Tumor-targeted statin derivative, pharmaceutical formulation and application
阅读说明：本技术 肿瘤靶向他汀类衍生物、药物配方及应用 (Tumor-targeted statin derivative, pharmaceutical formulation and application ) 是由 钟维国 朱家仪 张驿 程宇平 余波 廖凯 于 2019-08-16 设计创作，主要内容包括：本发明涉及肿瘤靶向他汀类衍生物(THSD),以及其在治疗、尤其是癌症治疗中的用途。这些THSD中包括下列三个部分：一个他汀部分,其中包括一个二羟基庚酸单元(DHHA),通过连接保持其开链结构；一个七甲川菁羰花青染料(HMCD)部分；一个将他汀部分中的DHHA连接到染料部分上的连接体。本发明还涉及向患者提供一种或多种治疗有效剂量的ELSD的给药方法,以及在协调给药机制中,ELSD和ALSD的共同给药方法。THSD及其用途的主要优点包括但不限于：能够提高疗效和剂量反应、减少他汀类药物的副作用。(The present invention relates to tumour-targeted statin derivatives (THSD), and their use in therapy, in particular in the treatment of cancer. These THSDs include the following three components: a statin moiety comprising a dihydroxyheptanoic acid unit (DHHA) which retains its open chain structure by ligation; a heptamethine cyanine carbocyanine dye (HMCD) moiety; a linker that links the DHHA in the statin moiety to the dye moiety. The invention also relates to methods of administering one or more therapeutically effective doses of ELSD to a patient, and methods of co-administering ELSD and ALSD in a coordinated dosing regime. Major advantages of THSD and its use include, but are not limited to: can improve curative effect and dosage response, and reduce side effect of statin drugs.)
1. An ester-linked statin derivative is characterized in that the molecular formula is represented by formula FIa:
wherein X is a halogen residue;
wherein R is1Is a residue and can be selected from the following functional groups: c1-C25Alkyl radical, C5-C25Aryl radical, C5-C25Indolyl, C5-C25Thienyl, C5-C25Phenyl radical, C5-C25Naphthyl, C1-C25Aralkyl radical, C1-C25Alkylsulfo group, C1-C25Alkyl carboxyl, C1-C25Alkylamino radical, C1-C25Omega-alkylamines, C1-C25Omega-alkynyl, one having (-CH)2-CH2-O-)2-20The PEGyl polyethylene chain of (A) has (-CH)2-CH2-O-)2-20PEGyl carboxylate of (I), with (-CH)2-CH2-O-)2-20Of omega-PEGyl amine, omega-acyl-NH, omega-acyl-lysyl, omega-acyl-triazole, with (-CH)2-CH2-O-)2-20Of omega-PEGyl carboxy-NH with (-CH)2-CH2-O-)2-20omega-PEGyl carboxy-lysyl, and a peptide having (-CH)2-CH2-O-)2-20omega-PEGyl carboxy-triazole;
wherein R is2And R3Are residues, which can be independently selected from the following functional groups: hydrogen atom, C1-C20Alkyl, sulfonic acid group, C1-C20Alkyl carboxyl, C1-C20Alkylamino radical, C1-C20Aryl, -SO3H、-PO3H、-OH、-NH2And a halogen residue;
wherein R is2And R3Can be attached to the following positions in a carbon ring: 3. 3 ', 4', 5 ', 6 and 6';
wherein A is-The functional group is a negatively charged anion compatible with the pharmaceutical agent;
wherein there is an ester linker LELinked to a statin residue R by an ester bondSThe above step (1);
wherein L isECan be selected fromE1And LE2Is selected from the functional groups of (1), wherein LE1Is- (CH)2)n-O-, wherein LE2Is- (CH)2)n–CO–NH–(CH2)m-O-, wherein n has a value of 4-9 and m has a value of 1-4;
wherein R isSIs a residue of a statin or statin derivative;
wherein R isSWith a dihydroxyheptanoic acid unit (DHHA) linked to the R constituting the remainder of the statin residueS*The above step (1);
wherein R isSThrough its open chain structure (-CO-CH) in DHHA2-COH-CH2-CHOH-RS*) Is connected to LEAbove.
2. The ester-linked statin derivative according to claim 1, wherein the statin is selected from the group consisting of: simvastatin, mevastatin, lovastatin, pravastatin, atorvastatin, fluvastatin, rosuvastatin, cerivastatin, pitavastatin, and other derivatives attached to the operably linked region in the statin.
3. The ester-linked statin derivative according to claim 1, wherein the statin is simvastatin, and the ester linker L isEIs LE2The molecular formula is shown as formula FIb:
wherein A is-、X、R1、R2、R3And LE2Both n and m in the linker are exactly as defined in FIa.
4. The ester-linked statin derivative of claim 1, wherein X is a chlorine atom.
5. The ester-linked statin derivative of claim 1, wherein R is1Is- (CH)2)n–SO3 -Alkylsulfo residues, where n in R1 may be 2, 3, 4,5, 6, 7 and 8.
6. The ester-linked statin derivative of claim 1, wherein R is1Is- (CH)2)4–SO3 -An alkyl sulfo residue.
7. The ester-linked statin derivative of claim 1, wherein R is2And R3Are all hydrogen atoms.
8. A pharmaceutical formulation comprising an ELSD and one or more pharmaceutical excipients, wherein the ELSD is selected from the group consisting of:
(i) the molecular formula of the ester bond connection statin derivative is shown as formula FIa:
wherein X is a halogen residue;
wherein R is1Is a residue and can be selected from the following functional groups: c1-C25Alkyl radical, C5-C25Aryl radical, C5-C25Indolyl, C5-C25Thienyl, C5-C25Phenyl radical, C5-C25Naphthyl, C1-C25Aralkyl radical, C1-C25Alkylsulfo group, C1-C25Alkyl carboxyl, C1-C25Alkylamino radical, C1-C25Omega-alkylamines, C1-C25Omega-alkynyl, one having (-CH)2-CH2-O-)2-20The PEGyl polyethylene chain of (A) has (-CH)2-CH2-O-)2-20PEGyl carboxylate of (I), with (-CH)2-CH2-O-)2-20omega-PEGyl ofAmines,. omega. -acyl-NH,. omega. -acyl-lysyl,. omega. -acyl-triazole, with (-CH)2-CH2-O-)2-20Of omega-PEGyl carboxy-NH with (-CH)2-CH2-O-)2-20omega-PEGyl carboxy-lysyl, and a peptide having (-CH)2-CH2-O-)2-20omega-PEGyl carboxy-triazole;
wherein R is2And R3Are residues, which can be independently selected from the following functional groups: hydrogen atom, C1-C20Alkyl, sulfonic acid group, C1-C20Alkyl carboxyl, C1-C20Alkylamino radical, C1-C20Aryl, -SO3H、-PO3H、-OH、-NH2And a halogen residue;
wherein R is2And R3Can be attached to the following positions in a carbon ring: 3. 3 ', 4', 5 ', 6 and 6';
wherein A is-The functional group is a negatively charged anion compatible with the pharmaceutical agent;
wherein there is an ester linker LELinked to a statin residue R by an ester bondSThe above step (1);
wherein L isECan be selected fromE1And LE2Is selected from the functional groups of (1), wherein LE1Is- (CH)2)n-O-, wherein LE2Is- (CH)2)n–CO–NH–(CH2)m-O-, wherein n has a value of 4-9 and m has a value of 1-4;
wherein R isSIs a residue of a statin or statin derivative;
wherein R isSWith a dihydroxyheptanoic acid unit (DHHA) linked to the R constituting the remainder of the statin residueS*The above step (1);
wherein R isSThrough its open chain structure (-CO-CH) in DHHA2-COH-CH2-CHOH-RS*) Is connected to LEAbove.
(ii) (ii) the ester bond-linked statin derivative described in (i), wherein the statin constituting the RS is selected from the following: simvastatin, mevastatin, lovastatin, pravastatin, atorvastatin, fluvastatin, rosuvastatin, cerivastatin, pitavastatin, and other derivatives attached to the operably linked region in the statin.
(iii) (ii) the ester bond in (i) is connected with statin derivatives, wherein the statin is simvastatin, and the ester linker LE is LE2(- (CH2) n-CO-NH- (CH2) m-O-) and the molecular formula is represented by formula FIb:
wherein A is-、X、R1、R2、R3And LE2N and m in the linker are all exactly as defined in FIa;
(iv) (ii) the ester linkage described in (i) to a statin derivative, wherein X is a chlorine atom;
(v) (iii) the ester bond described in (ii) is linked to a statin derivative, wherein X is a chlorine atom;
(vi) (iv) ester linkage of the statin derivative described in (iii), wherein X is a chlorine atom;
(vii) (ii) ester linkage to a statin derivative as described in (i) wherein R1 is- (CH2) n-SO 3-alkylsulfo residue, wherein n in R1 may be 2, 3, 4,5, 6, 7 and 8;
(viii) (iii) ester linkage to a statin derivative as described in (ii), wherein R1 is- (CH2) n-SO 3-alkylsulfo residue, wherein n in R1 may be 2, 3, 4,5, 6, 7 and 8;
(ix) (iv) ester linkage to a statin derivative as described in (iii), wherein R1 is- (CH2) n-SO 3-alkylsulfo residue, wherein n in R1 may be 2, 3, 4,5, 6, 7 and 8;
(x) (ii) ester linkage to a statin derivative as described in (i) wherein R1 is- (CH2) 4-SO 3-alkylsulfo residue;
(xi) (iii) ester linkage to a statin derivative as described in (ii), wherein R1 is- (CH2) 4-SO 3-alkylsulfo residue;
(xii) (iii) ester linkage to a statin derivative as described in (iii), wherein R1 is a- (CH2) 4-SO 3-alkylsulfo residue.
9. The pharmaceutical formulation of claim 8, comprising one or more ELSDs and further comprising an ALSD of the formula FIIa
Wherein X is a halogen residue;
wherein R is1Is a residue and can be selected from the following functional groups: c1-C25Alkyl radical, C5-C25Aryl radical, C5-C25Indolyl, C5-C25Thienyl, C5-C25Phenyl radical, C5-C25Naphthyl, C1-C25Aralkyl radical, C1-C25Alkylsulfo group, C1-C25Alkyl carboxyl, C1-C25Alkylamino radical, C1-C25Omega-alkylamines, C1-C25Omega-alkynyl, one having (-CH)2-CH2-O-)2-20The PEGyl polyethylene chain of (A) has (-CH)2-CH2-O-)2-20PEGyl carboxylate of (I), with (-CH)2-CH2-O-)2-20Of omega-PEGyl amine, omega-acyl-NH, omega-acyl-lysyl, omega-acyl-triazole, with (-CH)2-CH2-O-)2-20Of omega-PEGyl carboxy-NH with (-CH)2-CH2-O-)2-20omega-PEGyl carboxy-lysyl, and a peptide having (-CH)2-CH2-O-)2-20omega-PEGyl carboxy-triazole;
wherein R is2And R3Are residues, which can be independently selected from the following functional groups: hydrogen atom, C1-C20Alkyl, sulfonic acid group, C1-C20Alkyl carboxyl, C1-C20Alkylamino radical, C1-C20Aryl, -SO3H、-PO3H、-OH、-NH2And a halogen residue;
wherein R is2And R3Can be attached to the following positions in a carbon ring: 3. 3 ', 4', 5 ', 6 and 6';
wherein A is-The functional group is a negatively charged anion compatible with the pharmaceutical agent;
with an amide linker LALinked to statin residue R by amide bondSThe above step (1);
wherein L isACan be selected fromA1And LA2Is selected from the functional groups of (1), wherein LA1Is- (CH)2)n-NH-, wherein LA2Is- (CH)2)n–CO–NH–(CH2)m-NH-, wherein n has a value of 4-9 and m has a value of 1-4;
wherein R isSIs a residue of a statin or statin derivative;
wherein R isSWith a dihydroxyheptanoic acid unit (DHHA) linked to the R constituting the remainder of the statin residueS*The above step (1);
wherein R isSThrough its open chain structure (-CO-CH) in DHHA2-COH-CH2-CHOH-RS*) Is connected to LAAbove.
10. A method of treating cancer comprising administering to a patient a sufficient amount of one or more ELSDs sufficient to inhibit growth of, or to induce apoptosis of, cancer cells or precancerous cells, optionally in combination with one or more ALSDs
One or more of the ELSDs may be selected from the following:
(i) the molecular formula of the ester bond connection statin derivative is shown as formula FIa:
wherein X is a halogen residue;
wherein R is1Is a residue selected from the following functional groups：C1-C25Alkyl radical, C5-C25Aryl radical, C5-C25Indolyl, C5-C25Thienyl, C5-C25Phenyl radical, C5-C25Naphthyl, C1-C25Aralkyl radical, C1-C25Alkylsulfo group, C1-C25Alkyl carboxyl, C1-C25Alkylamino radical, C1-C25Omega-alkylamines, C1-C25Omega-alkynyl, one having (-CH)2-CH2-O-)2-20The PEGyl polyethylene chain of (A) has (-CH)2-CH2-O-)2-20PEGyl carboxylate of (I), with (-CH)2-CH2-O-)2-20Of omega-PEGyl amine, omega-acyl-NH, omega-acyl-lysyl, omega-acyl-triazole, with (-CH)2-CH2-O-)2-20Of omega-PEGyl carboxy-NH with (-CH)2-CH2-O-)2-20omega-PEGyl carboxy-lysyl, and a peptide having (-CH)2-CH2-O-)2-20omega-PEGyl carboxy-triazole;
wherein R is2And R3Are residues, which can be independently selected from the following functional groups: hydrogen atom, C1-C20Alkyl, sulfonic acid group, C1-C20Alkyl carboxyl, C1-C20Alkylamino radical, C1-C20Aryl, -SO3H、-PO3H、-OH、-NH2And a halogen residue;
wherein R is2And R3Can be attached to the following positions in a carbon ring: 3. 3 ', 4', 5 ', 6 and 6';
wherein A is-The functional group is a negatively charged anion compatible with the pharmaceutical agent;
wherein there is an ester linker LELinked to a statin residue R by an ester bondSThe above step (1);
wherein L isECan be selected fromE1And LE2Is selected from the functional groups of (1), wherein LE1Is- (CH)2)n-O-, wherein LE2Is- (CH)2)n–CO–NH–(CH2)m-O-, wherein n has a value of 4-9 and m has a value of 1-4;
wherein R isSIs a residue of a statin or statin derivative, via its open chain structure (-CO-CH) in DHHA2-COH-CH2-CHOH-RS*) Is connected to LEAbove, wherein R isS*Is the remainder of the statin residues.
(ii) (ii) the ester bond-linked statin derivative described in (i), wherein the statin constituting the RS is selected from the following: simvastatin, mevastatin, lovastatin, pravastatin, atorvastatin, fluvastatin, rosuvastatin, cerivastatin, pitavastatin, and other derivatives attached to the operably linked region in the statin;
(iii) (ii) the ester bond in (i) is connected with statin derivatives, wherein the statin is simvastatin, and the ester linker LE is LE2(- (CH2) n-CO-NH- (CH2) m-O-) and the molecular formula is shown in the following chart:
wherein A is-、X、R1、R2、R3And LE2Both n and m in the linker are exactly as defined in section (i) above at FIa;
(iv) (ii) the ester linkage described in (i) to a statin derivative, wherein X is a chlorine atom;
(v) (iii) the ester bond described in (ii) is linked to a statin derivative, wherein X is a chlorine atom;
(vi) (iv) ester linkage of the statin derivative described in (iii), wherein X is a chlorine atom;
(vii) (ii) ester linkage to a statin derivative as described in (i) wherein R1 is- (CH2) n-SO 3-alkylsulfo residue, wherein n in R1 may be 2, 3, 4,5, 6, 7 and 8;
(viii) (iii) ester linkage to a statin derivative as described in (ii), wherein R1 is- (CH2) n-SO 3-alkylsulfo residue, wherein n in R1 may be 2, 3, 4,5, 6, 7 and 8;
(ix) (iv) ester linkage to a statin derivative as described in (iii), wherein R1 is- (CH2) n-SO 3-alkylsulfo residue, wherein n in R1 may be 2, 3, 4,5, 6, 7 and 8;
(x) (ii) ester linkage to a statin derivative as described in (i) wherein R1 is- (CH2) 4-SO 3-alkylsulfo residue;
(xi) (iii) ester linkage to a statin derivative as described in (ii), wherein R1 is- (CH2) 4-SO 3-alkylsulfo residue;
(xii) (iv) ester linkage to a statin derivative as described in (iii), wherein R1 is- (CH2) 4-SO 3-alkylsulfo residue;
and wherein the optional one or more ALSDs may be selected from:
(xiii) ALSD with molecular formula shown as FIIa
With an amide linker LALinked to statin residue R by amide bondSThe above step (1);
wherein L isACan be selected fromA1And LA2Is selected from the functional groups of (1), wherein LA1Is- (CH)2)n-NH-, wherein LA2Is- (CH)2)n–CO–NH–(CH2)m-NH-, wherein n has a value of 4-9 and m has a value of 1-4;
wherein R isSIs a residue of a statin or statin derivative;
wherein R isSWith a dihydroxyheptanoic acid unit (DHHA) linked to the R constituting the remainder of the statin residueS*The above step (1);
wherein R isSThrough its open chain structure (-CO-CH) in DHHA2-COH-CH2-CHOH-RS*) Is connected to LAAbove;
wherein A is-、X、R1、R2、R3And LA2Both n and m in the linker are as in FIa in paragraph (i) aboveThe specifications are identical;
(xiv) The molecular formula of the ALSD is shown as FIIb, wherein the statin is simvastatin, and the connector is LA 2(- (CH2) n-CO-NH- (CH2) m-NH-):
wherein A is-、X、R1、R2、R3And LA2Both n and m in the linker are exactly as defined in section (i) above at FIa.
11. The method of claim 10, wherein the one or more ELSDs can be co-administered with the one or more ALSDs to the patient according to a coordinated dosing mechanism, using one or more combination dosage forms; wherein each dosage form comprises one or more ELSDs, one or more ALSDs, and one or more pharmaceutical excipients.
12. The method of claim 10, wherein the one or more ELSDs are co-administered with the one or more ALSDs to the patient according to a coordinated dosing mechanism;
wherein the administration mechanism comprises: at least one hour or more prior to administering one or more maintenance doses to the patient, requiring that a loading dose be administered to the patient first;
wherein the loading dose may comprise a single dosage form, may comprise one or more ELSDs and one or more pharmaceutical excipients, but may not comprise one or more ALSDs;
the following dosage forms should be included in one or more of the maintenance doses: contains one or more ALSDs and one or more pharmaceutical excipients, but optionally contains or does not contain ELSDs.
13. The method of claim 10, wherein the one or more ELSDs are co-administered to the patient with one or more co-drugs according to a coordinated dosing mechanism; wherein the one or more auxiliary drugs can be selected from the following substances: a hormone antagonist, an antiandrogen drug, abiraterone, enzalutamide, a chemotherapeutic drug, docetaxel, paclitaxel and cabazitaxel.
14. The method of claim 10, wherein a loading dose of one or more combination dosage forms, or one or more individual dosage forms, and one or more maintenance doses are co-administered to the patient according to a coordinated dosing mechanism;
wherein the combination dosage form comprises one or more ELSDs, one or more ALSDs, and one or more pharmaceutical excipients;
the following dosage forms should be included in one or more of the maintenance doses: contains one or more ALSDs and one or more pharmaceutical excipients, but optionally contains or does not contain ELSDs;
the following dosage forms should be included in one or more of the maintenance doses: contains one or more ALSDs and one or more pharmaceutical excipients, but optionally contains or does not contain ELSDs;
wherein co-administration means co-administration with one or more auxiliary drugs in the subsequent administration according to a coordinated administration mechanism:
wherein the one or more auxiliary drugs can be selected from the following substances: a hormone antagonist, an antiandrogen drug, abiraterone, enzalutamide, a chemotherapeutic drug, docetaxel, paclitaxel and cabazitaxel.
15. The method of claim 10, comprising administering one or more ELSDs to a patient having one or more genetic variations found in one or more gene codes for one or more tyrosine kinase receptors in cancer cells, precancerous lesions, tissues, tumors, or metastases thereof; wherein the tyrosine kinase receptors comprise: epidermal Growth Factor Receptor (EGFR), anaplastic lymphoma kinase receptor (ALF), and proto-oncogene tyrosine protein kinase receptor (ROS or ROS 1).
16. The method of claim 10, comprising administering one or more ELSDs to a patient whose cancer cells, precancerous lesions, tissues, tumors, or metastases have developed resistance to one or more Tyrosine Kinase Inhibitors (TKIs); these include patients who have undergone one or more TKI treatments prior to ELSD administration and for whom the effect of the previous TKI treatment has been inadequate.
17. The method of claim 15, wherein the TKI is selected from the group consisting of: epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI), ALK tyrosine kinase receptor inhibitors (ALK-TKI), and proto-oncogene tyrosine protein kinase ROS (ROS-TKI).
18. The method of claim 15, wherein the EGFR-TKI is selected from the group consisting of: gefitinib, erlotinib, bugatinib, dacomitinib, lapatinib, vandetanib, afatinib, axitinib (AZD9291), CO-1686, HM61713, nanozatinib (EGF816), Omtinib, PF-06747775, YH5448, Avertinib (AC0010), Rociletinib and cetuximab.
19. The method of claim 10, wherein the patient is a patient identified as having a drug-resistant cancer by drug exposure or genetic testing, wherein the drug-resistant cancer comprises: prostate cancer, pancreatic cancer, lung cancer, non-small cell lung cancer (NSCLC; which may include squamous cell carcinoma, adenocarcinoma (mucinous cystadenocarcinoma), large cell lung cancer, rod cell carcinoma, sarcoid-like carcinoma, carcinoid, salivary adenoid carcinoma, acanthoma, papillary adenocarcinoma, giant cell carcinoma), SCLC (small cell lung cancer), generalized small cell carcinoma, non-cancerous tumors of the lung (sarcoma, lymphoma, immature teratoma, and melanoma), kidney cancer, lymphoma, colorectal cancer, skin cancer, liver and breast cancer, lung squamous cell carcinoma, anal cancer, glioblastoma, epithelial cell tumors of the head and neck, and other cancers.
20. The method of claim 10, wherein the patient is a patient determined to have drug-resistant lung cancer by drug exposure or genetic testing, wherein the drug-resistant cancer comprises: small Cell Carcinoma Lung Cancer (SCCLC), non-small cell carcinoma lung cancer (NSCLC), comprehensive small cell carcinoma, squamous cell carcinoma, adenocarcinoma (AC, mucinous cystadenocarcinoma, mcalcl), large cell lung cancer, rod cell carcinoma, sarcoid, carcinoid, salivary adenoid, adenosquamous, papillary adenocarcinoma, giant cell carcinoma, non-cancerous tumors of the lung, sarcoma, lymphoma, immature teratoma, and melanoma.
Technical Field
The present invention relates generally to tumour-targeted statin derivatives (THSD), and their use in therapy, in particular in the treatment of cancer.
Background
A number of cancer therapeutics have been discovered and used in a variety of standard therapeutic approaches, such as Tyrosine Kinase Inhibitors (TKIs) and classical chemotherapeutic drugs. Many TKIs, such as gefitinib and erlotinib, and many chemotherapeutic drugs, such as platinum compounds and their derivatives, gemcitabine, doxorubicin, paclitaxel and docetaxel, can produce serious side effects and/or develop resistance. For example, platinum compounds, and their combination therapy with other drugs, are commonly used to treat advanced or metastatic cancers, or cancers that often undergo metastasis. The treatment of these cancers is very limited, especially after advanced stages/metastases. Moreover, these cancers often develop resistance after treatment with specific chemotherapeutic drugs. Many treatment regimes have therefore been followed which combine various traditional cancer drugs together, such as various chemotherapeutic drugs, and combinations of chemotherapeutic drugs and other drugs with specific anti-cancer effects.
Statins belong to HMG-CoA reductase inhibitors, have the efficacy of lowering cholesterol, and generally have certain side effects. The results of a plurality of researches show that the statins have potential anticancer effect. Positive responses have also been observed in some patients, especially in clinical treatment of certain types of tumors when statins are used in combination with traditional chemotherapeutic drugs. In other studies, adverse effects of statins on cancer risk or cancer treatment have been reported, such as increased risk of cancer onset, or increased risk of cancer recurrence. Specific problems with statins also include: its plasma circulation time is short, its residence time in the tumor is short, and it can be quickly inactivated in vivo.
Heptamethine cyanine carbocyanine dyes (HMCDs) are commonly used for imaging, for example in human imaging in various diagnostic procedures. Wherein some of the dyes can be used for both cancer imaging and cancer therapy.
Specific drug-conjugated HMCD dyes ("DZ 1") conjugated to simvastatin or other drugs susceptible to drug resistance for delivery to cancer cells are disclosed in WO 2018/075996, WO 2018/075994 and WO 2018/075993. These drug release conjugates are capable of re-sensitizing drug-resistant cancer cells and can be co-administered with a variety of easily drug-resistant chemotherapeutic agents, including but not limited to tyrosine kinase inhibitors (e.g., gefitinib and erlotinib), platinum compounds, gemcitabine, paclitaxel, docetaxel, and the antiandrogen agents enzalutamide and abiraterone. Such dye-drug conjugates include, but are not limited to: the dye is attached to simvastatin via an alkyl ester and ensures that the attached statin moiety is able to form a lactone structure. Such ester conjugates will be referred to hereinafter as "fast release esters" or "RREs".
According to WO2018075996, specific dye-simvastatin (NIR-SM) conjugates have been reported to reduce the expression of genes associated with aggressiveness in prostate cancer cells, leading to a reversal of phenotype associated with tumors with therapeutic/drug tolerance (re-sensitizing cells to previously administered drugs, ensuring that these re-sensitized cells are killed by the drug after re-administration). For example, simvastatin released from an alkyl ester linked NIR-SM conjugate (hereinafter referred to simply as "RRE") can re-sensitize cancer cells, ensuring successful re-administration of drugs such as enzalutamide, abiraterone, or docetaxel.
DZ-1 drug conjugates linked to cisplatin, simvastatin, or artemisinin and their use to sensitize various easily-resistant chemotherapeutic agents, including but not limited to cisplatin, gemcitabine, paclitaxel, and docetaxel, are reported in WO 2018/075994.
In WO 2018/075993, DZ 1-drug conjugates are reported, as well as their use in combination with Tyrosine Kinase Inhibitors (TKIs), such as gefitinib or erlotinib, to improve sensitivity of cancer cells to TKI treatment and to overcome TKI resistance.
Cancers treated with TKIs include cancers that are more aggressive and easily metastasize and/or develop drug resistance. Nevertheless, a major problem with TKI therapy is that cancer cells readily develop resistance to TKI after treatment. Another problem with partial TKIs is that they do not, or are not effective in, treating the brain, even when they are relatively small in size (MW: 300-. Other TKIs that can be used to treat the brain have a variety of side effects and can only be used for specific patient populations.
Nevertheless, there is a continuing need for improvements in cancer therapeutics and cancer treatments, including improvements in their efficacy. More specifically, there is a need for cancer therapeutic agents that inhibit/kill cancer cells more rapidly, while avoiding the development of resistance. In addition, there is a need to develop a cancer therapeutic drug and a therapeutic method with less side effects. In addition, there is a need to develop smaller doses of cancer therapeutics and methods of treatment. There is also a need to develop cancer therapeutics and treatments that effectively reduce the future risks, such as the risk of cancer onset, metastasis, and chemotherapy drug-induced disease onset. There is also a need to develop drugs and methods for treating cancer with less frequent administration. There is also a need to develop effective cancer therapeutic drugs and methods with less side effects and less frequent administration. There is also a need to develop one or more advanced therapeutic agents, such as reducing plasma or elimination half-life, increasing plasma circulation time, increasing tumor residence time (e.g., 1-4 weeks or more), reducing inactivation time, and having more favorable dose response curves. In addition, there is a need to develop therapeutic agents and methods suitable for more aggressive cancers, including those that reduce or avoid the administration of drugs that can produce side effects. There is also a need to develop advanced therapeutic agents and methods of treatment that do not require simultaneous administration with agents having serious side effects. More particularly, there is a need to develop advanced therapeutic agents and methods of treatment that do not require concurrent administration of chemotherapeutic agents with severe side effects, including severe side effects such as general cytotoxicity (including various non-cancerous cells). In addition, there is a need to develop therapeutic drugs and methods that are simultaneously applicable to a variety of cancers, especially drug-resistant cancers, metastatic cancers, rapidly developing cancers, and other cancers that are more aggressive. In addition, there is a need to develop advanced therapeutic drugs and methods suitable for prostate or lung cancer patients. In addition, there is a need to develop advanced therapeutic drugs and methods that can penetrate the Blood Brain Barrier (BBB), and can effectively treat brain tumors and metastases. For cancers such as lung cancer, which are commonly treated with TKIs, there is also a need to develop advanced therapeutic drugs and treatment methods that can avoid the formation of drug resistance. There is a need to develop treatments that overcome or are not affected by TKI resistance that has developed in patients who have previously been treated with TKIs, particularly non-small cell lung cancer (NSCLC) patients, including particularly lung Adenocarcinoma (AC) patients. For cancers that are commonly treated with chemotherapeutic drugs, such as lung cancer such as Small Cell Lung Cancer (SCLC), there is a need to develop therapeutic methods and drugs that avoid the development of drug resistance; there is a need to develop therapeutic agents and methods of treatment for patients who have previously been treated with chemotherapeutic agents that overcome or are unaffected by the resistance that has developed. These and other features and advantages of the present invention will be described in more detail below in the detailed description of the invention and in the summary of the invention, which will be apparent to those skilled in the art.
Disclosure of Invention
The present invention relates generally to tumour-targeted statin derivatives (THSD), and their use in therapy, in particular in the treatment of cancer. These THSDs comprise a heptamethine cyanine carbocyanine dye (HMCD) moiety linked to DHHA of the statin moiety via a linker and fix DHHA as an open chain structure. The linker may be attached to the open chain structure of DHHA via an ester bond ("ELSD") or via an amide bond ("ALSD"). Embodiments of the invention also include methods of synthesizing THSD, as well as methods of administering the same. More particularly, methods of administration include ELSD, or coordinated administration of ALSD with ESLD, or optionally, with one or more other drugs (e.g., chemotherapeutic drugs or hormone antagonist/antiandrogen drugs).
Surprisingly, the results of the study show that: unlike prodrugs, the tumor-targeted statin derivatives (THSD) of the present invention have substantially stable linkages, and thus have various advantages, such as improved growth inhibitory effects, dose response, and/or other therapeutic effects, while being relatively inactive for a longer period of time, and avoiding or mitigating various side effects. In fact, ALSD does not release statins; similarly, ELSDs release statins very slowly compared to RRE (see example 8 and fig. 8). ELSDs can provide a better dose response relationship, i.e., a more gradual dose response curve, than ALSDs (compare fig. 7D-E and 7H).
As can be seen from the comparative examples (see examples 1b, 1E and 7, and FIGS. 1A-E and 7A-I and 8), although statins are not released (or are released at a very slow rate for ELSDs), THSD is far more effective than unconjugated statins or unconjugated dyes, and even than therapeutic drugs such as docetaxel, and other cancer therapeutic drugs such as those including antiandrogen drugs (e.g., abiraterone and enzalutamide); and does not require administration at the same time as (and thus avoids the serious side effects of) these therapeutic agents.
Thus, THSD in the form of a couplet can provide an effective ingredient. In addition, it is surprising that even though it is relatively large in size, it can still bind to the relevant binding pocket, including the binding pocket of statins, and thereby achieve its various effects, including anticancer effects, as well as other effects of statins. In contrast, the fast release ester ("RRE") rapidly hydrolyzes and releases the statin (see comparative examples 1A-IV and 4A, and corresponding FIGS. 1A-IV, infra). Unlike RRE, THSD is more structurally stable and therefore does not systemically release statins in large quantities over a long period of time (e.g., hours, days, weeks, or even months) and is thus able to function in cancer cells in the form of couplets.
Also surprising is that ELSDs have a better dose response curve than ALSDs, with a more gradual dose response curve, meaning that the initial therapeutic effect can be achieved at lower doses and/or in shorter times (compare fig. 7D, 7E and 7H). Without being bound by current theory, it is believed that this is because ELSDs are not released systemically in the early stages and thus can directly provide some anti-cancer and statin effects in the first few hours, but the effects associated with the couplet and/or the released statin moiety need to be present after the ELSDs enter the cancer cells. In addition, these statin derivatives can also provide one or more statin effects to cancer cells, for example, reducing or avoiding the side effects typically associated with statins administered systemically or via a rapid release conjugate such as RRE.
In addition, it is also surprising that TSHD is effective in treating cancers that are susceptible to drug development, most aggressive, and other cancers that are susceptible to drug development, and that it is possible to achieve this without concurrent administration of drugs such as TKI or chemotherapy. Thus, THSD may be effective in treating cancers such as, but not limited to, prostate cancer, and lung cancer including non-small cell lung cancer (NSCLC), lung Adenocarcinoma (AC), and Small Cell Lung Cancer (SCLC). As can be seen from fig. 7A-I, fig. 8 and fig. 10, both ELSD and ALSD had growth inhibitory effects on cell lines representing prostate cancer, prostate adenocarcinoma, lung cancer, non-small cell lung cancer, lung adenocarcinoma, small cell lung cancer, including various resistant forms thereof, in a mouse model of human tumor obtained by implanting human cancer cells into a mouse. The use of THSD to treat cancer may avoid resistance that is normally associated with standard therapies such as TKI or chemotherapeutic drugs, or may overcome resistance that has developed, for example, resistance caused by previous cancer treatment drugs, for example Tyrosine Kinase Inhibitors (TKI) such as gefitinib, or standard chemotherapeutic drugs such as docetaxel and cisplatin (DDP).
In addition, it is also surprising that THSD can penetrate the blood brain barrier even when administered systemically rather than locally, and thus can be used to treat brain tumors and brain metastases.
Without being limited by current theory, it is believed that at least some of the effects and advantages of TSHD are related to the particular form of the coupling body, and more particularly, the particular form of the connector, the manner in which it is attached to different portions of the coupling body, and the particular form of different portions of the coupling body. I.e. DZ 1-backbone, LE/LAA linker, and the manner in which it is linked to the DHHA unit of the statin moiety (which is the key pharmacophore in the statin moiety) via an amide or ester bond; and more importantly the specific structure of the DHHA unit after attachment to the linker. Depending on their linker and/or structure of attachment (and their effect), the following three dye-statin conjugates can be distinguished: 1) ALSD; 2) ELSD; and 3) RRE. According to the connector/connection structure, the following effects can be achieved: 1) preventing statin release (e.g.: in the presence of an amide bond LAIn ALSD of linker); or 2) is able to release statins very slowly (e.g.: in ELSDs linked to the open chain structure of DHHA via an ester linkage); and 3) the ability to rapidly release a statin from a prodrug conjugate (e.g.: in an RRE linked to the lactone structure of DHHA via an ester bond). Unlike fast-release prodrugs, THSD form 1) or 2) above does not release statins, or only releases at a very slow rate.
Without being limited by current theory, it is believed that THSD (primarily "zero release" amide linked statin derivatives ("ALSD"), but also "slow release" ester linked statin derivatives ("ELSD")) can be tightly linked to proteins and nucleic acid molecules, which can render THSD less susceptible to inactivation, and can persist in cancer cells for extended periods of time, e.g., days, weeks, or even months; thereby enabling a substantial reduction in the frequency of administration, and reduction or even avoidance of side effects, such as those due to conjugated or unconjugated chemotherapeutic drugs, unconjugated statins, and "fast release" prodrug forms of conjugated statins. In particular for ELSDs, this tight junction may be the primary reason to prevent rapid release of statin and inactivation (as in the case of "rapid release" ester-linked HMCD statin conjugates), which allows for improved therapeutic efficacy (including growth inhibition/killing of cancer cells, and tumor healing) while avoiding one or more side effects.
Without being limited by current theory, it is found that the dose response curve of ELSDs is much flatter after comparing the growth inhibitory effect of ELSDs and ALSDs, indicating that it is capable of triggering initial non-maximal therapeutic effects at lower concentrations. For example, the total dose of ELSD or THSD is from about 0.1mg up to about 6mg per kg or less (e.g., up to about 2.5mg or even about 1.0mg per kg). Thus, one or more ELSDs can be used alone to treat cancer (lower therapeutic doses can be used due to their longer residence time in the tumor, which can exert non-maximal but sufficient therapeutic effect over a long period of time); it may also be used in combination with one or more ALSDs (to provide sufficient therapeutic effect and/or to reduce the dosage required to achieve sufficient or maximal therapeutic effect). Exemplary administration mechanisms are described below.
Drawings
Fig. 1A-I depict ELSD, RRE and ALSD linked to DHHA units in statins.
Fig. 1A-II depict the synthesis of HMCD dyes.
FIGS. 1A-III depict the synthesis of "zero release" ALSD (DZ2 a).
FIGS. 1A-IV depict the synthesis of a "slow release" ELSD (DZ2 v).
FIGS. 1A-V depict the "quick release ester" (RRE) (DZ2c) for comparison.
FIG.1B1 depicts the structures of DZ2a, DZ1, simvastatin, and docetaxel.
Figure 1B2 depicts the growth inhibitory effect of THSD on cancer cells.
Figure 1C depicts the growth inhibitory effect of THSD on various prostate cancer cells.
Figure 1D depicts the uptake and retention of THSD in human cancer cells grown in mice.
Figure 1E depicts the tumor healing effect of THSD in an in vivo setting.
Figure 2 depicts co-localization of THSD to mitochondria/lysosomes in cancer cells.
Figure 3A depicts the situation where THSD reduces the oxygen consumption rate OCR of cancer cells.
Figure 3B depicts the reduction in extracellular acidification rate (ECAR) by THSD.
Figure 3C depicts THSD binding to proteins in the cytosol or mitochondria.
Figure 3D depicts THSD prevention or elimination of polyubiquinone.
Fig. 4A depicts the rapid, complete inhibition of cancer cell growth by THSD.
Figure 4B depicts THSD inhibits growth of cancer cells resistant to abiraterone acetate.
Figure 4C depicts THSD inhibits growth of cancer cells with Enzalutamide (EZ) resistance.
FIG. 5 depicts RhoA/B staining of human tumors in a mouse model of lung cancer cells.
Figure 6B1 depicts the rapid depletion of THSD in rat plasma following systemic administration.
Fig. 6B2 depicts the rapid depletion of THSD in canine plasma following systemic administration.
FIG. 7A depicts the inhibition of 22Rv1 cancer cell growth by ALSD and ELSD.
Fig. 7B depicts the situation where ALSD and ELSD inhibit the growth of EZ resistant cancer cells.
FIG. 7C depicts the inhibition of growth of PC3 cancer cells by ALSD and ELSD.
Fig. 7D depicts the inhibition of a549 cancer cell growth by ALSD and ELSD.
FIG. 7E depicts the inhibition of the growth of A549DDP cancer cells by ALSD and ELSD.
FIG. 7F depicts the inhibition of growth of H1975 cancer cells by ALSD and ELSD.
FIG. 7G depicts the inhibition of growth of ELSD cancer cells by ALSD and ELSD.
FIG. 7H depicts the inhibition of growth of PC9 cancer cells by ALSD and ELSD.
FIG. 7I depicts the inhibition of H446 cancer cell growth by ALSD and ELSD.
Figure 8 depicts that ALSD is a stable/non-hydrolyzable drug, not a prodrug.
The growth inhibitory effect of TSHD in an in vivo model of third generation EGFR inhibitor resistant cancer cells is depicted in figure 9a 1.
Fig. 9a2 depicts a tumor weight comparison graph to illustrate the heterogeneity of THSD in vivo.
Fig. 9a3 depicts a tumor volume comparison graph to illustrate the heterogeneity of THSD in vivo.
A graph of body weight comparison of mice is depicted in fig. 9a4, indicating that no weight loss/acute toxicity phenomena occurred.
Figure 10 depicts the growth inhibitory effect of THSD in an in vivo model of H446 human cancer cells.
Figure 11 depicts a non-statin DZ1-DHA conjugate for comparison.
Detailed Description
In a specific embodiment, an ester-linked statin derivative is provided, which has a formula of FIa:
wherein X is a halogen residue; wherein R is1Is a residue, canTo select from the following functional groups: c1-C25Alkyl radical, C5-C25Aryl radical, C5-C25Indolyl, C5-C25Thienyl, C5-C25Phenyl radical, C5-C25Naphthyl, C1-C25Aralkyl radical, C1-C25Alkylsulfo group, C1-C25Alkyl carboxyl, C1-C25Alkylamino radical, C1-C25Omega-alkylamines, C1-C25Omega-alkynyl, one having (-CH)2-CH2-O-)2-20The PEGyl polyethylene chain of (A) has (-CH)2-CH2-O-)2-20PEGyl carboxylate of (I), with (-CH)2-CH2-O-)2-20Of omega-PEGyl amine, omega-acyl-NH, omega-acyl-lysyl, omega-acyl-triazole, with (-CH)2-CH2-O-)2-20Of omega-PEGyl carboxy-NH with (-CH)2-CH2-O-)2-20omega-PEGyl carboxy-lysyl, and a peptide having (-CH)2-CH2-O-)2-20omega-PEGyl carboxy-triazole; wherein R is2And R3Are residues, which can be independently selected from the following functional groups: hydrogen atom, C1-C20Alkyl, sulfonic acid group, C1-C20Alkyl carboxyl, C1-C20Alkylamino radical, C1-C20Aryl, -SO3H、-PO3H、-OH、-NH2And a halogen residue; wherein R is2And R3Can be attached to the following positions in a carbon ring: 3. 3 ', 4', 5 ', 6 and 6'; wherein A is-The functional group is a negatively charged anion compatible with the pharmaceutical agent; wherein there is an ester linker LELinked to a statin residue R by an ester bondSThe above step (1); wherein L isECan be selected fromE1And LE2Is selected from the functional groups of (1), wherein LE1Is- (CH)2)n-O-, wherein LE2Is- (CH)2)n–CO–NH–(CH2)m-O-, wherein n has a value of 4-9 and m has a value of 1-4; wherein R isSIs a residue of a statin or statin derivative, RSWith a dihydroxyheptanoic acid unit (DHHA) linked to the R constituting the remainder of the statin residueS*The above step (1); wherein R isSThrough its open chain structure (-CO-CH) in DHHA2-COH-CH2-CHOH-RS*) Is connected to LEAbove.
In a specific embodiment, an ELSD is provided wherein the statin is selected from the following functional groups: simvastatin, mevastatin, lovastatin, pravastatin, atorvastatin, fluvastatin, rosuvastatin, cerivastatin, pitavastatin, and other derivatives attached to the operably linked region in a statin.
In a specific embodiment, there is provided an ELSD, wherein the statin is simvastatin, and wherein the ester linker L isEIs LE2As shown in formula FIb:
and wherein A-,X,R1,R2,R3And LE2N and m in the linker are defined as formula FIa.
In a specific embodiment, an ELSD is provided of formula FIa or Fib, wherein X is a chlorine atom. Further examples are provided wherein R1Is- (CH)2)n–SO3-an alkylsulfonate residue, and wherein n in R1 is selected from 2, 3, 4,5, 6, 7 and 8. Other examples are also provided, wherein R1Is- (CH)2)4–SO3-an alkyl sulfonate residue. Further, examples are provided wherein R2And R3Is H.
In a specific embodiment, a pharmaceutical formulation is provided comprising an ELSD and one or more pharmaceutical excipients, wherein the ELSD is selected from the following functional groups: ELSD of the above formula FIa; FIa, wherein R is formedSThe statin (b) is selected from the following functional groups: simvastatin, mevastatin, lovastatin, pravastatinStatin, atorvastatin, fluvastatin, rosuvastatin, cerivastatin, pitavastatin and other derivatives attached to an operably linked region in a statin; ELSD of the above formula FIb, wherein the statin is simvastatin and the ester linker LEIs LE2(-(CH2)n–CO–NH–(CH2)m-O-, wherein LE2A of the linker-、X、R1、R2、R3And n and m are as defined in sub-formula FIa in (i) above; ELSD as defined herein, wherein X is a chlorine atom; ELSD as defined herein, wherein R1Is- (CH)2)n–SO3-an alkylsulfonate residue, and wherein n in R1 is selected from 2, 3, 4,5, 6, 7 and 8; ELSD as defined herein, wherein R1Is- (CH)2)4–SO3 -An alkyl sulfonate residue.
In a specific embodiment, a pharmaceutical formulation is provided comprising one or more ELSDs, and further comprising the following filia formula of ALSD:
wherein A is-、X、R1、R2And R3As defined herein FIa; and wherein the amide linker LATo statin residue R by amide bondS(ii) a Wherein L isAIs selected from LA1And LA2A linker of functional groups wherein LA1Is- (CH)2)n-NH-, and wherein LA2Is- (CH)2)n–CO–NH–(CH2)m-NH-and wherein n-4-9 and m-1-4; wherein Rs is the residue of a statin or a statin derivative, wherein Rs comprises a dihydroxyheptanoic acid unit (DHHA) linked to a residue Rs which is the residue of a statin, wherein R isSLigation to L by DHHA in its open chain formAThe open chain form is-CO-CH2-COH-CH2-CHOH-RS*。
In particular toIn embodiments, there is provided a method of treating cancer, wherein one or more ELSDs and optionally one or more ALSDs are administered to a patient in need thereof in a sufficient amount to inhibit growth of or induce apoptosis of cancer cells or precancerous cells in the patient, wherein the selected one or more ELSDs consist of the following functional groups: ELSD of the above formula FIa; FIa, wherein the statin of the RS formed is selected from the following functional groups: simvastatin, mevastatin, lovastatin, pravastatin, atorvastatin, fluvastatin, rosuvastatin, cerivastatin, pitavastatin and other derivatives attached to the operably linked region in the statin. ELSD of the above formula FIb, wherein the statin is simvastatin and the ester linker LE is LE2Is- (CH)2)n–CO–NH–(CH2)m-O-, and wherein, a-, X, R1、R2、R3And LE2 the N and M in the linker are as defined above for formula FIa in (i); in ELSD as defined herein, X is a chlorine atom; in ELSD as defined herein, R1Is- (CH)2)n–SO3 -Alkylsulfonic acid salts, and R1N in (1) is selected from 2, 3, 4,5, 6, 7 and 8; in ELSD as defined herein, R1Is- (CH)2)4–SO3 -An alkyl sulfonate residue; and wherein the optional one or more ALSDs are selected from the following functional groups: ALSD in formula FIIa as described herein; and ALSD of formula FIIb, wherein the statin is simvastatin and the linker is LA2(–(CH2)n–CO–NH–(CH2)m–NH-)：
Wherein, A-, X, R1、R2、R3And LA2The sum of the linkers n and m are as defined above for formula FIa.
In a specific embodiment, a method is provided wherein one or more ELSDs and one or more ALSDs are co-administered to a patient in a coordinated dosing regime of one or more combination dosage forms, wherein each dosage form comprises one or more ELSDs and one or more ALSDs and one or more pharmaceutical excipients.
In a specific embodiment, a method is provided wherein one or more ELSDs are co-administered with one or more ALSDs in a coordinated dosing mechanism; wherein the administration mechanism comprises: at least one hour or more prior to administering one or more maintenance doses to the patient, requiring that a loading dose be administered to the patient first; wherein the loading dose may comprise a single dosage form, may contain one or more pharmaceutical excipients, but may not contain one or more ELSDs; the following dosage forms should be included in one or more of the maintenance doses: contains one or more ALSDs and one or more pharmaceutical excipients, but optionally contains or does not contain ELSDs.
In a specific embodiment, a method is provided wherein one or more ELSDs are co-administered with one or more secondary drugs in a coordinated dosing mechanism, and wherein the one or more secondary drugs are selected from the group consisting of: hormone antagonists, antiandrogenic drugs, abiraterone, enzalutamide, chemotherapeutic drugs, docetaxel, paclitaxel and cabazitaxel.
In a specific embodiment, a method is provided wherein one or more combination dosage forms, or one or more individual dosage forms of one or more cargo amounts and one or more maintenance doses, are co-administered in a coordinated dosing regimen; wherein the combination dosage form comprises one or more ELSDs, one or more ALSDs, and one or more PHAs. A normal excipient, wherein the loading dose consists of separate dosage forms comprising one or more ELSDs and one or more pharmaceutical excipients, and does not comprise one or more ALSDs; and wherein the one or more maintenance doses consist of a dosage form comprising one or more ALSDs and one or more pharmaceutically acceptable excipients. ENT, and optionally ELSD; wherein the co-administration form is further co-administered with one or more secondary drugs on a coordinated dosing schedule, and wherein the one or more secondary drugs are selected from the following functional groups: hormone antagonist, antiandrogen, Abiraterone, Enzalutami A chemotherapeutic drug, docetaxel, paclitaxel and Cabazitaxel.
In a specific embodiment, a method is provided which comprises administering one or more ELSDs to a patient having one or more genetic variations found in one or more gene codes for one or more tyrosine kinase receptors in cancer cells, precancerous lesions, tissues, tumors, or metastases thereof; wherein the tyrosine kinase receptors comprise: epidermal Growth Factor Receptor (EGFR), anaplastic lymphoma kinase receptor (ALF), and proto-oncogene tyrosine protein kinase receptor (ROS or ROS 1).
In a specific embodiment, a method is provided which comprises administering one or more ELSDs to a patient whose cancer cells, precancerous lesions, tissues, tumors, or metastases have developed resistance to one or more Tyrosine Kinase Inhibitors (TKIs); these include patients who have undergone one or more TKI treatments prior to ELSD administration and for whom the effect of the previous TKI treatment has been inadequate.
In a specific embodiment, a method is provided wherein the TKI is selected from the group consisting of: epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI), ALK tyrosine kinase receptor inhibitors (ALK-TKI), and proto-oncogene tyrosine protein kinase ROS (ROS-TKI).
In a specific embodiment, a method is provided wherein the EGFR-TKI is selected from: gefitinib, erlotinib, bugatinib, dacomitinib, lapatinib, vandetanib, afatinib, axitinib (AZD9291), CO-1686, HM61713, nanozatinib (EGF816), Omtinib, PF-06747775, YH5448, Avertinib (AC0010), Rociletinib and cetuximab.
In a specific embodiment, a method is provided wherein the patient is identified as having a drug-resistant cancer by drug exposure or genetic testing, wherein the drug-resistant cancer comprises the following cancers: prostate cancer, pancreatic cancer, lung cancer, non-small cell lung cancer (NSCLC; which may include squamous cell carcinoma, adenocarcinoma (mucinous cystadenocarcinoma), large cell lung cancer, rod cell carcinoma, sarcoid-like carcinoma, carcinoid, salivary adenoid carcinoma, acanthoma, papillary adenocarcinoma, giant cell carcinoma), SCLC (small cell lung cancer), generalized small cell carcinoma, non-cancerous tumors of the lung (sarcoma, lymphoma, immature teratoma, and melanoma), kidney cancer, lymphoma, colorectal cancer, skin cancer, liver and breast cancer, lung squamous cell carcinoma, anal cancer, glioblastoma, epithelial cell tumors of the head and neck, and other cancers.
In a specific embodiment, a method is provided wherein the patient is a patient determined to have drug-resistant lung cancer by drug exposure or genetic testing, wherein the drug-resistant cancer comprises: small Cell Carcinoma Lung Cancer (SCCLC), non-small cell carcinoma lung cancer (NSCLC), comprehensive small cell carcinoma, squamous cell carcinoma, adenocarcinoma (AC, mucinous cystadenocarcinoma, mcalcl), large cell lung cancer, rod cell carcinoma, sarcoid, carcinoid, salivary adenoid, adenosquamous, papillary adenocarcinoma, giant cell carcinoma, non-cancerous tumors of the lung, sarcoma, lymphoma, immature teratoma, and melanoma.
In a specific embodiment, THSD consists essentially of three parts: statin moieties, Heptamethoxycarbocyanine (HMCD) dye moieties, and via ester or amide linkages (' L)E/LALinker "or" linker ") binds the statin DHHA to the linker moiety of the dye moiety. The dye is a heptamethine carbocyanine type near-infrared (near-infrared) dye comprising a central halogen cyclohexyl moiety. The linker can be considered to comprise the alkyl chain of the dye. In the conjugated statins, DHHA is immobilized to its open chain form by the linkage of a linker and a dye.
Examples include methods of preparing THSD; for example, without limitation, a dihydroheptanoic acid (DHHA) unit of a statin or a statin precursor can be reacted with hydroxylamine, and a hydroxyl group of the statin reaction product can be reacted with a carboxyl group of HMCD to form L having an ester linkageEA connector. Alternatively, the dye moiety precursor may be reacted with a diamine, and the amine group of the dye reaction product may be reacted with the carboxylic acid of statin DHHAThe radicals react to form an amide bond.
For example, in the case of ELSD, the linker may be an alkyl linker (- (CH)2)n-O-, also referred to herein as "LE1"), or may be a compound comprising a carboxamide (- (CH)2)n–CO–NH–(CH2) m-O-, referred to herein as "LE2") that can result from the reaction of, for example, a hydroxyl group (AMI) of an HMCD derivative. The group of the derivative resulting from the previous reaction of the carboxyl group of HMCD with hydroxylamine (e.g. ethanolamine) and the carboxyl group of the DHHA unit of the statin or statin derivative, thereby forming L linked to the DHHA unit of the statin by an ester bondEThe chain lactam of the linker (comparative examples such as FIGS. 1A-IV show illustrative examples of how ELSDs can be formed).
]In the case of ALSD, similarly, the linker may be an alkyl linker (- (CH)2)n-NH-, also referred to herein as "LA1"), or may be a compound comprising a carboxamide (- (CH)2)n–CO–NH–(CH2) m-NH-, also referred to herein as "LA2") formamide linker. L isA2The carboxamide of (a) can be generated by, but is not limited to, the reaction of an amine group of a statin derivative (the amine group of the derivative, e.g., the DHHA unit of the statin and the amine residue of a diamine (e.g., propane-1, 3-diamine) with the carboxyl group of HMCD to form thsd with an automobileA2) (e.g., the illustrative example in FIGS. 1A-III is how the ALSD is formed).
Thus, the linker of THSD can be via an amide or ester bond (L each)EOr LA) Linked to a statin.
Linking amide or ester linkages the dye moiety of THSD to the statin residue via the open chain DHHA of the statin; without wishing to be bound by theory, this particular linkage may help to enhance the efficacy of ELSD and ALSD and in the case of ester linkages result in slow ester release. Therefore, both ELSD and ALSD are relatively stable: one substantially zero release statin (amide linked SD or "ELSD") and the other release is very slow (ester linked SD or "ELSD") much slower than other fast release conjugates, particularly the fast release ester ("RRE") linked to DHHA. In the form of lactones
Amide linker (L)E2/LA2) May be a better therapeutic effect and a first choice in case the ester further improves stability/slow release. L isE2/LA2The linker may be of the formula- (CH)2)n–CO–NH–(CH2) m-NH-, wherein n ═ 4-9 and m ═ 1-4 (or "LE") for ALSD, and- (CH)2)n–CO–NH–(CH2) m-O-, wherein n ═ 4-9 and m ═ 1-4 (or "LA") for ELSD.
Preferably, it may be LEAnd LAN and m are chosen as follows: n-4-6 and m-1-4; most preferably, the total number of N (N + m) does not exceed 10, 9, 8, 7 or 6, for example N ═ 5 for 6-9 (i.e. THSD based on DZ1), i.e. not limited to: - (CH)2)5–CO–NH–(CH2)1–NH-,–(CH2)5–CO–NH–(CH2)2–NH-,–(CH2)5–CO–NH–(CH2)3–NH-,–(CH2)5–CO–NH–(CH2)4–NH-for LE(ii) a And- (CH)2)5–CO–NH–(CH2)1–O-,–(CH2)5–CO–NH–(CH2)2–O-,–(CH2)5–CO–NH–(CH2)3–O-,–(CH2)5–CO–NH–(CH2)4-O-for LA。
Surprisingly, it has been found that THSD provides an improvement in the form of a conjugate, rather than as a prodrug (where the drug moiety upon release will provide its effect). In the case of ALSD, there was essentially no or minimal hydrolysis of the statin, and surprisingly, under in vivo conditions, the amide bond was found to be essentially stable with essentially no detectable release of the statin. Also, in the case of ELSDs, hydrolysis is only very slow under "in vivo" conditions, and thus after the ELSD reaches the cancer cells or tissues
Since no statin release from statins is shown under in vivo conditions, such as in the serum of various mammals, see, e.g., examples 1A-V (ELSD stable in mouse serum at 37 ℃ as opposed to RRE), and example 6 (rat/dog; ALSD stable in vivo for at least 24h), the improved effects of THSD shown below are: essentially the conjugate itself, avoiding side effects, including those associated with the use of systemic statins.
Without wishing to be bound by theory, it is believed that the structure of THSD described herein, particularly the open chain form of the DHHA residue of statins, contributes to a variety of therapeutically relevant effects, including those not achieved by dyes or smaller statins themselves. This can be further improved by the presence of a carboxamide in the linker.
Surprisingly, the therapeutic efficacy of THSD is improved not only over unconjugated statins and unconjugated dyes, but also over the therapeutic efficacy of various chemotherapeutic drugs, some of which are highly cytotoxic and act at high doses in both resistant and non-resistant cancer cells or tumors. This effect of THSD is shown, for example, in comparative examples 1A-IV below and corresponding figures 1A-IV.
The improvement in THSD is illustrated in examples 1A-1E and fig. 1A-1E, which show that THSD can provide improved inhibition of cancer cell growth and tumor shrinkage in vivo compared to unconjugated statins by themselves and to unconjugated dyes (see, e.g., fig. 1A-C). These improvements can be found in different cancer cell lines and human tumors growing in mice (see, e.g., figure 1D), and are superior to docetaxel and like cancer drugs (see, e.g., figure 1E), even at high doses of 8mg/kg body weight, which have essentially no or very slight tumor suppression activity compared to THSD, which shows strong and durable tumor suppression at lower doses of 5 mg/kg. Docetaxel can therefore be replaced by THSD, avoiding its side effects, providing better activity/growth inhibition and tumor shrinkage.
In addition, as shown in examples 4A1-3, B1-3, and FIGS. 4A-C, it was shown that THSD provides improvements in the treatment of drug-resistant cancer cells. Various cancer drugs (abiraterone acetate, enzalutamide, docetaxel) are known to have only a very mild effect on reducing the cell survival rate and thus inhibiting the growth of drug-resistant cancer cells. However, when exposed to THSD, comparing fig. 4A-C, not only the growth of the parental (i.e. before induction of resistance) cancer cells could be completely inhibited (0% cell survival after 8 hours, see fig. 4A) but also the growth (0% cell survival after 8 hours, see fig. 4B and fig. 4C) at the same THSD concentration and within the same short time, at moderate concentrations of about 16 μm or less and only within 8 hours.
Furthermore, examples 7A-7I and fig. 9a1-4 demonstrate that THSD (including ELSD) provides improvements in inhibiting cell growth of various human cancer cell lines, including various types of prostate and lung cancer cell lines, including cell lines established from NSCLC and SCLC, and includes drug-resistant cell lines. The same applies to ALSD and ELSD, as both methods account for the reduction in cell viability in various human cancer cell lines, such as 22Rv1, MDVR (EZ-resistant C4-2B), and PC3 cancer cells as shown in figures 7A-7C, compared. The human drug-resistant lung cancer model (e.g. PC9AR AC/NSCLC cells resistant to EGFR-TKI, no acute toxicity of THSD (FIG. 9A1-4) combined with EGFR-TKI further slightly improved anticancer activity.
IC50And other cancer cell-based data, as well as weight/volume of tumor and other preliminary data, support that THSD causes greater cytotoxicity and fewer side effects in cancer cells (e.g., results are shown in fig. 1b-E, fig. 4A-C, fig. 7A-I).
Without wishing to be bound by theory, it is believed that the HMCD moiety and the statin moiety (especially via the open chain form of the statin DHHA) when linked as described are able to act synergistically to achieve at least an additive and possibly synergistic effect, and possibly provide or contribute to an improved therapeutic effect. TS of THSD. This may be due to one or more of the following three roles or functions of the THSD conjugate, but not to the unconjugated HMCD dye: 1) mitochondrial function, 2) lysosomal function, and 3) intercellular communication by protein pre-acylation. These three functions or effects occur independently, but together contribute to multiple mechanisms of THSD, each of which contributes to improved inhibition of cancer cell growth.
It is believed that the three functions of THSD shown (unlike the unconjugated HMCD dye or unconjugated statin) are supported by the results of example 2 shown in figure 2. Staining for 1) and 2) cancer cells showed that THSD is co-localized with mitochondria and lysosomes and therefore is capable of interfering with various mitochondrial and lysosomal functions in cancer cells. Although some of the proteins overlap, the bound protein differs in intensity and/or identity from the unconjugated dye-bound protein, as shown by the additional bands appearing in FIG. 3C. Examples 3A-D and FIGS. 3A-D show an incredible improvement in THSD in reducing cancer cell Oxygen Consumption Rate (OCR) compared to unconjugated dye/s. TATIN or control (THSD decreased from about 800-900pmol/min to less than 200pmol/min, unconjugated drug and control remained above 700, see FIG. 3A).
Also, for 1) and corresponding to a strong reduction in OCR, THSD also showed a reduction in extracellular acidification rate ("ECAR"), as shown in fig. 3B. ECAR corresponds to the use of anaerobic glycolysis, the anaerobic form of which produces and accumulates lactate extracellularly, and therefore, a higher rate of tracing cellular acidity from lactate. Although all samples showed a decrease in ECAR (anaerobic ATP production), the THSD-exposed cells ("DZ 2 a") showed the least decrease, which also appeared to be convex upward on the curve about 500 minutes before the decrease recovered; all other samples showed a gradual decline in ECAR with a negative control ("NT") curve of l. However, followed by unconjugated statins; the other curves (not visible) show similar decline and gradual trends; all these samples showed higher OCR (i.e. higher aerobic respiration) compared to THSD treated cells. A decrease in ECAR usually means a decrease in anaerobic glycolysis (and instead aerobic respiration). Anaerobic production of ATP, i.e. via the anaerobic glycolysis/lactate system, is an alternative to cell survival, and is particularly important for cancer cells, especially in solid tumors where oxygen may not be available. As shown, THSD affects physiological changes in cancer cells, including a strong reduction in oxygen, but also (after initial attempted switch) produces anaerobic ATP, which helps to inhibit the growth of cancer cells exposed to THSD.
With respect to 2), THSD prevented cancer cell ablation/lysosomal clearance of polyubiquitinated cellular proteins, as shown in fig. 3D.
With respect to 3), as shown by the results described in example 5, THSD can prevent RhoA/B from anchoring to the cell membrane by inhibiting the pre-acylation of RhoA/B, thereby blocking its downstream signal. Protein prenylation is a post-translational mechanism for protein modifications such as RhoA/B, helping to anchor these proteins to the membrane of cancer cells (e.g., lung and prostate cancer cells) for downstream cellular communication. THSD inhibits HMG-CoA reductase, and thus inhibits the production of cholesterol and isoprene. Cholesterol and isoprene are required for prenylation of the protein, but are absent due to THSD-mediated HGM CoA inhibition, thereby blocking downstream signaling of the prenylated/anchored protein.
Thus, unlike unconjugated statins that act primarily on the host liver HMG-CoA reductase (and are limited only to any effect on cancer cells), THSD may have at least three independent effects/functions (mitochondrial, lysosomal, and cell-cell communication). These may occur independently, but may collectively contribute to the inhibition of the growth of cancer cells.
Thus, without wishing to be bound by theory, THSD may provide various advantages compared to the unconjugated form of the statin, compared to unconjugated HMCD, and/or compared to bound statins that rapidly release the statin. These rapid-release conjugates include, inter alia, rapid-release ester conjugates, such as rapid-release ester conjugates (see DZ1-SIM in FIGS. 1A-V), directly linked to the lactone form of DHHA. THSD has many advantages over commonly used unconjugated dyes/statins, RREs, and/or other cancer drugs, possibly including one or more of the following: avoiding or reducing statin inactivation (especially in vivo), reduces general cytotoxicity of normal cells, while increasing cytotoxicity of cancer cells (as shown in panel E). E.g. by IC50) Improve and increaseAnti-cancer effects (e.g., growth inhibition, tumor shrinkage, more rapid growth inhibition/cancer cell death, e.g., less than 16, 12, 10, or 8 hours, even when tested on drug-resistant cells (see, e.g., example 4 and examples B1-B3 and fig. 4A-C), thereby avoiding development of drug resistance), shorten the duration. Administration of THSD reduces the frequency of THSD administration (including once, once a week, once every two weeks, once a month, etc.), reduces side effects (especially when administered systemically), reduces future risks (such as the risk of cancer, metastasis and chemotherapy-induced disease), reduces and/or slows drug inactivity. Improved plasma and/or elimination half-life (e.g., less than 8, 4, 2, 1 hour, or 30 minutes, such as about 1-2 hours), increased plasma circulation time, increased tumor residence time (e.g., longer than 1, 2, 3, 4 weeks, or longer), and improved dose-response curves, particularly less than steep sigmoid dose-response curves.
Without wishing to be bound by theory, ELSDs may have a longer tumor residence time than the Rapid Release Ester (RRE). Increased tumor residence time coupled with a steeper sigmoid dose-response curve (thereby reducing the dose at which the initial non-maximal effect occurs) may provide adequate therapeutic benefit for THSD. Thus, the ELSD may be administered alone, or with a dose of ALSD that is lower than the dose that provides the greatest effect when administered alone, or may be co-administered as described herein, e.g., as one or more subsequent maintenance doses following one or more initial ELSD loading doses.
Furthermore, particular advantages of ELSDs may include slow, sustained non-systemic statin release in cancer cells and/or tumor tissues, allowing systemic administration in the form of ELSDs, while avoiding or reducing statin side effects associated with systemic administration of unconjugated statins. Without wishing to be bound by theory, this may provide additional or improved anti-cancer effects, rather than being provided by "zero release" HMCD conjugates, including ALSD. Also, without wishing to be bound by theory, a particular LEThe linker may provide additional or improved anticancer effects in combination with the dye and statin moieties linked as described, including the statin DHHA immobilized in an open chain form.
Furthermore, surprisingly, ELSD appears to provide a much steeper sigmoid dose response curve as compared to ALSD, as shown by the preliminary data, as shown in fig. 7D, 7E and 7H. This means that the initial effect of ELSD at low doses for the range of doses that provides the initial effect, while the ALSD has not had any effect (but may cause side effects). Also, a high dose range was observed providing maximal effect, with ALSD providing maximal effect at low doses, while more ELSD was required. Thus, the use of ELSDs, alone or in combination with ALSDs, particularly according to the dosing and dosing schedules described herein, can provide improved therapies that provide enhanced inhibition of cancer growth and/or reduced side effects, and allow for the use of reduced doses of ALSDs. More toxic drug substitutes, including ALSD and/or less frequent drug doses, such as co-administering ELSD and ALSD in specific ratios, amounts, and frequencies to capture the initial effect of ELSD and the maximal effect of ALSD.
For example, the advantages and improvements of THSD allow improved dosing mechanisms with less dosing frequency, including combination dosing mechanisms that combine ALSD and ELSD together. For example, without limitation, the ELSD may be administered first, providing a loading dose, and then one or more subsequent maintenance doses of the ALSD.
In a specific embodiment, there is provided a THSD compound of formula FIa (also referred to herein as "ELSD") as shown below, wherein the a-group is a pharmaceutically acceptable negatively charged anion; wherein X is halogen; wherein R1 is an alkyl residue optionally finally substituted by a residue selected from SO3, CO2 or NH2, and wherein each R1 alkyl chain may optionally be branched, and the branches may constitute one or more of an alkyl chain, an aromatic ring, a heteroaryl group, an aralkyl group, and wherein one or more positions of the chain or its branches may be unsaturated; wherein R2 and R3 are independently selected from H, an Electron Withdrawing Group (EWG), or an electron donor. Group (EDG); wherein L isESelected from alkyl linkers LE1And a carboxamide linker LE2Of the group in which LE1Is- (CH)2)n-O-and wherein LE2Is- (CH)2)n–CO–NH–(CH2)m-O-, wherein n-4-9, e.g. 4-6, and m-1-4, and wherein LEAnd statin residue Rs are thus linked by an ester bond, and wherein Rs is a statin residue. By its-CO-CH2-COH-CH2-CHOH-R moiety attached:
in a specific embodiment, methods are provided for using THSD compounds of formula FIIa (also referred to as "ALSD") as shown below, wherein the residues are designated for formula FIa above, and wherein LA is selected from alkyl linker LA1And a carboxamide linker LA2Group of wherein LA1Is- (CH)2)n) -NH-and wherein LA2Is- (CH)2)n–CO–NH–(CH2)m-NH-, wherein n-4-9, e.g. 4-6, m-1-4, wherein la and the statin residue rs are thereby linked by an amide bond, wherein R isSIs through its-CO-CH2-COH-CH2-a statin residue linked by a CHOH-R moiety:
in a specific embodiment, there is provided THSD wherein n-5, and preferably, the linker is L with n-5 and m-2E2Or LA2A linker; alternatively, X, R may be selected as described above1、R2And R3Or selected as follows: x ═ Cl, R1＝(CH2)4-SO3 -,R2＝H,R3H (e.g., THSD based on DZ 1).
In a specific embodiment, there is provided THSD, wherein a-The radicals may be selected from the group comprising I-,Cl-,Br-,OSO2R-,BF4 -,ClO4A group of.
In the detailed descriptionIn (1), THSD is provided, wherein RSIs the residue of a statin or a derivative thereof, which retains the binding to the active binding site of a statin. Statins include dihydroxyheptanoic acid units (DHHA), typically 3, 5-dihydroxyha units, more particularly 3R, 5R-dihydroxyha units, which are the primary pharmacodynamic carrier of statins; DHHA may be in the inactive closed ring lactone form of statins or in the active open chain carboxylic acid form (i.e., COOH-CH)2-CHOH-CH2-CHOH-CH2-CH2-Rs*) Are present. -CH2-RS*(or when linked: HMCD-LE/A-CO-CH2-CHOH-CH2-CHOH-CH2-CH2-Rs*Where rs is the remainder of the statin. After formation of a THSD as described herein, an open chain form exists and is attached, or is formed and fixed on the attachment.
In the example of THSD, RS-statin residues can pass through the ester bond (L) through its DHHA unit residue in open chain carboxylic acid form, respectivelyE) Or amide bond (L)A) Is connected to (L)E)/(LA) A connector.
In a specific embodiment, an ELSD of formula FIb is provided, wherein the HMCD dye residue is via LE2Linker (- (CH)2)n–CO–NH–(CH2)m-O-) binds to statins having a dihydroxyheptanoic acid unit (DHHA), as shown below, i.e., the statin residue-CO-CH2-CO-CH2-CHOH-Rs*I.e. statin residue-CO-CH2-CO-CH2-CHOH-Rs*Wherein R iss*Represents the statin residue minus DHHA residue, thus the remainder of the statin molecule:
in a specific embodiment, an ELSD of the formula FIC is provided, wherein the linker is L as described aboveE2A linker, said statin being simvastatin:
in a specific embodiment, there is provided an ELSD of formula FId, wherein the statin is simvastatin as described above and the dye is a DZ 1-based dye, wherein R is1Is (CH)2)4-SO3 -Wherein said linker is L with n-5 and m-2E2A linker wherein X ═ Cl, wherein R2And R3Is H:
the synthesis of compounds of formula FId is described in examples 1A-IV below, comparative compound 14(DZ2 b).
In a specific embodiment, there is provided an ALSD of formula FIIb, wherein the FIIb dye residue is via LA2Linker (- (CH)2)n–CO–NH–(CH2)m-NH-) conjugation to DHHA of statins as shown in FIIb:
in a specific embodiment, provided is an ALSD of formula FIIb, wherein the HMCD residue is via LA2The linker was coupled to simvastatin as shown in FIic:
in particular embodiments, provided are ALSDs of formula FIb or FIIb, wherein the HMCD residue is via L, as described hereinALinker (e.g. L)A1Or LA2) Coupled with simvastatin, and wherein X ═ Cl.
In a specific embodiment, there is provided THSD of formula FIb or FIIb as described herein, wherein n ═ 5.
In a specific embodiment, there is provided THSD of formula FIb or FIIb as described herein, wherein R1Is (CH)2)4-SO3。
In a specific embodiment, there is provided THSD of formula FIb or FIIb as described herein, wherein R2/3are H。
In a specific embodiment, there is provided THSD of formula FIb or FIIb as described herein, wherein n ═ 5, R1Is (CH)2)4-SO3 -And R is2/3Is H (THSD based on DZ 1).
In a specific embodiment, there is provided THSD of formula FIb or FIIb as described herein, wherein n-5 and m-3.
As an example, ALSD is shown below for FIId, where HMCD passes through LA2The linker is coupled to simvastatin, where n ═ 5 and m ═ 3 (- (CH)2)5–CO–NH–(CH2)3-NH-), wherein X ═ Cl, R1Is (CH)2)4-SO3 -，R2/3Is H)
The synthesis of compounds of formula FIId is described in examples 1A-IV below, comparative compound 7 ("DZ 2 a").
For comparison purposes, the following formulas FIII and FIV show HMCD statin conjugates of the immediate release ester (RRE) type (FIV is compound 8 of comparative examples 1A-IV, also designated herein as "DZ 2 c"). Lack of more stable L of RREE/LAThe linker, instead, comprises a labile LRR linker (e.g., of the formula- (CH)2)n-OC-O-)) that forms a labile ester bond with a statin, e.g., in its lactone form with the remainder of the statin DHHA unit. Such unstable esters rapidly release the statin under typical in vivo conditions, with significant release occurring over a short period of time (e.g., 1 hour or less, such as 30 minutes or less, or even 10 minutes or less).
In particular embodiments, to form a THSD, the method may begin with a HMCD comprising a carboxyl group (e.g., DZ 1). The amine or diamine may react with the carboxyl group of HMCD or with the carboxyl group of the DHHA unit of a statin to form the L to be formedE/LAA portion of the linker and then reacting the resulting amide group with the carboxyl group of the remaining portion (i.e., the carboxyl group of the dye or statin) to provide the desired THSD and LE/LAThe linker links the HMCD to the statin.
For example, to form ELSDs, a hydroxyalkylamine may be reacted with HMCD to provide the desired L upon reaction with the carboxyl group of a statin DHHA unitEA connector. Statins can be activated accordingly, as shown in fig. 1A-IV, which illustrate a DZ 1-based approach to form an exemplary ELSD (compound 14, DZ2 b). For example, as a first step, a hydroxyethylamide derivative of HMCD is formed, e.g., by reacting HMCD with ethanolamine to form HMCD hydroxyethylamide. In a subsequent step, HMCD hydroxyethylamide is reacted with the methyl derivative of the desired statin. Exemplary methods for comparing DZ1 and simvastatin shown in FIGS. 1A-III, simultaneously.
For example, to form ALSD, an alkyl diamine can be reacted with the desired statin to provide the statin in the derivative, providing the desired L upon reaction with the carboxyl group of HMCDALinker, the carboxyl group being located in DZ1 and simvastatin in FIGS. 1A-III corresponding to LAThe position of (a). For example, as a first step, an amine derivative of a statin is formed, for example by reacting the statin with an alkyldiamine (e.g., propane-1, 3-diamine) to form the amine derivative. In a subsequent step, the amine group of the statin derivative introduced is reacted with the carboxyl group of HMCD, in particular the terminal carboxyl group at the R4 residue position (L which will become THSD after reaction with an alkyldiamineAA portion of a connector). Exemplary methods for comparing DZ1 and simvastatin shown in FIGS. 1A-III are also disclosed.
In an example, the THSD may be a THSD of formula FIa or FIa, and R1 may be selected, for example, as shown in the following illustrative list, and alternatively, X may be a halogen selected from, for example (but not limited to), Cl and Br.
Each alkyl chain may optionally be branched, and the branches may constitute one or more of an alkyl chain, an aromatic ring, a heteroaryl group, an aralkyl group; one or more positions of the chain or branch may be unsaturated.
**R2And R3The groups may be independently selected from H, Electron Withdrawing Groups (EWG) or Electron Donating Groups (EDG). The following table further indicates example R2/R3And (4) grouping.
In an example, there is provided THSD of formula FIa or FIIa, wherein R1＝(CH2)4-SO3 -And X ═ Cl. In an example, there is provided THSD of formula FIa or FIIa, wherein R1＝(CH2)4-SO3 -X ═ Cl and R2/R3Independently selected as follows.
In an example, there is provided THSD of formula FIa or FIIa, wherein the statin is related to L of THSDEOr LAConnection ofThe body residues are conjugated. Preferably, the statin is linked by its open chain form of the dihydroheptanoic acid unit, as shown in figures 1A-I.
In the examples, the statin residue is linked as residue RS via its dihydroheptanoic acid unit (DHHA) to a linker (L each)EOr LAAnd) are typically 3R,5R dihydroheptanoic acid units, the latter being the key pharmaceutical package. DHHA is a modified hydroxypentanoate component that is structurally similar to the endogenous substrate HMG-CoA. Some statins constitute the unit in the open ring acid form (e.g., atorvastatin, fluvastatin, rosuvastatin, cerivastatin, pravastatin, pitavastatin), and others in the closed ring lactone form (e.g., simvastatin, lovastatin, mevastatin). In the THSD of the invention, the statin is linked to the linker through the terminal carboxyl residue of its DHHA unit and is immobilized in a form corresponding to the "open acid" form of DHHA (as in fig. 1A-I), unlike the linkage that occurs in the RRE ester.
In an example, statins may be selected as shown in the left column of the table below to provide the R shown in the right columnS*And (c) a residue.
In an example, the THSD comprises a statin Residue (RS), wherein the corresponding statin forming the residue described herein may be selected from the group comprising simvastatin, mevastatin, lovastatin, pravastatin, atorvastatin, fluvastatin, rosuvastatin, cerivastatin, pitavastatin and other derivatives attached to an operably linked region in a statin. He is the active binding site for statins. Statins are a group of structurally related HMG-CoA analogs that competitively inhibit HMG-CoA reductase in terms of substrate HMG-CoA binding. Statins generally act by binding to the active site of the enzyme, sterically preventing substrate binding, i.e. the HMG-COA moiety of the respective statin binds to the HMG-COA binding moiety of the enzyme active site, while the enzyme rearranges so that the rigid hydrophobic ring structure of ST should provide accommodation.
The structure of statins is generally composed of three parts: 1) an HMG-CoA analog; 2) a hydrophobic ring structure involved in enzyme binding; 3) pendant groups on the ring that affect solubility and pharmacokinetic properties (e.g., simvastatin, atorvastatin, fluvastatin and lovastatin. Statins are relatively lipophilic, whereas pravastatin and rosuvastatin are more lipophilic).
Surprisingly, the much larger THSD can provide one or more statins (cholesterol lowering, risk of certain cancers reducing) and/or the additional effects described herein while avoiding one or more side effects commonly associated with statins, or combined effects with statins. Statins and one or more other drugs/secondary active drugs (especially chemotherapeutic drugs such as cisplatin and its derivatives, gemcitabine, doxorubicin, taxane drugs such as paclitaxel and docetaxel) and antiandrogenic drugs such as abiraterone and benzazolamide).
Side effects associated with statins include, but are not limited to: headache, needlestick, nausea, dizziness, lethargy, upset stomach, vomiting, abdominal pain, abdominal cramps, abdominal distension, diarrhea, difficulty falling asleep, sensory discomfort, rashes and rare, joint pain, arthritis, muscle pain, tenderness, pain or weakness. Knee (myalgia), tenderness, muscle spasm, muscle inflammation, flushing of the skin, increased risk of cataracts, abnormal testing of liver function, liver problems (elevated liver enzyme levels), liver failure, increased risk of diabetes, skeletal muscle damage, rhabdomyolysis (especially when statins are used in combination with other drugs). With a higher risk of rhabdomyolysis). Common side effects of statins include joint pain, diarrhea, nausea, vomiting, abdominal cramps or pain, dizziness, somnolence, stomach upset, headache, difficulty falling asleep, abnormal liver function, muscle pain, tenderness, pain or weakness (myalgia) and muscle cramps, flushing of the skin. Serious side effects of statins include rhabdomyolysis, liver problems/failure, and diabetes.
Drug-drug interactions (DDIs) associated with statin-drug combinations with other drugs include, but are not limited to: rhabdomyolysis, for example, due to combination with other drugs with a high risk of rhabdomyolysis, and/or with drugs that increase the concentration of statins in the blood.
In an example, methods are provided wherein one or more THSDs are administered to a patient or group of patients having a higher risk of developing a tumor or cancer to reduce their risk of developing a cancer or tumor, or to slow the growth of or prevent the growth and/or spread of an existing tumor. These may include patients/patient groups with or at increased risk of cancer (particularly lung cancer and breast cancer), as determined by biomarkers or risk profiles, including environmental risk (smoking, exposure to chemicals, occupational exposure to dust) or a family history of one or more specific cancers, such as breast cancer. Detection of cancer and/or potential precancerous lesions, such as breast nodules.
In an example, methods are provided wherein one or more THSDs are administered to a patient group that is normally statin prescribed to reduce the risk of developing one or more cancers, including but not limited to: esophageal cancer, colorectal cancer, gastric cancer, hepatocellular cancer, and prostate cancer. For these cancers, statins and similar THSDs may be associated with reduced risk of cancer, which may avoid side effects of statins while reducing the risk.
In an example, a method is provided in which one or more THSDs are administered to cancer patients experiencing (or at higher risk of) statin side effects, such as patients with liver disease, pregnant women, and lactating women.
In an example, a method is provided in which one or more THSDs are administered to a cancer patient that experiences (or is at higher risk of) side effects due to drug-drug interactions of statins with other drugs. These patients also include patients taking one or more other drugs that may interact with statins, including but not limited to: protease inhibitors (aids treatment), clarithromycin, erythromycin, itraconazole, clarithromycin, diltiazem, verapamil, grapefruit juice, niacin or fibril drugs (which also reduce cholesterol low density lipoprotein levels).
In an example, a method of treating cancer or reducing the risk of future cancer (e.g., by improving mitochondrial or lysosomal function, e.g., reducing mitochondrial Oxygen Consumption Rate (OCR), increasing extracellular acidification rate ("ECAR"), and reducing or preventing deubiquitination provides a protein).
In examples, the THSD and methods described herein may be applicable for treatment or risk reduction associated with one or more of the following cancers: prostate cancer, pancreatic cancer, lung cancer, NSCLC (non-small cell lung cancer), SCLC (small cell lung cancer), kidney cancer, lymphoma, colorectal cancer, skin cancer, HCC cancer and breast cancer, lung cancer squamous cell carcinoma, anal cancer, glioblastoma, head and neck epithelial tumors, and the like. Non-small cell lung cancer can include squamous cell carcinoma, adenocarcinoma (mucinous cystadenocarcinoma), large cell lung cancer, rhabdoid cancer, sarcomatoid cancer, carcinoid, salivary gland-like cancer, adenosquamous carcinoma, papillary adenocarcinoma, and giant cell carcinoma. Small cell lung cancer may include combined small cell carcinoma. Non-lung cancers may include sarcomas, lymphomas, immature teratomas, and melanomas. Without wishing to be bound by theory, it is believed that THSD has broad applicability to different types of cancer, tumors, and metastases thereof.
In embodiments, THSD may be particularly beneficial in treating lung cancer patients or patients with a higher than average risk of developing lung cancer (e.g., smokers). Lung cancer may include two major types of cancer, non-small cell lung cancer (NSCLC) and small cell lung cancer. The most common lung cancer is lung Adenocarcinoma (AC), which is one of three NSCLC types of lung cancer (the other two NSCLC cancers are squamous cell carcinoma and large cell carcinoma). Unlike Small Cell Lung Cancer (SCLC), non-small cell lung cancers, including AC, typically respond to a variety of mild non-chemotherapeutic treatment regimens, including TKI. SCLC is one of the most closely related forms of smoking, is more difficult to treat, usually requires chemotherapy, and is susceptible to development of resistance following chemotherapy. THSD allows for treatment despite acquired resistance (e.g., resistance to TKI and/or chemotherapeutic drugs) in patients, or avoids the development of resistance in these cancers after treatment.
In an example, a method of treating cancer by administering one or more THSDs to a patient exhibiting brain tumor or brain metastasis is provided. THSD can cross the Blood Brain Barrier (BBB) and thus it can provide its anti-cancer effect against brain tumors or brain metastases of various cancers, especially the cancers described above, which can be treated according to the methods of administration described herein, including in particular systemic methods of administration.
In an example, a pharmaceutical formulation comprising one or more THSDs is provided. Multiple THSDs may be administered in a single or corresponding multiple pharmaceutical formulations. As described below, multiple compositions may be co-administered in a coordinated dosing regimen. The pharmaceutical formulations are intended for human or veterinary use and comprise one or more compounds of the invention (or salts, solvates, metabolites or derivatives thereof) with one or more pharmaceutically acceptable carriers and/or one or more excipients and/or one or more active agents. One or more carriers, excipients, and/or active agents may be selected so as to be compatible with the other ingredients of the formulation and not to cause undue damage to the recipient. Such vectors are known in the art and may be selected as would be apparent to one of ordinary skill in the art.
In an example, a pharmaceutical kit is provided. The kit may comprise one or more THSDs or a composition comprising THSD, preferably in the form of a salt thereof, and typically a pharmaceutically acceptable carrier. The kit may further comprise conventional kit components, such as a needle for injecting the components, one or more vials for mixing the components, and the like, as will be apparent to those of ordinary skill in the art. Further, instructions may be included in the kit, for example, as inserts or labels, indicating the number of components, instructions for mixing the components, and medication/co-medication procedures. In particular, the kit may include instructions for the co-administration mechanism of multiple THSDs in a coordinated administration mechanism as described below.
In examples, the route of administration of the THSD and pharmaceutical formulation may be systemic (administration to the circulatory system to affect the entire body) or local/tissue specific, and may include, but is not limited to: oral, intraperitoneal, subcutaneous, intramuscular, transdermal, rectal, vaginal, subcutaneous UAL, intravenous, buccal, or inhalation. In some examples, the pharmaceutical formulations of the present invention contain pharmaceutically acceptable excipients suitable to render the compound or mixture administrable by the routes of administration described above. Alternatively, the compounds and pharmaceutical formulations may be administered via the urogenital tract (e.g., via internal organs, local lesions, skin patches or via instillation (e.g., bladder, vaginal catheter) access) or via implantation (e.g., periimplantation) of the vaginal channel.
Advantageously, in an example, THSD can be administered to a patient at a low frequency, avoiding the need for daily or multiple daily administrations. For example, depending on the dose, the dose may be administered once every 2, 3, 4,5, 6, or 7 days or longer intervals. Preferably once weekly. Even longer intervals, such as once every 2, 3 or 4 weeks, may be possible depending on the dose of THSD at each dose and the individual needs of the patient, including the degree of desired anti-cancer effect, the type of cancer and the rapidity of tumor growth or/and metastatic spread in the patient, and considerations of the level of side effects. And (6) acceptance. Without wishing to be bound by theory, it is believed that this is due to the tight binding of HMCD by this novel statin derivative, which allows it to persist in cancer cells for long periods of days, possibly weeks or months, thus providing long-term anti-cancer activity while preventing side effects.
Advantageously, in an example, the ELSD and the ALSD may be co-administered, e.g., administered in a coordinated dosing schedule. In particular, the first dose of ELSD may be followed by one or more subsequent maintenance doses of ALSD. Further doses of ALSD may then be administered to THSD as described above, e.g., once every 2-7 days, every week, or every 2-4 weeks.
In embodiments, the ELSD and ALSD may be co-administered simultaneously or subsequently in a coordinated dosing schedule. For example, a loading dose of an ELSD may be administered first during a first time interval, followed by one or more subsequent maintenance doses of the ALSD at the beginning of a second time interval. For example, depending on the route of administration, a first amount of about 0.1mg to about 10mg ELSD/kg body weight, preferably about 0.1mg to about 6mg/kg body weight (e.g., about 0.1mg to about 2.5mg or about 0.1mg to about 1.0mg/kg body weight) can be administered to the patient, e.g., intravenously or orally or by any convenient mode of administration (loading dose). The ELSD loading dose may be divided into multiple doses over a first time interval, e.g., 4 to 72 hours, e.g., about 4 hours, 8 hours, 16 hours, 24 hours, 32 hours, 48 hours, or 72 hours. Advantageously, the loading dose of ELSD is lower than the maintenance dose of ALSD. Without wishing to be bound by theory, it is believed that ELSD is effective at lower concentrations and allows the body to experience THSD effects first, as compared to ALSD, which, especially when used alone, may require higher doses (although higher doses may provide better effects as compared to ELSD).
In an example, one or more ALSD maintenance doses can be administered during a second, and optionally further, subsequent time interval that follows. For example, one or more maintenance doses may be administered in an amount of about 0.1mg to about 10mg ALSD/kg body weight, preferably about 1 to about 10mg/kg body weight per dose, e.g., about 1 to about 8mg, about 1 to about 6mg, about 1mg to about 4mg, or about 1 to about 2mg/kg may be administered to the patient, depending on the route of ADMI. Administration and administration frequency. The subsequent dose can be a single subsequent dose, or multiple subsequent doses at the same or different intervals (e.g., daily, over 2-7 days, weekly, etc.). Preferably, the ELSD loading dose may be administered on day 1, and after about 24 hours on day 2, one or more higher ALSD maintenance doses may be administered as described. Control, such as daily or less frequent intervals. Each maintenance dose of ALSD may be higher or lower than the loading dose of ELSD. ELSD: suitable ratios of ALSD may include, for example, from about 20: 1 to about 1: 20 (ELSD: ALSD), for example about 10: 1 to about 1: 10 (ELSD: ALSD), for example about 1: 10. 1: 9. 1: 8. 1: 7. 1: 6. 1: 5. 1: 4. 1: 3. 1: 2. 1: 1. 2: 1. 3: 1. 4: 1. 5: 1. 6: 1. 7: 1. 8: 1. 9: 1 or about 10: 1 (ELSD: ALSD). Advantageously, the loading dose of ELSD is lower than the maintenance dose of ALSD.
Alternatively, the ELSD loading dose may be administered concurrently with the ALSD maintenance dose during the first time interval, and one or more of the ALSD maintenance doses described above may be administered subsequently at the beginning of the second time interval.
Additionally, the ELSD and ALSD may be co-administered in specific proportions, whether for each dose (including the first dose in the first time interval) or only for the maintenance dose beginning in the second time interval. The ratio may be about 20: 1 to about 1: 20 (ELSD: ALSD)), for example, about 10: 1 to about 1: 10 (ELSD: ALSD), for example about 1: 10. 1: 9. 1: 8. 1: 7. 1: 6. 1: 5. 1: 4. 1: 3. 1: 2. 1: 1. 2: 1. 3: 1. 4: 1. 5: 1. 6: 1. 7: 1. 8: 1. 9: 1 or about 10: 1 (ELSD: ALSD).
Surprisingly, THSD has similar or better growth inhibitory effect on tumors in vivo compared to Tyrosine Kinase Inhibitors (TKIs), e.g., example 9 and fig. 9a 1-4. Thus, advantageously, THSD is useful for TKI-administered patients, which is often associated with rapid development of drug resistance in TKI-treated cancer cells; alternatively, THSD may be administered in place of TKI (thereby avoiding the development of resistance while providing similar or better growth inhibitory effects). In contrast to TKI), patient groups generally benefit from the use of TKI.
TKI is an agent or drug that inhibits tyrosine kinase. Tyrosine kinases are enzymes that activate many proteins through a signal transduction cascade, including EGFR (EGFR-TKI), ALK (ALK-TKI), and ROS1(ROS-TKI), among others. For example, proteins are activated by adding phosphate groups (phosphorylation) to the protein, which is a step in TKI inhibition. TKIs are commonly used as anticancer drugs to combat various cancers, to inhibit the growth of cancer cells (to arrest or slow the growth of tumors), and/or to induce apoptosis (cell death), often leading to tumor atrophy. Gene rearrangement events involving related tyrosine kinase receptor genes (such as the EGFR, ALK, and ROS1 genes) have been described to occur in various cancers, including lung cancer. Various cancers and tumors, including the lung (e.g., NSCLC/AC) respond to TKI, including one or more of EGFR-TKI, ALK-TKI, and ROS-1-TKI. In all TKIs, it is common for drug resistance to develop after TKIs given to cancer patients.
TKI, especially EGFR-TKI, is used for treating various cancers, especially non-small cell lung cancer (NSCLC).
The Epidermal Growth Factor Receptor (EGFR) is a member of the ErbB receptor family, and is a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2/neu (ErbB-2), Her 3(ErbB-3) and Her 4 (ErbB-4). Mutations that affect EGFR expression or activity can lead to various types of cancer, including, for example, NSCLC, lung Adenocarcinoma (AC), anal carcinoma, glioblastoma, head and neck epithelial tumors; oncogenic mutations include EGFRvIII (e.g., glioblastoma), and other aberrations include amplification or dysregulation. Major activating EGFR mutations include, but are not limited to, the L858R mutation, exon 19(Del19), and deletion (Del) of T790M; further mutations include, for example, E746-A750 deletions, L747-E749 deletions, A750P mutations, and C797S mutations, and the like. MET amplification is another resistance mechanism to EGFR-TKIs, including AZD9291 and CO 1686.
Anaplastic Lymphoma Kinase (ALK), also known as ALK tyrosine kinase receptor, is an enzyme encoded by the ALK gene. ALK abnormalities play a role in cancer, including, for example, anaplastic large cell lymphoma, NSCLC, lung Adenocarcinoma (AC), neuroblastoma, inflammatory myofibroblast tumor, renal cell carcinoma, esophageal squamous cell carcinoma, breast cancer (particularly of the inflammatory subtype), colon adenocarcinoma, glioblastoma multiforme, anaplastic thyroid carcinoma, and the like.
Proto-oncogene tyrosine protein kinase (ROS or ROS1) is an enzyme encoded by the ROS1 gene and has a structure similar to that of the ALK protein. ROS is a receptor tyrosine kinase, similar in structure to Anaplastic Lymphoma Kinase (ALK) protein. Gene rearrangement events involving ROS1 have been described in lung cancer and other cancers, and this tumor was found to respond to TKI.
THSD may be used in cancer patients, particularly in groups of cancer patients who respond or may respond to Tyrosine Kinase Inhibitor (TKI) treatment, depending on the type/grade of the tumor, the histology of the tumor, the pre-treatment, resistance to one or more TKIs, or various biomarkers, including mutations or aberrations of one or more TKIs. Or more genes that affect the activity of cellular tyrosine kinase receptors (including, for example, EGFR, ALK, ROS1, and BRAF). These patient groups are typically treated with corresponding inhibitors, including, for example, EGFR-TKI, ALK-TKI, ROS-TKI, and BRAF-TKI. For example, these patient groups include non-small cell lung cancer (NSCLC), particularly Adenocarcinoma (AC) NSCLC, which is a common type of lung cancer with increasing incidence. A subset of NSCLC patients contain one specific oncogenic driver, namely, an activating EGFR, ALK, or ROS1 aberration (including chromosomal rearrangements, translocations, or mutations); these carcinogenic drivers appear to be present almost exclusively at ac.
EGFR-TKI includes, but is not limited to: gefitinib, imatinib, erlotinib, brigatinib, dacrometinib, lapatinib, vandetanib, afatinib, aximin (AZD9291), CO-1686, HM61713, nanozatinib (EGF816), imatinib, PF-06747775, YH5448, avitinib (AC0010), rokitinib and cetuximab; can be used for treating various cancers, including partial cancers. Lung cancer, non-small cell lung cancer, colon cancer, metastatic colorectal cancer, head and neck cancer, and the like.
According to its mechanism, EGFR-TKI can be divided into three groups: first, second or third generation TKI/EGFR-TKI. Examples of first generation EGFR-TKIs include, for example, gefitinib, erlotinib, and erlotinib. Examples of second generation EGFR-TKIs include afatinib and dacrometinib. Second generation EGFR-TKIs generally bind irreversibly to EGFR and other ErbB family tyrosine kinases. Uses for second generation EGFR-TKIs include, for example, first line treatment of advanced NSCLC containing activating EGFR mutations. Examples of EGFR-TKI of generation 3 include, for example, oxitinib (AZD9291), Co-1686, HM61713, nanotinib (EGF816), Omtinib, PF-06747775, Afatinib avitinib (AC0010), and Rotinib.
TKIs of all generations show a tendency to develop resistance to treatment of tumor/cancer cells, and second and third generation drugs are commonly used to treat cancers with associated genetic mutations, particularly the Epidermal Growth Factor Receptor (EGFR) gene, and/or exhibit resistance to first generation TKIs. The grouping is based on the mechanism and corresponding patient group that may most likely benefit from the drug, and may also determine the risk of developing drug resistance, e.g., mutations that produce one or more different mutations, particularly EGFR mutations, or mutations that allow EGFR-related mechanisms to be bypassed. First generation EGFR-TKI was efficacious, for example, as a first line treatment for advanced non-small cell lung cancer containing activating EGFR mutations (exon 19(Del19) and exon 21L858R mutations); more mutations, particularly EGFR T790M resistance mutations (EGFR T790M), have occurred in these patients. The 2 nd and 3 rd generation EGFR-TKIs were designed to improve first generation drugs, in particular to more effectively inhibit EGFR and/or overcome various mutations produced in patients, particularly after first generation treatment, such as EGFR T790M.
Surprisingly, THSD was able to bypass TKI resistance and provide growth inhibition, e.g., in example 9 and fig. 9a1-4, for TKI-resistant tumors established in vivo by TKI-resistant human cancer cells; thus, THSD may be effective in patient groups exhibiting such resistance, particularly in patients and patient groups with the following characteristics: ergoline therapy using TKI inhibitors, including first, second or third generation TKI inhibitors, is particularly useful for patients who are suitable for or have been treated with third generation TKI inhibitors, or patients with TKI or third generation TKI resistant tumors. As shown in example 9, such evading TKI resistance may not require co-administration of TKIs (although co-administration may be beneficial for a given TKI). Thus, THSD alone may be effective in inhibiting cancers susceptible to drug resistance, particularly lung cancer, including but not limited to NSCLC. In particular, THSD alone (i.e. without TKI) is effective in providing growth inhibition in non-small cell lung cancer as an alternative treatment to third generation EGFR inhibitors, especially aximotinib (AZD9291) or following aximotinib treatment, especially following development of drug resistance.
Examples of first generation EGFR-TKIs include gefitinib and erlotinib. Examples of second generation EGFR-TKIs include afatinib and dacrometinib. Second generation EGFR-TKIs generally bind irreversibly to EGFR and other ErbB family tyrosine kinases. The EGFR-TKI used for the second generation comprises EGFR mutation activated by advanced NSCLC of first-line treatment. The third generation EGFR-TKI may include, but is not limited to, one or more of the following: oximetinib (AZD9291), Rotinib (CO-1686), HM61713, Nazaldini (EGF816), Omotinib (HM61713), PF-06747775, YH5448, Afatinib, Avertinib (AC0010) and ASP 8273. EGFR-TKI of generation 3 is generally effective in patients who develop TKI resistance of generation 1 or generation 2. Third generation EGFR-TKIs are typically selective EGFR mutants and EGFR wild type cells (WT) that remain, i.e., they have greater activity, e.g., at least 10, 100, 200-fold or more, on EGFR mutant cells than on EGFR Wild Type (WT) cells. In addition, the 3 rd generation EGFR-TKI has activity or inhibitory effect on both EGFR activating and resistance mutations (especially T790M resistance mutation). For example, the 3 rd generation EGFR-TKI (e.g., axitinib, CO-1686, and HM61713) selectively and irreversibly targets the sensitizing/activating EGFR mutation and the T790M resistance mutation while retaining the wild-type EGFR tyrosine kinase.
Oximetinib is a monoanilino pyrimidine that selectively and irreversibly targets sensitizing EGFR mutations and T790M resistance mutations while retaining wild-type EGFR tyrosine kinase. Specifically, oxitinib is much less effective in inhibiting EGFR phosphorylation in wild-type cell lines, e.g., L858R/T790M inhibits EGFR by 100-fold over 200-fold, and is useful for treating drug-resistant or drug-resistant-prone cancers, especially non-small cell lung cancer.
In an example, THSD can be administered to a cancer patient with resistance to TKIs as described herein to inhibit the growth of cancer cells while avoiding resistance. These cancers include advanced Epidermal Growth Factor Receptor (EGFR), ALK and/or ROS1 mutation-positive tumors, which are common in non-small cell lung cancer (NSCLC).
Thus, groups of patients who may benefit particularly from administration of THSD may include those patients with activating mutations of EGFR, ALF, or ROS1 and/or resistant mutations of EGFR, ALF, or ROS1, including especially mutations obtained during treatment with TKI, EGFR-, ALF-, or ROS1-TKI, including first-, second-, or third-generation TKIs or EGFR-TKIs. For example, patients who develop the T790M EGFR mutation are very common in advanced non-small cell lung cancer patients who progress after receiving first-line EGFR-TKI treatment, such as passage 1 or passage 2 EGFR-TKI.
In particular, THSD can be used in patient groups that are commonly treated with ALK-TKI (inhibitors of the ALK tyrosine kinase receptor or CD 246) to avoid development of resistance. Like other TKIs, resistance to ALK-TKI is also common. In addition, THSD can be administered to patients following treatment with ALK-TKI to overcome acquired resistance to ALK-TKI. The patient group includes those patients with inherited ALK-abnormally positive cancers, such as metastatic NSCLC. The group of patients with ALK-positive cancer, in particular NSCLC, typically includes non-smokers, younger patients, adenocarcinoma histology, women, and/or patients with pathological features including solid morphology and/or withdrawal of cells.
ALK aberrations may include chromosomal rearrangements leading to polymeric genes, as seen in ALCL and NSCLC. Other changes include increased ALK replication numbers and activation of ALK mutations. Aberrations such ALK mutations or translocations are known to occur in a variety of cancers, including NSCLC, Anaplastic Large Cell Lymphoma (ALCL), inflammatory myofibroblasts, diffuse large B-cell lymphoma, colon cancer, renal cell carcinoma, breast cancer, esophageal cancer, and neuroblastoma.
Examples of ALK-TKIs include brigatinib, crizotinib, cetitinib, altenib, and entinib (RXDX-101). Crizotinib (PF-02341066) is the first generation ALK-TKI for ALK-positive NSCLC and ROS 1-positive NSCLC, especially locally advanced and/or metastatic NSCLC. It has an IC50Against 250-300nm EML 4-ALK. For example, ceritinib is used for ALK-positive metastasis NSCLC.
Likewise, THSD can be used in a patient group for ROS-TKI (proto-oncogene tyrosine protein kinase ROS or ROS1 inhibitor) treatment. The patient group includes those cancer patients who are positive for a gene ROS1 aberration, such as a polymerization or mutation (e.g., metastatic NSCLC), and patients who develop resistance to ROS-TKI treatment. Cancers that may be useful for being positive for an abnormal ROS1 include, but are not limited to: glioblastoma, lung cancer including lung adenocarcinoma, ovarian cancer, sarcoma, cholangiocarcinoma, cholangiosarcoma, inflammatory myofibroblast carcinoma, gastric cancer, colorectal cancer, glenoid melanoma, angiosarcoma, etc.
Examples of ROS-1 inhibitors include crizotinib, entitinib, loratinib (PF-06463922), cetitinib, TPX-0005, DS-6051b, and carbozatinib. Ciclopirox for metastatic ROS1 positive NSCLC). Carboplatin is used for metastatic medullary thyroid carcinoma and renal cell carcinoma.
Some TKIs are useful for treating a variety of malignant tumor mechanisms, and may be used in a variety of patient groups, such as those EGFR-, ALK-, or ROS/ROS 1-positive (EGFR +, ALK +, ROS +), i.e., patients with genetic aberrations affecting these genes encoding the respective tyrosine kinase enzymes. For example, entiretiib (Entritinib) is a TKI containing three TRK proteins (encoded by three NTRK genes, respectively) as well as ROS1 and ALK receptor tyrosine kinases. Similarly, cyproconazole inhibits ALK and ROS 1.
In an example, the active ingredient (THSD and optional secondary drugs/active agents, such as chemotherapy or antiandrogens) may be mixed or compounded with conventional, pharmaceutically acceptable excipients. The mechanism of administration, carrier, excipient or vehicle should generally be substantially inert with respect to activity, as will be understood by those of ordinary skill in the art. Illustrative methods, carriers, excipients and carriers are described, for example, in Remington's pharmaceutical Sciences,18th ed. (1990), the disclosure of which is incorporated herein by reference. The excipient must be "acceptable", i.e., compatible with the other ingredients of the formulation and not deleterious to the recipient.
In an example, THSD (or a combination of ELSD and ALSD, co-administered as described above) can be co-administered with other drugs, either simultaneously or subsequently, in a coordinated schedule of administration. Such drugs for co-administration with THSD include, inter alia, drugs that readily induce drug resistance when administered alone, such as chemotherapy, e.g., cisplatin and its derivatives, gemcitabine and doxorubicin, antiandrogen drugs, e.g., abiraterone and enzalutamide, and taxane drugs, e.g., docetaxel and paclitaxel. Without wishing to be bound by theory, androgen blockade in combination with THSD therapy may provide an additive or synergistic therapeutic effect.
In the examples, taxanes used for co-administration (also known as taxanes) are structurally a class of diterpenes, originally identified from the genus taxus (yew wood), and are drugs used in chemotherapy; they comprise a core of a taxane, typically a nuclear ring of members 6/8/6 or 6/10/6. The paclitaxel may include one or more of docetaxel (taxotere), paclitaxel (taxol), and carboplatin. They may also include one or more arabixanes, a class of molecules with unconventional core 5/7/6-type ring structures, such as, but not limited to, taxol a. The core carbon backbone of conventional taxanes has a 6-membered a ring, an 8-membered B ring, and a 6-membered C ring, combined together. For the traditional side chain, the arabihexanes contains three altered ring structures, a 5-membered a ring, a 7-membered B ring and a 6-membered C ring (in combination with the traditional side chain). In addition to paclitaxel A, 11(15 → 1) arabixanes included brevifolic acid and TPI287 (formerly ARC-100). Other taxanes include paclitaxel B (an Obeotaxoid with a taxane ring of 11(15 → 1)).
In an example, pharmaceutical formulations can be conveniently prepared in unit dosage form by various methods well known in the medical arts, e.g., presenting the formulation in a form suitable for delivery, e.g., forming an aqueous suspension, formulating a tablet or encapsulating a powder in a capsule, e.g., releasing the powder at a particular time, stage or location of digestion, and/or protecting the powder from gastric acid. The dosage form may optionally contain one or more adjuvants or ancillary pharmaceutical ingredients useful in the formulation, including but not limited to mixtures, buffers, and solubilizers.
In examples, parenteral dosage forms of pharmaceutical formulations (i.e., dosage forms that bypass the gastrointestinal tract) include, but are not limited to, solutions ready for injection, dry products ready for dissolution or suspension in a pharmaceutically acceptable carrier for injection, suspensions ready for injection, and emulsions. In addition, controlled release parenteral administration forms can be prepared for administration to a patient, including, but not limited to, sustained release tablets, tablets or capsules for systemic or tissue-specific delivery,-type and other implantable forms of administration.
In examples, suitable carriers that can be used to provide parenteral dosage forms include, but are not limited to: sterile water; water for injection; brine; a glucose solution; aqueous vehicles (e.g., sodium chloride injection, ringer's injection, dextrose-sodium chloride injection, and lactated ringer's injection); water soluble carriers (e.g., ethanol, polyethylene glycol, and propylene glycol); and non-aqueous vehicles (e.g., corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate). Compounds that alter or modify the solubility of pharmaceutically acceptable salts of the compounds of the present invention as disclosed herein may also be incorporated into parenteral dosage forms of the invention, including conventional and controlled release parenteral dosage forms.
In an example, formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may further contain other agents, such as antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient. The formulations may include aqueous and non-aqueous sterile suspensions containing suspending agents and thickening agents.
In the examples, sterile injectable preparations, e.g., injectable aqueous or oleaginous suspensions, may be formulated as is well known in the art, e.g., using suitable dispersing or wetting agents and suspending agents. The injectable formulations include injectable solutions, suspensions or emulsions in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol. Acceptable carriers and solvents include water, ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or triglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
In an example, compositions for rectal or vaginal administration include suppositories, which can be prepared by mixing the active agent with a suitable non-irritating excipient or carrier which is solid at room temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity (e.g. cocoa butter, polyvinyl glycan) to release the active agent.
In examples, dosage forms suitable for oral or sublingual administration include tablets, troches, capsules, pellets, suspensions, syrups, wafers, chewing gums and the like, prepared as is well known in the art. The amount of active in such dosage forms can be adjusted by one of ordinary skill depending on the desired frequency of administration and whether a sustained release formulation is being prepared. Syrup formulations typically consist of a suspension or solution of the active agent or its salt in a liquid carrier (e.g., ethanol, glycerol, or water) and a flavoring or coloring agent.
In examples, solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active agent is mixed with at least one inert substance: a) fillers, extenders or diluents (e.g., starch, lactose, sucrose, glucose, mannitol, silicic acid and mixtures thereof), b) binders (e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, acacia and mixtures thereof), c) humectants (e.g., glycerin), d) disintegrating agents (e.g., agar-agar, calcium carbonate, potato starch, tapioca starch, alginic acid, certain silicates, sodium carbonate and mixtures thereof), e) solution retarders (e.g., paraffin), f) absorption enhancers (e.g., quaternary ammonium compounds and mixtures thereof), g) wetting agents (e.g., cetyl alcohol, glycerol monostearate and mixtures thereof), h) absorbents (e.g., kaolin, bentonite and mixtures thereof) and i) lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium carbonate, and mixtures thereof), Sodium lauryl sulfate and mixtures thereof) and mixtures thereof. Such dosage forms may also include other substances, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose, or buffering agents, especially in capsules, tablets and pills.
In the examples, similar solid ingredients may also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or high molecular weight polyethylene glycols. Alternatively, the active agent may be co-administered in microencapsulated form with one or more excipients.
Various solid dosage forms (e.g., tablets, dragees, capsules, pills, and granules) can be prepared with coatings and shells (e.g., enteric coatings, controlled release coatings, and other coatings well known in the pharmaceutical formulating art). They may optionally contain opacifying agents and may also be of a composition that they release the active agent in a delayed manner only, or preferentially, in a certain part of the intestinal tract. Entrapped agents that may be used for delayed or extended release include polymeric substances and waxes.
In an example, the active agent may be present in the form of a salt, which may be particularly suitable for use in the treatment of cancer. The salts of the invention may be administered to a patient in a variety of forms depending on the route of administration, the salt involved and the cancer being treated. For example, an aqueous component or suspension of the salt may be injected systemically or through a particular tissue (e.g., in the form of a drug matrix by injection or surgical implantation) at a desired location. For example, the particular technique of administering the drug to the substrate may depend on the shape and size of the substrate involved. In some examples, the salt is introduced substantially homogeneously into the tumor to reduce the incidence of the tumor in cold (untreated) areas. In certain examples, the salt is administered in combination with a pharmaceutically acceptable carrier. Various pharmaceutically acceptable carriers can be provided and can be combined with salts, as will be apparent to those of ordinary skill in the art.
In the examples, the effective amount, toxicity and therapeutic effect of the active agent and/or its dosage form can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., determining LD50(dose leading to death in half of the population) and ED50(dose with therapeutic effect on half population). The dosage may vary depending on the dosage form employed and the route of administration employed. The dose ratio of toxicity to therapeutic effect is a therapeutic index and can be expressed as the ratio LD50/ED 50. The therapeutically effective dose can be estimated initially from cell culture analysis. In addition, doses can be formulated in animal models to determine circulating plasma concentration ranges, including IC as determined in cell culture50I.e., the concentration of the compound of the invention that achieves half-maximal inhibition of symptoms in the case of cancer (e.g., inhibition of growth of cancer cells). In addition to cell culture, animals may also be usedModels, particularly mammals, including mice, rats, guinea pigs, rabbits, pigs, dogs, and other animals. Levels in plasma can be measured by High Performance Liquid Chromatography (HPLC). The effect of any particular dose can be monitored by biological tests well known in the medical arts.
In an example, the dosage of a pharmaceutical formulation as described herein can be determined by a physician and adjusted as necessary to accommodate the observed therapeutic effect. With respect to the duration and frequency of treatment, a skilled clinician will typically monitor the subject to determine when treatment provides a therapeutic benefit, and to determine whether to increase or decrease the dosage, increase or decrease the frequency of administration, stop the treatment, resume the treatment, or make other changes. A treatment regimen. The dosing schedule/regimen may vary, for example, once per week, once per day, or at specific predetermined intervals, depending on a number of clinical factors, including the sensitivity of the subject to each active agent.
In an example, a pharmaceutical formulation comprising one or more active agents (i.e., one or more of THSD, ALSD, and ELSD, optionally in combination with one or more other/secondary drugs) can be administered to a patient or to a tumor, cancer, or precancerous cell of the patient in vivo or in vitro at an effective dose, specifically, to inhibit tumor, cancer, or precancerous cell growth, or to optionally induce apoptosis, thereby treating the cancer or tumor, or reducing the risk of cancer development or increasing the risk of tumor growth, for example, in a particular group of patients having a higher risk of cancer and/or tumor development than average. One or more active agents may be administered simultaneously, or may be administered according to a particular dosing regimen, e.g., as described above. It will be apparent to one of ordinary skill that the dosing regimen will generally take into account factors such as the concentration of the active agent in the blood and the half-life of each active agent.
In an example, an effective dose of a pharmaceutical formulation comprising one or more activities may be administered to a patient one or more times. The pharmaceutical formulation may also be administered over a period of time, for example over a period of 5 minutes, 10 minutes, 15 minutes, 20 minutes or 25 minutes. If necessary, the administration may be repeated periodically, for example, 3 hours, 6 hours, 12 hours or more per hour, or one month, two months, three months, four months or more per two weeks (i.e., every two weeks). In some cases, subsequent treatments may be performed on a less frequent basis following the initial treatment regimen. For example, after three months of administration every two weeks, the administration may be repeated once a month for six months or one year or more. Administration of a composition comprising one or more components active in a coordinated dosing schedule may be adjusted accordingly to ensure exposure to multiple active agents, e.g., simultaneous exposure to two or more active agents (e.g., ALSD and ELSD, or one or more THSD and a secondary drug), e.g., a reduction in the level of a biomarker or one or more symptomatic consequences, e.g., inhibition of cancer or tumor cell growth, inhibition of tumor growth, reduction of tumor volume/tumor shrinkage, reduction in formation of metastases, or a reduction in risk thereof.
In an example, two or more active agents can be administered sequentially; for example, the ELSD and ALSD may be administered one after the other, e.g., the ELSD prior to the ALSD, or vice versa, for a period of time prior to the administration of the second active agent. Preferably, the ELSD is administered days, hours or minutes prior to the administration of the ALSD (or vice versa). For example, the period of time may be about 20-24 hours before, about 15-20 hours before, about 12-15 hours before, about 10-12 hours before, about 8-10 hours before, about 2-8 hours before, about 1-6 hours before, about 1-4 hours before, about 1-3 hours before, about 1-2 hours before, about 0.5-1.5 hours before, or about 45 minutes before. About 30 minutes, or 15 minutes prior to the second administration. Also and/or additionally, one or more THSDs (ELSD and/or ALSD, simultaneously or sequentially) may be administered prior to administration of the second drug, or vice versa.
In an example, the amount and/or concentration of the one or more active agents may depend on the typical dose of a particular formulation and its route of administration, and may be suitable for exposing a tumor, cancer, or precancerous cell to a concentration of about 0.1 to about 100 μm. For ALSD, such as DZ2a, the concentration can be about 0.5 to about 50 μm, such as about 16 μm, and can be combined with an ELSD (DZ2b) of about 0.5 to about 50 μm, such as about 16 μm. It will be apparent to the ordinarily skilled artisan that all amounts and concentrations will need to be adapted to factors including the individual patient's environment, the type of cancer, and the duration of treatment.
In examples, the amount and/or concentration of one or more active agents in a pharmaceutical formulation may be based on weight, moles, or volume. In an example, a pharmaceutical formulation may comprise about 0.01% -99%, 0.05% -90%, 0.1% -85%, 0.5% -80%, 1% -75%, 2% -70% or 3% -65%, 4% -60% or 5% -50% of an active agent. The pharmaceutical formulation may comprise at least 0.0001%, at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, or at least 15% of each active agent. Alternatively or additionally, the pharmaceutical formulation may contain up to a maximum of about 0.0001%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, or 15% of each active agent.
In an example, a dual-functional method of cancer therapy or cell suppression (inhibition of growth or development of cancer cells or precancerous cells, tumor shrinkage) and identification/localization (e.g., by imaging, particularly NIR imaging) is provided. Wherein the cancer cells, precancerous cells, or tumors are additionally identified, imaged, and/or localized in a patient in need of the treatment. The method may comprise providing and administering one or more THSDs to a patient, and performing optical imaging of the one or more THSDs. The method may comprise providing one or more ELSDs and administering them to any patient or sequentially further administering to one or more patients with an ALSD; and optical imaging for ELSD or ALSD or both. Preferably, the ALSD and ELSD are administered on a coordinated dosing schedule, and the optical imaging is performed after administration of at least one of the ALSD and ELSD. Preferably, the first loading dose is a dose of ELSD, and imaging can be performed after administration of the ELSD, or can be performed after one or more subsequent doses of the ELSD or an optional ALSD. This allows visual tracking of the progress of cytostatic or therapeutic treatment (e.g. growth arrest, disappearance or contraction of a tumour, cancer cell or precancerous lesion), in turn adjusting the dose of one or more THSDs and optional secondary active agents, and/or determining the location of a tumour and/or metastasis. The near infrared spectral region of THSD. In various examples, imaging can be performed, e.g., about 6 to 48 hours after administration, e.g., without limitation, injection. Imaging can be performed by comparing the near infrared signal of cancer/tumor cells with the background signal determined for imaging normal tissues/cells.
In an example, a dual-function method of in situ pharmacokinetic and pharmacodynamic analysis of an active agent in a tumor, cancer or tumor cell or normal cell or tissue is provided. The method may include providing one or more THSDs; contacting it with cancer/tumor cells or tumors, or with normal cells or normal tissues; and then imaging the THSD-exposed cells, followed by pharmacokinetic and/or pharmacodynamic analysis, e.g., determining the change in fluorescence (or changes thereof) over time.
Illustrative examples
Examples 1A-II-IV below describe the synthesis of ALSDs and ELSDs in the examples presented, including the synthesis of HMCD dye and its binding to statins, e.g., DZ1 bound to simvastatin, to form L in the resultant THSD (DZ1-SIM-ALSD, DZ1-SIM)E/LAA connector. For comparative examples with ELSD, in examples 1A-V, the quick release ester DZ1-SIM RRE (without L) is describedE/LALinker, and to statins that allow lactone formation). As an alternative to DZ1, another HMCD corresponding to dye moiety FIa or FIIa may be selected, as would be apparent to one of ordinary skill. Similarly, simvastatin can be replaced by another statin, and the replacement agent can be selected to produce a replacement stable LE/LAA linker, as will be apparent to one of ordinary skill. Reaction schemes corresponding to the synthetic methods are shown in FIGS. 1A-II, A-III and A-IV.
Examples 1A-I/general synthetic methods: simvastatin was purchased from Ark Pharm, Inc. All other chemicals and reagents were purchased from standard sources (e.g., SigmaAldrich) and were of the best quality. Deionized water (18.2 Ω) for solution preparation Milli-Q direct from Millipore (Billerica, Mass.) was usedAn ultrapure water system. All intermediates were characterized by nmr spectroscopy and mass spectrometry and analyzed for purity by hplc. 1H nmr data was collected on a Bruker 400MHz spectrometer using standard parameters; chemical shifts are expressed in ppm (δ) with reference to residual non-deuterated solvents. ESI Mass Spectrometry analysis of the new compounds was performed using the Thermo-Fisher LTQ Orbitrap Elite system in a Mass Spectrometry and Biomarker Discovery Core facility. Hereinafter, the following abbreviations or formulas are used: acetic acid ("HOAc" or "CH3COOH "), ethanol (" EtOH "), methanol (" MeOH "), equivalent (" eq. "), room temperature (" RT "), sodium acetate (" NaOAc "or" CH ")3COONa "), sodium bicarbonate (" NaHCO)3"), hydroxybenzotriazole (" HOBT "), 3-1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (" EDC "), 4-dimethylaminopyridine (" DMAP "), dichloromethane (" CH "), and mixtures thereof2Cl2") and p-toluenesulfonic acid monohydrate (" toluenesulfonic acid monohydrate "or" TsOH ").
Examples 1A-II: synthesis of HMC dye (as shown in DZ 1). The reaction schemes are shown in FIGS. 1A-II. An HMCD, here DZ1, is shown as a reactive carboxyl group attached to a dye. For DZ1, residue R1And R2Is H, R3Is- (CH)2)4SO3H, R4 (will become L)E/LAPart of a linker) is- (CH)2)5COOH. To be R1、R2And R3Generating THSD with different residues, different HMCD precipitates can be selected, and modified precipitates can be selected accordingly according to the reaction scheme, including the corresponding desired residue.
Compound 1a (Compound 1, wherein R3＝-(CH2)4SO3H) The synthesis of (2): a mixture of 2, 3, 3-trimethyl-3H-indole (5g, 31.4mmol) and 1, 4-butanesultone (5.1g, 37.7mmol) was heated while stirring with argon at 120 ℃ for 5H, the resulting mixture was cooled to room temperature and the solid was dissolved in 50ml of methanol. Ether (200ml) was added to the methanol solution, the precipitate was collected and ethyl acetateThe ester (15ml, 3 times) was washed and dried in vacuo to give the desired product (compound 1a, 6.8g, 73% yield) as a white solid.
Compound 1b (compound 1, wherein R ═ - (CH)2)5COOH) synthesis: to 6-bromohexanoic acid (2.5g, 13.0mmol) was added 2, 3, 3-trimethyl 3H-indole (2.5g, 15.7 mmol). The reaction mixture was stirred at 110 ℃ for 8h under argon while heating. The resulting dark red solid was dissolved in 50ml of methanol. Diethyl ether (150 ml) was added. The precipitate was filtered and washed with diethyl ether (15ml, 3 times) and acetone (15ml, 3 times). The product was obtained as a white solid (compound 1b, 2.7g, 58%).
Synthesis of Compound 3: to a mixture of 1a (2g, 6.78mmol) and compound 2(3g, 8.36mmol) in EtOH (100ml) was added sodium acetate (0.56g, 6.78 mmol). The resulting mixture was heated to reflux for 3 h. The reaction mixture was poured into 200ml of ice water. The precipitate formed was collected and crystallized from ethanol-acetone to give the desired product as a dark blue solid (compound 3, 2.1g, 58% yield). Mass Spectrometry (ESI)525.19[ M + H ]]+。
Synthesis of compound 4 ("DZ 1"): to a mixture of EtOH (20ml) consisting of 1b (0.67g, 1.9mmol) and Compound 3(1.0g, 1.9mmol) was added CH3COONa (156mg, 1.9 mmol). The resulting solution was heated to reflux for 3 h. The heated mixture was poured into 100ml of ice water. The solid was filtered and crystallized from methanol-water to give the desired product as a dark green solid (compound 4, 0.99g, yield 74%). Mass Spectrometry (ESI)705.31[ m + h ]]+。
Examples 1A-III: synthesis of Stable ALSD (here: DZ1-SIM, Compound 7, of formula FIId or "DZ 2 a"): FIGS. 1A-III are schematic reaction schemes. ALSD (where DZ 1/compound 4 was used as the HMCD dye and simvastatin/compound 5 was used as the statin) can be generated by reacting a diamine (e.g., propane-1, 3-diamine) with the statin to attach the resulting statin intermediate (e.g., compound 6) to the dye carboxyl group and form a substantially stable "zero release" ALSD (compound 7). To form alternative ALSDs of formula FIIa, i.e. with different R's as required1、R2、R3And R4Residue (will becomeIs LE/LALinker) the precipitates should be selected accordingly to form the desired respective residues, as will be apparent to the person of ordinary skill.
Synthesis of statin intermediate (compound 6): to a solution of simvastatin (compound 5, 1g, 2.39mmol, eq.1) in acetonitrile was added propane-1, 3-diamine (1ml, 11.95mmol, eq.5). The mixture was heated to reflux with continuous stirring for 4h, the solvent was removed under reduced pressure and dried under high vacuum overnight. The resulting product (compound 6) was used without further purification.
Synthesis of DZ1-SIM-ALSD ("DZ 2 a", Compound 7): HMCD having a carboxyl group, such as DZ1 (Compound 4, 500mg, 0.71mmol), 1-ethyl-3 (3-dimethylpropylamine) carbodiimide hydrochloride (204mg, 1.07mmol) and 1-hydroxy-7-azabenzotriazole (115mg, 0.85mmol) were dissolved in 10.0ml of CH2Cl2In solution, a mixture is formed. The mixture is stirred, for example for about 15 minutes, then compound 6(350mg, 0.71mmol) is added and stirred, for example at room temperature for an additional 2 hours. The solvent was removed under reduced pressure and the product was purified, for example by C18-RP reverse phase silica gel chromatography with methanol-water to give the desired product 7 as a dark green solid (compound 7, 327mg, 39%). Mass Spectrometry (ESI)1179.65[ M + H ]]+。
Examples 1A-IV: the DZ 1-based ELSD linked to statin (simvastatin) was synthesized by reacting a carboxylic dye (here DZ1) with ethanolamine. FIGS. 1A-IV are schematic reaction schemes. ELSD (where DZ 1/Compound 4 is used as the dye and simvastatin/Compound 5 is used as the statin) can be formed by reacting an amine (e.g., ethanolamine) with the carboxyl group of the dye to form a dye intermediate (e.g., Compound 9). The dye intermediate may then be reacted with dihydroxyheptanoic acid (DHHA, -CO-CH) of the statin drug2-CHOH-CH2-CHOH-CH2-CH2) The statin unit (in the case of simvastatin/compound 5, after conversion of the closed ring lactone form of DHHA to the open chain carboxylic acid form with two hydroxyl protecting agents, compound 12) reacts to form a substantially stable "sustained release" ELSD (compound 14, "DZ 2 b"). Alternative statins may be reacted accordingly with dye intermediates, e.g. with a derivativeThe chain carboxylic acid DHHA statins (shown as compound 12, compare fig. 1A-IV) can be reacted directly with dye intermediates. To form alternative ELSDs of formula FIa, i.e., with different R's as desired1、R2、R3And R4*Residue (to be L)E/LAPart of a linker) and different precipitates are selected as necessary to form the respective residues, as will be apparent to one of ordinary skill.
Synthesis of DZ 1-hydroxyethyl amide (Compound 9): the dye (wherein: DZ1, Compound 4, 600mg, 0.85mmol), 1-ethyl-3 (3-dimethylpropylamine) carbodiimide hydrochloride (245mg, 1.28mmol) and 1-hydroxy-7-azabenzotriazole (138mg, 1.02mmol) were dissolved in 10.0ml of CH2Cl2 solution. The resulting mixture was stirred for 15 minutes, then ethanolamine (57mg, 0.94mmol) was added at room temperature and stirred for an additional 2 hours. The solvent was removed under reduced pressure and the product was purified by C18-RP reverse phase silica gel chromatography eluting with methanol-water to afford the desired ED product (compound 9) as a dark green solid 331mg (52%). Mass Spectrometry (ESI)749.34[ M + H ]]+。
Synthesis of statin methyl ester (here: methyl derivative of simvastatin, Compound 10): to a solution of simvastatin (compound 5, 418mg, 1mmol) in methanol (20ml) at 0 deg.C was added 200. mu.l sulfuric acid, stirred at room temperature for 5h, and 10ml 10% NaHCO was added3To inhibit the reaction. The resulting mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and dried (Na)2SO4) And the solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography (ethyl acetate: hexane ═ 2: 1) to give compound 10(387 mg, 86% yield).
Synthesis of compound 11: a composition of 2, 2-dimethoxypropane (1.3ml, 1108mg, 10.639mmol) was added to a solution of compound 10(300mg, 0.666mmol), p-toluenesulfonic acid monohydrate (13mg, 0.067mmol) in dichloromethane (10 ml). The solution was stirred at reflux for 2h, then 10% NaHCO was added3(5ml) to inhibit the reaction. The resulting mixture was extracted with dichloromethane. The organic layer was washed with saturated brine and dried (Na)2SO4) At low pressureThe solvent is removed. The crude product was purified by silica gel chromatography (ethyl acetate: hexane ═ 1: 2) to give compound 11(301 mg, 92% yield).
Synthesis of statin derivatives having a carboxyl group, compound 12: NaOH (1M, 1ml) was added to a tetrahydrofuran solution (4ml) of compound 11(300mg, 0.600 mmol). The resulting mixture was stirred at room temperature for 18h, then 1M HCl (1.2ml) was added, followed by extraction with diethyl ether. The organic layer was washed with saturated brine, dried (Na2SO4) and evaporated to give compound 12(274mg, 94% yield). Mass Spectrometry (ESI)499.30[ M + Na ]]+
Synthesis of ELSD (here: DZ1-SIM ELSD, Compound 14, "DZ 2 b"): the dye intermediate having a hydroxyl group (compound 9, 25mg, 0.033mmol) and the statin derivative having a carboxyl group (SIM-derived compound 12, 16mg, 0.034mmol) were dissolved in 3ml of anhydrous CH2Cl2In (1). To the resulting solution was added EDC (9.5mg, 0.049mmol) and DMAP (2mg, 0.017mmol) and the resulting mixture was stirred at room temperature for 18h, the mixture was triturated in ether and the residue was treated with 80% aqueous HOAC (3ml) for 2 h. Purification by semi-preparative high performance liquid chromatography gave the product compound 14(14mg, 36%). Mass Spectrometry (ESI) M/z 1166.62[ M + H ]]+。
Comparative examples 1A-V: synthesis of DZ1-SIM quick release ester (RRE) and comparison with the stability of ELSD (here: DZ 1-SIM). A fast release ester (RRE) was synthesized as described below, and DZ1 was linked to simvastatin (DZ1-SIM RRE or "DZ 2 c", Compound 8) and tested in comparison to ELSD (here: DZ1-SIM or "DZ 2 b", Compound 14, synthesized as described above). Both compounds were incubated in mouse serum for 3 hours at 37 ℃, then samples were taken and tested for degradation of the conjugate by testing for unconjugated statins as described in example 6 below (LC-MS/MS-025 with positive ion, ESI, MRM detection and HPLC). ELSDs exhibit greater stability (i.e., reduced degradation/release of statin from conjugate) compared to 8 (RRE); specifically, the amount of ELSD was measured at time 0 and time 3 h. Time 0 for each sample has a value of 100. At 3h, ELSD has a value of 100 (i.e. no degradation/release) and at 3h, RRE has a value of 10 (i.e. 90% degradation).
Synthesis of DZ1-SIM-RRE (Compound 8, DZ2 c): DZ1 (Compound 4, 500mg, 0.71mmol) and simvastatin (Compound 5, 356mg, 0.85mmol) were dissolved in 10ml of anhydrous CH2Cl2In (1). To the resulting mixture was added EDC (204mg, 1.06mmol) followed by DMAP (40mg, 0.33mmol) and the resulting EDC/DMAP mixture was stirred at room temperature for 18 h. The reaction mixture was broken up in diethyl ether and the residue was dissolved in 2ml of methanol. The crude product was purified by C18-RP reverse phase silica chromatography eluting with methanol-water to give the desired product DZ1-SIM-RRE (compound 8, 329mg, 42%). Mass Spectrometry (ESI) M/z 1105.57[ M + H ]]+。
The formulations of the drugs tested in examples 1b-E described below are shown in FIGS. 1A-IV. In summary, the experiments described in the examples below demonstrate improved growth inhibition of various cancer cells, particularly tumor shrinkage/volume reduction, both in vitro and in vivo. Human tumors grown in mice with ALSD (formula FIId, "DZ 2a," Compound 7) compared to statins (simvastatin or "SIM" here) or unconjugated dye (DZ1 "here).
Method examples 1 b-E:
cell culture: in the following examples, all cell lines were purchased from the U.S. Standard cell Bank (ATCC) and cultured in the medium recommended by the U.S. Standard cell Bank (ATCC), using Fetal Bovine Serum (FBS) and 1 Xpenicillin/streptomycin at a final concentration, unless otherwise specified. Streptomycin was cultured in a cell culture incubator with 5% carbon dioxide at 37 ℃ unless otherwise specified. Unless otherwise specified, cultures were 2D. If the culture is 3D, a low attachment plate and the same medium are used. C4-2B (derived from a parental cell line and drug resistant cells derived therefrom) was cultured in RPMI-1640 containing 10% FBS. MDVR cells (an anti-enzyme active agent C4-2B prostate cancer cell formed as described in example 4 below) were cultured as indicated for parental C4-2B cells. 22Rv1 Prostate Cancer (PC) cells: (CRL-2505TMA human having an epithelial morphologyProstate-like cancer cell line) were cultured in RPMI-1640 containing 10% FBS. A549 cells (CCL-185TMA human lung cancer cell line with epithelial morphology) was cultured in F-12K medium containing 10% FBS. A549/DDP cells (described in DOI: 10.3892/mmr.2014.2163 and DOI: 10.7150/jca.19426; anti-cisplatin cells can be obtained by long-term exposure of parental A549 cells to cisplatin, MDRA 549/cisplatin lung adenocarcinoma cell line, drug-resistant) were cultured in F-12K medium containing 10% FBS. H1975 cells (CRL-5908TMAn epithelial-morphic human adenocarcinoma non-small cell lung cancer cell line) was cultured in RPMI-1640 medium containing 10% FBS; h1975 cells are a human lung cancer cell model for gefitinib, an EGFR inhibitor drug used to treat certain breast, lung and other cancers. H1650 cell (C)CRL-5883TMAn adenocarcinoma/bronchoalveolar carcinoma lung cancer epithelial cell line) was cultured in RPMI-1640 medium containing 10% FBS. PC9 cells (90071810, formerly PC-14, a lung adenocarcinoma/non-small cell lung carcinoma (NSCLC) cell line with mixed morphology of EGFR mutations, including round and spindle shaped cells, was cultured in RPMI1640 medium containing 2mM glutamine and 10% FBS. PC9AR cells, a cell line resistant to Oxitrinib (AZD9291), were published (DOI: 10.1158/1078-0432.CCR-17-1574), which the authors could obtain, but also from the parental cell line by exposing PC9 cells to Oxitrinib (also known as AZD9291), at concentrations increasing from 10nmol/L to 500nmol/L over a period of about 6 months; the resulting resistant cells were cultured as described for the parental PC9 cells. H446 cells (HTB-171TMHuman small cell carcinoma SCC lung cancer cell line originating from a metastatic site) were cultured as described for 22Rv1 cells. PC3 cells (CRL-1435TMAn epithelial-morphic human prostate cancer cell line, derived from bone metastases of quaternary prostate adenocarcinoma) was cultured in F-12K containing 10% FBS. LNCaP cells (LNCaP clone FGC,CRL-1740TMan epithelial-morphic prostate cancer cell line) was cultured in RPMI-1640 containing 10% FBS.
Cellular and tissue uptake: in the examples below, unless otherwise specified, the cellular and tissue uptake of THSD or HMCD controls was determined as follows. Mixing cells (1X 10)4Per well) were seeded on glass wall-coated four-well chamber slides (Nalgen Nunc) and incubated with corresponding growth media containing 5% fetal bovine serum (e.g., C4-B2 cells: t medium) for 24 hours. After the cells were fixed to the slide, the cells were washed with PBS and exposed to THSD/HMCD in a concentration of 20. mu. Mol/L of medium. The slides were incubated at 37 ℃ for 30 minutes, washed twice with PBS to remove excess dye, and the cells were fixed with 10% formaldehyde at 4 ℃. The slides were then washed twice with PBS and the glass coverslips were covered with aqueous mounting medium (Sigma-Aldrich). Image chromaticity was recorded by confocal laser microscopy (ZeissLSM 510META) using a 633nm excitation laser and 670-. To determine dye uptake in tissues, tissues isolated from tumor-bearing mice were placed in OCT (optimal cutting temperature) medium and frozen at-80 ℃. Frozen 5 μ M tissue sections were prepared using a microscope for histopathological observation as described above.
Subcellular localization (mitochondria, lysosomes): in the following examples, unless otherwise indicatedIt is specified that otherwise based on each compound constituting the dye or comprising the dye moiety, the uptake of THSD or HMCD control into mitochondria and lysosomes of cancer cells is determined in the following manner. Cells were treated overnight in a live cell imaging room (World Precision Instrument). Cells were exposed to different concentrations of dye (i.e., THSD/HMCD) and dye uptake was measured using a Perkin-Elmer hyperranging ERS rotating disk confocal microscope. The system was mounted on a ZeissAIXIOVERT 200m inverted microscope, equipped with a 37 ℃ practice heater, incubator, and continuously perfused with carbon dioxide. A 63X or 100X Zeiss oil objective (numerical aperture, 1.4) was used for real-time cell images and a piezoelectric Z stepper motor was used to create a Z stack. The 633-nm laser line of an argon ion laser (set at 60% power) was used to excite the THSD/HMCD. Light emission at 650nm, although not the optimal choice of dye, was found to be directly related to the concentration of dye in the cells. For comparative studies, exposure time and laser intensity were kept consistent for accurate intensity measurements. Pixel intensities were quantified using Metamorph 6.1 (general imaging) and the average pixel intensity was generated as a grayscale using a region statistics function on the software. To determine the dye uptake by mitochondria, the MitoTracker dye was usedTM Green FM(InvitrogenTMMolecular probeTM). To determine the location of the dye in lysosomes, the LysoTracker dye LysoTracker was usedTM Green DND-26(InvitrogenTMMolecular probeTM). The localization of mitochondrial and/or lysosomal dyes (THSD, HMCD) was imaged under confocal microscopy.
Uptake and accumulation of tumors in vivo: in the following examples, unless otherwise specified, the uptake and accumulation of THSD/HMCD in mouse tumors in vivo is determined as follows. Human cancer cells (1X 106) were transplanted into 4-6 week old nude mice (national cancer institute). When the tumor size reached 1-6mm in diameter (assessed by X-ray or palpation), the mice were injected intravenously or intravenously with HMCD at a dose of 0.375mg/kg or 10nmol/20g mouse body weight. Whole-body optical imaging was taken at 24 hours or using a 4000mm kodak imaging station equipped with a fluorescence filter set (excitation/emission, 800: 850nm), a 120mm diameter field of view, 2mW/cm2 near infrared excitation light frequency, and the following cameras. Setting: maximum gain, 2 × 2 pixel combination, 1024 × 1024 pixel resolution, exposure time 5 seconds. Live mice were imaged alternately/additionally by an Olympus OV100 whole mouse imaging system (excitation, 762 nm; emission, 800 nm; Olympus) comprising an MT-20 light source (Olympus biosystem) and a DP70CCD camera (Olympus). Prior to imaging, mice were anesthetized with chloraminone (75mg/kg) and maintained under anesthesia during imaging.
Example 1 b: prostate Cancer (PC) cells (22Rv1,-2505TMhuman prostate cancer cell line) and exposed to 0-50 μ M of the respective compound (THSD, unconjugated dye) for 24-48 hours. Cell viability was determined by counting live and dead cells, and distinguishing live and dead cells by trypan blue staining. The results are shown in FIG.1B 2, with the Y-axis representing the percentage of cell viability. The results show that the IC of ALSD of Compound 7, having the formula FIId "DZ 2 a", is useful for inhibiting cancer cells50Between about 3-4. mu.M, this is compared to unconjugated statins: (>50 μ M) and the unconjugated dye (referred to herein as "DZ 1"). The chemical formula of the test compound is shown in FIG.1B 1.
Example 1C: PC (22Rv1), LNCAP (LNCAP clone FGC: (II) (III)-1740TMHuman prostate; origin from the site of metastasis: left supraclavicular lymph node) and C4-2B (-3315TMHuman prostate cancer epithelial morphology cells) cancer cells were cultured as follows: 22Rv1 and lncap were cultured according to the protocol described above using RPMI culture; C4-2B was cultured in RPMI containing 10% FBS as described above. Cultured cells were exposed to ALSD of compound 7 of the formula FIId "DZ 2 a" for about 1h to 0-50 μ M. Cell viability was determined as described in example 1 b. The results are shown in FIG. 1C, with cells on the y-axisPercentage of viability, x-axis represents the dose of drug (unit: μ M). The results indicate that ALSD is equally effective in different types of cancer cells, IC50About 3-4. mu.M.
Example 1D: 22Rv1 human prostate cancer cells were cultured as described above, isolated from the culture medium, resuspended in 50% Phosphate Buffered Saline (PBS) and 50% BD MatrigelTM(BD Biosciences, CA), resuspension cells were then injected and grown subcutaneously in mice. Monitoring the uptake and retention of the drug by near infrared emission; tumors showed uptake and retention of ALSD for at least 2 to 4 weeks or more, especially in solid non-necrotic tumors/non-necrotic parts of tumors, see fig. 1D. The THSD decreased at 2 and 4 weeks, measured by near infrared signals, respectively, and was less than about 30%.
Example 1E: 22Rv1 human prostate cancer cells were cultured, injected and grown subcutaneously in mice as described in example 1D. Tumor volume was monitored by measuring caliper size and calculating the volume as described in example 9 below. Mice were injected intravenously with the following drugs: ALSD of the formula FIId ("DZ 2 a"), compound 7, the corresponding unconjugated dye (here DZ1), the statin unconjugated Simvastatin (SIM) and the cancer drug DOCetaxel (DOCetaxel), and mixtures of ALSD and DOC. For ALSD, DZ1 and SIM, the dose of each active drug was 5mg/kg, except for DOC, 8 mg/kg. The results are shown in FIG. 1E. A slower rate of tumor volume increase indicates tumor inhibitory activity. "vehicle PBS" served as a negative control, indicating that tumor growth was not inhibited. DZ1, SIM and DOC all have no obvious tumor inhibition activity; in contrast, ALSD exhibits tumor suppressor activity, significantly reduces tumor growth, and increases tumor volume at a slower rate. The addition of DOC and ALSD did not further increase the tumor growth inhibition of ALSD
Example 2: 22Rv1 human prostate cancer cells were cultured as described above and using DAPI (nuclear blue fluorescence), ALSD with the formula FIId "DZ 2 a", Compound 7 (yellow fluorescence), mitochondrial localizationTM(Green fluorescence) and lysosomal localizationTM(green fluorescence) treatment. As shown in FIG. 2, in the complex, the nucleus was stained blue with DAPI, while the red fluorescence of ALSD was associated with the cytoplasmic fraction of cancer cellsMitochondrial localization ofTMAnd lysosome localizationTMCo-exists as indicated by yellow fluorescence in the complex. Co-localization thus allows ALSD to interfere with mitochondrial and lysosomal functions of cancer cells.
Example 3A: the metabolic phenotype and changes in the metabolic phenotype of C2-4B MDVR cells following exposure to various drugs and controls as described below were determined by mitochondrial Oxygen Consumption Rate (OCR). Drug/controls included: negative control/vehicle only ("NT"), unconjugated dye ("DZ 1", 6 μ M), simvastatin, unconjugated statin ("SIM"), and cholesterol ("cholesterol", 20 μ M). OCR results are shown in FIG. 3A, ECAR results (with different controls) are shown in example 3B and FIG. 3B. OCR and ECAR correspond to two major energy pathways of the cell: mitochondrial respiration and glycolysis, OCR as an indicator of mitochondrial respiration (oxidative phosphorylation), and ECAR as an indicator of glycolysis and lactate production. The initial OCR for all drugs was in the range of 650-1000pmol/min, with highest SIM, lowest DZ1, and moderate OCR for the other drugs. ALSD of formula FIId ("DZ 2 a", compound 7) significantly reduced OCR compared to all other agents; after about 200-300 minutes, the rate dropped below NT, SIM, DZ1 or cholesterol, and continued to drop rapidly to a rate of less than 200-300pmol/min (compared to 650-950pmol/min for other drugs) within 600-800 minutes.
OCR and ECAR of live cells were measured in a metabolic chamber in 24-well plate format using a Seahorse XF analyzer (Seahorse XFE24, Agilent, CA). Reagents (drug/control) were added to the well plate before time 0 began recording, and the analyzer recorded the effect of the drug on primary mitochondrial function for approximately 12 hours (720 minutes). The reagents used were Agilent (Agilent) reagents. The cells were cultured by seeding in SeahorseXF RPMI medium at pH 7.4. Within about 24 hours after the start of the analysis, the seeding rate of the cells was 8 × 105, and a polymerization rate of 90% was reached. The procedure was performed essentially according to the manufacturer's standard procedure, removing the media, washing the cells once with XF real-time ATP rate analysis media, then pre-programming according to the instrument and subsequent real-time measurement of OCAR/ECAR.
Example 3B: the extracellular acidification rates ("ECAR") indicative of glucose consumption were determined as described in example 3A above. The results show that DZ2a shows an upward bump on the curve around 500 minutes, indicating the metabolic conversion of cells to glucose.
Example 3C: C2-4B MDVR cells were used and cultured as described above. Cells were denatured with β -mercaptoethanol as a denaturing agent, 20 μ g portions were loaded into channels of the denatured gel, proteins were separated by size, the gel was run, the separated proteins were transferred from the gel to a nitrocellulose membrane, and these proteins were hydrolyzed. ALSD (formula FIId, "DZ 2 a", compound 7) was compared to the unconjugated dye (DZ1) by near infrared signal. The results are shown in FIG. 3C. The figure shows that ALSD binds, possibly covalently, to multiple mitochondrial and cytosolic proteins in the cytosol and mitochondrial lysate. Compared to the unconjugated dye (DZ1), the signal intensity might indicate a higher affinity for certain proteins, as well as additional proteins that bind in the presence of additional bands.
Example 3D: as described in example 3C, the protein was denatured and isolated; performing western blot analysis on the membrane using an antibody targeting ubiquitin to detect polyubiquinated proteins; probes include molecular weight markers ("markers"), DMSO (vehicle as control), unconjugated statin (simvastatin), unconjugated dye (DZ1), and THSD (herein "DZ 2 a" with the formula FIId, compound 7). The results are shown in FIG. 3D. A strong signal for a large (rather than single/discrete band) protein smear in the DZ2a channel indicates the accumulation of polyubiquitinated protein, i.e., unfolded protein that is not cleared from the cell. Thus, studies have shown that THSD can prevent cancer cells from removing polyubiquitinated proteins by altering autophagy processes involving defective lysosomal function.
Example 3E: whole cell and mitochondria preparation/processing is as follows. 22Rv1 prostate cancer cell cultures are as described above. Cells were harvested, treated with Lithium Dodecyl Sulfate (LDS) detergent, then centrifuged to provide a whole cell fraction and a separated mitochondrial fraction, and the particles were treated to determine their cholesterol levels as follows: cholesterol was extracted with hexane/methanol mixture (7: 1, v/v) and a suitable amount of stable isotope labeled d7 cholesterol standard (anti-polar lipid) was added. The samples were sonicated in a water bath, spun, and centrifuged at 16000 Xg for 5 minutes at room temperature. The upper layer was dried in a rapid vacuum at room temperature. The sample was then incubated with 200. mu.l of 10mg/ml 4- (dimethylamino) phenyl isocyanate (DMAPI) in dichloromethane and 30. mu.l triethylamine in a hot mixer at 65 ℃ for 1.5 hours. The reaction was quenched with phosphate buffer (ph8.0), and then DMAPI-labeled cholesterol was extracted with hexane and concentrated in vacuo in a rapid vacuum. The dried residue was reconstituted in acetonitrile/isopropanol (1: 1) and analyzed by liquid chromatography selective ion monitoring (LC-SIM) using an Ultimate 3000XRS liquid chromatography system connected to an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific). After separation on a 5-cm platinum C18 column, DMAPI-derived endogenous cholesterol (m/z549.442) and D7 cholesterol (m/z 556.485) were analyzed using a time-schedule simulation assay. The obtained LC-SIM data were subjected to cholesterol quantification using Skyline.
The cholesterol quantification is shown in the table below.
The results show that ALSD (formula FIId, "DZ 2a," compound 7) significantly reduced cancer cell cholesterol levels to about 58% and mitochondrial cholesterol levels to 34% compared to the control group (see results in the table below). Compared with ALSD, statin simvastatin reduces cholesterol (cancer cells reduce 88%, mitochondria reduce 51%)
Example 4: cell analysis was performed and the cells cultured as described above were exposed to various drugs shown below for 72 hours. The following cells were used: parental C4-2B cells and anti-C4-B2 cells: C4-2B ABiR anti-abiraterone acetate, an anti-androgen drug for hormone therapy of prostate cancer, and enzalutamide resistant cells ("MDVR"). The cell designated as the "C4-2B parent" is a non-resistant C4-2B cell that has not been previously exposed to a cancer drug. All C4-B2-based cell cultures were used for parental cells as described above. Drug resistant C4-B2 cells are generated by exposure to the relevant drug. Cells were exposed to 2, 4, 8 or 16 μ M ALSD (formula FIId, "DZ 2 a", compound 7). The results are shown in fig. 4A (non-resistant parent C4-2B), fig. 4B (anti-abiraterone acetate or ABiR), and fig. 4C (anti-benzamil or MDVR). For all cell types, cell viability decreased to 0% after exposure to 16 μ MALSD for 8h, less than 50% after exposure for 3h, and less than 25% after exposure for 4 h. More cells survive during the experiment if less ALSD is used, e.g. 8 μ M ALSD reduces survival to below 25-50% within 4-8 hours, while less takes longer to reduce cell survival. The results indicate that ALSD reduces cell survival, inhibits the growth of abiraterone acetate and benzazamide resistant cells, and also inhibits the growth of non-resistant "parent" C4-2B cells. The amount of about 4-5 μ MALSD is generally sufficient to reduce cell survival/growth inhibition of these resistant cells compared to fig. 4B and 4C.
In addition, preliminary results indicate that an amount of ALSD of about 5-20 μ M (e.g., 14 μ M or less) reduces cell survival and can inhibit the growth of docetaxel drug-resistant cells.
ALSD can provide more rapid growth inhibition compared to other drugs (e.g., abiraterone acetate, enzalutamide, and docetaxel). For example, inhibition is typically achieved in less than 8-24 hours, typically begins in less than about 2 hours, and has a significant effect of reducing cell viability to below 50% within the first about 3-8 hours (e.g., at 8-16 μ M), compare fig. 4A-c. Complete inhibition (100%) can be achieved within 8-18 hours, especially at higher concentrations (e.g., about 16 μ M). The results show that ALSD provides rapid and complete (0% cell viability or 100% growth inhibition) within 8-24 hours, depending on the dose. Due to slow inactivation, for example, amounts of about 4-5 μ MALSD or less may be sufficient to reduce cell survival/growth inhibition of non-resistant or resistant cells over a longer period of time (e.g., 16-48 hours or about 24-32 hours). Higher amounts, e.g., about 5 to about 20 μ M or less, e.g., about 14 μ M, may be required for docetaxel-resistant cells to reduce cell viability and inhibit their growth (data not shown). Similarly, ELSD can provide rapid growth inhibition as compared to examples 7D and E, as is evident from its less steep dose-response curve.
Example 5: this example shows that THSD can prevent RhoA/B anchorage to the cell membrane, probably due to THSD inhibiting protein prenylation and its downstream cellular signaling. RhoA/RhoB membrane staining of tumor tissue from a mouse model of H-1975 human lung cancer cells, treated with THSD (here, ALSD of formula FIId, "DZ 2a," compound 7) in comparison to the corresponding unconjugated dye ("DZ 1") and gefitinib, an EGFR inhibitor drug used to treat certain breast, lung and other cancers, were performed as described below. The results showed that there was still staining on the cell membranes of DZ 1-and gefitinib-treated tumors, but not on the cell membranes of ALSD-and ALSD/gefitinib-treated tumors (see FIG. 5). This suggests that ALSD may prevent RhoA/B anchoring to the cell membrane, probably because ALSD inhibits prenylation of the protein.
H-1975 cells, a human lung cancer cell model with resistance to gefitinib, an EGFR inhibitor drug used to treat certain breast, lung and other cancers, were cultured as described above and injected subcutaneously into mice to form tumors. Mice and established tumors were exposed systemically to ALSD (here, the formula FIId, "DZ 2a," compound 7) by intravenous injection. Tumor-bearing mice were treated with either gefitinib (20mg/kg) or ALSD (here, formula iid, "DZ 2 a", compound 7, 5mg/kg), an unconjugated dye of formula III ("DZ 1", 5mg/kg), or a combination of ALSD and gefitinib. Tumor volume was significantly reduced following ALSD exposure, determined by visual examination after resection, tumor size was measured periodically and/or tumor volume was calculated with calipers (data not shown).
For immunostaining of tumor tissue, animals were sacrificed by the prescribed and approved euthanasia technique. Tumor tissue (maximum thickness 3 mm) was sectioned, formalin-fixed, embedded in paraffin blocks, sectioned and processed into paraffin section slides. For immunohistochemical staining, slides were dewaxed and rehydrated (heat, xylene, alcohol, 3% methanol in H2O2 solution, selective burst)Is exposed in RetrievaganA (pH6.0), BD PharmingenTMCat. # 550524). Glass slides were exposed to RhoA (26C4) monoclonal antibody 1: 1000(Santa Cruz, CAT SC-418). Alternatively, one or more other RhoA and/or RhoB monoclonal antibodies may be used. After blocking endogenous peroxidase, an HRP-based procedure (Hores Raddish peroxidase, catalytic chromogenic substrate for microscopic detection) can be used. Primary antibodies and negative control antibodies (e.g., RhoA 1: 1000 and negative control antibodies such as mouse Igg1 negative control, Agilent Dako, CA, 1: 100) are used. After exposure and washing, biotinylated secondary antibodies (e.g. LSAB2 kit, Agilent Dako, CA) were applied followed by streptavidin LSAB2 followed by 20ul/ml of DAB chromogenic reagent (Signet) and counter staining with hematoxylin 50. The stained slides were dehydrated and cleared with graded alcohol and xylene and then mounted and examined under a microscope appropriate for the chromophore used. For a discussion of the results, see above.
Example 6 a: in vivo administration of THSD was performed to mammals (rats, dogs). THSD (here ALSD, DZ1-SIM amide of formula FIId or "DZ 2 a") was administered to 4 animals (n 2m +2f), 4 beagle dogs (7.38-8.59kg) and 4 rats (220-. 1mg/kg (1ml/kg) by cephalic intravenous injection (dog) or dorsal plantar intravenous injection (rat), respectively. The animals are free to receive feed and water. Samples were taken at time points (HR)0.083, 0.25, 0.5, 1, 2, 4, 8, 24. At the time of blood collection at the indicated time points, each animal was manually restrained and approximately 150 microliters of blood were collected by cephalic venipuncture (dog) or caudal vein (rat) and continuously bled into EDTA-K2 sodium tubes. Blood samples were stored in wet ice and plasma was obtained by centrifugation (2000 g, 4 ℃,5 min) within 15 min after sampling. All plasma samples were diluted 2-fold in water with the same volume of 0.5% FA and stored at-70 ℃ until used for analysis. The concentration of THSD and unconjugated statin (simvastatin) was determined by analysis with LCMSMS-025(AB Sciex6500LC/MS/MS system). MS gives positive ion ESI. And carrying out MRM detection. The conditions of the high performance liquid chromatography are as follows: poroshell 120, EC-C18, 50 × 2.1M M, 2.7 μ M, flow rate of 0.50ml/min, column temperature of 45 ℃. Internal standard calibration curves (1.00-1000ng/ml THSD, 2.00-2000ng/ml simvastatin) were established. The sample matrix was animal plasma, and an equal amount of 0.5% formic acid was added to the aqueous solution. In ACN containing 0.1% FA, an aliquot of 30.0. mu.l of sample was added to 200. mu.l. The mixture was spun for 5 minutes and centrifuged at 13000rpm for 5 minutes. Mu.l of the mixture was injected into LC-MS/MS.
Representative results are shown in the table below. For dog and rat samples, large amounts of THSD were measured over 5 minutes, reaching peak concentrations in about 5-15 (dog) or 15-30 (rat) minutes, and then decreasing to 0 (no peak) over time after about 8 hours. In contrast, unconjugated statins were either not measured or only measured in minute amounts throughout the entire sampling period from 5 minutes to 24 hours, with the highest minor concentrations below 1ng/ml (0.094 ng/ml in the highest concentration rats, 0.46ng/ml in dogs). The results indicate that THSD zero-release statin as it was not detected in plasma.
Maximum value determined
Maximum of assay
Example 6B: THSD was administered in vivo to mammals (rats, dogs) and plasma concentrations were determined over time. The experiment was performed as described in example 6A, with 1mg/kg of THSD (here ALSD, DZ1-SIM amide of formula FIId or "DZ 2 a") injected intravenously; blood samples were taken from the animals every 24 hours and for each sample the THSD was quantitated as described above and plasma concentrations calculated. Results as shown in fig. 6B1 (mean concentration for female and male rats) and fig. 6B2 (mean concentration for female and male dogs), the measured values were below the quantifiable limit at 24 hours (not shown). These graphs show that the half-life of a mammal is about 1 hour, about 0.6 to 1.7 hours, for example, the half-life of female dogs is about half an hour and the half-life of male rats is about 2 hours. Relevant pharmacokinetic parameters are shown below.
Example 7: the experiment was performed by cell analysis essentially as described in example 4. All cell lines were human cancer cell lines and cultured as described above; it includes: 22Rv1 (prostate cancer cell line), MDVR (EZ resistant C4-2B prostate cancer cell line formed as described in example 4 above), PC3 (starting from grade IV prostate Adenocarcinoma (AC) bone metastasis, a549 cells (lung cancer cell line), a549/DDP cells (MDRA 549/cisplatin lung cancer cell line, drug resistant), H1975(AC/NSCLC cell line and TKI resistant cell models, especially gefitinib resistant), H1650(AC/NSCLC cell line), PC9(AC/NSCLC lung cancer cell line with mixed morphology of EGFR mutations), PC9AR cell, an ocitinib/AZD 9291-resistant cell line that can be grown to shape from parental cell line by exposing PC9 cells to ocitinib (also known as AZD929) and H446 (an SCLC cell line).
Cell lines PC9, H1975 and H1650 have the following EGFR activating mutations: PC9 has the EGFR activating mutation T790m, H1975 has the L858R/T790M double mutation, and EGFR exon 19 deletion (Del) of H1650. The following additional mutations are also included: in H446 cells, the c-myc DNA sequence was amplified 20-fold and the c-myc RNA was increased 15-fold relative to normal cells.
The cell lines were cultured according to the protocol described above and then each cell line was treated with ELSD (here molecular formula FId, "DZ 2 b" or compound 14) or ALSD (here molecular formula FIId, "DZ 2 a" or compound 7) for 48 h. For each cell line, the IC of each drug (i.e., ELSD, ALSD) and the corresponding unconjugated statin (simvastatin) was determined50The concentration is 0.1-100 μ M.
Cell viability (IC)50) The assay by MTT (thiazole blue colorimetry) was as follows: 100 μ l of 1X 104/ml cells were treated with increasing concentrations of drug or control (THSD, DZ1, SIM) for 24 hours. In a control group (not shown in the figure), cells were exposed to DMSO (vehicle)Body), to reach a final concentration equal to the highest concentration of the drug tested, the maximum concentration is less than 0.1% v/v. Mu.l of tetramethylazozole salt (3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2H-tetrazolium bromide, supplied by Sigma-Aldrich) was added to the well plate containing the cells 4 hours before the end of the incubation/addition of SDS. At the end of the culture, 100. mu.l of 10% sodium lauryl sulfate was added, and then the plate containing the cells was placed in a cell incubator at 37 ℃ for 8 hours. The absorbance density of the supernatant was read on a 96-well microplate reader with a wavelength of 595 nm. All IC50Are all relative IC50And based on curve fitting as shown.
The results are shown in the following table, with corresponding curves as shown in FIGS. 7A-I.
When comparing IC of THSD for three different cancer cell lines50IC with unconjugated statins or dyes50The trends were similar: IC of THSD (ELSD and ALSD)50IC of unconjugated statins in the range of about 2 to 12 μ M50Much higher, ranging from about 15 to about>50 μ M (i.e., more unconjugated statin is required to produce the same effect on cell viability). Thus, both ALSD and ELSD were more able to decrease cell viability and inhibit the growth of prostate cancer cells than unconjugated statins (compare the graphs in fig. 7A-I).
Example 8: a549 cells (lung cancer cell line) were used and treated with different concentrations of ALSD (here ALSD of formula FIId, "DZ 2a," compound 7), respectively, for 24, 72, or 120 hours, essentially as described in example 7 above, and cell viability was measured by MTT, as described below. The results (comparative graph shown in FIG. 8) show that ALSD has a strong growth inhibitory effect (IC) regardless of the culture time (24 to 120 hours)50About 5.7 μ M to 5.9 μ M), indicating that ALSD is a drug (rather than a prodrug) that can directly inhibit tumor growth without the dye group that statins release the enzyme from the amide bond to ALSD. Also, in the same manner as above,slow release ELSDs release statins only minimally (data not shown).
Example 9: TKI resistant cell lines are established by exposing sensitive cell lines to TKI, e.g., generation 3 EGFR-TKI AZD9291 or existing sensitive cell lines may be used. Other TKIs and EGFR-TKIs may be established and tested accordingly. AZD9291 was purchased from activated biochemicals. THSD "DZ-SIM" amide of molecular formula FId (compound 7, ALSD) was tested in vivo in a mouse xenograft model in comparison to unconjugated statins, unconjugated dyes, EGFR-TKI agents, and controls as described below. Human prostate cancer cells are PC9AR cells and are resistant to third generation Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor (TKI) drugs (EGFR-TKI). PC9AR cells were cultured as described above. 5X 106pc9ar cells were inoculated subcutaneously, together with one third of the matrix gel, flanking 5-week-old female mice. 7 days after inoculation, when the tumor size reached about 50mm3, the mice received 3 treatments per week for 16 days as follows: vector/negative controls (1% Tween80, i.p.), DZ1(5mg/kg, i.p.), simvastatin (5mg/kg, i.p.), DZ1-SIM (5mg/kg, i.p.), AZD9291(10mg/kg, o.g.), and a combination of DZ1-SIM (5mg/kg, i.p.) and third generation EGFR-TKI AZD9291(10mg/kg, o.g.). Tumor volume was measured with calipers and calculated using the formula 2a x b x 0.5, a and b being the two major dimensions of the tumor (width, length) and a < b, mice were sacrificed 16 days after treatment, tumor tissue was isolated, weighed and imaged. The photographs, weights and volumes of the tumors and the weight of the mice are shown in FIG. 9A 1-4. The standard deviation and confidence level are shown in the following figures: mean ± s.d. (n ═ 6). P <0.05, p <0.01, p <0.0005, p < 0.0001. The results are detailed in FIG. 9A1-4 and the above description. In summary, we determined that human tumor cell-derived tumors treated with THSD had significant growth inhibitory effects, but not in tumors treated with any other drug, by measurements of tumor size, weight and volume over time. The combined use of THSD and TKI did not increase the growth inhibitory effect of THSD.
Example 10: in vivo assay for THSD (ALSD and ELSD, respectively) in a mouse xenograft model of human lung cancerAnd (6) testing. This example was carried out essentially as described in example 9 with the following modifications: human SCLC H446 cancer cells were used, and the agents tested included two statin conjugates (compounds of molecular formula FId compound/compound 14 or "DZ 2 b" and FIId/compound 7 or "DZ 2 a", designated "DZ-SIM ester" or "DZ-SIM amide" in fig. 10, respectively), and a non-statin conjugate ("DZ-DHA"). 5-week-old nude mice were implanted bilaterally and subcutaneously with SCLC H446 cells (1.5X 10)6). 10 days after cell implantation, mice were treated twice weekly by intravenous injection with DZ1(2.8mg/kg), SIM (2mg/kg), DZ1-SIM amide/Compound 7 or DZ1-SIM ester/Compound 14(4mg/kg) and DZ1-DHA (4mg/kg, DZ 1-dihydroartemisinin ester "DZ 1-DHA" as shown in FIG. 11 (A) tumor size was measured every four days with calipers during 52 days of treatment and volume confidence levels were calculated as P.P.<0.05，**P<0.01，***P<0.0005, the results are shown in FIG. 10. In summary, both THSDs significantly enhanced growth inhibition of human cancer tissue compared to all other agents; growth inhibition was also slightly increased compared to the non-statin dye conjugates.
It should be noted that the features illustrated in the drawings and examples are not necessarily drawn to scale, and features of one example may be used with other examples, as the skilled artisan would recognize, even if not explicitly stated herein. Descriptions of well-known components and techniques may be omitted to avoid unnecessarily offsetting the center of gravity of the examples.
Other examples of the invention will be apparent to those skilled in the art from a reading of this detailed description when disclosing multiple examples. The invention is capable of numerous modifications in various obvious aspects, all without departing from the spirit and scope of the present invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
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