WO2002041829A2 - Oral nanosphere delivery - Google Patents
Oral nanosphere delivery Download PDFInfo
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- WO2002041829A2 WO2002041829A2 PCT/US2001/043299 US0143299W WO0241829A2 WO 2002041829 A2 WO2002041829 A2 WO 2002041829A2 US 0143299 W US0143299 W US 0143299W WO 0241829 A2 WO0241829 A2 WO 0241829A2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
- A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
Definitions
- the present invention relates to improved pharmaceutical formulations for administration of macromolecules or drugs not bioavailable using standard delivery platforms. More specifically, the invention relates to pharmaceutical formulations of controlled release rate dosages that may be administered orally using nanospheres or like particles.
- Oral delivery of drugs or long acting parenteral dosage forms of drugs is preferred by the patient and physician because of improved compliance and the inherent beneficial effect of constant pharmacodynamic action.
- biological macromolecules e.g. proteins, polypeptides, and some complex carbohydrates
- GI gastro- intestinal
- their injectable forms have short durations of action, requiring frequent injections, making the products unsuitable for use in the non-hospitalized patient.
- bioactive molecules including many cancer chemotherapeutic agents, are not orally bioavailable or are poorly absorbed from the GI tract.
- Insulin was delivered orally to rats in 145 nm nanocapsules composed of isobutyl cyanoacrylate.
- Poly(alkyl cyanoacrylate) nanospheres for oral administration of insulin C. Damge et al.; J. Pharm. Sci. Nol 86, No. 12, pgs. 1403-9, December 1995.
- the insulin effect was absent or lasted less than 2 days unless an oily medium was used to administer the nanospheres, in contrast to the present invention.
- attempts to obtain effective insulin delivery in humans with cyanoacrylate nanospheres were not successful.
- biodegradable nanospheres were examined for the delivery of heparin. See.
- the present invention relates to nanoparticulates and/or spheres formulated to include a cancer chemotherapeutic agent, such agent as can be used in conjunction with a related method for delivery in patients with cancer and particularly those with metastatic cancer in the lymphatic system.
- a cancer chemotherapeutic agent such agent as can be used in conjunction with a related method for delivery in patients with cancer and particularly those with metastatic cancer in the lymphatic system.
- Such nanoparticulates can comprise therapeutic agents and the structural delivery components described herein, as well as those agents and/or components known to those skilled in the art made aware of this invention.
- the nanoparticulates can be formulated and prepared as described below. Oral administration at predetermined dosage levels can facilitate lymphatic delivery and uptake of those particulates suitably dimensioned.
- the desired therapeutic effect includes sustained release post-dosage and/or stable plasma concentrations of the subject agent.
- lymphatic delivery and sustained release can provide a unique benefit in the treatment of various cancers and malignant disease of the lymphatic system. As described below, such delivery and/or sustained release can be controlled by way of nanoparticulate formulation.
- the present invention can also include various methods of using nanostructural components to modify the plasma profile of a therapeutic agent post- dosage. Such methods are especially useful for those agents, typically macromolecules, which heretofore have not been available through oral administration. Such methods include providing nanoparticulate rods or spheres formulated for the desired therapeutic effect. As mentioned above, such formulations can include anti-cancer agents, but also macromolecules including, but not limited to, heparin. Regardless, as described more fully below, the structural composition of a nanoparticulate/sphere can be altered to extend controlled release and/or enhance stable plasma levels over time.
- the present invention includes nanoparticulate structural components comprising a therapeutic dose of heparin and a polymeric structural component designed to extend and/or sustain controlled release.
- the structural component is a co-polymer of lactic acid and glycolic acid. The amount of this co- polymeric system in the structural component can be used to affect controlled release and thereby modify the plasma profile of heparin post-dosage.
- the co-polymer is present in amounts sufficiently distinguished over the prior art to provide the surprising and unexpected results described herein.
- the present invention obviates the problems of solvation as well as the need for classic absorption for delivery of those drugs where the molecular size is too large to be absorbed when the product is formulated and administered, and/or because of the deleterious effects of gastrointestinal enzymes, and other factors.
- the invention is directed to pharmaceutical formulations for effective controlled release of many drugs not now orally available. These formulations, properly adjusted, also may be administered by inhalation and achieve similar kinetic characteristics.
- the pharmaceutical formulations of this invention are made by entrapping the macromolecule and/or drug of choice in either an organic or water phase biodegradable polymer system to produce nanoparticles. These nanoparticles can, optionally, be coated or combined with one or more bioadhesive adjuvants to promote adherence of the particles and their associated active material or medications to the intestinal wall.
- Two pharmaceutical formulation embodiments can be used to form the nanoparticles:
- drugs that are rugged and can withstand organic solvents are treated by entrapment in single or combinations of biodegradable polymers.
- non-organic solvents may be used, but it is preferred to entrap drugs that need a water-based system, with secondary attachment to a cyclodextrin.
- This entrapped drug nanoparticle complex may then secondarily be entrapped in liposomes.
- the drugs can first be entrapped into a cyclodextrin for protection, and then granulated with water soluble or organic soluble polymers.
- the invention is therefore suitable for using both organic and non-organic solvents.
- An advantage of the nanoparticulate/sphere system can be, under appropriate conditions, absorption by the lymphatic system.
- the enzymes or degradative influences present in the gastrointestinal tract do not affect such a system.
- Bioadhesive polymers can ensure a more prolonged duration of time in which the nanoparticles are in contact with the intestinal mucosa, reducing the deleterious action of heightened gastrointestinal peristalsis. All of these compounds work in harmony to produce viable products that have demonstrated favorable and reproducible effects in animals.
- the invention thus describes simple and predictable methods for the preparation of oral or inhalation dosage forms of a macromolecule, drug and/or therapeutic agent in biodegradable polymers. The following combinations are representative of those, which can be used to formulate such a controlled release orally administered pharmaceutical product, in accordance with this invention:
- a controlled release pharmaceutical formulation with a biologically active molecule which is formed into nanoparticles with biodegradable polymers and then coated with one or more bioadhesive adjuvants.
- a controlled release pharmaceutical formulation with a biologically active molecule entrapped in a cyclodextrin which is then formed into nanoparticles with biodegradable water-soluble polymers, and then coated with one or more bioadhesive adjuvants.
- the sphere size in the nanoparticle will typically be in the range of lOOnm to 2.0 microns, so that the active medication entrapped, may be administered orally or by inhalation.
- the exact diameter of the nanoparticles may not be critical in some instances, but is sufficiently small for cell diffusion by the lymphatic system.
- the rate of active drug release from the entrapped spheres is dependent upon the basic kinetics of the drug administered, the amount of polymer, the cyclodextrin used and, in the case of parenteral administration, the method of administration, the area of deposition, and the vascularity of the body region.
- inhalation dosing it is not necessary to use the bioadhesive adjuvants.
- the invention is thus directed to controlled release rate formulations of biologically active molecules, especially macromolecules.
- Any therapeutic drug, biological active protein or polypeptide, polysaccharide molecule, and the like, referred to generally herein as “bioactive molecule”, “bioactive material,” “biologically active molecule,” or “drug,” can be formulated according to this invention.
- more than one drug can be formulated together according to this invention.
- poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles are used for controlling the release rate of drugs from their matrix. These products are used for several reasons. 1. They are biodegradable and form non-toxic monomers.
- the release kinetics of the active moiety can be controlled by varying the amount and nature, as well as the molecular weight and inherent viscosity of the polymer or polymers used.
- the polymer matrix protects proteins and peptides against the destructive conditions observed when these products are given orally.
- sugars and hydrophilic compounds may increase the entrapment rate significantly. It is known that the sugars stabilize the proteins by preferential exclusion, subsequently the proteins interact more strongly with water than with solvents.
- the amount of sugars added to the mixture will depend upon the product, but in general are in the range of 3%-15% by weight.
- drugs Primary candidates for development into controlled release oral formulations are the widely used drugs, particularly those drugs with low bioavailability and drugs currently only capable of being administered via parenteral means. These include, but are not limited to, therapeutic proteins such as insulin, GM-CSF and G-CSF, erythropoetin, interferon alpha, interferon beta, interferon gamma and other cytokines, IL-2 and other interleukins, antibodies, and peptide hormones. Other candidates for oral controlled release delivery of the invention include antiviral agents, antibacterial agents, antifungal agents, and fertility hormones.
- therapeutic proteins such as insulin, GM-CSF and G-CSF, erythropoetin, interferon alpha, interferon beta, interferon gamma and other cytokines, IL-2 and other interleukins, antibodies, and peptide hormones.
- Other candidates for oral controlled release delivery of the invention include antiviral agents, antibacterial agents, antifungal agents, and fertility hormones.
- heparin a classic large molecular weight drug that has been considered the gold standard for effectiveness of new drug delivery systems for large polypeptide or carbohydrate drugs, and has previously been unavailable by oral means for controlled release over sustained periods.
- these drugs are not only absorbed orally, but their pharmacological effects are prolonged.
- Heparin is a well-established drug that is a complex polysaccharide that is highly charged (electronegative). It has a molecular weight of 20,000-60,000 daltons, and is covalently attached to a core protein found in most secretory cells. Heparin is a multifaceted drug with primary actions as an anticoagulant. When venous or arterial thrombi occur and the patient survives, heparin is given at doses of 10,000-20,000 units every 4-6 hours to maintain blood levels at 0.5-1.5 anti-factor Xa units/mL, which should prevent thrombus enlargement and alleviate the possibility of embolic phenomena. After this, the heparin dose is usually maintained at 10,000 - 15,000 units every 4-6 hours to prevent further thromboembolic events.
- heparin has the ability to bind to the arterial wall following angioplasty and ameliorate the proliferation of smooth muscle cells and ameliorate the restenosis that so often occurs with this procedure. Also, heparin is used as a preventive agent for those patients that are at close risk of stroke or heart attack as well as the patients recovering from a heart attack. Heparin also has a clearing effect in the blood by activating lipoprotein lipase on the cell surface. This action clears hyperlipoproteinemia and lowers the low-density lipoprotein. In animals, it has reversed the athrogenic deposits on the arterial walls, which is a phenomenon of arteriosclerosis in humans.
- the following compounds can also be used in conjunction with a nanodimensioned system for sustained release oral delivery: such compounds and/or materials including but not limited to anticancer drugs or chemotherapeutic agents, including 5-fluorouracil, paclitaxel and covalent derivatives, docetaxel, taxotere, doxorubicin, daunorubicin, epirubicin, methotrexate, leucovorin, cisplatin, carboplatin, cyclophosphamide, vincristine, vinblastine, vinorelbine, BCNU, CCNU, camptothecin and covalent derivatives, irinotecan (CPT-11), lurtotecan, 9-nitrocamptothecin, lipophilic camptothecin BNP 1350, 9-methoxycamptothecin, topotecan; monoclonal antibodies and antibody fragments; anti- NEGF antibody and fragments, anti-VEGF aptamer,
- ⁇ anospheres/particulates may be produced by methods known in the art.
- the active medicinal agent is dissolved in a suitable solvent such as buffered water or purified water which may contain a surfactant.
- a suitable solvent such as buffered water or purified water which may contain a surfactant.
- the biodegradable polymers are most often dissolved in organic agents such as dichlomethane, acetone, ethyl acetate and the like or combinations of these solvents.
- the aqueous solution of active is then combined with the organic phase with rapid stirring to form a water in oil emulsion.
- This emulsion is then dispersed in purified water or buffer, typically including a surfactant.
- This solution is stirred, homogenized or sonicated to form nanoparticles or nanospheres in the desired size range.
- Isopropyl alcohol may be added to harden the nanospheres at this point.
- the organic solvent may be removed from the nanosphere emulsion by reduced pressure or by addition of the solution to a volume of water in which the solvent is partially soluble, and the nanospheres recovered by filtration. Alternately, the emulsion may be dried completely and the product recovered. Spray drying may be employed to remove the water and organic solvent to produce a dry powder.
- Biodegradable polymers include: poly (lactic acid), poly (glycolic acid), poly (d,l-lactide-co-glycolide) (poly (epsilon-caprolactone), poly (3-hydroxybutyrate), poly (3-hydroxyvalerate), poly (ortho esters), polyanhydrides, dextran, cross-linked polyvinyl alcohol, polyhydroxy methacrylate, polycyanoacrylates, poly (phosphoesters), poly (hydroxy butyrate co- valerate), poly (2-hydroxyethyl glutamate), polyvinylpyrrolidone, poly (ethylene glycol), poly (propylene glycol), chitosan, pullulan, zein, alginic acid and alginate salts.
- methacrylate polymers can be used specifically in water-soluble systems. These include, but are not limited to acrylic acid, methacrylacetic cyanoethyl methacrylic, aminoakyl methacrylate copolymers, ethoxyethyl methacrylate copolymers, polymethacrylic acid, methacrylate acid alkylamide copolymer, polymethacrylic acid, polyacrylic acid, methacrylic acid alkylamide copolymer, polymethacrylic acid (anhydride), and polymethyl methacrylate.
- the advantage of the acrylate series is that when properly compounded they are water-soluble or can be supplied as a 30% aqueous solution with varying degrees of solubility.
- polymers known to the art can be used in either the water or organic phase, if properly prepared.
- An example of this is alginic acid in intimate admixture with the drug, then cross-linking the alginic acid with known inorganic elements such as divalent ions of zinc, magnesium, calcium, or sodium salts.
- pectin, zein, and guar gum can be used and cross-linked with a metallic ion.
- Ethyl cellulose can also be used as a polymer to entrap the molecule and can be used in an aqueous or organic solvent system. Similar examples are polyvinyl acid phthalate, cellulose acid phthalate, cellulose acid trimaleate phthalate and similar cellulosic polymers such as hydroxypropyl methylcellulose phthalate and the like.
- Cyclodextrins are a class of compounds derived from corn. They are cyclic, non-reducing oligosaccharides built up from six, seven, and eight glucopyranose rings known respectively as alpha, beta, and gamma cyclodextrins. In addition, hydroxypropyl and other groups have been and are added to the molecule giving each series its own specific characteristics and pharmacological behavior.
- the cyclodextrins act as a biodegradable trapping agent, incorporating the active medication into its core and forming a unique molecular inclusion complex. These complexes provide an anchoring compound without the formation of covalent bonds. This guest-host complex protects the active drug, while not forming tight chemical bonds, and the present invention demonstrates its usefulness in extending the release of drug from the nanospheres.
- cyclodextrins can be used to entrap the drug, providing a guest-host interaction.
- the type of cyclodextrins used will again depend upon the molecule and the degree of protection required. These parameters can easily be determined by routine experimentation by one of skill in the art.
- Preferred bioadhesive adjuvant(s) that can be added to formulations of this invention include hydroxypropyl methylcellulose, methylcellulose, pectin, guar gum, xantham gums, gum acacia, gum dragon, hydroxypropyl alginate, sodium carboxymethyl cellulose, carbomers, acrylic acid derivatives and those of similar pharmaceutical characteristics and behavior.
- These adjuvants are pharmacopeial items and are blended in with the nanoparticle granulate at the final stage of production.
- the preferred dose is a single or combination of these adjuvants at a 50/50 W/W ratio and a percentage weight of the total granulate of 0.1-3% preferably in the range of 0.3-2.0% and most preferably 0.2-1.2% weight percentage.
- the enhanced results and/or therapeutic activities demonstrated by the present invention can be attributed to one or more of several factors not specifically disclosed, taught or suggested by the prior art.
- factors include but are not limited to nanoparticulate formulations incorporating a cyclodextrin component, the benefits of which are illustrated in several of the following examples.
- Other such factors include heat treatment or drying of the nanoparticulates after incorporation of the desired therapeutic agent, and the concentration of such an agent relative to the nanoparticulate matrix, with lower relative active concentrations providing extended therapeutic levels as compared to the prior art.
- Figures 1 A and IB are photomicrographs of the nanospheres of Examples 7 and 8, showing clearly passage through lymph ducts and into a lymph node, respectively;
- Figure 1C shows nanospheres adhered to the intestinal wall. The nanospheres were stained with fluorescein.
- Figures 2 A and 2B are photomicrographs of heparin nanospheres (600-800 nm), the spheres prepared in accordance with this invention and the micrographs taken as shown.
- the following examples describe the preparation of several novel nanoparticle formulations of the present invention and their administration (e.g., orally) to achieve delivery of a variety of therapeutic agents in vivo, particularly to the lymph system.
- the present invention enables for the first time the oral delivery of therapeutic agents in vivo at significant levels and duration, such that the agents reach the lymphatic system using nanoparticle formulations which include only biocompatible components safe for use in humans.
- nanoparticle formulations which include only biocompatible components safe for use in humans.
- Embodiments of the present invention in addition to those illustrated, will be readily understood by those skilled in the art and are included within the scope of the invention, including novel processes and products derived from the invention whether as individual features or in combination with each other to produce novel combinations.
- the invention is illustrated further by the following examples, which are not to be taken as limiting in any way.
- Examples 1 and 2 illustrate various aspects of the present invention, as can be described or inferred from the concentration of heparin in rabbit plasma over time after oral dosing with heparin-containing nanospheres suspended in a bioadhesive adjuvant.
- the nanospheres comprised a 50/50 (w/w) mixture of heparin and a biodegradable polymer poly(d,l-lactide-co-glycolide), PLGA.
- the formulation contained beta cyclodextrin in the aqueous phase.
- PLGA-only formulations compared to formulations in the prior art using other polymers (either alone or in conjunction with PLGA), is noteworthy.
- PLGA-only formulations have several advantages including a history of use in humans (as injectable dosage forms).
- the use of PVA is an advantage over the prior art, which doesn't disclose use of a surfactant. Use of a surfactant can provide physical stability of the emulsion during the process.
- Nanospheres were formed from 1 : 1 (w/w) poly(d,l- lactide-co-glycolide) and heparin with the emulsion prepared in an aqueous solution of beta-cyclodextrin and polyvinyl alcohol. Scanning Electron Microscopy showed aggregates made up of smaller, roughly spherical bodies with diameters in the range 500-800 nm. Materials and a method for preparing nanospheres are detailed in Example 2, below.
- Doses of 200mg/kg, 400 mg/kg and 600 mg/kg (based on weight of the heparin nanosphere granulate) were administered by oral gavage in aqueous bioadhesive polymer adjuvant solution to 2 rabbits at each dose level.
- Plasma was sampled at intervals up to 552 hrs following the single dose.
- Plasma heparin levels were determined using a factor Xa chromogenic assay with a quantitation limit of about 0.1 U/ml.
- a therapeutic level (0.39 factor Xa units/ml) was achieved at 2 hrs post dosing.
- the lower doses produced a roughly similar pattern of increase of plasma heparin levels between day 6 and 10.
- the plasma level dynamics for the present heparin nanosphere preparation is in contrast to that in the prior art where the heparin levels did not reach full therapeutic values by 2 hours, and the high levels that were reached by one day were tapering off at 6 days when the study was concluded.
- the present example illustrates the ability to achieve significant heparin plasma levels by 2 hrs post dosing, and to sustain levels to 10 days. Such a result was achieved in part by adjusting the relative amount of PLGA in the preparation and the use of PNA. No guidance is available from the prior art as to how to achieve these effects.
- Heparin Nanosphere Preparation provides another heparin nanosphere preparation of the sort, which can be used to prepare other compositions of this invention. Specifically it provides a preparation which includes the heparin nanospheres described in Example 1.
- Nanosphere Preparation This example illustrates the production of another nanosphere formulation of the present invention, which has an advantage over the prior art in that it involves entrapment of a biological macromolecule (heparin) in a nanosphere using a water-based procedure (compare to prior art in which Red-Lake dye, not a biological macromolecule, was used).
- the agent to be entrapped in the nanosphere may be sensitive to organic solvent in which case a water/water method can be utilized. This involves dispersion of a water-soluble agent in a suitable fluid media and producing cross linking of the agent, to form microparticles.
- alginic acid (1-5%)
- a suitable amount of water blended with a high shear mixer until there is complete dispersion of the alginate.
- calcium chloride solution is added (1-8%) until distinct bead formation occurs.
- the speed of blending is increased to a maximum rate which produces smaller size particle, i.e.: nanoparticles.
- These beads are blended at high speed for approximately 8-10 minutes and at the end of the time, 6mL of isopropyl alcohol is added to the mixture to harden the beads.
- the beads are then dried in vacuo and examined for size and structure, by a scanning electron microscope. After the beads are dry they are then ready for use.
- Beta cyclodextrin was added to 20 ml of purified water and medium heat was applied with continued stirring, until the solution was clear.
- 500mg of hydroxypropyl methylcellulose K4MP was added and stirred until clear.
- 500mg of sodium dextran was added and stirred until clear.
- 2mL of ethyl acetate was added with 5mL of glacial acetic acid and 500mg of chitosan with constant vigorous stirring. After all these agents were well blended l,000mg of heparin was added and blended into the solution.
- zein a prolamine of corn and heparin are placed into lOOmL water and methanol. Stir at high speed until opalescence occurs, check for bead formation under microscope. When material is completely stirred, add into Pyrex ® pan and heat at 60°C for 24 hours. Scrape the material from the plate when dry and weigh; then check for reaction of excess with protamine. The final weight was 1.97g, a yield of 93.81%.
- the heparin nanosphere formulation of this example was prepared by dissolving the polymers listed in the table above in acetone and dichloromethane. When particles were completely dispersed, aqueous heparin was added until mixed. The mixture was then poured into lOOmL of aqueous 0.05M KH 2 PO 4 . The mixture was stirred until an emulsion formed, then turned at high speed; spherical particles were seen by optical microscope. A scanning electron microscope showed nanospheric formation.
- HPLC quality dichloromethane, (50mL) and HPLC quality acetone (50mL) were added into a i50mL glass beaker with a magnetic stirring bar.
- the polymers shown above were dissolved in this mixture and separately the heparin was dissolved in 10 ml water.
- the heparin solution was added to the polymer solution, and the mixture was slowly added to the phosphate buffer in a 800 mL vessel to which has been added the polyvinyl alcohol.
- the Silverson ® mixer was run at low-medium speed. As particles started to form the fluid turned a milky white. At this point the speed was turned to high levels and a drop of the solution was taken out and examined under an optical microscope.
- polyvinyl alcohol can be added to the organic phase if there is an obvious separation of materials.
- the yield was 742 mg out of a theoretical yield of 1,400 mg.
- the resultant powder/granulate was weighed and titrated against protamine in normal saline to ensure that heparin was in fact entrapped in the spheres. To do this, the beads were suspended in normal saline and agitated by vortex. Protamine was then added to the solution and no precipitate was observed. The nanoparticles were assayed before degradation and found to have 0.75 U/mg of heparin.
- nanoparticles were blended with bioadhesive adjuvants. Nanoparticles were coated by dispersion in 20-mL of an aqueous adjuvant containing 0.5% Carbopol-934P and 0.5% hydroxyproplylmethylcellulose.
- Preferred nanosphere preparations of the invention are formulated to enhance lymphatic uptake and increase periods of stable plasma concentrations.
- nanosphere preparations can be used for lymphatic delivery of a variety of therapeutic agents, such as chemotherapeutics.
- therapeutic agents such as chemotherapeutics.
- the results provided illustrate (with the corresponding figures 1 A, IB and 1C) lymphatic delivery and uptake of the sort efficacious in the context of various other therapeutic agents.
- Nanoparticles containing fluorescent stains were prepared and administered orally with concomitant bioadhesive adjuvants, as a single dose to anesthetized rabbits, via a gastric tube. The rabbits were sacrificed 7 and 14 days after oral administration of the nanospheres. Both utraviolet light microscopy and direct vision revealed dye-containing spheres widely distributed throughout the animals' bodies.
- Nanoparticle formulations comprising polystyrene have been shown in the prior art to be taken up in the lymphatics after oral administration. However, these formulations have not demonstrated delivery of a therapeutic agent for diseases involving the lymphatic system. Moreover, the prior art does not teach as to the need to achieve lymphatic delivery for orally administered formulations in order to achieve the long term controlled release effect demonstrated by the present invention. It should also be noted that polystyrene oral formulations have safety concerns for use in humans due to biocompatibility issues. The present formulations of the invention, which are made with PLGA and other non-toxic materials, demonstrate lymphatic uptake and long term controlled release of a therapeutic agent with a therapeutic effect on a disease involving the lymph system.
- Lymphatic uptake has also been demonstrated for microparticles of PLGA in the size range 1 - 10 micrometers. These formulations were shown to be advantageous for generation of an immune response to the encapsulated agent. Such an outcome is counter to the aims of the present invention wherein nanoparticles in the size range 100 nm to 2 micrometers are employed in one embodiment for delivery of therapeutics via lymphatic uptake and treatment of a disease involving the lymph system.
- Nanosphere Absorption A number of factors influence the absorption of the nanosphere from the gastrointestinal tract. The primary and most important element is the size of the nanosphere. It has been demonstrated that microparticles in the small intestine should be restricted to a size of less than 10 microns and preferably less than 5 microns. Kinetic studies in the prior art regarding the fate of microsphere within the gastrointestinal lymphatic tract (GALT), demonstrated that particles larger than 5 microns were not transported in the efferent lymphatics, while particles smaller than 5 microns were readily transported through the lymphatic system to the lymph nodes.
- GALT gastrointestinal lymphatic tract
- the present invention relates to nanaospheres having a size smaller than 2 microns and larger than 100 nanometers, as demonstrated by the photomicrographs of 800nm fluorescent stained spheres. These photomicrographs resulted from an in vivo experiment, where the spheres were placed with adjuvant into a rabbit ileal pouch and serial sections taken of the spheres progress, thorough the lymphatic pores down the lymph duct and into the lymph nodes. Reference is made to Figures 1 A and IB. The particles of larger size are maintained on Peyers patches in the intestine. ( Figure 1C). Most importantly material entrapped in the nanospheres are distributed throughout the reticuloendothelial system before reaching the vascular highway.
- Adjuvants A number of adjuvants were evaluated for mucoadhesion and use with the macromolecules and agents described herein. The strongest to the weakest of a few of the more prominent agents studied were carbopol ⁇ alginic acid ⁇ xanthan gum ⁇ pectin ⁇ cellulose gums. A combination of carbopol 974-P and methocel K4MP in pH 6.8 0.05M monophosphate buffer as the adjuvant also was used in preparing several nanosphere preparations of the present invention; for instance, in 10% carbopol 974-P, 10% methocel K4MP, suspended in buffer as described, or formulated into capsules or tablets as the dry- adjuvant in combination with a bioactive molecule.
- Nanospheres of the present invention can be formulated using the therapeutic agents, polymers and other structural components described herein, such as those listed in the table below, or by using other techniques and structural components known in the art.
- Powder Portion 5FU Ingredients mg %
- the following formulation was prepared as a tablet matrix. Items 1-5 in the table above were blended and granulated with a small amount of beta cyclodextrin. Carbomer 974 -P was then blended into the granulate. The acrylate polymers were blended together and granulated over the rest of the powder. When the granulate was hard and dry after being dried in a fluid bed dryer, magnesium stearate was added as a flow agent and a lubricant. The material was then put through a granulator at size 093 and pressed into tablets weighing 63 lmg with a hardness of 10-15kg. The disintegration is generally over 4 hours or more.
- the preparation was given to a patient with cancer and titrated up to tolerability or definitive clinical response.
- the preparation was given to one 37 year old white male with recurrent metastatic carcinoma of the testicle. He had metastasis to the lung, brain, and also had lymphatic enlargement. The physician treating the patient said he had about 2 weeks to live.
- nanoparticulate formulations systems of the type described herein can be used to effect bioavailability, including lymphatic delivery under the conditions and parameters described above.
- chemotherapeutic agents may be formulated into (encapsulated by) the nanospheres of the present invention for oral delivery and may be used as appropriate for the particular cancer to be treated.
- Nanosphere Formulation Encapsulating the Chemotherapeutic Agent Paclitaxel for Treatment of Metastatic Cancer in Humans and Other Mammals.
- Nanospheres are prepared encapsulating paclitaxel as follows. 2 gm PLGA (50:50), inherent viscosity .4 dl/gm, are dissolved in 50 ml dichloromethane. To this mixture is added 200mg paclitaxel and the solution is blended at high speed with a Silverson homogenizer until , homogeneous. The mixture is poured slowly into a beaker containing 400 ml water to which has been added 2% (w/v) PNA.
- aqueous oil in water emulsion then is homogenized at high speed until light microscopy of a sample of the mixture shows uniform droplets of the required small size.
- the mixture is dried in a spray dryer and the dry powder is collected. 200 mg of the paclitaxel nanosphere powder are blended with 100 mg each of Carbopol 934-P and Methocel, and the combination is filled into gelatin capsules.
- the resulting paclitaxel nanospheres may be used to treat orally animals and humans with cancer including breast cancer, ovarian cancer, lung cancer and other metastatic cancers against which paclitaxel has activity.
- Nanospheres for Oral Administration of Growth Hormone Releasing Factor (GRF to Humans This example demonstrates that oral administration of nanospheres of the present invention can be used to deliver therapeutic bioactive molecules in vivo in order to produce a biological response in humans.
- the prior art offers no guidance with respect to this therapeutic goal.
- Nanospheres encapsulating GRF were prepared as described in Example 14, using mannitol as a protective agent.
- the growth hormone (GH) plasma levels gradually decreased, showing suppression of the short and long feed back loop, and providing evidence of drug absorption by these individuals. No one experienced adverse side effects. All the subjects showed a slight weight gain and some increase in upper body strength. They were followed weekly for 8 weeks without adverse effects from a single dose of the hormone.
- Nanospheres were made using a protein fragment (polypeptide) of growth hormone.
- Human Test Subjects (a)-(d) were orally administered a single dose of GRF nanospheres.
- the growth hormone (GH) blood level for each subject appeared suppressed after oral dosing.
- Subject (a) had an initial GH blood level of 1.7 ⁇ g/mL, which was measured at less than 0.4 ⁇ g/mL after eight weeks.
- Subject (a) received 25mg of the nanospheres.
- Each of Subjects (b)-(d) received a lOOmg dose, and an initial GH blood level for each (1.6, 1.3 and 1.5 ⁇ g mL, respectively) became undetectable after eight weeks. Suppression of GH levels is expected upon achievement of sufficient levels of GRF in the plasma.
- Each Subject reported more strength and endurance post dosage. A weight gain was also observed for each Subject, but bodybuilding activities may be a contributing factor.
- GRF Nanosphere Preparation GRF Nanospheres as described above in Example 13 were formulated as follows:
- Cyclodextrin and GRF were added to the 200mL of acidified phosphate buffer and stirred by the Silverson ® method using medium speed. Next the PVA was added. In a separate flask the Resomer was solvated using a magnetic stirring bar and dichloromethane. When the polymer was completely dispersed, the protein cyclodextrin mixture was added and blended at a high speed. Isopropyl alcohol was added to the blended mixture. Using a Buchi laboratory spray dryer, the entire flask of liquid material was spray dried and collected in the cyclone collection flask. Scanning electron microscopy showed nanosphere formation
- Theoretical Weight 4,400 mg Actual Weight - 3,680 mg
- Insulin powder was placed into a beaker with cyclodextrin then phospholipids were added. This was placed into a beaker with aqueous Eudragit RS ⁇ 50mL.
- the material was administered to streptozocin-induced diabetic rats and blood levels of glucose were monitored.
Abstract
Description
Claims
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AU2002239279A AU2002239279A1 (en) | 2000-11-20 | 2001-11-20 | Oral nanosphere delivery |
CA002429254A CA2429254A1 (en) | 2000-11-20 | 2001-11-20 | Oral nanosphere delivery |
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US25207000P | 2000-11-20 | 2000-11-20 | |
US60/252,070 | 2000-11-20 |
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AU (1) | AU2002239279A1 (en) |
CA (1) | CA2429254A1 (en) |
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Cited By (8)
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WO2002036169A2 (en) * | 2000-10-31 | 2002-05-10 | Pr Pharmaceuticals, Inc. | Methods and compositions for enhanced delivery of bioactive molecules |
EP1774971A1 (en) * | 2005-10-14 | 2007-04-18 | Advanced in Vitro Cell Technologies, S.L. | Chitosan and heparin nanoparticles |
EP1834636A1 (en) * | 2006-03-08 | 2007-09-19 | Sahajanand Medical Technologies PVT. ltd | Compositions comprising porous articles and uses in implantable medical devices |
WO2008129106A3 (en) * | 2007-04-20 | 2009-03-05 | Inst Cientifico Tecnol Navarra | Nanoparticles comprising a cyclodextrin and a biologically active molecule and uses thereof |
WO2010015688A1 (en) * | 2008-08-06 | 2010-02-11 | Bioalliance Pharma | Oral formulations of chemotherapeutic agents |
US20110293657A1 (en) * | 2008-03-24 | 2011-12-01 | Moti Harel | Encapsulated vaccines for the oral vaccination and boostering of fish and other animals |
US8778384B2 (en) | 2008-03-24 | 2014-07-15 | Advanced Bionutrition Corporation | Compositions and methods for encapsulating vaccines for the oral vaccination and boostering of fish and other animals |
US9040664B2 (en) | 2003-04-11 | 2015-05-26 | Antriabio, Inc. | Materials and methods for preparing protein-polymer conjugates |
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US6117455A (en) * | 1994-09-30 | 2000-09-12 | Takeda Chemical Industries, Ltd. | Sustained-release microcapsule of amorphous water-soluble pharmaceutical active agent |
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2001
- 2001-11-20 WO PCT/US2001/043299 patent/WO2002041829A2/en not_active Application Discontinuation
- 2001-11-20 CA CA002429254A patent/CA2429254A1/en not_active Abandoned
- 2001-11-20 AU AU2002239279A patent/AU2002239279A1/en not_active Abandoned
Patent Citations (1)
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US6117455A (en) * | 1994-09-30 | 2000-09-12 | Takeda Chemical Industries, Ltd. | Sustained-release microcapsule of amorphous water-soluble pharmaceutical active agent |
Cited By (20)
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WO2002036169A2 (en) * | 2000-10-31 | 2002-05-10 | Pr Pharmaceuticals, Inc. | Methods and compositions for enhanced delivery of bioactive molecules |
WO2002036169A3 (en) * | 2000-10-31 | 2003-07-31 | Pr Pharmaceuticals Inc | Methods and compositions for enhanced delivery of bioactive molecules |
US6706289B2 (en) | 2000-10-31 | 2004-03-16 | Pr Pharmaceuticals, Inc. | Methods and compositions for enhanced delivery of bioactive molecules |
US9789196B2 (en) | 2003-04-11 | 2017-10-17 | Antriabio, Inc. | Site-specific insulin-polymer conjugates |
US9040664B2 (en) | 2003-04-11 | 2015-05-26 | Antriabio, Inc. | Materials and methods for preparing protein-polymer conjugates |
EP1774971A1 (en) * | 2005-10-14 | 2007-04-18 | Advanced in Vitro Cell Technologies, S.L. | Chitosan and heparin nanoparticles |
WO2007042572A1 (en) * | 2005-10-14 | 2007-04-19 | Advanced In Vitro Cell Technologies, S.L. | Chitosan and heparin nanoparticles |
AU2006301162B2 (en) * | 2005-10-14 | 2012-04-05 | Advancell Advanced In Vitro Cell Technologies, S.A. | Chitosan and heparin nanoparticles |
EP1834636A1 (en) * | 2006-03-08 | 2007-09-19 | Sahajanand Medical Technologies PVT. ltd | Compositions comprising porous articles and uses in implantable medical devices |
WO2008129106A3 (en) * | 2007-04-20 | 2009-03-05 | Inst Cientifico Tecnol Navarra | Nanoparticles comprising a cyclodextrin and a biologically active molecule and uses thereof |
JP2010524902A (en) * | 2007-04-20 | 2010-07-22 | インスティトゥト シエンティフィコ イ テクノロジコ デ ナバッラ,ソシエダ アノニマ | Nanoparticles comprising cyclodextrin and bioactive molecules and uses thereof |
US9522197B2 (en) | 2007-04-20 | 2016-12-20 | Innoup Farma, S.L. | Nanoparticles comprising a cyclodextrin and a biologically active molecule and uses thereof |
RU2460518C2 (en) * | 2007-04-20 | 2012-09-10 | Институто Сьентифико И Текнолохико Де Наварра, С.А. | Nanoparticles containing cyclodextrin and biologically active molecule, and using them |
US20110293657A1 (en) * | 2008-03-24 | 2011-12-01 | Moti Harel | Encapsulated vaccines for the oral vaccination and boostering of fish and other animals |
US8329209B2 (en) * | 2008-03-24 | 2012-12-11 | Advanced Bionutrition Corporation | Encapsulated vaccines for the oral vaccination and boostering of fish and other animals |
US8778384B2 (en) | 2008-03-24 | 2014-07-15 | Advanced Bionutrition Corporation | Compositions and methods for encapsulating vaccines for the oral vaccination and boostering of fish and other animals |
US9205151B2 (en) | 2008-03-24 | 2015-12-08 | Advanced Bionutrition Corporation | Compositions and methods for encapsulating vaccines for the oral vaccination and boostering of fish and other animals |
JP2011529950A (en) * | 2008-08-06 | 2011-12-15 | ビオアリアンス ファルマ | Oral formulation of chemotherapeutic agent |
EP2153821A1 (en) * | 2008-08-06 | 2010-02-17 | BioAlliance Pharma | Oral formulations of camptothecin derivatives |
WO2010015688A1 (en) * | 2008-08-06 | 2010-02-11 | Bioalliance Pharma | Oral formulations of chemotherapeutic agents |
Also Published As
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CA2429254A1 (en) | 2002-05-30 |
WO2002041829A3 (en) | 2002-07-18 |
AU2002239279A1 (en) | 2002-06-03 |
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