WO2024123732A1 - Compositions and methods for treatment of prostate cancer - Google Patents

Compositions and methods for treatment of prostate cancer Download PDF

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Publication number
WO2024123732A1
WO2024123732A1 PCT/US2023/082430 US2023082430W WO2024123732A1 WO 2024123732 A1 WO2024123732 A1 WO 2024123732A1 US 2023082430 W US2023082430 W US 2023082430W WO 2024123732 A1 WO2024123732 A1 WO 2024123732A1
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cab
acid
map
conjugate
nanoparticles
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Robert J. Lamm
Richard Markus
Emily A. Wyent
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Dantari Inc
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Dantari Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D305/00—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms
    • C07D305/14—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems

Definitions

  • the present disclosure is in the field of chemotherapeutic compositions and vehicles for their delivery.
  • Certain prostate cancers are treated by castration, either physical or chemical.
  • Physical castration is generally accomplished by removal of one or both testicles (orchiotomy, orchidectomy).
  • Chemical castration is accomplished using, e.g., anti -androgenic drugs that slow or block the production of testosterone and/or dihydrotestosterone.
  • Prostate cancers that recur after either physical or chemical castration can be treated with docetaxel, but many mCRPCs eventually develop resistance to docetaxel.
  • Cabazitaxel is a tubulin-binding taxane with low affinity for P-glycoprotein, thereby lowering the chances for development of resistance, and is currently the treatment of choice for mCRPC. See, for example, Abidi (2013) J. Pharmacol. Pharmacother. 4:230-237.
  • compositions comprising cabazitaxel for use in the treatment of solid tumors such as prostate cancer.
  • reactive derivatives of cabazitaxel that are useful in the manufacture of these compositions.
  • the therapeutic compositions include a polymer, each of whose repeating units contains a polyol e.g., mucic acid) portion, a polyalkylene glycol (e.g., polyethylene glycol) portion, and one or more reactive groups.
  • Nanoparticles containing the polymers disclosed herein are also provided. Accordingly, some of the embodiments provided herein include:
  • * is -H or -OH or amino acid-CAB, or any combination of the foregoing.
  • conjugate of embodiment 4 wherein the targeting molecule is linked to the conjugate through a polyethylene glycol (PEG) polymer containing one or more nitrophenylboronic acid (NPBA) moieties.
  • PEG polyethylene glycol
  • NPBA nitrophenylboronic acid
  • a pharmaceutical composition comprising the nanoparticle of embodiment 9 and a pharmaceutically acceptable carrier or excipient.
  • a method for the treatment of a disease or disorder in a subject comprising administering the pharmaceutical composition of embodiment 10 to the subject.
  • Figure 1 shows kinetics of release of CAB from nanoparticles in mouse plasma in vitro.
  • the nanoparticles tested contained different amino acid linkers between the CAB and the MAP components of the nanoparticle, as follows: hexanoic acid (Hex), valine (Vai), gammaamino butyric acid (GABA), beta-alanine (
  • Figure 2 shows levels of total CAB in male Sprague Dawley rats at 0-120 hours after injection of CAB-containing nanoparticles with different amino acid linkers between the CAB and the MAP, as follows: gamma-amino butyric acid (GABA-CAB, inverted triangles, top line); P-alanine (P-ala-CAB, triangles, second line from top); alanine (Ala-CAB, squares, third line from top) and glycine (Gly-CAB, circles, bottom line).
  • GABA-CAB gamma-amino butyric acid
  • P-alanine P-ala-CAB, triangles, second line from top
  • alanine Ala-CAB, squares, third line from top
  • glycine Gly-CAB, circles, bottom line
  • Figure 3 shows levels of total CAB in male Sprague Dawley rats at 0-24 hours after injection of CAB-containing nanoparticles with different amino acid linkers between the CAB and the MAP, as follows: gamma-amino butyric acid (GABA-CAB, inverted triangles, top line); P-alanine (P-ala-CAB, triangles, second line from top); alanine (Ala-CAB, squares, third line from top) and glycine (Gly-CAB, circles, bottom line).
  • GABA-CAB gamma-amino butyric acid
  • P-alanine P-ala-CAB, triangles, second line from top
  • alanine Ala-CAB, squares, third line from top
  • glycine Gly-CAB, circles, bottom line
  • Figure 4 shows levels of free (unconjugated) CAB in male Sprague Dawley rats at 0-120 hours after injection of CAB-containing nanoparticles with different amino acid linkers between the CAB and the MAP, as follows: alanine (Ala-CAB, squares, top line); glycine (Gly- CAB, circles, second line from top); P-alanine (P-ala-CAB, triangles, third line from top) and gamma-amino butyric acid (GABA-CAB, inverted triangles, bottom line).
  • alanine Al-CAB, squares, top line
  • Gly- CAB circles, second line from top
  • P-alanine P-ala-CAB, triangles, third line from top
  • GABA-CAB gamma-amino butyric acid
  • FIG. 5 shows the results of two studies assessing the maximum tolerated dose (MTD) of CAB and CAB-containing nanoparticles in male NSG mice.
  • Mice were dosed with unconjugated cabazitaxel (Cabazitaxel, leftmost set of data points); MAP-Gly-CAB nanoparticles (Gly-Cab, next set of data points to the right); MAP-Ala-CAB nanoparticles (Ala- Cab, next set of data points to the right); MAP-P-Ala-CAB nanoparticles (P-Ala-Cab, next set of data points to the right); and MAP-GABA-CAB nanoparticles (GABA-Cab, rightmost set of data points).
  • Tolerated doses are indicated by circles; non-tol erated doses are indicated by x’s.
  • compositions and methods of making and using refer to compositions and methods of making and using said compositions. That is, where the disclosure describes or claims a feature or embodiment associated with a composition or a method of making or using a composition, it is appreciated that such a description or claim is intended to extend these features or embodiment to embodiments in each of these contexts (i.e., compositions, methods of making, and methods of using).
  • “about x” means within + 0.5% of x. In other embodiments, “about x” means within +1% of x. In other embodiments, “about x” means within +2% of x. In other embodiments, “about x” means within +3% of x. In other embodiments, “about x” means within +4% of x. In other embodiments, “about x” means within +5% of x. In other embodiments, “about x” means within +6% of x. In other embodiments, “about x” means within +7% of x. In other embodiments, “about x” means within +8% of x.
  • “about x” means within +9% of x. In other embodiments, “about x” means within +10% of x. In other embodiments, “about x” means within +15% of x. In other embodiments, “about x” means within +20% of x. In other embodiments, “about x” means within +25% of x. In other embodiments, “about x” means within +50% of x.
  • references to alcohols, aldehydes, amines, carboxylic acids, ketones, or other similarly reactive functional groups also includes their protected analogs.
  • reference to hydroxy or alcohol also includes those substituents wherein the hydroxy is protected by an acetyl (Ac), benzoyl (Bz), benzyl (Bn, Bnl), -methoxyethoxymethyl (MEM), dimethoxytrityl (DMT), methoxymethyl (MOM), monomethoxytrityl (MMT), p-methoxybenzyl (PMB), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl (e.g., trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso- propylsilyloxymethyl
  • Reference to amines also includes those substituents wherein the amine is protected by a tertbutyloxycarbonyl (Boc), carbobenzyloxy (Cbz), p-methoxybenzyl carbonyl (Moz or MeOZ), 9- fluorenylmethyloxycarbonyl (Fmoc), acetyl (Ac), benzoyl (Bz), benzyl (Bn, Bnl), carbamate, p- m ethoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts), or sulfonamide (Nosyl & Nps) group.
  • Boc tertbutyloxycarbonyl
  • Cbz carbobenzyloxy
  • Moz or MeOZ p-methoxybenzyl carbonyl
  • 9- fluorenylmethyloxycarbonyl Fmoc
  • substituent containing a carbonyl group also includes those substituents wherein the carbonyl is protected by an acetal or ketal, acylal, or dithiane group.
  • substituent containing a carboxylic acid or carboxylate group also includes those substituents wherein the carboxylic acid or carboxylate group is protected by its methyl ester, benzyl ester, tert -butyl ester, an ester of 2,6-disubstituted phenol (e.g. 2,6- dimethylphenol, 2,6-diisopropylphenol, 2,6-di-tert-butylphenol), a silyl ester, an orthoester, or an oxazoline.
  • 2,6-disubstituted phenol e.g. 2,6- dimethylphenol, 2,6-diisopropylphenol, 2,6-di-tert-butylphenol
  • silyl ester e.g. 2,6- dimethylphenol, 2,
  • Protecting groups for imidazole groups include trityl (Trt); monomethoxytrityl (Mmt); 4-methyltrityl (Mtt); benzyloxymethyl (Bom); and 2,4-dinitrophenyl (Dnp).
  • Trt trityl
  • Mmt monomethoxytrityl
  • Mtt 4-methyltrityl
  • Bom benzyloxymethyl
  • Dnp 2,4-dinitrophenyl
  • BBB blood-brain barrier
  • Boc-Lys(Boc)-OSu bis-Boc-L-lysine-N-hydroxysuccinimide ester
  • BTB blood-tumor barrier
  • CAB cabazitaxel
  • DIPEA N,N'-diisopropylethyamine diSPA-PEG: di(succinimidyl propionate)-PEG
  • EGFR epidermal growth factor receptor ESI: electrospray ionization
  • GABA gamma-amino butyric acid
  • Gly glycine, glycyl
  • Hex hexanoic acid, //-caproic acid
  • HPLC-UV HPLC with ultraviolet absorbance detection
  • Im-cMAP-Im cationic mucic acid flanked by imidazole moieties
  • IPA isopropyl alcohol kDa: kilodalton
  • MAM polymerizable mucic acid monomer
  • Mmt monomethoxytrityl mol: mole
  • mPEG methoxy polyethylene glycol
  • mRNA messenger ribonucleic acid
  • N-Boc diamine N-Boc-ethylenediamine
  • NPBA 3-carboxy-5-nitrophenylboronic acid, nitrophenylboronic acid
  • PAA poly (amino acid)
  • PAMAM poly(amidoamine)
  • PBS phosphate-buffered saline
  • PEG polyethylene glycol
  • PEGm methoxy polyethylene glycol
  • Pent pentanoic acid; zz-valeric acid
  • PEI polyethyleneimine
  • PrOAc isopropyl acetate
  • PVDF polyvinylidene fluoride, polyvinylidene difluoride
  • RNA ribonucleic acid
  • siRNA short interfering ribonucleic acid, silencing ribonucleic acid
  • TBDPSC1 tert-butlyl(chloro)diphenylsilane
  • TfR transferrin receptor
  • TFA trifluoroacetate, trifluoroacetic acid
  • TIPS triisopropyl silane
  • trastuzumab Herceptin
  • Trt trityl
  • UV ultraviolet
  • exemplary solvents include but are not limited to chlorinated solvents (e.g., dichloromethane, chloroform, 1,2-di chloroethane), ethers (e.g., diethyl ether, tert-butyl methyl ether, cyclopentyl methyl ether, diglyme, 1,4-dioxane, 2-methyltetrahydrofuran), alcohols (e.g., methanol, ethanol, isopropanol, tert-butanol), alkanes (e.g., pentane, hexanes, heptanes), glycols (e.g., ethylene glycol, polyethylene glycol), polar aprotic solvents (e.g., dimethylacetamide, acetonitrile, dimethyl sulfoxide, dimethyl formamide, acetone, N-methyl-2- pyrrolidone, ), and polar protic solvents (
  • Exemplary acids include but are not limited to mineral acids (e.g., hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid) and organic acids (e.g., acetic acid, malonic acid, methanesulfonic acid, propionic acid, thioacetic acid, p-toluenesulfonic acid, tribromoacetic acid, trichloroacetic acid, trifluoroacetic acid).
  • mineral acids e.g., hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid
  • organic acids e.g., acetic acid, malonic acid, methanesulfonic acid, propionic acid, thioacetic acid, p-toluenesulfonic acid, tribromoacetic acid, trichloroacetic acid, trifluoroacetic acid.
  • Exemplary bases include but are not limited to amino bases (e.g. 1,4- diazabicyclo[2.2.2]octane, diethylamine, triethylamine, N,N-diisopropylethylamine, lithium amide, lithium bis(trimethylsilyl)amide, morpholine, piperidine), alkoxides (e.g., barium tert- butoxide, lithium tert-butoxide, sodium methoxide), hydroxides (e.g., tetrabutylammonium hydroxide, sodium hydroxide, potassium hydroxide), organometallic bases (e.g., n-butyllithium, tert-butyllithium, butyl magnesium chloride), pyridines (e.g., 4-dimethylaminopyridine, 2,6- lutidine, pyridine), carbonates (e.g., lithium carbonate, sodium carbonate, magnesium carbonate, potassium carbonate), and
  • Exemplary coupling agents include but are not limited to carbodiimide reagents (e.g., N,N'-diisopropylcarbodiimide, l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclopentylcarbodiimide), additives for carbodiimide reagents (e.g., 1- hydroxy-7-azabenzotriazole, 6-chl oro-1 -hydroxy benzotri azole, N-hydroxysuccinimide, 1- hydroxy-2-pyridinone, 6-chloro-N-hydroxy-2-phenylbenzimidazole, ethyl 2-cyano-2- (hydroxyimino)acetate), anhydride-based or -forming reagents (e.g., ditertbutyl carbonate, acetic anhydride, 2-ethoxy-l-ethoxycarbonyl-l,2-di
  • a nanoparticle as described herein contains, as part of its structure, a polymer whose repeat units comprise a polyol containing at least two vicinal diols, a polyalkylene glycol segment, and one or more reactive groups.
  • the polyol is mucic acid:
  • certain of the polymers disclosed herein contain repeating units containing:
  • the polyalkylene glycol segment is made up of repeating units of polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • certain of the polymers disclosed herein contain repeating units of (-O-CH2-CH2-) , as part of a repeat unit that also contains mucic acid or another polyol.
  • the reactive group is a carbonyl group, a carboxyl group or an amine group.
  • a mucic acid polymer has the following structure: wherein the end groups represented by * can be -H, -OH or -R, wherein R is aminoacyl- cabazitaxel such as:
  • the number of polymer repeat units, m in Compound 20, is between 5 and 200 (or any integral values therebetween; e.g., between 10 and 150, between 20 and 120, between 50 and 100, or between 10 and 25). In certain embodiments, m is about 16. In other embodiments, m is 16.
  • the number of polyethylene glycol repeat units, n in Compound 20, is between 20 and 200 (or any integral value therebetween, e.g., between 25 and 150, between 30 and 120, or between 50 and 100).
  • n is chosen so as to provide a number average molecular weight for the PEG portion of Compound 20 (i.e., -O-CH2-CH2-) in a range from about 500 Da to about 50,000 Da.
  • n is about 114 (PEG 5k).
  • n is about 80 (PEG 3.5k).
  • n is about 46 (PEG 2k).
  • n is 46, or n is 80 or n is 114.
  • the weight average molecular weight of the MAP is between 5 and 150 kDa or any integral values therebetween, e.g., between 20 and 120 kDa, and the values of m and n are chosen accordingly.
  • values of m and n are chosen such that, after assembly of the MAP, or the CAB-conjugated MAP, into a nanoparticle, the size of the nanoparticle is between 10 and 900 nm or any integral values therebetween, e.g., between 100 and 800 nm, between 200 and 500 nm, between 400 and 700 nm, or between 20 and 100 nm.
  • the length of a polymer can be determined as described in US Patent No. 11,738,092.
  • Additional polymers to which cabazitaxel can be conjugated using the methods and compositions described herein include neutral and cationic polymers such as those described in US Patent No. 10,342,879; US Patent No.9, 446, 149; US Patent No.11,285,212; US Patent No.10,182,986; US Patent No. 11,041,050; US Patent No. 11,738,092 and U.S. provisional patent application No. 63/588,162 (filed October 5, 2023).
  • the disclosures of all of the foregoing U.S. patents, and of the foregoing patent application are incorporated by reference in their entireties, for the purposes of describing polymers containing vicinal diols, their synthesis, their properties and their uses.
  • Cabazitaxel has the structure: (Compound 1).
  • cabazitaxel is reacted with a carboxybenzyl-amino acid (e.g., Cbz-Gly, Cbz-Ala, Cbz-P-Ala, Cbz-Val, Cbz-(GABA), or Cbz- Hex) to form a CAB-2'-amino acid-Cbz derivative (Example 1).
  • a carboxybenzyl-amino acid e.g., Cbz-Gly, Cbz-Ala, Cbz-P-Ala, Cbz-Val, Cbz-(GABA), or Cbz- Hex
  • CAB-2'-amino acid-Cbz derivative is reacted with methanesulfonic acid to form the CAB-2'-amino acid-methanesulfonic acid salt (Example 2).
  • reactive CAB derivatives contain an amino acid spacer.
  • an amino acid, or portion thereof is used as a spacer molecule in the drug- polymer conjugates disclosed herein.
  • an amino acid can be used as a spacer between a cabazitaxel molecule and a mucic acid polymer.
  • the twenty essential amino acids have the general structure: in which Ri is a functional group that is specific to each particular amino acid. Functional groups for the twenty essential amino acids are given in Table 1. Chemical structures for a number of non-essential amino acids are given in Table 2.
  • the left column provides the name of the ammo acid and its three -letter and one-letter abbreviations.
  • the right column shows the chemical structure of the functional group (Ri) for each ammo acid, except for proline.
  • a CAB-2'-amino acid-methanesulfonic acid salt is combined with a polyol/alkylene glycol polymer (e.g., a mucic acid polymer such as Compound 20) in the presence of dimethyl sulfoxide, PyAOP and DIPEA.
  • the amino group of the MSA salt reacts with one or more carboxyl groups of the polymer, forming an amide bond (Example 3).
  • the product is purified, and appropriate size fractions are obtained, e. ., by dialysis. Percent CAB in the MAP-CAB conjugate is determined as described in Example 4.
  • a MAP-CAB conjugate contains at least 5% CAB by weight, at least 10% CAB by weight, at least 15% CAB by weight, at least 20% CAB by weight, at least 25% CAB by weight, at least 30% CAB by weight, at least 35% CAB by weight, at least 40% CAB by weight, at least 45% CAB by weight, at least 50% CAB by weight, at least 55% CAB by weight, or at least 60% CAB by weight,.
  • a MAP-CAB conjugate contains between 5-10% CAB by weight, between 10-15% CAB by weight, between 15-20% CAB by weight, between 20-25% CAB by weight, between 25-30% CAB by weight, between 30-35% CAB by weight, between 35-40% CAB by weight, between 40-45% CAB by weight, between 45-50% CAB by weight, between 50-55% CAB by weight, or between 55-60% CAB by weight.
  • Conjugates of CAB-containing polymers with nitrophenylboronic acid-containing polymers are also provided.
  • Polymers containing conjugated cabazitaxel can also have conjugated to them one or more boronic acid-containing polymers. These can include boronic acid-containing polymers, phenyl boronic acid-containing polymers; nitrophenyl boronic acid-containing polymers; and polymers containing one or a plurality of phenyl-boronic acid- or nitrophenyl boronic acid molecules.
  • Boronic acid-containing polymers including nitrophenylboronic acid-containing polymers, are described, for example, in US Patent No. 10,342,879; US Patent No. 9,446,149; US Patent No. 11,285,212; US Patent No. 10,182,986; US Patent No. 11,041,050; US Patent No.
  • cabazitaxel -containing polymers are conjugated to polyethylene glycol (PEG) polymers containing one or more nitrophenylboronic acid (NPBA) moieties as described, for example, in co-owned U.S. provisional patent application No. 63/588,185.
  • PEG polyethylene glycol
  • NPBA nitrophenylboronic acid
  • a NPBA-PEG polymer can have a structure as shown in the formulabelowl, in which the NPBA portion of the polymer is to the left of the spacer in the formula, and the PEG portion of the polymer is to the right of the spacer: wherein:
  • L3 is a linking group, or linking groups, between the one or more boronic acid moieties and the spacer;
  • L4 is a linking group, or linking groups, between the spacer and the one or more polyethylene oxide linkages
  • Spacer is a moiety containing at least one terminal amine and/or at least one terminal carboxylic acid; q is independently at each occurrence 0, 1 , 2, 3, or 4; r is 1, 2, or 3; s is independently at each occurrence 20-1200; t is 1, 2, or 3; and
  • Xi is a terminal functional group.
  • Xi is either of:
  • Xi comprises a targeting molecule or a therapeutic molecule.
  • the spacer is an amino acid.
  • a NPBA-PEG polymer can be synthesized, for example, by combining an amine- functionalized PEG (e.g., PEG2k, PEG3.5k, PEG5k, PEGlOk) with a carboxy NPBA to form a NPBA-PEG conjugate joined by an amide linkage.
  • an amine- functionalized PEG e.g., PEG2k, PEG3.5k, PEG5k, PEGlOk
  • CAB -containing nanoparticles contain a targeting molecule, or targeting agent, which facilitates homing of a nanoparticle to a target (e.g., a tumor cell) after systemic delivery.
  • Targeting molecules include, for example, small molecules e.g., vitamins, e.g., folate), saccharides (e.g., mannose, allose, altrose, glucose, gulose, idose, galactose, talose, di saccharides, trisaccharides, oligosaccharides), peptides (e.g., RGD), polypeptides (e.g., antibodies, proteins that bind cell-surface receptors such as transferrin), nucleic acids (e.g, aptamers), peptoids and peptide nucleic acids (PNAs).
  • vitamins, e.g., folate include, for example, small molecules e.g., vitamins, e.g., folate), saccharides (e.g.
  • a targeting molecule is a protein, a peptide, an aptamer, or a ligand for a cellular receptor.
  • a targeting molecule that is a protein can be, for example, an antibody or fragment thereof.
  • the target is the prostate gland
  • the targeting agent binds to a molecule present on the surface of a prostate cancer cell, such as prostate-specific antigen (PSA) or prostate-specific membrane antigen (PSMA).
  • a targeting molecule is an antibody (or fragment thereof) directed to PSA, or an antibody (or fragment thereof) directed to PSMA, or an antibody (or fragment thereof) directed to B7-H3, or an antibody (or fragment thereof) directed to B7-H4, or an antibody (or fragment thereof) directed to STEAP1, or an antibody (or fragment thereof) directed to HER2, or an antibody (or fragment thereof) directed to HER3, or an antibody (or fragment thereof) directed to TROP2.
  • one or more small molecule targeting molecules can be used. These include, but are not limited to, carbohydrates (such as, for example, fructose, glucose, mannose, levan; or a derivative thereof), vitamins (such as, for example, riboflavin, biotin, thiamine, vitamin B 12, and folic acid), and amino acids (such as, for example, glycine, alanine, arginine lysine and tryptophan).
  • Additional small molecule targeting agents include adenosine, anisamide, anacardic acid, phenylboronic acid and methotrexate. See, for example, Kaur et al. (2023) ./. Controlled Release 355:417-433.
  • a targeting molecule is covalently coupled to a boronic acid containing polymer, such as, for example a NPBA-PEG polymer as described in co-owned United States provisional patent application No. 63/588,162 filed October 5, 2023; the disclosure of which is hereby incorporated by reference in its entirety for the purposes of describing NPBA- PEG polymers, their structures, their synthesis and their uses.
  • a targeting molecule can be part of the Xi moiety of the NPBA-PEG polymer, e.g., the targeting molecule is a covalent conjugate of one of the functional groups that are specified United States provisional patent application No. 63/588,162 filed October 5, 2023.
  • a targeting molecule is attached to a NPBA-PEG polymer by way of reaction with a pentafluorophenyl (PFP)-activated ester of the NPBA-PEG polymer, as described, for example, in U.S. Patent No. 11,738,092 and in co-owned U.S. provisional patent application No. 63/588,162; the disclosures of which are incorporated by reference for the purpose of describing methods for attaching polypeptides to NPBA-PEG polymers.
  • PFP pentafluorophenyl
  • the targeting agent is attached to the CAB-MAP conjugate, prior to formation of the nanoparticle, by methods described in, for example, U.S. Patents 9,466,149; 10,182,986; 10,342,879; 11,041,050; 11,285,212 and 11,738,092.
  • the disclosures of all of the aforementioned patents are hereby incorporated by reference in their entireties for the purposes of describing targeting molecules and methods for their attachment to MAP-containing polymers and to polyol/PEG-containing polymers.
  • cabazitaxel-containing polymers as described herein, comprise a therapeutic molecule (e.g., a large molecule therapeutic).
  • a therapeutic molecule can be conjugated to a NPBA-PEG polymer in the manner described above for conjugation of a targeting molecule to a NPBA-PEG polymer; i.e., by reaction between the therapeutic molecule and a pentafluorophenyl (PFP)-activated ester of the NPBA-PEG polymer.
  • a therapeutic molecule is part of the Xi moiety of the NPBA-PEG polymer described in co-owned U.S. provisional patent applications 63/588,162 and 63/588,185.
  • Large molecule therapeutics are known in the art and include, for example, therapeutic polypeptides (e.g., antibodies, enzymes, and bioactive proteins) and nucleic acids.
  • Exemplary therapeutic polypeptides include, but are not limited to, antibodies such as trastuzumab (Herceptin, an anti-Her2 antibody), anti-Her3, anti-Trop2, anti-PSMA, anti- LIV-1, anti-FOLRl, anti-DLL3, anti-PDGF, anti-FRalpha, anti-PTK7, anti-mesothelin, anti-c- MET, anti-MUCl, anti-CD70, anti-CD74, anti-CD30, anti-CD33, anti-FLT3, anti-CD22, anti- CD20, and anti-CD19, anti-CD4, anti-CD8, and antibodies directed against B7-H3, B7-H4, Nectin-4, Claudin 18.2, R0R1, R0R2, BCMA, EpCAM, Claudin 6, CDH6, CEACAM5, and Integrin beta-6.
  • trastuzumab Herceptin, an anti-Her2 antibody
  • anti-Her3, anti-Trop2, anti-PSMA anti- LIV-1
  • polypeptide therapeutics such as antibodies directed to tumor markers like those exemplified in the previous paragraph, can also be used as targeting molecules.
  • nanoparticles as disclosed herein comprise a cabazitaxel- containing polymer and a biological agent.
  • Biological agents include amino acids, peptides, polypeptides, nucleotides, and nucleic acids, such as oligonucleotides and polynucleotides.
  • Exemplary nucleic acids include DNAs such as genomic DNA, cDNA, plasmids and antisense oligonucleotides.
  • Additional exemplary nucleic acids include RNAs, such as, for example, mRNA, siRNA, circular RNA, shRNA, mi RNA, antisense RNA, guide RNA, and tracr RNA. Any of the biological agents mentioned above can be linear, circular or branched (e.g., cyclic peptides, or circular nucleic acids, such as circular RNA).
  • RNA interference methods are siRNAs targeted to certain cancer-related genes (e.g., oncogenes) such as, for example, c-myc, c-myb, mdm2, PKA-I, Abl-1, Bcl2, Ras, c-Raf kinase, CDC25 phosphatases, cyclins, cyclin dependent kinases, telomerase, PDGF/sis, erb-B, fos, jun, mos, src, the Bcr/Abl fusion gene, EGFR, BRAF, RRM2, HER2, VEGF, FGFR2, K-RAS, Claudin 18.2, EpCAM, STAT3, mesothelin/MSLN and BCRP.
  • oncogenes e.g., oncogenes
  • cancer-related genes e.g., oncogenes
  • oncogenes e.g., oncogenes
  • cancer-related genes e
  • the biological agent can be negatively charged, such as a nucleic acid.
  • the nucleic acid can be DNA, RNA or a peptide nucleic acid (PNA).
  • Biological agents that are RNA can be low molecular weight RNAs such as antisense RNA, miRNA and siRNA, which can be introduced into a target cell to regulate (e.g., repress) expression of an endogenous target gene; or high molecular weight RNAs such as mRNAs, which can be used to encode an exogenous protein in a target cell.
  • a biological agent can be any of a guide RNA and/or a tracr RNA, and/or a RNA encoding the Cas9 nuclease, for use in gene editing using the CRISPR-Cas9 system.
  • Biological agents can also be DNA molecules encoding any of the RNAs described above.
  • a biological agent is a small molecule therapeutic, such as a chemotherapeutic.
  • chemotherapeutics include camptothecin and its derivatives and metabolites (such as SN38); taxanes e.g., taxol, docetaxel) and epithilones.
  • Nanoparticles are formed by diluting CAB-MAP conjugates (or conjugates of CAB with other polyol/polyalkylene glycol polymers) in water or dilute saline.
  • the CAB-MAP conjugate (or CAB-polyol/polyalkylene glycol conjugate) may optionally also comprise one or more of a targeting molecule, a therapeutic molecule and/or a biological agent, wherein the targeting molecule, therapeutic molecule and/or biological agent is optionally joined to the MAP polymer via a NPBA-PEG polymer to which the targeting molecule, therapeutic molecule and/or biological agent is covalently joined.
  • the ionic strength of the nanoparticle solution is adjusted if desired, e.g., by the addition of saline (Example 6).
  • the size of the nanoparticles ranges from about 10 to about 60 nm in diameter.
  • Exemplary nanoparticle diameters are 15 nm, 16 nm, 17 nm, 18 nm, 19, nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm or larger.
  • Exemplary nanoparticle diameters are 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, or any value within the range of 20-60 nm. Specific embodiments can also describe these nanoparticles as having diameters in a range of from about 20 nm to about 30 nm, from about 30 nm to about 40 nm, from about 40 nm to about 50 nm, from about 50 nm to about 60 nm, or a combination of two or more of these ranges. In certain embodiments, the nanoparticles are spherical and have a diameter of 20-40 nm as determined by DLS and Cryo-transmission electron microscopy.
  • the total size of the nanoparticle is from about 20 nm to about 60 nm. In some embodiments the size of the nanoparticle is from about 20 nm to about 30 nm. In some embodiments the size of the nanoparticle is from about 20 nm to about 40 nm. In some embodiments the size of the nanoparticle is from about 20 nm to about 50 nm. In some embodiments the size of the nanoparticle is from about 20 nm to about 60 nm. In some embodiments the size of the nanoparticle is from about 30 nm to about 40 nm. In some embodiments the size of the nanoparticle is from about 30 nm to about 50 nm.
  • the size of the nanoparticle is from about 30 nm to about 60 nm. Tn some embodiments the size of the nanoparticle is from about 40 nm to about 50 nm. In some embodiments the size of the nanoparticle is from about 40 nm to about 60 nm.
  • the malignancy to be treated can include any solid tumor for which cabazitaxel is an effective treatment.
  • examples include, but are not limited to, prostate cancer, metastatic castration-resistant prostate cancer (mCRPC), breast cancer, lung cancer, uterine cancer, cervical cancer, bladder cancer, head and neck cancer, and sarcomas, including Kaposi’s sarcoma.
  • nanoparticles comprising cabazitaxel, and further comprising a targeting agent directed to PSMA can be used for the treatment of prostate malignancies such as, for example, prostate cancer and mCRPC.
  • compositions comprising nanoparticles as disclosed herein are also provided.
  • Such compositions typically comprise the nanoparticles and a pharmaceutically acceptable carrier or excipient.
  • Supplementary active compounds such as targeting molecules, therapeutic molecules, and/or biological agents, can also be incorporated into nanoparticle compositions.
  • compositions disclosed herein are useful for, inter alia, treating cancer, cancer metastases and other disorders of the brain and central nervous system.
  • a “therapeutically effective amount” of a composition comprising nanoparticles is any amount that reduces symptoms or, e.g., stimulates tumor regression.
  • dosage amounts of nanoparticles can vary from about 0.1-1.0 mg/kg body weight, or from about 0.5 to 2.0 mg/kg body weight or from about 1-5 mg/kg body weight or from about 1 mg/kg body weight to about 10 mg/kg body weight or more (or any integral value therebetween); with a frequency of administration of, e.g., hourly, twice per day, once per day, twice per week, once per week, twice per month, once per month, depending upon, e.g., body weight, route of administration, severity of disease, etc.
  • a therapeutically effective amount can comprise a plurality of administrations of the same amount, or different amounts, of nanoparticles.
  • a single administration of nanoparticles is a therapeutically effective amount.
  • nanoparticles are administered at a dosage of 1-20 mg nanoparticle (or any integral or decimal value therebetween) per square meter of the surface area of the body of the subject, as is typical for dosages of chemotherapeutics.
  • a dosage can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 122, 13, 14, 15, 16, 17, 18, 19, or 20 mg/m 2 ; or from 1-5, 2-7, 5-10, 8-12, 10-15, 13-17, or 15-20 mg/m 2 .
  • dosage amounts and schedules are determined empirically and may be different from those listed above.
  • compositions and techniques for their preparation and use are known to those of skill in the art in light of the present disclosure.
  • suitable pharmacological compositions and techniques for their administration one may refer to texts such as Remington's Pharmaceutical Sciences, 17th ed. 1985; Brunton et al., “Goodman and Gilman’s The Pharmacological Basis of Therapeutics,” McGraw-Hill, 2005; University of the Sciences in Philadelphia (eds.), “Remington: The Science and Practice of Pharmacy,” Lippincott Williams & Wilkins, 2005; and University of the Sciences in Philadelphia (eds.), “Remington: The Principles of Pharmacy Practice,” Lippincott Williams & Wilkins, 2008.
  • the nanoparticles described herein can be suspended in a physiologically compatible carrier for administration.
  • physiologically compatible carrier refers to a carrier that is compatible with the nanoparticles and with any other ingredients of the formulation, and is not deleterious to the recipient thereof.
  • suitable carriers include water (e.g., pH 4 water), phosphate-buffered saline, Hank’s balanced salt solution+/- glucose (HBSS), and multiple electrolyte solutions such as, e.g., Plasma-LyteTM A (Baxter).
  • the volume of a nanoparticle suspension administered to a subject will vary depending on the site of administration, treatment goal and number of nanoparticles in solution.
  • the amount of nanoparticles administered will be a therapeutically effective amount.
  • a “therapeutically effective amount” or “effective amount” refers to the number of administered nanoparticles which are required to effect treatment of the particular disorder; i.e., to produce a reduction in the amount and/or severity of the symptoms associated with that disorder.
  • administration of a therapeutically effective amount of nanoparticles results in regression of the cancer.
  • Therapeutically effective amounts vary with the type and extent of malignancy, and can also vary depending on the overall condition of the subject; and can be determined by one of skill in the art using established methods.
  • compositions or vehicle can also include pharmaceutically acceptable materials, compositions or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, i.e., carriers.
  • carriers can, for example, stabilize the nanoparticles and/or facilitate the retention of the nanoparticles in the body.
  • Each carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • materials which can serve as pharmaceutically- acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol and polyethylene glycol; polyols, such as glycerin, sorbitol and mannitol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • Nanoparticles can be administered to a subject by any suitable route, including, but not limited to, inhalation, topically, nasally, orally, parenterally (e.g., intravenously, intraperitoneally, intravesically or intrathecally) or rectally in a vehicle comprising one or more pharmaceutically acceptable carriers, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard practice.
  • Administration of the compositionds described herein can be carried out using any method known in the art.
  • administration may be transdermal, parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intracerebroventricular, intrathecal, intranasal, aerosol, by suppositories, or by oral administration.
  • a pharmaceutical composition of the nanoparticles described herein can be for intravenous administration or for administration by injection, or for oral, pulmonary, nasal, transdermal, or ocular administration.
  • Exemplary formulations include, but are not limited to, those suitable for parenteral administration, e.g., intrapulmonary, intravenous, intra-arterial, intra-ocular, intracranial, sub-meningeal, or subcutaneous administration, including formulations encapsulated in micelles, liposomes or drug-release capsules (active agents incorporated within a biocompatible matrix designed for slow-release); ingestible formulations; formulations for topical use, such as eye drops, creams, ointments and gels; and other formulations such as inhalants, aerosols and sprays.
  • the dosage of the compositions of the disclosure will vary according to the extent and severity of the need for treatment, the activity of the administered composition, the general health of the subject, and other considerations well known to the skilled artisan.
  • the compositions described herein are delivered intracranially at or near a site of brain injury or metastasis.
  • Such localized delivery allows for the delivery of the composition non-systemically, thereby reducing the body burden of the composition as compared to systemic delivery.
  • Local delivery can be achieved, for example, by intra-cranial injection, or through the use of various medically implanted devices including, but not limited to, stents and catheters, or can be achieved by inhalation, phlebotomy, or surgery.
  • Methods for coating, implanting, embedding, and otherwise attaching desired agents to medical devices such as stents and catheters are established in the art and contemplated herein.
  • kits for carrying out the administration of nanoparticles optionally in combination with another therapeutic agent, to a subject.
  • a kit comprises a composition of nanoparticles formulated in a pharmaceutical carrier, suitable for administration, e.g., by injection.
  • Example 1 Synthesis of cabazitaxel-2'-carboxybenzyl-glycinate (CAB-2'-Gly-Cbz) (Compound 3) and related carboxybenzyl-protected cabazitaxel amino acid derivatives (Compounds 9-13)
  • reaction solution was washed with 0.1 M HCI (3 x 3 mL) to remove the EDC and DMAP, with 0.1 M NaHCO.i (3 x 3 mL) to remove the excess Cbz-Gly-OH, and then with brine (1 x 3 mL).
  • the residue was dried over Na2SO4, filtered, and dried under a steam of argon. The material was dried further under high vacuum.
  • Cbz-Gly-OH can be substituted with other Cbz-protected amino acids in the above procedure.
  • Cbz-Ala-OH Compound 4
  • Cbz-P-Ala-OH Compound 5
  • Cbz-Val-OH Compound 6
  • Cbz-(GABA)-OH Compound 7
  • Cbz-Hex-OH Compound 8
  • CAB-2'-Ala-Cbz Compound 9
  • CAB-2'-P-Ala-Cbz Compound 10
  • CAB-2'-Val-Cbz Compound 11
  • CAB-2'-GABA-Cbz Compound 12
  • CAB-2'-Hex- Cbz Compound 13
  • Example 2 Synthesis of cabazitaxel-2'-glycinate.methane sulfonic acid (CAB-2’- Gly.MSA) (Compound 14) and related methane sulfonic acid salts of cabazitaxel (Compounds 15-19)
  • Example 3 Synthesis of MAP cabazitaxel glycinate conjugate (MAP-Gly-CAB) (Compound 21) and related MAP-cabazitaxel (MAP-CAB) conjugates (Compounds 22-26)
  • CAB-2'-Gly.MSA (14) was conjugated to a mucic acid polymer (MAP, as described, for example, in US Patent No. 11,738,092) as follows. 40 mg (0.019 mmol COOH) of MAP (compound 20, identified as compound 6 in US 11,738,092) and 16.2 mg (0.016 mmol) of CAB-2'-Gly.MSA (14) were combined in an 8 mL reaction vial with a septum and a magnetic stirring bar. The vial was charged with 3mL anhydrous DMSO. In a separate vial, 30.1 mg (0.058 mmol) PyAOP and 1 mb anhydrous DMSO were combined to generate a solution.
  • MAP mucic acid polymer
  • the PyAOP solution was added to the reaction vial. Then, 12 mL (0.069 mmol) DIPEA was added, with stirring. The reaction mixture was stirred at ambient temperature overnight; then dialyzed against DMF (4 x 900 mL) and water (4 x 900 mL) using a 10 kDa membrane. The last water dialysis product was frozen, and lyophilized to afford 46.7 mg MAP-Gly-CAB (21).
  • MAP polymers Before conjugation of CAB, MAP polymers can have a number of terminal structures, as shown below.
  • the polymer can contain amine groups at both ends (top); an amine group at one end and a carboxylic acid group at the other end (middle); or carboxylic acid groups at both ends (bottom).
  • carboxylic acid end groups of the MAP polymers may be derivatized in the resulting MAP-CAB conjugates, as described by the structures below:
  • Additional MAP-amino acid-CAB conjugates are produced by the same method using CAB-2'-Ala.MSA (15), CAB-2'-P-Ala.MSA (16), CAB-2'-Val.MSA (17), CAB-2'- GABA.MSA (18), and CAB-2'-Hex.MSA (19) as starting material, instead of CAB-2'-Gly.MSA, to generate MAP-Ala-CAB (Compound 22), MAP-P-Ala-CAB (Compound 23), MAP-Val-CAB (Compound 24), MAP-GAB A-C AB (Compound 25), and MAP -Hex -CAB (Compound 26), respectively.
  • n can range from 20 to 200 and m can range from 5 to 200.
  • n is 114 or about 114 (PEG molecular weight about 5 kDa).
  • n is 80 or about 80 (PEG molecular weight about 3.5 kDa).
  • n is 46 or about 46 (PEG molecular weight about 2 kDa).
  • m is 16 or about 16.
  • Weight percent CAB was then determined using the following formula:
  • Weight percent CAB 100% x [(concentration of total CAB)/concentration of MAPCAB conjugate)].
  • Reference solutions of cabazitaxel were prepared by dissolving different concentrations of CAB in dimethyl formamide (DMF). 10 pL of each reference solution was added to 6.7 pL 0.5 N NaOH and the mixture was incubated for 3 h at room temperature. 6.7 pL 0.5 N HC1 was subsequently added and the mixture was incubated for 30 min at room temperature. After the incubation with HC1, 23.4 pL of acetonitrile (ACN) was added. The absorbance at 230 nm (A230) was measured for each reference solution and the absorbance values were used to construct a standard curve (calibration curve).
  • DMF dimethyl formamide
  • MAP-Gly-CAB (21) 75 mg was dissolved in 15 m of pH 4 ultrapure water. The solution was stirred at room temperature for approximately 3 hours. 1.7 m of 9 % saline, pH 4, was added and stirring was continued for 1 hour. The nanoparticle solution was concentrated using a 10 kDa membrane, passed through a 0.22 pm fdter, and frozen.
  • MAP-amino acid-CAB conjugates i.e., MAP- Ala-CAB (22), MAP-0-Ala-CAB (23), MAP-Val-CAB (24), MAP-GAB A-CAB (25), and MAP- Hex-CAB (26) are converted to nanoparticles in similar fashion, i.e., by dissolving in water or dilute saline, at pH 4, to a concentration of 1-10 mg/mL.
  • Example 7 Nanoparticle characterization
  • Nanoparticle size (Z avg ) and poly dispersity index (PDI) were determined using dynamic light scattering (DLS), which was performed on a Malvern Zetasizer Nano ZS instrument. Nanoparticles made from MAP-Gly-CAB were diluted to a concentration of 2.0 mg/mL in pH 4 saline. The results of 3 measurements, 10 runs each, were averaged.
  • DLS dynamic light scattering
  • MAP-Gly-CAB nanoparticles The surface charge, or zeta potential, of MAP-Gly-CAB nanoparticles was measured using a Malvern Zetasizer Nano ZS instrument. 75 LLL of 2.0 mg/mL of MAP-Gly- CAB nanoparticle solution was mixed with 675 pL of 1 mM potassium chloride. The results of 3 measurements, 10 runs each, were averaged.
  • the Z avg of MAP-Gly-CAB nanoparticles was determined to be 19 nm; the PDI of MAP-Gly-CAB nanoparticles was determined to be 0.21; and the zeta potential of MAP-Gly-CAB nanoparticles was determined to be -0.08 mV.
  • Z avg , PDI and zeta potential of nanoparticles made from MAP-Ala-CAB, MAP- - Ala-CAB, MAP-Val-CAB, MAP-GABA-CAB, and MAP-Hex-CAB are determined in similar fashion.
  • Release of CAB from nanoparticles is determined by incubating solutions of nanoparticles in various media and subsequently measuring the amount of unconjugated and total CAB present in solution at different timepoints.
  • nanoparticles were diluted to a concentration of 1.0 mg/mL in various release media (pH 5.5 phosphate-buffered saline (PBS), pH 7.4 PBS, mouse plasma, rat plasma, or human plasma).
  • PBS phosphate-buffered saline
  • This 1.0 mg/mL stock of particles in release media was separated into 14 microcentrifuge tubes with 2 holes poked in the top of each cap. These tubes were subsequently incubated in a Thermo Forma Series II water-jacketed CO2 incubator at 5 % CO2 and 37 °C. At 0, 2, 4, 6, 12, 24, 48, 72, 96, and 120 h, two tubes were removed from the incubator, frozen on liquid nitrogen, and stored at -80°C until analysis.
  • 3-Ala-CAB, and MAP-GABA-CAB nanoparticles were administered via bolus intravenous injection at 7.5 mg/kg (CAB basis) through a femoral vein canula in male Sprague Dawley rats. At predetermined time points, blood was collected via a jugular vein canula. Samples were kept on ice until centrifugation at 2000 x g for 10 min at 5°C within 1 h of collection. Plasma was directly transferred to cluster tubes and stored at -80°C until analysis.
  • Analyses for total CAB and unconjugated CAB were as follows. To determine the amount of unconjugated CAB, 10 pL of sample was mixed with 13.4 pL 0.5 N HC1 and incubated for 30 min at room temperature. 23.4 pL ACN was subsequently added and incubated for 3 h at room temperature. After incubation with ACN, the mixture was centrifuged at 14,000 x g for 10 min at 4°C and the supernatant was recovered and filtered through a 0.20 pm filter. To measure the total amount of CAB, 10 pL of sample was mixed with 6.7 pL 0.5 N NaOH and incubated for 3 h.
  • 6.7 pL 0.5 N HC1 was subsequently added and incubated for 30 min at room temperature, followed by addition of 23.4 pL ACN and incubation for 3 h at room temperature. After incubation with ACN, the mixture was centrifuged at 14,000 x g for 10 min at 4°C and supernatant was filtered with a 0.20 pm filter. Filtered samples were analyzed using an Agilent 1200 Series HPLC system as described in Example 4 and compared to a calibration curve of reference CAB solutions, prepared as described in Example 5.
  • Example 10 In vivo mouse maximum tolerated dose (MTD) study: unconjugated CAB and MAP-Gly-CAB nanoparticles
  • Example 11 In vivo mouse MTD study: unconjugated CAB, and nanoparticles containing MAP-Ala-CAB, MAP-P-Ala- CAB and MAP-GABA-CAB
  • mice Health of the mice was monitored and recorded daily for 4 weeks after the start of treatment. Body weight was monitored and recorded about every second or third day. MTD was defined as the highest dose resulting in less than 20% body weight loss or with less than 20% treatment-related deaths. Animals were euthanized when the criteria for the MTD was exceeded or at the end of the study.
  • CAB at 1 or 3 mg/kg was tolerated, resulting in no deaths and weight change in the treatment windows of -2.9% and -5.8%.
  • Administration of MAP-Ala-C AB nanoparticles at 8, 12 or 16 mg/kg (CAB basis), MAP-P-Ala-CAB nanoparticles at 8, 16, 24, or 32 mg/kg (CAB basis), or MAP-GAB A-CAB at 16, 24 or 32 mg/kg (CAB basis) were all tolerated. Weight change in the treatment window across all groups ranged from -6.7% to 3.9%. There were no deaths in the treatment window with the exception of one animal that received MAP-P-Ala-CAB nanoparticles at 24 mg/kg (CAB basis).
  • Figure 5 summarizes the results of both MTD studies, showing that MAP-Gly- CAB nanoparticles are tolerated better than unconjugated cabazitaxel; while nanoparticles containing MAP-Ala-CAB, MAP-P-Ala-CAB and MAP-GABA-CAB are even more well tolerated that MAP-Gly-CAB-containing nanoparticles.

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Abstract

Provided herein are compositions comprising cabazitaxel for use in the treatment of prostate cancer; and reactive derivatives of cabazitaxel that are useful in the manufacture of these compositions.

Description

COMPOSITIONS AND METHODS FOR TREATMENT OF PROSTATE CANCER
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of United States provisional patent application No. 63/386,076; entitled “Compositions and Methods for Treatment of Prostate Cancer;” filed December 5, 2022 and of United States provisional patent application No. 63/512,296; entitled “Compositions and Methods for Treatment of Prostate Cancer;” filed July 7, 2023. The disclosures of both of the foregoing applications, including all text and figures, are incorporated by reference in their entireties, for all purposes.
STATEMENT REGARDING FEDERAL SUPPORT
[0002] Not applicable.
FIELD
[0003] The present disclosure is in the field of chemotherapeutic compositions and vehicles for their delivery.
BACKGROUND
[0004] Certain prostate cancers are treated by castration, either physical or chemical. Physical castration is generally accomplished by removal of one or both testicles (orchiotomy, orchidectomy). Chemical castration is accomplished using, e.g., anti -androgenic drugs that slow or block the production of testosterone and/or dihydrotestosterone.
[0005] Prostate cancers that recur after either physical or chemical castration (metastatic castration-resistant prostate cancer, mCRPC) can be treated with docetaxel, but many mCRPCs eventually develop resistance to docetaxel. Cabazitaxel is a tubulin-binding taxane with low affinity for P-glycoprotein, thereby lowering the chances for development of resistance, and is currently the treatment of choice for mCRPC. See, for example, Abidi (2013) J. Pharmacol. Pharmacother. 4:230-237.
[0006] However, cabazitaxel is not well-tolerated. Accordingly, more stable and well- tolerated formulations of cabazitaxel are needed. SUMMARY
[0007] The present disclosure provides compositions comprising cabazitaxel for use in the treatment of solid tumors such as prostate cancer. Also provided are reactive derivatives of cabazitaxel that are useful in the manufacture of these compositions. The therapeutic compositions include a polymer, each of whose repeating units contains a polyol e.g., mucic acid) portion, a polyalkylene glycol (e.g., polyethylene glycol) portion, and one or more reactive groups. Nanoparticles containing the polymers disclosed herein are also provided. Accordingly, some of the embodiments provided herein include:
[0008] 1 Cabazitaxel-2'-carboxybenzyl-glycinate having the structure:
Figure imgf000003_0001
[0009] 2 Cabazitaxel-2'-glycinate. methane sulfonic acid having the structure:
Figure imgf000003_0002
[0010] 3 A mucic acid polymer cabazitaxel glycinate conjugate having the structure:
Figure imgf000004_0001
wherein: m is from 5 to 200 inclusive; and n is from 20 to 200 inclusive. In certain embodiments, * is -H or -OH or amino acid-CAB, or any combination of the foregoing.
[0011] 4. The conjugate of embodiment 3, further comprising one or more targeting molecules.
[0012] 5. The conjugate of embodiment 4, wherein the targeting molecule is selected from the group consisting of a protein, a peptide, an aptamer, and a ligand for a cellular receptor.
[0013] 6 The conjugate of embodiment 5, wherein the protein is an antibody or an antibody fragment.
[0014] 7 The conjugate of embodiment 6, wherein the antibody or antibody fragment is directed to prostate-specific membrane antigen (PSMA), HER2, or TROP2.
[0015] 8. The conjugate of embodiment 4, wherein the targeting molecule is linked to the conjugate through a polyethylene glycol (PEG) polymer containing one or more nitrophenylboronic acid (NPBA) moieties. [0016] 9. A nanoparticle comprising the conjugate of any of embodiment 3-8.
[0017] 10. A pharmaceutical composition comprising the nanoparticle of embodiment 9 and a pharmaceutically acceptable carrier or excipient.
[0018] 11. A method for the treatment of a disease or disorder in a subject, the method comprising administering the pharmaceutical composition of embodiment 10 to the subject.
[0019] 12. The method of embodiment 11, wherein the disorder is a malignancy.
[0020] 13. The method of embodiment 12, wherein the malignancy is a solid tumor.
[0021] 14. The method of embodiment 13, wherein the solid tumor is a prostate tumor, a breast tumor, or a lung tumor.
[0022] 15. The method of embodiment 14, wherein the prostate tumor is metastatic castration-resistant prostate cancer (mCRPC).
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 shows kinetics of release of CAB from nanoparticles in mouse plasma in vitro. The nanoparticles tested contained different amino acid linkers between the CAB and the MAP components of the nanoparticle, as follows: hexanoic acid (Hex), valine (Vai), gammaamino butyric acid (GABA), beta-alanine (|3-Ala), alanine (Ala) and glycine (Gly).
[0024] Figure 2 shows levels of total CAB in male Sprague Dawley rats at 0-120 hours after injection of CAB-containing nanoparticles with different amino acid linkers between the CAB and the MAP, as follows: gamma-amino butyric acid (GABA-CAB, inverted triangles, top line); P-alanine (P-ala-CAB, triangles, second line from top); alanine (Ala-CAB, squares, third line from top) and glycine (Gly-CAB, circles, bottom line).
[0025] Figure 3 shows levels of total CAB in male Sprague Dawley rats at 0-24 hours after injection of CAB-containing nanoparticles with different amino acid linkers between the CAB and the MAP, as follows: gamma-amino butyric acid (GABA-CAB, inverted triangles, top line); P-alanine (P-ala-CAB, triangles, second line from top); alanine (Ala-CAB, squares, third line from top) and glycine (Gly-CAB, circles, bottom line).
[0026] Figure 4 shows levels of free (unconjugated) CAB in male Sprague Dawley rats at 0-120 hours after injection of CAB-containing nanoparticles with different amino acid linkers between the CAB and the MAP, as follows: alanine (Ala-CAB, squares, top line); glycine (Gly- CAB, circles, second line from top); P-alanine (P-ala-CAB, triangles, third line from top) and gamma-amino butyric acid (GABA-CAB, inverted triangles, bottom line).
[0027] Figure 5 shows the results of two studies assessing the maximum tolerated dose (MTD) of CAB and CAB-containing nanoparticles in male NSG mice. Mice were dosed with unconjugated cabazitaxel (Cabazitaxel, leftmost set of data points); MAP-Gly-CAB nanoparticles (Gly-Cab, next set of data points to the right); MAP-Ala-CAB nanoparticles (Ala- Cab, next set of data points to the right); MAP-P-Ala-CAB nanoparticles (P-Ala-Cab, next set of data points to the right); and MAP-GABA-CAB nanoparticles (GABA-Cab, rightmost set of data points). Tolerated doses are indicated by circles; non-tol erated doses are indicated by x’s.
DETAILED DESCRIPTION
[0028] The present disclosure may be understood more readily by reference to the following description taken in connection with the accompanying Examples, all of which form a part of this disclosure. It is to be understood that this disclosure is not limited to the specific products, methods, conditions or parameters described or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of any claimed invention. Similarly, unless specifically otherwise stated, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosure herein is not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement. Throughout this text, it is recognized that the descriptions refer to compositions and methods of making and using said compositions. That is, where the disclosure describes or claims a feature or embodiment associated with a composition or a method of making or using a composition, it is appreciated that such a description or claim is intended to extend these features or embodiment to embodiments in each of these contexts (i.e., compositions, methods of making, and methods of using).
[0029] In the present disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a material” is a reference to at least one of such materials and equivalents thereof known to those skilled in the art, and so forth. [0030] When a value is expressed as an approximation by use of the descriptor “about,” it will be understood that the particular value forms another embodiment. In general, use of the term “about” indicates approximations that can vary depending on the desired properties sought to be obtained by the disclosed subject matter and is to be interpreted in the specific context in which it is used, based on its function. The person skilled in the art will be able to interpret this as a matter of routine. In some cases, the number of significant figures used for a particular value may be one non-limiting method of determining the extent of the word “about.” In other cases, the gradations used in a series of values may be used to determine the intended range available to the term “about” for each value. Where present, all ranges are inclusive and combinable. That is, references to values stated in ranges include every value within that range.
[0031] In certain embodiments, when the term “about” is used with respect to a value x, “about x” means within + 0.5% of x. In other embodiments, “about x” means within +1% of x. In other embodiments, “about x” means within +2% of x. In other embodiments, “about x” means within +3% of x. In other embodiments, “about x” means within +4% of x. In other embodiments, “about x” means within +5% of x. In other embodiments, “about x” means within +6% of x. In other embodiments, “about x” means within +7% of x. In other embodiments, “about x” means within +8% of x. In other embodiments, “about x” means within +9% of x. In other embodiments, “about x” means within +10% of x. In other embodiments, “about x” means within +15% of x. In other embodiments, “about x” means within +20% of x. In other embodiments, “about x” means within +25% of x. In other embodiments, “about x” means within +50% of x.
[0032] It is to be appreciated that certain features of the disclosure which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. That is, unless obviously incompatible or specifically excluded, each individual embodiment is deemed to be combinable with any other embodiment s) and such a combination is considered to be another embodiment. Conversely, various features of the disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination. Finally, while an embodiment may be described as part of a series of steps or part of a more general structure, each said step may also be considered an independent embodiment in itself, combinable with others. [0033] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.” Likewise, a term such as Cl -3 alkyl also includes, as separate embodiments, Cl alkyl, C2 alkyl, C3 alkyl, Cl -2 alkyl, and C2-3 alkyl.
[0034] Reference to alcohols, aldehydes, amines, carboxylic acids, ketones, or other similarly reactive functional groups also includes their protected analogs. For example, reference to hydroxy or alcohol also includes those substituents wherein the hydroxy is protected by an acetyl (Ac), benzoyl (Bz), benzyl (Bn, Bnl), -methoxyethoxymethyl (MEM), dimethoxytrityl (DMT), methoxymethyl (MOM), monomethoxytrityl (MMT), p-methoxybenzyl (PMB), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl (e.g., trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso- propylsilyloxymethyl (TOM), triisopropyl silyl (TIPS)), or ethoxyethyl (EE) group. Reference to amines also includes those substituents wherein the amine is protected by a tertbutyloxycarbonyl (Boc), carbobenzyloxy (Cbz), p-methoxybenzyl carbonyl (Moz or MeOZ), 9- fluorenylmethyloxycarbonyl (Fmoc), acetyl (Ac), benzoyl (Bz), benzyl (Bn, Bnl), carbamate, p- m ethoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts), or sulfonamide (Nosyl & Nps) group. Reference to substituent containing a carbonyl group also includes those substituents wherein the carbonyl is protected by an acetal or ketal, acylal, or dithiane group. Reference to substituent containing a carboxylic acid or carboxylate group also includes those substituents wherein the carboxylic acid or carboxylate group is protected by its methyl ester, benzyl ester, tert -butyl ester, an ester of 2,6-disubstituted phenol (e.g. 2,6- dimethylphenol, 2,6-diisopropylphenol, 2,6-di-tert-butylphenol), a silyl ester, an orthoester, or an oxazoline. Protecting groups for imidazole groups (e.g., histidine) include trityl (Trt); monomethoxytrityl (Mmt); 4-methyltrityl (Mtt); benzyloxymethyl (Bom); and 2,4-dinitrophenyl (Dnp).
Abbreviations
[0035] The following abbreviations and acronyms may be used in this disclosure:
AA: acetic acid Ab: antibody
ACN: acetonitrile
ARS: alizarin red S
Asp: aspartate, aspartic acid
BBB: blood-brain barrier
Boc: tert-butyloxycarbonyl
Boc-Lys(Boc)-OSu: bis-Boc-L-lysine-N-hydroxysuccinimide ester
Bom: benzyloxymethyl
BTB: blood-tumor barrier
CAB: cabazitaxel
Cbz: carboxybenzyl
CPME: cyclopentyl methyl ether
CPT: camptothecin
C V : column volume cMAP: cationic mucic acid polymer, cationic mucic acid-containing polymer cP: cationic polymer
Da: dalton
DCM: dichloromethane, methylene chloride
DF: diafiltration
DIC: N,N'-diisopropylcarbodiimide
DIPEA: N,N'-diisopropylethyamine diSPA-PEG: di(succinimidyl propionate)-PEG
DLS: dynamic light scattering
DMA: dimethylacetamide
DMAP: 4-dimethylaminopyridine
DMF: N,N-dimethyl -formamide
DMSO: dimethyl sulfoxide
Dnp: 2,4-dinitrophenyl
EDC: 1 -(3 -dimethylaminopropyl)-3 -ethylcarbodiimide hydrochloride
EEDQ: N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline
EGFR: epidermal growth factor receptor ESI: electrospray ionization
EtOAc: ethyl acetate
FPLC: fast protein liquid chromatography g: gram
GABA: gamma-amino butyric acid
Gly: glycine, glycyl
GPC: gel permeation chromatography h: hour
HBSS: Hank’s balanced salt solution
HC1: hydrochloric acid
HEPES: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
Hex: hexanoic acid, //-caproic acid
HIC: hydrophobic interaction chromatography
HOPO: hydroxypyridine-N-oxide
HPLC: high-performance liquid chromatography
HPLC-UV: HPLC with ultraviolet absorbance detection
Im: imidazole, imidazolium
Im-cMAP-Im: cationic mucic acid flanked by imidazole moieties
Im.Na: 4-imidazoleacetic acid, sodium salt hydrate
IPA: isopropyl alcohol kDa: kilodalton
L: liter
LAR: linker-to-antibody ratio
MAM: polymerizable mucic acid monomer
MAP: mucic acid polymer
MeOH: methanol
MES: 2-(N-morpholino)ethanesulfonic acid, 2-ethanesulfonic acid mg: milligram mL: milliliter mmol: millimole
Mmt: monomethoxytrityl mol: mole mPEG: methoxy polyethylene glycol mRNA: messenger ribonucleic acid
MTBE: methyl tert-butyl ether
Mtt: 4-methyltrityl
MWCO: molecular weight cut-off
N-Boc diamine: N-Boc-ethylenediamine
NHS: N-hydroxy succinimide
NMM: N-methylmorpholine
NP: nanoparticle
NPBA: 3-carboxy-5-nitrophenylboronic acid, nitrophenylboronic acid
PAA: poly (amino acid)
PAMAM: poly(amidoamine)
PBAE: poly(P-amino ester)
PBS: phosphate-buffered saline
Pd/C: palladium on carbon catalyst
PEG: polyethylene glycol
PEGm: methoxy polyethylene glycol
Pent: pentanoic acid; zz-valeric acid
PEO: polyethylene oxide
PES: poly ethersulfone
PEI: polyethyleneimine
PFP: pentaflurorphenyl
(PFPO)2O: bis(pentafluorophenyl) carbonate
PrOAc: isopropyl acetate
PVDF : polyvinylidene fluoride, polyvinylidene difluoride
PyAOP. (7-azabenzotriazol- l-yloxy)tripyrrolidinophosphonium hexafluorophosphate
RNA: ribonucleic acid siRNA: short interfering ribonucleic acid, silencing ribonucleic acid
SN38 : 7-ethyl- 10-hydroxy-camptothecin
SpP: strands per particle TBDPS: tert-butyl diphenyl si lane; tert-butyldiphenylsilyl
TBDPSC1: tert-butlyl(chloro)diphenylsilane
TEA: triethylamine
Tf: holo-transferrin
TfR: transferrin receptor
TFA: trifluoroacetate, trifluoroacetic acid
THF : tetrahydrofuran
TIPS: triisopropyl silane
Tras: trastuzumab (Herceptin)
Trt: trityl
UF/DF: ultrafiltration & diafiltration pg: microgram pL: microliter pmol: micromole
UV: ultraviolet
Vai: valine, valyl
Reaction components
[0036] The procedures described herein include standard solvents and catalysts as are known in the art. Although particular compounds (e.g., acids, bases, coupling agents) are recited in the disclosure, it is clear to one of skill in the art that different reagents can be used.
[0037] To that end, exemplary solvents include but are not limited to chlorinated solvents (e.g., dichloromethane, chloroform, 1,2-di chloroethane), ethers (e.g., diethyl ether, tert-butyl methyl ether, cyclopentyl methyl ether, diglyme, 1,4-dioxane, 2-methyltetrahydrofuran), alcohols (e.g., methanol, ethanol, isopropanol, tert-butanol), alkanes (e.g., pentane, hexanes, heptanes), glycols (e.g., ethylene glycol, polyethylene glycol), polar aprotic solvents (e.g., dimethylacetamide, acetonitrile, dimethyl sulfoxide, dimethyl formamide, acetone, N-methyl-2- pyrrolidone, ), and polar protic solvents (e.g., water, ethanol, acetic acid, propionic acid).
[0038] Exemplary acids include but are not limited to mineral acids (e.g., hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid) and organic acids (e.g., acetic acid, malonic acid, methanesulfonic acid, propionic acid, thioacetic acid, p-toluenesulfonic acid, tribromoacetic acid, trichloroacetic acid, trifluoroacetic acid).
[0039] Exemplary bases include but are not limited to amino bases (e.g. 1,4- diazabicyclo[2.2.2]octane, diethylamine, triethylamine, N,N-diisopropylethylamine, lithium amide, lithium bis(trimethylsilyl)amide, morpholine, piperidine), alkoxides (e.g., barium tert- butoxide, lithium tert-butoxide, sodium methoxide), hydroxides (e.g., tetrabutylammonium hydroxide, sodium hydroxide, potassium hydroxide), organometallic bases (e.g., n-butyllithium, tert-butyllithium, butyl magnesium chloride), pyridines (e.g., 4-dimethylaminopyridine, 2,6- lutidine, pyridine), carbonates (e.g., lithium carbonate, sodium carbonate, magnesium carbonate, potassium carbonate), and hydrides (e.g., sodium hydride, calcium hydride, potassium hydride).
[0040] Exemplary coupling agents include but are not limited to carbodiimide reagents (e.g., N,N'-diisopropylcarbodiimide, l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclopentylcarbodiimide), additives for carbodiimide reagents (e.g., 1- hydroxy-7-azabenzotriazole, 6-chl oro-1 -hydroxy benzotri azole, N-hydroxysuccinimide, 1- hydroxy-2-pyridinone, 6-chloro-N-hydroxy-2-phenylbenzimidazole, ethyl 2-cyano-2- (hydroxyimino)acetate), anhydride-based or -forming reagents (e.g., ditertbutyl carbonate, acetic anhydride, 2-ethoxy-l-ethoxycarbonyl-l,2-dihydroquinoline, ethyl chloroformate), acylazoles (e.g., carbonyl diimidazole), acid halide generating reagents (e.g., thionyl chloride, phosgene, cyanuric chloride, benzyltriphenylphosphonium dihydrogen trifluoride), phosphonium salt coupling reagents (e.g., benzotriazol-l-yloxytris(diemthylamino)phosphonium hexafluorophosphate), tetramethyl aminium reagents (e.g., 2-(2-oxo-l(2H)-pyridyl-l, 1,3,3- tetramethyluronium tetrafluoroborate), aminium reagents (e.g., 2-chloro-l,3- dimethylimidazolidinium hexafluorophosphate), oxyma salts (e.g., l-((l-cyano-2-ethoxy-2- oxoethylideneaminooxy)(morpholino)methylene)pyrrolidinium hexafluorophosphate), antimonate uranium salts (e.g., benzotriazol- l-yloxuy-N,N-dimethyl-methaniminium hexachloroantimonate), organophosphorus reagents (e.g., diethylcyanophosphonate, 1 -oxochlorophospholane, 2-propanephosphonic acid anhydride), triazine based reagents (e.g., 2- chloro-4,6-dimethoxy-l,3,5-triazine), organosulfur reagents (e.g., pentafluorophenyl-4- nitrobenzenesulfonate), pyridinium reagents (e.g., 2-chl oro-1 -methylpyridinium iodide), polymer bound reagents (e.g., polymer supported N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline). Polymers
[0041] A nanoparticle as described herein contains, as part of its structure, a polymer whose repeat units comprise a polyol containing at least two vicinal diols, a polyalkylene glycol segment, and one or more reactive groups. In certain embodiments, the polyol is mucic acid:
HOOC-CH(OH)-CH(OH)-CH(OH)-CH(OH)-COOH
Thus, certain of the polymers disclosed herein contain -O-C(=O)-CH(OH)-CH(OH)-CH(OH)- CH(OH)-C(=O)-O- as part of a repeating unit.
[0042] In other embodiments, certain of the polymers disclosed herein contain repeating units containing:
Figure imgf000014_0001
These structures are rotational isomers of each other and for present purposes are functionally equivalent. As used herein, representation of one of these structures connotes either or both of these structures in the context of their use.
[0043] In certain embodiments, the polyalkylene glycol segment is made up of repeating units of polyethylene glycol (PEG). Thus, certain of the polymers disclosed herein contain repeating units of (-O-CH2-CH2-) , as part of a repeat unit that also contains mucic acid or another polyol.
[0044] In certain embodiments, the reactive group is a carbonyl group, a carboxyl group or an amine group.
[0045] In certain embodiments, a mucic acid polymer (MAP) has the following structure:
Figure imgf000014_0002
wherein the end groups represented by * can be -H, -OH or -R, wherein R is aminoacyl- cabazitaxel such as:
Figure imgf000015_0001
(see, e.g., Example 3).
[0046] The number of polymer repeat units, m in Compound 20, is between 5 and 200 (or any integral values therebetween; e.g., between 10 and 150, between 20 and 120, between 50 and 100, or between 10 and 25). In certain embodiments, m is about 16. In other embodiments, m is 16.
[0047] The number of polyethylene glycol repeat units, n in Compound 20, is between 20 and 200 (or any integral value therebetween, e.g., between 25 and 150, between 30 and 120, or between 50 and 100). In certain embodiments, n is chosen so as to provide a number average molecular weight for the PEG portion of Compound 20 (i.e., -O-CH2-CH2-) in a range from about 500 Da to about 50,000 Da. In certain embodiments, n is about 114 (PEG 5k). In other embodiments, n is about 80 (PEG 3.5k). In additional embodiments, n is about 46 (PEG 2k). In certain embodiments, n is 46, or n is 80 or n is 114.
[0048] In certain embodiments, the weight average molecular weight of the MAP is between 5 and 150 kDa or any integral values therebetween, e.g., between 20 and 120 kDa, and the values of m and n are chosen accordingly.
[0049] In additional embodiments, values of m and n are chosen such that, after assembly of the MAP, or the CAB-conjugated MAP, into a nanoparticle, the size of the nanoparticle is between 10 and 900 nm or any integral values therebetween, e.g., between 100 and 800 nm, between 200 and 500 nm, between 400 and 700 nm, or between 20 and 100 nm.
[0050] If desired, the length of a polymer can be determined as described in US Patent No. 11,738,092.
[0051] Any and all combinations of two or more of the preceding embodiments are also contemplated as additional embodiments of the compositions disclosed herein.
[0052] Certain methods for the synthesis of mucic acid polymers are provided in coowned US Patent Number 11,738,092; the disclosure of which is incorporated by reference in its entirety for the purposes of describing synthesis of mucic acid polymers, properties of mucic acid polymers, and uses of mucic acid polymers.
[0053] Additional polymers to which cabazitaxel can be conjugated using the methods and compositions described herein include neutral and cationic polymers such as those described in US Patent No. 10,342,879; US Patent No.9, 446, 149; US Patent No.11,285,212; US Patent No.10,182,986; US Patent No. 11,041,050; US Patent No. 11,738,092 and U.S. provisional patent application No. 63/588,162 (filed October 5, 2023). The disclosures of all of the foregoing U.S. patents, and of the foregoing patent application, are incorporated by reference in their entireties, for the purposes of describing polymers containing vicinal diols, their synthesis, their properties and their uses.
Reactive derivatives of cabazitaxel [0054] Cabazitaxel has the structure:
Figure imgf000016_0001
(Compound 1).
[0055] In preparation for its conjugation to a polymer, cabazitaxel is reacted with a carboxybenzyl-amino acid (e.g., Cbz-Gly, Cbz-Ala, Cbz-P-Ala, Cbz-Val, Cbz-(GABA), or Cbz- Hex) to form a CAB-2'-amino acid-Cbz derivative (Example 1). The CAB-2'-amino acid-Cbz derivative is reacted with methanesulfonic acid to form the CAB-2'-amino acid-methanesulfonic acid salt (Example 2).
Amino acid spacers
[0056] As noted above, reactive CAB derivatives contain an amino acid spacer. Thus, in certain embodiments, an amino acid, or portion thereof, is used as a spacer molecule in the drug- polymer conjugates disclosed herein. For example, an amino acid can be used as a spacer between a cabazitaxel molecule and a mucic acid polymer.
[0057] The twenty essential amino acids have the general structure:
Figure imgf000017_0001
in which Ri is a functional group that is specific to each particular amino acid. Functional groups for the twenty essential amino acids are given in Table 1. Chemical structures for a number of non-essential amino acids are given in Table 2.
Table 1: Essential amino acid functional groups
Figure imgf000018_0001
Legend: The left column provides the name of the ammo acid and its three -letter and one-letter abbreviations. The right column shows the chemical structure of the functional group (Ri) for each ammo acid, except for proline.
* For proline, the structure of the entire amino acid is shown, since Ri cannot be represented independently. Table 2: Some Non-essential amino acids and their chemical structures
Figure imgf000019_0001
Synthesis of MAP-CAB conjugates
[0058] To form a CAB-polymer conjugate, a CAB-2'-amino acid-methanesulfonic acid salt is combined with a polyol/alkylene glycol polymer (e.g., a mucic acid polymer such as Compound 20) in the presence of dimethyl sulfoxide, PyAOP and DIPEA. The amino group of the MSA salt reacts with one or more carboxyl groups of the polymer, forming an amide bond (Example 3). The product is purified, and appropriate size fractions are obtained, e. ., by dialysis. Percent CAB in the MAP-CAB conjugate is determined as described in Example 4. In certain embodiments, a MAP-CAB conjugate contains at least 5% CAB by weight, at least 10% CAB by weight, at least 15% CAB by weight, at least 20% CAB by weight, at least 25% CAB by weight, at least 30% CAB by weight, at least 35% CAB by weight, at least 40% CAB by weight, at least 45% CAB by weight, at least 50% CAB by weight, at least 55% CAB by weight, or at least 60% CAB by weight,. In additional embodiments, a MAP-CAB conjugate contains between 5-10% CAB by weight, between 10-15% CAB by weight, between 15-20% CAB by weight, between 20-25% CAB by weight, between 25-30% CAB by weight, between 30-35% CAB by weight, between 35-40% CAB by weight, between 40-45% CAB by weight, between 45-50% CAB by weight, between 50-55% CAB by weight, or between 55-60% CAB by weight. Combined and/or overlapping combinations of the above concentrations and ranges are also provided. Conjugates of CAB-containing polymers with nitrophenylboronic acid-containing polymers
[0059] Polymers containing conjugated cabazitaxel can also have conjugated to them one or more boronic acid-containing polymers. These can include boronic acid-containing polymers, phenyl boronic acid-containing polymers; nitrophenyl boronic acid-containing polymers; and polymers containing one or a plurality of phenyl-boronic acid- or nitrophenyl boronic acid molecules. Boronic acid-containing polymers (including nitrophenylboronic acid-containing polymers, are described, for example, in US Patent No. 10,342,879; US Patent No. 9,446,149; US Patent No. 11,285,212; US Patent No. 10,182,986; US Patent No. 11,041,050; US Patent No. 11,738,092 and U.S. provisional patent application No. 63/588,185; the disclosures of all of which are incorporated by reference in their entireties for the purposes of describing the synthesis, properties and uses of boronic acid-containing polymers, nitrophenyl boronic acidcontaining polymers; di-boronic acid-containing polymers and di-nitrophenyl boronic acidcontaining polymers.
[0060] In certain embodiments, cabazitaxel -containing polymers, as described above, are conjugated to polyethylene glycol (PEG) polymers containing one or more nitrophenylboronic acid (NPBA) moieties as described, for example, in co-owned U.S. provisional patent application No. 63/588,185. For example, a NPBA-PEG polymer can have a structure as shown in the formulabelowl, in which the NPBA portion of the polymer is to the left of the spacer in the formula, and the PEG portion of the polymer is to the right of the spacer:
Figure imgf000020_0001
wherein:
L3 is a linking group, or linking groups, between the one or more boronic acid moieties and the spacer;
L4 is a linking group, or linking groups, between the spacer and the one or more polyethylene oxide linkages;
Spacer is a moiety containing at least one terminal amine and/or at least one terminal carboxylic acid; q is independently at each occurrence 0, 1 , 2, 3, or 4; r is 1, 2, or 3; s is independently at each occurrence 20-1200; t is 1, 2, or 3; and
Xi is a terminal functional group.
[0061] In certain embodiments, Xi is either of:
(a) -Ci-6 alkyl, optionally substituted with -OH, -COOH, -C(=O)O(alkyl), - C(=O)O(aryl), -NH2, -NH(alkyl), orN(alkyl)2; or
(b) a salt or protected analog of any of the groups of (a); or
Xi comprises a targeting molecule or a therapeutic molecule. In certain embodiments, a targeting molecule (as described elsewhere herein) is a covalent conjugate of one of the functional groups of (a) or (b) above, such that Xi has the structure -C-C(=O)-NH-targeting molecule. In additional embodiments, a therapeutic molecule (as described elsewhere herein) is a covalent conjugate of one of the functional groups of (a) or (b) above, such that Xi has the structure -C-C(=O)-NH-therapeutic molecule.
[0062] In certain embodiments, the spacer is an amino acid. In a preferred embodiment, q is 1, r is 2, s is 50-350, t is 1, L3 is -C(=O)-NH-, L4 is -C(=O)-NH-, Xi is -CH3, and the spacer is a lysyl group. In an additional embodiment, q is 1, r is 2, s is 50-350, t is 1, L3 is -C(=O)- NH-, L4 is -C(=O)-NH-, Xi is -C-C(=O)-OH, and the spacer is a lysyl group.
[0063] A NPBA-PEG polymer can be synthesized, for example, by combining an amine- functionalized PEG (e.g., PEG2k, PEG3.5k, PEG5k, PEGlOk) with a carboxy NPBA to form a NPBA-PEG conjugate joined by an amide linkage. See, for example, U.S. Patent No.
11,738,092 and U.S. provisional patent application No. 63/588,162; the disclosures of which are incorporated by reference for the purposes of describing synthesis, properties and uses of NPBA- PEG polymers.
Targeting molecules
[0064] In certain embodiments, CAB -containing nanoparticles contain a targeting molecule, or targeting agent, which facilitates homing of a nanoparticle to a target (e.g., a tumor cell) after systemic delivery. Targeting molecules are known in the art and include, for example, small molecules e.g., vitamins, e.g., folate), saccharides (e.g., mannose, allose, altrose, glucose, gulose, idose, galactose, talose, di saccharides, trisaccharides, oligosaccharides), peptides (e.g., RGD), polypeptides (e.g., antibodies, proteins that bind cell-surface receptors such as transferrin), nucleic acids (e.g, aptamers), peptoids and peptide nucleic acids (PNAs).
[0065] In additional embodiments, a targeting molecule is a protein, a peptide, an aptamer, or a ligand for a cellular receptor. A targeting molecule that is a protein can be, for example, an antibody or fragment thereof.
[0066] In certain embodiments, the target is the prostate gland, and the targeting agent binds to a molecule present on the surface of a prostate cancer cell, such as prostate-specific antigen (PSA) or prostate-specific membrane antigen (PSMA). In certain embodiments, a targeting molecule is an antibody (or fragment thereof) directed to PSA, or an antibody (or fragment thereof) directed to PSMA, or an antibody (or fragment thereof) directed to B7-H3, or an antibody (or fragment thereof) directed to B7-H4, or an antibody (or fragment thereof) directed to STEAP1, or an antibody (or fragment thereof) directed to HER2, or an antibody (or fragment thereof) directed to HER3, or an antibody (or fragment thereof) directed to TROP2.
[0067] In further embodiments, for targeting to a tumor cell, one or more small molecule targeting molecules can be used. These include, but are not limited to, carbohydrates (such as, for example, fructose, glucose, mannose, levan; or a derivative thereof), vitamins (such as, for example, riboflavin, biotin, thiamine, vitamin B 12, and folic acid), and amino acids (such as, for example, glycine, alanine, arginine lysine and tryptophan). Additional small molecule targeting agents include adenosine, anisamide, anacardic acid, phenylboronic acid and methotrexate. See, for example, Kaur et al. (2023) ./. Controlled Release 355:417-433.
[0068] Additional targeting molecules are described in co-owned U.S. provisional patent application No. 63/588,162; the disclosure of which is incorporated by reference for the purposes of describing targeting molecules and methods for their inclusion in therapeutic nanoparticles.
[0069] In certain embodiments, a targeting molecule is covalently coupled to a boronic acid containing polymer, such as, for example a NPBA-PEG polymer as described in co-owned United States provisional patent application No. 63/588,162 filed October 5, 2023; the disclosure of which is hereby incorporated by reference in its entirety for the purposes of describing NPBA- PEG polymers, their structures, their synthesis and their uses.. For example, a targeting molecule can be part of the Xi moiety of the NPBA-PEG polymer, e.g., the targeting molecule is a covalent conjugate of one of the functional groups that are specified United States provisional patent application No. 63/588,162 filed October 5, 2023.
[0070] In certain embodiments, a targeting molecule is attached to a NPBA-PEG polymer by way of reaction with a pentafluorophenyl (PFP)-activated ester of the NPBA-PEG polymer, as described, for example, in U.S. Patent No. 11,738,092 and in co-owned U.S. provisional patent application No. 63/588,162; the disclosures of which are incorporated by reference for the purpose of describing methods for attaching polypeptides to NPBA-PEG polymers. Inasmuch as primary amine groups in proteins react with the PFP-activated ester, attachment of a polypeptide (such as, for example, an antibody) to the NPBA-PEG- pentafluorophenyl ester is achieved by combining the ester with the polypeptide. Progress of the reaction can be monitored by high-performance liquid chromatography (HPLC), and the NPBA- PEG-polypeptide conjugate can be purified by hydrophobic interaction chromatography (HIC).
[0071] The targeting agent is attached to the CAB-MAP conjugate, prior to formation of the nanoparticle, by methods described in, for example, U.S. Patents 9,466,149; 10,182,986; 10,342,879; 11,041,050; 11,285,212 and 11,738,092. The disclosures of all of the aforementioned patents are hereby incorporated by reference in their entireties for the purposes of describing targeting molecules and methods for their attachment to MAP-containing polymers and to polyol/PEG-containing polymers.
Therapeutic molecules
[0072] In certain embodiments, cabazitaxel-containing polymers, as described herein, comprise a therapeutic molecule (e.g., a large molecule therapeutic). For example, a therapeutic molecule can be conjugated to a NPBA-PEG polymer in the manner described above for conjugation of a targeting molecule to a NPBA-PEG polymer; i.e., by reaction between the therapeutic molecule and a pentafluorophenyl (PFP)-activated ester of the NPBA-PEG polymer. Thus, in certain embodiments, a therapeutic molecule is part of the Xi moiety of the NPBA-PEG polymer described in co-owned U.S. provisional patent applications 63/588,162 and 63/588,185. Large molecule therapeutics are known in the art and include, for example, therapeutic polypeptides (e.g., antibodies, enzymes, and bioactive proteins) and nucleic acids.
[0073] Exemplary therapeutic polypeptides include, but are not limited to, antibodies such as trastuzumab (Herceptin, an anti-Her2 antibody), anti-Her3, anti-Trop2, anti-PSMA, anti- LIV-1, anti-FOLRl, anti-DLL3, anti-PDGF, anti-FRalpha, anti-PTK7, anti-mesothelin, anti-c- MET, anti-MUCl, anti-CD70, anti-CD74, anti-CD30, anti-CD33, anti-FLT3, anti-CD22, anti- CD20, and anti-CD19, anti-CD4, anti-CD8, and antibodies directed against B7-H3, B7-H4, Nectin-4, Claudin 18.2, R0R1, R0R2, BCMA, EpCAM, Claudin 6, CDH6, CEACAM5, and Integrin beta-6.
[0074] Certain polypeptide therapeutics, such as antibodies directed to tumor markers like those exemplified in the previous paragraph, can also be used as targeting molecules.
Biological Agents
[0075] In certain embodiments, nanoparticles as disclosed herein comprise a cabazitaxel- containing polymer and a biological agent. Biological agents include amino acids, peptides, polypeptides, nucleotides, and nucleic acids, such as oligonucleotides and polynucleotides. Exemplary nucleic acids include DNAs such as genomic DNA, cDNA, plasmids and antisense oligonucleotides. Additional exemplary nucleic acids include RNAs, such as, for example, mRNA, siRNA, circular RNA, shRNA, mi RNA, antisense RNA, guide RNA, and tracr RNA. Any of the biological agents mentioned above can be linear, circular or branched (e.g., cyclic peptides, or circular nucleic acids, such as circular RNA).
[0076] Exemplary biological agents for use in RNA interference methods are siRNAs targeted to certain cancer-related genes (e.g., oncogenes) such as, for example, c-myc, c-myb, mdm2, PKA-I, Abl-1, Bcl2, Ras, c-Raf kinase, CDC25 phosphatases, cyclins, cyclin dependent kinases, telomerase, PDGF/sis, erb-B, fos, jun, mos, src, the Bcr/Abl fusion gene, EGFR, BRAF, RRM2, HER2, VEGF, FGFR2, K-RAS, Claudin 18.2, EpCAM, STAT3, mesothelin/MSLN and BCRP.
[0077] In additional embodiments, the biological agent can be negatively charged, such as a nucleic acid. In further embodiments, the nucleic acid can be DNA, RNA or a peptide nucleic acid (PNA). Biological agents that are RNA can be low molecular weight RNAs such as antisense RNA, miRNA and siRNA, which can be introduced into a target cell to regulate (e.g., repress) expression of an endogenous target gene; or high molecular weight RNAs such as mRNAs, which can be used to encode an exogenous protein in a target cell. In addition, a biological agent can be any of a guide RNA and/or a tracr RNA, and/or a RNA encoding the Cas9 nuclease, for use in gene editing using the CRISPR-Cas9 system. Biological agents can also be DNA molecules encoding any of the RNAs described above. [0078] In further embodiments, a biological agent is a small molecule therapeutic, such as a chemotherapeutic. Exemplary chemotherapeutics include camptothecin and its derivatives and metabolites (such as SN38); taxanes e.g., taxol, docetaxel) and epithilones.
Nanoparticles
[0079] Nanoparticles are formed by diluting CAB-MAP conjugates (or conjugates of CAB with other polyol/polyalkylene glycol polymers) in water or dilute saline. The CAB-MAP conjugate (or CAB-polyol/polyalkylene glycol conjugate) may optionally also comprise one or more of a targeting molecule, a therapeutic molecule and/or a biological agent, wherein the targeting molecule, therapeutic molecule and/or biological agent is optionally joined to the MAP polymer via a NPBA-PEG polymer to which the targeting molecule, therapeutic molecule and/or biological agent is covalently joined. The ionic strength of the nanoparticle solution is adjusted if desired, e.g., by the addition of saline (Example 6).
[0080] The size of the nanoparticles (z.e., cross-sectional diameter) ranges from about 10 to about 60 nm in diameter. Exemplary nanoparticle diameters are 15 nm, 16 nm, 17 nm, 18 nm, 19, nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm or larger. Exemplary nanoparticle diameters are 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, or any value within the range of 20-60 nm. Specific embodiments can also describe these nanoparticles as having diameters in a range of from about 20 nm to about 30 nm, from about 30 nm to about 40 nm, from about 40 nm to about 50 nm, from about 50 nm to about 60 nm, or a combination of two or more of these ranges. In certain embodiments, the nanoparticles are spherical and have a diameter of 20-40 nm as determined by DLS and Cryo-transmission electron microscopy.
[0081] In some embodiments, the total size of the nanoparticle is from about 20 nm to about 60 nm. In some embodiments the size of the nanoparticle is from about 20 nm to about 30 nm. In some embodiments the size of the nanoparticle is from about 20 nm to about 40 nm. In some embodiments the size of the nanoparticle is from about 20 nm to about 50 nm. In some embodiments the size of the nanoparticle is from about 20 nm to about 60 nm. In some embodiments the size of the nanoparticle is from about 30 nm to about 40 nm. In some embodiments the size of the nanoparticle is from about 30 nm to about 50 nm. In some embodiments the size of the nanoparticle is from about 30 nm to about 60 nm. Tn some embodiments the size of the nanoparticle is from about 40 nm to about 50 nm. In some embodiments the size of the nanoparticle is from about 40 nm to about 60 nm.
Treatment methods
[0082] Also provided are methods for treating malignancies with the nanoparticles disclosed herein. The malignancy to be treated can include any solid tumor for which cabazitaxel is an effective treatment. Examples include, but are not limited to, prostate cancer, metastatic castration-resistant prostate cancer (mCRPC), breast cancer, lung cancer, uterine cancer, cervical cancer, bladder cancer, head and neck cancer, and sarcomas, including Kaposi’s sarcoma. In one embodiment, nanoparticles comprising cabazitaxel, and further comprising a targeting agent directed to PSMA, can be used for the treatment of prostate malignancies such as, for example, prostate cancer and mCRPC.
Formulations, kits, and routes of administration
[0083] Therapeutic compositions comprising nanoparticles as disclosed herein are also provided. Such compositions typically comprise the nanoparticles and a pharmaceutically acceptable carrier or excipient. Supplementary active compounds, such as targeting molecules, therapeutic molecules, and/or biological agents, can also be incorporated into nanoparticle compositions.
[0084] The therapeutic compositions disclosed herein are useful for, inter alia, treating cancer, cancer metastases and other disorders of the brain and central nervous system. Accordingly, a “therapeutically effective amount” of a composition comprising nanoparticles is any amount that reduces symptoms or, e.g., stimulates tumor regression. For example, dosage amounts of nanoparticles can vary from about 0.1-1.0 mg/kg body weight, or from about 0.5 to 2.0 mg/kg body weight or from about 1-5 mg/kg body weight or from about 1 mg/kg body weight to about 10 mg/kg body weight or more (or any integral value therebetween); with a frequency of administration of, e.g., hourly, twice per day, once per day, twice per week, once per week, twice per month, once per month, depending upon, e.g., body weight, route of administration, severity of disease, etc. Thus, a therapeutically effective amount can comprise a plurality of administrations of the same amount, or different amounts, of nanoparticles. In certain embodiments, a single administration of nanoparticles is a therapeutically effective amount.
[0085] In certain embodiments, nanoparticles are administered at a dosage of 1-20 mg nanoparticle (or any integral or decimal value therebetween) per square meter of the surface area of the body of the subject, as is typical for dosages of chemotherapeutics. For example, a dosage can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 122, 13, 14, 15, 16, 17, 18, 19, or 20 mg/m2; or from 1-5, 2-7, 5-10, 8-12, 10-15, 13-17, or 15-20 mg/m2.
[0086] In certain embodiments, dosage amounts and schedules are determined empirically and may be different from those listed above.
[0087] Various pharmaceutical compositions and techniques for their preparation and use are known to those of skill in the art in light of the present disclosure. For a detailed listing of suitable pharmacological compositions and techniques for their administration one may refer to texts such as Remington's Pharmaceutical Sciences, 17th ed. 1985; Brunton et al., “Goodman and Gilman’s The Pharmacological Basis of Therapeutics,” McGraw-Hill, 2005; University of the Sciences in Philadelphia (eds.), “Remington: The Science and Practice of Pharmacy,” Lippincott Williams & Wilkins, 2005; and University of the Sciences in Philadelphia (eds.), “Remington: The Principles of Pharmacy Practice,” Lippincott Williams & Wilkins, 2008.
[0088] The nanoparticles described herein can be suspended in a physiologically compatible carrier for administration. As used herein, the term “physiologically compatible carrier” refers to a carrier that is compatible with the nanoparticles and with any other ingredients of the formulation, and is not deleterious to the recipient thereof. Those of skill in the art are familiar with physiologically compatible carriers. Examples of suitable carriers include water (e.g., pH 4 water), phosphate-buffered saline, Hank’s balanced salt solution+/- glucose (HBSS), and multiple electrolyte solutions such as, e.g., Plasma-LyteTM A (Baxter).
[0089] The volume of a nanoparticle suspension administered to a subject will vary depending on the site of administration, treatment goal and number of nanoparticles in solution. Typically the amount of nanoparticles administered will be a therapeutically effective amount. As used herein, a “therapeutically effective amount” or “effective amount” refers to the number of administered nanoparticles which are required to effect treatment of the particular disorder; i.e., to produce a reduction in the amount and/or severity of the symptoms associated with that disorder. For example, in the case of mCRPC, administration of a therapeutically effective amount of nanoparticles results in regression of the cancer. Therapeutically effective amounts vary with the type and extent of malignancy, and can also vary depending on the overall condition of the subject; and can be determined by one of skill in the art using established methods.
[0090] The disclosed therapeutic compositions can also include pharmaceutically acceptable materials, compositions or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, i.e., carriers. These carriers can, for example, stabilize the nanoparticles and/or facilitate the retention of the nanoparticles in the body. Each carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically- acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol and polyethylene glycol; polyols, such as glycerin, sorbitol and mannitol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
[0091] Nanoparticles can be administered to a subject by any suitable route, including, but not limited to, inhalation, topically, nasally, orally, parenterally (e.g., intravenously, intraperitoneally, intravesically or intrathecally) or rectally in a vehicle comprising one or more pharmaceutically acceptable carriers, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard practice. Administration of the compositionds described herein can be carried out using any method known in the art. For example, administration may be transdermal, parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intracerebroventricular, intrathecal, intranasal, aerosol, by suppositories, or by oral administration. A pharmaceutical composition of the nanoparticles described herein can be for intravenous administration or for administration by injection, or for oral, pulmonary, nasal, transdermal, or ocular administration.
[0092] Exemplary formulations include, but are not limited to, those suitable for parenteral administration, e.g., intrapulmonary, intravenous, intra-arterial, intra-ocular, intracranial, sub-meningeal, or subcutaneous administration, including formulations encapsulated in micelles, liposomes or drug-release capsules (active agents incorporated within a biocompatible matrix designed for slow-release); ingestible formulations; formulations for topical use, such as eye drops, creams, ointments and gels; and other formulations such as inhalants, aerosols and sprays. The dosage of the compositions of the disclosure will vary according to the extent and severity of the need for treatment, the activity of the administered composition, the general health of the subject, and other considerations well known to the skilled artisan.
[0093] In additional embodiments, the compositions described herein are delivered intracranially at or near a site of brain injury or metastasis. Such localized delivery allows for the delivery of the composition non-systemically, thereby reducing the body burden of the composition as compared to systemic delivery. Local delivery can be achieved, for example, by intra-cranial injection, or through the use of various medically implanted devices including, but not limited to, stents and catheters, or can be achieved by inhalation, phlebotomy, or surgery. Methods for coating, implanting, embedding, and otherwise attaching desired agents to medical devices such as stents and catheters are established in the art and contemplated herein.
[0094] Another aspect of the present disclosure relates to kits for carrying out the administration of nanoparticles, optionally in combination with another therapeutic agent, to a subject. In one embodiment, a kit comprises a composition of nanoparticles formulated in a pharmaceutical carrier, suitable for administration, e.g., by injection.
EXAMPLES
Example 1: Synthesis of cabazitaxel-2'-carboxybenzyl-glycinate (CAB-2'-Gly-Cbz) (Compound 3) and related carboxybenzyl-protected cabazitaxel amino acid derivatives (Compounds 9-13)
[0095] 0.108 g (0.51 mmol) of Cbz-Gly-OH (Compound 2), 0.120 g (0.98 mmol) of DMAP, 0.282 g (1.47 mmol) of EDC, and 0.411 g (0.49 mmol) of cabazitaxel (CAB, Compound 1) were measured into an 8 mL vial with a magnetic stir bar and septum. The headspace of the vessel was purged with positive argon pressure. To above mixture of solids was added anhydrous DCM to generate a solution. The reaction was allowed to stir at room temperature overnight. HPLC indicated completion of the reaction. The reaction solution was washed with 0.1 M HCI (3 x 3 mL) to remove the EDC and DMAP, with 0.1 M NaHCO.i (3 x 3 mL) to remove the excess Cbz-Gly-OH, and then with brine (1 x 3 mL). The residue was dried over Na2SO4, filtered, and dried under a steam of argon. The material was dried further under high vacuum.
[0096] 0.450 g CAB-2'-Gly-Cbz (3) was obtained as a white solid (0.438 mmol, 89% yield). ’HNMR (600 MHz, DMSO-r76): 7.98 (d, 2H), 7.91 (d, 1H). 7.78 (t, 1H), 7.73 (d, 1H), 7.68 (t, 2H), 7.42 (t, 2H), 7.38 (m, 6H), 7.32 (m, 1H), 7.19 (t, 1H). 5.81 (t, 1H), 5.39 (d, 1H), 5.12 (d, 1H), 5.10 (m, 1H), 5.07 (dd, 2H,), 4.96 (d, 1H), 4.70 (s, 1H), 4.50 (s, 1H), 4.01 (dd, 2H), 3.91 (m, 2H), 3.74 (dd, 1H), 3.58 (d, 1H), 3.27 (s, 3H), 3.20 (s, 3H), 2.55 (m, 1H), 2.23 (s, 3H), 1.78 (m, 4H), 1.51 (m, 5H), 1.39 (s, 9H), 0.97 (d, 6H). ESI/MS: 1027.4466. [M+H1].
Figure imgf000030_0002
Figure imgf000030_0001
[0097] Cbz-Gly-OH can be substituted with other Cbz-protected amino acids in the above procedure. By substituting Cbz-Ala-OH (Compound 4), Cbz-P-Ala-OH (Compound 5), Cbz-Val-OH (Compound 6), Cbz-(GABA)-OH (Compound 7), and Cbz-Hex-OH (Compound 8), the same procedure yields CAB-2'-Ala-Cbz (Compound 9), CAB-2'-P-Ala-Cbz (Compound 10), CAB-2'-Val-Cbz (Compound 11), CAB-2'-GABA-Cbz (Compound 12), and CAB-2'-Hex- Cbz (Compound 13), respectively.
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
[0098] It will be apparent that additional Cbz-protected amino acids can be used (see, e.g., Tables 1 and 2), in the procedure described in this example, to provide additional Cbz- protected CAB-amino acid derivatives.
Example 2: Synthesis of cabazitaxel-2'-glycinate.methane sulfonic acid (CAB-2’- Gly.MSA) (Compound 14) and related methane sulfonic acid salts of cabazitaxel (Compounds 15-19)
[0099] 0.066 mg Pd/C (10%) and 0.319 g (0.331 mmol) of CAB-2'-Gly-Cbz (3) were weighed into an 8 mL vial with a septum and a magnetic stir bar. Air was evacuated under vacuum through a needle. Isopropyl alcohol (IP A, 4mL) was added, and a H2 balloon with a long needle was inserted into the reaction mixture, allowing H2 to bubble through the reaction mixture with a vent (short needle) through the septum. Immediately upon insertion of the H2 balloon, 1 mL of a solution of 0.030 g (0.31 mmol) of methanesulfonic acid in IPA was added via syringe. After stirring for 5 mins, the venting needle was removed, and the reaction mixture was stirred vigorously for 2 hrs. HPLC indicated completion of the reaction. 0.6 g Celite was added to the reaction mixture. The resulting mixture was stirred for 2 mins, then filtered through a Celite plug. The filtrate was washed with IPA (3 x 0.5 mL). The combined filtrates were dried using a steam of argon, and dried further under high vacuum.
[0100] 0.277 g CAB-2'-Gly.MSA (14) was obtained as a white solid (0.28 mmol, 90% yield). JH NMR (600 MHz, DMSC ,): 8.01 (d, 2H), 7.91 (d, 1H), 7.73 (t, 1H), 7.63 (t, 2H), 7.44 (t, 2H), 7.38 (d, 1H, 2H), d, 1H)7.21 (t, 1H), 5.87 (t, 1H), 5.39 (d, 1H), 5.18 ((d, 1H), 5.13 (t, 1H), 4.45 (d, 1H), 4.70 (s, 1H), 4.54 (s, 1H), 4.02 (dd, 2H), 3.87 (dd, 2H), 3.74 (s, 3H)m, 1H), 3..59 (d, 1H), 3.29 (s, 3H), 3.20 (s. 3H), 2.65 (m, 1H), 2.29 (s, 3H), 2.27 (s, 3H), 1.85 (m, 1H), 1.83 (s, 3H), 1.64 (m, 1H), 1.51 (s, 3H), 1.48 (m, 1H), 1.34 (s, 9H), 0.95 (d, 6H). ESI/MS: 893.4103 [M+H+],
Figure imgf000034_0002
[0101] Substitution, in the method described above, of CAB-2'-Gly-Cbz with CAB-2'- Ala-Cbz (9), CAB-2' -P-Ala-Cbz (10), CAB-2'-Val-Cbz (11), CAB-2'-GABA-Cbz (12), and CAB-2'-Hex-Cbz (13) yields CAB-2'-Ala.MSA (Compound 15), CAB-2'-P-Ala.MSA (Compound 16), CAB-2'-Val.MSA (Compound 17), CAB-2'-GABA.MSA (Compound 18), and CAB-2'-Hex.MSA (Compound 19), respectively.
Figure imgf000034_0001
Figure imgf000035_0001
[0102] It will be apparent that additional Cbz-protected CAB-amino acid derivatives can be used (see, e.g, Tables 1 and 2), in the procedure described in this example, to provide additional MSA salts of CAB.
Example 3: Synthesis of MAP cabazitaxel glycinate conjugate (MAP-Gly-CAB) (Compound 21) and related MAP-cabazitaxel (MAP-CAB) conjugates (Compounds 22-26)
[0103] CAB-2'-Gly.MSA (14) was conjugated to a mucic acid polymer (MAP, as described, for example, in US Patent No. 11,738,092) as follows. 40 mg (0.019 mmol COOH) of MAP (compound 20, identified as compound 6 in US 11,738,092) and 16.2 mg (0.016 mmol) of CAB-2'-Gly.MSA (14) were combined in an 8 mL reaction vial with a septum and a magnetic stirring bar. The vial was charged with 3mL anhydrous DMSO. In a separate vial, 30.1 mg (0.058 mmol) PyAOP and 1 mb anhydrous DMSO were combined to generate a solution. The PyAOP solution was added to the reaction vial. Then, 12 mL (0.069 mmol) DIPEA was added, with stirring. The reaction mixture was stirred at ambient temperature overnight; then dialyzed against DMF (4 x 900 mL) and water (4 x 900 mL) using a 10 kDa membrane. The last water dialysis product was frozen, and lyophilized to afford 46.7 mg MAP-Gly-CAB (21).
[0104] 'H NMR (600 MHz, DMSO-t/6): 8.33 (s, br, 1H), 8.02 (d, , 1H), 7.98 (d, 2H), 7.89 (s, br, 1H), 7.78 (s, br, 1H), 7.72 (t, 2H), 7.65 (t, 3H), 7.41 (t, 2H), 7.34 (d, 2H), 7.18 (t, 1H), 5.81 (t, 1H), 5.36 (d, 1H), 5.12 (t, 1H), 5.08 (dd, 2H), 4.92 (d, 1H), 4.69 (s, 1H), 4.52 (dd, 1H), 4.47 (s, 1H), 4.39 (s, br, 1H), 4.12 (d, 1H), 4.0 (m, 4H), 3.81 (s, br, 1H), 3.73 (t, 1H), 3.29 (s, 3H), 3.21 (s, 3H), 2.63 (m, 2H), 2.37 (m, 2H), 2.23 (s, 3H), 1.80 (m, 1H), 1.79 (s, 3H), 1.51
(m, 2H), 1.50 (s, 3H), 1.37 (s, 9H), 0.95 (d, 6H).
Figure imgf000036_0001
[0105] Before conjugation of CAB, MAP polymers can have a number of terminal structures, as shown below. The polymer can contain amine groups at both ends (top); an amine group at one end and a carboxylic acid group at the other end (middle); or carboxylic acid groups at both ends (bottom).
Figure imgf000037_0001
[0106] After conjugation of CAB, carboxylic acid end groups of the MAP polymers, as well as the carboxylic acid groups within the polymer repeat unit, may be derivatized in the resulting MAP-CAB conjugates, as described by the structures below:
Figure imgf000038_0001
[0107] Additional MAP-amino acid-CAB conjugates are produced by the same method using CAB-2'-Ala.MSA (15), CAB-2'-P-Ala.MSA (16), CAB-2'-Val.MSA (17), CAB-2'- GABA.MSA (18), and CAB-2'-Hex.MSA (19) as starting material, instead of CAB-2'-Gly.MSA, to generate MAP-Ala-CAB (Compound 22), MAP-P-Ala-CAB (Compound 23), MAP-Val-CAB (Compound 24), MAP-GAB A-C AB (Compound 25), and MAP -Hex -CAB (Compound 26), respectively.
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
[0108] It will be apparent that additional MSA salts of CAB-amino acid derivatives (see, e.g., Tables 1 and 2) can be used, in the procedure described in this example, to provide additional MAP-amino acid-CAB conjugates.
[0109] In compounds 20-26, n can range from 20 to 200 and m can range from 5 to 200. In certain embodiments, n is 114 or about 114 (PEG molecular weight about 5 kDa). In other embodiments, n is 80 or about 80 (PEG molecular weight about 3.5 kDa). In further embodiments, n is 46 or about 46 (PEG molecular weight about 2 kDa). In certain embodiments, m is 16 or about 16.
Example 4: CAB content in MAP-CAB conjugates
[0110] Weight percent of CAB in MAP-amino acid-CAB conjugates was determined by high-performance liquid chromatography with ultraviolet absorbance detection (HPLC-UV). MAP-Gly-CAB conjugate was dissolved in dimethyl formamide (DMF) at 1 mg/mL. 10 pL of conjugate solution was added to 6.7 pL 0.5 N NaOH and incubated for 3 h at room temperature. 6.7 pL 0.5 N HC1 was subsequently added and incubated for 30 min at room temperature. After incubation with HC1, 23.4 pL of acetonitrile (ACN) was added to complete sample preparation. [0111] Samples were analyzed using an Agilent 1200 Series HPLC system. Using an isocratic mobile phase of 57:43 Water + 0.01 % tri fluoroacetic acid (TFA):ACN + 0.01 % TFA, 20 pL of sample was injected onto a Phenomenex Synergi Hydro-RP (4 pm, 80 A) reverse-phase column. The amount of CAB in the conjugate solution was quantified by measuring absorbance at 230 nm and comparing to a calibration curve of reference CAB solutions, prepared as described in Example 5.
[0112] Weight percent CAB was then determined using the following formula:
Weight percent CAB = 100% x [(concentration of total CAB)/concentration of MAPCAB conjugate)].
Example 5: Preparation of reference CAB solutions
[0113] Reference solutions of cabazitaxel (CAB) were prepared by dissolving different concentrations of CAB in dimethyl formamide (DMF). 10 pL of each reference solution was added to 6.7 pL 0.5 N NaOH and the mixture was incubated for 3 h at room temperature. 6.7 pL 0.5 N HC1 was subsequently added and the mixture was incubated for 30 min at room temperature. After the incubation with HC1, 23.4 pL of acetonitrile (ACN) was added. The absorbance at 230 nm (A230) was measured for each reference solution and the absorbance values were used to construct a standard curve (calibration curve).
Example 6: Formation of nanoparticles
[0114] 75 mg of MAP-Gly-CAB (21) was dissolved in 15 m of pH 4 ultrapure water. The solution was stirred at room temperature for approximately 3 hours. 1.7 m of 9 % saline, pH 4, was added and stirring was continued for 1 hour. The nanoparticle solution was concentrated using a 10 kDa membrane, passed through a 0.22 pm fdter, and frozen.
[0115] Any of the other MAP-amino acid-CAB conjugates described above (i.e., MAP- Ala-CAB (22), MAP-0-Ala-CAB (23), MAP-Val-CAB (24), MAP-GAB A-CAB (25), and MAP- Hex-CAB (26) are converted to nanoparticles in similar fashion, i.e., by dissolving in water or dilute saline, at pH 4, to a concentration of 1-10 mg/mL. Example 7. Nanoparticle characterization
[0116] Nanoparticle size (Zavg) and poly dispersity index (PDI) were determined using dynamic light scattering (DLS), which was performed on a Malvern Zetasizer Nano ZS instrument. Nanoparticles made from MAP-Gly-CAB were diluted to a concentration of 2.0 mg/mL in pH 4 saline. The results of 3 measurements, 10 runs each, were averaged.
[0117] The surface charge, or zeta potential, of MAP-Gly-CAB nanoparticles was measured using a Malvern Zetasizer Nano ZS instrument. 75 LLL of 2.0 mg/mL of MAP-Gly- CAB nanoparticle solution was mixed with 675 pL of 1 mM potassium chloride. The results of 3 measurements, 10 runs each, were averaged.
[0118] Using the methods described above, the Zavg of MAP-Gly-CAB nanoparticles was determined to be 19 nm; the PDI of MAP-Gly-CAB nanoparticles was determined to be 0.21; and the zeta potential of MAP-Gly-CAB nanoparticles was determined to be -0.08 mV.
[0119] Zavg, PDI and zeta potential of nanoparticles made from MAP-Ala-CAB, MAP- - Ala-CAB, MAP-Val-CAB, MAP-GABA-CAB, and MAP-Hex-CAB are determined in similar fashion.
Example 8: In vitro release studies
[0120] Release of CAB from nanoparticles is determined by incubating solutions of nanoparticles in various media and subsequently measuring the amount of unconjugated and total CAB present in solution at different timepoints.
[0121] In a typical experiment, nanoparticles were diluted to a concentration of 1.0 mg/mL in various release media (pH 5.5 phosphate-buffered saline (PBS), pH 7.4 PBS, mouse plasma, rat plasma, or human plasma). This 1.0 mg/mL stock of particles in release media was separated into 14 microcentrifuge tubes with 2 holes poked in the top of each cap. These tubes were subsequently incubated in a Thermo Forma Series II water-jacketed CO2 incubator at 5 % CO2 and 37 °C. At 0, 2, 4, 6, 12, 24, 48, 72, 96, and 120 h, two tubes were removed from the incubator, frozen on liquid nitrogen, and stored at -80°C until analysis.
[0122] Frozen samples were thawed and analyzed for total CAB and free (unconjugated) CAB as described in Example 9. Standards were prepared as described in Example 5. Release of CAB from nanoparticles is expressed as the ratio of free CAB to total (z.e., the sum of free and conjugated) CAB. Exemplary results, showing in vitro release rates of CAB from nanoparticles in mouse plasma, are shown in Figure 1 . The results indicate that the release rate depends on the amino acid linker present between the CAB and the MAP components of the nanoparticle.
Example 9: In vivo rat pharmacokinetics (PK) study
[0123] MAP-Gly-CAB, MAP-Ala-CAB, MAP-|3-Ala-CAB, and MAP-GABA-CAB nanoparticles, formulated in 0.9% saline, pH 4, were administered via bolus intravenous injection at 7.5 mg/kg (CAB basis) through a femoral vein canula in male Sprague Dawley rats. At predetermined time points, blood was collected via a jugular vein canula. Samples were kept on ice until centrifugation at 2000 x g for 10 min at 5°C within 1 h of collection. Plasma was directly transferred to cluster tubes and stored at -80°C until analysis.
[0124] Analyses for total CAB and unconjugated CAB were as follows. To determine the amount of unconjugated CAB, 10 pL of sample was mixed with 13.4 pL 0.5 N HC1 and incubated for 30 min at room temperature. 23.4 pL ACN was subsequently added and incubated for 3 h at room temperature. After incubation with ACN, the mixture was centrifuged at 14,000 x g for 10 min at 4°C and the supernatant was recovered and filtered through a 0.20 pm filter. To measure the total amount of CAB, 10 pL of sample was mixed with 6.7 pL 0.5 N NaOH and incubated for 3 h. 6.7 pL 0.5 N HC1 was subsequently added and incubated for 30 min at room temperature, followed by addition of 23.4 pL ACN and incubation for 3 h at room temperature. After incubation with ACN, the mixture was centrifuged at 14,000 x g for 10 min at 4°C and supernatant was filtered with a 0.20 pm filter. Filtered samples were analyzed using an Agilent 1200 Series HPLC system as described in Example 4 and compared to a calibration curve of reference CAB solutions, prepared as described in Example 5.
[0125] The results, shown in Figures 2-4, indicate that nanoparticles display a biphasic profile with a fast redistribution phase and a long elimination phase. The elimination phase for the nanoparticles was prolonged for increasingly stable linkers, such as GABA and -ala. The amount of unconjugated (free) CPT was low at all timepoints and for all nanoparticles.
Example 10: In vivo mouse maximum tolerated dose (MTD) study: unconjugated CAB and MAP-Gly-CAB nanoparticles
[0126] In a first MTD study, male NSG mice were randomly divided into 7 groups containing 4 mice each. CAB at 8, 12, and 16 mg/kg was administered on days 1, 8 and 15 via intravenous tail vein injection. MAP-Gly-CAB nanoparticles at 8, 12, 16, and 24 mg/kg (CAB basis) were administered on days 1, 8, and 15 via intravenous tail vein injection. CAB was formulated in 1 : 1 : 18 ethanol :polysorbate 80:5% glucose, and nanoparticles were formulated in 0.9% saline, pH 4. Health of the mice was monitored and recorded daily for 4 weeks after the start of treatment. Body weight was monitored and recorded about every second or third day. MTD was defined as the highest dose resulting in less than 20% body weight loss or with less than 20% treatment-related deaths. Animals were euthanized when the criteria for the MTD was exceeded or at the end of the study.
[0127] Administration of CAB at 8, 12, or 16 mg/kg was at or near the MTD. All three weekly doses were completed in these groups. However, there was a 25.0% incidence of deaths in the treatment window in each group. The treatments were associated with weight changes of - 15.9%, -13.7% and -15.3%, respectively. There was a trending recovery of the lost body weight in all animals that survived until study end. Administration of MAP-Gly-CAB nanoparticles at 12, 16 or 24 mg/kg (CAB basis) was not tolerated. The incidences of deaths in the treatment window were 25.0%, 25.0% and 75.0%, respectively. The treatments were associated with a - 12.2%, -15.5% and -19.9% weight change in the treatment window, respectively. Due to the degree of body weight loss in these groups, the final dose of the scheduled three weekly doses was cancelled. Treatment with MAP-Gly-CAB nanoparticles at 8 mg/kg (CAB basis) was tolerated, resulting in no deaths and a -6.2% weight change in the treatment window. All three weekly doses were completed in this group.
Example 11: In vivo mouse MTD study: unconjugated CAB, and nanoparticles containing MAP-Ala-CAB, MAP-P-Ala- CAB and MAP-GABA-CAB
[0128] In a second MTD study, male NSG mice were randomly divided into 12 groups containing 4 mice each. CAB at 1 and 3 mg/kg was administered on days 1, 8 and 15 via intravenous tail vein injection. MAP-Ala-CAB nanoparticles at 8, 12, and 16 mg/kg (CAB basis); MAP-P-Ala-CAB nanoparticles at 8, 16, 24, and 32 mg/kg (CAB basis); and MAP- GABA-CAB nanoparticles at 16, 24, and 32 mg/kg (CAB basis) were administered on days 1, 8, and 15 via intravenous tail vein injection. CAB was formulated in 1 : 1 : 18 ethanol :polysorbate 80:5% glucose, and nanoparticles were formulated in 0.9% saline, pH 4. Health of the mice was monitored and recorded daily for 4 weeks after the start of treatment. Body weight was monitored and recorded about every second or third day. MTD was defined as the highest dose resulting in less than 20% body weight loss or with less than 20% treatment-related deaths. Animals were euthanized when the criteria for the MTD was exceeded or at the end of the study.
[0129] Administration of CAB at 1 or 3 mg/kg was tolerated, resulting in no deaths and weight change in the treatment windows of -2.9% and -5.8%. Administration of MAP-Ala-C AB nanoparticles at 8, 12 or 16 mg/kg (CAB basis), MAP-P-Ala-CAB nanoparticles at 8, 16, 24, or 32 mg/kg (CAB basis), or MAP-GAB A-CAB at 16, 24 or 32 mg/kg (CAB basis) were all tolerated. Weight change in the treatment window across all groups ranged from -6.7% to 3.9%. There were no deaths in the treatment window with the exception of one animal that received MAP-P-Ala-CAB nanoparticles at 24 mg/kg (CAB basis). It should be noted that this single death due to excessive weight loss appears to be an outlier in comparison to the other animals in this group, and the overall tolerance of MAP-P-Ala-CAB nanoparticles is further supported by the test article being tolerated at a higher dose.
[0130] Figure 5 summarizes the results of both MTD studies, showing that MAP-Gly- CAB nanoparticles are tolerated better than unconjugated cabazitaxel; while nanoparticles containing MAP-Ala-CAB, MAP-P-Ala-CAB and MAP-GABA-CAB are even more well tolerated that MAP-Gly-CAB-containing nanoparticles.

Claims

CLAIMS What is claimed is:
1. Cabazitaxel-2'-carboxybenzyl-glycinate having the structure:
Figure imgf000047_0001
2. Cabazitaxel-2'-glycinate. methane sulfonic acid having the structure:
Figure imgf000047_0002
3. A mucic acid polymer cabazitaxel glycinate conjugate having the structure:
Figure imgf000048_0001
wherein: m is from 5 to 200 inclusive; and n is from 20 to 200 inclusive.
4. The conjugate of claim 3, further comprising one or more targeting molecules.
5. The conjugate of claim 4, wherein the targeting molecule is selected from the group consisting of a protein, a peptide, an aptamer, and a ligand for a cellular receptor.
6. The conjugate of claim 5, wherein the protein is an antibody or an antibody fragment.
7. The conjugate of claim 6, wherein the antibody or antibody fragment is directed to prostate-specific membrane antigen (PSMA), HER2, or TR0P2.
8. The conjugate of claim 4, wherein the targeting molecule is linked to the conjugate through a polyethylene glycol (PEG) polymer containing one or more nitrophenylboronic acid (NPBA) moieties.
9. A nanoparticle comprising the conjugate of any of claims 3-8.
10. A pharmaceutical composition comprising the nanoparticle of claim 9 and a pharmaceutically acceptable carrier or excipient.
11. A method for the treatment of a disease or disorder in a subject, the method comprising administering the pharmaceutical composition of claim 10 to the subject.
12. The method of claim 11, wherein the disorder is a malignancy.
13. The method of claim 12, wherein the malignancy is a solid tumor.
14. The method of claim 13, wherein the solid tumor is a prostate tumor, a breast tumor, or a lung tumor.
15. The method of claim 14, wherein the prostate tumor is metastatic castration-resistant prostate cancer (mCRPC).
PCT/US2023/082430 2022-12-05 2023-12-05 Compositions and methods for treatment of prostate cancer Ceased WO2024123732A1 (en)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110105598A1 (en) * 2009-11-04 2011-05-05 Emcure Pharmaceuticals Limited Process for Preparation of Taxane Derivatives
US20110268658A1 (en) * 2009-03-30 2011-11-03 Crawford Thomas C Polymer-agent conjugates, particles, compositions, and related methods of use
US20130345298A1 (en) * 2010-12-22 2013-12-26 Nektar Therapeutics Multi-Arm Polymeric Prodrug Conjugates of Cabazitaxel-Based Compounds
US20140024845A1 (en) * 2010-12-22 2014-01-23 Nektar Therapeutics Non-Ring Hydroxy Substituted Taxanes and Methods for Synthesizing the Same
US20210170049A1 (en) * 2019-12-04 2021-06-10 Dantari, Inc. Methods and compositions for synthesis of therapeutic nanoparticles
US20220378735A1 (en) * 2019-12-03 2022-12-01 Shenyang Pharmaceutical University Cabazitaxel weakly- alkaline derivative and formulation thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110268658A1 (en) * 2009-03-30 2011-11-03 Crawford Thomas C Polymer-agent conjugates, particles, compositions, and related methods of use
US20110105598A1 (en) * 2009-11-04 2011-05-05 Emcure Pharmaceuticals Limited Process for Preparation of Taxane Derivatives
US20130345298A1 (en) * 2010-12-22 2013-12-26 Nektar Therapeutics Multi-Arm Polymeric Prodrug Conjugates of Cabazitaxel-Based Compounds
US20140024845A1 (en) * 2010-12-22 2014-01-23 Nektar Therapeutics Non-Ring Hydroxy Substituted Taxanes and Methods for Synthesizing the Same
US20220378735A1 (en) * 2019-12-03 2022-12-01 Shenyang Pharmaceutical University Cabazitaxel weakly- alkaline derivative and formulation thereof
US20210170049A1 (en) * 2019-12-04 2021-06-10 Dantari, Inc. Methods and compositions for synthesis of therapeutic nanoparticles

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