (W
esternRagweed) (Ambrosia psilostachya), Prairie Sage (Artemisia iudovician)
a), Common Sagebrush (Artemisia tridentata), Annual Wormwood (Artemisia)
annua), Marsh Elder and High-Water Shrub.

To safely administer the pollen grains of the present invention to animals, especially humans, the pollen grains are first processed to remove antigenic substances. This significantly reduces the risk of allergic reactions or anaphylactic shock. Although all humans have been exposed to pollen grains, it will be seen that only those susceptible to its allergic inclusions will respond.

The method of the present invention comprises the steps of extracting the natural pollen grains, ie, dissolving and reacting with the components normally contained in the cellulose shell of the pollen grains and decomposing or degrading them.
A step of removing this component by treating with a substance to be downed, that is, performing an extraction step. The lipid, fat or oil component is usually extracted with an organic solvent, preferably ethyl ether, but any other suitable such as petroleum ether, acetone, hexane, benzene, toluene, kerosene, chloroform, carbon tetrachloride, diethylformamide, etc. Volatile organic solvents can also be used. The same components can also be removed, for example, by saponification, ie treatment with alkali. However, this degreasing treatment is well known, and defatted pollen grains are actually commercially available. When intended for use in plants, it is usually not necessary to remove the antigenic components from the pollen grains, and the porous cellulose shell of the pollen grains without further processing of any desired biologically active plant treatment substance. Can be blended. Any desired biologically active plant treatment material, such as herbicides, insecticides, larvicides, nematicides, pesticides, growth regulators, etc., is incorporated into the pollen grains, The loaded pollen grains can be delivered to the plant in any desired manner, such as aerial spraying, spraying, irrigation, or by natural pollination using bees or the like.

For animal use, delipidation is also usually required, as fat in pollen grains hinders the desired extraction or removal of the antigenic, proteinaceous and other components of the pollen grains. The remaining non-fat components of the pollen grains can likewise be extracted, preferably by treatment at room temperature and / or with hot water and / or alcoholic liquid, optionally alternating and / or continuously. it can. After continuous use of saline solution, preferably isotonic extraction solution at room temperature and high temperature, washing repeatedly with water and / or alcohol, drying and autoclaving at high temperature and pressure to completely denature the antigenic protein Preferably, the extracted pollen grains are sterilized. This treatment effectively removes almost all the original non-fat components of the pollen grains. Optionally, extraction with ethyl ether or another suitable volatile organic solvent as described above can be performed to remove selected non-fat components.

Example of pretreatment of pollen grains: Commercially defatted pollen grains are continuously extracted with isotonic saline (0.9% NaCl) for 12 hours. The pollen grains are then filtered and continuously extracted with hot isotonic saline for a further 12 hours (50-60 ° C). These extractions should be sufficient to remove any antigenic proteins or other foreign substances other than the cellulose shell. The ninhydrin test can be used to test for residual protein. Next, filter the pollen grains and use deionized water 2-3 times,
Wash 2-3 times with alcohol and dry (at 80-90 ° C) for 1-4 hours. Other organic solvents suitable for extraction include those described above. By drying in addition to removing excess water, the antigenic protein that has not been extracted is also denatured. The extracted and dried pollen grains are then autoclaved (15 pounds pressure and 121 ° C. for about 30 minutes) to further denature and sterilize. After cooling, drugs, chemicals or other biologically active substances can be incorporated into the pretreated pollen grains.

Example of a drug substance formulation: Extracted and dried pollen grains are placed in a vacuum chamber (about 50 mmHg) and the volatile solvent of a biologically active drug, chemical or other pharmacologically active substance or other A concentrated dispersion, such as a solution (preferred), suspension or emulsion, in a removable liquid carrier is introduced. The concentration of the material used depends on its nature, solubility and the amount of material per mg of material incorporated into the pollen grains, and can be about 5 to 40% by weight. Processing is usually about 5 to 50% by weight of the total composition, preferably about 10 to 30%.
% Of the biologically active substance, but not so much as to coat the surface of the pollen grains and effectively obscure the spiny or bumpy nature of said surface. After the biologically active substance is incorporated into the cellulose shell of the pollen grains, the pollen grains are dried and (when the substance is insoluble in water) with deionized water before being incorporated into the final dosage form for administration. Wash once.

Examples of preparation of the final form to be administered: The treatment of the pollen grains depends mainly on the stability and other properties of the drug, chemical or other biologically active substance used. As mentioned above, the shells of pollen grains are mainly composed of cellulose, which are stable to temperature and chemical treatment within reasonable limits. When the substance selected is sensitive to heat or high or low pH, treatment of the pollen grains avoids high temperatures, extreme changes in pH or any fluctuations that adversely affect the activity of the biologically active substance .

A wide variety of biologically active substances can be incorporated into the pollen grains of the present invention, as described below. They include simple and complex chemicals, drugs and other pharmacologically active substances.

 Examples of drugs include: Local anesthetics: xylocaine, eugenol, and the like.

Analgesics: ibuprofen and the like.

Antibacterials Antibiotics: neomycin, tetracycline, etc.

Antitumor agents: Fluoride (used for toothpaste).

Anti-inflammatory drugs: cortisone acetate, prednisolone, etc.

Antivirals Fragrances: cinnamon, clove oil, etc.

Biocides Cytotoxic drugs (for local antitumor treatment), mitomycin, etc.

Flavoring agents: menthol (during toothpaste) and the like.

Hormones proteins and peptides: insulin, calcitonin, etc.

Steroids Examples of biologically active substances for plant treatment include: Herbicides: 2,4-D, 2,4,5-T, TCDD, Alaclar, Amiben, Barben , Diuron, paraquat, propanil, propham and so on.

Insecticides: benzyl benzoate, organic phosphorus compounds, etc.

Larvicides: synthetic pyrethroids and the like.

Nematicides Pesticide: DDT, aldrin, benzene hexachloride, chlordane, dieldrin, heptachlor, methyl bromide, phosphine, rotenone and the like.

Plant growth regulators Precise dosage forms (tablets, suspensions, parenteral drugs, inhalants,
Creams) vary with the pharmacokinetics and pharmacodynamic properties of the drug. The release characteristics of a substance into a target body are very similar to first-order kinetic release and vary, in particular, with the solubility of this substance in the environment in question and the degree of access of the solvent to the pollen.

In addition, the pollen granules of the present invention may be of any type of formulation or dosage form for administration to humans or animals, such as aerosols, buccal and sublingual tablets, oral tablets, capsules, caplets, surgical implants, lozenges ,
It can be incorporated into nasal sprays, creams and ointments, injectables, parenteral drugs, transdermal patches, solutions, mouthwashes, nasal drops, dental floss, and the like. They can also be provided with an enteric coating.

They can also be used to introduce substances into or onto plants by pollination.

The following examples illustrate the schemes considered the best for carrying out the invention. Unless otherwise indicated, all amounts and percentages in the specification and claims are by weight and temperatures are in ° C.

Example I (a) 100 g of pollen grains first in a hot air open at 55-60 ° C
And dry for 24 hours. (B) Next, the dried pollen grains are degreased by extracting with anhydrous ethyl ether for 12 hours using a magnetic stirrer. 1 g of each pollen is extracted with 50 cc of ethyl ether. This process is repeated 2-3 times at room temperature. (C) Next, the defatted pollen was extracted again with a magnetic stirrer, again for 12 hours at room temperature using 100 ml of a normal physiological solution (isotonic saline, 0.9% NaCl) for every 2 g of pollen. And extracted with hot isotonic saline.
The separation in steps (b) and (c) is performed by simple filtration in vacuo through a 0.45 millipore filter. (D) Next, the pollen grains are washed 2-3 times with deionized water and 95% alcohol to desalinate the pollen grains. Then
This is dried in a hot air oven at 90 ° C for 2-4 hours.
(C) Next, the extracted, washed and dried pollen grains are
After autoclaving at 15 ° C. at 15 ° C. for 30 minutes, transfer to a vacuum desiccator (about 5 mmHg) and store at room temperature for 24 hours before incorporating the biologically active substance into the pollen grains. This step (e) and the previous drying step, individually and collectively, denature any residual protein and make it sterile. The compounded material is then dissolved, suspended or emulsified in a suitable, usually volatile, aqueous or non-aqueous solvent or other liquid carrier to produce a concentration of typically 10-20%. Add the solution, suspension or emulsion to the pollen. The incorporation of the substance into the cellulose shell of the pollen grains takes place under vacuum at a pressure of 5 mmHg (about 1 hour). The pollen carrier is then dried (at 45-50 ° C for 24 hours or at lower temperatures for heat sensitive drugs such as proteins).
24 hours) The pollen carrier can then be introduced into a suitable formulation or dosage form to release the substances contained therein over a prolonged period of time.

Example II 100 g of ragweed pollen grains were treated with anhydrous diethyl ether.
Degreasing using 500 ml 3 times in succession. Dry the defatted pollen at room temperature until the ether odor disappears (6-24
time). The pollen grains are then extracted for 12 hours in a continuous extraction device using isotonic saline (2-3 liters). This process is repeated with hot (50-60 ° C) isotonic saline for an additional 12 hours. Next, the pollen grains are filtered, washed three times with 500 ml each of deionized water, subjected to the same treatment using anhydrous alcohol, and then dried in a hot air oven (85 to 90 ° C, 1 ° C).
~ 4 hours). The pollen grains are then autoclaved (15 pounds pressure, 121 ° C. for 30 minutes). After cooling, the pollen grains are placed in a vacuum chamber with a 20% solution of insulin in water. After a good vacuum is obtained (about 50 mmHg), the insulin solution is mixed well with the pollen grains. After reaching equilibrium, the vacuum is released. The pollen grains are then filtered and lyophilized at low temperature and low pressure (temperature below 0 ° C. and pressure below 50 mm Hg) to remove excess liquid. Once all the liquid has been removed, the pollen grains are ready for use.

Example III Tablets consist of the following ingredients: Ingredients: Avicel Ph.102 (carboxymethylcellulose and microcrystalline cellulose) 70 mg Cabosil (silica) 0.2 mg Magnesium stearate 0.5 mg Pollen drug carrier 10 mg Total with mannitol To 100 mg.

Preparation: Tablets mix the ingredients, directly compress the mixture to 100m
Prepared by making tablets of g. Each tablet disintegrates in less than 20 seconds and contains 2 mg or 48 units of insulin. Pollen drug carriers are prepared by adding porcine, a soluble insulin at a concentration of 20 mg / ml to 80 mg of pollen pretreated to a buffered aqueous solution at pH 7. The pretreatment (extraction) of the pollen grains and the incorporation of insulin therein are carried out according to the treatment methods disclosed in Examples I and II above. The water is then removed by freeze-drying (freeze-drying) or vacuum evaporation (0.5 mm Hg and 10 ° C.).

In general, the present invention relates to a composition comprising pollen grains modified by incorporating a biologically active substance into the pollen grains, the substance being releasable in or on a plant or animal. And is different from naturally occurring pollen grains. The present invention is a method for modifying pollen grains,
It also relates to a method for enabling delivery of a substance that is different from pollen grains naturally present in or on plants and animals. This method generally defattes pollen grains,
At least a portion of the extractable substances normally found in pollen grains are extracted by solvent means and modified by incorporating a substance into the defatted and extracted pollen grains, and at least one of these pollen grains is modified. A portion of the compound in a form suitable for introduction into or on a plant or into a human or animal body. It should be noted that when using pollen grains in plants, a defatting step may not be necessary. In such a case, the extraction step provides a space for the extractable material commonly found in naturally occurring pollen grains for the amount of biologically active material incorporated into the pollen grains. Only needed to remove a sufficient amount or volume.

It will be understood that the foregoing description, completed by way of example, is merely illustrative of the invention and its principles. However, many modifications and variations of the details of this disclosure will occur to those skilled in the art to which this invention pertains and remain within the scope and principles of the invention. For example, an exemplary embodiment of the present invention deals primarily with the incorporation of a drug into pretreated pollen grains. However, it is clear that the principles of the present invention can be applied to the formulation of other drugs, chemicals and the biologically active substances described above. In a broad sense, the principles of the present invention apply to the use of modified pollen grains to deliver a substance incorporated therein, wherein the substance is heterogeneous to naturally occurring pollen grains, It is formulated for special purposes which are later released in a suitable environment. More specifically, the present invention is specifically directed to the use of modified pollen grains to deliver biologically or pharmacologically active substances to target flower beds or organs of plants, humans or animals. Applied. The invention has application to providing a controlled release of the formulated substance over an extended period of time as well as a method of incorporating the substance into pollen grains. Therefore, in essence, the present invention provides
It relates to providing a "reservoir" for the controlled release of drugs, chemicals and other biologically or pharmacologically active substances into plants and humans and animals over a long period of time. Finally, the present invention preferably removes at least some naturally occurring extractable substances from defatted pollen grains, and then replaces them with drugs, chemicals and other pharmacologically active substances. It is applied to the administration of these pollen grains to plants, humans or animals.

Although the present invention has been disclosed in terms of a preferred embodiment, those skilled in the art can make changes and modifications which fall within the spirit and scope of the present application and claims. Will be understood.

Claims (13)

(57) [Claims] Claims: 1. A method for releasing a biologically active substance, comprising a porous cellulose pollen kernel and a biologically active substance, wherein the pollen kernel is used as a carrier or excipient. A composition comprising 5 to 50% by weight of said biologically active substance, capable of releasing said biologically active substance into or on a plant or animal, said substance comprising: A composition that is different from naturally occurring pollen grains. 2. The method according to claim 1, wherein the pollen grains are defatted.
The composition according to Item. 3. The method according to claim 1, wherein said biologically active substance is a plant growth regulator, a pesticide, an anesthetic, an analgesic, an antibacterial, an antibiotic, an anti-cariogenic agent, an anti-inflammatory agent, or an antiviral agent. Claims 1 to 4, comprising a fragrance, a biocide, a cytotoxic agent, a flavoring agent, a hormone, a protein and a peptide, an insulin, a steroid or a mixture of two or more thereof.
The composition according to Item. 4. The composition according to claim 3, wherein said biologically active substance is a pesticide which is a larvicide, a nematicide or a herbicide. 5. The method according to claim 5, wherein said pollen grains are ragweed, linden, corn, onyx fir, goldenrod, poberty weed, mackerel ragweed, false ragweed, oobtaxa, kogatabakusa, slender ragweed, southern ragweed, western ragweed, prairie sage common, 2. The composition according to claim 1, which is a pollen grain of brush, annual warm wood, marsh elder or high water shrub. 6. The method according to claim 1, wherein said porous cellulose cereal comprises 10% to 30% by weight of a biologically active substance.
A composition according to any one of the preceding claims. 7. A composition suitable for introduction into an animal, comprising an aerosol, an oral tablet, a capsule, a caplet, a surgical implant, a lozenge, a nasal spray, a cream, an ointment, an injection, a parenteral drug, a parenteral drug. A composition comprising a composition according to any one of claims 1 to 6, in the form of a skin patch, solution, mouthwash, nasal drop, dental floss. 8. A method for producing a composition comprising a modified pollen grain for delivering a biologically active substance different from pollen grains naturally present in or on a plant or animal, comprising: Defatting the pollen grains, extracting at least a portion of extractable substances commonly found in pollen grains, and extracting the defatted and extracted pollen grains with biologically active substances Modifying by incorporating it into the extracted porous cellulosic cereal. 9. The method of claim 1, wherein the biologically active substance incorporated in the pollen grains is dispersed in a suitable aqueous or non-aqueous solvent. 9. The method according to claim 8, comprising incorporating the substance into the pollen grains in addition to the cellulose grains and drying the pollen grains. 10. The method according to claim 9, wherein the concentration of the biologically active substance in the suitable solvent is 5 to 40%.
The method described in the section. 11. The method according to claim 11, wherein said biologically active substance is an anesthetic,
Analgesics, antibacterials, antibiotics, antineoplastic agents, anti-inflammatory agents,
11. The method according to claims 8 to 10, which is an antiviral agent, a fragrance, a biocide, insulin, a cytotoxic agent, a flavoring agent, a hormone, a protein and a peptide, a steroid, a plant growth regulator, or a pesticide. A method according to any one of the preceding claims. 12. A method for preparing pollen grains containing a biologically active substance, comprising mixing a biologically active substance different from pollen grains naturally present in porous cellulose grains of the pollen grains. A method, comprising: 13. The method according to claim 13, wherein said pollen grains are formulated in a form suitable for introduction into an animal, said form being an aerosol, an oral tablet,
Capsules, caplets, surgical implants, lozenges, nasal sprays, creams, ointments, injectables, parenteral drugs, transdermal patches, solutions, gargles, nasal drops or tental floss. A method according to any one of claims 8 to 12.