WO2017081606A1

WO2017081606A1 - Methods of determining features of polymeric nanoparticles that include a therapeutic agent

Info

Publication number: WO2017081606A1
Application number: PCT/IB2016/056716
Authority: WO
Inventors: Gregory Charles TROIANO; Donald Maxwell PARSONS
Original assignee: Pfizer Inc
Current assignee: Pfizer Inc
Priority date: 2015-11-10
Filing date: 2016-11-08
Publication date: 2017-05-18
Anticipated expiration: 2018-05-10

Abstract

The present disclosure generally relates to methods of determining the structural features corresponding to the release rate of a therapeutic agent in a therapeutic nanoparticle. Such nanoparticles may include the therapeutic agent. In particular, the application discloses a method of determining the structural features corresponding to the release rate of a therapeutic agent in a therapeutic nanoparticle that includes the therapeutic agent and a hydrophobic counterion, comprising: obtaining a Raman spectra of the nanoparticle; comparing the Raman spectra of the nanoparticle to a Raman spectra of at least one of the therapeutic agent alone, the hydrophobic counterion alone, a nanoparticle that does not include the therapeutic agent, and a salt formed from the therapeutic agent and the hydrophobic counterion to determine whether the therapeutic agent and the hydrophobic counterion in therapeutic nanoparticle occur as a salt.

Description

METHODS OF DETERMINING FEATURES OF POLYMERIC NANOPARTICLES THAT INCLUDE A THERAPEUTIC AGENT

BACKGROUND

[0001] Systems that deliver certain drugs to a patient (e.g. , targeted to a particular tissue or cell type or targeted to a specific diseased tissue but not normal tissue) or that control release of drugs have long been recognized as beneficial.

[0002] For example, therapeutics that include an active drug and that are, e.g. , targeted to a particular tissue or cell type or targeted to a specific diseased tissue but not to normal tissue, may reduce the amount of the drug in tissues of the body that are not targeted. This is particularly important when treating a condition such as cancer where it is desirable that a cytotoxic dose of the drug is delivered to cancer cells without killing the surrounding non-cancerous tissue. Effective drug targeting may reduce the undesirable and sometimes life threatening side effects common in anticancer therapy. In addition, such therapeutics may allow drugs to reach certain tissues they would otherwise be unable to reach.

[0003] Therapeutics that offer controlled release and/or targeted therapy also must be able to deliver an effective amount of drug, which is a known limitation in other nanoparticle delivery systems. For example, it can be a challenge to prepare nanoparticle systems that have an appropriate amount of drug associated with each nanoparticle, while keeping the size of the nanoparticles small enough to have advantageous delivery properties.

[0004] In general, it can be difficult to understand and/or obtain information of the structure-property relationship of therapeutic nanoparticles. For example, there are limited or no analytic tools to obtain nanoparticle information such as whether the therapeutic agent is in a substantially homogenous solid solution or is substantially phase separated within a therapeutic nanoparticle such as a polymeric therapeutic nanoparticle.

[0005] Accordingly, a need exists for methods of structural features of a polymeric nanoparticle having a therapeutic agent and/or a counterion. . SUMMARY

[0006] Provided herein in part is a method of determining the structural features corresponding to the presence of and/or release rate of a therapeutic agent in a therapeutic nanoparticle that includes the therapeutic agent and a hydrophobic counterion, comprising: obtaining a Raman spectra of the nanoparticle; comparing the Raman spectra of the nanoparticle to a Raman spectra of at least one of the therapeutic agent alone, the

hydrophobic counterion alone, a nanoparticle that does not include the therapeutic agent, and a salt formed from the therapeutic agent and the hydrophobic counterion to determine whether the therapeutic agent and the hydrophobic counterion in therapeutic nanoparticle occur as a salt.

[0007] For example, provided herein is a method of determining the structural features of a polymeric nanoparticle having a therapeutic agent and a hydrophobic counterion, comprising: determining whether a salt of the therapeutic agent and the hydrophobic counterion exists in the polymeric nanoparticle; optionally determining the stoichiometry of the salt if present; optionally determining the number or presence of one or more of di-salts of the hydrophobic counterion, free therapeutic agent , and free hydrophobic counterion; and optionally determining whether the polymeric nanoparticle is a substantially homogenous solid solution or substantially phase-separated.

[0008] A contemplated method, in an embodiment, includes determining whether a salt of the therapeutic agent and the hydrophobic counterion exists in the polymeric nanoparticle and comprises obtaining a Raman spectra of the nanoparticle and/or determining the number or presence of one or more of di-salts of the hydrophobic counterion, free therapeutic agent , and free hydrophobic counterion comprising obtaining a Raman spectra of the nanoparticle. The method is further directed to, in some embodiments, determining whether the polymeric nanoparticle is a substantially homogenous solid solution or substantially phase-separated may include for example, by obtaining a XRPD analysis.

[0009] A disclosed method may further comprise correlating the peak intensities of the Raman spectrum with the mole ratios of the therapeutic agent and hydrophobic acid used to prepare the therapeutic nanoparticles and/or further comprise determining the structural components that correlate with therapeutic release rates from nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 depicts Raman spectra of single components, their mixtures,

Compound A-pamoate salt, placebo nanoparticles, and nanoparticles containing Compound A and pamoic acid.

[0011] Figure 2 depicts XRPD patterns from the individual nanoparticles components.

[0012] Figure 3 A depicts overlay plot of XRPD patterns nanoparticles containing compound A and pamoic acid.

[0013] Figure 3B depicts overlay plot of XRPD patterns from nanoparticles containing compound A and pamoic acid

[0014] Figure 4 depicts an example of background subtraction.

[0015] Figure 5 depicts the processed data sets from the placebo nanoparticles (RX-

9064 and RXl-9065), the PLA-PEG copolymer (RXl-9056) and the nanoparticles containing compound A and pamoic acid (RX 1-9060, RX 1-9061, RX 1-9063, RX 1-9071, RX 1-9072, RX 1-9073, RX 1-9093, RX 1-9094, RX 1-9095, RX 1-9096, and RX 1-9097) were evaluated using a variance component, least squares analysis method. Two components, designated VCl(PLA-PEG) and VC2 (non-crystalline constituent) were returned.

[0016] Figure 6 depicts plots of VC2 coefficients against concentrations of

(compound A) and pamoic acid.

[0017] Fif re 7 depicts spectra of RC1 and RC2.

[0018] Fif lure 8 depicts spectra of RC1 and RC2.

[0019] Fif lure 9 depicts spectra of RC1 and RC2.

[0020] Fif lure 10 depicts graph of salt v. amount released.

[0021] Fif lure 11 depicts graph of salt v. amount released.

[0022] Fif lure 12 depicts graph of salt v. amount released

[0023] Fif lure 13 depicts a plot of Raman peak ratios against moles of pamoic acid used to prepare nanoparticle samples. DETAILED DESCRIPTION

[0024] Provided herein, in part, is a method of determining the structural features corresponding to the presence of and/or release rate of a therapeutic agent in a therapeutic nanoparticle that includes the therapeutic agent and a hydrophobic counterion, comprising: obtaining a Raman spectra of the nanoparticle; comparing the Raman spectra of the nanoparticle to a Raman spectra of at least one of the therapeutic agent alone, the

[0025] For example, provided herein is a method of determining the structural features of a polymeric nanoparticle having a therapeutic agent and a hydrophobic counterion, comprising: determining the presence of a salt of the therapeutic agent and the hydrophobic counterion existing in the polymeric nanoparticle; optionally determining the stoichiometry of the salt if present; optionally determining the number or presence of one or more of di- salts of the hydrophobic counterion, free therapeutic agent , and free hydrophobic counterion; and optionally determining whether the polymeric nanoparticle is a substantially homogenous solid solution or substantially phase-separated. For example, a contemplated method includes, in an embodiment, determining whether a salt of the therapeutic agent and the hydrophobic counterion exists in the polymeric nanoparticle and comprises obtaining a Raman spectra of the nanoparticle and/or determining the number or presence of one or more of di-salts of the hydrophobic counterion, free therapeutic agent , and /orfiree hydrophobic counterion in the nanoparticle. For Contemplated methods can include determining whether the polymeric nanoparticle is a substantially homogenous solid solution or substantially phase-separated and may include for example obtaining a XRPD analysis. A disclosed method further comprises, in an embodiment, correlating the peak intensities of the Raman spectrum with the mole ratios of the therapeutic agent and hydrophobic acid used to prepare the therapeutic nanoparticles and/or further comprise determining the structural components that correlate with therapeutic release rates from nanoparticles.

[0026] Disclosed methods of determining the structural features of a polymeric nanoparticle having a therapeutic agent and a hydrophobic counterion can include

determining nanoparticles including one, two, three or more biocompatible and/or biodegradable polymers. For example, a disclosed method may be used to measure the structural features of a nanoparticle that include about 35 to about 99.75 weight percent, in some embodiments about 50 to about 99.75 weight percent, in some embodiments about 50 to about 99.5 weight percent, in some embodiments about 50 to about 99 weight percent, in some embodiments about 50 to about 98 weight percent, in some embodiments about 50 to about 97 weight percent, in some embodiments about 50 to about 96 weight percent, in some embodiments about 50 to about 95 weight percent, in some embodiments about 50 to about 94 weight percent, in some embodiments about 50 to about 93 weight percent, in some embodiments about 50 to about 92 weight percent, in some embodiments about 50 to about 91 weight percent, in some embodiments about 50 to about 90 weight percent, in some embodiments about 50 to about 85 weight percent, in some embodiments about 60 to about 85 weight percent, in some embodiments about 65 to about 85 weight percent, and in some embodiments about 50 to about 80 weight percent of one or more block copolymers that include a biodegradable polymer and poly(ethylene glycol) (PEG), and about 0 to about 50 weight percent of a biodegradable homopolymer. Nanoparticles used in the disclosed methods include about 0.2 to about 35 weight percent, about 0.2 to about 20 weight percent, about 0.2 to about 10 weight percent, about 0.2 to about 5 weight percent, about 0.5 to about 5 weight percent, about 0.75 to about 5 weight percent, about 1 to about 5 weight percent, about 2 to about 5 weight percent, about 3 to about 5 weight percent, about 1 to about 20 weight percent, about 2 to about 20 weight percent, about 5 to about 20 weight percent, about 1 to about 15 weight percent, about 2 to about 15 weight percent, about 3 to about 15 weight percent, about 4 to about 15 weight percent, about 5 to about 15 weight percent, about 1 to about 10 weight percent, about 2 to about 10 weight percent, about 3 to about 10 weight percent, about 4 to about 10 weight percent, about 5 to about 10 weight percent, about 10 to about 30 weight percent, or about 15 to about 25 weight percent of an therapeutic agent, e.g. a chemotherapeutic agent such as a kinase inhibitor or a taxane such as docetaxel.

[0027] In certain embodiments, nanoparticles used in the disclosed methods comprise a hydrophobic acid and/or are prepared by a process that includes a hydrophobic acid, such as oleic acid, xinafoic acid, cholic acid, deoxycholic acid, dioctylsulfosuccinic acid, or pamoic acid. In some cases, a contemplated hydrophobic acid may include a mixture of two or more acids. For example, in certain embodiments, the hydrophobic acid may comprise a mixture of two substantially hydrophobic acids, in some embodiments a mixture of three substantially hydrophobic acids, in some embodiments a mixture of four substantially hydrophobic acids, or in some embodiments five substantially hydrophobic acids or more. In some cases, a salt of a hydrophobic acid may be used in a nanoparticlesused in the disclosed methods. The hydrophobic acid present in nanoparticles may have an acid dissociation constant in water (pK_a) of about -5 to about 7, in some embodiments about -3 to about 5, in some embodiments about -3 to about 4, in some embodiments about -3 to about 3.5, in some embodiments about -3 to about 3, in some embodiments about -3 to about 2, in some embodiments about -3 to about 1, in some embodiments about -3 to about 0.5, in some embodiments about -0.5 to about 0.5, in some embodiments about 1 to about 7, in some embodiments about 2 to about 7, in some embodiments about 3 to about 7, in some embodiments about 4 to about 6, in some embodiments about 4 to about 5.5, in some embodiments about 4 to about 5, and in some embodiments about 4.5 to about 5, determined at 25 °C. Such hydrophobic acids may be chosen, at least in part, on the basis of the difference between the pKg of the hydrophobic acid and the pK_a of a therapeutic agent. For example, in some instances, the difference between the pK_a of the hydrophobic acid and the pK_a of a therapeutic agent may be between about 1 pK_a unit and about 15 pKa units, in some embodiments between about 1 pK_a unit and about 10 pK_a units, in some embodiments between about 1 pK_a unit and about 5 pK_a units, in some embodiments between about 1 pK_a unit and about 3 pK_a units, in some embodiments between about 1 pK_a unit and about 2 pK_a units, in some embodiments between about 2 pK_a units and about 15 pK_a units, in some embodiments between about 2 pK_a units and about 10 pK_a units, in some embodiments between about 2 pK_a units and about 5 pK_a units, in some

embodiments between about 2 pK_a units and about 3 pK_a units, in some embodiments between about 3 pK_a units and about 15 pK_a units, in some embodiments between about 3 pK_a units and about 10 pK_a units, in some embodiments between about 3 pK_a units and about 5 pK_a units, in some embodiments between about 4 pK_a units and about 15 pK_a units, in some embodiments between about 4 pK_a units and about 10 pK_a units, in some embodiments between about 4 pK_a units and about 6 pK_a units, in some embodiments between about 5 pK_a units and about 15 pK_a units, in some embodiments between about 5 pKa units and about 10 pK_a units, in some embodiments between about 5 pK_a units and about 7 pK_a units, in some embodiments between about 7 pK_a units and about 15 pK_a units, in some embodiments between about 7 pK_a units and about 9 pK_a units, in some embodiments between about 9 pK_a units and about 15 pK_a units, in some embodiments between about 9 pKa units and about 11 pK_a units, in some embodiments between about 11 pK_a units and about 13 pK_a units, and in some embodiments between about 13 pK_a units and about 15 pK_a units, determined at 25 °C.

[0028] In some embodiments, a contemplated hydrophobic acid may have a phase transition temperature that is advantageous and/or detectable using contemplated methods for example, for improving the properties of the therapeutic nanoparticles. [0029] In one embodiment, a method of determining the structural features of a polymeric nanoparticle includes a nanoparticle having a targeting ligand, e.g. , a low- molecular weight ligand. In certain embodiments, the low-molecular weight ligand is conjugated to a polymer, and the nanoparticle comprises a certain ratio of ligand-conjugated polymer (e.g., PLA-PEG-Ligand) to non-functionalized polymer (e.g., PLA-PEG or PLGA- PEG).

[0030] In general, a "nanoparticle" refers to any particle having a diameter of less than 1000 nm, e.g. , about 10 nm to about 200 nm. Disclosed therapeutic nanoparticles include nanoparticles having a diameter of about 60 to about 120 nm, or about 70 to about 120 nm, or about 80 to about 120 nm, or about 90 to about 120 nm, or about 100 to about 120 nm, or about 60 to about 130 nm, or about 70 to about 130 nm, or about 80 to about 130 nm, or about 90 to about 130 nm, or about 100 to about 130 nm, or about 1 10 to about 130 nm, or about 60 to about 140 nm, or about 70 to about 140 nm, or about 80 to about 140 nm, or about 90 to about 140 nm, or about 100 to about 140 nm, or about 1 10 to about 140 nm, or about 60 to about 150 nm, or about 70 to about 150 nm, or about 80 to about 150 nm, or about 90 to about 150 nm, or about 100 to about 150 nm, or about 1 10 to about 150 nm, or about 120 to about 150 nm.

[0031] Methods contemplated herein may be applied to any polymeric nanoparticles that include polymers and a therapeutic agent. In some embodiments, a therapeutic agent and/or targeting moiety (for example, a low-molecular weight ligand) is associated with at least part of the polymeric matrix and such features are detectable using the disclosed methods. For example, in some embodiments, a targeting moiety (for example, a ligand) is covalently associated with the surface of a polymeric matrix. In some embodiments, covalent association is mediated by a linker. For example, methods contemplated herein are applied to any polymeric nanoparticles with a therapeutic agent e.g., associated with the surface of, encapsulated within, surrounded by, and/or dispersed throughout the polymeric

matrixPolymers can be natural or unnatural (synthetic) polymers. Polymers can be homopolymers or copolymers comprising two or more monomers. In terms of sequence, copolymers can be random, block, or comprise a combination of random and block sequences. Typically, polymers are organic polymers. The term "polymer," as used herein, is given its ordinary meaning as used in the art, that is a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. The repeat units may all be identical, or in some cases, there may be more than one type of repeat unit present within the polymer. In some cases, the polymer can be biologically derived, that is, a biopolymer. Non-limiting examples include peptides or proteins. In some cases, additional moieties may also be present in the polymer, for example, biological moieties such as those described below. If more than one type of repeat unit is present within the polymer, then the polymer is said to be a "copolymer." It is to be understood that in any embodiment employing a polymer, the polymer being employed may be a copolymer in some cases. The repeat units forming the copolymer may be arranged in any fashion. For example, the repeat units may be arranged in a random order, in an alternating order, or as a block copolymer comprising one or more regions each comprising a first repeat unit (for example a first block), and one or more regions each comprising a second repeat unit (for example, a second block), etc. Block copolymers may have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks. Contemplated methods include detecting featurs of nanoparticles having polymers that may be polyesters, including copolymers comprising lactic acid and glycolic acid units, such as poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide), collectively referred to herein as "PLGA"; and homopolymers comprising glycolic acid units, referred to herein as "PGA," and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as "PLA. " In some embodiments, exemplary polyesters include, for example, polyhydroxyacids; PEGylated polymers and copolymers of lactide and glycolide (e.g. , PEGylated PLA, PEGylated PGA, PEGylated PLGA, and derivatives thereof). In some embodiments, polyesters include, for example, polyanhydrides, poly(ortho ester) PEGylated poly(ortho ester), poly(caprolactone), PEGylated poly(caprolactone), polylysine, PEGylated polylysine, poly(ethylene imine), PEGylated poly(ethylene imine), poly(L-lactide-co-L- lysine), poly(serine ester), poly(4-hydroxy-L-proline ester), poly[a-(4-aminobutyl)-L-glycolic acid], and derivatives thereof, or the nanoparticles may include polymers with one or more acrylic polymers. In certain embodiments, acrylic polymers include, for example, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl

methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamide copolymer, polyrinethyl methacrylate), poly(methacrylic acid polyacrylamide, amino alkyl methacrylate copolymer, glycidyl methacrylate copolymers, polycyanoacrylates, and combinations comprising one or more of the foregoing polymers. The acrylic polymer may comprise fully-polymerized copolymers of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups. [0032] Methods contemplated herein are applied to nanoparticles that can for example comprise a diblock copolymer of PEG and PL(G)A, wherein for example, the PEG portion may have a number average molecular weight of about 1,000-20,000, e.g., about 2,000- 20,000, e.g., about 2 to about 10,000, and the PL(G)A portion may have a number average molecular weight of about 5,000 to about 20,000, or about 5,000-100,000, e.g., about 20,000- 70,000, e.g., about 15,000-50,000, e.g., applied to an exemplary therapeutic nanoparticle that includes about 10 to about 99 weight percent poly(lactic) acid-poly(ethylene)glycol copolymer or poly(lactic)-co-poly (glycolic) acid-poly(ethylene)glycol copolymer, or about 20 to about 80 weight percent, about 40 to about 80 weight percent, or about 30 to about 50 weight percent, or about 70 to about 90 weight percent poly(lactic) acid-poly(ethylene)glycol copolymer or poly(lactic)-co-poly (glycolic) acid-poly(ethylene)glycol copolymer.

Exemplary poly(lactic) acid-poly(ethylene)glycol copolymers can include a number average molecular weight of about 15 to about 20 kDa, or about 10 to about 25 kDa of poly(lactic) acid and a number average molecular weight of about 4 to about 6, or about 2kDa to about 10 kDa of poly(ethylene)glycol.

[0033] Disclosed nanoparticles optionally include about 1 to about 50 weight percent poly(lactic) acid or poly(lactic) acid-co-poly (glycolic) acid (which does not include PEG), or optionally include about 1 to about 50 weight percent, or about 10 to about 50 weight percent or about 30 to about 50 weight percent poly(lactic) acid or poly(lactic) acid-co-poly

(glycolic) acid. For example, poly(lactic) or poly(lactic)-co-poly(glycolic) acid may have a number average molecule weight of about 5 to about 15 kDa, or about 5 to about 12 kDa. Exemplary PLA have a number average molecular weight of about 5 to about 10 kDa.

Exemplary PLGA have a number average molecular weight of about 8 to about 12 kDa.

[0034] A therapeutic nanoparticle may, in some embodiments, contain about 10 to about 30 weight percent, in some embodiments about 10 to about 25 weight percent, in some embodiments about 10 to about 20 weight percent, in some embodiments about 10 to about 15 weight percent, in some embodiments about 15 to about 20 weight percent, in some embodiments about 15 to about 25 weight percent, in some embodiments about 20 to about 25 weight percent, in some embodiments about 20 to about 30 weight percent, or in some embodiments about 25 to about 30 weight percent of poly(ethylene)glycol, where the poly(ethylene)glycol may be present as a poly(lactic) acid-poly(ethylene)glycol copolymer, poly(lactic)-co-poly (glycolic) acid-poly(ethylene)glycol copolymer, or poly(ethylene)glycol homopolymer. In certain embodiments, the polymers of the nanoparticles can be conjugated to a lipid. The polymer can be, for example, a lipid-terminated PEG. [0035] Methods contemplated herein can be applied to polymeric nanoparticle s that

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof, wherein the NH₂, OH, or SH groups serve as the point of covalent attachment to the nanoparticle (e.g. , -N(H)-PEG, -O-PEG, or -S-PEG) or indicates the point of attachment to the nanoparticle, wherein n is 1, 2, 3, 4, 5, or 6, and wherein R is independently selected from the group consisting of NH₂, SH, OH, CO₂H, Ci-6-alkyl that is substituted with NH₂, SH, OH, or CO₂H, and phenyl that is substituted with N¾, SH, OH, or CO₂H, and wherein R serves as the point of covalent attachment to the nanoparticle (e.g. , -N(H)-PEG, -S-PEG, -O- PEG, or CO₂-PEG). These compounds may be further substituted with NH₂, SH, OH, CO₂H, Ci-6-alkyl that is substituted with NH₂, SH, OH, or CO₂H, or phenyl that is substituted with NH₂, SH, OH or CO₂H, wherein these functional groups can also serve as the point of covalent attachment to the nanoparticle. In an embodiment, disclosed methods are be applied to a therapeutic nanoparticle may include a polymer-drug conjugate. For example, a drug may be conjugated to a disclosed polymer or copolymer (e.g., PLA-PEG), and such a polymer-drug conjugate may form part of a disclosed nanoparticle. For example, a disclosed therapeutic nanoparticle may optionally include about 0.2 to about 30 weight percent of a PLA-PEG or PLGA-PEG, wherein the PEG is functionalized with a drug (e.g., PLA-PEG- Drug).

EXAMPLES

[0036] The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments, and are not intended to limit the invention in any way.

[0037] 2-[3-[[7-[3-[ethyl(2-hydroxyethyl)amino]propoxy]quinazolin-4-yl]amino]-lH- pyrazol-5-yl]-N-(3-fluorophenyl)acetamide, compound A) was provided as an anhydrous free base. PLA-PEG was received from Evonik (part #100 DL mPEG 5000 3.5CE), which has a number-average molecular weight of approximately 16 kDa for PLA and approximately 5 kDa for PEG (determined via nuclear magnetic resonance, NMR), with a polydispersity index of approximately 1.25 (determined via gel permeation chromatography). Ethyl acetate, benzyl alcohol, trifluoroacetic acid (TFA), and dimethyl sulfoxide (DMSO) were from EMD Millipore (Billerica, MA, USA). Oleic acid, l-hydroxy-2 -naphthoic acid, cholic acid, deoxycholic acid, dioctyl sulfosuccinate sodium salt (docusate sodium salt), pamoic acid, citric acid, sodium dihydrogen phosphate, di-sodium hydrogen phosphate dihydrate, and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich Ltd. (St. Louis, MO, USA). All reagents were analytical or high-performance liquid chromatography (HPLC) grade and were used without further purification.

EXAMPLE 1 : Preparation of drug encapsulated nanoparticles

[0038] Nanoparticles were created via a nanoemulsion process using the modified o/w emulsification solvent extraction method as described by J. Hrkach, D. Von Hoff, M.M. Ali, E. Andrianova, J. Auer, T. Campbell, D. De Witt, M. Figa, M. Figueiredo, A. Horhota, S. Low, K. McDonnell, E. Peeke, B. Retnarajan, A. Sabnis, E. Schnipper, J.J. Song, Y.H. Song, J. Summa, D. Tompsett, G. Troiano, T. Van Geen Hoven, J. Wright, P. LoRusso, P.W.

Kantoff, N.H. Bander, C. Sweeney, O.C. Farokhzad, R. Langer, S. Zale, Preclinical development and clinical translation of a PSMA-targeted docetaxel nanoparticle with a differentiated pharmacological profile, Sci Transl Med, 4 (2012) 128 and 16-5 PLA-PEG in a EA/BA solvent mixture was rapidly mixed and dispersed with an immiscible aqueous phase containing the optimal concentration of surfactant for particle size control. First, a coarse emulsion is formed by a handheld rotor/stator homogenizer. This coarse emulsion was subsequently processed by a high-pressure microfludizer (Microfluidics, Inc., Westwood, MA, USA) to form a fine nanoemulsion. The emulsions were quenched by addition into cold water or buffer solutions, resulting in extraction of solvent from the organic phase and hardening of nanoparticles. Subsequent processing included tangential flow filtration, which removes unencapsulated API and processing aids, and addition of sucrose for stabilization and cryopreservation of the nanoparticle suspension.

As cholic acid has relatively low solubility in the organic phase (EA/BA), the cholic acid formulations were created by dissolving sodium cholate in the aqueous phase while using TFA to solubilize the API in the organic phase. Dioctylsulfosuccinic acid was prepared by two-immiscible liquid phase extraction method using dioctyl sodium sulfosuccinate. Briefly, sodium dioctylsulfosuccinate was dissolved in BA, and then concentrated hydrochloric acid solution and water were added to acidify the solution. The mixture was vortex-mixed and after phase separation, BA containing protonated dioctylsulfosuccininc acid on the top layer was aspirated and used as is. Due to the low solubility of pamoic acid in the organic phase, pamoic acid formulations were created using DMSO as a cosolvent in the organic phase and TFA as a drug solubilizer. Hydrophobic counterions having various acidity, molecular weight, shape/bulkiness, and lipophilicity were characterized for pKa, Log P, and Log D7.5 and incorporated into Compound A nanoparticles for characterization of release rate. The physicochemical properties of the representative counterions measured by SiriusT3.

EXAMPLE 2: Spectral identification A-PAM supporting ion pair formation in nanoparticles

[0039] Raman spectroscopy was used to obtain reference spectra of the salts, free

Compound A alone, pamoic acid alone, and placebo nanoparticles as well as nanoparticles formulated with Compound A and pamoic acid. As shown in Fig. 1, Raman spectra of nanoparticles that include an active agent (compound A) with pamoic acid as counterion have several features observed in the spectra of the salt; while spectral features of free active agent and free pamoic acid are not observed. This supports a showing that the ion pair complex/salt formation occurs during the process of nanoparticle preparation. EXAMPLE 3 : Raman

[0040] The Rigaku Smart-Lab X-ray diffraction system was configured for reflection

Bragg-Brentano geometry using a line source X-ray beam. The x-ray source is a Cu Long Fine Focus tube that was operated at 40 kV and 44 mA. That source provides an incident beam profile at the sample that changes from a narrow line at high angles to a broad rectangle at low angles. Beam conditioning slits are used on the line X-ray source to ensure that the maximum beam size is less than 10 mm both along the line and normal to the line. The Bragg-Brentano geometry is a para-focusing geometry controlled by passive divergence and receiving slits with the sample itself acting as the focusing component for the optics. The inherent resolution of Bragg-Brentano geometry is governed in part by the diffractometer radius and the width of the receiving slit used. Typically, the Rigaku Smart-Lab is operated to give peak widths of 0.1 °2Θ or less. The axial divergence of the X-ray beam is controlled by 5.0-degree Soller slits in both the incident and diffracted beam paths.

[0041] Powder samples were prepared in a low background Si holder using light manual pressure to keep the sample surfaces flat and level with the reference surface of the sample holder. Each sample was analyzed from 2 to 70 °2Θ using a continuous scan of 2 °2Θ per minute with an effective step size of 0.04 °2Θ. Each sample was spun at 50 rpm during data collection.

[0042] Fourier transform (FT) Raman spectra were acquired on a Nicolet model 6700 spectrometer interfaced to a Nexus Raman accessory module. The instrument is configured with a Nd:YAG laser operating at 1024 nm, a CaF2 beam splitter, and a indium gallium arsenide detector. OMNIC 8.1 software was used for control of data acquisition and processing of the spectra. Samples were packed into 3-inch glass NMR tubes for analysis. Each spectrum consisted of 512 scans at 2 cm-1 resolution. The Omnic 8.2 software package (Thermo -Nicolet) was used to acquire, process, and evaluate the spectral data.

[0043] Processing and chemometric analyses of XRPD data were carried out using

Microsoft Excel, Professional Plus 2010 version 14.0.7128.5000.

[0044] Processing and principal component analyses of Raman data were carried out using Unscrambler X version 10.2.

[0045] XRPD patterns from the individual nanoparticles components are shown in

Figure 2. There are no detectable crystalline components visible in any of the patterns from the nanoparticles containing Compound A and pamoic acid. Instead, only the PLA-PEG copolymer pattern is seen. The concentrations of compound A and pamoic acid are high enough to expect to see peaks in the patterns if either of those components were present in crystalline form. The conclusion is that those components are in non-crystalline form in the nanoparticles. See Figures 2, 3A and 3B. In order to carry out chemometric analyses, each data set (XRPD pattern) was processed by subtraction of instrumental background contribution and normalization to a common intensity scale. An example of background subtraction is shown in FIG. 4.

[0046] The processed data sets from the placebo nanoparticles (RX-9064 and RX1 -

9065), the PLA-PEG copolymer (RX1-9056) and the nanoparticles containing compound A and pamoic acid (RX1-9060, RX1-9061, RX1-9063, RX1-9071, RX1-9072, RX1-9073, RX1-9093, RX1-9094, RX1-9095, RX1-9096, and RX1-9097) were evaluated using a variance component, least squares analysis method. Two components, designated VCl and VC2, were returned (Figure 5). VCl describes the PLA-PEG copolymer and VC2 describes an added non-crystalline constituent.

[0047] The normalized data sets from the nanoparticle samples having known amounts of compound a and pamoic acid (TCL3006-TCL3012) are described using VCl and VC2 as shown in Table 1 A. The numbers in that table are the coefficients assigned to each component that minimize the difference between measured and calculated patterns. Note that the use of variance components can result in negative numbers. Plots of VC2 coefficients against the concentrations of active agent and pamoic acid (determined post lyophilization) are reasonably linear (Figure 6), which supports that VC2 represents those constituents. Figure 6 depicts lots of VC2 coefficients against concentrations of (compound A) and pamoic acid.

Table 1A. Variance Component Description of Data Sets

[0048] Forcing the variance components to be positive resulted in the projection of real components (Figure 7). RC1 is essentially identical to the measured, processed XRPD pattern from the PLA-PEG copolymer, giving confidence that that extracted component is meaningful (Figure 8). On the other hand, RC2 is somewhat different from the measured, processed XRPD pattern from non-crystalline compound A pamoate (Figure 9). That difference may be indicative of the presence of salts with other stoichiometrics, the presence of free compound A, or a change in the local environment of compound A pamoate because it is dissolved in the PLA-PEG copolymer.

[0049] A preliminary investigation into the reasons for the difference between RC2 and the measured and processed XRPD pattern from non-crystalline compound A pamoate was carried out. A real-component least-squares analysis was performed using three components, RCl, RC2, and the measured and processed XRPD pattern from non-crystalline compound A pamoate. The results describe normalized data sets from the nanoparticles samples having known amounts of compound A and pamoic acid (TCL3006-TCL3012), as well as the measured, processed data sets from PLA-PEG copolymer, nanoparticle placebo, and compound A pamoate samples, as shown in Table 2.

[0050] The numbers in Table 2 are the coefficients assigned to each component that minimize the difference between measured and calculated patterns. Since positive, real components were used the sum of the coefficients for each data set is approximately 1. The coefficients may be multiplied by 100 to yield approximate percentages. Note that the PLA- PEG copolymer and placebo data sets are described only by RC 1. Similarly, the compound A pamoate data sets are described only by the measured, processed data set from that salt. RC2 can be considered a non-crystalline component that is different from the type of compound A pamoate that exists in the standard sample provided.

[0051] Table 2. Real Component Description of Data Sets

[0052] Linear data fits for plots of compound A pamoate coefficients against release rate, for plots of the sum of compound A pamoate and RC2 coefficients against release rate, and for plots of RC2 coefficients against release rate. RC2 is correlated to release rate.

[0053] It is possible that the peak at about 1350 cm-1 arises from free compound A.

Overlay plots of the spectrum from compound A and component 1 show that the most intense peak in the compound A spectrum at 1347 cm-1 may correspond to the 1350 cm-1 peak in the component 1 spectrum. This suggests that free compound A is present in nanoparticles. In the spectra from the nanoparticles, other peaks arising from free compound A would likely be obscured by peaks from compound A pamoate.

[0054] The relevant peak intensities were correlated with compound A/pamoic acid mole ratios used to prepare the nanoparticles. Examination shows that there is a peak at about 1367 cm-1 that is present in the component 1 spectrum (and consequently in the spectra of the nanoparticles) that is not present in the spectrum of compound A. For each nanoparticle spectrum (RmF.377 through RmF.383) the ratio of the intensities of the peaks at 1367 cm-1 and 1350 cm-lwere ratioed. The resulting ratios were plotted against the moles of pamoic acid relative to one mole of compound A used to prepare the nanoparticle samples (Figure 13). A trend is apparent in which the lower peak ratios (lower amounts of compound A pamoate) correspond to lower amounts of pamoic acid. That is consistent with the idea that, in the nanoparticles using less pamoic acid results in less salt formation, leaving more free compound A, which is evident from increased intensities of the 1347 cm-1 peak in the Raman spectra.

a. Ratio of the intensity of the compound a pamoate peak at 1637 cm-1

to the peak in the nanoparticle spectrum at 1350 cm^"1.

b. Moles of pamoic acid per mole of compound.

[0055] Tentative Raman band assignments were made, and the most informative bands are in the carbonyl stretching region. The typical C=0 stretching band for free carboxylic acid (about 1644 cm-1) is present in the spectrum of pamoic acid but absent in the spectra of compound A pamoate and the nanoparticles. Instead, the salt spectra exhibit lower-frequency bands at about 1574 cm-1 (weaker) and 1366 cm-1 (stronger) that are likely C=0 antisymmetric and symmetric stretching vibrations, respectively. Such a change in the C=0 stretching vibrations on going from a free carboxylic acid to a salt are typical.

[0056] The 1 : 1 compound A does not exhibit a free carboxylic acid C=0 stretching band (arising from the unsaltified C02H group of pamoic acid) in its Raman spectrum. This would suggest the absence of such a band in the spectra of the nanoparticle means that all carboxylic acid groups are in salt forms which, based on the fact that the stoichiometrics of compound A and pamoic acid used provide an excess of carboxylic acid groups, would mean some 1 :2 compound A pamoate was present.

EQUIVALENTS

[0057] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

INCORPORATION BY REFERENCE

[0058] The entire contents of all patents, published patent applications, websites, and other references cited herein are hereby expressly incorporated herein in their entireties by reference.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of determining the structural features corresponding to the release rate of a therapeutic agent in a therapeutic nanoparticle that includes the therapeutic agent and a hydrophobic counterion, comprising:

obtaining a Raman spectra of the nanoparticle;

comparing the Raman spectra of the nanoparticle to a Raman spectra of at least one of the therapeutic agent alone, the hydrophobic counterion alone, a nanoparticle that does not include the therapeutic agent, and a salt formed from the therapeutic agent and the hydrophobic counterion to determine whether the therapeutic agent and the hydrophobic counterion in therapeutic nanoparticle occur as a salt.

2. A method of determining the structural features of a polymeric nanoparticle having a therapeutic agent and a hydrophobic counterion, comprising:

determining whether a salt of the therapeutic agent and the hydrophobic counterion exists in the polymeric nanoparticle;

optionally determining the stoichiometry of the salt if present;

optionally determining the number or presence of one or more of di-salts of the hydrophobic counterion, free therapeutic agent , and free hydrophobic counterion;

optionally determining whether the polymeric nanoparticle is a substantially homogenous solid solution or substantially phase-separated.

3. The method of claim 2, wherein determining whether a salt of the therapeutic agent and the hydrophobic counterion exists in the polymeric nanoparticle comprises obtaining a Raman spectra of the nanoparticle.

4. The method of claim 2 or 3, wherein determining the number or presence of one or more of di-salts of the hydrophobic counterion, free therapeutic agent , and free hydrophobic counterion comprises obtaining a Raman spectra of the nanoparticle.

5. The method of any one of claims 2-4, wherein determining whether the polymeric nanoparticle is a substantially homogenous solid solution or substantially phase -separated comprises obtaining a XRPD analysis.

6. The method of any one of claims 2-5, further comprising correlating the peak intensities of the Raman spectrum with the mole ratios of the therapeutic agent and hydrophobic acid used to prepare the therapeutic nanoparticles.

7. The method of any one of claims 2-6, further comprising determining the structural components that correlate with therapeutic release rates from nanoparticles.