Patent Application
20230398077
[Not Applicable]
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with 5-year survival of ˜8%[1]. As surgery is only suitable for ˜15% of PDAC patients upon diagnosis, the best clinically available option is to use chemotherapy in the majority of PDAC patients[2]. This can be accomplished by the use of a gemcitabine (GEM)/Nab-paclitaxel combination or a four-drug regimen, FOLFIRINOX, which includes folinic acid, 5-fluorouracil, irinotecan, and oxaliplatin[3]. While the recent breakthrough of a modified FOLFIRINOX regimen that reduces the irinotecan dose has yielded promising results in a phase 3 study, the use of the alternative option is still limited due to the occurrence of toxic side effects and chemo-resistance[3c]. In order to develop a nanocarrier with reduced irinotecan side effects, we have previously developed mesoporous silica nanoparticles (MSNP), coated with a lipid bilayer for safer and more efficacious irinotecan delivery[4]. The success of this carrier, also known as a “silicasome”, was largely attributed to the ability of a proton-generating trapping agent for remote import of the weak-basic molecule, irinotecan, into the large packaging space of the porous interior[4]. This resulted in improved irinotecan delivery, which was accompanied by a significant improvement in treatment efficacy for PDAC, prolonged survival, and reduced toxicity compared to the free drug or a liposomal carrier, ONIVYDE®[4b].
Oxaliplatin is another active pharmaceutical ingredient (API) of the FOLFIRINOX regimen that is very potent but exhibits major, and dose-limiting toxicity (e.g., bone marrow)[5]. In addition, oxaliplatin is known to exert immunogenic effects that could be potentially useful to supplement its chemotherapeutic effects[6]. However, since Pt drugs are coordination compounds, it is not possible to do remote loading in MSNPs to obtain high loading capacity, which has limited the utility of this carrier for Pt drugs. It would be of great advantage to develop MSNPs carriers for efficient Pt drug loading and delivery from the perspective that ˜50% of all cancer patients undergoing chemotherapy receive at least one type of Pt-based treatment[7].
Pt-based antineoplastic molecules (
Herein, we report an intravenous (IV) injectable tailored-designed silicasome carrier for Pt drug encapsulation. This was achieved by introducing active Pt drugs that can be efficiently loaded under a carefully designed complexation conditions, making use of weak basic pH conditions. Instead of using a post-grafting approach, we took advantage of the pH-dependent properties of the surface silanol groups for electrostatic and coordination binding of DACHPt, followed by applying a uniform lipid coat to seal the MSNP pores. This strategy, which can also be adapted to load other types of Pt payloads, led to high-loading and colloidally stable nanocarriers. The availability of a DACHPt silicasome allowed us to perform efficacy and safety studies in an orthotopic Kras PDAC model. It was also possible to use the silicasome-encapsulated DACHPt to test its immunogenic effects for the development of a chemo-immunotherapy approach in combination with an anti-PD-1 antibody in an orthotopic PDAC survival study.
Accordingly, various embodiments contemplated herein may include, but need not be limited to, one or more of the following:
The terms “subject,” “individual,” and “patient” may be used interchangeably and refer to humans, as well as non-human mammals (e.g., non-human primates, canines, equines, felines, porcines, bovines, ungulates, rodents, lagomorphs, and the like). In various embodiments, the subject can be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, as an outpatient, or other clinical context. In certain embodiments, the subject may not be under the care or prescription of a physician or other health worker.
As used herein, the phrase “a subject in need thereof” refers to a subject, as described infra, that suffers from, or is at risk for a cancer as described herein. Thus, for example, in certain embodiments the subject is a subject with a cancer (e.g., pancreatic ductal adenocarcinoma (PDAC), breast cancer (e.g., drug-resistant breast cancer), colon cancer, brain cancer, and the like). In certain embodiments the methods described herein are prophylactic and the subject is one in whom a cancer is to be inhibited or prevented. In certain embodiments the subject for prophylaxis is one with a family history of cancer and/or a risk factor for a cancer (e.g., a genetic risk factor, an environmental exposure, and the like).
The term “treat” when used with reference to treating, e.g., a pathology or disease refers to the mitigation and/or elimination of one or more symptoms of that pathology or disease, and/or a delay in the progression and/or a reduction in the rate of onset or severity of one or more symptoms of that pathology or disease, and/or the prevention of that pathology or disease. The term “treat” can refer to prophylactic treatment which includes a delay in the onset or the prevention of the onset of a pathology or disease.
The terms “coadministration” or “administration in conjunction with” or “cotreatment” when used in reference to the coadministration of a first compound (or component) (e.g., a Platinum (Pt)-based drug) and a second compound (or component) (e.g., a different cancer therapeutic) indicates that the first compound (or component) and the second compound (or component) are administered so that there is at least some chronological overlap in the biological activity of first compound and the second compound in the organism to which they are administered. Coadministration can include simultaneous administration or sequential administration. In sequential administration there may even be some substantial delay (e.g., minutes or even hours) between administration of the first compound and the second compound as long as their biological activities overlap. In certain embodiments, the coadministration is over a time frame that permits the first compound and second compound to produce an enhanced therapeutic or prophylactic effect on the organism. In certain embodiments the enhanced effect is a synergistic effect.
The terms “nanocarrier”, “nanoparticle drug carrier”, and “drug delivery vehicle” are used interchangeably and refer to a submicron structure (e.g., a nanostructure) having one or a plurality of cavities, e.g., a porous interior. In various embodiments, the cavities contain a cargo that is to be delivered, e.g., to a target cell. In certain embodiments the nanoparticle is a porous silica nanoparticle (e.g., mesoporous silica nanoparticle or “MSNP”). In certain embodiments the nanocarrier comprises a lipid bilayer encasing (or surrounding or enveloping) the core particle.
As used herein, the term “lipid” refers to conventional lipids, phospholipids, cholesterol, chemically functionalized lipids for attachment of PEG, pharmaceutically active ingredients, ligands, etc.
As used herein, the terms “lipid bilayer” or “LB” refers to any double layer of oriented amphipathic lipid molecules in which the hydrocarbon tails face inward to form a continuous non-polar phase.
An activated platinum (activated PT) drug refers to the form of a platinum drug that is pharmaceutically active (e.g., due to the high reactivity of coordinated crosslinking to DNA which stops cancer growth). Platinum drugs exist as an equilibrium of “neutral” or “cationic” species in an aqueous solution. The binding equilibrium is dependent on the Cl— ion concentration (CCl—) as well as pH. While the neutral drug version is dominant in the blood circulation due to a high CCl— concentration (˜150 mM), the formation of an intracellular cationic version is facilitated due to a lower CCl— concentration (˜30 mM). Moreover, the cationic formulation is regarded as pharmaceutically active due to the high reactivity of coordinated crosslinking to DNA, which stops cancer growth.
As used herein, the term “selective targeting” or “specific binding” refers to use of targeting ligands on the surface of a drug delivery nanocarrier (e.g., a LB-coated nanoparticle). In certain embodiments the targeting ligand(s) are on the surface of a lipid bilayer or LB-coated nanoparticle. Typically, the ligands interact specifically/selectively with receptors or other biomolecular components expressed on the target, e.g., a cell surface of interest. The targeting ligands can include such molecules and/or materials as peptides, antibodies, aptamers, targeting peptides, polysaccharides, and the like.
A “silica nanoparticle” refers to a nanoparticle that comprises silica or that consists of silica. In certain embodiments, the silica nanoparticle can include, e but need not be limited to a nanoparticle comprising a functionalized silica.
A coated silica nanoparticle, having targeting ligands can be referred to as a “targeted nanoparticle or a targeted drug delivery nanocarrier, or a targeted silicasome when the nanoparticle is coated with a lipid bilayer.
The term “about” or “approximately” as used herein refers to being within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e. the limitations of the measurement system, i.e. the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5% and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.
The term “drug” as used herein refers to a chemical entity of varying molecular size, small and large, naturally occurring or synthetic, that exhibits a therapeutic effect in animals and humans. A drug may include, but is not limited to, an organic molecule (e.g., a small organic molecule), a therapeutic protein, peptide, antigen, or other biomolecule, an oligonucleotide, an siRNA, a construct encoding CRISPR cas9 components and, optionally one or more guide RNAs, and the like.
A “pharmaceutically acceptable carrier” as used herein is defined as any of the standard pharmaceutically acceptable carriers. The pharmaceutical compositions of the subject invention can be formulated according to known methods for preparing pharmaceutically useful compositions. The pharmaceutically acceptable carrier can include diluents, adjuvants, and vehicles, as well as carriers, and inert, non-toxic solid or liquid fillers, diluents, or encapsulating material that does not react with the active ingredients of the invention. Examples include, but are not limited to: phosphate buffered saline, physiological saline, water, and emulsions, such as oil/water emulsions. The carrier can be a solvent or dispersing medium containing, for example, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Formulations are described in a number of sources that are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Sciences (Martin E W [1995] Easton Pa., Mack Publishing Company, 19th ed.) describes formulations that can be used in connection with the drug delivery nanocarrier(s) (e.g., liposomes or nanoparticles encapsulated with a lipid bilayer) described herein.
As used herein, an “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes or derived therefrom that is capable of binding (e.g., specifically binding) to a target (e.g., to a target polypeptide). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.
Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)′2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab′)2 dimer into a Fab′ monomer. The Fab′ monomer is essentially a Fab with part of the hinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab′ fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. Certain preferred antibodies include single chain antibodies (antibodies that exist as a single polypeptide chain), more preferably single chain Fv antibodies (sFv or scFv) in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide. The single chain Fv antibody is a covalently linked VH-VL heterodimer which may be expressed from a nucleic acid including VH- and VL-encoding sequences either joined directly or joined by a peptide-encoding linker. Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. While the VH and VL are connected to each as a single polypeptide chain, the VH and VL domains associate non-covalently. The first functional antibody molecules to be expressed on the surface of filamentous phage were single-chain Fv's (scFv), however, alternative expression strategies have also been successful. For example Fab′ molecules can be displayed on a phage if one of the chains (heavy or light) is fused to g3 capsid protein and the complementary chain exported to the periplasm as a soluble molecule. The two chains can be encoded on the same or on different replicons; the important point is that the two antibody chains in each Fab molecule assemble post-translationally and the dimer is incorporated into the phage particle via linkage of one of the chains to, e.g., g3p (see, e.g., U.S. Pat. No. 5,733,743). The scFv antibodies and a number of other structures converting the naturally aggregated, but chemically separated light and heavy polypeptide chains from an antibody V region into a molecule that folds into a three-dimensional structure substantially similar to the structure of an antigen-binding site are known to those of skill in the art (see e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,956,778). In certain embodiments antibodies should include all that have been displayed on phage (e.g., scFv, Fv, Fab and disulfide linked Fv (see, e.g, Reiter et al. (1995) Protein Eng. 8: 1323-1331) as well as affibodies, unibodies, and the like.
The term “specifically binds”, as used herein, when referring to a biomolecule (e.g., protein, nucleic acid, antibody, etc.), refers to a binding reaction that is determinative of the presence of a biomolecule in heterogeneous population of molecules (e.g., proteins and other biologics). Thus, under designated conditions (e.g. immunoassay conditions in the case of an antibody or stringent hybridization conditions in the case of a nucleic acid), the specified ligand or antibody binds to its particular “target” molecule and does not bind in a significant amount to other molecules present in the sample.
The term “immunogenic cell death” or “ICD” refers to a unique form of cell death caused by some cytostatic agents such as anthracyclines (Obeid et al. (2007) Nature Med., 13(1): 54-61), anthracenedione (mitoxantrone, aka MTX), oxaliplatin, irinotecan, and bortezomib, or radiotherapy and/or photodynamic therapy (PDT). Unlike regular apoptosis, which is mostly non-immunogenic or even tolerogenic, immunogenic apoptosis of cancer cells can induce an effective antitumor immune response through activation of dendritic cells (DCs) and consequent activation of specific T cell response (Spisek and Dhodapkar (2007) Cell Cycle, 6(16): 1962-1965). Endoplasmic reticulum (ER) stress, reactive oxygen species (ROS) production and induction of autophagy are key intracellular response pathways that govern ICD (Krysko et al. (2012) Nat. Rev. Canc. 12(12): 860-875). In addition to facilitating tumor cell death that facilitates antigen presentation by dendritic cells, ICD is characterized by secretion or release of damage-associated molecular patterns (DAMPs), which exert additional immune adjuvant effects. Calreticulin (CRT), one of the DAMP molecules, which is normally in the lumen of the ER, is translocated to the surface of dying cell where it functions as an “eat me” signal for phagocytes. Other important surface exposed DAMPs are heat-shock proteins (HSPs), namely HSP70 and HSP90, which under stress conditions are also translocated to the plasma membrane. On the cell surface they have an immunostimulatory effect, based on their interaction with number of antigen-presenting cell (APC) surface receptors like CD91 and CD40 and also facilitate cross-presentation of antigens derived from tumor cells on MHC class I molecule, which then triggers CD8+ T cell-activation and expansion. Other important DAMPs, characteristic for ICD are secreted, high-mobility group box 1 (HMGB1) protein and ATP (see, e.g., Apetoh et al. (2007) Nature Med. 13(9): 1050-1059; Ghiringhelli et al. (2009) Nature Med. 15(10): 1170-1178). HMGB1 is considered to be a late apoptotic marker and its release to the extracellular space appears to be required for the optimal release and presentation of tumor antigens to dendritic cells. It binds to several pattern recognition receptors (PRRs) such as Toll-like receptor (TLR) 2 and 4, which are expressed on APCs. The most recently found DAMP released during immunogenic cell death is ATP, which functions as a “find-me” signal for monocytes when secreted and induces their attraction to the site of apoptosis (see, e.g., Garg et al. (2012) EMBO J. 31(5): 1062-1079). ATP binds to purinergic receptors on APCs. An inducer of immunogenic cell death is referred to as an ICD inducer.
The terms “IDO inhibitor”, “IDO pathway inhibitor”, and “inhibitor of the IDO pathway) are used interchangeably and refer to an agent (a molecule or a composition) that either partially or fully blocks the activity of indoleamine-2,3-dioxygenase (IDO) and/or partially or fully suppresses the post-enzymatic signaling cascade(s) in the IDO pathway. IDO is an intracellular heme-containing enzyme that initiates the first and rate-limiting step of tryptophan degradation along the kynurenine pathway. The indoleamine 2,3-dioxygenase (IDO) pathway regulates immune response by suppressing cytotoxic T cell function, enhancing regulatory T cell activity (Tregs) and enabling tumor immune escape, either at the tumor or regional lymph node sites. An IDO pathway inhibitor can inhibit the IDO enzyme directly or by interfering or perturbing IDO effector pathway components. Such components include, but are not limited to: IDO2, tryptophan 2,3-dioxygenase (TDO), the mammalian target of rapamycin (mTOR) pathway, aryl hydrocarbon receptor (AhR) pathway, the general control nonderepressible 2 (GCN2) pathway, and the AhR/IL-6 autocrine loop.
As described herein a porous silica nanoparticle (e.g., a mesoporous silica nanoparticle) based platform for the high dose loading and delivery of a range of metal-based therapeutic agents. In certain embodiments, the metal-based therapeutic agents comprise one or more activated platinum chemotherapeutic agents. In various embodiments, illustrative, but non-limiting embodiments, the activated platinum (Pt) chemotherapeutic agents are attached to the silica nanoparticle (e.g., within the pores of a mesoporous silica nanoparticle through the use of electrostatic and coordination chemistry under weak-basic pH conditions). Moreover, in certain embodiments, the nanoparticles are encapsulated in a lipid bilayer thereby forming a “silicasome”. Without being bound to a particular theory, it is believed the presence of the lipid bilayer (LB) improves colloidal stability after intravenous (IV) injection.
The porous silica nanoparticles (e.g., mesoporous silica nanoparticles (MSNPs) have a large interior packaging space for drugs against the walls of the porous interior. This leads to a substantial increase in loading capacity and stable retention until the carrier enters the tumor site to deliver its payload. The presence of a supported lipid bilayer (LB), provides for stable drug encapsulation by an intact surface coat. The LB-coated MSNPs have been labeled “silicasomes” to distinguish them from liposomes, which also contain (a non-supported) LB that encapsulates a fluid space and its content (e.g., a drug).
As described in Example, 1, the PT-loaded silicasomes described herein show improved pharmacokinetics and intratumor delivery of encapsulated oxaliplatin ((1,2-diaminocyclohexane)platinum(II) (DACHPt)), over free drug in an orthotopic Kras-derived pancreatic cancer (PDAC) model. Not only did IV injection of the DACHPt silicasome provide more efficacious cytotoxic tumor cell killing, but could also demonstrate that chemotherapy-induced cell death is accompanied by the features of immunogenic cell death (ICD) as well as a dramatic reduction in bone marrow toxicity. Subsequent performance of a survival experiment demonstrated that the DACHPt silicasome generate a significant improved survival outcome, which could be extended by co-administration of an anti-PD-1 antibody.
In view of the high loading achieved with platinum-based therapeutic agents, it is recognized that the same loading methods can be used with any of a number of other metal-based drugs, e.g., as described herein.
In certain embodiments, the PT-loaded silicasomes described herein can comprise one or more additional therapeutic agents. Such agents can be disposed within the silica nanoparticle or within the lipid bilayer or conjugated to the lipid bilayer. Thus, for example, in certain embodiments the silicasomes described herein can additionally contain one or more inhibitor(s) of the indoleamine 2,3-dioxygenase (IDO) pathway (IDO pathway inhibitor). Without being bound to a particular theory it is believed that such IDO pathway inhibitors can synergize with loaded platinum-based chemotherapeutics.
In certain embodiments, the silicasomes described herein comprise a hydrophobic therapeutic moiety disposed in the lipid bilayer. Thus, for example, in certain embodiments the additional therapeutic moiety can comprise paclitaxel.
Additionally method of making the drug delivery nanoparticles are provided as well as methods of use of the nanoparticles, e.g., in the treatment of a cancer.
The direct loading approach for platinum (Pt)-based drugs typically results in a very low loading capacity. Thus, for example, using the direct drug encapsulation approach, it is only possible to make an oxaliplatin (OX)-laden silicasome with a maximum loading capacity of ˜5% (OX/MSNP w/w) and with a loading efficiency of ˜5% (i.e., 95% of the offered drug was wasted). Without being bound to a particular theory, it is believed that the low loading efficiency/capacity is principally due to the poor water solubility of OX and the lack of specific interaction between OX and the MSNP silica surface.
In view of these concerns a novel approach to achieve a high loading capacity for an active version of Pt-based drug (e.g., OX) into the silicasome was developed. Instead of passive encapsulation of the Pt drug, we made use of cationic, activated Pt drugs (e.g., 1,2-cyclohexanediamine platinum (II), a.k.a. DACHPt), for drug loading by interacting with the silanol groups in the walls of the MSNP pores. A conceptualization of the final product is demonstrated in
The loading of three common Pt drugs is illustrated in Example 1, however, it will be recognized that using these teachings, other Pt-based drugs can readily be loaded. As proof of principle, Example 1 illustrates loading of oxaliplatin, cisplatin and dichloro (ethylenediamine) platinum (Pt(en)Cl2) (
These findings prompted us to consider loading cationic, activated Pt drugs into MSNP rather than working with pristine drugs. This involves the use of “neutral” Pt drugs where the X2ligand is represented by Cl− ions. Thus, for example, commercially available dichloro(1,2-diaminocyclohexane) platinum(II) (structure 5 in
The PT-loaded silicasomes described herein need not be limited to oxaliplatin, cisplatin, and dichloro(ethylenediamine) platinum. Activated cationic versions of numerous other Pt-based therapeutics, can readily be prepared and loaded using the teachings provided herein. Illustrative, but non-limiting additional platinum-based therapeutics include, but are not limited to carboplatin, nedaplatin, heptaplatin, lobaplatin, iproplatin, tetraplatin, satraplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and setraplatin Illustrative activated cationic versions of these drugs appears in
Additionally, it will be recognized that using the methods described herein, other metal-based drugs can be loaded into the drug delivery vehicles described and provide a high degree of drug loading. In particular, the loading methods are well suited to other metal-based drugs that exhibit similar metal complexation structure. Generally, as long as the metal-based drug can bind to a surface of the nanoparticle through similar electrostatic/coordination interactions it can readily be incorporated into the drug-delivery vehicles described herein.
Numerous metal-based drugs are known and well suited to incorporation into the drug delivery vehicles described herein. For example, such metal-based drugs include, but are not limited to gold-based rugs (e.g., such as auranofin used for rheumatoid arthritis), technetium and rhenium which can be used as radiopharmaceuticals for imaging and radiotherapy, ruthenium which is an anticancer drug. Other possibilities include, but are not limited to metal-based drugs comprising palladium, gadolinium, cobalt, lithium, bismuth, iron, calcium, lanthanum, gallium, tin, arsenic, rhodium, copper, zinc, aluminum, lutetium, vanadium, manganese, and the like (see, e.g., Jurka, et al. (2017) Metal Complexes of Pharmaceutical Substances, Spectroscopic Analyses—Developments and Applications, Eram Sharmin and Fahmina Zafar, IntechOpen, DOI: 10.5772/65390; Sodhi & Paul (2019) Canc. Therapy & Oncol. Int. J. 14(2): 555883. DOI:10.19080/CTOI; and the like).
“Metal containing drugs are important for a few medical applications including diagnosis and treatment. For example, platinum based compounds have been shown to specifically affect head and neck tumors. These coordination complexes are thought to act cross-link DNA in tumor cells. Gold salt complexes have been used to treat Rheumatoid Arthritis. The gold salts are believed to interact with albumin and eventually be taken up by immune cells, triggering anti-mitochondrial effects and eventually cell apoptosis. Lithium (Li2CO3) can be used to treat prophylaxis of manic-depression behavior. Zinc can be used topically to heal wounds and Zn+ can be used to treat Herpes and other viruses. Silver has been used to prevent infection at the burn site for burn wound patients. Phosphine ligand compounds containing gold, silver, and/or copper have anti-cancer properties. Lanthanum carbonate often used under the trade name Fosrenol is used as a phosphate binder in patients suffering from chronic kidney disease. Bismuth subsalicylate is used as an antacid. Platinum, Titanium, Vanadium, Iron: cis DDP (cisdiaminedichoroplatinum), titanium, vanadium, and iron have been shown to react with DNA specifically in tumor cells to treat patients with cancer. Barium has been used for X-ray diagnoses, while gadolinium, and manganese are used for magnetic resonance imaging.
Illustrative metal-based drugs that can be incorporated in the nanoparticle drug delivery systems alone or in combination include, but are not limited to the platinum-based drugs described above, as drugs comprising a metal selected from the group consisting of palladim, gold, ruthenium, titanium, technetium and rhenium galdolinium, cobalt, lithium, bismuth, iron, calcium, lanthanum, gallium, tin, arsenic, rhodium, copper, zinc, aluminum, lutetium, vanadium, and manganese. In certain embodiments, the metal-based drug comprises a metal-based drug selected from the group consisting of a palladium complex drug, a gold complex drug, a ruthenium complex drug, and a titanium complex drug.
In certain embodiments, the metal-based drugs include, but are not limited to anti-cancer palladium complexes such as rans-[PdCl2(2-dqmp)] (2-dqmp=diethyl-2-quinolmethylphosphonate, and glycoconjugated Pd(II) complex, [PdCl2(L)] (L=2-deoxy-2-[(2-pyridinylmethylene) amino]-a-D-glucopyranose (see, e.g., Table 1, compounds 1-2; Lazarević, et al. (2017) Eur. J. Med. Chem., 142: 8-31; and the like).
In certain embodiments, the metal-based drugs include, but are not limited to anti-cancer gold complexes. These can include for example, a number of Au(III) complexes with multidentate ligands, namely [Au(en)Cl2][Cl], [Au(dien)Cl][Cl2], [Au(cyclam)][ClO4]2Cl, [Au(terpy)Cl][Cl2], [Au(phen)Cl2][Cl], and the like (see, e.g., Table 1, compounds 3-7; Messori, et al. (2000) J. Med. Chem. 43:3541-3548; Eur J Med Chem. (2017) 142:8-31; Lazarević, et al. (2017) Eur. J. Med. Chem., 142: 8-31; and the like).
It is also possible to load Au(III) complexes that contain functionalized bipyridine ligands of the general formula [Au(N—N)Cl2][PF6] where N—N=2,2′-bipyridine, or 4,4′-dimethyl-2,2′-bipyridine, or 4,4′-dimethoxy-2,2′-bipyridine; or 4,4′-diamino-2,2′-bipyridine) (see, e.g., Table 1, compounds 8-11).
Other possibilities include, but are not limited to Au(III) complexes of the type [Au(dach)(pn)]Cl3 where dach=cis-; trans-1,2-; and S,S-1,2-diaminocyclohexane and pn=1,3-diaminopropane (see, e.g., Table 1, compounds 12-14).
In certain embodiments, the metal-based drugs include, but are not limited to anti-cancer ruthenium complexes (see, e.g., Table 1, compounds 15-18; Ndagi et al. (2017) Drug Design, Development and Therapy, 11: 599-616; and the like).
In certain embodiments, the metal-based drugs include, but are not limited to anti-cancer titanium complexes, such as titanocenes (see, e.g., Table 1, compounds 19-20; Ndagi et al. (2017) Drug Design, Development and Therapy, 11: 599-616; and the like).
The foregoing metal-based drugs are illustrative and non-limiting. Using the teaching provided herein, drug delivery vehicles as described herein carrying numerous other metal-based drugs will be available to one of skill in the art.
Nanoparticle Fabrication.
In various embodiments the drug delivery vehicles described herein comprise a solid silica nanoparticle or a silica nanoparticle containing one or more cavities where the nanoparticle is disposed within and fully encapsulated by a lipid bilayer.
In certain embodiments the nanoparticle comprise a porous silica nanoparticle. In certain embodiments the porous silica nanoparticle comprises a mesoporous silica nanoparticle (MSN), a mesoporous organosilica nanoparticle (MON), and/or a periodic mesoporous organosilica (PMO) nanoparticle.
MSNs, MONs, and PMOs are commonly fabricated using sol-gel processes in aqueous solutions (Croissant et al. (2015) Nanoscale, 7: 20318-20334; Wu et al. (2013) Chem. Soc. Rev. 42: 3862-3875; Yano & Fukushima (2004) J. Mater. Chem. 14: 1579-1584; Nakamura et al. (2007) J. Phys. Chem. C, 111: 1093-1100). The conventional sol-gel synthesis has been studied extensively and allows precise control of nanoparticle properties such as size, pore size and geometry, particle modification, and/or surface functionalization (see, e.g., Wu et al. (2013) Chem. Soc. Rev. 42: 3862-3875). In one illustrative sol-gel synthesis, silica particles are formed via hydrolysis of various silanes and/or silicates with a subsequent silica condensation:
—Si—O-+HO-Si—→—Si—O—Si-+OH—
In one illustrative, but non-limiting embodiment, synthesis takes place in an aqueous solution and can involve alcohol and ammonia or other catalysts (see, e.g., Yano & Fukushima (2004) J. Mater. Chem. 14: 1579-1584). The speed of the synthesis reaction depends on the pH value with the maximum silica condensation rate at normal pH conditions. The types and concentrations of the synthesis reagents affect the resulting particle size. Tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate (TMOS) and other compounds can be used as silicon sources. To inhibit silica growth and, thus, obtain smaller MSNs, surface-protection agents can be used, such as triethanolamine (TEA), poly (ethylene glycol) (PEG) and/or a second nonionic surfactant (see, e.g., Möller et al. (2007) Adv. Funct. Mater. 17: 605-612). These agents can also be useful for isolation of the growing silica particles from each other, preventing their aggregation and the growth of silica bridges between neighboring particles.
In certain embodiments, to obtain MSNs, micelles can be used as a soft template to form the mesoporous structure. In one illustrative, but non-limiting embodiment, the silica particles are grown on the templates as starting points for the condensation. Surfactants such as cetyltrimethylammonium bromide (CTAB) or cetyltrimethylammonium chloride (CTACl) can be added to the solution as well. At low concentrations just above the critical micellar concentration, the surfactant molecules bind together and form small spherical micelles. At higher concentrations, micelles can have cylindrical or other shapes. These micelles are positively charged and attract negatively charged silanes, facilitating their condensation. Addition of the second surfactant can lead to the formation of the more complicated micellar structures, allowing further modification of the MSNs pore structure. Similar to the micelles, vesicles can be used as templates for the MSN growth (see, e.g., Yeh et al. (2006) Langmuir, 22: 6-9). In certain embodiments, inorganic nanoparticles, such as metal (Au, Pt) or metal oxide (Fe3O4) nanoparticles could be incorporated into the structure of MSNs as desired (see, e.g., Kneževi' et al. (2013) RSC Adv. 3: 9584-9593; Timin et al. (2016) Mater. Chem. Phys. 183: 422-429; Ott et al. (2015) Chem. Mater. 2015, 27: 1929-1942). They can be used as the templates for the MSNs growth as well. Such “hybrid” nanoparticles can be capable of both carrying a drug load and acting as contrast agents for bioimaging. In certain embodiments to produce larger pore sizes to accommodate higher quantities of molecules or simply larger molecules (e.g., biomolecules, such as DNA and proteins a swelling agent can be utilized. Several swelling agents can be used to increase the pore sizes, e.g., trimethylbenzene (TMB) (see, e.g., Zhang et al. (2011) J. Colloid Interface Sci. 361: 16-24). Another way to increase the size of the pores is the use of the block-polymers as templates (see, e.g., Han & Ying (2005) Angew. Chem. 117: 292-296).
In one illustrative, but non-limiting synthesis protocol, MSNPs are synthesized by a sol/gel procedure, similar to the method described by Liu et al. (2016) ACS Nano, 10(2): 2702-2715. Thus, for example, to synthesize a batch of ˜100 g MSNP, 17.1 L pure water is added to a 20 L beaker. 0.9 L CTAC solution (25 wt. % in H2O) is gently added while stirring at e.g., 185 rpm, using an overhead shaft for stirring. The solution is heated to 85° C. while stirring and then 72 g triethanolamine in 300 mL H2O is added when the solution reaches a temperature of 85° C. After stirring the solution for another 30 min at 85° C., 600 mL TEOS at 85° C. is gently added, followed by stirring at the same temperature for another ˜4 hr. This yields a milky particle suspension, which is allowed to cool down to room temperature. Six L of ethanol is added to the suspension to precipitate the silica particles, followed by centrifugation at 10,000 rpm for 10 mins. To remove the CTAC, the particles pellets can be resuspended in acidic ethanol (HCl/ethanol, 4:100 v/v) by sonication, followed by repetitive centrifugation (10,000 rpm×60 mins) and resuspension, which is repeated, e.g., 5 times. This is followed by particle washing in pure ethanol, e.g., for 3 times. The purified MSNPs are spun down and resuspended in H2O for the next step of activated PT-drug loading.
The mixture of silane [usually tetraethyl orthosilicate(TEOS)] and an organosilane induces the formation of MONs and PMO. In this case, in certain embodiments, the surfactant templates can be removed with less aggressive extraction procedures, in order not to destroy the inorganic-organic framework of MONs and PMO. In general, harsh pH and temperature conditions are usually employed for the extracting process. The silica-etching chemistry [alkaline or hydrofluoric acid (HF) etching] can be introduced into the synthesis to form the hollow PMO structure (see e.g., Chen et al. (2013) Adv. Mater. 25: 3100-3105). For this, the PMO layer can be directly deposited onto the surface of silica particles in order to form well-defined solid silica core/PMO shell.
The chemical stability of some families of PMOs is higher than for the silica particles under etching. Therefore, the silica core can be selectively removed under alkaline or HF etching conditions, producing hollow periodic mesoporous structure. Illustrative, but non-liming examples of fabrication protocols are described by Wu et al. (2013) Chem. Soc. Rev. 42: 3862-3875 and by Chen et al. (2014) J. Am. Chem. Soc. 136: 16326-16334.
Uniform mesoporous silica particles of different diameters can be prepared using various synthetic conditions (e.g., controlling pH values or time of reaction). For instance, a simple method for tailoring the size of well-ordered and dispersed MSNs by adjusting the pH of the reaction medium, which leads to the series of MSNs with diameter sizes ranging from 30 to 280 nm is described by Lu et al. (2009) Small, 5: 1408-1413. It also possible to control particle growth at different times of the reaction. Smaller particles (140 nm) emerged for 160 s into the reaction process grew to their final size (500 nm) in 600 s.
In one illustrative, but non-limiting embodiment, mesoporous silica nanoparticles (MSNPs) are synthesized as a large batch, as previously described by Liu et al. (2019) ACS Nano. 13(1): 38-53. By way of non-limiting illustration, in certain embodiments, this can involve the addition of 0.9 L of 25 wt % CTAC in water to 17.1 L pure water in a beaker, stirred at 85° C. 72 g triethanolamine is added, followed by 600 mL TEOS. After stirring for 4 hours and cooling to room temperature, the bare MSNPs are precipitated with ethanol and CTAC is removed by washing in acidic ethanol, with sonication. MSNPs at 80 mg/mL in ethanol are centrifuged at 21,000×g for 15 minutes to pellet the nanoparticles. After removal of the ethanol supernatant, the MSNP pellet is resuspended in 123 mM ammonium sulfate in water by bath sonication.
Potential bioaccumulation is one of the biggest limitations for silica nanodrug delivery systems in cancer. Accordingly, in certain embodiments, the porous silica nanoparticles described herein (e.g., mesoporous silica nanoparticles) are modified to improve degradation and clearance. In one illustrative, but non-limiting example, the nanoparticles comprise a mesoporous silica/hydroxyapatite (MSNs/HAP) hybrid drug carrier, that provides enhanced biodegradability of silica. Synthesis of such nanoparticles is described by Hao et al. (2015) ACS Nano, 9(10): 9614-9625.
Other approaches for improving silica nanoparticle degradation include, but are not limited to noncovalent organic doping of silica, covalent incorporation of either hydrolytically stable or redox- and enzymatically cleavable silsesquioxanes, as well as bridged silsesquioxane (BS), and periodic mesoporous organosilica (PMO) NPs. Inorganically doped silica particles such as calcium-, iron-, manganese-, and zirconium-doped NPs, can also be used (see, e.g., Croissant et al. (2017) Adv. Mater., 29: 1604634).
In certain embodiments the mesoporous silica nanoparticles can be imine-doped silica nanoparticles. These nanoparticles contain imine groups embedded within the silica framework (see, e.g., Travaglini et al. (2019) Mater. Chem. Front., 3: 111-119). These methods of increasing degradability of silica nanoparticles are illustrative and non-limiting. Using the teaching provided herein, numerous other porous silica nanoparticles modified for enhanced biodegradation will be available to one of skill in the art.
Illustrative mesoporous silica nanoparticles include, but are not limited to MCM-41, MCM-48, and SBA-15 (see, e.g., Katiyar et al. (2006) J. Chromatog. 1122(1-2): 13-20).
In various embodiments the nanoparticles comprising the drug delivery vehicles described herein (e.g., “core” silica nanoparticles) can include particles as large (e.g., average or median diameter (or other characteristic dimension) as about 1000 nm. However, in various embodiments the nanoparticles are typically less than 500 nm or less than about 300 nm as, in general, particles larger than 300 nm may be less effective in entering living cells or blood vessel fenestrations. In certain embodiments the nanoparticles range in size from about 40 nm, or from about 50 nm, or from about 60 nm up to about 100 nm, or up to about 90 nm, or up to about 80 nm, or up to about 70 nm. In certain embodiments the nanoparticles range in size from about 60 nm to about 70 nm. Some embodiments include nanoparticles having an average maximum dimension between about 50 nm and about 1000 nm. Other embodiments include nanoparticles having an average maximum dimension between about 50 nm and about 500 nm. Other embodiments include nanoparticles having an average maximum dimension between about 50 nm and about 200 nm. In some embodiments, the average maximum dimension is greater than about 20 nm, greater than about 30 nm, greater than 40 nm, or greater than about 50 nm. Other embodiments include nanoparticles having an average maximum dimension less than about 500 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm or less than about 75 nm. As used herein, the size of the nanoparticle refers to the average or median size of the primary particles, as measured by transmission electron microscopy (TEM) or similar visualization technique.
In certain embodiments the drug delivery vehicles (including lipid bilayer) have an average hydrodynamic diameter ranging from about 30 nm up to about 300 nm, or from about 40 nm up to about 200 nm, or from about 50 up to about 100 nm, or from about 60 nm up to about 90 nm, or from about 70 nm up to about 90 nm, or from about 80 nm up to about 90 nm by DLS. In certain embodiments, the drug delivery vehicles have an average hydrodynamic diameter ranging from about 79 nm up to about 86 nm by DLS. In certain embodiments, the drug delivery vehicles have an average diameter ranging from about 30 nm up to about 300 nm, or from about 50 nm up to about 250 nm, or from about 70 nm up to about 200 nm, or from about 90 nm up to about 150 nm, or from about 110 nm up to about 150 nm by cryoEM. In certain embodiments, the vehicles have an average diameter ranging from about 136 nm up to about 139 nm by cryoEM.
Illustrative mesoporous silica nanoparticles include, but are not limited to MCM-41, MCM-48, and SBA-15 (see, e.g., Katiyar et al. (2006) J. Chromatog. 1122(1-2): 13-20).
Using the teachings provided herein, silica nanoparticles are readily available to those of skill in the art and, using the teaching described herein, can be used in the fabrication of the drug delivery vehicles described herein.
Pt-Drug Loading of Nanoparticle.
In various embodiments the silica nanoparticles are loaded with platinum-based drugs using a combination of coordination and electrostatic interactions. Since the silanol group density on the silica nanoparticle surface(s) (e.g., pore surfaces) an important role in the surface binding of the activated platinum compound (e.g., DACHPt) (see
Various key parameters that govern successful drug loading are outlined in
This optimization of PT-based drug loading is illustrative and non-limiting. Using the teaching provided herein a combination of electrostatic and coordination interactions can be provided for loading of essentially any cationic, activated platinum drug.
Lipid bilayer (LB)
Bilayer composition.
The drug carrier nanoparticles described herein comprise a silica nanoparticle comprising one or more cavities, e.g., a porous nanoparticle such as a mesoporous silica nanoparticle (MSNP)), coated with a lipid bilayer. In certain embodiments the bilayer composition is optimized to provide a rapid and uniform particle coating, to provide colloidal and circulatory stability, and to provide effective cargo retention, while also permitting a desirable cargo release profile.
In certain embodiments the lipid bilayer comprises a combination of a phospholipid, and cholesterol, and in certain embodiments, a pegylated lipid (e.g., PE-PEG2000, DSPE-PEG2000), or a factionalized pegylated lipid (e.g., DSPE-PEG2000-maleimide) to facilitate conjugation with targeting moieties or other moieties including, for example, a drug.
In certain illustrative, but non-limiting embodiments the lipid bilayer can comprise: 1) one or more saturated fatty acids with C14-C20 carbon chain, such as phosphatidylethanolamine (PE), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and diactylphosphatidylcholine (DAPC); and/or 2) One or more unsaturated fatty acids with a C14-C20 carbon chain, such as 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine; and/or 3) natural lipids comprising a mixture of fatty acids with C12-C20 carbon chain, such as Egg PC, and Soy PC, sphingomyelin, and 4) cholesterol (CHOL) and/or a modified cholesterol (e.g., cholesterol hemisuccinate (CHEMS)) the like. It is noted that, in certain embodiments, in order to compensate a positive charge, it is possible to use cholesteryl hemisuccinate (CHEMS) that carries one negative charge at pH >6.5 in the formulation. These lipids are illustrative but non-limiting and numerous other lipids are known and can be incorporated into a lipid bilayer for formation of a drug delivery nanocarrier (e.g., a bilayer-coated nanoparticle).
In certain embodiments the drug carrier comprises bilayer comprising a lipid (e.g., a phospholipid), cholesterol, and a PEG functionalized lipid (e.g., a mPEG phospholipid). In certain embodiments the mPEG phospholipids comprises a C14-C18 phospholipid carbon chain from, and a PEG molecular weight from 350-5000 (e.g., MPEG 5000, MPEG 3000, MPEG 2000, MPEG 1000, MPEG 750, MPEG 550, MPEG 350, and the like). In certain embodiments the mPEG phospholipid comprises DSPE-PEG5000, DSPE-PEG3000, DSPE-PEG2000, DSPE-PEG1000, DSPE-PEG750, DSPE-PEG550, or DSPE-PEG350, PE-PEG5000, PE-PEG3000, PE-PEG2000, PE-PEG1000, PE-PEG750, PE-PEG550, PE-PEG350, and the like. MPEGs are commercially available (see, e.g.,//avantilipids.com/product-category/products/polymers-polymerizable-lipids/mpeg-phospholipids).
In certain embodiments lipid bilayer comprises an mPEG phospholipid with a phospholipid C14-C18 carbon chain, and a PEG. In certain embodiments, the PEG molecular weight ranges from about 350 Da to about 5000 Da. In certain embodiments the lipid bilayer comprises PE-PEG2K. In certain embodiments the lipid bilayer comprises PE-PEG5K.
In certain embodiments, the said lipid bilayer comprises DPSC, cholesterol, and PE-PEG. In certain embodiments, the ratio of DPSC:cholesterol:PE-PEG ranges from 40-90% DSPC:10%-50% Chol:1%-10% PE-PEG (molar ratio). In certain embodiments, the bilayer comprises DSPC:cholesterol:PE-PEG at a molar ratio of about 3:2:0.15 for DSPC, cholesterol, and PE-PEG, respectively.
In certain embodiments, the lipid bilayer comprises a cholesterol derivative selected from the group consisting of cholesterol hemisuccinate (CHEMS), lysine-based cholesterol (CHLYS), and PEGylated cholesterol (Chol-PEG). In certain embodiments, the lipid bilayer comprises CHEMS. In certain embodiments, the bilayer comprises CHEMS ranging from about 5% (mol percent) up to about 30% total lipid. In certain embodiments, the bilayer comprises about 10% or about 20% CHEMS or about 30% CHEMS or about 40% CHEMS. In certain embodiments, the cholesterol derivative is used in place of said cholesterol.
The foregoing lipid bilayer compositions are illustrative, but non-limiting. Using the teachings provided herein numerous other lipid bilayer compositions will be available to one of skill for incorporation into the silicasomes described herein.
Encapsulation of Silica Nanoparticle by Lipid Bilayer.
In various embodiments, the silica nanoparticles are coated (encapsulated) with a lipid bilayer by an ethanol exchange method that results in the formation of the bilayer encapsulated nanoparticle. The ethanol exchange s bilayer method provides rapid and uniform pore sealing, capable of entrapping drug payloads of ˜70% into the porous interior (see, e.g.,
In one illustrative, but non-limiting embodiment, following the synthesis of the drug-soaked (e.g., DACHPt soaked) in bare particles, the MSNPs are subsequently coated by a lipid bilayer (LB) as follows: Briefly, a mixture of lipids (e.g., 16 mg DSPC, 5.4 mg, cholesterol (Chol) and 2.8 mg DSPE-PEG2000), yielding a DSPC/Chol/DSPE-PEG2000 molar ratio of 3:2:0.15) is dissolved in 50 μL pure ethanol at ˜65° C. The drug-laden MSNPs (e.g., DACH-Pt laden MSNPs), are resuspended in 500 μL preheated (˜65° C.). Dextrose/HEPES buffer (e.g., 5% dextrose, 5 mM HEPES, pH7.4), is added to the lipid solution by pipette mixing. The mixture is treated by probe sonication (e.g., power=52 W) using, e.g., a 15 s/5 s on/off cycle for ˜10 min. The coated DACHPt silicasomes are washed (e.g., 3 times using a HEPES-buffered dextrose solution (5% dextrose, 5 mM HEPES, pH7.4)). The sample is processed by filtration using a 0.2 μm filter for sterilization. Using this method, or minor variations thereof, lipid bilayers of numerous different formulations can readily be formed on drug-containing silica nanoparticles.
In certain embodiments, to attach a surface LB coating, a coated lipid film procedure can be utilized in which nanoparticle (e.g., MSNP) suspensions are added to a large lipid film surface, coated on, e.g., a round-bottom flask. Using different lipid bilayer compositions, a series of experiments can be performed to find a composition and optimal lipid/particle ratio that provides rapid and uniform particle wrapping, coating and effective cargo retention and/or release upon sonication. It is believed that this lipid composition and wrapping cannot be achieved by liposomal fusion to the particle surface under low energy vortexing conditions.
In certain embodiments the drug delivery vehicles described herein can contain an additional cargo (in addition to a platinum-based drug, or other metal-based drug as described above) on the surface and/or in the cavities of the nanoparticle (when such cavities are present). In certain embodiments, the additional cargoes comprise an additional metal-based drug, as described above. In certain embodiments such additional cargoes comprise one or more cancer therapeutic agents. In certain embodiments the additional agents are cancer therapeutic agents capable of being loaded, e.g., according to the methods described herein. In certain embodiments, the additional agents comprise anti-cancer therapeutic agents that can be functionalized to be capable of being loaded, e.g., according to the methods described herein.
Additional illustrative, but non-limiting additional therapeutic agents include, but are not limited to alkaloids (e.g. irinotecan, topotecan, 10-hydroxycamptothecin, belotecan, rubitecan, vinorelbine, LAQ824, vinblastine, vincristine, homoharringtonine, trabectedin), anthracyclines (e.g. doxorubicin, epirubicin, pirarubicin, daunorubicin, rubidomycin, valrubicin, amrubicin), alkaline anthracenediones (e.g. mitoxantrone), alkaline alkylating agents (e.g. cyclophosphamide, mechlorethamine, temozolomide), purine or pyrimidine derivatives (e.g. 5-fluorouracil, 5′-deoxy-5-fluorouridine, gemcitabine, capecitabine) and protein kinase inhibitors (e.g., pazopanib, enzastaurin, vandetanib erlotinib, dasatinib, nilotinib, sunitinib, osimertinib, palbociclib, ribociclib), and the like.
In certain embodiments, embodiments the additional therapeutic agent comprise an inhibitor of the IDO pathway. Without being bound by a particular theory, it is believed that an IDO inhibitor will synergize with an inducer of cell death such as indoximod and the like (see, e.g., PCT Patent Application No: PCT/US2018/033265. In certain embodiments, the IDO pathway inhibitor comprises an agent selected from the group consisting of of D-1-methyl-tryptophan (indoximod, D-1MT), L-1-methyl-tryptophan (L-1MT), a mixture of D-1MT and L-1MT, 1-methyl-L-tryptophan (L-1MT), methylthiohydantoin-dl-tryptophan (MTH-Trp, Necrostatin), β-carbolines (e.g., 3-butyl-p-carboline), Naphthoquinone-based (e.g., annulin-B), S-allyl-brassinin, S-benzyl-brassinin, N-[2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[2-(benzo[b]thiophen-3-yl)ethyl]-S-methyl-dithiocarbamate, N-[3-(Indol-3-yl)propyl]-S-methyl-dithiocarbamate, S-hexyl-brassinin, N-[2-(indol-3-yl)ethyl]-S-benzyl-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S[(naphth-2-yl)methyl]-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-3-yl)methyl]-dithiocarbamate, N-[2-(indol-3-yl)ethyl]-S-[(pyrid-4-yl)methyl]-dithiocarbamate, 5-bromo-brassinin, Phenylimidazole-based IDO inhibitors (e.g., 4-phenylimidazole), Exiguamine A, imidodicarbonimidic diamide,N-methyl-N′-9-phenanthrenyl-monohydrochloride (NSC401366), INCB024360 (epacadostat), 1-cyclohexyl-2-(5H-imidazo[5,1-a]isoindol-5-yl)ethanol (GDC-0919), IDO1-derived peptide, NLG919, Ebselen, Pyridoxal Isonicotinoyl Hydrazone, Norharmane, CAY10581, 2-Benzyl-2-thiopseudourea hydrochloride, and 4-phenylimidazole. In certain embodiments, the IDO pathway inhibitor comprises 1-methyl-tryptophan. In certain embodiments, the IDO pathway inhibitor comprises a “D” enantiomer of 1-methyl-tryptophan (indoximod, 1-MT). In certain embodiments, the IDO pathway inhibitor comprises an “L” enantiomer of 1-methyl-tryptophan (L-MT).
In certain embodiments, the IDO pathway inhibitor, is disposed in a lipid comprising said vesicle and/or conjugated to a lipid comprising said vesicle. In certain embodiments, the IDO inhibitor is conjugated to a component of the lipid bilayer (e.g., lipid, PHGP, vitamin E, cholesterol, a fatty acid, etc.). In certain embodiments, the IDO inhibitor is conjugated to cholesterol. In certain embodiments, the IDO inhibitor is conjugated to a cholesterol derivative.
Alternatively, or additionally, in certain embodiments, hydrophobic compounds can be incorporated into the lipid bilayer surrounding the nanoparticle. Thus, for example, paclitaxel can be incorporated in the lipid bilayer.
The foregoing compounds are illustrative and non-limiting. Using the teachings provided herein, numerous other additional cargoes can be incorporated in the drug delivery vehicles described herein.
In certain embodiments the drug delivery vehicles described herein can be conjugated to one or more targeting ligands, e.g., to facilitate specific delivery in endothelial cells, to cancer cells, to fusogenic ligands, e.g., to facilitate endosomal escape, ligands to promote transport across the blood-brain barrier, and the like.
In one illustrative, but non-limiting embodiment, the delivery vehicles described herein is conjugated to a fusogenic peptide such as histidine-rich H5WYG (H2N-GLFHAIAHFIHGGWHGLIHGWYG-COOH, (SEQ ID NO:1)) (see, e.g., Midoux et al., (1998) Bioconjug. Chem. 9: 260-267).
In certain embodiments delivery vehicles described herein are conjugated to one or more targeting ligand(s) that can include antibodies as well as targeting peptides. Targeting antibodies include, but are not limited to intact immunoglobulins, immunoglobulin fragments (e.g., F(ab)′2, Fab, etc.) single chain antibodies, diabodies, affibodies, unibodies, nanobodies, and the like. In certain embodiments antibodies will be used that specifically bind a cancer marker (e.g., a tumor associated antigen). A wide variety of cancer markers are known to those of skill in the art. The markers need not be unique to cancer cells, but can also be effective where the expression of the marker is elevated in a cancer cell (as compared to normal healthy cells) or where the marker is not present at comparable levels in surrounding tissues (especially where the chimeric moiety is delivered locally).
Illustrative cancer markers include, for example, the tumor marker recognized by the ND4 monoclonal antibody. This marker is found on poorly differentiated colorectal cancer, as well as gastrointestinal neuroendocrine tumors (see, e.g., Tobi et al. (1998) Cancer Detection and Prevention, 22(2): 147-152). Other important targets for cancer immunotherapy are membrane bound complement regulatory glycoproteins CD46, CD55 and CD59, which have been found to be expressed on most tumor cells in vivo and in vitro. Human mucins (e.g. MUC1) are known tumor markers as are gp100, tyrosinase, and MAGE, which are found in melanoma. Wild-type Wilms' tumor gene WT1 is expressed at high levels not only in most of acute myelocytic, acute lymphocytic, and chronic myelocytic leukemia, but also in various types of solid tumors including lung cancer.
Acute lymphocytic leukemia has been characterized by the TAAs HLA-Dr, CD1, CD2, CD5, CD7, CD19, and CD20. Acute myelogenous leukemia has been characterized by the TAAs HLA-Dr, CD7, CD13, CD14, CD15, CD33, and CD34. Breast cancer has been characterized by the markers EGFR, HER2, MUC1, Tag-72. Various carcinomas have been characterized by the markers MUC1, TAG-72, and CEA. Chronic lymphocytic leukemia has been characterized by the markers CD3, CD19, CD20, CD21, CD25, and HLA-DR. Hairy cell leukemia has been characterized by the markers CD19, CD20, CD21, CD25. Hodgkin's disease has been characterized by the Leu-M1 marker. Various melanomas have been characterized by the HMB 45 marker. Non-Hodgkins lymphomas have been characterized by the CD20, CD19, and Ia marker. And various prostate cancers have been characterized by the PSMA and SE10 markers.
In addition, many kinds of tumor cells display unusual antigens that are either inappropriate for the cell type and/or its environment, or are only normally present during the organisms' development (e.g., fetal antigens). Examples of such antigens include the glycosphingolipid GD2, a disialoganglioside that is normally only expressed at a significant level on the outer surface membranes of neuronal cells, where its exposure to the immune system is limited by the blood-brain barrier. GD2 is expressed on the surfaces of a wide range of tumor cells including neuroblastoma, medulloblastomas, astrocytomas, melanomas, small-cell lung cancer, osteosarcomas and other soft tissue sarcomas. GD2 is thus a convenient tumor-specific target for immunotherapies.
Other kinds of tumor cells display cell surface receptors that are rare or absent on the surfaces of healthy cells, and which are responsible for activating cellular signaling pathways that cause the unregulated growth and division of the tumor cell. Examples include (ErbB2) HER2/neu, a constitutively active cell surface receptor that is produced at abnormally high levels on the surface of breast cancer tumor cells.
Other useful targets include, but are not limited to CD20, CD52, CD33, epidermal growth factor receptor and the like.
An illustrative, but not limiting list of suitable tumor markers is provided in Table 2. Antibodies to these and other cancer markers are known to those of skill in the art and can be obtained commercially or readily produced, e.g. using phage-display technology. Such antibodies can readily be conjugated to the drug delivery vehicles (e.g., LB-coated nanoparticle) described herein, e.g., in the same manner that iRGD peptide is conjugated in Example 3.
Any of the foregoing markers can be used as targets for the targeting moieties comprising delivery vehicles described herein. In certain embodiments the target markers include, but are not limited to members of the epidermal growth factor family (e.g., HER2, HER3, EGF, HER4), CD1, CD2, CD3, CD5, CD7, CD13, CD14, CD15, CD19, CD20, CD21, CD23, CD25, CD33, CD34, CD38, 5E10, CEA, HLA-DR, HM 1.24, HMB 45, 1a, Leu-M1, MUC1, PMSA, TAG-72, phosphatidyl serine antigen, and the like.
The foregoing markers are intended to be illustrative and not limiting. Other tumor associated antigens will be known to those of skill in the art.
Where the tumor marker is a cell surface receptor, a ligand to that receptor can function as targeting moieties. Similarly, mimetics of such ligands can also be used as targeting moieties. Thus, in certain embodiments peptide ligands, and other ligands, can be used in addition to or in place of various antibodies. An illustrative, but non-limiting list of suitable targeting ligands is shown in Table 3. In certain embodiments any one or more of these peptides can be conjugated to a drug delivery vehicle described herein.
In certain embodiments the nanoparticle drug delivery vehicles described herein can be conjugated to moieties that facilitate stability in circulation and/or that hide the drug delivery vehicle from the reticuloendothelial system (RES) and/or that facilitate transport across a barrier (e.g., a stromal barrier, the blood brain barrier, etc.), and/or into a tissue. In certain embodiments the drug delivery vehicle is conjugated to transferrin or ApoE to facilitate transport across the blood brain barrier. In certain embodiments the drug delivery vehicle is conjugated to folate.
Methods of coupling the nanoparticle drug delivery vehicle to targeting (or other) agents are well known to those of skill in the art. Examples include, but are not limited to the use of biotin and avidin or streptavidin (see, e.g., U.S. Pat. No. 4,885,172 A), by traditional chemical reactions using, for example, bifunctional coupling agents such as glutaraldehyde, diimide esters, aromatic and aliphatic diisocyanates, bis-p-nitrophenyl esters of dicarboxylic acids, aromatic disulfonyl chlorides and bifunctional arylhalides such as 1,5-difluoro-2,4-dinitrobenzene; p,p′-difluoro m,m′-dinitrodiphenyl sulfone, sulfhydryl-reactive maleimides, and the like. Appropriate reactions which may be applied to such couplings are described in Williams et al. Methods in Immunology and Immunochemistry Vol. 1, Academic Press, New York 1967.
In one illustrative but non-limiting approach a peptide (e.g., iRGD) is coupled to the nanoparticle drug delivery vehicle by a lipid coupled to a linker (e.g., DSPE-PEG2000-maleimide), allowing thiol-maleimide coupling to the cysteine-modified peptide. It will also be recognized that in certain embodiments the targeting (and other) moieties can be conjugated to other moieties comprising the lipid bilayer. In certain embodiments possible to improve tumor delivery of the Pt-based drug loaded nanoparticle through co-administration (not conjugated) of the iRGD peptide to enhance particle transcytosis.
The former conjugates and coupling methods are illustrative and non-limiting. Using the teachings provided herein, numerous other moieties can be conjugated to the nanoparticle drug delivery vehicles described herein by any of a variety of methods.
In some embodiments, the nanoparticle drug delivery vehicles described herein are administered alone or in a mixture with a physiologically-acceptable carrier (such as physiological saline or phosphate buffer) selected in accordance with the route of administration and standard pharmaceutical practice. For example, when used as an injectable, the nanoparticle drug delivery vehicles can be formulated as a sterile suspension, dispersion, or emulsion with a pharmaceutically acceptable carrier. In certain embodiments normal saline can be employed as the pharmaceutically acceptable carrier. Other suitable carriers include, e.g., water, buffered water, 0.4% saline, 0.3% glycine, 5% glucose and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. In compositions comprising saline or other salt-containing carriers, the carrier is preferably added following nanoparticle drug delivery vehicle formation. Thus, after the nanoparticle drug delivery vehicle is formed and loaded with suitable drug(s), the vehicles can be diluted into pharmaceutically acceptable carriers such as normal saline.
The pharmaceutical compositions may be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solutions, suspensions, dispersions, emulsions, etc., may be packaged for use or filtered under aseptic conditions. In certain embodiments the nanoparticle drug delivery vehicles described herein are lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may also contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
Additionally, in certain embodiments, the pharmaceutical formulation may include lipid-protective agents that protect lipids against free-radical and lipid-peroxidative damage on storage. Lipophilic free-radical quenchers, such as alpha-tocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.
The concentration of the nanoparticle drug delivery vehicles in the pharmaceutical formulations can vary widely, e.g., from less than approximately 0.05%, usually at least approximately 2 to 5% to as much as 10 to 50%, or to 40%, or to 30% by weight and are selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. For example, the concentration may be increased to lower the fluid load associated with treatment. This may be particularly desirable in patients having atherosclerosis-associated congestive heart failure or severe hypertension. Alternatively, nanoparticle drug delivery vehicles composed of irritating lipids may be diluted to low concentrations to lessen inflammation at the site of administration. The amount of nanoparticle drug delivery vehicles administered will depend upon the particular drug used, the disease state being treated and the judgment of the clinician but will generally be between approximately 0.01 and approximately 50 mg per kilogram of body weight, preferably between approximately 0.1 and approximately 5 mg per kg of body weight.
In some embodiments, it is desirable to include polyethylene glycol (PEG)-modified phospholipids in the LB-coated nanoparticles or vesicles. Alternatively, or additionally, in certain embodiments, PEG-ceramide, or ganglioside GMI-modified lipids can be incorporated in the nanoparticle drug delivery vehicles described herein. Addition of such components helps prevent delivery vehicle aggregation and provides for increasing circulation lifetime and increasing the delivery of the loaded delivery vehicles to the target tissues.
In some embodiments, overall nanoparticle drug delivery vehicle charge is an important determinant in clearance of the vehicle from the blood. It is believed that highly charged delivery vehicles (e.g., zeta potential >+35 mV) will be typically taken up more rapidly by the reticuloendothelial system (see, e.g., Juliano (1975), Biochem. Biophys. Res. Commun. 63: 651-658 discussing liposome clearance by the RES). Drug delivery vehicles with prolonged circulation half-lives are typically desirable for therapeutic uses. For instance, in certain embodiments, drug delivery nanoparticle drug delivery vehicles that are maintained from 8 hrs, or 12 hrs, or 24 hrs, or greater are desirable.
In another example of their use, the nanoparticle drug delivery vehicles can be incorporated into a broad range of topical dosage forms including but not limited to gels, oils, emulsions, and the like, e.g., for the treatment of a topical cancer. For instance, in some embodiments the suspension containing the drug delivery vehicles is formulated and administered as a topical cream, paste, ointment, gel, lotion, and the like.
In some embodiments, pharmaceutical formulations comprising the nanoparticle drug delivery vehicles described herein additionally incorporate a buffering agent. The buffering agent may be any pharmaceutically acceptable buffering agent. Buffer systems include, but are not limited to citrate buffers, acetate buffers, borate buffers, and phosphate buffers. Examples of buffers include, but are not limited to citric acid, sodium citrate, sodium acetate, acetic acid, sodium phosphate and phosphoric acid, sodium ascorbate, tartaric acid, maleic acid, glycine, sodium lactate, lactic acid, ascorbic acid, imidazole, sodium bicarbonate and carbonic acid, sodium succinate and succinic acid, histidine, and sodium benzoate, benzoic acid, and the like.
In some embodiments, pharmaceutical formulations comprising the nanoparticle drug delivery vehicles described herein additionally incorporate a chelating agent. The chelating agent may be any pharmaceutically acceptable chelating agent. Chelating agents include, but are not limited to ethylene diaminetetraacetic acid (also synonymous with EDTA, edetic acid, versene acid, and sequestrene), and EDTA derivatives, such as dipotassium edetate, disodium edetate, edetate calcium disodium, sodium edetate, trisodium edetate, and potassium edetate. Other chelating agents include citric acid (e.g., citric acid monohydrate) and derivatives thereof. Derivatives of citric acid include anhydrous citric acid, trisodiumcitrate-dihydrate, and the like. Still other chelating agents include, but are not limited to, niacinamide and derivatives thereof and sodium deoxycholate and derivatives thereof.
In some embodiments, pharmaceutical formulations comprising the nanoparticle drug delivery vehicles described herein additionally incorporate an antioxidant. The antioxidant may be any pharmaceutically acceptable antioxidant. Antioxidants are well known to those of ordinary skill in the art and include, but are not limited to, materials such as ascorbic acid, ascorbic acid derivatives (e.g., ascorbylpalmitate, ascorbylstearate, sodium ascorbate, calcium ascorbate, etc.), butylated hydroxy anisole, buylated hydroxy toluene, alkylgallate, sodium meta-bisulfate, sodium bisulfate, sodium dithionite, sodium thioglycollic acid, sodium formaldehyde sulfoxylate, tocopherol and derivatives thereof, (d-alpha tocopherol, d-alpha tocopherol acetate, dl-alpha tocopherol acetate, d-alpha tocopherol succinate, beta tocopherol, delta tocopherol, gamma tocopherol, and d-alpha tocopherol polyoxyethylene glycol 1000 succinate) monothioglycerol, sodium sulfite and N-acetyl cysteine. In certain embodiments such materials, when present, are typically added in ranges from 0.01 to 2.0%.
In some embodiments, pharmaceutical formulations comprising the nanoparticle drug delivery vehicles described herein are formulated with a cryoprotectant. The cryoprotecting agent may be any pharmaceutically acceptable cryoprotecting agent. Common cryoprotecting agents include, but are not limited to, histidine, polyethylene glycol, polyvinyl pyrrolidine, lactose, sucrose, mannitol, polyols, and the like.
In some embodiments, pharmaceutical formulations comprising the nanoparticle drug delivery vehicles described herein are formulated with an isotonic agent. The isotonic agent can be any pharmaceutically acceptable isotonic agent. This term is used in the art interchangeably with iso-osmotic agent, and is known as a compound that is added to the pharmaceutical preparation to increase the osmotic pressure, e.g., in some embodiments to that of 0.9% sodium chloride solution, which is iso-osmotic with human extracellular fluids, such as plasma. Illustrative isotonicity agents include, but are not limited to, sodium chloride, mannitol, sorbitol, lactose, dextrose and glycerol.
In certain embodiments pharmaceutical formulations of the the nanoparticle drug delivery vehicles described herein may optionally comprise a preservative. Common preservatives include, but are not limited to, those selected from the group consisting of chlorobutanol, parabens, thimerosol, benzyl alcohol, and phenol. Suitable preservatives include but are not limited to: chlorobutanol (e.g., 0.3-0.9% w/v), parabens (e.g., 0.01-5.0%), thimerosal (e.g., 0.004-0.2%), benzyl alcohol (e.g., 0.5-5%), phenol (e.g., 0.1-1.0%), and the like.
In some embodiments, pharmaceutical formulations comprising the nanoparticle drug delivery vehicles described herein are formulated with a humectant, e.g., to provide a pleasant mouth-feel in oral applications. Humectants known in the art include, but are not limited to, cholesterol, fatty acids, glycerin, lauric acid, magnesium stearate, pentaerythritol, and propylene glycol.
In some embodiments, an emulsifying agent is included in the formulations, for example, to ensure complete dissolution of all excipients, especially hydrophobic components such as benzyl alcohol. Many emulsifiers are known in the art, e.g., polysorbate 60.
For some embodiments related to oral administration, it may be desirable to add a pharmaceutically acceptable flavoring agent and/or sweetener. Compounds such as saccharin, glycerin, simple syrup, and sorbitol are useful as sweeteners.
Administration
The nanoparticle drug delivery vehicles described herein can be administered to a subject (e.g., patient) by any of a variety of techniques.
In certain embodiments the nanoparticle drug delivery vehicles and/or pharmaceutical formulations thereof are administered parenterally, e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In some embodiments, the pharmaceutical compositions are administered intravenously, intraarterially, or intraperitoneally by a bolus injection (see, e.g., U.S. Pat. Nos. 3,993,754; 4,145,410; 4,235,871; 4,224,179; 4,522,803; and 4,588,578 describing administration of liposomes). Particular pharmaceutical formulations suitable for this administration are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). Typically, the formulations comprise a solution of the nanoparticle drug delivery vehicles suspended in an acceptable carrier, preferably an aqueous carrier. As noted above, suitable aqueous solutions include, but are not limited to physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiological (e.g., 0.9% isotonic) saline buffer and/or in certain emulsion formulations. The solution(s) can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. In certain embodiments the active agent(s) can be provided in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. For transmucosal administration, and/or for blood/brain barrier passage, penetrants appropriate to the barrier to be permeated can be used in the formulation. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc., e.g., as described above.
In other methods, the pharmaceutical formulations containing the nanoparticle drug delivery vehicles described herein may be contacted with the target tissue by direct application of the preparation to the tissue. The application may be made by topical, “open” or “closed” procedures. By “topical” it is meant the direct application of the pharmaceutical preparation to a tissue exposed to the environment, such as the skin, oropharynx, external auditory canal, and the like. Open procedures are those procedures that include incising the skin of a patient and directly visualizing the underlying tissue to which the pharmaceutical formulations are applied. This is generally accomplished by a surgical procedure, such as a thoracotomy to access the lungs, abdominal laparotomy to access abdominal viscera, or other direct surgical approaches to the target tissue. Closed procedures are invasive procedures in which the internal target tissues are not directly visualized, but accessed via inserting instruments through small wounds in the skin. For example, the preparations may be administered to the peritoneum by needle lavage. Likewise, the pharmaceutical preparations may be administered to the meninges or spinal cord by infusion during a lumbar puncture followed by appropriate positioning of the patient as commonly practiced for spinal anesthesia or metrizamide imaging of the spinal cord. Alternatively, the preparations may be administered through endoscopic devices. In certain embodiments the pharmaceutical formulations are introduced via a cannula.
In certain embodiments the pharmaceutical formulations comprising the nanoparticle drug delivery vehicles described herein are administered via inhalation (e.g., as an aerosol). Inhalation can be a particularly effective delivery route for administration to the lungs and/or to the brain. For administration by inhalation, the nanoparticle drug delivery vehicles are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
In certain embodiments, the nanoparticle drug delivery vehicles described herein are formulated for oral administration. For oral administration, suitable formulations can be readily formulated by combining the drug delivery vehicles with pharmaceutically acceptable carriers suitable for oral delivery well known in the art. Such carriers enable the active agent(s) described herein to be formulated as tablets, pills, dragees, caplets, lozenges, gelcaps, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. For oral solid formulations such as, for example, powders, capsules and tablets, suitable excipients can include fillers such as sugars (e.g., lactose, sucrose, mannitol and sorbitol), cellulose preparations (e.g., maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose), synthetic polymers (e.g., polyvinylpyrrolidone (PVP)), granulating agents; and binding agents. If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, solid dosage forms may be sugar-coated or enteric-coated using standard techniques. The preparation of enteric-coated particles is disclosed for example in U.S. Pat. Nos. 4,786,505 and 4,853,230.
In various embodiments the nanoparticle drug delivery vehicles described herein can be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. Methods of formulating active agents for rectal or vaginal delivery are well known to those of skill in the art (see, e.g., Allen (2007) Suppositories, Pharmaceutical Press) and typically involve combining the active agents with a suitable base (e.g., hydrophilic (PEG), lipophilic materials such as cocoa butter or Witepsol W45), amphiphilic materials such as Suppocire AP and polyglycolized glyceride, and the like). The base is selected and compounded for a desired melting/delivery profile.
The route of delivery of the nanoparticle drug delivery vehicles described herein can also affect their distribution in the body. Passive delivery of the drug delivery vehicles involves the use of various routes of administration e.g., parenterally, although other effective administration forms, such as intraarticular injection, inhalant mists, orally active formulations, transdermal iontophoresis, or suppositories are also envisioned. Each route produces differences in localization of the drug delivery vehicle.
Because dosage regimens for pharmaceutical agents are well known to medical practitioners, the amount of the liposomal pharmaceutical agent formulations that is effective or therapeutic for the treatment of a disease or condition in mammals and particularly in humans will be apparent to those skilled in the art. The optimal quantity and spacing of individual dosages of the formulations herein will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and such optima can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, e.g., the number of doses given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.
Typically, the nanoparticle drug delivery vehicles described herein and/or pharmaceutical formations thereof described herein are used therapeutically in animals (including man) in the treatment of various cancers. In certain embodiments the drug delivery vehicles and/or pharmaceutical formations thereof described herein are particularly well suited in conditions that require: (1) repeated administrations; and/or (2) the sustained delivery of the drug in its bioactive form; and/or (3) the decreased toxicity with suitable efficacy compared with the free drug(s) in question. In various embodiments the nanoparticle drug delivery vehicles and/or pharmaceutical formations thereof are administered in a therapeutically effective dose. The term “therapeutically effective” as it pertains to the nanoparticle drug delivery vehicles described herein and formulations thereof means that the metal-based drug(s) (e.g., platinum-based chemotherapeutic agents) inhibitor contained therein, alone or in combination with other drugs, produces a desirable effect on the cancer. Such desirable effects include, but are not limited to slowing and/or stopping tumor growth and/or proliferation and/or slowing and/or stopping proliferation of metastatic cells, reduction in size and/or number of tumors, and/or elimination of tumor cells and/or metastatic cells, and/or prevention of recurrence of the cancer following remission.
Exact dosages will vary depending upon such factors as the particular metal-based drug and the desirable medical effect, as well as patient factors such as age, sex, general condition, and the like. Those of skill in the art can readily take these factors into account and use them to establish effective therapeutic concentrations without resort to undue experimentation.
For administration to humans (or to non-human mammals) in the curative, remissive, retardive, or prophylactic treatment of diseases the prescribing physician will ultimately determine the appropriate dosage of the drug for a given human (or non-human) subject, and this can be expected to vary according to the age, weight, and response of the individual as well as the nature and severity of the patient's disease. In certain embodiments the dosage of the drug provided by the nanoparticle drug delivery vehicles can be approximately equal to that employed for the free drug. However as noted above, the nanoparticle drug delivery vehicles described herein can significantly reduce the toxicity of the drug(s) administered thereby and significantly increase a therapeutic window. Accordingly, in some cases dosages in excess of those prescribed for the free drug(s) will be utilized.
In certain embodiments, the dose of each of the drug(s) (e.g., PT-based drugs)) administered at a particular time point will be in the range from about 1 to about 1,000 mg/m2/day, or to about 800 mg/m2/day, or to about 600 mg/m2/day, or to about 400 mg/m2/day. For example, in certain embodiments a dosage (dosage regiment) is utilized that provides a range from about 1 to about 350 mg/m2/day, 1 to about 300 mg/m2/day, 1 to about 250 mg/m2/day, 1 to about 200 mg/m2/day, 1 to about 150 mg/m2/day, 1 to about 100 mg/m2/day, from about 5 to about 80 mg/m2/day, from about 5 to about 70 mg/m2/day, from about 5 to about 60 mg/m2/day, from about 5 to about 50 mg/m2/day, from about 5 to about 40 mg/m2/day, from about 5 to about 20 mg/m2/day, from about 10 to about 80 mg/m2/day, from about 10 to about 70 mg/m2/day, from about 10 to about 60 mg/m2/day, from about 10 to about 50 mg/m2/day, from about 10 to about 40 mg/m2/day, from about 10 to about 20 mg/m2/day, from about 20 to about 40 mg/m2/day, from about 20 to about 50 mg/m2/day, from about 20 to about 90 mg/m2/day, from about 30 to about 80 mg/m2/day, from about 40 to about 90 mg/m2/day, from about 40 to about 100 mg/m2/day, from about 80 to about 150 mg/m2/day, from about 80 to about 140 mg/m2/day, from about 80 to about 135 mg/m2/day, from about 80 to about 130 mg/m2/day, from about 80 to about 120 mg/m2/day, from about 85 to about 140 mg/m2/day, from about 85 to about 135 mg/m2/day, from about 85 to about 135 mg/m2/day, from about 85 to about 130 mg/m2/day, or from about 85 to about 120 mg/m2/day. In certain embodiments the does administered at a particular time point may also be about 130 mg/m2/day, about 120 mg/m2/day, about 100 mg/m2/day, about 90 mg/m2/day, about 85 mg/m2/day, about 80 mg/m2/day, about 70 mg/m2/day, about 60 mg/m2/day, about 50 mg/m2/day, about 40 mg/m2/day, about 30 mg/m2/day, about 20 mg/m2/day, about 15 mg/m2/day, or about 10 mg/m2/day.
In certain embodiments, the dose administered may be higher or lower than the dose ranges described herein, depending upon, among other factors, the bioavailability of the composition, the tolerance of the individual to adverse side effects, the mode of administration and various factors discussed above. Dosage amount and interval may be adjusted individually to provide plasma levels of the composition that are sufficient to maintain therapeutic effect, according to the judgment of the prescribing physician. Skilled artisans will be able to optimize effective local dosages without undue experimentation in view of the teaching provided herein.
Multiple doses (e.g., continuous or bolus) of the compositions as described herein may also be administered to individuals in need thereof of the course of hours, days, weeks, or months. For example, but not limited to, 1, 2, 3, 4, 5, or 6 times daily, every other day, every 10 days, weekly, monthly, twice weekly, three times a week, twice monthly, three times a month, four times a month, five times a month, every other month, every third month, every fourth month, etc.
In various embodiments methods of treatment using the PT-drug loaded nanoparticle drug delivery vehicles described herein and/or pharmaceutical formulation(s) comprising the nanoparticle drug delivery vehicles described herein are provided. In certain embodiments the method(s) comprise a method of treating a cancer. In certain embodiments the method can comprise administering to a subject in need thereof an effective amount of a nanoparticle drug delivery vehicle described herein, and/or a pharmaceutical formulation comprising the nanoparticle drug delivery vehicles.
In certain embodiments the nanoparticle drug delivery vehicles described herein (containing one or more platinum-based drug(s)) and/or pharmaceutical formulation is a primary therapy in a chemotherapeutic regimen. In certain embodiments the nanoparticle drug delivery vehicle and/or pharmaceutical formulation is a component in an adjunct therapy in addition to chemotherapy using one or more other chemotherapeutic agents, and/or surgical resection of a tumor mass, and/or radiotherapy.
In certain embodiments the nanoparticle drug delivery vehicles and/or pharmaceutical formulation thereof is a component in a multi-drug chemotherapeutic regimen. In certain embodiments the multi-drug chemotherapeutic regimen comprises at least two drugs selected from the group consisting of irinotecan (IRIN), oxaliplatin (OX), 5-fluorouracil (5-FU), and leucovorin (LV). In certain embodiments the multi-drug chemotherapeutic regimen comprises at least three drugs selected from the group consisting of irinotecan (IRIN), oxaliplatin (OX), 5-fluorouracil (5-FU), and leucovorin (LV). In certain embodiments the multi-drug chemotherapeutic regimen comprises at least irinotecan (IRIN), oxaliplatin (OX), 5-fluorouracil (5-FU), and leucovorin (LV).
In various embodiments the nanoparticle drug delivery vehicles and/or pharmaceutical formulation(s) thereof described herein are effective for treating any of a variety of cancers. In certain embodiments the cancer is pancreatic ductal adenocarcinoma (PDAC). In certain embodiments the cancer is a cancer selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi sarcoma, lymphoma), anal cancer, appendix cancer, astrocytomas, atypical teratoid/rhabdoid tumor, bile duct cancer, extrahepatic cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytomas, glioblastoma, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, breast cancer, bronchial tumors, burkitt lymphoma, carcinoid tumors (e.g., childhood, gastrointestinal), cardiac tumors, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, duct cancers e.g. (bile, extrahepatic), ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma), fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancers, extragonadal cancers, central nervous system), gestational trophoblastic tumor, brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, langerhans cell cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kaposi sarcoma, kidney cancer (e.g., renal cell, Wilm's tumor, and other kidney tumors), langerhans cell histiocytosis, laryngeal cancer, leukemia, acute lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myelogenous (CML), hairy cell, lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer (e.g., childhood, non-small cell, small cell), lymphoma (e.g., AIDS-related, Burkitt (e.g., non-Hodgkin lymphoma), cutaneous T-Cell (e.g., mycosis fungoides, Sézary syndrome), Hodgkin, non-Hodgkin, primary central nervous system (CNS)), macroglobulinemia, Waldenström, male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma (e.g., childhood, intraocular (eye)), merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, chronic myeloid leukemia (CML), multiple myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity cancer, lip and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., Ewing, Kaposi, osteosarcoma, rhadomyosarcoma, soft tissue, uterine), Sézary syndrome, skin cancer (e.g., melanoma, merkel cell carcinoma, basal cell carcinoma, nonmelanoma), small intestine cancer, squamous cell carcinoma, squamous neck cancer with occult primary, stomach (gastric) cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilm's tumor.
In various embodiments, the Pt-drug loaded drug delivery vehicles (silicasomes) described herein are effective to treat a cancer that is routinely treated using a platinum-based therapeutic. Illustrative cancers include, but are not limited to pancreatic cancer, colorectal cancer, and cervical cancer.
In certain embodiments the nanoparticle drug delivery vehicles described herein are not conjugated to an iRGD peptide and the drug delivery vehicles are administered in conjunction with an iRGD peptide (e.g., the drug delivery vehicle and the iRGD peptide are co-administered as separate formulations).
In certain embodiments, the drug delivery vehicles described herein are administered as a component FOLFIRINOX protocol that additionally includes folinic acid, 5-fluorouracil, and irinotecan.
In certain embodiments, the drug delivery vehicles described herein are administered in conjunction with a checkpoint inhibitor (e.g., a PD-L1 inhibitor, a PD-1 inhibitor, a CTLA-4 inhibitor, etc.). In certain embodiments, the checkpoint inhibitor comprises one or more PD-L1 inhibitors. In certain embodiments, the checkpoint inhibitor comprises an anti-PD-L1 antibody. In certain embodiments, the checkpoint inhibitor comprises an anti-PD-L1 antibody selected from the group consisting of Atezolizumab, Avelumab, Durvalumab, BMS-936559, RG-7446. MPDL3280A, MEDI-4736, and MSB0010718C. In certain embodiments, the checkpoint inhibitor comprises a peptidic PD-L1 inhibitor. In certain embodiments, the PD-L1 inhibitor comprise a moiety selected from the group consisting of AUNP12, CA-170, and BMS-986189.
In certain embodiments, the checkpoint inhibitor comprises a PD1 inhibitor. In certain embodiments, the checkpoint inhibitor comprises an anti-PD1 antibody. In certain embodiments, the checkpoint inhibitor comprises an anti-PD1 antibody selected from the group consisting of Nivolumab, Pembrolizumab, Cemiplimab, avelumab, durvalumab, and atezolizumab.
In certain embodiments, the checkpoint inhibitor comprises an fc fusion with PD-L2. In certain embodiments, the checkpoint inhibitor comprises AMP224.
In certain embodiments, the checkpoint inhibitor comprises CTLA-4 inhibitor. In certain embodiments, the CTLA-4 inhibitor comprises Ipilimumab.
In certain embodiments, the checkpoint inhibitor comprises a bispecific antibody that binds to two checkpoint inhibitors, or an antibody that binds to a checkpoint inhibitor attached to a cytokine. In certain embodiments, the checkpoint inhibitor comprises a bispecific antibody that binds to two checkpoint inhibitors. In certain embodiments, the bispecific antibody comprises an antibody that binds to PD-1 attached to an antibody that binds to PD-L1, or an antibody that binds to PD-1 attached to an antibody that binds to CTLA4, or an antibody that binds to PD-L1 attached to an antibody that binds to CTLA4. In certain embodiments, the bispecific antibody comprises an antibody that binds to PD-1 attached to an antibody that binds to CTLA4. In certain embodiments, the checkpoint inhibitor comprises a cytokine attached to an antibody that binds to a checkpoint inhibitor. In certain embodiments, the checkpoint inhibitor comprises a cytokine attached to an antibody selected from the group consisting of anti-PD-1, anti-PD-L1, and CTLA4. In certain embodiments, the checkpoint inhibitor comprises cytokine attached to an anti-PD-1 antibody. In certain embodiments, the checkpoint inhibitor comprises an IL-7 attached to an anti-PD-1 antibody.
In various embodiments of these treatment methods, the Pt-based drug loaded nanoparticle drug delivery vehicles described herein and/or pharmaceutical formulations are administered via a route selected from the group consisting of intravenous administration, intraarterial administration, intracerebral administration, intrathecal administration, oral administration, aerosol administration, administration via inhalation (including intranasal and intratracheal delivery, intracranial administration via a cannula, and subcutaneous or intramuscular depot deposition. In certain embodiments the drug delivery vehicles and/or pharmaceutical formulations thereof are administered as an injection, from an IV drip bag, or via a drug-delivery cannula. In various embodiments the subject is a human and in other embodiments the subject is a non-human mammal.
While the drug delivery vehicles described herein are often used in the treatment of cancer, depending on the metal-based drug(s) loaded into the vehicle the drug delivery vehicles find utility in a number of other indications such as autoimmune disease (e.g., rheumatoid arthritis), systemic bacterial, fungal, or viral infection, as imaging reagents, and the like.
In certain embodiments, kits are provided containing reagents for the practice of any of the methods described herein. In certain embodiments the kit comprises a container containing a drug delivery vehicle described herein.
Additionally, in certain embodiments, the kits can include instructional materials disclosing the means of the use of the nanoparticle drug delivery vehicles described herein as a cancer therapeutic.
In addition, the kits optionally include labeling and/or instructional materials providing directions (e.g., protocols) for the use of the materials described herein, e.g., alone or in combination for the treatment of various cancers. Instructional materials can also include recommended dosages, description(s) of counter indications, and the like.
While the instructional materials in the various kits typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
The following examples are offered to illustrate, but not to limit the claimed invention.
In this example we describe the development of a mesoporous silica nanoparticle (MSNP) based platform for high-dose loading of a range of activated platinum (Pt) chemo agents that could be attached to the porous interior through the use of electrostatic and coordination chemistry under weak-basic pH conditions. In addition to the design feature for improving drug delivery, the MSNP could also be encapsulated in a coated lipid bilayer (silicasome), to improve the colloidal stability after intravenous (IV) injection. We demonstrate improved pharmacokinetics and intratumor delivery of encapsulated oxaliplatin (DACHPt) over free drug in an orthotopic Kras-derived pancreatic cancer (PDAC) model. Not only did IV injection of the DACHPt silicasome provide more efficacious cytotoxic tumor cell killing, but could also demonstrate that chemotherapy-induced cell death is accompanied by the features of immunogenic cell death (ICD) as well as a dramatic reduction in bone marrow toxicity. The added features of an immunogenic response were reflected by calreticulin and HMGB1 expression, along with increased CD8+/FoxP3+ T-cell ratios and evidence of perforin and granzyme B release at the tumor site. Subsequent performance of a survival experiment demonstrated that the DACHPt silicasome generate a significant improved survival outcome, which could be extended by co-administration of an anti-PD-1 antibody.
Three common Pt drug payloads were used in this study to develop our drug loading strategy, namely oxaliplatin, cisplatin and dichloro (ethylenediamine) platinum (Pt(en)Cl2) (
The synthesis procedure for deriving the Pt-silicasomes nanocarriers is schematically outlined in
Following the synthesis of the DACHPt soaked-in bare particles, the MSNPs were subsequently coated by a lipid bilayer (LB), which were accomplished by an ethanol exchange method that results in the formation of a bilayer with a molar ratio of 3:2:0.15 for DSPC:cholesterol:PE-PEG2K, respectively[4b]. The LB provided rapid and uniform pore sealing, capable of entrapping drug payloads of ˜70% into the porous interior (
In order to confirm the LC % analysis, we conducted elemental mapping at nanostructural level through performance of energy-dispersive X-ray spectroscopy (EDS) in combination with scanning transmission electron microscope (STEM) (
In order to show the broader application of our loading approach, we also carried out drug loading studies for DAPt (activated cisplatin) and EDAPt (activated Pt(en)Cl2) at pH 8.5, followed by lipid coating. ICP-MS analysis confirmed that the LC % and EE % were improved 5.6 to 8.5-fold, respectively, compared to passive drug loading (
In order to investigate the in vivo relevance of improved DACHPt delivery by silicasomes in an animal tumor model, a freshly prepared batch of DACHPt silicasomes were prepared and characterized, as shown in
To further investigate the impact of the silicasome carrier in an orthotopic PDAC model, KPC cells were surgically implanted into the tail of the pancreas to establish a primary cancer that develops metastatic spread and resembles human PDAC in the expression of a robust dysplastic stroma and poor anti-PDAC immunity[20]. For ease of tumor visualization, the KPC cells were stably transfected with a luciferase vector, as previously described[4a, 21] In order to assess the pharmacokinetics (PK) of the DACHPt silicasome, the plasma Pt content was quantitatively assessed by ICP-MS in animals receiving a single IV injection of 50 mg/kg MSNPs that contain Pt drug equal to 10 mg/kg oxaliplatin. IV injection of non-encapsulated oxaliplatin (at identical Pt molar dose) served as the free drug control because free DACHPt leads to drug precipitates when interacting with Cl ions in the blood[7b]. Blood collection was performed at 5 mins, 3 h, 6 h, 24 h and 48 h after IV injection, followed by ICP-MS quantification. Circulatory half-life (t1/2) was calculated to be 10.4±1.3 h and 0.35±0.17 h for the silicasome vs. the free drug, respectively, in making use of a one-compartment model[4] (
An efficacy study was performed by IV injection of the silicasome carrier eight days after orthotopic KPC implantation in the pancreas of B6129SF1/J mice (
Overcoming chemotherapy side effects is an important objective of nano-based chemo drug delivery[22]. In order to determine if treatment safety also applies to the DACHPt silicasome, histological analysis was performed on all the main organs and the bone marrow. In contrast to the major (>50%) reduction in cellularity of the hemopoetic cells by the free drug in the bone marrow, there was no noticeable hypocellularity during treatment with the DACHPt silicasome, similar to the effect of saline administration (
While clearly efficacious for chemotherapy delivery in the orthotopic PDAC model, we were also interested to see whether encapsulated DACHPt exerts an immunogenic cell death (ICD) effect, similar to what was described for oxaliplatin[23]. ICD represents a unique form of apoptotic cell death that is accompanied by a chemo-induced cell stress response that is characterized by the expression of calreticulin (CRT) and the release of adjuvant stimuli[23a, 24]. While CRT expression on the dying tumor cell surface provides an “eat-me” signal to antigen-presenting cells (APC)[23], the subsequent release of high-mobility group box 1 (HMGB1) from the dying tumor cells provide an adjuvant stimulus for APC maturation (
Given this background, we proceeded to the performance of IHC staining for ICD markers, and immune activation in the tumor tissues harvested from the animals treated with the DACHPt silicasomes and oxaliplatin in
To confirm the therapeutic benefit of the DACHPt silicasome in the efficacy study, we also performed a survival outcome study in the KPC orthotopic model, using the same dosimetry, and frequency of DACHPt silicasome administration as for
In this study, we developed a facile and effective drug loading approach to allow Pt drug delivery by a silicasome nanocarrier. This required the use of activated Pt drugs for efficient loading, and electrostatic/coordination attachment to the porous interior under weak basic conditions. Additional coating of the MSNPs surface by a lipid bilayer allowed secured entrapment of the Pt drug molecules led to providing colloidal stability and successful systemic biodistribution. This was demonstrated by the improved PK profile and intratumoral drug delivery by the DACHPt laden silicasome over free drug in an orthotopic KPC model. IV injection of DACHPt silicasome also demonstrated efficacy in the chemotherapy response of the tumor along with a significant reduction in bone marrow toxicity. In addition, the tumor killing response was associated with an immunogenic cell death response that was reflected by increased biomarker for ICD and cytotoxic T-cell generated tumor cell death. In a separate survival experiment, IV injection of DACHPt silicasome led to a significantly improved survival benefit compared to the free drug. Moreover, the efficacy of the chemo-immunotherapy response was further enhanced by the co-administration of anti-PD-1 antibody. Collectively, the successful development of a facile and versatile Pt silicasome offers great promise in improving the therapeutic index of Pt-based chemotherapy agents, both as a monotherapy as well as for combination therapy with the new checkpoint inhibitors, based on the additional dimension of an immunogenic effect of the delivered drugs.
Materials
Tetraethylorthosicate (TEOS), triethanolamine (TEA-ol), triethylamine (TEA) cetyltrimethylammonium chloride solution (CTAC, 25 wt % in water), silver nitrate(AgNO3), nitric acid (HNO3), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), dextrose, dichloro(1,2-diaminocyclohexane)platinum(II) (DACHPtCl2), cis-diammineplatinum(II) dichloride (cisplatin), and dichloro(ethylenediamine)platinum(II) (Pt(en)Cl2) were purchased from Sigma-Aldrich, USA. 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phospho-ethanol amine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000), and cholesterol (Chol) were purchased from Avanti Polar Lipids, USA. Oxaliplatin was purchased from LC Laboratories, USA. Murine anti-PD-1 antibody (#BE0146) and InVivoPure pH 7.0 dilution buffer (#IP0070) were purchased Bio X Cell (New Hampshire, USA). Penicillin, streptomycin, Dulbecco's modified Eagle medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 Medium were purchased from Invitrogen. Fetal bovine serum (FBS) was purchased from Gemini Bio Products. Matrigel™ Matrix Basement Membrane was purchased from BD Bioscience.
Preparation of Cationic and Activated Pt Drugs: DACHPt, DAPt and EDAPt
To achieve efficient drug loading, a list of cationic and activated Pt drugs were prepared as detailed in the literature, with minor modification[29]. Taking DACHPt for example, DACHPtCl2 (506 mg, 1.33 mmol) and AgNO3 (406 mg, 2.39 mmol) were added in 9.37 mL DI H2O. The molar ratio of AgNO3:DACHPtCl2 was 1.8:1. Subsequently, 93.7 μL of a 5% HNO3 solution was added to the mixture to achieve an acidic pH of <2. The suspension was wrapped in aluminum foil and placed in a 70° C. oil bath, with stirring overnight (˜16 h). The mixture was cooled on ice and stored in a 4° C. refrigerator overnight. The sample was spun down at 4,000 rpm for 10 min and the supernatant filtrated through a 0.22 μm syringe filter to obtain the final product, DACHPt. The DAPt and EDAPt aqueous complexes were prepared in similar fashion from commercially available cisplatin and Pt(en)Cl2, respectively. The ratio of AgNO3:cisplatin (or Pt(en)Cl2) remained the same (1.8:1), corresponding to 51 mg AgNO3 plus 50 mg cisplatin or 47 mg AgNO3 plus 50 mg Pt(en)Cl2, respectively. The Pt concentration was determined by ICP-MS (NexION 2000, PerkinElmer). We also used ICP-MS to measure the Ag ion concentration to ensure the removal of AgCl from our samples.
Synthesis, Purification, and Characterization of Cationic and Activated Pt Drugs Laden Silicasomes
Sixty-five nm bare MSNPs were synthesized at 18 L scale and purified by extensive acidic ethanol washing to remove the CTAC detergent, as reported previously[4b]. In order to determine the optimal loading condition, we experimented with multiple rounds of drug loading to find the optimal pH, incubation time, sonication condition, and particle/drug feed ratio, etc. Details of the protocol optimization appear online (
The Pt drug content of the final synthesized products was determined by ICP-OES or ICP-MS by diluting the sample in 2% HNO3. The encapsulation efficiency was defined as EE %=[the total amount of encapsulated Pt drug (m1)]/[the total amount of Pt drug (m0)]×100%. Drug loading capacity was defined as LC %=[the total amount of encapsulated Pt drug (mdrug)]/[the total amount of particle (mMSNP)]×100%. Particle hydrodynamic size and zeta potential were measured by a ZETAPALS instrument (Brookhaven Instruments Corporation). The final product was visualized by cryoEM (TF20 FEI Tecnai-G2) to confirm the uniformity and integrity of the coated lipid bilayer. The energy-dispersive X-ray spectroscopy (EDS) and element mapping were performed by scanning transmission electron microscopy (STEM) in a FEI Titan 80-300 kV TEM.
Cell Culture
The KPC pancreatic adenocarcinoma cell line, which was derived from a spontaneous tumor originating in a transgenic KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx-1-Cre mouse (B6/129 background)[4, 20b, 21], was cultured in DMEM, containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine and 1 mM sodium pyruvate. To allow bioluminescence imaging, the KPC cells were permanently transfected with a luciferase-based lentiviral vector in the UCLA vector core facility, followed by a limiting dilution cloning as we previously described[4a].
Cytotoxicity MTS Assay
Cytotoxicity testing of free Pt drugs or drug-laden silicasomes was performed by using a standard MTS assay (CellTiter 96© AQueous One Solution Cell Proliferation Assay, Promega). PDAC cells were plated at a density of 5×103 cells per well in a 96-well plate and cultured for 24 h before the medium was replaced with fresh medium containing free OX, free DACHPt or DACHPt laden silicasome at indicated concentrations. Non-treated cells were used as control. After treatment for 48 h, the medium was replaced with 100 μL of fresh medium containing MTS solution (5:1, v/v medium/CellTiter 96© Aqueous stock solution), and the cells were further cultured at 37° C. for 1 h. The absorbance of the culture wells at 490 nm was recorded by a microplate reader (M5e, Molecular Device, USA). Wells receiving the MTS solution without cells were used as blank. The relative cell viability (%) is [(the absorption of treated well−blank)/(the absorption of control well−blank)]×100%.
Animal Purchase and Study Permission
Female B6129SF1/J mice (JAX 101043) were purchased from The Jackson Laboratory, and maintained under pathogen-free conditions. All animal experiments were performed according to protocols (#2009-134) approved by the UCLA Animal Research Committee.
PK Study
The PK study was performed on 10-12-week-old healthy female B6129SF1/J mice. The animals received a single IV injection of free OX or DACHPt silicasome at a Pt dose of 4.95 mg/kg (equal to oxaliplatin dose of 10 mg/kg), followed by collection of blood samples at 5 min, 3, 6, 24, and 48 hrs. After separation of the plasma fraction, the plasma samples were digested with HCl:HNO3 3:1, v/v) in a hot-block, before replenishment in 2% HNO3 for ICP-MS analysis of the Pt content. The PK data were analyzed by PKSolver software, using a one-compartment model[4].
Tumor Drug Content and Biodistribution Study
An orthotopic KPC tumor model in immunocompetent B6129SF1/J mouse was established as described previously[4, 21]. Briefly, 30 μL of DMEM/Matrigel (1:1 v/v), containing ˜1×106 KPC-luc cells, was injected into the tail of the pancreas in female B6129SF1/J mice (8˜10 weeks) by a sort surgical survival procedure[4, 21]. To determine the tumor drug content and biodistribution, tumor bearing mice received a single IV injection of free OX or DACHPt silicasome at a Pt dose of 4.95 mg/kg. Animals were sacrificed 48 h post-injection, followed by tumor and tissue collections. These samples were accurately weighed, and followed by digestion using aqua regia in a hot-block and reconstructed in 2% HNO3 for ICP-MS measurement to determine the Pt content.
Assessment of Anti-PDAC Efficacy by the DACHPt Silicasome in the Orthotopic KPC Model Described Above
KPC-luc cells (˜1×106) were orthotopically injected into the pancreas in mice. Eight-day post-surgery, the tumor-bearing mice received IV injections of DACHPt silicasome at Pt dose of 2 mg/kg. The control includes saline as well as free oxaliplatin. This dose arrangement is in agreement with the literature[27]. Tumor-bearing mice received IV injection of the indicated therapy every 3 days for a total of 3 administrations. Before animal sacrifice (72 h post the last IV injection), the mice received intraperitoneal injection of D-luciferin, followed by ex vivo bioluminescence imaging using an IVIS imaging system. Primary tumor and major organs (e.g. sternum, heart, liver, spleen, lung and kidneys) were harvested and fixed in 10% formalin, followed by paraffin embedding and sectioning to provide 4 μm slices for histological analysis in the UCLA Translational Pathology Core Laboratory (TPCL). H&E staining was performed to look at the pathological abnormality in mice receiving different treatments. The H&E slides for toxicity assessment were read in a blinded fashion by an experienced veterinary pathologist.
Identification of DACHPt as an ICD-Inducing Agent
Surface CRT expression was visualized by immunofluorescence (IF) staining using an anti-CRT primary antibody, followed by the incubation with Alex488 conjugated secondary antibody. Briefly, ˜1.5×104 KPC cells were seeded into an 8-well confocal chamber slide. After 24 h, the cell culture medium was replaced with fresh medium containing the chemo agents, following which the cells were incubated for another 24 h. The cells were washed twice in cold PBS and fixed with 4% paraformaldehyde (PFA) at room temperature (r.t.) for 15 mins. After fixation, the cells were washed twice with cold PBS and blocked with 1% BSA in PBS for 0.5 h. The cells were incubated with anti-CRT primary antibody (ab2907, 1:200) in 200 μL blocking solution at 4° C. overnight, followed by washing with PBS and staining with secondary antibody (Alex488 conjugated goat anti-rabbit secondary antibody, A-11008, 1:1000) together with the nuclear dye, Hoechst 33342, at r.t. for 1 h. The cells were washed with PBS, then imaged by using a Leica SP8-MD confocal microscope under the 100× objective lens.
Surface CRT expression was measured by flow cytometry as previously described[24a]. Briefly, 7.5×104 KPC cells were seeded into 24-well plates. After cell attachment, KPC cells were treated with free oxaliplatin and DACHPt (500 μM), for 24 h. The loosely attached cells were combined with trypsin-treated at adherent cells. The cells were washed in cold PBS and then stained on ice with a primary anti-CRT antibody (Abcam, ab2907, 1:140) in 200 μL BD staining buffer for 0.5 h. The cells were washed in cold PBS and stained with an Alexa Fluor® 680-conjugated secondary antibody (LifeScience Technologies #A21244) for 30 min on ice. After washing in cold PBS, the cells were assessed in a LSRII flow cytometer (BD Biosciences). In the same experiment, the cell culture media were spun down to collect the supernatants for HMGB1 detection by an ELISA kit (Catalog #ST51011, IBL International GmbH), according to the manufacture's instruction.
Moreover, we also validated the ICD effect of DACHPt in a vaccination experiment, using a published protocol[26]. Briefly, eight million KPC cells were seeded in a tissue culture dish. After cellular attachment, DACHPt or free oxaliplatin (500 μM) were added for 24 h. Cells were collected and washed before being resuspended in 0.8 mL cold PBS. For vaccination, each mouse received subcutaneous injection (SC) of a 100 μL suspension of chemo-treated cells in the right flank. Control animals received PBS only. The vaccination was repeated after 7 days. Fourteen days after the 1st vaccination, the same mice received the SC injection of healthy KPC cells (1×106 cells) in the contralateral side. Tumor growth was measured by a digital caliper every 2-3 days. At the conclusion of the vaccination experiment (Day 26), animals were sacrificed and the tumors collected for IHC immunophenotyping of CD8+ T cells and FoxP3+ Treg cells. Primary antibodies to CD8 (#14-0808-82, 1:100) and FoxP3 (#13-5773-82, 1:200) were purchased from ThermoFisher. IHC staining was performed in the UCLA Translational Pathology Core Laboratory (TPCL). The slides were scanned and images were assessed by using Aperio ImageScope software (Leica).
Investigation of ICD and Immune Activation by Silicasome Encapsulated DACHPt in the Orthotopic KPC Model
The tumor tissues in the efficacy study (
Assessment of the Survival Outcome Using DACHPt Silicasome w/Wo Anti-PD-1 in an Orthotopic KPC Tumor Model
Tumor-bearing mice were randomly assigned into 6 groups (n=5-7) and received IV injection of Pt drug formulations twice per week as designed in
Statistical Analysis
Comparative analysis of differences between groups was performed using the 2-tailed Student's t-test (Excel software, Microsoft) for two-group comparison. One-way ANOVA followed by a Tukey's test (Origin software, OriginLab) was performed for multiple group comparisons. Data were expressed as mean±SD or SEM, as stated in the figure legends. The survival analysis was performed by Log Rank testing (Mantel-Cox), using GraphPad Prism 7.00 software. A statistically significant difference was considered at *p<0.05.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
This application claims benefit of and priority to U.S. Ser. No. 63/108,172, filed on Oct. 30, 2020, which is incorporated herein by reference in its entirety for all purposes.
This invention was made with government support under Grant Number CA198846, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/057122 | 10/28/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63108172 | Oct 2020 | US |
