CN116568335A

CN116568335A - Folate receptor targeted nanoparticle drug conjugate and application thereof

Info

Publication number: CN116568335A
Application number: CN202180073503.XA
Authority: CN
Inventors: 马恺; A·M·文卡特桑; 陈风; 吴菲; M·Z·图尔克; T·C·加迪尼尔二世; G·J·小杰曼诺; G·P·亚当斯; F·Y·F·李
Original assignee: Yida Pharmaceutical Co
Current assignee: Yida Pharmaceutical Co
Priority date: 2020-10-27
Filing date: 2021-10-26
Publication date: 2023-08-08
Also published as: CN116669773A

Abstract

The present disclosure relates to Nanoparticle Drug Conjugates (NDCs) comprising ultra-small nanoparticles, a Folate Receptor (FR) targeting ligand, and a linker-drug conjugate, as well as methods of making and using the same to treat cancer.

Description

Folate receptor targeted nanoparticle drug conjugate and application thereof

Cross-reference to related application

The present application claims the benefits of U.S. provisional application number 63/105,995 filed on 10 month 27, U.S. provisional application number 63/116,393 filed on 11 month 20, U.S. provisional application number 63/117,110 filed on 11 month 23, U.S. provisional application number 63/155,043 filed on 3 month 1 2021, U.S. provisional application number 63/222,181 filed on 7 month 15 2021, U.S. provisional application number 63/242,201 filed on 9 month 2021, and U.S. provisional application number 63/254,837 filed on 10 month 12 2021, each of which is incorporated herein by reference in its entirety.

Background

Targeted delivery of therapeutic agents (e.g., cytotoxic drugs) to cancer cells is an emerging approach for cancer treatment. By selectively delivering drugs to a target disease area, toxicity of the delivered therapeutic agent to healthy tissues or organs of the subject can be greatly reduced, resulting in improved therapeutic results. Antibody Drug Conjugates (ADCs) are a common platform for targeted drug delivery, typically characterized by a covalent linkage of highly toxic drug substances to monoclonal antibodies that can target cancer, where the toxic drug substances are released upon targeting the cancer. However, conventional targeted drug delivery platforms, such as ADCs, still present many challenges including production difficulties, drug loading capacity limitations, poor tumor penetration, and lack of ability to overcome tumor heterogeneity.

The university of kanell and commemorative ston-ketteline cancer center developed subminiature sub-10 nm silica-organic hybrid nanoparticles, called kanell dots (Cornell prime Dot, C' Dot), which have great potential in diagnostic and therapeutic applications. For example, the C' Dot may be conjugated with an epidermal growth factor receptor inhibitor, such as gefitinib, a cancer targeting agent that inhibits cancer growth (WO 2015/183882 A1). However, the mechanism of action (MOA) of EGFR inhibitors requires binding to epidermal growth factor receptor activity and therefore requires a sustained high concentration of payload in the targeted cancer cells to effectively inhibit cancer cell proliferation. MOAs of this type are generally incompatible with the fast blood circulation half-life of C' Dot.

Folate receptor alpha (fra), also known as FOLR1, has received great attention in the scientific community as a potential target for cancer treatment, and other isoforms of FR have also been identified as potential biological targets. See, e.g., targeting Folate Receptor Alpha For Cancer Treatment, cheung, a., et al Oncotarget (2016) 7 (32): 52553; targeting the folate receptor: diagnostic and therapeutic approaches to personalize cancer treatments, ledermann, j.a. et al, annals of Oncology (2015), 26:2034-2043; each of which is incorporated herein by reference in its entirety. Folate Receptor (FR) is an ideal target for cancer treatment, as FR can be overexpressed in tumors, such as those of the ovary, endometrium, breast, colon and lung, but its distribution in normal tissues is low and limited. Emerging insights have shown that FR may exhibit cell growth regulation and signaling functions in addition to its use as a folate receptor and transporter. Together, these features make FR an attractive therapeutic target.

Folate is transported into cells by a variety of mechanisms, the most common of which is mediated by folate receptors, of which there are four glycopeptide members (FR alpha [ FOLR1], FR beta [ FOLR2], FR gamma [ FOLR3] and FR delta [ FOLR4 ]). Among these four members, the α isoform (FR alpha or FR alpha) is a Glycosyl Phosphatidylinositol (GPI) anchored membrane protein with high affinity for the active form of 5-methyltetrahydrofolate (5 MTF) that binds and transports folic acid. The α isoform has been reported to be overexpressed in certain solid tumors, for example, in ovarian, fallopian tube, primary peritoneal, uterine, renal, lung, brain, gastrointestinal and breast cancers. The alpha isoform is also overexpressed in certain hematological malignancies, which can be developed for the treatment of these malignancies, for example, for the treatment of Acute Myelogenous Lymphomas (AML), including pediatric AML. This low and limited distribution in normal tissues or cells, along with new insights into their tumor promoting function and their association of expression with patient prognosis, together make fra an attractive therapeutic target. Furthermore, β isoforms (FR β) are overexpressed in certain cancers, e.g., hematological malignancies, such as Acute Myelogenous Leukemia (AML) and Chronic Myelogenous Leukemia (CML), providing an opportunity to develop targeted therapies for these cancers.

Although many FR-targeted drug delivery platforms have been developed and tested in the past for cancer treatment, e.g., using ADC and small molecule drug conjugates, none have been successfully approved for clinical use due to limited therapeutic results (EP 0624377 A2, US 9194682 B2, leamon, et al, "Comparative preclinical activity of the Folate-targeted Vinca alkaloid conjugates EC140 and EC145, int. J. Cancer (2007) 121:1585-1592; leamon et al," form-Vinca Alkaloid Conjugates for Cancer Therapy: ASstructure-Activity Relationships, bioconjugate Chemistry (2014) 25:560-568; scaranti, M., et al Exploiting the Folate receptor. Alpha. In oncology. Nat Rev Clin Oncol (2020) 17:349-359).

Thus, successful development of FR targeted drug delivery platforms is still highly desirable.

Disclosure of Invention

The present disclosure provides nanoparticle-drug conjugates (NDCs) comprising: (a) Silica nanoparticles and polyethylene glycol (PEG) covalently bonded to the surface of the nanoparticles;

(b) A targeting ligand comprising folic acid or a derivative or salt thereof, wherein the targeting ligand is attached to the nanoparticle directly or indirectly through a spacer group; and (c) a linker-payload conjugate, wherein:

(i) The payload is isatecan (exatecan); (ii) The linker-payload conjugate is attached to the nanoparticle directly or indirectly through a spacer group; (iii) the linker is a protease cleavable linker; and (iv) release of i Sha Tikang after cleavage of the linker.

The present disclosure also relates to nanoparticle-drug-conjugates (NDCs) comprising: (a) A nanoparticle comprising a silica-based core and a silica shell surrounding at least a portion of the core; polyethylene glycol (PEG) covalently bonded to the surface of the nanoparticle and a fluorescent compound covalently encapsulated within the nanoparticle core; (b) A targeting ligand that binds to a Folate Receptor (FR), wherein the targeting ligand comprises folic acid or a folate receptor binding derivative thereof, and wherein the targeting ligand is linked to the nanoparticle directly or indirectly through a spacer group; (c) A linker-payload conjugate, wherein the payload is a cytotoxic agent; wherein the linker-payload conjugate is attached to the nanoparticle directly or indirectly through a spacer group; wherein the cytotoxic agent is released after cleavage of the linker; wherein the linker in the linker-payload conjugate is a protease cleavable linker; and wherein the NDC has an average diameter of between about 1nm and about 10nm, such as between about 3nm and about 8nm, or between about 3nm and about 6 nm. The cytotoxic agent may be irinotecan.

In NDCs of the present disclosure, the average nanoparticle to payload ratio may be 1 to 80, such as 1 to 21 (e.g., 1 to 13, or 1 to 12), and the average nanoparticle to targeting ligand ratio may be 1 to 50, such as 1 to 25 (e.g., 1 to 11).

NDCs of the present disclosure may have an average diameter of about 1nm to about 10nm, for example about 5nm to about 8nm, about 3nm to about 8nm, or about 3nm to about 6 nm.

NDCs of the present disclosure may comprise any suitable dye or detectable compound, such as fluorescent compounds. For example, in NDCs of the present disclosure, the fluorescent compound may be Cy5. The fluorescent compound may be encapsulated within the nanoparticle (e.g., covalently linked to the silica core). NDCs of the present disclosure may comprise targeting ligands that bind to the Folate Receptor (FR). The targeting ligand may comprise folic acid or a derivative thereof. It is understood that "folic acid" can encompass amides or esters of folic acid, e.g., folic acid can be conjugated to the nanoparticle (or spacer group) at its carboxy terminus via an amide or ester linkage. For example, "folic acid" can refer to folic acid amide present in the exemplary NDC shown in fig. 1.

NDCs of the present disclosure may comprise structure (S-1):

wherein the payload comprises ixabepilone; the linker comprises a protease cleavable linker; and the silicon atoms are part of the nanoparticle.

For example, an NDC of the present disclosure may comprise the structure (S-1 a):

wherein the silicon atom is part of the nanoparticle.

NDCs of the present disclosure may comprise structure (S-2):

wherein the targeting ligand is folic acid or a folate receptor binding derivative thereof and the silicon atom is part of a nanoparticle.

For example, an NDC of the present disclosure may comprise the structure (S-2 a):

wherein the silicon atom is part of the nanoparticle.

The NDC of the present disclosure can comprise a combination of structures (S-1) and (S-2). For example, the NDC can include a structure (S-1 a) and a structure (S-2 a), e.g., as shown in FIG. 1. The structures S-1, S-1a, S-2, or S-2a may be present in the NDC in any desired ratio, for example, in the ratios disclosed herein.

The disclosure also relates to NDCs comprising: a nanoparticle comprising a silica-based core and a silica shell surrounding at least a portion of the core; polyethylene glycol (PEG) covalently bonded to the surface of the nanoparticle; fluorescent compounds covalently encapsulated within nanoparticle cores; a targeting ligand, wherein the targeting ligand is folic acid; a linker-payload conjugate, wherein the linker-payload conjugate is a protease cleavable linker capable of undergoing hydrolysis at the C-terminus upon protease binding, thereby releasing the payload from the nanoparticle, wherein the protease comprises a serine protease or a cysteine protease, wherein the payload in the linker-payload conjugate is irinotecan or an analog of irinotecan; and wherein the fluorescent compound is Cy5.

The disclosure also relates to NDCs comprising: a nanoparticle comprising a silica-based core and a silica shell surrounding at least a portion of the core; polyethylene glycol (PEG) covalently bonded to the surface of the nanoparticle; cy5 dye covalently encapsulated within the core of the nanoparticle; a targeting ligand that binds to a folate receptor, wherein the targeting ligand is folic acid, and wherein the targeting ligand is indirectly linked to the nanoparticle through a spacer group; a linker-payload conjugate, wherein the linker-payload conjugate is indirectly attached to the nanoparticle through a spacer group, wherein the linker-payload conjugate isThe loaded conjugate comprises a containing structureA compound of (a); and wherein the NDC has an average diameter between about 1nm and about 10nm (e.g., between about 1 and about 6 nm).

The present disclosure also relates to Nanoparticle Drug Conjugates (NDCs) comprising: (a) A silica nanoparticle comprising a silica-based core and a silica shell surrounding at least a portion of the core; and polyethylene glycol (PEG) covalently bound to the surface of the nanoparticle; (b) An isatecan-linker moiety comprising the structure of formula (NP-3):

wherein x is 4 and y is 9; and (c) a targeting ligand moiety comprising the structure of formula (NP-2)

Wherein x is 4 and y is 3, and wherein the ixabepilone-linker moiety and the targeting ligand moiety are each conjugated to the nanoparticle surface. NDC may comprise a fluorescent dye (e.g., cy 5) covalently encapsulated within the core of the nanoparticle.

The present disclosure also provides a method of treating a Folate Receptor (FR) -expressing cancer (e.g., a Folate Receptor (FR) -expressing tumor) comprising administering to a subject in need thereof an effective amount of an NDC described herein. The method may comprise intravenously administering NDC to a subject in need thereof. In the methods of the present disclosure, the subject may have a cancer selected from the group consisting of: ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, breast cancer (including, for example, her2+ breast cancer, hr+ breast cancer, HR-breast cancer, and triple negative breast cancer), lung cancer (e.g., non-small cell lung cancer (NSCLC)), mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer, and gastric cancer), pancreatic cancer, bladder cancer, renal cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, testicular cancer, acute myelogenous leukemia (AML, e.g., pediatric AML), and Chronic Myelogenous Leukemia (CML). NDCs of the present disclosure may also be used to target tumor-associated macrophages, which may be used as a means of altering the immune status of a tumor in a subject. NDCs of the present disclosure are useful in methods of treating advanced, recurrent, or refractory solid tumors.

The present disclosure provides the use of NDC for treating a Folate Receptor (FR) expressing cancer (e.g., a Folate Receptor (FR) expressing tumor). The use may comprise intravenous administration of NDC to a subject in need thereof. In the use of NDC, a subject may have a cancer selected from the group consisting of: ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, breast cancer (including, for example, her2+ breast cancer, hr+ breast cancer, HR-breast cancer, and triple negative breast cancer), lung cancer (e.g., non-small cell lung cancer (NSCLC)), mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer, and gastric cancer), pancreatic cancer, bladder cancer, renal cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, testicular cancer, acute myelogenous leukemia (AML, e.g., pediatric AML), and Chronic Myelogenous Leukemia (CML). In the use of NDC, the cancer may be an advanced, recurrent or refractory solid tumor.

The present disclosure provides NDCs for use in the manufacture of a medicament for the treatment of a Folate Receptor (FR) expressing cancer (e.g., a Folate Receptor (FR) expressing tumor). The use in the preparation of a medicament may comprise intravenous administration of NDC to a subject in need thereof. The use in the preparation of a medicament may comprise administering NDC to a subject, wherein the subject has a cancer selected from the group consisting of: ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, breast cancer (including, for example, her2+ breast cancer, hr+ breast cancer, HR-breast cancer, and triple negative breast cancer), lung cancer (e.g., non-small cell lung cancer (NSCLC)), mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer, and gastric cancer), pancreatic cancer, bladder cancer, renal cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, testicular cancer, acute myelogenous leukemia (AML, e.g., pediatric AML), and Chronic Myelogenous Leukemia (CML). NDCs of the present disclosure may be used in the preparation of a medicament for the treatment of advanced, recurrent or refractory solid tumors.

The present disclosure also relates to pharmaceutical compositions comprising NDC and a pharmaceutically acceptable excipient. The pharmaceutical compositions disclosed herein are useful for treating a Folate Receptor (FR) expressing cancer (e.g., a Folate Receptor (FR) expressing tumor).

Drawings

Fig. 1 shows a representative chemical structure of nanoparticle-drug conjugates (NDCs).

FIG. 2 depicts a flow chart for synthesizing an exemplary functionalized nanoparticle (dibenzocyclooctyne (DBCO) -functionalized C' Dot).

FIG. 3 depicts a synthesis flow diagram for synthesizing an exemplary NDC (FA-CDC) comprising a C' Dot functionalized with Folic Acid (FA) and irinotecan.

FIG. 4 shows representative UV-Vis absorption spectra of exemplary functionalized nanoparticles (DBCO-functionalized C' Dot). The absorption peak at 648nm corresponds to the Cy5 dye covalently encapsulated within the C' Dot core. The absorption peaks around 270-320nm correspond to DBCO groups.

Fig. 5 shows representative UV-Vis absorption spectra of an exemplary NDC (folic acid (FA) -functionalized C' Dot (FA-CDC) comprising isatecan). The absorption peak at 648nm corresponds to the Cy5 dye covalently encapsulated within the C' Dot core. The absorption peak around 330-400nm corresponds to that of irinotecan.

Fig. 6 depicts Fluorescence Correlation Spectroscopy (FCS) correlation curves of an exemplary NDC (folic acid (FA) -functionalized irinotecan-linker conjugated C' Dot (FA-CDC)), fitted by a single-mode FCS correlation function. The average hydrodynamic diameter is obtained by fitting FCS correlation curves.

Fig. 7 depicts a chromatogram showing elution of an exemplary NDC (folic acid (FA) -functionalized irinotecan-linker conjugated C' Dot (FA-CDC)) by Gel Permeation Chromatography (GPC). The elution of FA-CDC (striped line under the curve) was compared to the elution time of protein standards with different molecular weights (dashed line).

FIG. 8 depicts a reverse phase HPLC chromatogram of purified exemplary NDC (folic acid (FA) -functionalized irinotecan-linker conjugated C' Dot (FA-CDC)) at 330 nm. This wavelength can be used to monitor FA-CDC and impurities that may be present after synthesis or due to any degradation of NDC.

Fig. 9 shows the UV-Vis absorption spectra of exemplary ixabepilone-payload conjugates. I Sha Tikang has a maximum absorption near 360 nm.

Fig. 10A-10B show representative HPLC chromatograms providing analysis of exemplary NDCs prepared according to example 3 with folic acid as a targeting ligand and ixatide Kang Zhuige as a payload (NDCs prepared using the ixatikang-linker conjugate precursor 202 from example 1). Fig. 10A depicts a representative HPLC chromatogram of an uncleaved NDC at 360nm, showing a single peak at an elution time of about 6.3 minutes, which peak corresponds to the unreleased payload remaining on the NDC. Fig. 10B depicts a representative HPLC chromatogram of cleaved NDC at 360nm, showing additional peaks at elution times of about 3 to 4 minutes, which peaks correspond to the payload of released irinotecan. The area under the curve (AUC) of the released payload and the reserved payload is used to calculate the percentage of payload released.

FIGS. 11A-11C are graphs showing drug release assays at various time points after incubation with cathepsin-B for exemplary NDC loaded with folic acid and protease (cathepsin-B) cleavable irinotecan-linker conjugates as targeting ligands. FIG. 11A depicts reverse phase HPLC chromatograms of NDC B at various time points after incubation with cathepsin-B. FIG. 11B depicts reverse phase HPLC chromatograms of NDC C at various time points after incubation with cathepsin-B. Fig. 11C depicts reverse phase HPLC chromatograms of NDC D (prepared using the ixabepilone-linker conjugate precursor 202 from example 1) at different time points after incubation with cathepsin-B.

FIGS. 12A-12C are graphs showing drug release kinetics at various time points after incubation with cathepsin-B enzyme for exemplary NDC loaded with protease (cathepsin-B) cleavable irinotecan-linker conjugates, and depict time to release half of the payload, i.e., T _1/2 . FIG. 12A depicts T of NDC B _1/2 For 2.9 hours. FIG. 12B depicts T of NDC C _1/2 For 2.6 hours. FIG. 12C depicts T of NDC D _1/2 1.4 hours.

FIG. 13 depicts competitive binding of exemplary NDC (folate (FA) -functionalized drug-linker conjugated C' Dot (FA-CDC)) in a FR alpha positive (KB) cell line when compared to free folate.

Fig. 14 depicts flow cytometry of representative NDCs (two Folate (FA) -functionalized drug-linker conjugated C' Dot (FA-CDC)) in KB cell lines with different folate ligand densities (0, 12, or 25 folate molecules per nanoparticle average). The ixabepilone-linker conjugate precursors (compound 202) used to prepare each NDC used in the study are described in example 1. Blocking in the blocking group was achieved using 1mM free folic acid. CDC without folic acid but with the same amount of the ixabepilone-linker conjugate was used as a negative control group.

Fig. 15 depicts flow cytometry of representative NDCs (trilobate acid (FA) -functionalized drug-linker conjugated C' Dot (FA-CDC)) in KB cell lines with different drug/particle ratios (DPR). Example 1 describes an ixabepilone-linker conjugate precursor (compound 202) used to prepare NDCs for use in the study. Blocking in the blocking group was achieved using 1mM free folic acid. All FA-CDCs contain 12 to 22 folic acid moieties. FA-CDC with high drug-particle ratio (DPR) comprises between 35 and 50 ixabepilone-linker conjugate groups. FA-CDC with moderate DPR contains between 17 and 25 ixabepilone-linker conjugate groups. FA-CDC with low DPR has between 5 and 10 ixabepilone-linker conjugate groups. CDC without folic acid and with 17 to 25 drug linkers was used as a negative control group.

FIG. 16 depicts flow cytometry of a representative NDC (folate (FA) -functionalized drug-linker conjugated C' Dot (FA-CDC)) of 1nM pre-incubated with different amounts of human plasma for 24 hours. Blocking in the blocking group was achieved with 1mM free folic acid. The ixabepilone-linker conjugate precursor (compound 202) used to prepare the NDCs used in the study is described in example 1 to provide an average of 25 ixabepilone molecules per nanoparticle. The average number of folate ligands per nanoparticle was 15. CDC without folic acid but with the same amount of drug linker was used as a negative control group.

Figure 17 shows that at 1 hour and 24 hours, in KB (+++) and TOV-in the 112D (-) cell line, the cell line, confocal microscopy images of exemplary NDCs (folic acid (FA) -functionalized drug-linker conjugated C' Dot (FA-CDC), shown in the examples as NDC B). Blocking in blocking groups was achieved using 0.1mM free folic acid. The average number of folate ligands on the FA-CDC (NDC B) was 12 and the number of irinotecan-linker conjugates was 40). Lysosomes for useGreen staining, the latter is a Green fluorescent dye used to label and track acid organelles in living cells. In a color image (not shown), CDC appears red, lysosomes appear green, and nuclei appear blue due to fluorescence.

Fig. 18 is a Z-stacked confocal microscopy image of comparative FR-targeted nanoparticles (FA-C' Dot), FR-targeted ADC or corresponding FR-targeted antibodies without payload treated with exemplary folate-receptor (FR) -targeted NDC (NDC D, prepared according to example 3 using the ixatikang-linker conjugate precursor 202) at 37 ℃ for 4 hours followed by washing of KB tumor spheres. Scale bar: 200 μm.

FIG. 19A depicts injections at 1, 24, 48 and 72 hours post-injection ⁸⁹ Representative Maximum Intensity Projection (MIP) PET/CT imaging of healthy nude mice with Zr-DFO-FA-CDC.

Figure 19B shows in healthy nude mice at 2 and 24 hours post injection ⁸⁹ Biodistribution pattern of Zr-DFO-FA-CDC (n=3). The ixabepilone-linker conjugate precursor (compound 202) used to prepare the NDC used in the study is described in example 1; the average number of folate ligands per NDC (FA-CDC) was 12; and the average number of irinotecan-linker conjugates per NDC was 25.

Figures 20A-20F depict in vivo tumor growth inhibition studies of six exemplary folate receptor targeted NDCs (NDCs a-F) in KB tumor bearing mice (n=7). NDC-se:Sub>A each nanoparticle comprises about 19 drug-linker conjugate groups and about 18 folate ligands. NDC B contains about 25 drug-linker groups and about 15 folate ligands per nanoparticle. NDC C each nanoparticle comprises about 19 drug-linker conjugate groups and about 13 folate ligands. NDC D contains about 25 drug-linker conjugate groups per nanoparticle and about 12 folate ligands. NDC E contains about 17 drug-linker conjugate groups per nanoparticle and about 17 folate ligands. NDC F contains about 23 drug-linker conjugate groups per nanoparticle and about 20 folate ligands.

FIGS. 21A-21B depict the IC of exemplary NDC in irinotecan resistant and naive KB cells compared to unconjugated irinotecan ₅₀ A curve. FIG. 21A shows the IC of irinotecan in conventional KB cells (naive cells) and KB cells treated 4 times with irinotecan (irinotecan resistant cells) _s0 A curve. FIG. 21B shows the IC of exemplary NDC in naive and irinotecan resistant cells ₅₀ A curve. The ixabepilone-linker conjugate precursor (compound 202) used to prepare the exemplary NDCs in this study is described in example 1.

FIGS. 22A-22B depict the IC of exemplary NDC in irinotecan resistant and naive KB cells compared to unconjugated irinotecan ₅₀ A curve. FIG. 21A shows the IC of Sha Tikang in conventional KB cells (naive cells) and KB cells treated 4 or 7 times with irinotecan (irinotecan resistant cells) ₅₀ A curve. FIG. 22A shows the IC of exemplary NDC in naive and irinotecan resistant cells (4 and 7 rounds of pretreatment) ₅₀ A curve. The ixabepilone-linker conjugate precursor (compound 202) used to prepare the exemplary NDCs in this study is described in example 1.

Figure 23 provides a table demonstrating cytotoxicity of exemplary folate receptor targeted NDCs ("FA-CDC") with different drug-to-particle ratios (DPR) in different FR-a overexpressing cancer cell lines compared to unconjugated ixabencan. The ixabepilone-linker conjugate precursor (compound 202) used to prepare the exemplary NDCs in this study is described in example 1.

Figure 24 provides a table showing cytotoxicity of exemplary NDCs in various 3D patient-derived platinum-resistant tumor spheres. The ixabepilone-linker conjugate precursor (compound 202) used to prepare the exemplary NDCs in this study is described in example 1.

FIGS. 25A-25D provide a schematic representation of an exemplary FR-targeted NDC (prepared according to example 3 using the ixatikang-linker conjugate precursor 202 of example 1) on IGFV-1 (FR alpha positive human ovarian cancer) and an engineered AML MV4 overexpressing FR alpha; flow cytometry histograms of specific Folate Receptor (FR) alpha targeting ability of 11 cell lines. FIG. 25A is a flow cytometry histogram of FR-targeted NDC (10 nM) and non-targeted NDC (negative control; 10 nM) in an IGF-1 cell line. FIG. 25B is a flow cytometry histogram of anti-FR alpha antibody-PE and isotype antibody-PE (negative control) in an IGF-1 cell line. FIG. 25C is an engineered AML MV4 overexpressing FR alpha; flow cytometry histograms of FR-targeted NDC (10 nM) and non-targeted NDC (negative control; 10 nM) in 11 cell lines. FIG. 25D is an engineered AML MV4 overexpressing FR alpha; flow cytometry histograms of anti-FR alpha antibody-PE and isotype antibody-PE (negative control) in 11 cell lines.

FIGS. 26A-26B are diagrams showing exemplary NDCs (prepared according to example 3 using the irinotecan-linker conjugate precursor compound 202 of example 1) in an IGROV-1 (FR alpha positive human ovarian cancer) cell line (FIG. 26A) and MV4 overexpressing FR alpha; 11 engineering AML MV4;11 (fig. 26B) in vitro cytotoxicity profile in cell line, non-targeted NDC was used as negative control.

FIG. 27 is a graph providing the change in body weight over time in FR alpha over-expressing AML mice after treatment with physiological saline or exemplary NDC (prepared according to example 3 using the irinotecan-linker conjugate precursor compound 202 of example 1) at three different dosage regimens (0.33 mg/kg, Q3Dx6 (represented by squares), 0.50mg/kg, Q3Dx3 (represented by diamonds), or 0.65mg/kg, Q3Dx3 (represented by triangles).

FIG. 28 provides three different dosage regimens (0.33 mg/kg, Q3Dx 6) in physiological saline or exemplary NDC (prepared according to example 3 using the irinotecan-linker conjugate precursor compound 202 of example 1); 0.50mg/kg, Q3Dx3; or 0.65mg/kg, Q3Dx 3) in vivo bioluminescence imaging (BLI) images of FR alpha over-expression AML mice treated.

FIG. 29 is a graph providing three different dosage regimens (0.33 mg/kg, Q3Dx 6) in physiological saline or exemplary NDC (prepared according to example 1 using the irinotecan-linker conjugate precursor compound 202 of example 1); 0.50mg/kg, Q3Dx3; or 0.65mg/kg, Q3Dx 3) treated fα over-expressing AML mice.

FIG. 30 is a graph showing leukemia detected in bone marrow aspirate at day 42 post leukemia cell injection, obtained from three different dosage regimens (0.33 mg/kg, Q3Dx 6) with physiological saline or exemplary NDC (prepared according to example 3 using the irinotecan-linker conjugate precursor compound 202 of example 1); 0.50mg/kg, Q3Dx3; or 0.65mg/kg, Q3Dx 3).

FIG. 31 is a graph of the doses used to prepare mice overexpressing FR.alpha.AML and at three different dosage regimens (0.33 mg/kg, Q3Dx 6); 0.50mg/kg, Q3Dx3; or 0.65mg/kg, Q3Dx 3) to mice, and a graphical representation of a timeline of imaging mice with bioluminescence imaging (BLI) using the ixabepilone-linker conjugate precursor compound 202 of example 1 according to example 3. Each day of administration is represented by triangles (i.e., on days 46, 49 and 52 for all dose groups, and also on days 55, 58 and 62 for the 0.33mg/kg Q3Dx6 dose group).

Figure 32 shows that after 1 hour and 24 hours, in KB (+++) and TOV-in the 112D (-) cell line, the cell line, confocal microscopy images of exemplary NDC (folic acid (FA) -functionalized drug-linker conjugated C' Dot (FA-CDC) prepared using the ixatikang-linker conjugate precursor 202, shown in the examples as NDC DGreen staining, the latter is a Green fluorescent dye used to label and track acid organelles in living cells. In a color image (not shown), CDC appears red, lysosomes appear green, and nuclei appear blue due to fluorescence.

Fig. 33A-33B are graphs demonstrating the stability of exemplary NDCs prepared using the methods disclosed herein. Fig. 33A compares the stability of NDC produced using diene-based functionalized nanoparticles (i.e., based on the protocol outlined in example 3) and comparative NDC produced using amine-based functionalized nanoparticles in human serum at 37 ℃ over 7 days. Fig. 33B compares the stability of NDC produced using a diene-based bifunctional precursor and a comparative NDC produced using an amine-based bifunctional precursor in mouse serum at 37 ℃ over 7 days.

Detailed description of the preferred embodiments

Described herein are Nanoparticle Drug Conjugates (NDCs) comprising nanoparticles (e.g., silica nanoparticles, such as multimodal silica-based nanoparticles) that allow conjugation to targeting ligands and cytotoxic payloads for detection, prevention, monitoring, and/or treatment of diseases, e.g., cancer.

The disclosure provides compositions and methods of using Nanoparticle Drug Conjugates (NDCs) comprising: a nanoparticle; targeting ligands that bind to a folate receptor (e.g., folic acid or derivatives or salts thereof) and linker-payload conjugates that can include an ixabepilone and a protease cleavable linker.

Conjugation of targeting ligands and linker-drug conjugates to nanoparticles can be achieved by efficient "click chemistry" reactions, which are rapid, simple to operate, versatile, and result in high product yields. The payload may be a cytotoxic agent comprising irinotecan or a salt or analog thereof, linked to the nanoparticle by a cleavable linker group. When the NDC is internalized into a cancer cell (e.g., a tumor cell), such as an endosome or lysosomal compartment of the cell, the cleavable linker group can be cleaved, resulting in release of the active cytotoxic agent from the NDC. Cleavage may be catalyzed by proteases (e.g., cathepsin B).

NDCs disclosed herein provide an optimal platform for drug delivery due in part to their physical properties. For example, NDCs comprise nanoparticles of ultra-small diameter (e.g., average diameter between about 1nm and about 10nm, such as between about 5nm and about 8 nm) and benefit from Enhanced Permeability and Retention (EPR) effects in tumor microenvironments while maintaining desired clearance and pharmacokinetic properties.

NDCs described herein have certain benefits relative to other drug delivery platforms (e.g., ADCs, such as FR-targeted ADCs and FR-targeted small molecule drugs (e.g., chemotherapeutic agents)). For example, a single NDC of the present disclosure may contain up to about 80 drug molecules (e.g., 80 ixabetecan molecules) on each nanoparticle. In contrast, in conventional ADCs, only about 4 to 8 therapeutic agents/drug molecules may be attached to the antibody, and conventional FR-targeted small molecule drugs are limited to a single therapeutic agent/drug molecule. Thus, the NDCs described herein can carry at least 10-fold more drug molecules NDCs relative to conventional drug delivery platforms, and deliver relatively higher drug loads to cells.

While conventional Folate Receptor (FR) -targeted drug-delivery platforms (e.g., ADC and FR-targeted small molecule chemotherapeutic agents) generally exhibit high efficacy in cancer cells with high receptor expression levels, their efficacy in cancer cells with moderate or low FR expression levels is limited. In contrast, NDCs of the present disclosure can effectively target cancer cells with high and low FR expression levels and provide effective therapies for cancers with low FR expression (see, e.g., the relevant assays described in fig. 23 and the examples).

Without wishing to be bound by any particular theory of mechanism, it is believed that because NDCs disclosed herein may include multiple FR targeting ligands on a single nanoparticle, there is a multivalent or affinity effect on binding to several FR on the cell surface. In contrast, a single ADC can typically only bind to at most two FR on the cell surface, and a single FR-targeted chemotherapeutic drug can only bind to one FR on the cell surface. Thus, the multivalent effect of FR-targeted NDCs of the present disclosure can significantly enhance the binding of NDCs to FR-expressing cells, resulting in improved targeting efficiency and therapeutic outcome. The multivalent effect may also make NDCs of the present disclosure suitable for treating cancers with low FR expression, which cannot be effectively treated using conventional FR-targeted drug delivery platforms, such as ADC or FR-targeted chemotherapeutic drugs.

The efficacy of ADCs in the treatment of solid tumors is often greatly limited by their poor tumor penetration. In contrast, FR-targeted NDCs disclosed herein exhibit highly effective tumor penetration, allowing therapeutic agents to be delivered to the entire tumor after administration, which improves the therapeutic outcome of treating solid tumors relative to the use of ADCs.

NDCs of the present disclosure have smaller dimensions than conventional drug delivery platforms, such as ADCs. Notably, the NDC of the present disclosure is smaller than the cutoff particle size for renal clearance, such that the NDC is renal clearance. Thus, NDC administered to a subject but not entering cancer cells (i.e., non-targeted NDC) can be rapidly cleared from the body by renal elimination. The targeting and/or clearance method reduces toxicity of NDC compared to conventional drug delivery platforms (e.g., ADCs) and prevents undesirable accumulation of NDC (or its payload) in healthy tissues or organs. NDCs of the present disclosure exhibit improved biodistribution compared to conventional drug delivery platforms (e.g., ADCs), resulting in reduced side effects and toxicity.

Nanoparticles

The present disclosure relates to NDCs comprising nanoparticles, such as silica nanoparticles. The nanoparticle may comprise a silica-based core and a silica shell surrounding at least a portion of the core. Alternatively, the nanoparticle may have only a core and no shell. The core of the nanoparticle may contain the reaction product of a reactive fluorescent compound and a co-reactive organosilane compound. For example, the core of the nanoparticle may contain the reaction product of a reactive fluorescent compound and a co-reactive organosilane compound, as well as silica. In a preferred aspect of the present disclosure, the nanoparticle is a core-shell particle.

The diameter of the core may be about 0.5nm to about 100nm, about 0.1nm to about 50nm, about 0.5nm to about 25nm, about 0.8nm to about 15nm, or about 1nm to about 8nm. For example, the diameter of the core may be about 3nm to about 8nm, or 3nm to about 6nm, e.g., the diameter of the core may be about 3nm to about 4nm, about 4nm to about 5nm, about 5nm to about 6nm, about 6nm to about 7nm, or about 7nm to about 8nm.

The shell of the nanoparticle may be the reaction product of a silica forming compound, such as a tetraalkyl orthosilicate, e.g., tetraethyl orthosilicate (TEOS). The shell of the nanoparticle may have a series of layers. For example, the silica shell may be about 1 to about 20 layers, about 1 to about 15 layers, about 1 to about 10 layers, or about 1 to about 5 layers. For example, the silica shell may comprise about 1 to about 3 layers. The thickness of the shell may be about 0.5nm to about 90nm, about 0.5nm to about 40nm, about 0.5nm to about 20nm, about 0.5nm to about 10nm, or about 0.5nm to about 5nm, such as about 1nm, about 2nm, about 3nm, about 4nm, or about 5nm. For example, the thickness of the silica shell may be about 0.5nm to about 2nm. The silica shell of the nanoparticle may cover only a portion of the nanoparticle or the entire particle. For example, the silica shell may cover from about 1% to about 100%, from about 10% to about 80%, from about 20% to about 60%, or from about 30% to about 50% of the nanoparticles. For example, the silica shell may cover about 50% to about 100%. The silica shell may be solid, i.e., substantially non-porous, mesoporous, semi-porous, or the silica shell may be porous. The silica nanoparticles may be solid, i.e., substantially non-porous, mesoporous, semi-porous, or the silica nanoparticles may be porous. In some embodiments, the nanoparticle is a non-mesoporous nanoparticle, such as a non-mesoporous silica core-shell nanoparticle.

The nanoparticle surface may be modified to introduce at least one functional group. The organic polymer may be attached to the nanoparticle and may be modified by any technique known in the art to introduce at least one functional group. Functional groups may include, but are not limited to, dibenzocyclooctyne (DBCO), maleimide, N-hydroxysuccinimide (NHS) ester, diene (e.g., cyclopentadiene), amine, or thiol. For example, a difunctional group comprising silane on one end and DBCO, maleimide, NHS ester, diene (e.g., cyclopentadiene), amine, or thiol on the other end may be condensed onto the silica nanoparticle surface by a silane group. The introduction of functional groups may also be achieved by techniques known in the art, for example using "click chemistry", amide coupling reactions, 1, 2-addition such as Michael addition or Diels-Alder (2+4) cycloaddition reactions. This introduction allows various targeting ligands, contrast agents and/or therapeutic agents to be attached to the nanoparticle.

Organic polymers that may be attached to the nanoparticle include, but are not limited to, poly (ethylene glycol) (PEG), polylactic acid esters, polylactic acid, sugar, lipids, polyglutamic acid (PGA), polyglycolic acid, poly (lactic-co-glycolic acid) (PLGA), polyvinyl acetate (PVA), and combinations thereof. In a preferred aspect of the present disclosure, the organic polymer is poly (ethylene glycol) (PEG).

In a preferred aspect of the present disclosure, the nanoparticle surface is functionalized. For example, nanoparticle surfaces may have functional groups (e.g., -OH groups (generated from terminal Si-OH groups on the nanoparticle surface) and PEG groups (generated from Si-OH groups on the nanoparticle surface) different from those generated by synthesis of the nanoparticle.

The nanoparticle may comprise a non-porous surface and a porous surface. In one embodiment, at least a portion of the individual nanoparticle pore surfaces and at least a portion of the individual nanoparticle pore surfaces are functionalized. In one embodiment, at least a portion of the nanoparticle non-porous surface and at least a portion of the pore surface have different functionalization. The bore surface is also referred to herein as the interior surface. The nanoparticles may also have a non-porous surface (or a non-porous surface). The nonporous surface is also referred to herein as an external nanoparticle surface.

The pore surface (e.g., at least a portion of the pore surface) and/or the non-porous surface (e.g., at least a portion of the non-porous surface) of the nanoparticle may be functionalized. For example, the nanoparticle may be reacted with a compound such that the functional group of the compound is present (e.g., covalently bound) on the nanoparticle surface. The surface may be functionalized with hydrophilic groups (e.g., polar groups such as ketone, carboxylic acid, carboxylate, and ester groups) that provide the surface with hydrophilic properties or hydrophobic groups (e.g., non-polar groups such as alkyl, aryl, and alkylaryl groups) that provide the surface with hydrophobic properties. Such functionalization is known in the art. For example, diethoxydimethylsilane (dehs) may condense on at least a portion of the pore surface, such that the pore surface has hydrophobic properties, allowing for increased loading performance of the hydrophobic cytotoxic payload relative to the nanoparticle not so functionalized.

In a preferred aspect of the present disclosure, the nanoparticle surface is at least partially functionalized with polyethylene glycol (PEG) groups. Attachment of PEG to the nanoparticle may be accomplished by covalent or non-covalent bonds, such as by ionic, hydrogen, hydrophobic, coordination, adhesion, and physical adsorption.

In certain aspects, PEG groups are attached (e.g., covalently attached) to the nanoparticle surface. In core-shell nanoparticles, PEG groups are covalently bound to the silica of the shell surface and/or the silica in the core through Si-O-C bonds. In core nanoparticles, PEG groups are covalently bound to silica in the core.

In a preferred aspect, the nanoparticle is a core-shell nanoparticle wherein the PEG groups are covalently bound to the silica of the shell surface through Si-O-C bonds. PEG groups on the nanoparticle surface can prevent serum proteins from adsorbing to the nanoparticle in a physiological environment (e.g., in a subject), and can promote efficient urinary excretion and reduce aggregation of the nanoparticle (see, e.g., burns et al, "Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine", nano Letters (2009) 9 (1): 442-448).

The PEG groups may be derived from PEG polymers having molecular weights (Mw) of 400g/mol to 2000g/mol, including all integer g/mol values and ranges therebetween. In one embodiment, the PEG groups are derived from PEG polymers having Mw of 460g/mol to 590g/mol, which contain 6 to 9 ethylene glycol units. In various embodiments, at least 50%, at least 75%, at least 90%, or at least 95% of the nanoparticles are functionalized with PEG groups. In one embodiment, the nanoparticle is functionalized with a PEG group having the greatest number of PEG groups such that the pore maintains accessibility (e.g., the pore may be functionalized). In one embodiment, the pore surface is a silica surface having terminal silanol (si—oh) groups.

The polyethylene glycol units disclosed herein may be functionalized with functional groups, such as "click chemistry" groups, such as Dibenzocyclooctyne (DBCO) or azide, diene (e.g., cyclopentadiene), maleimide, NHS ester, amine, thiol, or activated acetylene moieties, such asAlthough DBCO can be used, the functional group can also be another alkyne, e.g., anotherStrained alkynes (e.g., DIBO or derivatives thereof, or derivatives of DBCO). Furthermore, the functional group may be a nitrone or a nitrile oxide.

Alternatively, or in addition to the above, functional groups may be introduced into NDC without the need for PEG groups. For example, NDC may be functionalized with functional groups such as "click chemistry" groups, e.g., dibenzocyclooctyne (DBCO) or azide; dienes (e.g., cyclopentadiene); a maleimide; NHS esters; an amine; a mercaptan; or activated acetylene moieties, e.gIt may comprise any suitable linker, or may not have a linker. While DBCO may be used to functionalize the nanoparticle, the functional group may also be another alkyne, such as another strained alkyne (e.g., DIBO or derivative thereof, or derivative of DBCO). Furthermore, the functional group may be a nitrone or a nitrile oxide.

For example, a DBCO-functionalized linker may be incorporated into a nanoparticle (e.g., a PEGylated C 'Dot) by reacting a silane group on the DBCO-linker-silane compound with a silanol group on the surface of the nanoparticle (e.g., under a PEG layer on the C' Dot surface). Similarly, a diene-functionalized precursor (e.g., a cyclopentadiene functionalized precursor) can be introduced into a nanoparticle (e.g., a pegylated C 'Dot) by reacting a silane group on a diene-linker-silane or diene-silane precursor compound with a silanol group on the nanoparticle surface (e.g., under a PEG layer on the C' Dot surface), followed by functionalizing the diene on the nanoparticle with a second precursor comprising a reactive group (e.g., DBCO) by a dienophile. The linker group in the DBCO-linker-silane or diene-linker-silane may comprise any structure (or substructure) including, but not limited to, PEG, carbon chains (e.g., alkylene), heteroalkylene, and the like. The diene-functionalized linker covalently attached to the nanoparticle may be further modified, for example, by reaction with a DBCO-functionalized group. For example, a diene-functionalized linker covalently attached to a nanoparticle may be contacted with a DBCO-linker-maleimide compound (or other suitable DBCO-linker-dienophile) to form a cycloadduct between the diene and maleimide, resulting in an NDC comprising DBCO groups attached to its surface, e.g., using cycloaddition chemistry such as Diels-Alder cycloaddition.

Conjugation of a suitably functionalized FR targeting ligand and/or functionalized drug payload (e.g., an azide functionalized FR targeting ligand and/or azide functionalized drug payload) to a nanoparticle is facilitated by a coupling reaction (e.g., by click chemistry, (3+2) cycloaddition reactions, amide coupling, or Diels-Alder reactions), such as one of the functional groups described above, such as DBCO or cyclopentadiene. The functionalization approach also improves the versatility of formulation chemistry and stability of FR-targeted NDC constructs.

The benefit of the NDCs disclosed herein is that they can be prepared using relatively stable linkers or spacers or precursors thereof. The linker or spacer or precursor thereof may avoid premature or undesired cleavage, which may occur using other linkers or precursors. For example, some methods of functionalizing nanoparticles use amine-silane precursors (to provide amine-functionalized nanoparticles) that are modified at the amine groups to conjugate other moieties to the nanoparticles. However, amine-silane precursors may be unstable and may self-condense during the reaction, resulting in undesired aggregation. Aggregates can be very difficult to separate from functionalized nanoparticles. Furthermore, amine groups on the nanoparticle surface can promote undesirable reactivity, which can lead to premature release of the payload or undesirable release of the targeting ligand.

NDCs disclosed herein can be prepared using relatively stable precursors, and NDCs are stable and of high purity. For example, the nanoparticles of NDCs of the present invention can be prepared with a silane-diene precursor (e.g., a silane-cyclopentadiene precursor) to provide nanoparticles functionalized with one or more diene groups. The dienyl group can then be reacted with a second precursor, such as a dienophile-containing precursor (e.g., a PEG-maleimide derivative, e.g., DBCO-PEG-maleimide), resulting in the formation of a stable cycloadduct. The resulting functionalized nanoparticle comprising a cycloadduct can optionally be reacted with one or more subsequent precursors (e.g., targeting ligand precursors and/or payload-linker conjugate precursors described herein) to further functionalize the nanoparticle. The produced diene-silane precursors and cycloadducts do not exhibit the undesirable properties of other functionalized nanoparticles, e.g., they have relatively high serum stability and can be produced in high yields and purity (e.g., free of aggregated precursors). See, for example, FIGS. 33A-33B. Furthermore, since the nanoparticle functionalization approach is highly modular, any desired proportion of payload, targeting ligand, or other substance can be incorporated into the nanoparticle. Examples of nanoparticles prepared using these methods and their benefits are provided in the examples.

NDCs of the present disclosure may comprise a structure of formula (NP):

wherein x is an integer from 0 to 20, for example 4; wherein the silicon atom is part of a nanoparticle; and wherein adjacent to the triazole moietyIndicating the point of attachment to the targeting ligand or payload-linker conjugate directly or indirectly, e.g., through a linker or spacer group, e.g., a PEG moiety. For example, the linkage may be a linker or spacer group, e.g., a linker of a linker-payload conjugate, or a linker or spacer group of a folate receptor targeting ligand, e.g., a PEG moiety. NDCs of the present disclosure can be prepared from diene (e.g., cyclopentadiene) functionalized nanoparticles, for example, by conjugating a linker moiety (e.g., a linker comprising a dienophile such as maleimide) to a diene using a cycloaddition reaction.

The silica shell surface of the nanoparticle may be modified by introducing surface functional groups using known cross-linking agents. Crosslinking agents include, but are not limited to, divinylbenzene, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, N' -methylene-bis-acrylamide, alkyl ethers, sugars, peptides, DNA fragments or other known functionally equivalent agents.

To allow nanoparticles to be detectable not only by optical imaging (e.g., fluorescence imaging), but also by other imaging techniques, such as Positron Emission Tomography (PET), single Photon Emission Computed Tomography (SPECT), computed Tomography (CT), and Magnetic Resonance Imaging (MRI), nanoparticles may also be conjugated to a contrast agent, such as a radionuclide.

The nanoparticle may incorporate any suitable fluorescent compound, such as fluorescent organic compounds, dyes, pigments, or combinations thereof. Such fluorescent compounds may be incorporated into the silica matrix of the nanoparticle core. A wide variety of suitable chemically reactive fluorescent dyes/fluorophores are known, see for example MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, 6 th edition, r.p. haugland, editions (1996). In a preferred aspect of the present disclosure, the fluorescent compound is covalently encapsulated within the core of the nanoparticle.

In some aspects, the fluorescent compound may be, but is not limited to, a near infrared fluorescent (NIRF) dye located within the silica core of the nanoparticle, which may provide greater brightness and fluorescence quantum yield relative to the free fluorescent dye. Near infrared emission probes are known to exhibit reduced tissue attenuation and autofluorescence (Burns et al, "Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine", nano Letters (2009) 9 (1): 442-448).

Fluorescent compounds that may be used in the present disclosure (e.g., encapsulated by NDC) include, but are not limited to, cy5, cy5.5 (also known as Cy 5++), cy2, fluorescein Isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin, cy7, fluorescein (FAM), cy3, cy3.5 (also known as Cy 3++), texas red (sulfonyl rhodamine 101 acyl chloride), and, 640、/>705. Tetramethyl rhodamine (TMR), rhodamine derivatives (ROX), hexachloro fluorescein (HEX), rhodamine 6G (R6G), rhodamine derivatives JA133. Alexa fluorochromes (e.g. ALEXA->488、ALEXA/>546、ALEXA/>633、ALEXA 555 and ALEXA->647 4', 6-diamidino-2-phenylindole (DAPI), propidium iodide, aminomethylcoumarin (AMCA), spectrum Green, spectrum Orange, spectrum Aqua LISSAMINE) ^TM And fluorescent transition metal complexes such as europium.

Fluorescent compounds that can be used also include fluorescent proteins such as GFP (green fluorescent protein), enhanced GFP (EGFP), blue fluorescent proteins and derivatives (BFP, EBFP, EBFP, blue copper mine, mKalama 1), cyan fluorescent proteins and derivatives (CFP, ECFP, celadon, cyPet) and yellow fluorescent proteins and derivatives (YFP, yellow crystals, venus, YPet) (WO 2008/142571, WO2009/056282, WO 1999/22026).

In a preferred aspect of the present disclosure, the fluorescent compound is selected from Cy5 and Cy5.5. In a preferred aspect, the fluorescent compound is Cy5.

Fluorescent nanoparticles can be synthesized by the following steps: (1) Covalently conjugating a fluorescent compound (e.g., a reactive fluorescent dye (e.g., cy 5)) with a reactive moiety (including, but not limited to, maleimide, iodoacetamide, thiosulfate, amine, N-hydroxysuccinimide ester, 4-sulfo-2, 3,5, 6-tetrafluorophenyl (STP) ester, sulfosuccinimidyl ester, sulfodichlorophenol ester, sulfonyl chloride, hydroxyl, isothiocyanate, carboxyl) to an organosilane compound (e.g., a co-reactive organosilane compound) to form a fluorescent silica precursor, and reacting the fluorescent silica precursor to form a fluorescent core; or (2) reacting the fluorescent silica precursor with a silica forming compound, such as a tetraalkoxysilane, to form a fluorescent core. The fluorescent core may then be reacted with a silica forming compound, such as a tetraalkoxysilane, to form a silica shell on the core, thereby providing the fluorescent nanoparticle.

Fluorescent silica nanoparticles are known in the art and are described in US 8298677 B2, US 9625456 B2, US 10548997 B2, US 9999694 B2, US 10039847 B2 and US 10548998 B2, the contents of each of which are incorporated herein by reference in their entirety.

In a preferred aspect of the present disclosure, the NDC comprises a nanoparticle comprising a silica-based core and a silica shell surrounding at least a portion of the core, and polyethylene glycol (PEG) is covalently bound to the nanoparticle surface, and the fluorescent compound is covalently encapsulated within the core of the nanoparticle.

Targeting ligands

NDCs of the present disclosure may comprise a targeting ligand attached to a nanoparticle directly or indirectly through a spacer group. NDC with targeting ligands can enhance internalization of payload/drug in tumor cells and/or delivery of drug into tumor cells due to increased permeability and targeting ability of NDC. Targeting ligands may allow nanoparticles to target specific cell types through specific binding between the ligand and the cell components. Targeting ligands may also facilitate nanoparticle transport into cells or barriers, for example, for assaying the intracellular environment.

The targeting ligands of the present disclosure are capable of binding to receptors on tumor cells. Specifically, the targeting ligand can bind to Folate Receptors (FR), including all four human isoforms of FR, including FR alpha (FR alpha, also known as FOLR 1), FR beta (FR beta, also known as FOLR 2), FR gamma (FR gamma, also known as FOLR 3), and FR delta (FR delta, also known as FOLR 4). Conjugation of FR targeting ligands to the nanoparticle surface of the present disclosure allows targeted therapy of cells, tissues and tumors of cancers that overexpress FR. For example, NDCs of the present disclosure comprising a targeting ligand that can bind to folate receptor alpha (froc) (e.g., folic acid) can be used to target ovarian cancer, endometrial cancer, fallopian tube cancer, peritoneal cancer, cervical cancer, breast cancer, lung cancer, mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer, and gastric cancer), pancreatic cancer, bladder cancer, renal cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, and testicular cancer, acute myelogenous leukemia (AML, e.g., pediatric AML). NDCs of the present disclosure comprising targeting ligands that can bind folate receptor beta (frβ) can be used to target acute myelogenous leukemia (AML, e.g., pediatric AML), chronic Myelogenous Leukemia (CML), and tumor-associated macrophages. Tumor-associated macrophages can be targets for means of modifying tumor immune status. Without wishing to be bound by theory, the binding affinity of FR-targeted NDC to the folate receptor may be enhanced due to multivalent effects.

Folate receptors can be highly expressed in solid tumor cells including ovarian, renal, lung, brain, endometrial, colorectal, pancreatic, gastric, prostate, breast, and non-small cell lung cancer. FR is overexpressed in other cancers, including fallopian tube cancer, cervical cancer, mesothelioma, uterine cancer, esophageal cancer, gastric cancer, bladder cancer, liver cancer, head and neck cancer, thyroid cancer, skin cancer, and testicular cancer. FR is also overexpressed in hematological malignancies, such as Acute Myelogenous Leukemia (AML) and Chronic Myelogenous Leukemia (CML).

In a preferred aspect of the disclosure, the targeting ligand binds to folate receptor alpha (fra), folate receptor beta (fra), or both.

The present disclosure provides FR targeting ligands capable of binding to specific cell types with elevated levels of FR alpha, such as, but not limited to, cancers of the uterus, ovary, breast, cervix, kidney, colon, testis (e.g., testicular choriocarcinoma), brain (e.g., ependymal brain tumor), malignant pleural mesothelioma, and non-functional pituitary adenocarcinoma (e.g., adenocarcinoma). The present disclosure also provides FR targeting ligands capable of targeting acute myelogenous leukemia (AML, e.g., pediatric AML), chronic Myelogenous Leukemia (CML), and tumor-associated macrophages. The targeting ligand may be any suitable molecule capable of binding FR (e.g., froc), such as a small organic molecule (e.g., folic acid or folic acid analog), an antigen binding portion of an antibody (e.g., fab fragment, fab ' fragment, F (ab ') 2 fragment, scFv fragment, fv fragment, dsFv duplex (diabody), dAb fragment, fd ' fragment, fd fragment, or an isolated Complementarity Determining Region (CDR) region), an antibody mimetic (e.g., aptamer, affibody, affinity protein (affilin), affinity peptide (affimer), anti-cardiolipin (anticalin), avimer, darpin, etc.), a nucleic acid, a lipid, and the like.

In aspects of the disclosure, the targeting ligand is folic acid or a folate receptor binding derivative thereof. It is to be understood that "folic acid" can include any amide or ester derivative of folic acid. For example, free folic acid can be modified to be conjugated to the nanoparticle through a spacer group, such as PEG or a PEG derivative (e.g., by forming an amide bond between the terminal carboxylic acid of folic acid and the nitrogen atom of the spacer group).

FR-targeted NDCs can not only accumulate in cancer cells or tumors, but can also penetrate tumor tissue and deliver a payload to the entire tumor tissue for optimal therapeutic efficacy. Without wishing to be bound by any particular theory or mechanism, it is believed that the targeting ligand binds to specific receptor groups on the surface of cancer cells, resulting in receptor-mediated NDC cellular uptake. The receptor-mediated uptake of NDC by cells occurs through endocytic processes and eventually transports NDC into the endosomes and lysosomes of cancer cells.

In aspects of the disclosure, the NDC comprises a targeting ligand attached to the nanoparticle directly or indirectly through a spacer group. For example, the targeting ligand may be directly attached (i.e., covalently bound) to the nanoparticle through the silica of the nanoparticle. In a preferred aspect, the targeting ligand is indirectly attached to the nanoparticle through a suitable spacer group.

The spacer group can be any group that can act as a spacer (e.g., as a spacer between the targeting ligand and the nanoparticle) and attach the targeting ligand to the nanoparticle. The spacer group may be a divalent linker, for example, comprising a divalent linker having a chain length of between about 5 and about 200 atoms (e.g., carbon atoms, heteroatoms, or combinations thereof), for example, between about 5 and about 100 atoms, between about 5 and about 80 atoms, between about 10 and about 70 atoms, between about 10 and about 30 atoms, between about 20 and about 30 atoms, between about 30 and about 80 atoms, or between about 30 and about 60 atoms. Suitable spacer groups can include alkylene, alkenylene, alkynylene, heteroalkylene (e.g., PEG), carbocyclyl, heterocyclyl, aryl, heteroaryl, or combinations thereof. For example, the spacer group may comprise a PEG group, an alkylene group, or a combination thereof. The spacer group may be substituted or unsubstituted, for example, the spacer group may comprise a substituted alkylene group, a substituted heteroalkylene group, or a combination thereof. For example, the spacer group may comprise a PEG group (or PEG spacer group), an alkylene group (or alkylene spacer group), one or more heteroatoms, and/or one or more cyclic groups (e.g., a heterocycloalkylene group, such as piperazine).

Targeting ligands, such as folic acid, may be indirectly linked to the nanoparticle through a PEG spacer. Folic acid can be present in NDC as an amide, e.g., to facilitate conjugation to a PEG spacer or other divalent linker, e.g., as shown in fig. 1. The number of PEG monomers in the PEG spacer may range from 2 to 20, 2 to 10, 2 to 8, or 2 to 5. In a preferred aspect, the number of PEG groups as spacers in the functionalized FR targeting ligand is 3.

The average nanoparticle to targeting ligand (e.g., folic acid) ratio can be in the range of about 1 to about 50, about 1 to about 40, about 1 to about 30, or about 1 to about 20. For example, the average nanoparticle to targeting ligand (e.g., folic acid) ratio may be about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:40, or 1:50. For example, the average nanoparticle to targeting ligand ratio may be in the range of about 1 to about 20, e.g., the average number of folate molecules per nanoparticle may be between about 5 to about 10, between about 10 to about 15, or between about 15 to about 20, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 folate molecules per nanoparticle. NDCs disclosed herein may comprise about 10 folate molecules. NDCs disclosed herein may comprise about 11 folate molecules. NDCs disclosed herein may comprise about 12 folate molecules. NDCs disclosed herein may comprise about 13 folate molecules. NDCs disclosed herein may comprise about 14 folate molecules. NDCs disclosed herein may comprise about 15 folate molecules.

The smaller number of targeting ligands attached to the nanoparticle may help maintain the hydrodynamic diameter of the nanoparticle, e.g., to meet the renal clearance cutoff size range (Hilderbrand et al, near-infrared fluorescence: application to in vivo molecular imaging, curr. Opin. Chem. Biol., (2010) 14:71-79). The measured amount of targeting ligand may be the average amount of targeting ligand attached to more than one nanoparticle. Alternatively, one nanoparticle may be measured to determine the number of linked targeting ligands.

The amount of targeting ligand attached to the nanoparticle may be measured by any suitable method, such as, but not limited to, optical imaging, fluorescence Correlation Spectroscopy (FCS), UV-Vis, chromatography, mass spectrometry, or indirect enzymatic analysis.

The targeting ligand may be attached to the nanoparticle by silica covalently bound to the nanoparticle (e.g., indirectly through a spacer group). The ligand may be conjugated to the nanoparticle described herein (e.g., via a functional group on the nanoparticle surface), for example, using a coupling reaction, click chemistry (e.g., a 3+2 click chemistry reaction), cycloaddition (e.g., a 3+2 or 2+4 cycloaddition reaction, using a suitable functional group), or via a carboxylate, ester, alcohol, urea, aldehyde, amine, sulfur oxide, nitrile oxide, nitrone, nitroxide, halide, or any other suitable compound known in the art.

In a preferred aspect of the present disclosure, conjugation of FR targeting ligands can be achieved by a "click chemistry" reaction using a Diarylcyclooctyne (DBCO) group. Any suitable reaction mechanism may be suitable for "click chemistry" in the present disclosure, as long as an easy and controlled attachment of the targeting ligand to the nanoparticle can be achieved.

In some aspects, a triple bond (e.g., an alkyne, e.g., a terminal alkyne) is introduced onto the nanoparticle surface (e.g., by PEG covalently conjugated to the shell of the nanoparticle, or by another suitable linker or spacer group). Separately, azide bonds or other groups that react with triple bonds are introduced onto the desired targeting ligand. For example, folic acid can be modified by conjugating a terminal carboxylic acid of folic acid with a spacer group (e.g., a PEG moiety) that includes an azide at one terminus. Nanoparticles (e.g., pegylated nanoparticles) comprising a free triple bond and a targeting ligand (comprising a group that reacts with the triple bond) may be mixed (with or without copper or other metal catalyst) to effect cycloaddition of the group that reacts with the triple bond (e.g., azide) to the triple bond, resulting in conjugation of the targeting ligand to the nanoparticle (e.g., "click chemistry"). Many variations of this method may also be used, as will be apparent to those of ordinary skill in the art.

The azide-functionalized FR-ligand (where the FR-ligand may comprise a spacer group and the spacer group may have an azide group) may be directly or indirectly attached to the nanoparticle through an alkyne (e.g., a DBCO group). A spacer group, such as but not limited to a PEG group, may be present in the FR-targeting ligand precursor and may have a terminal group (e.g., azide) to facilitate conjugation with the nanoparticle, and after conjugation, the spacer group may be located between the targeting ligand and the nanoparticle. For example, the FR-targeting ligand precursor may comprise a structure of formula (D-1):

where y is an integer from 0 to 20 (e.g., 3). For example, y may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, such as 2, 3 or 4.

In some aspects, FR targeting ligands can be functionalized with suitable end groups (such as, but not limited to, azides). The azide-functionalized FR-ligand may be attached to the nanoparticle directly or indirectly through a DBCO group. A spacer group, such as but not limited to a PEG group, may be present between the azide-functionalized FR-ligand and the nanoparticle. In a preferred aspect, the FR targeting ligand is functionalized to include a spacer group, such as, but not limited to, a PEG group terminated with an azide group, which reacts with a DBCO group on the nanoparticle surface.

Functionalization of FR targeting ligands can include hydrophilic PEG groups as spacers, which can enhance solubility in water, and can reduce or eliminate aggregation and precipitation of nanoparticles.

In aspects of the disclosure, the number of PEG groups as spacers that may be present in the functionalized FR targeting ligand may be in the range of 2 to 20, 2 to 10, 2 to 8, or 2 to 5. In a preferred aspect, the number of PEG groups as spacers in the functionalized FR targeting ligand is 3.

NDCs of the present disclosure comprising targeting ligands may comprise a structure of formula (NP-2):

where x is an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, e.g., 4), and y is an integer from 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 3), and the silicon atom is part of a nanoparticle (e.g., associated with the silica shell of a core-shell silica nanoparticle). For example, x may be 4 and y may be 3. Each nanoparticle of the NDCs disclosed herein may comprise more than one molecule of formula (NP-2), e.g., the nanoparticle may comprise from about 1 to about 20 molecules of formula (NP-2), e.g., from about 5 to about 20 molecules of formula (NP-2), from about 8 to about 15 molecules of formula (NP-2), from about 10 to about 15 molecules of formula (NP-2), e.g., from about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 molecules of formula (NP-2). The NDCs disclosed herein may comprise about 12 molecules of formula (NP-2). The NDCs disclosed herein may comprise about 13 molecules of formula (NP-2).

Linker-payload conjugates

NDCs of the present disclosure may also comprise a linker-payload conjugate attached directly or indirectly to the nanoparticle through a spacer group. In a preferred aspect, the linker-payload conjugate is attached to the nanoparticle through a spacer group. The payload may be irinotecan or a salt or analog thereof.

The spacer group can be any group that can act as a spacer (e.g., as a spacer between the payload/linker conjugate and the nanoparticle) and attach the linker-payload conjugate to the nanoparticle. The spacer group may be a divalent linker, for example, comprising a divalent linker having a chain length of between about 5 and about 200 atoms (e.g., carbon atoms, heteroatoms, or combinations thereof), for example, between about 5 and about 100 atoms, between about 5 and about 80 atoms, between about 10 and about 70 atoms, between about 10 and about 30 atoms, between about 20 and about 30 atoms, between about 30 and about 80 atoms, or between about 30 and about 60 atoms. Suitable spacer groups can include alkylene, alkenylene, alkynylene, heteroalkylene (e.g., PEG), carbocyclyl, heterocyclyl, aryl, heteroaryl, or combinations thereof. For example, the spacer group may comprise a PEG group, an alkylene group, or a combination thereof. The spacer group may be substituted or unsubstituted, for example, the spacer group may comprise a substituted alkylene group, a substituted heteroalkylene group, or a combination thereof. For example, the spacer group may comprise a PEG group (or PEG spacer), an alkylene group (or alkylene spacer), one or more heteroatoms, and/or one or more cyclic groups.

It will be appreciated that the payload may be chemically modified to facilitate reaction of the payload with the linker for the purposes of preparing the conjugates of the present disclosure. For example, a functional group, such as an amine, hydroxyl, or thiol, can be pendant to the payload (e.g., irinotecan) at a position that has minimal or acceptable impact on the activity or other properties of the payload (e.g., irinotecan). Alternatively, the functional group (e.g., pendant amine group) present on the payload may be the point of attachment to the linker. For example, irinotecan contains amine functional groups suitable for coupling to a linker moiety.

The payload (e.g., the irinotecan payload, or a salt or analog thereof) can be cleaved from the nanoparticle, e.g., by an enzyme, within the cell or cell organelle, thereby releasing the irinotecan, e.g., within the cell or organelle. Irinotecan is a topoisomerase 1 (Topo-1) inhibitor that stabilizes the complex of DNA and Topo-1 enzyme, preventing DNA regulation and inducing lethal DNA strand breaks. The creation of these DNA lesions is effective for killing cancer cells, allowing NDCs of the present disclosure to achieve the desired therapeutic effect.

In a preferred aspect of the present disclosure, the payload is irinotecan or a salt thereof. In other preferred aspects of the present disclosure, the payload is an analog of ixabepilone or a salt thereof.

In aspects of the disclosure, the average nanoparticle to payload ratio ranges from 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 12, preferably 1 to 10. For example, the average nanoparticle to payload (e.g., isaatikang or a salt thereof or analog thereof) ratio may be about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:32, 1:34, 1:36, 1:38, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, or 1:80). For example, the average number of irinotecan molecules on each nanoparticle can be between about 5 and about 10, between about 10 and about 15, between about 15 and about 20, between about 20 and about 25, or between about 25 and about 30, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 irinotecan molecules per nanoparticle. The NDCs disclosed herein may comprise about 18 ixabepilone molecules. The NDCs disclosed herein may comprise about 19 ixabepilone molecules. The NDCs disclosed herein may comprise about 20 ixabepilone molecules. The NDCs disclosed herein may comprise about 21 ixabepilone molecules. The NDCs disclosed herein may comprise about 22 ixabepilone molecules. The NDCs disclosed herein may comprise about 23 ixabepilone molecules. The NDCs disclosed herein may comprise about 24 ixabepilone molecules. The NDCs disclosed herein may comprise about 25 ixabepilone molecules. The NDCs disclosed herein may comprise about 26 ixabepilone molecules. The NDCs disclosed herein may comprise about 27 ixabepilone molecules.

The vintakolide developed by Endocyte and Merck & co. Is a small molecule drug conjugate consisting of a small molecule targeting the folate receptor (which is overexpressed on certain cancers such as ovarian cancer) and the chemotherapeutic drug vinblastine (US 7601332 B2 and US 1002942 B2). However, vintakolide is only capable of carrying a single payload molecule, linked to a targeting moiety by a pH-cleavable linker. In contrast, in the present disclosure, several cytotoxic payloads (e.g., ixabepilone molecules) may be incorporated onto the surface of a single nanoparticle.

The linker in the linker-payload conjugate may be a self-destructing linker capable of releasing the active payload in vitro as well as in vivo under conditions sufficient for enzymatic release of the active payload (e.g., conditions for presentation of an enzyme capable of catalytic release).

The linkers described herein may be used, for example, to attach a cytotoxic drug payload (e.g., irinotecan) to a carrier and/or targeting moiety (e.g., nanoparticle) that binds to a cancer cell (e.g., binds to a receptor on the surface of a cancer cell) and internalize into the cell (e.g., through endosomal and lysosomal compartments). Once internalized, the linker can be cleaved or degraded to release the active cytotoxic drug. In particular, the protease cleavable linker may release its payload under the action of a protease, such as a cathepsin, trypsin or other protease, in the lysosomal compartment of the cell.

The cleavable linkers described herein may comprise a structure of formula (F):

wherein [ AA ]]Is a natural or unnatural amino acid residue; z is an integer from 1 to 5; w is an integer from 1 to 4 (e.g., 2 or 3); and each ofThe indication being attached to, e.g. a spacerA point of a bolus (e.g., PEG) or another portion of a linker or a point of attachment to an ixabepilone molecule. For example, - [ AA] _w May comprise Val-Lys, val-Cit, phe-Lys, trp-Lys, asp-Lys, val-Arg or Val-Ala and z may be 2, one of which>Indicates an oxygen atom attached to the PEG group, and the other +.>Indicating the nitrogen atom attached to the ixabepilone. For example, - [ AA] _w Val-Lys may be included.

The cleavable linkers described herein may comprise a structure of formula (F-1):

one of which is a platePoints indicating oxygen atom attached to PEG group and the other +.>Indicating the point of attachment of the nitrogen atom of irinotecan.

The linkers of the present disclosure may be prepared from linker precursors containing reactive groups at one or both ends of the molecule. The reactive groups may be selected to allow conjugation to the ixabepilone or analogue thereof at one end and also to facilitate conjugation to the nanoparticle at the other end. It is desirable that the payload contain an amine, hydroxy, hydrazone, hydrazide or thiol group to facilitate conjugation to the linker. For example, irinotecan contains primary amine groups that can facilitate its conjugation to the linker.

The linker-payload conjugate precursor may be attached to the nanoparticle using any suitable technique and method, and many such techniques are well known in the art. See, for example, WO 2017/189961, WO 2015/183882, WO 2013/192609, WO 2016/179260, and WO 2018/213851, each of which is incorporated herein by reference in its entirety, which describe that silica-based core-shell or silica-based core nanoparticles can be used to prepare targeted nanoparticle-based drug delivery systems. Furthermore, the linker-payload conjugate precursor or ligand-linker precursor may be attached to the nanoparticle using a reaction or method described in Kolb et al angel.chem.int.ed. (2001) 40:2004-2021, which is incorporated herein by reference in its entirety.

The linker-payload conjugate may be directly or indirectly attached to the nanoparticle through a spacer group (e.g., a spacer group as described herein). Suitable spacer groups include, but are not limited to, divalent linkers (e.g., divalent linkers described herein), such as PEG spacer groups or alkylene spacer groups (e.g., methylene spacer groups), which may further comprise heteroatoms or cyclic groups (e.g., heterocycloalkylene groups). The linker-payload conjugate may be absorbed into voids or pores of a silica shell or coated onto a silica shell of a nanoparticle, e.g., a fluorescent nanoparticle (e.g., covalently attached to the nanoparticle surface). In other aspects, the linker-payload conjugate may be associated with the fluorescent core, for example, by physical absorption or by binding interactions, without the silica shell covering all surfaces of the nanoparticle.

In some aspects, the linker-payload conjugate may also be associated with a PEG group covalently bound to the nanoparticle surface. For example, the linker-payload conjugate may be attached to the nanoparticle by PEG. The PEG may have multiple functional groups for attachment to the nanoparticle and the linker-payload conjugate.

In particular aspects of the disclosure, the linker-payload conjugate (or linker-payload conjugate precursor) may be functionalized with a hydrophilic PEG spacer. The linker-payload conjugate precursor may be functionalized with a hydrophilic PEG spacer and/or a suitable end group such as, but not limited to, an azide group to facilitate covalent attachment (e.g., by the spacer) of the linker-payload conjugate to the nanoparticle surface, e.g., by reaction with a DBCO group on the nanoparticle surface. Other end groups may include, for example. Nitrile oxide or nitrone for conjugation to a suitable group (e.g., diene moiety) on the nanoparticle by a 3+2 cycloaddition reaction.

The number of PEG groups as spacers that may be present in the functionalized linker-payload conjugate (or precursor thereof) may be in the range of 0 to 20, e.g., 2 to 20, 2 to 10, or 5 to 8, e.g., 5, 6, 7, 8, 9, 10, 11, or 12. In a preferred aspect, the number of PEG groups as spacers in the functionalized linker-payload conjugate is 9.

For example, isatecan may be conjugated to a protease cleavable linker to form a linker-payload conjugate. The linker-conjugate can be prepared from a precursor functionalized with a PEG spacer having a terminal reactive group, such as azide, for further conjugation to the nanoparticle surface, e.g., via a DBCO group.

The protease cleavable linker may be designed to be unstable to cathepsin B (Cat-B), an enzyme that is overexpressed in malignant tumors, thereby affecting the release of cytotoxic agents (e.g., isatecan) through a self-destructing process.

The linker payload conjugate precursor may comprise a structure of formula (E-1):

wherein y is an integer from 0 to 20, e.g., 5 to 15, e.g., 9.

The linker and linker-payload conjugates described in this disclosure have several benefits over conventional drug delivery platforms, linkers, or linker-payload conjugates, ranging from excellent serum stability to faster release kinetics mechanisms. Furthermore, the ability of these linkers to pair with a variety of chemical groups provides the opportunity to selectively release free payloads/drugs with minimal derivatization, which is a significant benefit.

In a preferred aspect of the present disclosure, the linker in the linker-payload conjugate is a protease cleavable linker.

NDCs of the present disclosure comprising a payload-linker moiety may comprise a structure of formula (NP-3):

where x is an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 4), and y is an integer from 0 to 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 9), and the silicon atom is part of a nanoparticle (e.g., associated with the silica shell of a core-shell silica nanoparticle). For example, x may be 4 and y may be 9. The NDCs disclosed herein may comprise more than one molecule of formula (NP-3), e.g., the nanoparticle may comprise between about 1 and about 80 molecules of formula (NP-3), e.g., between about 1 and about 60 molecules of formula (NP-3), between about 1 and about 40 molecules of formula (NP-3), between about 1 and about 30 molecules of formula (NP-3), between about 10 and about 30 molecules of formula (NP-3), between about 15 and about 25 molecules of formula (NP-3), e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 molecules of formula (NP-3).

Upon contact with a protease (e.g., within a cancer cell, such as within the lysosome of the cancer cell), the NDCs of the present disclosure can undergo cleavage to release free irinotecan. Cleavage of NDC disclosed herein may be accompanied by release of i Sha Tikang, carbon dioxide and 4-aminobenzyl alcohol from NDC. For example, cleavage of the exemplary NDCs disclosed herein is provided in scheme 1 below.

Scheme 1. Exemplary cleavage mechanism of NDCs disclosed herein.

The NDCs disclosed herein may comprise molecules of formula (NP-2) and molecules of formula (NP-3), for example, each NDC may comprise about 1 to about 20 molecules of formula (NP-2) and about 1 to about 30 molecules of formula (NP-3). For example, each NDC may comprise from about 10 to about 15 molecules of formula (NP-2) and from about 15 to about 25 molecules of formula (NP-3). The NDC disclosed herein can comprise an average of 13 molecules of formula (NP-2) and an average of 21 molecules of formula (NP-3); an average of 12 molecules of formula (NP-2) and an average of 25 molecules of formula (NP-3); on average 12 molecules of formula (NP-2) and on average 20 molecules of formula (NP-3).

The present disclosure provides compositions and methods relating to nanoparticle-drug conjugates (NDCs) comprising: a nanoparticle; a targeting ligand that binds to a folate receptor; and a linker-payload conjugate, wherein the NDC has an average diameter between about 1nm and about 10 nm. For example, nanoparticles comprising folic acid as a targeting ligand and linker-payload conjugates comprising ixabepilone conjugated by a protease cleavable linker, wherein NDC has an average diameter between about 1nm to about 10 nm.

Fig. 1 shows a representative nanoparticle-drug conjugate (NDC) having an average diameter of about 6nm, comprising: nanoparticles comprising a silica-based core and a silica shell surrounding at least a portion of the core, polyethylene glycol (PEG) covalently bonded to the surface of the nanoparticle, and a fluorescent compound (Cy 5) covalently encapsulated within the nanoparticle core, folic Acid (FA) as a targeting ligand that can bind to a folate receptor, and a linker-payload conjugate comprising a protease-cleavable linker-irinotecan conjugate. It will be understood that "folic acid" is intended to encompass any amide or ester derivative of folic acid, for example, as shown in fig. 1, wherein folic acid is covalently linked to a spacer group (PEG) through an amide group.

NDCs may have an average diameter between about 5nm to about 8nm or between about 6nm to about 7 nm. The average diameter of NDC may be measured by any suitable method, such as, but not limited to, fluorescence Correlation Spectroscopy (FCS) (see, e.g., fig. 6) and Gel Permeation Chromatography (GPC) (fig. 7).

NDCs of the present disclosure may include nanoparticles that may be functionalized with contrast agents for Positron Emission Tomography (PET), single Photon Emission Computed Tomography (SPECT), computed Tomography (CT), magnetic Resonance Imaging (MRI), and optical imaging (e.g., fluorescence imaging, including near infrared fluorescence (NIRF) imaging, bioluminescence imaging, or a combination thereof).

Contrast agents, such as radionuclides (radiolabels), include, but are not limited to ⁸⁹ Zr、 ⁶⁴ Cu、 ⁶⁸ Ga、 ⁸⁶ Y、 ¹²⁴I and ¹⁷⁷ lu, can be attached to the nanoparticle. Alternatively, the nanoparticle may be linked to a chelator moiety suitable for binding radionuclides, e.g., DFO, DOTA, TETA and DTPA. Such nanoparticles may be detected by PET, SPECT, CT, MRI or optical imaging (e.g., fluorescence imaging, including near infrared fluorescence (NIRF) imaging, bioluminescence imaging, or a combination thereof).

Radionuclides may additionally be used as therapeutic agents for the production of multiple therapeutic platforms. The coupling allows for delivery of the therapeutic agent to a particular cell type by specific binding between the targeting ligand and the cell component.

Protease cleavable linker-payload conjugates

The linker-payload conjugate may comprise a compound of formula (I)

Or a salt thereof, wherein,

the line represents the linkage to the nanoparticle through the spacer group; a is a dipeptide selected from Val-Cit, phe-Lys, trp-Lys, asp-Lys, val-Arg and Val-Ala, or A is an alpha amino acid selected from Val-Phe-Gly-Sar, val-Cit-Gly-Sar, val-Lys-Gly-Sar, val-Phe-Gly-Pro, val-Cit-Gly-Pro, val-Lys-Gly-Pro, val-Ala-Gly-Pro, val-Cit-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, val-Lys-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, val-Phe-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, val-Ala-Gly-any natural or unnatural N-alkyl substituted alpha amino acid Tetrapeptides of the group consisting of amino acids, phe-Lys-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, and Trp-Lys-Gly-any natural or unnatural N-alkyl substituted alpha amino acid; the payload is ixabepilone, and the primary amine group of ixabepilone is represented by Z; r is R ¹ and R² Independently at each occurrence hydrogen, substituted or unsubstituted C _1-6 Alkyl or substituted or unsubstituted C _1-6 Alkoxy or hydroxy; r is R ³ and R⁴ Independently at each occurrence hydrogen, halogen, substituted or unsubstituted C _1-6 Alkyl or substituted or unsubstituted C _1-6 An alkoxy group; r is R ⁵ Selected from hydrogen, substituted or unsubstituted C _1-6 An alkyl group; substituted or unsubstituted C _3-7 Cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted C _5-6 A heterocycloalkyl group; with the proviso that when A is a dipeptide, R ⁵ Is H; r is R ^a 、R ^b and R^c Independently at each occurrence hydrogen or substituted or unsubstituted C _1-6 An alkyl group; x is absent, -O-, -CO-, or-NR ^a -; y is not present and is not present, wherein />Wherein the carbonyl group is attached to Z;

provided that when Y isWhen X is absent and n is 1; when Y is +.>When X is absent and n is 0; when Y is +.>When X is absent and n is 0; and/or whenWhen X is-CO-, Y is absent and n is 0; x is X ₁ and X₂ Independently is-CH-or-N-; x is X ₃ is-CH-; x is X ₄ is-CH-; z is-NR ^c -or-O-; n is 0 or 1; q is 1 to 3.

In a preferred aspect of formula (I), A is Val-Lys; r is R ¹ -R ⁵ Each independently is hydrogen; x is absent; y is wherein />Wherein the carbonyl group is attached to Z; n is 1; x is X ₁ 、X ₂ 、X ₃ and X₄ Each independently is-CH-; z is-NR ^c-, wherein R^c Is hydrogen, and wherein N is the nitrogen atom present in the ixabepilone payload.

In the linker-payload conjugate of formula (I), the payload may be irinotecan, which has a functional group bound to the linker, wherein the functional group is an amine (which is a secondary amine when the irinotecan is bound to the linker and once released (or prior to conjugation), i.e. as a separate molecular entity, the amine of the irinotecan is a primary amine).

Exemplary linker-payload conjugates: representative linker-payload conjugates of the present disclosure include, but are not limited to, the following substructures, whereinThe line represents a direct bond to the nanoparticle or an indirect bond to the nanoparticle through a spacer group. Suitable spacer groups include, but are not limited to, PEG spacers or alkylene spacers (e.g., methylene spacers), which may further comprise heteroatoms or cyclic groups (e.g., heterocycloalkylene). In a preferred aspect, the spacer group is a PEG spacer.

Exemplary linker-payload conjugates of formula (I) of the present disclosure include the following substructures:

a linker and a precursor thereof:the linkers of the present disclosure and/or precursors thereof may contain reactive groups at both ends of the molecule. The reactive groups may be selected to allow conjugation to the irinotecan or salt or analog thereof at one end, and also facilitate conjugation to the nanoparticle (e.g., through a spacer group) at the other end. For example, the linker may be attached to the irinotecan through a chemically reactive functional group that is part of the irinotecan, such as a primary amine of the irinotecan (which becomes a secondary amine when conjugated to the linker).

The linker may be linked to the functionalized polyethylene glycol or C by a chemically reactive functional group, such as a primary or secondary amine or carboxyl group, as part of the linker ₅ -C ₆ Alkyl chain conjugation.

Protease cleavable linkers:proteases are involved in all stages of cancer disease from tumor cell growth and survival to angiogenesis and invasion. Thus, it can be used as a selective trigger for the activation of the linker/payload system for the treatment of cancer. The present disclosure relates to cleavable linkers by the action of a protease, thereby releasing a free payload (e.g., isatecan). In the context of prodrug development, lysosomal proteases such as cathepsin B and serine proteases such as cathepsin a or tripeptidylpeptidase I have been extensively studied. Proteolytic enzymes such as caspases are also well known as biological triggers for selectively activating payloads or delivering specific cargo to target cells such as cancer cells.

The linker (or precursor thereof) may comprise a compound of formula (I-A)

Wherein: a is a dipeptide selected from Val-Cit, phe-Lys, trp-Lys, asp-Lys, val-Arg and Val-Ala, or A is a dipeptide selected from Val-Phe-Gly-Sar, val-Cit-Gly-Sar, val-Lys-Gly-Sar, val-Ala-Gly-Sar, val-Phe-Gly-Pro, val-Cit-Gly-Pro, val-Lys-Gly-Pro, val-Ala-Gly-Pro-Pro, val-Cit-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, val-Lys-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, val-Phe-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, val-Ala-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, phe-Lys-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, and Trp-Lys-Gly-any natural or unnatural N-alkyl substituted alpha amino acid; r is R ¹ and R² Independently at each occurrence hydrogen, substituted or unsubstituted C _1-6 Alkyl or substituted or unsubstituted C _1-6 Alkoxy or hydroxy; r is R ³ and R⁴ Independently at each occurrence hydrogen, halogen, substituted or unsubstituted C _1-6 Alkyl or substituted or unsubstituted C _1-6 An alkoxy group; r is R ⁵ Selected from hydrogen, substituted or unsubstituted C _1-6 An alkyl group; substituted or unsubstituted C _3-7 Cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted C _5-6 Heterocycloalkyl, provided that when A is a dipeptide, R ⁵ Is H; r is R ¹ '、R ² '、R ³ '、R ⁴' and R⁵ ' independently at each occurrence hydrogen, substituted or unsubstituted C _1-6 Alkyl or substituted or unsubstituted C _1-6 Cycloalkyl; x is absent, -O-, -CO-, or-NR ^a -; y is absent, wherein /> Is bound to Z ₁ Provided that when Y is +>When X is absent and n is 1; provided that when Y is +>When X is absent and n is 0; provided that when Y is +>When X is absent and n is 0 or 1; provided that when X is-CO-, Y is absent and n is 0; x is X ₃ is-CH-; x is X ₄ is-CH-; z is Z ₁ Is selected from halogen, hydroxy, -OSO ₂ -CH ₃ 、-OSO ₂ CF ₃ Functional groups of 4-nitrophenoxy, -COCl and-COOH; z is Z ₂ Is selected from NH ₂ 、-NHR ^c and-COOH functional groups; or Z is ₂ is-C (O) -T ₁ ；T ₁ Is functionalized polyethylene glycol or C ₅ -C ₆ Alkyl chain having a group selected from azide groups,/->Is a terminal group of (2); r is R ^a 、R ^b and R^c Independently at each occurrence hydrogen or substituted or unsubstituted C _1-6 An alkyl group; n is 0 or 1; and q is 1 to 3.

In certain aspects of formula (I-A), A is Val-Lys; r is R ¹ -R ⁵ Each independently is hydrogen; x is absent; y is wherein />Wherein the carbonyl group is attached to Z; n is 1; x is X ₁ 、X ₂ 、X ₃ and X₄ Each independently is-CH-; z is Z ₁ Is selected from halogen, hydroxy, -OSO ₂ -CH ₃ 、-OSO ₂ CF ₃ Functional groups of 4-nitrophenoxy, -COCl and-COOH; z is Z ₂ Is selected from-NH ₂ 、-NHR ^c and-COOH functional groups or Z ₂ is-C (O) -T ₁, wherein T₁ Such as(I-A).

Pharmaceutical composition

The present disclosure further provides pharmaceutical compositions for treating a disease (e.g., a cancer, such as a cancer associated with a tumor that expresses a folate receptor), wherein the composition comprises an effective amount of an NDC described herein.

In particular aspects of the disclosure, pharmaceutical compositions comprising NDC are useful for treating cancers selected from the group consisting of: ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, breast cancer, lung cancer, mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer, and gastric cancer), pancreatic cancer, bladder cancer, kidney cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, testicular cancer, acute myelogenous leukemia (AML, e.g., pediatric AML), and Chronic Myelogenous Leukemia (CML). Pharmaceutical compositions comprising NDC can also be used to target tumor-associated macrophages, for example, to alter the immune state of a tumor in a subject.

The pharmaceutical compositions of the present disclosure may comprise pharmaceutically acceptable excipients, such as non-toxic carriers, adjuvants, diluents or carriers, which do not negatively affect the pharmacological activity of the NDC formulated therewith. Pharmaceutically acceptable excipients that can be used to prepare the pharmaceutical compositions of the present disclosure are any of those well known in the pharmaceutical formulation arts and can include inert diluents, dispersants and/or granulating agents, surfactants and/or emulsifying agents, disintegrants, binders, preservatives, buffers, lubricants, and/or oils. Pharmaceutically acceptable excipients that may be used to prepare the pharmaceutical compositions of the present disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphate salts), glycine, sorbic acid, potassium sorbate, glyceride mixtures (e.g., mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin.

The pharmaceutical compositions of the present disclosure may be administered orally in the form of suitable pharmaceutical unit dosage forms. The pharmaceutical compositions of the present disclosure may be prepared in a number of forms, including tablets, hard or soft gelatin capsules, aqueous solutions, suspensions, liposomes and other sustained release formulations, such as molded polymer gels.

Suitable modes of administration of NDC or compositions include, but are not limited to, oral, intravenous, rectal, sublingual, mucosal, nasal, ocular, subcutaneous, intramuscular, transdermal, spinal, intrathecal, intra-articular, intra-arterial, subarachnoid, bronchial, lymphatic administration, intratumoral, and other routes suitable for systemic delivery of the active ingredient.

The pharmaceutical compositions herein may be administered by any method known in the art, including but not limited to transdermal (passive by patch, gel, cream, ointment, or iontophoresis); intravenous (bolus infusion); subcutaneous (infusion, depot); transmucosal (buccal and sublingual, e.g., orodispersible tablets, rapidly disintegrating tablets (wafers), films, and effervescent formulations); conjunctiva (eye drops); rectal administration (suppositories, enemas)); or intradermally (bolus, infusion, depot). The composition may be delivered topically.

Oral liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product (for constitution with water or other suitable vehicle before use). Such liquid pharmaceutical compositions may contain conventional additives such as suspensions, emulsifiers, non-aqueous carriers (which may include edible oils) or preservatives.

The pharmaceutical compositions of the present disclosure may also be formulated for parenteral administration (e.g., by injection, such as bolus injection or continuous infusion) and may be presented as unit dosage forms in ampoules, prefilled syringes, infusion containers (e.g., small volume infusion containers), or multi-dose containers, which may contain an added preservative.

The pharmaceutical compositions may take the form of suspensions, solutions or emulsions, for example, in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the pharmaceutical compositions of the present disclosure may be in the form of a powder obtained by sterile isolation of a sterile solid or by lyophilization from solution for formulation with a suitable carrier, e.g., sterile, pyrogen-free water, prior to use.

For topical administration (e.g., to the epidermis), the pharmaceutical composition may be formulated as an ointment, cream or lotion, or as the active ingredient of a transdermal patch. Suitable transdermal delivery systems are disclosed in Fisher et al (U.S. Pat. No. 4,788,603) and R.Bawa et al (U.S. Pat. Nos. 4,931,279;4,668,506; and 4,713,224), which are incorporated herein by reference in their entirety. For example, ointments and creams may be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. Pharmaceutical compositions may also be delivered by ionophoresis, such as, for example, U.S. patent No. 4,140,122;4,383,529; or 4,051,842, which are incorporated herein by reference in their entirety.

Pharmaceutical compositions suitable for topical administration in the oral cavity include unit dosage forms, such as lozenges, comprising the pharmaceutical composition of the present disclosure in a flavored base (e.g., sucrose and acacia or tragacanth); a pill comprising a pharmaceutical composition in an inert matrix (e.g., gelatin and glycerin or sucrose and acacia); mucoadhesive gels and mouthwashes (which comprise a pharmaceutical composition in a suitable liquid carrier).

For topical ocular administration, the pharmaceutical compositions may be administered as drops, gels (s.chrai et al, U.S. patent No. 4,255,415), gums (s.l.lin et al, U.S. patent No. 4,136,177), or by prolonged release ocular inserts (a.s. michaels, U.S. patent No. 3,867,519 and h.m. haddad et al, U.S. patent No. 3,870,791), each of which is incorporated herein by reference in its entirety.

The above-described pharmaceutical compositions may be adapted to provide sustained release of the therapeutic compound(s) employed, when desired, for example, by combination with certain hydrophilic polymer matrices (e.g., comprising natural gels, synthetic polymer gels, or mixtures thereof).

Pharmaceutical compositions suitable for rectal administration wherein the carrier is a solid, most preferably present as a unit dose suppository. Suitable carriers include cocoa butter and other materials commonly used in the art, and suppositories may be conveniently formed by mixing the pharmaceutical composition with the softened or melted carrier, followed by cooling and shaping in a mold.

Pharmaceutical compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to nanoparticles and therapeutic agents a carrier. Such vectors are well known in the art.

For administration by inhalation, the pharmaceutical compositions according to the present disclosure are conveniently delivered from an insufflator, nebulizer or pressurized pack or other convenient means of delivering an aerosol spray. The pressurized packs may contain a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, the pharmaceutical compositions of the present disclosure may take the form of a dry powder composition, for example, a powder mix of the pharmaceutical composition and a suitable powder base (e.g., lactose or starch). The powder composition may be present in unit dosage form, for example in a capsule or cartridge or in a package, for example gelatin or blisters, from which the powder may be administered by means of an inhaler or insufflator.

For intranasal administration, the pharmaceutical compositions of the present disclosure may be administered by liquid spray, for example by a plastic bottle nebulizer. Of which is typically (Isopropylepinephrine inhaler-Wintrop)(isopropyl epinephrine inhaler-linker).

The pharmaceutical compositions of the present disclosure may also contain other adjuvants, such as flavoring agents, coloring agents, antimicrobial agents, or preserving agents.

It will be further appreciated that the amount of pharmaceutical composition suitable for treatment will vary not only with the therapeutic agent selected, but also with the route of administration, the nature of the condition being treated and the age and condition of the patient, and will ultimately be at the discretion of the attendant physician or clinician. For an assessment of these factors, see j.f. brien et al, europ.j. Clin.pharmacol.,14,133 (1978); and Physics' Desk Reference, charles E.Baker, jr., pub, medical Economics Co., oradell, N.J. (41 th edition, 1987), each of which is incorporated herein by Reference in its entirety.

Methods of administration and treatment

NDCs of the present disclosure may be administered to a subject. The subject may be a mammal, preferably a human. Mammals include, but are not limited to, mice, rats, rabbits, apes, cattle, sheep, pigs, dogs, cats, farm animals, sports animals, pets, horses, and primates.

NDC may be administered to a subject by, but is not limited to, the following routes: oral, intravenous, nasal, subcutaneous, topical, intramuscular or transdermal administration. For example, NDCs of the present disclosure may be administered intravenously to a subject.

The methods and compositions of the present disclosure can be used to assist a physician or surgeon in identifying and characterizing a disease region, such as a cancer, including, but not limited to, a cancer that overexpresses FR; distinguishing diseased tissue from normal tissue, for example, detecting tumor margins that are difficult to detect using conventional surgical microscopes, for example, in brain surgery; help guide therapeutic or surgical intervention, for example by determining that the lesion is cancerous and should be removed or non-cancerous and left alone, or by staging the disease through surgery.

The methods and compositions of the present disclosure are useful for, but are not limited to, metastatic disease detection, therapeutic response monitoring, and targeted delivery of payloads, including across the blood brain barrier.

The methods and compositions of the present disclosure may also be used to detect, characterize, and/or determine the localization of a disease, including early stage disease, severity of disease or disease-related disorder, stage of disease, and/or monitor disease. The presence, absence or level of the transmitted signal may be indicative of a disease state.

The methods and compositions of the present disclosure may also be used to monitor and/or direct various therapeutic interventions, such as surgery and catheter-based procedures, as well as to monitor drug therapies, including cell-based therapies. The methods of the present disclosure may also be used for prognosis of a disease or disease condition. Using the methods and compositions of the present disclosure, cell subsets, such as stem cell-like cells ("cancer stem cells") and/or inflammatory/phagocytic cells, located within or at the edges of a disease site can be identified and characterized.

With respect to each of the foregoing, examples of such diseases or conditions that can be detected or monitored (before, during, or after treatment) include cancers (e.g., melanoma, thyroid cancer, colorectal cancer, ovarian cancer, lung cancer, breast cancer, prostate cancer, cervical cancer, skin cancer, brain cancer, gastrointestinal cancer, oral cancer, renal cancer, esophageal cancer, bone cancer), which can be used to identify subjects having increased susceptibility to developing cancer and/or malignancy, i.e., who are prone to developing cancer and/or malignancy, inflammation (e.g., inflammatory conditions induced by the presence of cancerous lesions), cardiovascular diseases (e.g., vascular atherosclerosis and inflammatory conditions, ischemia, stroke, thrombosis), skin diseases (e.g., kaposi's sarcoma, psoriasis), ophthalmic diseases (e.g., macular degeneration, diabetic retinopathy), infectious diseases (e.g., bacterial, viral, fungal, and parasitic infections, including acquired immunodeficiency syndrome (AIDS)), immune diseases (e.g., autoimmune disorders, lymphomas, multiple sclerosis, rheumatoid arthritis, diabetes), central nervous system diseases (e.g., neurodegenerative diseases such as parkinson's disease or alzheimer's disease), genetic diseases, metabolic diseases, environmental diseases (e.g., lead, mercury and radiation poisoning, skin cancer), bone related diseases (e.g., osteoporosis, primary and metastatic bone tumors, osteoarthritis), and neurodegenerative diseases.

Thus, the methods and compositions of the present disclosure can be used, for example, to determine the presence and/or location of tumors and/or coexisting stem cell-like cells ("cancer stem cells"), the presence and/or location of inflammatory cells, including the presence of activated macrophages, for example, in peri-tumor regions, the presence and location of vascular disease, including regions of coronary and peripheral arteries at risk of acute infarction (i.e., vulnerable plaque), aneurysmal distended regions, unstable plaque in carotid arteries, and ischemic regions. The methods and compositions of the present disclosure can also be used to identify and evaluate cell death, injury, apoptosis, necrosis, hypoxia, and angiogenesis (PCT/US 2006/049222).

The methods of the present disclosure comprise administering to a subject in need thereof an effective amount of an NDC described herein. For example, NDC may be administered intravenously to a subject in need thereof. An "effective amount" is an amount of NDC that elicits the desired biological or medicinal response under the conditions of administration, e.g., reduces the sign and/or symptom of the disease or disorder being treated, e.g., reduces tumor size or tumor burden. The actual amount administered can be determined by a skilled clinician based on, for example, the age, weight, sex, general health and tolerance of the subject to the drug, severity of the disease, the dosage form selected, the route of administration, and other factors.

In a specific aspect of the method, the subject has a cancer selected from the group consisting of: ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, breast cancer, lung cancer, mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer, and gastric cancer), pancreatic cancer, bladder cancer, renal cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, testicular cancer, acute myelogenous leukemia (AML, e.g., pediatric AML), and Chronic Myelogenous Leukemia (CML).

The disclosure also includes the use of NDC for treating tumors that express folate receptors. For example, the use of NDC may comprise intravenous administration to a subject in need thereof.

The disclosure also relates to the use of NDCs in subjects with cancer selected from ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, breast cancer, lung cancer, mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer and gastric cancer), pancreatic cancer, bladder cancer, kidney cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, testicular cancer, acute myelogenous leukemia (AML, e.g., pediatric AML), and Chronic Myelogenous Leukemia (CML).

NDCs of the present disclosure are also useful for the preparation of a medicament for treating a tumor expressing a folate receptor, wherein the NDC is administered intravenously to a subject in need thereof, and wherein the subject has a cancer selected from the group consisting of: ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, breast cancer, lung cancer, mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer, and gastric cancer), pancreatic cancer, bladder cancer, renal cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, testicular cancer, acute myelogenous leukemia (AML, e.g., pediatric AML), and Chronic Myelogenous Leukemia (CML).

The compositions and methods disclosed herein can include compositions and methods comprising NDCs as disclosed herein administered in combination with one or more additional anti-cancer agents. In this case, the NDC may be administered before, substantially simultaneously with, or after the additional agent or agents. Suitable additional agents include, for example, chemotherapeutic agents, such as nitrogen mustard, cyclophosphamide, melphalan, chlorambucil, ifosfamide, busulfan, N-nitroso-N-methylurea, carmustine, lomustine, semustine, fotemustine, streptozotocin, dacarbazine, mitozolomide, temozolomide, thiotepa, mitomycin, deaquinone, cisplatin, carboplatin, oxaliplatin, procarbazine, hexamethylmelamine, methotrexate, pemetrexed, fluorouracil (e.g., 5-fluorouracil), capecitabine, cytarabine, gemcitabine, decitabine, azacytidine, and the like fludarabine, nelarabine, cladribine, clofarabine, spinosad, thioguanine, mercaptopurine, vincristine, vinblastine, vinorelbine, vindesine, vinflunine, paclitaxel, docetaxel, irinotecan, topotecan, camptothecin, etoposide, mitoxantrone, teniposide, neomycin, mevalonate, doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, doxorubicin, mitomycin C, actinomycin, bleomycin, bicubicin, cytarabine, and the like. Other anti-cancer agents that may be used with NDCs in the compositions and methods disclosed herein include immune checkpoint inhibitors (e.g., anti-PD 1, anti-PDL 1, anti-CTLA 4 antibodies), hormone receptor antagonists, other chemotherapeutic conjugates (e.g., in the form of antibody-drug conjugates, nanoparticle drug conjugates, etc.), and the like.

The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of embodiments of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

When referring to a numerical value, the term "about" means a value of ±20% or ±10%. Furthermore, when used in conjunction with one or more numbers or numerical ranges, the term "about" is understood to mean all such numbers, including all numbers within the range, and the range is modified by extending the boundaries above and below the listed numbers. The recitation of numerical ranges by endpoints includes all numbers subsumed within that range, for example, including integers and fractions thereof (e.g., the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, and fractions thereof such as 1.5, 2.25, 3.75, 4.1, etc.) and any range within that range.

Throughout the specification and claims, the term "comprising" is used in a non-exclusive sense unless the context requires otherwise. Also, the term "include" and grammatical variants thereof are intended to be non-limiting such that recitation of items in a list is not to the exclusion of other like items that may be substituted for or added to the listed items.

Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently described subject matter belongs.

It is to be understood that, in the detailed description and in the appended claims, abbreviations and terms employed are standard terms in amino acid and peptide chemistry.

Abbreviations used in this disclosure are as follows, unless otherwise indicated:

fmoc: fluorenylmethoxycarbonyl groups

MeOH: methanol

Cit-OH: l-citrulline

DCM: dichloromethane (dichloromethane)

EEDQ: 2-ethoxy-1- (ethoxycarbonyl) -1, 2-dihydroquinoline

THF: tetrahydrofuran (THF)

And (3) NMR: nuclear magnetic resonance

DMSO: dimethyl sulfoxide

LCMS: liquid chromatography-mass spectrometry

TEA: triethylamine

HATU: (1- [ bis (dimethylamino) methylene ] -1H-1,2, 3-triazolo [4,5-b ] pyridinium 3-oxide hexafluorophosphate

DMF: dimethylformamide

DIPEA: n, N-diisopropylethylamine

TMSCN: trimethyl silicon cyanide

RP HPLC reversed phase high pressure liquid chromatography

SFC: supercritical fluid chromatography

CAN: acetonitrile

NMP: n-methylpyrrolidone

r.t: room temperature

TEA: triethylamine

TFA: trifluoroacetic acid

MTBE: methyl tert-butyl ether

EtOAC: acetic acid ethyl ester

PyBOP: (Benzotriazol-1-yl-oxy-tripyrrolidine hexafluorophosphate)

Definition of the definition

As used herein, the term "alkyl" refers to a monovalent aliphatic hydrocarbon radical ("C") that may contain from 1 to 18 carbon atoms, for example from 1 to about 12 carbon atoms or from 1 to about 6 carbon atoms _1-18 Alkyl "). The alkyl group may be a straight chain,Branched, monocyclic or polycyclic moieties, or combinations thereof. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like. Each instance of alkyl may independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkyl") or substituted ("substituted alkyl") with one or more substituents (e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent).

As used herein, the term "alkenyl" refers to a monovalent straight or branched hydrocarbon radical having 2 to 18 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds ("C _2-18 Alkenyl "). Alkenyl groups may have 2 to 8 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. One or more of the carbon-carbon double bonds may be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). Examples of alkenyl groups include vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, butadienyl, pentenyl, pentadienyl, hexenyl, heptenyl, octenyl, xin San alkenyl and the like. Each instance of alkenyl may independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkenyl") or substituted ("substituted alkenyl") with one or more substituents (e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent).

As used herein, the term "alkynyl" refers to a monovalent straight or branched hydrocarbon radical ("C") having 2 to 18 carbon atoms, one or more carbon-carbon triple bonds _2-18 Alkynyl "). Alkynyl groups may have 2 to 8 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. One or more carbon-carbon triple bonds may be internal (e.g., in 2-butynyl) or terminal (e.g., in 1-butynyl). Examples of alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl and the like. Each instance of an alkynyl group may independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkynyl") or substituted ("substituted alkynyl") with one or more substituents (e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent).

As used herein, the term "heteroalkyl" refers to an acyclic stable straight or branched chain comprising at least one carbon atom and at least one heteroatom selected from O, N, P, si and S, or a combination thereof, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N, P, S and Si can be located anywhere in the heteroalkyl group.

Unless otherwise indicated, the terms "alkylene", "alkenylene", "alkynylene" or "heteroalkylene", alone or as part of another substituent, mean a divalent radical derived from an alkyl, alkenyl, alkynyl or heteroalkyl group, respectively. Unless otherwise indicated, the term "alkenylene" by itself or as part of another substituent means a divalent radical derived from an olefin. Alkylene, alkenylene, alkynylene or heteroalkylene may be described as, for example, C _1-6 -a meta-alkylene, C _1-6 -a meta alkenylene group, C _1-6 -meta-alkynylene or C _1-6 -a meta-heteroalkylene, wherein the term "meta" refers to a non-hydrogen atom within the moiety. In the case of heteroalkylene groups, the heteroatom can also occupy one or both of the chain ends (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Furthermore, for alkylene and heteroalkylene linking groups, the direction of writing of the formula of the linking group does not imply the direction of the linking group. For example, -C (O) ₂ R' -can represent-C (O) ₂ R '-and-R' C (O) ₂ -. Each instance of alkylene, alkenylene, alkynylene, or heteroalkylene may independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkylene") or substituted ("substituted heteroalkylene") with one or more substituents.

As used herein, the terms "substituted alkyl", "substituted alkenyl", "substituted alkynyl", "substituted heteroalkyl", "substituted heteroalkenyl", "substituted heteroalkynyl", "substituted cycloalkyl", "substituted heterocyclyl", "substituted aryl" and "substituted heteroaryl" refer to alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl moieties, respectively, that have substituents that replace one or more hydrogen atoms on one or more carbons or heteroatoms of the moiety. Such substituents may include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate (carboxylate), alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthio, alkoxy, phosphate (phospho), phosphonooxy, phosphinyloxy, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylaryl amino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, mercapto, alkylthio, arylthio, thiocarboxylate, sulfate (sulfo), alkylsulfinyl, sulfonic acid, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or aromatic or heteroaromatic moieties. Cycloalkyl groups may be further substituted with, for example, the substituents described above.

As used herein, the term "alkoxy" refers to a group of formula-O-alkyl. The term "alkyloxy" or "alkoxy" includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom. Examples of alkyl oxy groups or alkoxy radicals include, but are not limited to, methoxy, ethoxy, isopropoxy, propoxy, butoxy, and pentoxy. Examples of substituted alkoxy groups include haloalkoxy groups. Alkoxy groups may be substituted with groups such as alkenyl, alkynyl, halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthio carbonyl, alkoxy, phosphate, phosphonooxy, phosphinoyloxy, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, mercapto, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, sulfamoyl, sulfinylamino, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Examples of halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy and trichloromethoxy.

As used herein, the term "aryl" refers to a stable aromatic ring system, which may be monocyclic or polycyclic, wherein all ring atoms are carbon, and which may be substituted or unsubstituted. The aromatic ring system may have, for example, 3 to 7 ring atoms. Examples include phenyl, benzyl, naphthyl, anthracenyl, and the like. Each instance of aryl may independently be optionally substituted, i.e., unsubstituted ("unsubstituted aryl") or substituted by one or more substituents ("substituted aryl").

As used herein, the term "heteroaryl" refers to an aryl group that includes one or more ring heteroatoms. For example, heteroaryl groups may include stable 5-, 6-, or 7-membered monocyclic or 7-, 8-, or 9-membered bicyclic aromatic heterocycles consisting of carbon atoms and one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur. The nitrogen atom may be substituted or unsubstituted (e.g., N or NR ₄, wherein R₄ Is H or other substituents as defined). Examples of heteroaryl groups include pyrrole, furan, indole, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.

As used herein, the terms "cycloalkylene", "heterocyclylene", "arylene" and "heteroarylene", alone or as part of another substituent, mean divalent radicals derived from cycloalkyl, heterocyclyl, aryl and heteroaryl, respectively. Each occurrence of cycloalkylene, heterocyclylene, arylene, or heteroarylene may be independently optionally substituted, i.e., unsubstituted ("unsubstituted arylene") or substituted with one or more substituents ("substituted heteroarylene").

As used herein, the term "cycloalkyl" is intended to include non-aromatic cyclic hydrocarbon rings, such as hydrocarbon rings having 3 to 8 carbon atoms in their ring structure. Cycloalkyl groups may include cyclobutyl, cyclopropyl, cyclopentyl, cyclohexyl, and the like. Cycloalkyl groups may be monocyclic ("monocyclic cycloalkyl") or contain fused, bridged or spiro ring systems, for example bicyclic systems ("bicyclic cycloalkyl"), and may be saturated or may be partially unsaturated. "cycloalkyl" also includes ring systems wherein a cycloalkyl ring as defined above is fused with one or more aryl groups, wherein the point of attachment is on the cycloalkyl ring, and in such cases the carbon number continues to represent the number of carbons in the cycloalkyl ring system. Each instance of cycloalkyl can independently be optionally substituted, i.e., unsubstituted ("unsubstituted cycloalkyl") or substituted by one or more substituents ("substituted cycloalkyl").

As used herein, the term "heterocyclyl" refers to a monovalent cyclic molecular structure (i.e., a heterocyclic radical) comprising atoms of at least two different elements in one or more rings. Additional references are made to: oxford Dictionary of Biochemistry and Molecular Biology, oxford University Press, oxford,1997, as proof that heterocycles are well known terms in the field of organic chemistry.

The term "dipeptide" as used herein refers to a peptide consisting of two amino acid residues, which may be denoted herein as-A ₁ -A ₂ -. For example, the dipeptide used to synthesize the protease cleavable linker-payload conjugate of the present disclosure may be selected from Val-Cit, phe-Lys, trp-Lys, asp-Lys, val-Lys, and Val-Ala.

As used herein, the term "functionalized polyethylene glycol" refers to polyethylene glycol comprising functional groups. For example, the functionalized polyethylene glycol may be prepared with a polymer selected from azides, wherein R is a terminal functionalized polyethylene glycol ¹ '、R ² '、R ³ '、R ⁴' and R⁵ ' independently at each occurrence hydrogen, substituted or unsubstituted C _1-6 Alkyl or substituted or unsubstituted C _1-6 NaphtheneA base. In a preferred aspect, R ¹ '、R ² '、R ³ '、R ⁴' and R⁵ ' hydrogen at each occurrence. In a preferred aspect, R ¹ '、R ² '、R ³ '、R ⁴' and R⁵ ' is methyl at each occurrence.

In some aspects of the present disclosure, the term "functionalized polyethylene glycol" refers to, but is not limited to, the following structure.

As used herein, T ₁ Can be referred to as having a structure selected from the group consisting of azides, Functional polyethylene glycol or C of the terminal group of (C) ₅ -C ₆ Alkyl chain, wherein R ¹ '、R ² '、R ³ '、R ⁴' and R⁵ ' independently at each occurrence hydrogen, substituted or unsubstituted C _1-6 Alkyl or substituted or unsubstituted C _1-6 Cycloalkyl groups. At T ₁ R is a preferred aspect of (2) ¹ '、R ² '、R ³ '、R ⁴' and R⁵ ' hydrogen at each occurrence. At T ₁ R is a preferred aspect of (2) ¹ '、R ² '、R ³ '、R ⁴' and R⁵ ' is methyl at each occurrence. In a preferred aspect T ₁ Is C having azide end groups ₅ -C ₆ Alkyl chains. Polyethylene glycol (PEG) repeat units (-O-CH) ₂ -CH ₂ (-) may be in the range of 5-20 units, preferably 5-15 units, and more preferably 6-12 units.

As used herein, T ₁ Can be referred to as having a structure selected from the group consisting of azides, C of the terminal group of (C) ₅ -C ₆ Alkyl chain, wherein R ¹ '、R ² '、R ³ '、R ⁴' and R⁵ ' independently at each occurrence hydrogen, substituted or unsubstituted C _1-6 Alkyl or substituted or unsubstituted C _1-6 Cycloalkyl groups. In a preferred aspect, R ¹ '、R ² '、R ³ '、R ⁴' and R⁵ ' hydrogen at each occurrence. In a preferred aspect, R ¹ '、R ² '、R ³ '、R ⁴' and R⁵ ' is methyl at each occurrence.

Monofunctional azide-capped PEG and monofunctional azide-capped C ₅ -C ₆ The alkyl chains can be prepared from PEG using known procedures and suitable reagents (e.g., those disclosed in the schemes provided herein).

As used herein, the term "halo" or "halogen" refers to F, cl, br or I.

Aryl or heteroaryl groups described herein may be substituted at one or more ring positions with such substituents as described above, for example, alkyl, alkenyl, alkynyl, halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthio carbonyl, alkoxy, phosphate, phosphonooxy, phosphinoyloxy, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylaryl amino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, mercapto, alkylthio, arylthio, thiocarboxylic acid, sulfate, alkylsulfinyl, sulfonic acid, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

As used herein, the term "hydroxyl" refers to a group of formula-OH.

As used herein, the term "hydroxyl" refers to hydroxyl radicals (.oh).

As used herein, the phrase "optionally substituted" means unsubstituted or substituted. In general, the term "substituted" means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with an allowable substituent (e.g., a substituent that upon substitution results in a stable compound). The term "substituted" may include substitution with all permissible substituents of organic compounds such as any of the substituents described herein which result in the formation of stable compounds. For purposes of this disclosure, a heteroatom such as nitrogen may have a hydrogen substituent and/or any suitable substituent described herein that satisfies the valences of the heteroatom and results in the formation of a stable moiety.

The term "tetrapeptide" as used herein refers to a peptide consisting of four amino acid residues, which may be denoted herein as-a ₁ -A ₂ -A ₃ -A ₄ -. The tetrapeptides useful for synthesizing the disclosed protease cleavable linker-payload conjugates are selected from the group consisting of Val-Phe-Gly-Sar, val-Cit-Gly-Sar, val-Lys-Gly-Sar, val-Ala-Gly-Sar, val-Phe-Gly-Pro, val-Cit-Gly-Pro, val-Lys-Gly-Pro, val-Ala-Gly-Pro, val-Cit-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, val-Lys-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, val-Phe-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, val-Ala-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, phe-Lys-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, and Trp-Lys-Gly-any natural or unnatural N-alkyl substituted alpha amino acid.

Furthermore, one of ordinary skill in the art will appreciate that synthetic methods utilize a variety of protecting groups, as described herein. As used herein, the term "protecting group" refers to a specific functional moiety that is temporarily blocked, e.g., O, S or N, so that the reaction can be selectively performed at another reactive site in the polyfunctional compound. The protecting groups may be introduced and removed at appropriate stages during the synthesis of the compounds using methods known to those of ordinary skill in the art. Protecting groups were applied according to standard organic synthesis methods described in the literature (Theodora W.Greene and Peter G.M.Wuts (2007) Protecting Groups in Organic Synthesis, 4 th edition, john Wiley and Sons, incorporated by reference for protecting groups).

Exemplary protecting groups include, but are not limited to, oxygen, sulfur, nitrogen, and carbon protecting groups. For example, oxygen protecting groups include, but are not limited to, methyl ethers, substituted methyl ethers (e.g., MOM (methoxymethyl ether), MTM (methylthiomethyl ether), BOM (benzyloxymethyl ether), PMBM (heptyloxybenzyloxymethyl ether), optionally substituted ethyl ethers, optionally substituted benzyl ethers, silyl ethers (e.g., TMS (trimethylsilyl ether), TES (triethylsilyl ether), TIPS (triisopropylsilyl ether), TBDMS (tert-butyldimethylsilyl ether), tribenzylsilyl ethers, TBDPS (tert-butyldiphenylsilyl ether), esters (e.g., formate, acetate, benzoate (Bz), trifluoroacetate, dichloroacetate), carbonates, cyclic acetals and ketals).

Throughout this disclosure, nanoparticle-drug-conjugates (NDCs) may sometimes be referred to as CDCs (C' Dot-drug-conjugates), such as FA-CDCs.

The following examples are provided to further illustrate embodiments of the invention but are not intended to limit the scope of the invention. Although they are typical of those that may be used, other procedures, methods, or techniques known to those skilled in the art may alternatively be used.

Examples

The following examples are set forth in order to provide a more thorough understanding of the invention described herein. These examples are provided to illustrate nanoparticle drug conjugates, methods of use, and methods of preparation, and should not be construed in any way as limiting the scope thereof.

The compounds provided herein can be prepared from readily available starting materials using modifications to the specific synthetic schemes set forth below that are well known to those skilled in the art. It will be appreciated that other process conditions may be used, unless otherwise indicated, given typical or preferred process conditions (i.e., reaction temperature, time, molar ratios of reactants, solvents, pressures, etc.). The optimal reaction conditions may vary with the particular reactants or solvents used, but such conditions may be determined by one skilled in the art by routine optimization procedures.

Furthermore, it will be apparent to those skilled in the art that conventional protecting groups may be required to prevent certain functional groups from undergoing undesired reactions. The selection of suitable protecting groups for a particular functional group and suitable conditions for protection and deprotection are well known in the art. For example, greene et al Protecting Groups in Organic Synthesis, second edition, wiley, new York, 1991, and the references cited therein describe a number of protecting groups, and their incorporation and removal.

General procedure

The methods for preparing the compounds discussed herein are set forth in the examples below and summarized herein. Those of skill in the art will recognize that these embodiments may be suitable for preparing linker-payload conjugates, linkers and payloads according to the present disclosure, and pharmaceutically acceptable salts thereof. In the reaction, a reactive functional group such as a hydroxyl group, an amino group, an imino group, a thio group, or a carboxyl group may be protected as needed, for example, to avoid an unnecessary reaction. Conventional protecting groups may also be used according to standard practice and synthetic techniques. The materials required to synthesize the novel linker carrying a payload (e.g., isatecan) are commercially available, the corresponding analogs of which are prepared as disclosed in the examples below.

Reagents were purchased from commercial suppliers (Combi-Blocks/SIGMA-ALDRICH) and used without further purification. All non-aqueous reactions were performed in flame-dried glassware under positive argon pressure. Anhydrous solvents were purchased from commercial suppliers (RANKEM). All amino acids, e.g., cit, val, phe, lys, trp, asp, are naturally occurring amino acids having the S-configuration. In several embodiments, tetrapeptides and unnatural amino acids can also be used. Flash chromatography was performed on 230-400 mesh silica gel using the indicated solvent system. Proton nuclear magnetic resonance spectra were recorded at 400MHZ on Bruker Spectrometer using DMSO as solvent. The peak positions are given in parts per million starting from the low field of tetramethylsilane as internal standard. J values are expressed in hertz. Mass analysis was performed on an (Agilent/Shimadzu) spectrometer using Electrospray (ES) technique. HPLC analysis was performed on an (Agilent/Waters) PDA-UV detector equipped with Gemini C-18 (1000X 4.6 mm;5 u) and all compounds tested were determined to be >95% pure using this method. As can be seen in many protease cleavable linker-payload conjugates, the two peaks are separated at the end of the reaction. Peak-A (or Peak-1) is the desired compound with the stereochemistry shown.

Compounds prepared according to the procedures described herein may be isolated by preparative HPLC methods. Representative HPLC conditions and methods are provided below:

agilent UPLC-MS; column: column-YMC Triart C18 (2.1X33 mm,3 u)

Gradient conditions: flow rate: 1.0ml/min; column temperature: 50 ℃; solvent a: 0.01% HCOOH in water and solvent B: CH (CH) ₃ 0.01% HCOOH in CN; mobile phase: 95% [ 0.01% HCOOH in Water ]]And 5% [ CH ] ₃ 0.01% HCOOH in CN]For 0.50min, then to 1% [ 0.01% HCOOH in water ] within 3.00min]And 99% [ CH ] ₃ 0.01% HCOOH in CN]The conditions were maintained for up to 4.00min, and finally returned to the initial conditions within 4.10min, and maintained for 4.50min (table 1).

Example 1: synthesis of the Isatikang-linker conjugate precursor

The ixabepilone-linker conjugate precursors suitable for use in preparing NDCs of the present disclosure can be synthesized according to the following schemes. Since the ixatikang-linker conjugate precursor comprises a terminal azide group, it is suitable for attachment to nanoparticles functionalized with alkyne moieties (e.g., DBCO) using click chemistry.

(S) -2-amino-N- (4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) -6- ((diphenyl (p) Synthesis of tolyl) methyl) amino) hexanamide (161)

a) TBDPSCl, imidazole, DMF, 0 ℃ to room temperature, 16h; b) (149), HATU, DIPEA, DMF, room temperature, 16h; c) 30% piperidine, DMF, room temperature, 3h

Scheme 2: synthesis of Compound (161).

Synthesis of 4- (((tert-butyldiphenylsilyl) oxy) methyl) aniline (159): imidazole (5.54 g,81.22 mmoL) was added to a solution of (4-aminophenyl) methanol (75) (5.0 g,40.61 mmoL) in DMF (25 mL) at 0deg.C, then tert-butyl (chloro) diphenylsilane (13.39 g,48.73 mmoL) was added and the reaction mixture was stirred at room temperature for 16h. The progress of the reaction was monitored by TLC. After completion of the starting material, the reaction mixture was quenched with water (20 mL) and extracted with EtOAC (2×200 mL). The combined organic layers were treated with anhydrous Na ₂ SO ₄ Dried, concentrated under reduced pressure, and purified by column chromatography over silica gel (230-400 mesh) eluting with 10% EtOAc in petroleum ether to give 4- (((tert-butyldiphenylsilyl) oxy) methyl) aniline (159; 6.6 g) as a gum. LCMS: m/z 362.31[ (M+H) ⁺ ]；R _t :2.58min; purity 93.68%.

Synthesis of (9H-fluoren-9-yl) methyl (S) - (1- ((4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) amino) -6- ((diphenyl (p-tolyl) methyl) amino) -1-oxohexan-2-yl) carbamate (160): diisopropylethylamine (4.18 mL,24 mmol), HATU (6.08 g,16 mmol) and 4- (((tert-butyldiphenylsilyl) oxy) methyl) aniline (159) (2.89) g,8 mmol) was added to a solution of N2- (((9H-fluoren-9-yl) methoxy) carbonyl) -N6- (diphenyl (p-tolyl) methyl) -L-lysine (149) (5.0 g,8 mmol) in DMF (50 mL) and the reaction mixture was stirred at room temperature for 16H. The progress of the reaction was monitored by TLC. After completion of the starting materials, the reaction mixture was quenched with ice water. The precipitated solid was filtered and dried in vacuo to give (9H-fluoren-9-yl) methyl (S) - (1- ((4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) amino) -6- ((diphenyl (p-tolyl) methyl) amino) -1-oxohexan-2-yl) carbamate (160; 5.5 g) as a solid. LCMS: m/z 990.37[ (M+H) ⁺ ]；R _t :2.84min; the purity was 96.79%.

Synthesis of (S) -2-amino-N- (4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) -6- ((diphenyl (p-tolyl) methyl) amino) hexanamide (161):

piperidine (16.5 mL) was added to a solution of (9H-fluoren-9-yl) methyl (S) - (1- ((4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) amino) -6- ((diphenyl (p-tolyl) methyl) amino) -1-oxohexan-2-yl) carbamate (160) (5.5 g,5.68 mmoL) in DMF (38.5 mL) at room temperature and the reaction mixture was stirred at room temperature for 3H. The progress of the reaction was monitored by TLC. After completion of the starting material, the reaction mixture was concentrated under reduced pressure and purified by column chromatography over silica gel (230-400 mesh) eluting with 100% EtOAc to give gummy (S) -2-amino-N- (4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) -6- ((diphenyl (p-tolyl) methyl) amino) hexanamide (160; 3.5 g). LCMS: m/z744.24[ (M-H). ] A ]；R _t :2.20min; the purity was 90.16%.

4- ((32S, 35S) -1-azido-35- (4- ((diphenyl (p-tolyl) methyl) amino) butyl) -32-isopropyl Base-30, 33-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-diazatricetyl-36-amide Synthesis of benzyl (4-nitrophenyl) carbonate (191).

(a) Fmoc-Val-OH, HATU, DIPEA, DMF, room temperature; (b) piperidine, DMF, 0deg.C to room temperature; (c) azido-PEG 9-acid (86), HATU, DIPEA, DMF, room temperature; (d) NH (NH) ₄ F. MeOH, room temperature; (e) P-nitrophenyl chloroformate, pyridine, DCM, 0 ℃ to room temperature

Scheme 3: synthesis of Compound (191).

Synthesis of (9H-fluoren-9-yl) methyl ((S) -1- (((S) -1- ((4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) amino) -6- ((diphenyl (p-tolyl) methyl) amino) -1-oxohexan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) carbamate (187) _： Diisopropylethylamine (1.54 mL,8.83 mmoL), HATU (2.24 g,5.89 mmoL) and (S) -2-amino-N- (4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) -6- ((diphenyl (p-tolyl) methyl) amino) hexanamide (161) (2.19 g,2.94 mmoL) were added to a solution of ((N- (9-fluorenylmethoxycarbonyl) -L-valine (1 g,2.94 mmoL) in DMF (20 mL) at 0 ℃ C.) and the reaction mixture was stirred at room temperature for 3 hours. After completion of the starting material by TLC the reaction mixture was monitored, the precipitated solid was filtered and dried in vacuo to give solid ((9H-fluoren-9-yl) methyl ((S) -1- ((4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) amino) -6-diphenyl (p-tolyl) methyl) oxy) methyl) amino) -6-2-oxo-2-butan-2-yl amino-2- (-7H-fluorenyl methyl) methyl 1- (-7-methyl) oxy) amino-2-butan-2-yl 2-7-yl methyl (5 MH-2-oxo amino) ester ⁺ 1067, retention time 2.42min.

Synthesis of (S) -2- ((S) -2-amino-3-methylbutanamido) -N- (4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) -6- ((diphenyl (p-tolyl) methyl) amino) hexanamide (188): a30% solution of piperidine in DMF (4.5 mL) was added to a solution of (((9H-fluoren-9-yl) methyl ((S) -1- (((S) -1- ((4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) amino) -6- ((diphenyl (p-tolyl) methyl) amino) -1-oxohexan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) carbamate (187) (1.5 g,1.40 mmoL) in DMF (6 mL) at room temperature and the reaction mixture was stirred at room temperature for 2H byTLC monitored the progress of the reaction. After completion of the starting material, the reaction mixture was concentrated under reduced pressure and purified by flash chromatography eluting with 100% EtOAc to provide (S) -2- ((S) -2-amino-3-methylbutanamido) -N- (4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) -6- ((diphenyl (p-tolyl) methyl) amino) hexanamide (188; 1.1 g) as a solid. ¹ H NMR(400MHz，DMSO-d ₆ )：δ10.07(s，1H)，7.64-7.63(d，4H)，7.56-7.54(d，2H)，7.46-7.35(m，9H)，7.27-7.24(m，8H)，7.185-7.11(m，2H)，7.05-7.03(d，2H)，4.71(s，2H)，4.44(d，1H)，3.25-3.16(d，1H)，3.01-3.00(m，1H)，2.21(s，3H)，1.98-1.93(m，2H)，1.68-1.38(m，4H)，1.15(s，10H)，LCMS：MH ⁺ 845, retention time 3.63min.

Synthesis of 1-azido-N- ((S) -1- (((S) -1- ((4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) amino) -6- ((diphenyl (p-tolyl) methyl) amino) -1-oxohexan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) -3,6,9, 12, 15, 18, 21, 24, 27-nonaoxatriacontane-30-amide (189): diisopropylethylamine (0.49 mL,2.83 mmoL), HATU (719.47 mg,1.89 mmoL) and (S) -2- ((S) -2-amino-3-methylbutanoylamido) -N- (4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) -6- ((diphenyl (p-tolyl) methyl) amino) hexanamide (188) (800 mg,0.94 mmoL) were added to a solution of 1-azido-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxatriacontane-30-oic acid (86) (284 mg,0.94 mmoL) in DMF (8 mL) at 0deg.C and the reaction mixture was stirred at room temperature for 6h. The progress of the reaction was monitored by TLC. After completion of the starting material, the reaction mixture was quenched with water (15 mL) and extracted with EtOAC (2 x 30 mL). The combined organic layers were treated with anhydrous Na ₂ SO ₄ Dried, concentrated under reduced pressure, and purified by flash chromatography with 3% MeOH in DCM to give gummy 1-azido-N- ((S) -1- (((S) -1- ((4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) amino) -6- ((diphenyl (p-tolyl) methyl) amino) -1-oxohexan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) -3,6,9, 12, 15, 18, 21, 24, 27-nonaoxatriacontan-30-amide (189; O.60 g). ¹ H NMR(400MHz，DMSO-d ₆ )：δ9.91(s，1H)，8.02-8.00(d，2H)，7.95(s，2H)，7.87-7.85(d，1H)，7.64-7.63(d，4H)，7.57-7.55(d，2H)，7.46-7.32(m，11H)，7.26-7.24(m，8H)，7.15-7.11(t，2H)，7.05-7.03(d，2H)，4.71(s，2H)，4.35-4.33(m，1H)，4.19(s，1H)，3.59-3.36(m，38H)，2.68-2.38(m，6H)，2.22(s，3H)，1.98-1.92(m，2H)，1.47-1.17(m，4H)，1.02(s，9H)，0.85-0.80(m，6H)。LCMS：MH ⁺ 1338, retention time 2.92min.

Synthesis of 1-azido-N- ((S) -1- (((S) -6- ((diphenyl (p-tolyl) methyl) amino) -1- ((4- (hydroxymethyl) phenyl) amino) -1-oxohexan-2-yl) amino) -3-methyl-l-oxobutan-2-yl) -3,6,9, 12, 15, 18, 21, 24, 27-nonaoxatriacontan-30-amide (190): NH at room temperature ₄ F (166 mg,4.48 mmoL) was added to a solution of 1-azido-N- ((S) -1- (((S) -1- ((4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) amino) -6- ((diphenyl (p-tolyl) methyl) amino) -1-oxohexan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) -3,6,9, 12, 15, 18, 21, 24, 27-nonaoxatriacontan-30-amide (189) (600 mg,0.44 mmoL) in methanol (10 mL) and the reaction mixture was stirred at room temperature for 6h. The progress of the reaction was monitored by TLC. After completion of the starting material, the reaction mixture was concentrated under reduced pressure, and the obtained residue was diluted with water (15 mL) and extracted with EtOAC (2×20 mL). The combined organic layers were treated with anhydrous Na ₂ SO ₄ Dried, concentrated under reduced pressure, and purified by flash chromatography with 5% MeOH in DCM to give 1-azido-N- ((S) -1- (((S) -6- ((diphenyl (p-tolyl) methyl) amino) -1- ((4- (hydroxymethyl) phenyl) amino) -1-oxohexan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) -3,6,9, 12, 15, 18, 21, 24, 27-nonaoxatriacontan-30-amide (190; 0.40 g) as a gum. ¹ H NMR(400MHz，DMSO-d ₆ )：δ9.81(s，1H)，7.96-7.94(d，1H)，7.84-7.81(d，1H)，7.53-7.51(d，2H)，7.37-7.35(d，4H)，7.26-7.12(m，9H)，7.096-7.04(d，2H)，5.06-5.04(t，1H)，4.43-4.41(d，2H)，4.35(m，1H)，4.18-4.16(t，1H)，3.60-3.46(m，33H)，3.39-3.36(t，2H)，2.50-2.23(m，2H)，2.23(s，3H)，2.23-1.93(m，2H)，1.48-1.23(m，6H)，0.85-0.80(m，6H)。LCMS：MH ⁺ 1100, retention time 3.72min.

Synthesis of 4- ((32S, 35S) -1-azido-35- (4- ((diphenyl (p-tolyl) methyl) amino) butyl) -32-isopropyl-30, 33-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-ditolyl-tricetyl-36-acylamino) benzyl (4-nitrophenyl) carbonate (191): pyridine (0.14 mL,1.80 mmol) and 4-nitrophenyl chloroformate (14) (145 mg,0.72 mmol) were added to a solution of 1-azido-N- ((S) -1- (((S) -6- ((diphenyl (p-tolyl) methyl) amino) -1- ((4- (hydroxymethyl) phenyl) amino) -1-oxohexan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) -3,6,9, 12, 15, 18, 21, 24, 27-nonaoxatriacontan-30-amide (190) (400 mg,0.36 mmol) in DCM (10 mL) at 0 ℃ and the reaction mixture was stirred at room temperature for 6h. The progress of the reaction was monitored by TLC. After completion of the starting material, the reaction mixture was concentrated under reduced pressure and purified by flash chromatography eluting with 3% meoh in DCM to give gummy 4- ((32 s,35 s) -1-azido-35- (4- ((diphenyl (p-tolyl) methyl) amino) butyl) -32-isopropyl-30, 33-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-diazatricetyl-36-amido) benzyl (4-nitrophenyl) carbonate (191; 0.34 g). LCMS: MH (Mobile band) ⁺ 1265, retention time 1.33min.

4- ((32S, 35S) -35- (4-aminobutyl) -1-azido-32-isopropyl-30, 33-trioxo-3, 6,9, 12 15, 18, 21, 24, 27-nonaoxa-31, 34-diazatricetyl-36-acylamino) benzyl ((1S, 9S) -9-ethyl- 5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2, 3,9, 10, 13, 15-hexahydro 1H, 12H-benzo [ de ]]Pyrano [3', 4′：6，7]indolo [1,2-b ]]Synthesis of quinolin-1-yl) carbamate (202).

(a) (16), NMP, et3N, room temperature; (b) 1% tfa, DCM, 0 ℃ to room temperature;

scheme 4: protease may cleave the synthesis of linker-payload conjugate precursors (202).

4- ((32S, 35S) -l-azido-35- (4- ((diphenyl (p-tolyl) methyl) amino) butyl) -32-isopropyl-30, 33-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-diazatricetyl-36-acylamino) benzyl ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2, 3,9, 10, 13, 15-hexahydro-1H, 12H-benzo [ de ]]Pyrano [3',4':6,7]Indolo [1,2-b ]]Synthesis of quinolin-1-yl) carbamate (201): at 0deg.C, triethylamine (0.09 mL,0.62 mmoL) and (1R, 9R) -1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1, 2,3,9, 12, 15-hexahydro-10H, 13H-benzo [ de ] ]Pyrano [3',4':6,7]Indolo [1,2-b ]]Quinoline-10, 13-dione methanesulfonate (i Sha Tikang methanesulfonate; 16;131mg,0.25 mmoL) was added to a solution of 4- ((32S, 35S) -1-azido-35- (4- ((diphenyl (p-tolyl) methyl) amino) butyl) -32-isopropyl-30, 33-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-diazatricetyl-36-amido) benzyl (4-nitrophenyl) carbonate (191; 311mg,0.25 mmoL) in NMP (2.5 mL) and the mixture was stirred at room temperature for 8h. The progress of the reaction was monitored by LCMS. After completion of the starting material, the reaction mixture was quenched with water (15 mL) and extracted with 10% methanol in chloroform (2×20 mL). The combined organic layers were washed with anhydrous sodium sulfate (Na ₂ SO ₄ ) Dried, and concentrated under reduced pressure. Diethyl ether was added to the crude material, the resulting precipitate was filtered and purified using column chromatography (Combi-Flash) eluting with 5% meoh in DCM to give 4- ((32 s,35 s) -1-azido-35- (4- ((diphenyl (p-tolyl) methyl) amino) butyl) -32-isopropyl-30, 33-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-diazatricetyl-36-amido) benzyl ((1 s,9 s) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2, 3,9, 10, 13, 15-hexahydro-1 h,12 h-benzo [ de ] solid ]Pyrano [3',4':6,7]Indolo [1,2-b ]]Quinolin-1-yl) carbamate (201) (0.3 g). LC (liquid Crystal) deviceMS：MH ⁺ 1561, retention time 2.18min.

4- ((32S, 35S) -35- (4-aminobutyl) -1-azido-32-isopropyl-30, 33-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-diazatricetyl-36-acylamino) benzyl ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2, 3,9, 10, 13, 15-hexahydro-1H, 12H-benzo [ de ]]Pyrano [3',4':6,7]Indolo [1,2-b ]]Quinolin-1-yl]Synthesis of carbamate (202): 1% solution of trifluoroacetic acid (TFA) in DCM was added to 4- ((32S, 35S) -1-azido-35- (4- ((diphenyl (p-tolyl) methyl) amino) butyl) -32-isopropyl-30, 33-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-diazatricetyl-36-acylamino) benzyl ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2, 3,9, 10, 13, 15-hexahydro-1H, 12H-benzo [ de ]]Pyrano [3',4':6,7]Indolo [1,2-b ]]Quinolin-1-yl) carbamate (201; 300mg,0.19 mmoL) in DCM (5 mL) and the reaction mixture was stirred at room temperature for 1h. The progress of the reaction was monitored by LCMS. After completion of the starting material, the reaction mixture was concentrated under reduced pressure and the residue was triturated with diethyl ether and purified by RP-prep-HPLC to give 4- ((32 s,35 s) -35- (4-aminobutyl) -1-azido-32-isopropyl-30, 33-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-diazatricetyl-36-acylamino) benzyl ((1 s,9 s) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2, 3,9, 10, 13, 15-hexahydro-1 h,12 h-benzo [ de ] as a solid ]Pyrano [3',4':6,7]Indolo [1,2-b ]]Quinolin-1-yl]Carbamate (202) (70 mg). ¹ H NMR(400MHz，DMSO-d ₆ )：δ9.96(s，1H)，8.12-8.10(q，2H)，7.89-7.87(d，1H)，7.76-7.61(d，1H)，7.59-7.31(m，7H)，6.51(s，1H)，5.44(s，2H)，5.29(s，3H)，5.09(s，2H)，4.37-4.20(m，1H)，4.18-4.16(t，1H)，3.49-3.44(m，4H)，3.12-2.55(m，39H)，2.40-1.34(m，15H)，0.89-0.82(m，9H)，LCMS：MH ⁺ 1305, retention times 5.33 and 5.47 minutes.

Example 2: synthesis of folic acid conjugate precursors

Folic acid conjugate precursors suitable for use in preparing the folic acid receptor-targeted NDCs disclosed herein can be prepared according to one of the following synthetic schemes. Since the folate conjugate precursor comprises a terminal azide group, it is suitable for linking to nanoparticles functionalized with alkyne moieties (e.g. DBCO) using click chemistry.

(S) -16- (4- (((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) amino) benzoylamino) -1- Synthesis of azido-13-oxo-3, 6, 9-trioxa-12-aza-heptadecane-17-acid (606)

Preparation of compound 600:compound 599 (160 g,512 mmol) was dissolved in TFAA (800 mL) at 25℃and stirred under nitrogen for 5 hours in the dark. The solvent was then removed in vacuo at 50 ℃ to give the crude product. The crude was triturated with MTBE (750 mL) for 60min and then filtered to give compound 600 as a solid (203 g, crude) which was used in the next step without further purification. LC-MS: ¹ H NMR:(400MHz,CDCl ₃ )δ12.74(br s,1H),8.88(s,1H),7.97-8.05(m,2H),7.66-7.74(m,2H),5.26(s,1H)。

preparation of compound 602: -TBTU (238 g,740 mmol) and DIPEA (95.7 g,740 mmol) were added to a solution of compound 601 (225 g,529 mmol) in DMF (2.25L). After stirring at 20℃for 30 minutes, 2- (2- (2- (2-azidoethoxy) ethoxy) ethyl-1-amine (reagent A;121g, 55mmol) was added and the mixture stirred at 50℃for 12 hours. The two reaction mixtures were combined and worked up, the residue was taken up with H ₂ O (3L) was diluted and extracted with ethyl acetate (1500 mL. Times.3). The combined organic layers were washed with brine (800 mL. Times.3), and dried over Na ₂ SO ₄ Dried, filtered and concentrated under reduced pressure, and purified by column chromatography (SiO ₂ Petroleum ether/ethyl acetate=100/1 to 1/1) to give 602 (590 g) as an oil. ¹ H NMR:(400MHz,CDCl ₃ )δ7.76-7.78(m,2H),7.63-7.60(m,2H),7.41-7.27(m,4H),6.43(s,1H),5.70(s,1H),4.42-4.38(m,2H),4.24-4.23(m,2H),3.63-3.36(m,16H),2.28-2.18(m,3H),1.98-1.96(m,1H),1.48(s,9H)。

Preparation of compound 603:n-ethyl ethylamine (1.27 kg,17.4 mol) was added to a solution of compound 602 (435 g,695 mmol) in DCM (4.35L) and the mixture stirred at 25℃for 3 h. The solvent was then removed in vacuo at room temperature and the residue was purified by flash column chromatography (DCM/meoh=100/1 to 1/1) to give compound 603 (245 g) as an oil. ¹ H NMR:(400MHz,CDCl ₃ )δ6.55(s,1H),3.67-3.30(m,17H),2.34-2.30(m,2H),2.10-2.06(m,1H),1.87(s,2H),1.77-1.73(m,1H),1.44(s,9H)。

Preparation of compound 604:TBTU (119 g,372 mmol) and DIEA (160 g,1.24 mol) were added to a solution of compound 600 (101 g,248 mmol) in DMF (900 mL) and the mixture was stirred for 30 min. Compound 603 (100 g,248 mmol) in DMF (100 mL) is then added. The mixture was stirred at 25℃for 12 hours. The two reaction mixtures were combined and concentrated, and the residue was taken up in H ₂ O (2.5L) was diluted and extracted with ethyl acetate (1 L.times.5). The combined organic layers were washed with brine (600 mL. Times.3), and dried over Na ₂ SO ₄ Drying, filtration and concentration under reduced pressure afforded compound 4 (420 g, crude) as a solid, which was used in the next step without further purification.

Preparation of compound 605:will K ₂ CO ₃ (585 g,4.23 mol) was added to compound 604 (420 g,529 mmol) in THF (4.2 mL) and H ₂ A solution in O (500 mL) and the mixture was stirred at 60℃for 0.5 h. The reaction mixture was concentrated under reduced pressure to remove THF, and the residue was taken up with H ₂ O (500 mL) and pH was adjusted to 3 with HCl (m=1), filtered and concentrated under reduced pressure to give compound 605 (260 g, crude) as a solid, which was used without purification.

Preparation of compound 606:trifluoroacetic acid (2.12 kg,18.6 mol) was added in one portion to compound 605 (260 g,373 mmol) at 20℃under nitrogen ₂ Cl ₂ (2.6L) and stirring the mixture at 20℃for 5 hours. Mixing the reactionThe compound was concentrated under reduced pressure and purified by HPLC (column Agela DuraShell C, 250 x 80mM x 10um; mobile phase: [ water (10 mM NH ₄ HCO ₃ )-MeOH]The method comprises the steps of carrying out a first treatment on the surface of the B%:5% -40%,20 min) to give compound 606 as a solid (52.5 g,81.82mmol, yield 21.96%). (m+h) 642.80; IR 2107 (N) ₃ A key).

(S) -38- (4- (((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) amino) benzoylamino) -1-azido-30, 35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31, 34-diaza-tridecanone-39-acid (472)

(a) EDC, HOBT, EA, DCM, room temperature; (b) TFA, DCM, room temperature; (c) PyBOP, DIPEA, DMF, room temperature; (d) piperidine, DMF, 0deg.C to room temperature; (e) (469), pyBOP, DIPEA, DMF, room temperature; (f) TFA, DCM, room temperature; (g) Liquid ammonia, DMF, room temperature, then RP-prep-HPLC.

Scheme 5: synthesis of folic acid conjugate precursors (472).

Synthesis of tert-butyl (1-azido-30-oxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31-aza-tridecan-33-yl) carbamate (465): triethylamine (0.36 mL,2.64 mmoL), EDC (218 mg,1.14 mmoL), HOBT (154 mg,1.14 mmoL) and tert-butyl (2-aminoethyl) carbamate (464) (124 mg,0.881 mmoL) were added to a solution of 1-azido-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxatriacontane-30-acid (86; 450mg,0.881 mmoL) in DCM (20 mL) at 0deg.C and the reaction mixture was stirred at room temperature for 16h. The progress of the reaction was monitored by TLC. After consumption of the starting material, the reaction mixture was extracted with DCM and water and taken up in Na ₂ SO ₄ The organic layer was dried and evaporated under vacuum. The residue was purified by flash chromatography and dried under vacuum to give liquid (1-azido-30-oxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31-aza-tridecan-33-yl) carbamic acid tert-butyl ester (465; 0.45 g). ¹ H NMR(400MHz，DMSO-d ₆ )：7.83(t，1H)，6.75(t，1H)，3.61-3.31(m，38H)，3.02-2.97(t，4H)，2.28(t，2H)，1.37(s，9H)。

Synthesis of N- (2-aminoethyl) -1-azido krypton-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxatriacontane-30-amide (466): a solution of tert-butyl (1-azido-30-oxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31-aza-tridecan-33-yl) carbamate (465) (350 mg, 0.463mmol) in DCM was cooled to 0 ℃ and TFA was added dropwise, and the reaction mixture was stirred at room temperature for 16h. The progress of the reaction was monitored by TLC. After consumption of the starting material, the reaction mixture was concentrated under reduced pressure and azeotroped with DCM (3 times) to give crude product (466), which was purified by flash chromatography eluting with 5% MeOH in DCM to give liquid N- (2-aminoethyl) -1-azido-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxatriacontane-30-amide (466; 0.25 g). ¹ H NMR(400MHz，DMSO-d ₆ )：8.02(t，1H)，7.73(t，2H)，3.71-3.26(m，40H)，2.86(t，2H)，2.35(t，2H)。

Synthesis of (S) -38- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -1-azido-3035-dioxo-3, 6,9, 12, 15, 18,2l,24, 27-nonaoxa-31, 34-diaza-tridecanone-39-oic acid tert-butyl ester (467): diisopropylethylamine (0.174 mL,1.0 mmol), pyBOP (416 mg,0.8 mmol) and N- (2-aminoethyl) -1-azido-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxatriacontan-30-amide (466) (331 mg,0.6 mmol) were added to a solution of (S) -4- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -5- (tert-butoxy) -5-oxopentanoic acid (170 mg,0.4 mmol) in DMF (5 mL) at 0 ℃ and the reaction mixture was stirred at room temperature for 16 hours. The progress of the reaction was monitored by TLC. After consumption of the starting material, the reaction mixture was evaporated under vacuum at low temperature and purified by flash chromatography eluting with 5% meoh in DCM to give (S) -38- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -1-azido-30, 35-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-diaza-trioctadecan-39-oic acid tert-butyl ester (467; 0.35 g) as a liquid. MH (Mobile band) ⁺ 962, retention time 1.81min.

(S) -38-amino-1-azido-3035-dioxo-3, 6,9, 12,1 Synthesis of 5,18, 21, 24, 27-nonaoxa-31, 34-diaza-tridecyl-39-oic acid tert-butyl ester (468): a 30% solution of piperidine in DMF (1 ml) was added to (S) -38- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -1-azido-30, 35-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-diaza-trioxadecan-39-oic acid at room temperatureT-butylA solution of the ester (467; 350mg,3.65 mmoL) in DMF (5 mL) was stirred at room temperature for 3 hours. The progress of the reaction was monitored by TLC. After completion of the starting material, the reaction mixture was concentrated under reduced pressure to give (S) -38-amino-1-azido-30, 35-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-diaza-tridecanone-39-acid tert-butyl ester (468; 250 mg), which was used in the next step without further purification. MH (Mobile band) ⁺ 739, retention time 1.50min.

Synthesis of (S) -38- (4- (N- ((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) -2, 2-trifluoroacetylamino) benzamido-1-azido-30, 35-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-diaza-tridecanone-39-acid tert-butyl ester (470): diisopropylethylamine (0.107 mL, 0.313 mmoL), pyBOP (254 mg,0.49 mmoL) and (S) -38-amino-1-azido-30, 35-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-diaza-tridecen-39-oic acid tert-butyl ester (468) (271 mg, 0.178 mmoL) were added to a solution of 4- (N- ((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) -2, 2-trifluoroacetamido) benzoic acid (469; 100mg,0.245 mmol) in DMF (5 mL) at 0deg.C and the reaction mixture was stirred at room temperature for 16h. To give (S) -38- (4- (N- ((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) -2, 2-trifluoroacetylamino) benzoylamino-1-azido-30, 35-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-diaza-trioxadecan-39-oic acid tert-butyl ester (470; 180 mg) MH ⁺ 1129, retention time 2.61min.

(S) -38- (4- (N- ((2-amino)-synthesis of 4-oxo-3, 4-dihydropteridine 6-yl) methyl) -2, 2-trifluoroacetamido) benzoylamino) -1-azido-30, 35-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-diaza-tridecanone-39-acid (471): trifluoroacetic acid (0.123 ml,1.59 mmol) was added to a solution of (S) -38- (4- (N- ((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) -2, 2-trifluoroacetamido) benzoylamino-1-azido-30, 35-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-diaza-tridecyldecane-39-oic acid tert-butyl ester (470) (180 mg,0.16 mmol) in DCM at room temperature and the reaction mixture was stirred at room temperature for 16h. After completion of the starting material by TLC, the reaction mixture was concentrated under reduced pressure and azeotroped with DCM (3 times) to give crude product (471; 100 mg) which was used in the next step MH without further purification ⁺ 1073, retention time 2.34min.

Synthesis of (S) -38- (4- (((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) amino) benzoylamino) -1-azido-30, 35-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-3 l, 34-diaza-tridecanone-39-acid (472): at 0 ℃, aqueous NH ₃ (dissolved in DMF) (0.01 mL,0.71 mmoL) was added to a solution of (S) -38- (4- (N- ((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) -2, 2-trifluoroacetylamino) benzoylamino) -1-azido-30, 35-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-diaza-tridecylethane-39-acid (471; 80mg, 0.071mmoL) in DMF (3 mL) and the reaction mixture was stirred at room temperature for 6h. After completion of the starting material, the reaction mixture was concentrated under reduced pressure and the residue was purified by RP-prep-HPLC to give (S) -38- (4- (((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) amino) benzoylamino) -1-azido-30, 35-dioxo-3, 6,9, 12, 15, 18, 21, 24, 27-nonaoxa-31, 34-diaza-tridecanone-39-acid (472; 15 mg) as a solid. ¹ H NMR(400MHz，DMSO-d ₆ )：8.62(S，1H)，8.01(d，1H)，7.98(t，1H)，7.64(d，2H)，6.64(d，2H)，4.47(d，2H)，4.21(t，1H)，3.68-3.35(m，38H)，3.07(t，4H)，2.32-2.11(t，6H)，1.86(t，1H).LCMS：MH ⁺ 977, retention time 1.96min.

LCMS method: column-YMC TRIART C (33 x 2.1mm,3 u); (mobile phase: 95% [ 0.1% HCOOH in water)]And 5% [ CH ] ₃ 0.1% HCOOH in CN]For 0.50min, then to 1% [ 0.1% HCOOH in water ] within 3.0min]And 99% [ CH ] ₃ 0.1% HCOOH in CN]The composition was maintained for up to 4.00min and finally returned to the original condition within 4.10min for 4.50 min). Flow rate-1.0 ml/min.

Example 3: synthesis of Nanoparticle Drug Conjugates (NDCs)

Preparation of nanoparticles

Aqueous synthetic methods can be used for the preparation and functionalization of the ultra-small nanoparticles of the present disclosure. For example, methods based on the procedures outlined in WO 2016/179260 A1 and WO 2018/213851 A1 (the contents of which are incorporated herein by reference in their entirety) may be used.

For example, a fluorescent compound such as, but not limited to, cy5, may be functionalized with maleimide groups to provide a maleimide-functionalized fluorescent compound having a net positive charge. This can be conjugated with a thiol-silane, such as (3-mercaptopropyl) trimethoxysilane (MPTMS), to produce a silane-functionalized fluorescent compound, such as Cy 5-silane. Conjugation can be performed overnight (16-24 hours) in Dimethylsulfoxide (DMSO) in a glove box at room temperature (18-25 ℃) under an inert atmosphere.

The next day, the next step of the synthesis may be performed in a suitable chamber, such as a glass flask, vessel or reactor, and may involve stirring deionized water, having a pH of about 8.5-10.5, which may be accomplished using an aqueous ammonium hydroxide solution having a pH of 7.5-8.5. The silica precursor, e.g., a tetraalkyl orthosilicate such as tetramethyl orthosilicate (TMOS), can then be added to the reaction chamber at room temperature with vigorous stirring, followed immediately by the addition of a silane-functionalized fluorescent compound, e.g., cy 5-silane. The reaction can be stirred at room temperature overnight (1-48 hours) to provide a silica core encapsulating fluorescent compounds such as Cy5 dye.

The next day, PEG-silane may be added to the reaction at room temperature with stirring to coat the silica core with PEG molecules, and the reaction may be maintained under stirring for 1-48 hours. This step may be followed by heating for 1-48 hours at a temperature between 75-85 ℃. The reaction may then be cooled to room temperature and purified (e.g., including sterile filtration to remove aggregates and bacteria formed as reaction byproducts, if present). Further functionalization of the nanoparticles can then be performed.

Nanoparticle functionalization

Nanoparticles prepared using the methods disclosed herein can be further functionalized, for example, using the methods outlined in fig. 2 or 3 or in scheme 6 below. For example, (3-cyclopentadienyl propyl) triethoxysilane ("diene-silane") can be used to functionalize the nanoparticle (e.g., C' Dot) with cyclopentadienyl groups, and then DBCO-PEG-maleimide can be reacted with the diene-functionalized nanoparticle to provide a DBCO-functionalized nanoparticle.

Scheme 6. Exemplary method of functionalizing nanoparticles with DBCO.

For example, in a round bottom flask with a stir bar, cy5-C' Dot (which can be prepared using the methods described herein) is diluted to the desired concentration with deionized water, typically between 15 and 30. Mu.M. (3-cyclopentadienyl propyl) triethoxysilane (cyclopentadiene) was first diluted 100x in DMSO and then added to the reaction with stirring to achieve the desired molar ratio of particles to cyclopentadiene. After overnight reaction, 10 x PBS was added to the reaction to reach the final concentration of 1 x PBS. Next, a DBCO-maleimide precursor (e.g., DBCO-PEG 4-maleimide) is dissolved in DMSO and added to the reaction to achieve the desired particle to DBCO molar ratio. After mixing for about 30min to 1 hour, the reaction mixture was heated to 80 ℃ while stirring overnight. The reaction solution was then concentrated and purified using Gel Permeation Chromatography (GPC) to give diene-based DBCO-C' Dot.

Purification can be performed based on the principle of size separation. Aggregates and free small molecules having a molecular weight different from that of the pegylated nanoparticles are separated using a gel permeation chromatography column (GPC) or Tangential Flow Filtration (TFF) system. Two different membranes were used, with a cut-off size of 300kDa and 50kDa, to remove large aggregates and free small molecules, respectively. Both GPC and TFF systems can be used to transfer aqueous media to water, saline, and the like. Purified DBCO-C' Dot in deionized water can be again sterile filtered and subjected to a Quality Control (QC) step and then stored in a refrigerator at 2-8deg.C.

Without wishing to be bound by theory, it is believed that the neutral charge of the cyclopentadienyl group avoids hydrolysis of the amide bond in the bond, which may be accelerated by other types of precursors (e.g., primary amine groups may cause hydrolysis when amine-silane is used instead of diene-silane). Thus, NDCs produced using this method are highly stable (see, e.g., comparisons in fig. 33A-33B). Furthermore, the use of diene-functionalized nanoparticles (e.g., cyclopentadiene-functionalized nanoparticles) in the preparation of NDCS greatly reduces self-condensation of silane during the reaction and improves stability, size uniformity, reaction yield, and purity of the functionalized nanoparticles relative to other methods (e.g., using amine-silane).

Targeting NDC systems

NDCs of the present disclosure comprising nanoparticles (also referred to as C' Dot), targeting ligand (folic acid), and linker-drug conjugates can be prepared as outlined in the flow chart presented in fig. 3 and scheme 7 below. By adjusting the amount of targeting ligand precursor used in the functionalization step, the desired amount of targeting ligand per nanoparticle can be achieved. For example, the nanoparticles of the present disclosure may be functionalized to contain from about 10 to about 20 folic acid moieties, such as about 10, about 11, about 12, about 13, about 14, or about 15 folic acid moieties. Similarly, by adjusting the amount of payload-linker conjugate precursor used in the functionalization step, a desired number of payload portions/nanoparticle can be achieved. For example, the nanoparticles of the present disclosure may be functionalized to contain from about 10 to about 40 irinotecan-linker moieties, e.g., about 20, about 21, about 22, about 23, about 24, or about 25 irinotecan moieties.

Scheme 7. Exemplary method of functionalizing nanoparticles with folic acid moieties and ixabepilone-linker moieties.

Synthesis of folic acid conjugated nanoparticles: DBCO-C 'Dot (referred to as C' Dot in FIG. 3) was diluted to a concentration of 15-45. Mu.M using deionized water. After the temperature of the DBCO-C 'Dot solution is about 18-25 ℃, the Folate Receptor (FR) -targeting ligand precursor, e.g., folic Acid (FA) functionalized with azide (compound 606 prepared in example 2) is dissolved in DMSO (0.021M) and then added to the reaction with stirring at room temperature to give C' Dot functionalized with FA through DBCO groups on the surface. The reaction ratio between DBCO-C' Dot and FA was maintained at 1:5 to 1:30, and the solution is stirred at a temperature of 18-25 ℃ for 16-24 hours. Sterile filtration, purification and QC testing were performed after FR targeting ligand addition to produce FA-C 'Dot (referred to as C' Dot intermediate in fig. 3) and could be stored in a refrigerator at 2-8 ℃. FA-C' Dot comprises a portion of DBCO groups that can be used for further click reactions (e.g., with molecules having azide functional groups). It is understood that a folate-targeting ligand (e.g., folic acid) can be conjugated to the nanoparticle after conjugation to, for example, a payload-linker conjugate.

The volume of the FR-targeting ligand conjugation reaction can be in the range of 5mL to 30L and the concentration of DBCO-C' Dot can be in the range of 15 to 45 μm. The following parameters are given for a typical reaction volume of 600mL and DBCO-C' Dot concentration of 25. Mu.M. The ratio of DBCO-C' Dot to FR-targeting ligand is precisely controlled to achieve the desired amount of FR-targeting ligand/particle and can generally range from 1:5 to 1:30. For a typical ratio of 1:12, folic acid-PEG-azide was dissolved in DMSO to a concentration of 0.021M, and 8.571mL of folic acid-PEG-azide/DMSO solution was added to the reaction. After stirring overnight at room temperature, the reaction mixture was purified to give FA-C 'Dot, or if the purity of FA-C' Dot was not less than 95%, the next conjugation step was directly continued. The conversion of FR targeting ligand is typically higher than 95%.

The number of folic acid groups attached to each FA-C' Dot was characterized by UV-Vis, and a representative UV-Vis absorption spectrum is shown in fig. 4. The number of DBCO groups per C 'Dot can be calculated using the extinction coefficients of the C' Dot and the DBCO groups.

Synthesis of FA-targeted NDC (or FA-CDC) comprising isatecan: FA-C' Dot was diluted to a concentration of 15-45. Mu.M using deionized water. After the FA-C' Dot solution temperature reached about 18-25 ℃, the precursor of the ixabepilone-linker conjugate (e.g., compound 202 described in example 1) dissolved in DMSO (0.04M) was added to the reaction under stirring at room temperature. This step functionalizes the FA-C' Dot with the linker-drug conjugate through available DBCO groups on the surface. The reaction ratio between FA-C' Dot and linker-drug conjugate was maintained at about 1:10 to 1:50 and the solution was stirred for 16-24 hours. After addition of the linker-drug conjugate, sterile filtration and purification were performed. QC testing was performed on FA-CDC (also known as NDC) in deionized water and stored in a refrigerator at 2-8deg.C.

The volume of cleavable irinotecan conjugation reaction can be in the range of 5mL to 30L, and the concentration of FA-C' Dot can be in the range of 15 to 45 μm. The following parameters are given for a typical reaction volume of 600mL and FA-C' Dot concentration of 25. Mu.M. The ratio of FA-C' Dot to cleavable irinotecan is precisely controlled to obtain the desired amount of cleavable irinotecan/particle and can typically be in the range of 1:10 to 1:60. For 1:40, the cleavable irinotecan was dissolved in DMSO to a concentration of 0.04M, and 15mL of the cleavable irinotecan/DMSO solution was added to the reaction. After stirring overnight at room temperature, the reaction mixture was purified to give FA-CDC.

The amount of ixabepilone payload attached to each NDC was characterized by UV-Vis, such as Folic Acid (FA) -functionalized drug-linker conjugated C' Dot (FA-CDC). Representative UV-Vis absorption spectra are shown in FIG. 5. After subtracting the absorbance of folic acid at the same wavelength, the number of irinotecan payloads on each C 'Dot can be calculated using the extinction coefficients of C' Dot and i Sha Tikang at 360 nm.

As described above, the nanoparticles can be functionalized with the folate receptor targeting ligand and the payload-linker conjugate in any order (e.g., the protocol outlined above for functionalizing the nanoparticles with isatecan can be performed prior to the protocol for conjugating folic acid).

Particle size measurement: the average diameter of NDC may be measured by any suitable method, such as, but not limited to, fluorescence Correlation Spectroscopy (FCS) (fig. 6) and Gel Permeation Chromatography (GPC) (fig. 7).

FCS detects fluorescence fluctuations caused by particle diffusion through the focal point on the objective lens. Particle dispersion information is then extracted from the autocorrelation of the signal intensity fluctuations, whereby the average hydrodynamic particle size can be obtained by fitting an autocorrelation curve using a unimodal FCS correlation function. The average hydrodynamic diameter of NDC is about 6nm to about 7nm (fig. 6).

GPC is a molecular sieve chromatography in which the separation mechanism is based on the size of the analyte (here NDC). The elution time of NDC was compared to a series of proteins with different molecular weights. The results show that the elution time of NDC is comparable to the elution time of protein standards with molecular weights between 158kDa and 44kDa, consistent with a particle size average hydrodynamic size of about 6.4nm (fig. 7).

Purity analysis: the purity of NDC was analyzed using reverse phase HPLC (RP-HPLC). RP-HPLC was used in combination with a photodiode array detector using a commercially available Waters Xbridge Peptide BEH C column. RP-HPLC separates molecules of different polarity and is suitable as an analytical method for NDC due to its ultra-small sub-10 nm particle size. Using RP-HPLC, the nanoparticles are sufficiently separated from aggregates and other chemical moieties, such as targeting ligands that are non-covalently bound to the nanoparticles and degradation products. The different chemical moieties were identified based on their elution times and unique UV/Vis spectra. The photodiode array detector collects UV-Vis spectra from 210 to 800nm and measures target impurities at 330 nm. The representative chromatogram of NDC shown in fig. 8 shows that the purity of NDC of the present disclosure is greater than 99.0%.

Example 4: drug release assay

NDCs of the present disclosure include linker-payload conjugates, e.g., protease cleavable linkers, such as cathepsin-B (Cat-B) cleavable linkers. NDC can release a payload (i.e., ixabepilone) upon contact with a protease. Drug release profiles and the stability of linker-drug conjugates on nanoparticles were tested according to the following protocol.

NDC was prepared using the method described in example 3 and then incubated under release conditions required for release kinetics testing. NDC tested in the assay is provided in table 2 below.

Table 2: exemplary NDCs for use in drug release assays.

The number of FA ligands per particle is between 12 and 22; the number of linker-drug conjugates per particle is between 17 and 25. Each payload-linker was conjugated to NDC through a DBCO moiety (prepared according to the protocol outlined in example 3).

The i Sha Tikang showed maximum absorption at a wavelength of about 360nm (fig. 9), and this signal could be used to track the payload in High Performance Liquid Chromatography (HPLC) for release and stability studies. The amount of released drug relative to the amount of unreleased drug was measured by analyzing the area under the curve (AUC) using reverse phase HPLC (fig. 10A and 10B).

The general method comprises the following steps: using a particle size of 4.6mm by 50mm, 5um andwaters Xbridge Peptide BEH C18 column of apertures (part number 186003622). Acetonitrile (VWR HiPerSolv Chromanorm, UHPLC grade) was used as is, without further preparation, a deionized water solution of 0.01% trifluoroacetic acid was prepared by adding 1mL trifluoroacetic acid (HPLC grade, millipore-Sigma) to 999mL of 18.2M omega cm deionized water using the IQ7000 Millipore deionized water systemProduced and filtered through a 0.2 μm filter prior to use. The sealing wash used in the system consisted of 90%18.2 M.OMEGA.cm deionized water and 10% methanol (HPLC grade, VWR). The needle was washed with a mixture of 25% 18.2mΩ·cm deionized water, 25% acetonitrile, 25% methanol, and 25% 2-propanol by volume. The concentration range of the sample preparation is 0.25 to 2 mu M, and the sample injection volume ranges from 60 mu L to 10 mu L respectively. If the detector signal is low, a higher sample concentration may be used. The vial for all injections was a new Waters Total Recovery vial with a threaded cap with a pre-cut PTFE septum (part number 186000385C).

The PDA lamp was turned on and allowed to warm up for at least 30 minutes before any sample injection was initiated. After the PDA lamp was preheated, the system and column were equilibrated with 95%0.01% TFA, 5% acetonitrile in deionized water at a flow rate of 1.0mL/min for at least 10 minutes. Prior to injecting any sample for analysis, two blank injections were made, with an injection volume of 10 μl, containing only 18.2mΩ·cm deionized water. The gradient used starts from 95%0.01% TFA and 5% acetonitrile in deionized water and changes linearly over 8 minutes to 15%0.01% TFA, 85% acetonitrile in deionized water. Acetonitrile composition was increased to 95% over an additional 1 minute and maintained at 95% for an additional 2 minutes to ensure that any strongly retained compounds eluted. The composition of the solvent was then changed back to the initial composition of the gradient within an additional minute and allowed to equilibrate for 3 minutes before another injection was started. A blank injection was performed between the two sample injections to ensure that no residue occurred.

For a typical cathepsin B (Cat-B) protease cleavage study, 300. Mu.L of activation buffer (25mM MES,5mM DTT,pH 5.0) was first added to 2. Mu.L, 0.33. Mu.g/. Mu.L Cat-B (sigma Aldrich), to form 2.2. Mu.g/mL Cat-B. The mixture was maintained at room temperature for 15 minutes before use. After activation, 100 μl of 2 μΜ drug-nanoparticle-conjugate was mixed with 100 μl of activated Cat-B. The mixture was then transferred to 37 ℃. To monitor cleavage kinetics, 10 μl of the mixture was sampled at selected post-incubation time points (e.g., 2, 4, 24 h) and injected into HPLC (TFA/acetonitrile). For analysis of the cut data, the peak areas of all relevant components were determined using the Empower 3 apertrack integration.

FIGS. 11A-11C depict RP-HPLC chromatograms of three representative NDCs (NDC B, NDC C and NDC D), respectively, at different time points after incubation with cathepsin-B. Under specific experimental conditions, half the time of the payload release from each NDC, i.e., T, is analyzed by fitting _1/2 And are shown in fig. 12A-12C, respectively. FIG. 12A depicts T of NDC B _1/2 For 2.9 hours. FIG. 12B depicts T of NDC C _1/2 For 2.6 hours. FIG. 12C depicts T of NDC D _1/2 1.4 hours.

Stability test: To evaluate the drug release profile and stability of linker-drug conjugates under non-cleavage conditions, exemplary NDCs were incubated in Phosphate Buffered Saline (PBS) buffer or animal serum at 37 ℃. NDC was prepared according to example 3 using the ixatikang-linker conjugate precursor 202 from example 1).

For a typical stability test in PBS buffer, 600 μl of PBS mixture was prepared (drug-nanoparticle-conjugate concentration was maintained at 2 μΜ, while the volume percent of PBS was maintained at 50%) and maintained at 37 ℃. To monitor the stability of the linker-drug conjugate attached to the nanoparticle, 10 μl of the mixture was sampled at selected post-incubation time points (e.g., 4, 24, 48, and 72 h) and injected into HPLC (TFA/acetonitrile). For analysis of the cut data, the peak areas of all relevant components were determined using the Empower 3 Apex Track integral.

For typical stability tests in plasma from different species (e.g., mice, rats, dogs, monkeys, and humans), 600 μl of plasma mixture was prepared (drug-nanoparticle-conjugate concentration was maintained at 2 μΜ, while the volume percentage of plasma was maintained at 62.5%) and maintained at 37 ℃. To monitor the stability of the linker-drug conjugate, at selected time points after incubation (e.g., 4, 24, 48, and 72 h), 80 μl of the mixture was first mixed with 80 μl of cold acetonitrile and then centrifuged at 10,000rmp for 30 minutes. After removal of the protein, 60 μl of supernatant was carefully sampled and injected into HPLC. For cathepsin-B cleavable NDC, TFA/acetonitrile was used. For analysis of the cut data, the peak areas of all relevant components were determined using the Empower 3 apertrack integration.

The linker-payload conjugate of NDC (prepared according to example 3 using the ixabepilone-linker conjugate precursor 202 from example 1) is stable in that 5% or less of the ixabepilone is released from the linker drug conjugate under non-cleavage conditions, i.e., after 24 hours when maintained in PBS, human serum, or mouse serum.

Example 5: in vitro flow cytometry cell binding studies

The cell binding activity of NDCs disclosed herein was tested according to the following protocol. The NDC used was prepared according to example 3 using the ixabepilone-linker conjugate precursor 202 of example 1. The amount of folic acid per nanoparticle and the amount of irinotecan per nanoparticle can be adjusted according to the protocol outlined in example 3.

Cell and cell culture: human KB cell lines, SKOV-3 cells and TOV-112 cell lines were purchased from ATCC. I-GROV1 human ovarian cancer cell lines were purchased from EMD Millipore. Unless otherwise indicated, cells were maintained in RPMI1640 medium without folic acid/10% fbs and 1% penicillin/streptomycin. Prior to the study, cancer cells were cultured in folate-free medium (RPMI 1640, thermoFisher, GIBCO) for at least one week. Cell binding studies (n=3) were performed by incubating 5×105 cells (total 500 μl,1 million/mL) in cold PBS (with 1% bsa) together with FA-CDC (concentration: 1 nM) prepared in example 3 at 4 ℃ for 60 min. Thereafter, the cell suspension was subjected to viability kit (LIVE/DEAD) ^TM Fixable Violet Dead Cell Stain Kit, thermo Fisher) for 10-15 minutes. The cells were then centrifuged (2000 rpm,5 min), washed (2-3 times) with cold PBS (containing 1% BSA), and then resuspended in PBS (containing 1% BSA). Samples were analyzed in triplicate on an LSRFortessa flow cytometer (BD Biosciences) (Cy 5 channel, 633nm/647nm, live/dead cell staining, 405 nm). Results were processed using Flowjo and Prism 7 software (GraphPad).

Competitive binding studies were performed using NDC of example 3 (fig. 13). By incubation together in the presence of 1mM free folic acid, active targeting of NDC can be completely blocked.

Competitive binding studies showed that NDC binding capacity was > 40-fold enhanced when compared to free folic acid, indicating that there was a multivalent effect when multiple folic acid ligands were conjugated on each ultra-small C' Dot (fig. 13).

These results demonstrate the benefit of conjugating multiple small tumor-targeting ligands on the surface of the nanoparticle (C' Dot) to enhance targeting ability with multivalent effects. Folate receptor targeting can be blocked by competitive binding of free folate, for example by incubation in the presence of 1mM free folate.

Flow cytometry showed comparable folate receptor targeting efficacy of two NDC formulations with different folate ligand densities in KB cell lines. The linker-ixabetecan conjugate precursors used to prepare NDCs in this study are described in example 1 (compound 202). The blocking group had 1mM free folic acid (FIG. 14).

The results show that when the folate ligand density is increased from 0 to 12 (i.e. 12 folate molecules per nanoparticle), the folate receptor-alpha activity targeting is significantly increased (MFI >300 fold), whereas when the density is further increased to 25 folate molecules per nanoparticle, the observed differences are small.

Flow cytometry showed comparable folate receptor targeting efficacy of three NDCs with different drugs/particles (DPR) (i.e., number of ixabepilone molecules/nanoparticle) in KB cell lines. Blocking groups involved blocking the receptor with 1mM free folic acid. NDCs with different ratios of irinotecan per nanoparticle were prepared using compound 202 described in example 1 and the results of the study are provided in fig. 15. All FA-CDCs contain 12 to 22 folic acid moieties. FA-CDC with high drug-particle ratio (DPR) comprises between 35 and 50 ixabepilone-linker conjugate groups. FA-CDC with moderate DPR contains between 17 and 25 ixabepilone-linker conjugate groups. FA-CDC with low DPR has between 5 and 10 ixabepilone-linker conjugate groups.

These results, together with the almost unchanged FCS dimensional changes of the three NDs, demonstrate the robust surface chemistry and sustained folate receptor targeting ability of the NDCs disclosed herein, which surprisingly are not disturbed by the loading capacity of the altered payload, and demonstrate the significant benefits of the NDCs disclosed herein relative to other drug delivery platforms.

Pre-incubation of NDC in human plasma does not negatively affect folate receptor targeting ability. The study was aimed at testing the possible negative effects of human plasma on NDC, such as the formation of protein crowns (protein cobrona). The formation of protein crowns and their negative impact on the active targeting ability of drug delivery system design is well documented in the literature. The results of this flow cytometry study are depicted in fig. 16, which shows little change in the folate receptor targeting efficacy of NDC at 1nM after 24 hours of pre-incubation with varying amounts of human plasma. NDC was prepared according to the procedure of example 3 using the ixabepilone-payload conjugate precursor of example 1 (compound 202). Blocking groups included blocking with 1mM free folic acid. This study clearly shows that the formation of protein crowns on NDCs (if any) has little negative effect on the in vitro targeting ability of NDCs.

Example 6: in vitro cell viability assay:

NDCs disclosed herein were tested for cytotoxicity in vitro in cancer cells. Prior to the study, cancer cells were cultured in folate-free medium (RPMI 1640, thermoFisher, GIBCO) for at least one week. Cells were plated at 3X10 per well ³ Individual cell densities (90 mL total) were seeded in opaque 96-well plates and allowed to ligate overnight. The next day, cells were treated with NDC (prepared according to example 3) at a concentration ranging from 0-50nM (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50 nM) by adding 10mL of 10 Xstock FA-CDC solution.

Cells are treated for a predetermined exposure time (e.g., 4-6 hours or 7 days, depending on the study design). In the case of short exposure time viability studies, the cancer cells in each well were washed with 100mL PBS and refreshed with 100mL cell culture medium. After washing, the plates were returned to the 37℃incubator for 7 days, and then subjected to viability assay. In the case of the 7 day exposure time viability study, no additional washing step was performed. After 7 days, cell viability was assessed using the CellTiter-Glo2.0 assay (Promega) according to the manufacturer's instructions. Data on viability and proliferation were plotted using Prism7 software (GraphPad). Table 3 provides representative cell viability results for six FA-CDCs with similar folate targeting ligands and drug linker surface densities.

Table 3. Representative cell viability results for NDCs with similar folate targeting ligand and linker-drug conjugate surface densities.

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Example 7: two-dimensional (2D) confocal imaging of NDC in cancer cells

Using two exemplary NDCs, 2D confocal imaging studies were performed to determine targeting to cells with different levels of folate receptor availability. Cells with high folate receptor expression (denoted as++) are KB cells. Cells without FR expression (denoted (-)) are the TOV-112D cell line. FR blocked cells were also used.

KB cells were maintained in folate-free RPMI 1640 medium containing 10% FBS, 1% penicillin/streptomycin. TOV-112D cells were maintained in 1:1 insect cell culture with MCDB 105 medium having a final concentration of 1.5g/L sodium bicarbonate and medium 199 having a final concentration of 2.2g/L sodium bicarbonate, and supplemented with 15% FBS and 1% penicillin/streptomycin. Cells were digested with trypsin and 1.0X10 per well ⁵ Individual cell densities were seeded on 8-well Lab-Tek chamber coverslips and incubated overnight to allow ligation.

NDC was prepared according to example 3 and is shown in table 4 below. NDC D was prepared using the linker-payload conjugate (202) described in example 1.

Table 4. Exemplary NDCs used in 2D confocal imaging assays.

Cells were washed once with RPMI 1640 medium without folic acid prior to incubation with NDC. NDC was added to folate-free RPMI 1640 medium to a final concentration of 50nM. For blocking conditions, folic acid (20 mM stock solution in 0.1M NaOH) was added to a final concentration of 0.1mM and incubated with NDC. Cells were incubated with NDC for 1 or 24 hours at 37 ℃. After incubation, the cells were washed three times. For staining lysosomes LysoTracker Green DND-26 (Thermo Fisher Cat.L7526, ex/em504/511 nM) was added to folate-free RPMI 1640 medium with 10% FBS, 1% P/S (final concentration 100 nM) and incubated for 45 min at 37 ℃. Cells were washed once to remove residual lysosomal tracer (lysotracker) dye. For staining nuclei, hoechst 33342 solution (Thermo Fisher product No. 62249, 20 mM) was diluted 1:4000 in RPMI 1640 medium with 10% FBS, 1% P/S without folic acid and incubated at 37℃for 10 min. Cells were washed once and medium was exchanged for RPMI 1640 medium without phenol red, using a Nikon rotary disc confocal microscope, 60x objective, 405nm, 488nm, 640nm laser line confocal imaging, exposure time for 405 channels of 100ms, exposure time for 488 channels of 500ms, exposure time for 640 channels of 600ms.

Time point of 1 hour to KB% ++) and TOV-112D (-) results of confocal microscopy imaging of NDCs in cell lines showed that, NDC is predominantly present on the cell membrane of KB cells expressing high levels of folate receptor, but is not expressed in blocking conditions or in the folate negative cell line TOV-112D, indicating that NDC specifically binds to folate receptor. After 24 hours, membrane-bound NDC was internalized, with a significant increase in the amount of internalized NDC compared to the 1 hour time point. Internalized NDC localizes in acidic organelles stained by LysoTracker, indicating that transport of NDC occurs through the endolysosomal pathway. The effect of serum on NDC binding capacity was also studied by pre-incubating NDC overnight in medium supplemented with 10% fbs before incubating NDC with cells, and no significant differences were observed (data not shown), indicating that the presence of serum had no effect on NDC binding capacity.

Fig. 17 provides these results for confocal microscopy of NDC B, and fig. 32 provides the results for NDC D. These images demonstrate highly specific active targeting and lysosomal trafficking of NDCs of the present disclosure, indicating that once FA-targeted NDCs bind to cells, they internalize into folate receptor positive cell lines, where the irinotecan payload can be cleaved (e.g., by cathepsin-B) to release free irinotecan in cancer cells.

Example 8: confocal imaging of FA-CDC in 3D tumor spheroid model in KB cells

A 3D tumor spheroid model assay was performed to determine tumor penetration of NDCs disclosed herein. This assay compares an exemplary NDC (prepared according to example 3 using the ixabepilone-linker conjugate precursor 202 of example 1) with FA-targeted nanoparticles without payload (also prepared according to example 3, using only FA precursor and no ixabepilone-payload conjugate precursor); folate Receptor (FR) -targeted ADC; and corresponding FR-targeted antibodies without payload. FR-targeting antibodies were prepared based on the sequence of the published mirvetuximab (provided as huMov19 in U.S. Pat. No. 9,637,547; the contents of which are incorporated herein by reference in their entirety). ADCs were prepared with the same antibodies and conjugated to maytansinoids DM4 (prepared from Syngene International ltd.) via a 4- (pyridin-2-yldisulfonyl) -2-sulfo-butanoic acid (spdb) linker (based on the linker used in us patent No. 9,637,547). By reaction with Cy5-NHS ester, ADC and antibody were each conjugated with Cy5 organic dye and purified by PD-10 column.

Corning ultra-low connection surface 96 Kong Qiuti microplates were used to inoculate KB cells to give KB spheres with a cell density of 10,000 per well. Single cell suspensions were generated from trypsin digested monolayers and diluted to 100,000 cells/mL using RPMI medium (without folic acid). 100mL of the cell suspension was dispensed into each well of the microplate. The plates were maintained in the incubator for 24 hours to allow the cells to form spheres. KB cell spheres were easily observed with a 10X objective microscope.

After overnight incubation in ultra-low 96 well microwell plates 3D KB spheres were formed. NDC (prepared according to example 3 using the ixabepilone-linker conjugate precursor 202 of example 1), folate-targeted nanoparticles ("FA-C' Dot"), FR-targeted ad+ or FR-targeted antibodies without payload were added to the wells (n=3) at a final concentration of 50nM and incubated for 4 hours at 37 ℃. Each treated KB sphere and control sphere were washed three times with PBS and then carefully transferred into a glass bottom 96 well plate (Cellvis) for observation by a Nikon A1R-STED confocal microscope using a 640nm laser line, 20X objective. The Z-stack is obtained by taking two-dimensional images, each of which is separated by 1um in the Z-direction.

FIG. 18 depicts the results of Z-stacked confocal microscopy imaging of KB tumor spheres treated with NDC, FA-C' Dot, FR-targeting ADC and FR-targeting antibody without payload. The results show the permeability and good diffusion of NDC and FA-C' Dot in tumor spheres of > 800mm throughout. In contrast, the labeled antibodies and ADCs accumulated only around the tumor sphere, not inside the tumor sphere. The ability of the NDCs disclosed herein to achieve effective tumor penetration is highly advantageous and shows significant improvements over conventional drug delivery platforms.

Example 9: DFO-FA-CDC ⁸⁹ Zr radiolabelling and in vivo static PET/CT and biodistribution studies

Radiolabeling assays were performed to determine the in vivo biodistribution of the folate receptor targeted NDCs of the present disclosure. NDC for assay was conjugated with the chelator Desferrioxamine (DFO) followed by radionuclide ⁸⁹ Zr) bonds.

For a typical case ⁸⁹ Zr-labelling, about 1nmol of DFO-conjugated NDC was reacted with 1mCi in HEPES buffer (pH 8) at 37 ℃ ⁸⁹ Zr-oxalate (produced and supplied by University of Wisconsin-Madison Cyclotron group) was mixed for 60min; the final labeling pH was maintained at 7-7.5. The labeling yield can be monitored by using radioactive real time thin layer chromatography (ilc). An ethylenediamine tetraacetic acid (EDTA) excitation procedure was then introduced to remove any non-specific binding from the particle surface ⁸⁹ Zr. Then purifying the synthesized marked NDC by using a PD-10 column ⁸⁹ Zr-DFO-FA-CDC). The final radiochemical purity was quantified by using ilc.

For PET/CT imaging, healthy nude mice (n=3) were injected intravenously with 200-300 μci (7.4-11.1 MBq) ⁸⁹ Zr-DFO-FA-CDC. About 5 minutes before PET/CT images were acquired, mice were anesthetized by inhalation of a 2% isoflurane/oxygen mixture and placed on a scanner bed; anesthesia was maintained using a 1% isoflurane/gas mixture. PET/CT imaging was performed in a small animal PET/CT scanner (Inveon microPET/microCT) 1-2, 24, 48 and 72 hours after injection. An energy window of 350-700keV and a coincidence timing window of 6ns are used. The data were classified as 2D histograms by fourier reconstruction and the transverse images were reconstructed by filtered back projection into 128 x 63 (0.72 x 1.3mm 3) matrices. Normalizing the PET/CT imaging data to correct for response non-uniformities, dead time count loss, positron branch ratio, and physical decay to injection time; no attenuation, scattering or partial volume average correction is applied. By using a slave containing ⁸⁹ The count rate in the reconstructed image was converted to active concentration (percent injected dose per gram of tissue,% ID/g) by the systematic calibration factor obtained in imaging of the mouse-sized water equivalent model of Zr (water-equivalent phantom). Target Region (ROI) analysis was performed on PET data using IRW software. Organs from each mouse were collected 72 hours after injection, wet weighed and gamma counted (Automatic Wizard2 gamma-Counter, perkinElmer). ⁸⁹ Uptake of Zr-DFO-FA-CDC is expressed in% ID/g (mean.+ -. SD).

The NDCs of the present disclosure are capable of achieving accurate tumor targeting, deep tumor penetration, and high tumor killing efficacy. NDC is rapidly and effectively cleared from the body, which reduces the likelihood of off-target toxicity and results in improved safety. NDCs disclosed herein, comprising a targeting ligand (folic acid) and a payload (isatecan), can be administered to a subject and through the circulation of the blood stream, target cancers (e.g., tumors), diffuse, penetrate, internalize, and cleave the isatecan payload, thereby killing cancer cells.

In this study, the renal clearance and biodistribution pattern of FA-CDC were tested. As shown in fig. 19A, after intravenous injection, ⁸⁹ Zr-DFO-FA-CDC circulates in the blood stream of healthy nude mice as indicated by the high radioactivity signals from the heart and arteries. A significant radioactivity signal was also seen from the mouse bladder, indicating renal clearance of NDC. After 24 hours, most of the injections were given ⁸⁹ Zr-DFO-FA-CDC was cleared from mice in vivo. Fig. 19B also shows the change in biodistribution pattern at 2 hours and 24 hours after injection. As expected, NDC may circulate in the blood stream through the primary renal clearance pathway while avoiding clearance of the Mononuclear Phagocyte System (MPS) (i.e., liver and spleen).

Example 10: human KB tumor model and in vivo efficacy study

In vivo efficacy assays of NDC were performed using a human KB tumor mouse model. This assay compares the use of the ixabepilone-payload conjugate precursor 202 according to example 3 (example 1; labeled D herein and shown in fig. 20D, NDCs are shown in table 5 below (NDCs a-C and E-F.) NDCs are compared to control and free ixabepilone and NDCs E and F are compared to free ixabepilone and irinotecan (CPT-11).

Table 5 exemplary NDCs used in vivo efficacy studies.

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Unless otherwise indicated, human KB cell lines were purchased from ATCC and maintained in folate-free RPMI 1640 medium/10% FBS and 1% penicillin/streptomycin. Once the KB cells were cultured to a sufficient cell count, cell viability was confirmed by a hemocytometer and trypan blue staining assay. For subcutaneous implantation, KB cells at a density of 2X106 cells/mouse were injected on the left underside of the thigh of each mouse, each injection of 0.1mL matrigel/cell dilution volume. Once the subcutaneous tumor volume reached a reachable 75 to 150mm in the number of mice required for the study ³ To randomly assign mice to each treatment cohort, resulting in comparable tumor volume statistics. Following randomization and study cohort assignment, each dose cohort was treated according to route of administration, dose, and schedule.

In the efficacy study, two dose levels of each NDC B-D (only one dose level of NDC a, E and F) were used. Tumor volume measurements were made every two days during dose therapy using calibrated calipers, followed by twice weekly measurements during recovery in the in vivo phase, and tumor volumes were determined using the formula length (mm) x width (mm) x 0.50. Body weight measurements were taken every two days during dose treatment, followed by two measurements per week during recovery in the in vivo phase. When the study reaches 1000mm ³ At the end of (2), mice were euthanized. Tumors were obtained and tumor sizes were measured. Tumors were surgically excised and stored frozen rapidly at-80 ℃ until future analysis.

Figures 20A-20F depict in vivo tumor growth inhibition studies of six folate receptor targeted NDCs in KB tumor bearing mice (n=7). The tumor growth plot depicted for the in vivo efficacy study shows an explicit response to tumor growth inhibition in mice treated with NDC prepared according to example 3 using the ixabepilone-payload conjugate precursor 202 (from example 1), which is shown in fig. 20D. Similarly, growth inhibition was observed in NDC a (fig. 20A), NDC B (fig. 20B), and NDC (fig. 20C). In contrast, mice treated with NDC E (fig. 20E) and NDC F (fig. 20F) showed no significant inhibition of tumor growth. Figures 20A-20F provide doses of NDC. In mice treated with NDC a-D, a clear response to tumor growth inhibition was observed. Control mice received normal saline following the same Q3DX3 dosing regimen.

Example 11: NDC activity in drug resistant cell lines

An assay was performed using the NDCs disclosed herein to determine their efficacy in drug-resistant cancer cells (specifically,irinotecan antibodies Sex KB cells and irinotecan resistant KB cells) Is a compound of the formula (I). NDC used in this assay was prepared according to example 3 using the ixabepilone-linker conjugate precursor 202 (from example 1).

TOP1 inhibitors resistant to the development of folate receptor alpha positive cancer cells.

The naive human KB cell line was purchased from ATCC and maintained in folic acid free RPMI 1640 medium/10% FBS and 1% penicillin/streptomycin. To develop TOP1 inhibitor resistant KB cells, cells in flasks (50-60% confluence) were repeatedly treated with increasing concentrations of either i Sha Tikang, topotecan, SN-38 or irinotecan for more than 4 months. IC with TOP1 inhibitor initial treatment concentration near KB cells ₉₀ Values. After each treatment, the cells were carefully washed with fresh RPMI 1640 medium and allowed to proliferate for 2-3 days until 50-60% confluence was reached. The next round of TOP1 inhibitor treatment started with a 2-10X higher TOP1 inhibitor concentration.

₅₀ Resistance factors and IC assays.

Naive and TOP1 inhibitor resistant KB cells were cultured in folate-free medium (RPMI 1640, thermoFisher, GIBCO). Cells were plated at 3X10 per well ³ Individual cell densities (90 μl total) were seeded in opaque 96-well plates and allowed to ligate overnight. The following day, cells are treated with selected TOP1 inhibitors (e.g., free irinotecan) or NDCs in the appropriate concentration range. After exposure of the TOP1 inhibitor for the same period of time as the two types of cells, the fines were assessed using the CellTiter-G1o2.0 assay (Promega) according to the manufacturer's instructionsCell viability. Data on viability and proliferation were plotted using Prism7 software (GraphPad). The resistance factor can be calculated by using the following equation:

irinotecan resistant KB cell line and NDC efficacy test

FIG. 21A shows IC of irinotecan in naive and resistant KB cells ₅₀ A curve demonstrating the successful development of 5X irinotecan-resistant KB cells, wherein IC of free irinotecan in irinotecan-resistant KB cells compared to 668nM in naive cells ₅₀ Is 3,618nM. FIG. 21B provides a schematic representation of the cell population in naive KB cells (IC ₅₀ IC of NDC (FA-CDC) (prepared according to example 3 using the ixabepilone-linker conjugate precursor 202 of example 1) in =0.27 nM) and resistant KB cells (ic50=0.26 nM) ₅₀ The curve shows that NDC has uniformly high efficacy in naive KB cells and TOP1 inhibitor resistant KB cells.

Efficacy test of irinotecan resistant KB cell lines and NDC

FIG. 22A shows the IC of I Sha Tikang in naive and resistant KB cells ₅₀ The curve, which shows that irinotecan resistant KB cells > 8X were successfully developed, wherein the IC of I Sha Tikang in conventional KB cells was compared to 4nM in KB cells pretreated with irinotecan for 4X and 16.9nM in KB cells pretreated with irinotecan for 7X ₅₀ 2nM. FIG. 22B shows the IC of NDC (FA-CDC) (prepared according to example 3 using the irinotecan-linker conjugate precursor 202 of example 1) in naive and resistant KB cells (4 x or 7x pretreatment) ₅₀ Curves, wherein IC of FA-CDC ₅₀ 0.27nM, 0.28nM and 0.30nM, respectively. The results indicate that NDC has uniformly high efficacy in naive and resistant KB cells.

Example 12: NDC activity in cancer cells with different folate receptor expression levels

Assays were performed to determine cytotoxicity of exemplary NDC (FA-CDC) with different levels of drug-particle ratio in different FR-a over-expressing cancer cell lines as compared to unconjugated ixabencan. NDC was prepared according to example 3 using the payload-linker conjugate precursor 202 of example 1. The NDC (FA-CDC) tested had drug-particle ratios (DPR) of 43, 20, 8 and 1 (i.e., 43, 20, 8 and 1 irinotecan-linker groups per nanoparticle).

Prior to the study, the number of samples, will have different levels of FR alpha expression (KB (+++)) IGROV-1 (++), SK-OV-3 (++), HCC827 (++) a549 (-) and BT549 (-)) in folate-free medium (RPMI 1640, thermo fisher, gibco) for at least one week. Measurements of 7 day exposure and 6 hour exposure were performed.

Cells were seeded in opaque 96-well plates at a cell density of 3×103 cells per well (90 μl total) and allowed to ligate overnight. The following day, cells were treated with NDCs at different drug-to-particle ratios (DPR) ranging from 0-50nM (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50 nM) by adding 10 μl of 10x stock compound.

For the 6 hour exposure viability study, cells were treated for 6 hours and washed with 100 μl PBS (3×). Then 100. Mu.L of fresh cell culture medium was added to each well and the plates were incubated for a further 7 days at 37℃and then carried out according to the manufacturer's instructionsCytotoxicity assay (Promega). Fig. 23 presents the results of a 7 day exposure assay, which demonstrates that NDC is highly effective in all cell lines despite varying levels of FR expression in the cells.

For a 7 day exposure viability study, cells were incubated with compound for an entire 7 day period, then subjected to Cytotoxicity assay. Half maximal Inhibitory Concentration (IC) was plotted using Prism7 software (GraphPad) ₅₀ ) Is a data of (a) a data of (b). FIG. 23 shows the results of a 6 hour exposure assay demonstrating that NDC is highly potent in all cell lines despite differences in FR expression levels in the cellsIs effective.

Example 13: cytotoxicity of NDC in Pt-resistant tumor cell lines derived from patients

Assays were performed to determine cytotoxicity of exemplary NDCs (prepared according to example 1 using the ixabepilone-linker conjugate precursor compound 202 from example 1) in various patient-derived pt-resistant tumor cell lines, as compared to unconjugated ixabepilone. Cell lines were obtained from ovarian cancer, non-small cell lung cancer (NSCLC), breast cancer (hr+, her2+; and HR-, her2+; and Triple Negative Breast Cancer (TNBC), endometrial cancer and head and neck cancer (H & N). The assay results are provided in fig. 24.

Using KIYA-PREDIT ^TM The assay measures cytotoxic efficacy by KIYATEC. Tumor tissues from platinum-resistant ovarian cancer, endometrial cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, head and neck cancer patients were scored by XenoSTART for fα Immunohistochemistry (ICH) using a Biocare Medical FRa IHC assay kit (product number BRI4006 KAA) according to manufacturer's instructions. Based on IHC scores, a total of 28 PDX models were selected from different indications and provided to KIYATEC for KIYA-PREDIT ^TM And (5) measuring. Briefly, frozen stored PDX tumors were thawed and enzymatically dissociated into single cells and inoculated into 384-well spherical microwell plates (Corning). Flow cytometry was also performed to assess the fα levels between the different PDX models. NDC or control was added at the designed concentration range 24 hours after sphere formation and incubated for 7 days. Thereafter, by3D (Promega) measures cell viability. Data were analyzed in Microsoft Excel and GraphPad Prism.

Example 14: exemplary in vitro and in vivo efficacy of NDC in pediatric acute myelogenous leukemia models

Assays were performed to determine the in vitro and in vivo efficacy of exemplary NDCs (prepared according to the protocol in example 3 using the ixatikang-linker conjugate precursor 202 from example 1) in a folate-receptor alpha-positive pediatric acute myelogenous leukemia model.

In vitro flow cytometry cell binding studies

Cancer cells (IGROV-1 and AML MV4;11 cell lines) were cultured in folate-free medium (RPMI 1640, thermoFisher, GIBCO) for at least one week prior to the study. By mixing 5x10 in cold Phosphate Buffered Saline (PBS) with 1% Bovine Serum Albumin (BSA) ⁵ Individual cells (total 500 μl,1 million/mL) were incubated with exemplary NDC or antibodies conjugated to anti-fra Phycoerythrin (PE) (concentration: 10 nM) for 60 minutes at 4 ℃ for cell binding studies (n=3). Non-targeting CDC and isotype antibody-PE served as negative controls for exemplary NDC and anti-fra antibody-PE, respectively. The cell suspension was then stained with the viability kit (LIVE/DEADTM Fixable Violet Dead Cell Stain Kit, thermo Fisher) for 10-15 minutes. The cells were then centrifuged (2000 rpm, 5 min), washed (2-3 times) with cold PBS (with 1% bsa), and resuspended in PBS (with 1% bsa). Samples were analyzed in triplicate on an LSRFortessa flow cytometer (BD Biosciences) (Cy 5 channel, 633nm/647nm, live/dead cell staining, 405 nm). Results were processed using FlowJo and Prism 7 software (GraphPad).

Flow cytometer histograms for exemplary NDCs and anti-fra antibodies-PE are shown in fig. 25A-25D, as compared to corresponding negative controls (non-targeted NDCs or isotype antibodies-PE). Flow studies demonstrated that exemplary NDCs were specific for IGROV-1 (fαpositive human ovarian cancer) and AML MV4;11 cell line specific fra targeting ability.

In vitro Cytotoxicity assays

Cancer cells (IGROV-1 and AML MV4;11 cell lines) were cultured in folate-free medium (RPMI 1640, thermoFisher, GIBCO) for at least one week prior to the study. Cells were plated at 3X10 per well ³ Individual cell densities (90 μl total) were seeded in opaque 96-well plates and allowed to ligate overnight. The next day, 10. Mu.L of 10 Xstock NDC solution was used with a concentration of 0-100nMThe cells were treated with sexual NDC. For the shorter exposure viability study, cells were treated for 4 hours and washed with 100 μl PBS (3×). mu.L of fresh cell culture medium without NDC was then added to each well and the plates were incubated for an additional 5 days at 37℃and then carried out according to the manufacturer's instructionsCytotoxicity assay (Promega). Half maximal Inhibitory Concentration (IC) was plotted using Prism 7 software (GraphPad) ₅₀ ) Is a data of (a) a data of (b).

FIGS. 26A-26B show FR alpha positive human ovarian cancer and MV4;11 Exemplary NDC in AML cell lines in vitro specific cytotoxic activity. In progress Prior to cytotoxicity assays, cells were treated with the indicated concentrations of exemplary NDCs, incubated for 4 hours at 37 ℃, washed, and returned to the incubator for an additional 5 days.

CBFA2T3-GLIS2 fusion-positive AML cell line-derived xenograft model

The in vivo anti-tumor killing activity of exemplary NDCs was evaluated in a cell line derived xenograft (CDX) model. NOD Scidγ (NSG) mice were fed a folate-free diet for 1 week prior to injection of AML cell lines. Then 1-5 million fusion-positive cell lines (M07 e, WSU-AML) and engineered cells transduced with the Luciferase reporter gene (MV 4;11 FOLR+) were transplanted into NSG mice by tail vein injection. Leukemia load and response to treatment were monitored using non-invasive bioluminescence imaging (front and back of mice) and flow cytometry analysis of mouse peripheral blood drawn through submandibular hemorrhage was performed every two weeks starting with the first week of CDC treatment. Mice were monitored for disease symptoms (including shortness of breath, humpback, sustained weight loss, fatigue, and hindlimb paralysis). Mice from the saline control group (cohort 1) were euthanized (tissues including blood, bone marrow, thymus, liver, lung and spleen were obtained at necropsy and analyzed for the presence of leukemia cells) due to high AML load at day 44 post leukemia injection. Mice from the treatment group (cohorts 2-4) continued to receive weekly bioluminescence imaging and weight monitoring. An illustration of the timeline of mouse preparation, treatment, and imaging is provided in fig. 31.

All mice were randomly grouped and weighed prior to dosing to provide the correct design dose according to table 6 below. Leukemia burden and response to treatment were monitored weekly using non-invasive bioluminescence imaging. Body weight was measured every other day. Mice were sacrificed if their body weight was reduced by more than 20%.

TABLE 6 dose design (n=5/group)

Figure 27 provides changes in body weight of AML mice treated with physiological saline and exemplary NDC at the three dose levels shown in table 6. Saline group (cohort 1) showed weight loss within 20%, mainly due to leukemia load. In the 0.33mg/kg (Q3 Dx 6) dose group (cohort 2), 4 of 5 mice were well tolerated for NDC (loss < 20%), and body weight increased after 6 doses; while the remaining mice lost > 20% of their body weight after the 5 th dose and more after the 6 th dose. In the 0.50mg/kg (Q3 Dx 3) dose group (cohort 3), all 5 mice were well tolerated for NDC (loss < 20%) and gained weight after 3 doses. In the 0.65mg/kg (Q3 Dx 3) group (cohort 4), 2 of 5 mice were well tolerated for NDC (loss < 20%), and body weight increased after 3 doses, while 3 of 5 mice showed a body weight loss > 20% after the 3 rd dose.

Fig. 28 provides in vivo bioluminescence images (BLI) obtained from AML mice treated with physiological saline or exemplary NDC at each dose regimen (i.e., cohorts 1-4 from table 6). Figure 29 provides quantitative in vivo bioluminescence imaging analysis of cohorts 1-4 (i.e., AML mice treated with physiological saline or exemplary NDC at each dose regimen outlined in table 6). In the normal saline group (cohort 1), leukemia load continued to develop, with an average systemic BLI signal > 90-fold increase over 34 days, while rapid and dose-dependent inhibition of leukemia load was achieved in all 3 treatment groups (cohorts 2-4). The 0.5mg/kg (Q3 Dx 3) dose group (cohort 3) showed 11-fold lower leukemia load on day 34 compared to the load on day 1 after leukemia injection. When comparing the 0.33mg/kg (Q3 Dx 6) dose group (cohort 2) with the 0.65mg/kg (Q3 Dx 3) dose group (cohort 4), 0.33mg/kg was better tolerated with slightly better response. Taken together, these data demonstrate that exemplary NDCs successfully inhibit leukemia burden in fra-positive AML mice and exhibit a rapid and dose-dependent response.

Fig. 30 provides a graph illustrating bone marrow aspirate results for cohorts 1-4 (i.e., mice treated with saline or exemplary NDC at each dose group shown in table 6) at day 42 post leukemia injection. Leukemia was detected in the group of mice treated with physiological saline (cohort 1), whereas no detectable leukemia load was observed in any mice from the treatment group (cohorts 2-4).

EXAMPLE 15 stability of diene-derived linker

To determine the stability of NDCs disclosed herein prepared using a diene-based functionalization process, the stability of NDCs prepared using a diene-based functionalization process is compared to the stability of NDCs prepared using an amine-based functionalization process.

NDC was incubated in 0.9% saline, PBS, human plasma (10%) and mouse plasma (10%) for different periods of time at 37 ℃ in an oscillating dry bath. Prior to analysis, plasma proteins in the samples were removed by adding an equal volume of cold acetonitrile for precipitation, and then centrifuged at 10000rpm in an Eppendorf 5425 microcentrifuge. After centrifugation, the clarified supernatant was transferred from the centrifuge tube to a clarified total recovery HPLC vial. The supernatant, free of any visible aggregates, was diluted with an equal volume of deionized water to adjust the sample matrix to match the starting conditions for HPLC separation and avoid sensitivity loss. The purity and impurities of each sample were then quantified by RP-HPLC.

The targeted NDCs produced using the diene-silane precursors exhibited high stability in mice and human plasma and exhibited significant stability improvements using the method described in example 3 relative to the corresponding NDCs produced using the amine-silane precursors (see fig. 33A and 33B). In NDCs prepared using diene-silane precursors, as obtained by UV-Vis spectroscopy of the NDC peak in RP-HPLC chromatograms, more than 95% of the irinotecan drug remained on NDCs in mouse and human plasma for up to 7 days. At the same time, monitoring an independent RP-HPLC assay for free irinotecan indicated that the released irinotecan was below the detection limit of RP-HPLC, i.e., 0.02%, and that no undesired free drug was present, further demonstrating its high plasma stability. The targeted NDCs also exhibit high storage stability in 0.9% saline at 4 ℃. The purity, particle size distribution and hydrodynamic diameter were characterized by RP-HPLC, SEC and FCS, respectively, and maintained unchanged under storage conditions for 6 months. Such high storage stability is another key parameter important for both clinical transformation (clinical translation) and commercial preparation.

EXAMPLE 16 pharmacokinetic and toxicology Studies

The pharmacokinetics and toxicology of exemplary NDCs were evaluated in rat and canine models. NDC used in this study was prepared according to example 3 using the ixabepilone-linker conjugate precursor compound 202 of example 1. As shown in the examples above, this exemplary NDC is highly stable in plasma and elicits anti-tumor efficacy in a variety of cell lines and PDX-derived tumor models in vitro and in vivo.

In repeated dose toxicology and Toxicology (TK) studies performed in Wistar Han rats and Beagle dogs for 15 days, when administered by 1 hour infusion in a QWx regimen, the NDC tolerance of rats was up to 0.87 mg/kg/day and the NDC tolerance of dogs was up to 0.174 mg/kg/day based on conjugated ixabepilone concentrations. The dose-related toxicity observed for both species is limited to bone marrow and gastrointestinal tract. These are the same organs as those observed when the free payload (ixabepilone) was administered, indicating that delivery of ixabepilone conjugated to NDC did not broaden the tissue toxicity profile. At the end of the recovery period of two weeks, a recovery or significant reduction in toxicity was observed. No drug-related liver, kidney, lung or eye toxicity was observed in the repeat dose toxicity study, and no drug-related death.

TK parameters evaluated in GLP studies for 15 days showed similar plasma exposure values for NDC, total irinotecan (conjugated and released) and released irinotecan in males and females. The average circulatory half-life of NDC was about 15 to 20 hours in rats and about 24 to 29 hours in dogs, no accumulation of NDC, total irinotecan or free irinotecan was observed from day 1 to day 15. Based on AUC _0-last The levels of payload released in the rat and dog circulation were lower than about 0.3% and 0.1% of the total payload level, respectively (hours ng/mL). NDC anti-drug antibodies were not induced in either species. In summary, NDC has good non-clinical safety/TK characteristics.

While the invention has been particularly shown and described with reference to a particular preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A nanoparticle-drug conjugate (NDC), comprising:

(a) Silica nanoparticles and polyethylene glycol (PEG) covalently bonded to the surface of the nanoparticles;

(b) A targeting ligand comprising folic acid or a derivative or salt thereof, wherein the targeting ligand is directly or indirectly linked to the nanoparticle through a spacer group; a kind of electronic device with high-pressure air-conditioning system

(c) A linker-payload conjugate, wherein:

(i) The payload is irinotecan;

(ii) The linker-payload conjugate is attached to the nanoparticle through a spacer group;

(iii) The linker is a protease cleavable linker; and is also provided with

(iv) Releasing the irinotecan after cleavage of the linker.

2. The NDC of claim 1, wherein the nanoparticle comprises a silica-based core and a silica shell surrounding at least a portion of the core.

3. The NDC of claim 1 or 2, wherein the NDC has an average diameter of between about 1nm to about 10 nm.

4. The NDC of any one of the preceding claims, wherein the NDC has an average diameter of between about 5nm to about 8 nm.

5. The NDC of any one of the preceding claims, wherein the average nanoparticle to payload ratio is from about 1:1 to about 1:80, such as about 1:60, about 1:40, about 1:30, about 1:28, about 1:26, about 1:25, about 1:24, about 1:23, about 1:22, about 1:21, about 1:20, about 1:19, or about 1:18.

6. The NDC of any one of the preceding claims, wherein the average nanoparticle to targeting ligand ratio is about 1:1 to about 1:50, such as about 1:40, about 1:30, about 1:25, about 1:20, about 1:15, about 1:14, about 1:13, about 1:12, about 1:11, or about 1:10.

7. The NDC of any one of the preceding claims, wherein the average nanoparticle to targeting ligand (e.g., nanoparticle to isafutecan to folic acid) ratio is about 1:20:10, 1:20:11, 1:20:12, 1:20:13, 1:20:14, 1:20:15, 1:21:10, 1:21:11, 1:21:12, 1:21:13, 1:21:14, 1:21:15, 1:22:10, 1:22:11, 1:22:12, 1:22:13, 1:22:14, 1:22:15, 1:23:10, 1:23:11, 1:23:12, 1:23:13, 1:23:14, 1:23:15, 1:24:10, 1:24:11, 1:24:12, 1:24:13, 1:24:14, 1:24:15, 1:25:10, 1:25:25, 1:25:25:25:25:25:25:25).

8. The NDC of any one of the preceding claims, comprising a fluorescent compound covalently encapsulated within the nanoparticle (e.g., within the core of the nanoparticle).

9. The NDC of claim 7, wherein the fluorescent compound is selected from Cy5 and Cy5.5.

10. The NDC of any one of the preceding claims, comprising a structure of formula (NP):

wherein the method comprises the steps of

x is an integer from 0 to 20 (e.g., 4);

a silicon atom is part of the nanoparticle; and is also provided with

Adjacent to the triazole moietyIndicating the point of attachment to the targeting ligand or payload-linker conjugate, either directly or indirectly, e.g., through a linker or spacer group, e.g., a PEG moiety.

11. NDC according to any one of the preceding claims, comprising a structure of formula (S-1):

wherein the method comprises the steps of

The payload is irinotecan;

the linker is a protease cleavable linker (e.g., a cathepsin-B cleavable linker), and

the silicon atom is part of the nanoparticle.

12. NDC according to any one of the preceding claims, comprising a structure of formula (S-2):

wherein the method comprises the steps of

The targeting ligand is folic acid or a derivative or salt thereof; and is also provided with

The silicon atom is part of the nanoparticle.

13. The NDC of any one of the preceding claims, wherein the linker-payload conjugate comprises a structure of formula (I):

or a salt thereof,

wherein,

represents a bond to the nanoparticle through a spacer group;

a is Val-Lys;

the payload is a residue of ixabepilone, wherein Z is the nitrogen atom of ixabepilone;

R ¹ 、R ² 、R ³ 、R ⁴ and R is ⁵ Independently at each occurrence hydrogen;

x is absent;

y isWherein->Is bonded with Z;

X ₁ 、X ₂ 、X ₃ and X ₄ Each independently is-CH-;

z is-NR ^c –；

R ^c Is hydrogen; and is also provided with

n is 1.

14. The NDC of any one of the preceding claims, wherein the linker-payload conjugate is a protease cleavable linker capable of undergoing hydrolysis at the C-terminus upon protease binding, thereby releasing the payload from the nanoparticle.

15. The NDC of claim 14, wherein the protease comprises a serine protease or a cysteine protease.

16. A nanoparticle-drug conjugate (NDC), comprising:

(a) Nanoparticles comprising a silica-based core and a silica shell surrounding at least a portion of the core; polyethylene glycol (PEG) covalently bonded to the surface of the nanoparticle, and Cy5 dye covalently encapsulated within the core of the nanoparticle;

(b) A targeting ligand that binds to the folate receptor,

wherein the targeting ligand is folic acid, and wherein the targeting ligand is indirectly linked to the nanoparticle through a spacer group;

(c) A linker-payload conjugate, wherein the linker-payload conjugate is indirectly attached to the nanoparticle through a spacer group; wherein the linker-payload conjugate comprisesAnd is also provided with

Wherein the NDC has an average diameter between about 1nm and about 6 nm.

17. A nanoparticle-drug conjugate (NDC) comprising

(a) Nanoparticles comprising a silica-based core and a silica shell surrounding at least a portion of the core; and polyethylene glycol (PEG) covalently bonded to the surface of the nanoparticle;

(b) A structure of formula (NP-3):

(NP-3) wherein x is 4 and y is 9, and wherein the silicon atom is part of the nanoparticle; a kind of electronic device with high-pressure air-conditioning system

(c) Structure of formula (NP-2)

Wherein x is 4 and y is 3, and a silicon atom is part of the nanoparticle.

18. NDC according to claim 17, comprising a fluorescent dye (e.g. Cy 5) covalently encapsulated within the core of the nanoparticle.

19. A method of treating a folate receptor expressing cancer (e.g., a folate receptor expressing tumor), comprising administering to a subject in need thereof an effective amount of an NDC of any one of the preceding claims.

20. The method of claim 19, wherein the NDC is administered intravenously to the subject in need thereof.

21. The method of claim 19 or 20, wherein the subject has a cancer selected from ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, breast cancer, lung cancer, mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer, and gastric cancer), pancreatic cancer, bladder cancer, renal cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, testicular cancer, acute myelogenous leukemia (AML, e.g., pediatric AML), and Chronic Myelogenous Leukemia (CML).

22. The NDC of any one of claims 1-18, for use in treating a cancer that expresses a folate receptor (e.g., a tumor that expresses a folate receptor).

23. The use of claim 22, wherein the NDC is administered intravenously to a subject in need thereof.

24. The use of claim 22 or 23, wherein the cancer is selected from ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, breast cancer, lung cancer, mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer and gastric cancer), pancreatic cancer, bladder cancer, renal cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, testicular cancer, acute myelogenous leukemia (AML, e.g., pediatric AML), and Chronic Myelogenous Leukemia (CML).

25. NDC according to any one of claims 1-18, for use in the manufacture of a medicament for the treatment of a folate receptor expressing cancer (e.g. a folate receptor expressing tumour).

26. The NDC of claim 25, wherein the cancer is selected from ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, breast cancer, lung cancer, mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer, and gastric cancer), pancreatic cancer, bladder cancer, renal cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, testicular cancer, acute myelogenous leukemia (AML, e.g., pediatric AML), and Chronic Myelogenous Leukemia (CML).

27. A pharmaceutical composition comprising the NDC of any one of claims 1-18 and a pharmaceutically acceptable excipient.

28. The pharmaceutical composition of claim 27, wherein the pharmaceutical composition is suitable for intravenous administration.

29. The pharmaceutical composition of claim 27 or 28, wherein the composition is in unit dosage form, e.g., in an ampoule, prefilled syringe, infusion container, or multi-dose container.