Targeted delivery of bioactive factors to the systemic skeleton

Tag : glass desiccator


Abstract
The invention provides methods and compositions for the delivery ofbioactive factors to the systemic skeleton. The methods of theinvention allow targeted delivery of bioactive factors to boneusing nanocapsules. Timed release of bioactive factors may also beused to increase the efficacy of treatment. The methods of theinvention have wide applicability for the treatment or preventionof bone-associated maladies.
Description of the Invention
The invention overcomes the deficiencies of the prior art byproviding compositions comprising nanocapsules for the targeteddelivery of therapeutic agents to the systemic skeleton. Thecompositions and methods of the invention can be used for thedelivery of potentially any bioactive factor to bone. The bioactivefactors are delivered in nanocapsules comprising a payload of thebioactive factor and that are targeted for specific delivery tobone. The nanocapsules can be delivered non-invasively as atherapeutic or as a countermeasure designed to prevent developmentof bone abnormalities. The invention has application for a varietyof conditions associated with bone loss, bone abnormalities or bonedamage including, but not limited to osteoporosis, osteoarthritis,Paget's disease, osteohalisteresis, osteomalacia, periodontaldisease, bone loss resulting from multiple myeloma and other formsof cancer, bone loss resulting from side effects of other medicaltreatment (such as steroids), bone loss resulting from fractures,and age-related or weightlessness-related loss of bone mass.

By allowing targeted delivery of bioactive factors, the inventionallows localized delivery of the bioactive factors at the sitewhere the factors are needed, e.g., to the affected bone matrix.Localized delivery increases the potency without requiring anincrease in the dosage, and also reduces side-effects incurred bysystemic exposure. The nanocapsules of the invention can also bedesigned for controlled or triggered release of payloads, forexample, by capsule degradation and payload diffusion or inresponse to external signals or physiologic signals occurring thebone microenvironment. This allows therapeutic release specificallyto sites where treatment is needed. Targeted and/or timed deliveryof bioactive factors also allows administration of a lower overalldose of the bioactive factor to the patient, minimizing thepotential for adverse side effects due to narrow therapeutic-toxicwindows.

The nanocapsule drug vehicles can be targeted by fabricating thenanocapsules to contain both surface-bound bone-specific targetingligands and payloads comprising bioactive therapeutic agents,compounds or countermeasures. Such surface-bound bone targetingligands can specifically target the bone mineral phase. Examples oftargeting ligands that may be used include bisphosphonates andoligopeptides, which have been shown to preferentially bind tobone.

Targeting to bone can also be achieved by surface-functionalizingnanocapsules with hydroxyapatite (HAp) binding residues (e.g.,bisphosphonates, peptide residues, etc.). Therefore, thenanocapsules can be comprised of targeting ligands, a membranecomponent and a therapeutic payload. The targeting ligands can beattached to a nanocapsule membrane and can selectively bind totargeted sites within the systemic skeleton.

One application of the invention is in the delivery of bioactivefactors to maintain skeletal health by preventing bone loss. Suchfactors may prevent bone resorption, for example, to treatosteoporosis or as a maintenance program for the prophylacticprevention of bone loss. Such prophylactic treatment may find use,for example, in preventing bone loss during manned spaceflight.Bone mass loss is also a growing problem for the rapidly agingpopulation, and could be prevented by administration of thetargeted nanocapsules provided by the invention. One such bioactivefactor that may be used is transforming growth factor beta(TGF-.beta.), which activates cell proliferation and metabolicpathways in osteoblast-like cells in vitro. Other non-limitingexamples of bioactive peptides that may be used are othersub-classes of the TGF-.beta. family of peptides and the bonemorphogenetic proteins. Still other bioactive factors that may beused are compounds that stimulate expression of bone morphogeneticprotein 2. Other non-limiting examples of such BMP-2 expressionstimulators are statins. All these molecules are well known in theart.

Specific targeting of nanocapsules also allows targeting ofskeletal structures, especially areas of high bone turnover (e.g.,areas undergoing active resorption due to disease or non-loadeduse). The targeted nanocapsules can also be used to delivery otherselected therapeutics to bone, including, for example, treatment ofinfection or tumors in bone via antibiotics or cancer therapies,respectively. Similarly, the nanocapsules may also find use offracture repair therapies and tissue engineering applications.

I. Targeted Delivery of Bioactive Factors

A. Proposed Countermeasure Methods

One aspect of the invention is set forth, for illustrative purposesonly, in FIG. 1 (see Original Patent). In this approach, abioactive factor is incorporated into a nanocapsule vehicle. Thenanocapsule is designed to specifically target bone, for example,by targeting the hydroxyapatite (HAp) component of the skeletalmatrix. In vivo, the targeted nanocapsules preferentially bind toexposed HAp surfaces (e.g., especially bone matrix locationsundergoing osteoclastic resorption), whereupon they subsequentlydisrupt to release the therapeutic payload contained in thenanocapsules.

In a second approach, the nanocapsules may be designed to releasetheir therapeutic payload temporally in response to a specificstimulus. This stimulus could be an externally applied signal, acomplementary factor administered in schedule, or may be abiochemical signal present in the bone microenvironment. In thisway, delivery can be made at locations where needed or otherwiseappropriate. If the nanocapsules are not exposed to the appropriatesignal or factor, the nanocapsules remain intact and are eventuallyexpelled by the body through normal metabolic activities.Therefore, any side effects associated with the bioactive factor(s)contained in the nanocapsules may be avoided when treatment is notneeded. Still further, lower effective doses of the bioactivefactor in the locally affected bone microenvironment will bereceived by the patient when the treatment is needed.

The bioactive factor could be one or more of many agents that havebeen shown to impact the formation of new bone matrix (e.g., BMPs,protein fragments, statins, estrogens, molecular conjugates, etc.)or to have any other desired therapeutic or preventative effectwith respect to any bone-associated malady. As indicated, thebioactive factors are encapsulated in nanocapsules (for example,liposomes, niosomes, self-assembled molecular cages, etc.) that maybe designed to have controlled temporal release in the appropriatephysiological environment. Some examples of bioactive factors thatcould be delivered with the invention include insulin-like growthfactors (IGF), bone morphogenetic proteins (BMP), heparin-bindingfibroblast growth factor (FGF), platelet-derived growth factors(PDGF), TGF-.beta., parathyroid hormone (PTH), fluoride, andstatins.

The nanocapsules could be introduced systemically by intravenousinjection or non-invasively by intranasal uptake, or topicalapplication. In vivo, the nanocapsules would preferentially bind toexposed HAp surfaces (e.g., especially bone matrix locationsundergoing osteoclastic resorption), whereupon they wouldsubsequently disrupt to release their therapeutic payload.

B. Bone Loss

As described above, one application of the methods of the inventionis in the prevention or treatment of bone loss. The aging globalpopulation translates to ever-increasing demand for suchcountermeasures to skeletal deterioration resulting from theincreasing fragility of skeletal structures with age. Demographictrends in the United States, Europe and Japan are similar, with thepercentage of the population over the age of 65 increasingdramatically (FIG. 2 (see Original Patent)) (see, e.g., USDepartment of Health and Human Services, Administration on Aging,www.aoa.dhhs.gov). This underscores the importance of theinvention. Maintaining or restoring bone volume is therefore amajor health care issue for an increasingly vast segment of thegeneral population (see, e.g., Trends in Orthopedics, 2000;Orthopedic Industry, 2000). The need for a therapeutic protocolthat targets the systemic skeleton to maintain and/or increase bonevolume cannot be overstated.

Yet another application of this technology is with regard tofracture healing and prevention of bone loss during fractures.Targeted delivery of bone healing agents, such as anabolic agents,to the bone can minimize the time of bone-healing and also preventloss of existing bone tissue. In addition, the invention iscontemplated useful in the process of prosthetic fixation.

Another possible source of bone loss which could be treated withthe invention is spaceflight. Astronaut health during long-termspace flight is a major concern and central to this general healthconcern are the effects of space flight on skeletal tissues.Skeletal degradation can dramatically affect the ability to performboth rudimentary tasks and critical extravehicular activitiesduring long-term space missions. Astronauts experience a 1-2%decrease in bone volume per month at selected skeletal sites andthis bone loss is generally not fully recovered on return to Earth(National Geographic, January, 2001; Vico et al., 2000). This rateof bone volume loss could cause decreases in bone mineral density(BMD) of more than 50% during a 2-3 year Mars mission,significantly impairing an astronaut's abilities during spaceflight and on entry into gravitational environments. The consensusis that bone loss countermeasures are necessary for continued spaceexploration.

Knowledge of human adaptation to long-term space flight lags behindthe technical knowledge required for such travel (Turner, 2000).Experience in near-earth space flight suggests that most biologicaleffects on the skeletal system result from changes in physicalloading of the skeleton. This stems from the fact that an astronautin near-earth orbit, though still within the gravitational field,experiences free-fall as an adjunct of spacecraft velocity. Changesin bone biology and the associated loss in bone volume begin tooccur within a few days after leaving Earth. MIR space stationstudies clearly show that individuals experience decreases in BMDin load-bearing areas such as the lumbar spine, proximal femur, andcalcaneus, while non-load-bearing areas, such as the cranium,distal radius, and ribs, experience increases in BMD (McCarthy etal., 2000). Experiments have shown a decrease in the expression ofselected bone matrix cytokines (Carmeliet et al., 1998), such asTGF-.beta. and insulin-like growth factor-1 (IGF-1), both of whichare known to regulate bone formation (Mundy, 1996). Similarfindings have been reported in cell culture studies whereinosteoblast activity and associated deposition of new skeletalmatrix both decrease. Thus, it is clear that the normal boneremodeling process is profoundly altered during space flight.Osteoblastic bone formation decreases and osteoclastic resorptionactivity either remains unchanged or slightly increases. The netresult is the onset of osteopenia (Holick, 2000).

Infusion of IGF-1 stimulates bone growth in normally loaded bonesbut in unloaded bones, an extremely high dose of 2 mg/kg/day isrequired to demonstrate any protective effect against unloading(Bikle et al., 1994). Decreases in TGF-.beta. message levels havebeen observed in three different models of skeletal unloading:spaceflight, sciatic neurotomy, and hindlimb unloading (Westerlindand Turner, 1995). These results arc consistent with a growing bodyof evidence suggesting that reduced bone formation duringspaceflight is due to decreased osteoblast function (Harris et al.,2000). Significantly, infusion of TGF-.beta. (2 .mu.g/kg/day)corrects the decrease in bone mass, calcium content, osteoblastnumber and mineralization rate induced by hindlimb unloading inrats, but has no effect on bone formation in control animals.Further, TGF-.beta. infusion decreases the indices of boneresorption in both normal and unloaded rats (Machwate et al.,1995). Effective targeting and delivery of TGF-.beta. to increasedconcentrations locally in the bone microenvironment would be adesirable goal of any drug delivery countermeasure (Mundy, 2000).

C. Targeted Delivery of Nanocapsules

Targeted delivery of therapeutics via nanocapsules can occur byeither passive or active mechanisms. Passive targeting occurs whennanocapsules extravasate through damaged vasculature to accumulatein tumors and inflamed tissues (Wu et al., 1993). Accumulationincreases by improving circulation half-life and by preventingnanocapsules interaction with serum components. In contrast, activetargeting is achieved through specific interaction betweennanocapsule-bound or -associated ligands and complementary bindingagents at the targeted site. This approach has clear opportunityfor improved, efficacious delivery of biactive agents, since manydo not target bone, have narrow therapeutic-toxic windows whenadministered systemically, and require close proximity to targetcells to exert their biological activity.

Prior techniques employing liposome-based targeting approaches haveused one of a handful of methods, including receptor targeting,cell adhesion molecules, extracellular matrix molecules, selecting,and antibody ligands (Forssen and Willis, 1998). Lee and Huang(1995) demonstrated a 45-fold increase in doxorubicin uptake fromfolate-modified liposomes in to epithelial cancer cells, whichover-express folate receptors. Kamps, et al. (1997), demonstratedthat liposomes modified with anionized albumin were taken up byhepatic endothelial cells, whereas nearly all non-modifiedliposomes remained in circulation 30 minutes post-injection.

Bone offers several potential sites for targeted delivery ofbioactive agents. Bone is a composite matrix comprised of organicand inorganic constituents. The organic portion of the matrixconsists of a mixture of collagen, bone proteins, water, and cells(Rho et al., 1998). The inorganic portion of the matrix consistschiefly of hydroxyapatite (HAp). While it is possible toselectively bind bioactive constituents to specific receptorslocated either on bone cells or on bone proteins, such targetingmay not be sufficiently specific. Similar receptors may exist onother cell phenotypes and many bone proteins can be found externalto the bone matrix. Site-specific targeting requires targetingreceptors quantitatively distinct from receptor sites found inother tissues. For this reason, HAp, which occurs normally only inhard tissues, provides one attractive targeting site for theselective delivery of bioactive agents to bone.

1. Targeting Ligands

In certain aspects of the invention, ligands having an affinitywith bone are linked to nanocapsules. Two ligands in particularthat may be used for targeting of nanocapsules to, for example, theHAp portion of the bone matrix without an associated therapeuticeffect are methylene bisphosphonate (MBP) and an aspartic acidpeptide residue. MBP is well known for its predilection to boneremodeling sites. For this reason, it has been used extensively incombination with Technetium-99m (.sup.99mTc) as a diagnosticimaging tool in the study of bone pathology (Davis and Jones, 1976;Lantto et al., 1987; Cronhjort et al., 1999). MBP has further beenstudied as a bone matrix-targeting moiety for osteotropic drugdelivery. Fujisaki, et al., (1995 and 1996) have conjugated variousmodel materials and prodrug candidates to MBP and demonstratedtheir efficacy in vivo. Estradiol conjugated to MBP has been shownto be rapidly taken up in bone and then to be released from MBPeither by enzymatic or chemical hydrolysis of the ester conjugationlinkage. Uludag, et al. (2000), have demonstrated the osteotropicdelivery of model proteins conjugated to MBP by similar chemistry.The chemical formula for MBP is given below -- see Original Patent.

Several bone noncollagenous proteins, such as osteopontin and bonesialoprotein, are also known to contain amino residue sequencesthat bind specifically to HAp. For example, Fujisawa, et al.,determined that a six-residue aspartic acid oligopeptide(Asp.sub.6) would preferentially bind to the calcified matrix invivo (Kasugai et al., 2000). They further showed that thistargeting ligand could deliver an estradiol prodrug in vivo(Yokogawa et al., 2000). The chemical formula of this targetingligand is given below: (Asp).sub.6n=6

Yet another ligand that could be used is 1-amino-1,1-diphosphonatemethane (ABP). The amino functionalized analogue of methylenediphosphonate can be synthesized by published methods (Kontoci etal, 1996) as modified by Uludag, et al (2000). Once synthesized,the molecular structure of ABP can be confirmed by 1H, 13C, and 31PNMR. A description of the technique for synthesis ofABP-phospholipid and ABP-PEGylated phospholipid conjugates is setforth in FIG. 3 and Example 1 (see Original Patent). Still anotherligand that can be used is alendronic acid.

2. Linking Targeting Ligands to Nanocapsules

Bifunctional cross-linking reagents represent one means forattaching a targeting ligand to a nanocapsule that may be used withthe invention. Bifunctional cross-linking reagents have beenextensively used for a variety of purposes including preparation ofaffinity matrices, modification and stabilization of diversestructures, identification of ligand and receptor binding sites,and structural studies and can be used for linking targeting agentsto nanocapsules. Homobifunctional reagents that carry two identicalfunctional groups have proved to be highly efficient in inducingcross-linking between identical and different macromolecules orsubunits of a macromolecule, and linking of polypeptide ligands totheir specific binding sites. Heterobifunctional reagents containtwo different functional groups. By taking advantage of thedifferential reactivities of the two different functional groups,cross-linking can be controlled both selectively and sequentially.The bifunctional cross-linking reagents can be divided according tothe specificity of their functional groups, e.g., amino,sulfhydryl, guanidino, indole, carboxyl specific groups. Of these,reagents directed to free amino groups have become especiallypopular because of their commercial availability, ease of synthesisand the mild reaction conditions under which they can be applied. Amajority of heterobifunctional cross-linking reagents contains aprimary amine-reactive group and a thiol-reactive group.

Exemplary methods for cross-linking ligands to nanocapsules aredescribed in U.S. Pat. No. 5,603,872 and U.S. Pat. No. 5,401,511,each specifically incorporated herein by reference in its entirety.Various ligands can be covalently bound to a nanocapsule surfacethrough the cross-linking of amine residues. The inclusion ofphosphatidylethanolamine (PE) in a liposome provides an activefunctional residue, a primary amine, on the liposomal surface forcross-linking purposes.

Ligands can be bound covalently to discrete sites on nanocapsulesurfaces. The number and surface density of these sites will bedictated by the nanocapsule type. The nanocapsule surfaces may alsohave sites for non-covalent association. To form covalentconjugates of ligands and nanocapsule, cross-linking reagents havebeen studied for effectiveness and biocompatibility. Cross-linkingreagents include glutaraldehyde (GAD), bifunctional oxirane (OXR),ethylene glycol diglycidyl ether (EGDE), and a water solublecarbodiimide, preferably 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Through the complex chemistry of cross-linking,linkage of the amine residues of the recognizing substance andnanocapsules is established.

In another example, heterobifunctional cross-linking reagents andmethods of using the cross-linking reagents are described (U.S.Pat. No. 5,889,155, specifically incorporated herein by referencein its entirety). The cross-linking reagents combine a nucleophilichydrazide residue with an electrophilic maleimide residue, allowingcoupling in one example, of aldehydes to free thiols. Thecross-linking reagent can be modified to cross-link variousfunctional groups and is thus useful for cross-linking polypeptidesand sugars. Table 1 (see Original Patent) details examples ofcertain hetero-bifunctional cross-linkers that may be used inaccordance with the invention.

It has been shown that surface-bound targeting ligands can beshielded by other liposome components or surface-adsorbed species(Allen et al., 1995). However, recent research has shown that whentargeting ligands are tethered to surface-bound spacers, theprobability of these ligands to capture binding sites and thedistance at which site recognition occurs are both increased as thetether length is increased (Jeppesen et al., 2001).

II. Nanocapsules

In certain aspects of the invention, nanocapsules are providedwhich are targeted to bone. Potentially any type of nanocapsulecould be used. For example, liposomes or carbon-based cagestructures could be used. Cage-like structures can be formed of,for example, ultrafine fullerene such as C.sub.60 crystallitehaving diameters in the range of 5 to 50 nm. Bioactive payloads,such as agents that heal fractures, prevent bone loss, build bonetissue etc., are enclosed in these structures. Methods forproducing the structures are disclosed in U.S. Pat. No. 5,648,056,the entire disclosure of which is specifically incorporated hereinby reference. Nanocapsules may also be formed of charged particlesof materials including clay and other pillared compounds, which canbe linked with short-chain linking molecules to form secondarycage-like strictures. Similarly, niosomes may be used. Some methodsfor producing niosomes are described in U.S. Provisional PatentApplication Ser. No. 60/351,701, filed Jan. 24, 2002 and entitled"Compositions and Methods for Targeted Drug Delivery" theentire disclosure of which application is specifically incorporatedherein by reference.

A. Liposomes

In certain embodiments of the invention, liposomes could be used asnanocapsules. A "liposome" is a generic term encompassinga variety of single and multilamellar lipid vehicles formed by thegeneration of enclosed lipid bilayers or aggregates. Liposomes maybe characterized as having vesicular structures with a bilayermembrane, generally comprising a phospholipid, and an inner mediumthat generally comprises an aqueous composition.

Liposomes can range in size from several nanometers to severalmicrometers in diameter. Liposome morphological types are broadlycategorized as either multilamellar, unilamellar or micellar.Typically, small unilamellar vesicles (SUV) find application indrug delivery applications. The formulation, preparation,stability, and utility of the various liposome types have been thetopic of considerable research culminating in severalliposome-based drug formulations (Forssen and Willis, 1998). Thekey to success of liposome-based therapies has been the choice ofthe lipid components to achieve vesicle stability and theimprovement in liposome circulation half-life in vivo.

Liposomes are inherently thermodynamically unstable due to both thehigh radius of curvature of the lipid bilayer and inefficientpacking of the phospholipids (Lasic, 1996). The constituentphospholipids exhibit a gel-liquid crystalline phase transitiontemperature (T.sub.c), below which the lipids are organized in alamellar gel state. Stable liposomes are most commonly comprised ofphospholipids having a T.sub.c well above physiologic temperatures.Similarly, phospholipid type plays a role in the fluidity of thelipid bilayer. The naturally occurring phospholipids, such asegg-derived phosphatidylcholine, produce more fluid bilayers, whilesynthetic phospholipids, such as distearoylphosphatidylcholine,produce more ridge bilayers. This stems from differences in thesaturation of the pendent alkyl chains of the natural and syntheticphospholipids. It has been shown that liposomes with ridge bilayerssignificantly reduce the burst release of encapsulated proteins invivo (Van Slooten et al., 2001).

Bilayer additives, such as cholesterol and .alpha.-tocopherol,further improve liposome stability by essentially filling andhardening the lipid bilayer. These additives also decrease bilayerpermeability (Gregoriadis and Davis, 1979), reduce phospholipidexchange (Kirby et al., 1980), and increase oxidation stability,making liposomes viable drug delivery candidates. However, in vivo,liposomes exhibit short circulation longevity due to both bilayerlipid exchange with systemic lipids and rapid elimination by thereticuloendothelial system (RES). Short longevity results indecreased therapeutic effects of encapsulated drugs. Allen, et al.(1991), developed liposomes containing PEGylated phospholipidconjugates and showed that these vesicles greatly improvedcirculation half-lives by effectively introducing steric hindrancebarriers to liposome breakdown.

A multilamellar liposome has multiple lipid layers separated byaqueous medium. They form spontaneously when lipids comprisingphospholipids are suspended in an excess of aqueous solution. Thelipid components undergo self-rearrangement before the formation ofclosed structures and entrap water and dissolved solutes betweenthe lipid bilayers (Ghosh and Bachhawat, 1991). Lipophilicmolecules or molecules with lipophilic regions may also dissolve inor associate with the lipid bilayer.

In other embodiments, phospholipids from natural sources, such asegg or soybean phosphatidylcholine, brain phosphatidic acid, brainor plant phosphatidylinositol, heart cardiolipin and plant orbacterial phosphatidylethanolamine could be used, although aregenerally preferably not used as the primary phosphatide, i.e.,constituting 50% or more of the total phosphatide composition or aliposome, because of the instability and leakiness of the resultingliposomes.

In particular embodiments, a bioactive factor may be, for example,encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome viaa linking molecule that is associated with both the liposome andthe bioactive factor, entrapped in a liposome, complexed with aliposome, etc.

A liposome used according to the present invention can be made bydifferent methods, as would be known to one of ordinary skill inthe art. Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio oflipid to water. At low ratios the liposome is the preferredstructure.

For example, a phospholipid (Avanti Polar Lipids, Alabaster, Ala.),such as for example the neutral phospholipiddioleoylphosphatidylcholine (DOPC), is dissolved in tert-butanol.The lipid(s) is then mixed with the bioactive factor and/or othercomponent(s). Tween 20 is added to the lipid mixture such thatTween 20 is about 5% of the composition's weight. Excesstert-butanol is added to this mixture such that the volume oftert-butanol is at least 95%. The mixture is vortexed, frozen in adry ice/acetone bath and lyophilized overnight. The lyophilizedpreparation is stored at -20.degree. C. and can be used up to threemonths. When required the lyophilized liposomes are reconstitutedin 0.9% saline.

Alternatively, a liposome can be prepared by mixing lipids in asolvent in a container, e.g., a glass, pear-shaped flask. Thecontainer should have a volume ten-times greater than the volume ofthe expected suspension of liposomes. Using a rotary evaporator,the solvent is removed at approximately 40.degree. C. undernegative pressure. The solvent normally is removed within about 5min. to 2 hours, depending on the desired volume of the liposomes.The composition can be dried further in a desiccator under vacuum.The dried lipids generally are discarded after about 1 week becauseof a tendency to deteriorate with time.

Dried lipids can be hydrated at approximately 25-50 mM phospholipidin sterile, pyrogen-free water by shaking until all the lipid filmis resuspended. The aqueous liposomes can be then separated intoaliquots, each placed in a vial, lyophilized and sealed undervacuum.

In other alternative methods, liposomes can be prepared inaccordance with other known laboratory procedures (e.g., seeBangham et al., 1965; Gregoriadis, 1979; Deamer and Uster 1983,Szoka and Papahadjopoulos, 1978, each incorporated herein byreference in relevant part). These methods differ in theirrespective abilities to entrap aqueous material and theirrespective aqueous space-to-lipid ratios.

The dried lipids or lyophilized liposomes prepared as describedabove may be dehydrated and reconstituted in a solution ofinhibitory peptide and diluted to an appropriate concentration witha suitable solvent, e.g., DPBS. The mixture is then vigorouslyshaken in a vortex mixer. Unencapsulated additional materials, suchas agents including but not limited to hormones, drugs, nucleicacid constructs and the like, are removed by centrifugation at29,000.times.g and the liposomal pellets washed. The washedliposomes are resuspended at an appropriate total phospholipidconcentration, e.g., about 50-200 mM. The amount of additionalmaterial or active agent encapsulated can be determined inaccordance with standard methods. After determination of the amountof additional material or active agent encapsulated in the liposomepreparation, the liposomes may be diluted to appropriateconcentrations and stored at 4.degree. C. until use. Apharmaceutical composition comprising the liposomes will usuallyinclude a sterile, pharmaceutically acceptable carrier or diluent,such as water or saline solution.

The size of a liposome varies depending on the method of synthesis.Liposomes in the present invention can be a variety of sizes. Inaccordance with the invention, it will be desired that nanocapsulesare sufficiently small to cross the blood vessel wall. Suchnanocapsules will generally have a size of about 100 nm or smaller,including about 90 nm, about 80 nm, about 70 nm, about 60 nm, orless than about 50 nm in external diameter. In preparing suchliposomes, any protocol described herein, or as would be known toone of ordinary skill in the art may be used. Additionalnon-limiting examples of preparing liposomes are described in U.S.Pat. Nos. 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282,4,310,505, and 4,921,706; International Applications PCT/US85/01161and PCT/US89/05040; U.K. Patent Application GB 2193095 A; Mayer etal., 1986; Mayhew et al., 1984; each incorporated herein byreference).

A liposome suspended in an aqueous solution is generally in theshape of a spherical vesicle, having one or more concentric layersof lipid bilayer molecules. In aqueous suspension, the concentriclayers are arranged such that the hydrophilic moieties tend toremain in contact with an aqueous phase and the hydrophobic regionstend to self-associate. For example, when aqueous phases arepresent both within and without the liposome, the lipid moleculesmay form a bilayer, known as a lamella. Aggregates of lipids mayform when the hydrophilic and hydrophobic parts of more than onelipid molecule become associated with each other. The size andshape of these aggregates will depend upon many differentvariables, such as the nature of the solvent and the presence ofother compounds in the solution.

The production of lipid formulations often is accomplished bysonication or serial extrusion of liposomal mixtures after (I)reverse phase evaporation (II) dehydration-rehydration (III)detergent dialysis and (IV) thin film hydration. In one aspect, acontemplated method for preparing liposomes in certain embodimentsis heating sonicating, and sequential extrusion of the lipidsthrough filters or membranes of decreasing pore size, therebyresulting in the formation of small, stable liposome structures.

Once manufactured, lipid structures can be used to encapsulatebioactive factors for targeting to bone, as is described herein.Liposomes interact with cells to deliver agents via four differentmechanisms: Endocytosis by phagocytic cells of thereticuloendothelial system such as macrophages and/or neutrophils;adsorption to the cell surface, either by nonspecific weakhydrophobic and/or electrostatic forces, and/or by specificinteractions with cell-surface components; fusion with the plasmacell membrane by insertion of the lipid bilayer of the liposomeinto the plasma membrane, with simultaneous release of liposomalcontents into the cytoplasm; and/or by transfer of liposomal lipidsto cellular and/or subcellular membranes, and/or vice versa,without any association of the liposome contents. Varying theliposome formulation can alter which mechanism is operative,although more than one may operate at the same time.

B. Timed or Triggered Release of Nanocapsule Payloads

In certain embodiments of the invention, the use of nanocapsulesdesigned for sustained, triggered or timed release is contemplated.For example, nanocapsules may be designed to release payloads ofbioactive factors upon contact with a given signal, for example,that is released by bone. In this way, nanocapsule payloads aretargeted not only to bone but also to bone in need of treatmentwith the given bioactive factor. Such a signal could be endogenousor externally administered. An external signal could be used tocause release of the nanocapsule payloads by, for example, using achemical signal or physical signal. Examples of physical signalsinclude administration of ultrasound or heat. In this manner thesignal could be administered only to the site where treatment withthe bioactive factor is needed, maximizing delivery of the factorto the site where needed and minimizing exposure to other parts ofthe body. Sustained release nanocapsule formulations could also beused. In this manner the efficacy of treatment may be maximized bymaintaining therapeutic levels of the bioactive factor over time,without the need for continual administrations of the nanocapsules.

Temporally pulsed release of nanocapsules is also specificallycontemplated. This could be achieved, for example, byadministration of several types of nanocapsules having differentdelayed release characteristics. Such temporally pulsed techniquesmay yield benefits beyond those available with sustained releaseformulations. For example, increased bone matrix generationactivity is observed in systems subjected to periodic exposure tobioactive factors in contrast to systems subjected to sustainedexposure to the bioactive factor. This increased matrix generationmay be due to the unhindered completion of the natural matrixgeneration cascade triggered by a spiked dosage of bioactive factorat the site. Also, sustained release may cause a physiologicacclimation that suppresses the triggered response to the spikedelevations in the bone factor concentration.

C. Kits

Nanocapsules prepared in accordance with the invention may becomprised in a kit. In a non-limiting example, nanocapsulestargeted to bone and containing payloads comprising one or morebioactive factors may be comprised in a kit. The kits will thuscomprise, in suitable container means, nanocapsules of the presentinvention.

The kits may comprise the nanocapsules in a suitably aliquotedcomposition of the present invention. The components of the kitsmay be packaged either in aqueous media or in lyophilized form. Thecontainer means of the kits will generally include at least onevial, test tube, flask, bottle, syringe or other container means,into which a component may be placed, and preferably, suitablyaliquoted. Where there are more than one components in the kit, thekit also will generally contain a second, third or other additionalcontainer into which the additional components may be separatelyplaced. However, various combinations of components may becomprised in a vial. The kits of the present invention also willtypically include a means for containing the nanocapsules and anyother reagent containers in close confinement for commercial sale.Such containers may include injection or blow-molded plasticcontainers into which the desired vials are retained.

Therapeutic kits of the present invention may includepharmaceutical compositions for delivery of bioactivefactor-containing nanocapsules targeted to bone. Such kits willgenerally contain, in suitable container means, a pharmaceuticallyacceptable formulation of nanocapsules. The kit may have a singlecontainer means, and/or it may have distinct container means foreach compound.

When the components of the kit are provided in one and/or moreliquid solutions, the liquid solution is an aqueous solution, witha sterile aqueous solution being particularly preferred. Thenanocapsule compositions may also be formulated into a syringeablecomposition. In which case, the container means may itself be asyringe, pipette, and/or other such like apparatus, from which theformulation may be injected into an animal, and/or even applied toand/or mixed with the other components of the kit.

However, the components of the kit may be provided as driedpowder(s). When reagents and/or components are provided as a drypowder, the powder can be reconstituted by the addition of asuitable solvent. It is envisioned that the solvent may also beprovided in another container means.

Irrespective of the number and/or type of containers, the kits ofthe invention may also comprise, and/or be packaged with, aninstrument for assisting with the injection/administration and/orplacement of the ultimate nanocapsule containing composition withinthe body of an animal. Such an instrument may be a syringe,pipette, forceps, and/or any such medically approved deliveryvehicle.

III. Pharmaceutical Compositions

In certain aspects of the current invention, pharmaceuticalcompositions are provided for delivering nanocapsules containingbioactive factors to patients or subjects in need thereof.Pharmaceutical compositions of the present invention thus comprisean effective amount of one or more bioactive factors contained innanocapsules in addition to any other desired components dissolvedor dispersed in a pharmaceutically acceptable carrier.

An "effective amount" is the amount of an bioactive ortherapeutic compound, agent or factor that is sufficient to treator prevent a bone related condition or disease associated in apatient or subject. Thus and "effective amount" is onethat preferably reduces the amount of symptoms of the condition inthe infected patient by at least about 20%, more preferably by atleast about 40%, even more preferably by at least about 60%, andstill more preferably by at least about 80% relative to untreatedsubjects. For example, the efficacy of a compound can be evaluatedin an animal model system that may be predictive of efficacy intreating the disease in humans, such as the model systems such asthose described in the examples or any of those known to one ofskill in the art.

The phrases "pharmaceutical or pharmacologicallyacceptable" refers to molecular entities and compositions thatdo not produce an adverse, allergic or other untoward reaction whenadministered to an animal, such as, for example, a human, asappropriate. The term "bioactive factor" or "isintended to refer to a chemical entity, whether in the solid,liquid, or gaseous phase which is capable of providing a desiredtherapeutic effect when administered to a subject in accordancewith the invention. The term "bioactive factor" includessynthetic compounds, natural products and macromolecular entitiessuch as polypeptides, polynucleotides, or lipids and also smallentities such as neurotransmitters, ligands, hormones or elementalcompounds. The term also includes such compounds whether in a crudemixture or purified and isolated.

The preparation of a pharmaceutical composition that contains atleast one nanocapsule or other ingredients will be known to thoseof skill in the art in light of the present disclosure, asexemplified by Remington's Pharmaceutical Sciences, 18th Ed. MackPrinting Company, 1990, incorporated herein by reference. Moreover,for animal (e.g., human) administration, it will be understood thatpreparations should meet sterility, pyrogenicity, general safetyand purity standards as required by FDA Office of BiologicalStandards.

As used herein, "pharmaceutically acceptable carrier"includes any and all solvents, dispersion media, coatings,surfactants, antioxidants, preservatives (e.g., antibacterialagents, antifungal agents), isotonic agents, absorption delayingagents, salts, preservatives, drugs, drug stabilizers, gels,binders, excipients, disintegration agents, lubricants, sweeteningagents, flavoring agents, dyes, such like materials andcombinations thereof, as would be known to one of ordinary skill inthe art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporatedherein by reference). Except insofar as any conventional carrier isincompatible with the nanocapsules of bioactive factors, its use inthe therapeutic or pharmaceutical compositions is contemplated.

The nanocapsule-containing pharmaceutical composition may becomprised in different types of carriers depending on whether it isto be administered in solid, liquid or aerosol form, and whether itneed to be sterile for such routes of administration as injection.The nanocapsules can potentially be administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, intraperitoneally, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularally, orally, topically, locally,inhalation (e.g. aerosol inhalation), injection, infusion,continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in cremes, in lipidcompositions (e.g., liposomes), or by other method or anycombination of the forgoing as would be known to one of ordinaryskill in the art (see, for example, Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated hereinby reference). In certain aspects of the invention, non-invasiveadministration techniques in particular may be used advantageously,for example, intranasal administration.

The actual dosage amount of a pharmaceutical composition of thepresent invention administered to a patient can be determined byphysical and physiological factors such as body weight, severity ofcondition, the type of disease being treated, previous orconcurrent therapeutic interventions, idiopathy of the patient andon the route of administration. This amount may also be adjustedbased on the targeting agent used for the nanocapsules. One advanceof the current invention is that targeting allows usage of doseslower than required using non-targeted treatments.

The practitioner responsible for administration will, in any event,determine the concentration of bioactive factor(s) and nanocapsulesin a composition and appropriate dose(s) for the individualsubject. In certain embodiments, pharmaceutical compositions maycomprise, for example, an overall concentration of at least about0.1% of an active compound, including, for example, about 0.1% toabout 75%, 0.1% to about 50%, 0.1% to about 25%, 0.1% to about 10%,0.1% to about 5%, 0.1% to about 3%, 0.1% to about 1%, 1% to about10% and about 5% to about 15%.

In other non-limiting examples, a dose may also comprise, innanocapsule carriers, about 1 microgram/kg/body weight, about 5microgram/kg/body weight, about 10 microgram/kg/body weight, about50 microgram/kg/body weight, about 100 microgram/kg/body weight,about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about10 milligram/kg/body weight, about 50 milligram/kg/body weight,about 100 milligram/kg/body weight, about 200 milligram/kg/bodyweight, about 350 milligram/kg/body weight, about 500milligram/kg/body weight, and about 1000 mg/kg/body weight or moreper administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered in nanocapsulepayloads, based on the numbers described above.

In addition to nanocapsules, the composition may comprise variousantioxidants to retard oxidation of one or more component.Additionally, the prevention of the action of microorganisms can bebrought about by preservatives such as various antibacterial andantifungal agents, including but not limited to parabens (e.g.,methylparabens, propylparabens), chlorobutanol, phenol, sorbicacid, thimerosal or combinations thereof.

The bioactive factor that is used may be formulated into acomposition in a free base, neutral or salt form. Pharmaceuticallyacceptable salts, include the acid addition salts, e.g., thoseformed with the free amino groups of a proteinaceous composition,or which are formed with inorganic acids such as for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such asfor example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine,trimethylamine, histidine or procaine.

In embodiments where the nanocapsule composition is in a liquidform, a carrier can be a solvent or dispersion medium comprisingbut not limited to, water, ethanol, polyol (e.g., glycerol,propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g.,triglycerides, vegetable oils, liposomes) and combinations thereof.It will be necessary that such a carrier does not disrupt thenanocapsules prior to delivery to a patient. The proper fluidity ofthe composition can be maintained, for example, by the use of acoating, such as lecithin; by the maintenance of the requiredparticle size by dispersion in carriers such as, for example liquidpolyol or lipids; by the use of surfactants such as, for examplehydroxypropylcellulose; or combinations thereof such methods. Insome cases, it will be preferable to include isotonic agents, suchas, for example, sugars, sodium chloride or combinations thereof

In other embodiments of the invention, one may use eye drops, nasalsolutions or sprays, aerosols or inhalants in the presentinvention. Such compositions are generally designed to becompatible with the target tissue type. In a non-limiting example,nasal solutions are usually aqueous solutions designed to beadministered to the nasal passages in drops or sprays. Nasalsolutions are prepared so that they are similar in many respects tonasal secretions, so that normal ciliary action is maintained.Thus, in preferred embodiments the aqueous nasal solutions usuallyare isotonic or slightly buffered to maintain a pH of about 5.5 toabout 6.5. In addition, antimicrobial preservatives, similar tothose used in ophthalmic preparations, drugs, or appropriate drugstabilizers, if required, may be included in the formulation. Forexample, various commercial nasal preparations are known andinclude drugs such as antibiotics or antihistamines.

In certain embodiments the nanocapsules are prepared foradministration by such routes as oral ingestion. In theseembodiments, the solid composition may comprise, for example,solutions, suspensions, emulsions, tablets, pills, capsules (e.g.,hard or soft shelled gelatin capsules), sustained releaseformulations, buccal compositions, troches, elixirs, suspensions,syrups, wafers, or combinations thereof. Oral compositions may beincorporated directly with the food of the diet. Preferred carriersfor oral administration comprise inert diluents, assimilable ediblecarriers or combinations thereof. In other aspects of theinvention, the oral composition may be prepared as a syrup orelixir. A syrup or elixir, and may comprise, for example, at leastone active agent, a sweetening agent, a preservative, a flavoringagent, a dye, a preservative, or combinations thereof.

In certain further embodiments an oral composition may comprise oneor more binders, excipients, disintegration agents, lubricants,flavoring agents, and combinations thereof. Such a composition maycomprise, for example, one or more of the following: a binder, suchas, for example, gum tragacanth, acacia, cornstarch, gelatin orcombinations thereof; an excipient, such as, for example, dicalciumphosphate, mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, cellulose, magnesium carbonate or combinations thereof;a disintegrating agent, such as, for example, corn starch, potatostarch, alginic acid or combinations thereof; a lubricant, such as,for example, magnesium stearate; a sweetening agent, such as, forexample, sucrose, lactose, saccharin or combinations thereof; aflavoring agent, such as, for example peppermint, oil ofwintergreen, cherry flavoring, orange flavoring, etc.; orcombinations thereof the foregoing. When the dosage unit form is acapsule, it may contain, in addition to materials of the abovetype, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical formof the dosage unit. For instance, tablets, pills, or capsules maybe coated with shellac, sugar or both.

Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are soliddosage forms of various weights and shapes, usually medicated, forinsertion into the rectum, vagina or urethra. After insertion,suppositories soften, melt or dissolve in the cavity fluids. Ingeneral, for suppositories, traditional carriers may include, forexample, polyalkylene glycols, triglycerides or combinationsthereof. In certain embodiments, suppositories may be formed frommixtures containing, for example, the active ingredient in therange of about 0.5% to about 10%, and preferably about 1% to about2%.

Sterile injectable solutions are prepared by incorporatingbioactive factors, e.g., in nanocapsules, in the required amount inthe appropriate solvent with various of the other ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the varioussterilized active ingredients into a sterile vehicle which containsthe basic dispersion medium and/or the other ingredients. In thecase of sterile powders for the preparation of sterile injectablesolutions, suspensions or emulsion, the preferred methods ofpreparation are vacuum-drying or freeze-drying techniques whichyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessaryand the liquid diluent first rendered isotonic prior to injectionwith sufficient saline or glucose. The preparation of highlyconcentrated compositions for direct injection is alsocontemplated, where the use of DMSO as solvent is envisioned toresult in extremely rapid penetration, delivering highconcentrations of the active agents to a small area.

The composition must be stable under the conditions of manufactureand storage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciatedthat endotoxin contamination should be kept minimally at a safelevel, for example, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminummonostearate, gelatin or combinations thereof.

Nanocapsules can generally entrap compounds in a stable and/orreproducible way. To avoid side effects due to intracellularpolymeric overloading, such ultrafine particles (sized around 0.1.mu.m) should be designed using polymers able to be degraded invivo. Biodegradable polyalkyl-cyanoacrylate nanocapsules that meetthese requirements are contemplated for use in the presentinvention, and/or such particles may be easily made.

Claim 1 of 70 Claims
1. A nanocapsule encapsulating at least a first bioactive factor,wherein said nanocapsule is bound to a bisphosphonate targetingligand having specificity for a component of the systemic skeletonand wherein said nanocapsule is capable of releasing said bioactivefactor due to an externally applied signal, a complementary factoradministered in schedule or a biochemical signal present in thebone microenvironment, wherein said nanocapsule, when not releasingsaid bioactive factor, is capable of remaining intact and beingexpelled by normal metabolic activity, wherein said bisphosphonatetargeting ligand prior to binding with the nanocapsule has astructure selected from the following -- see Original Patent.
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