Cationic polymer nanocarriers for inhibiting chemotherapy-induced cancer metastasis and cognitive impairment

Abstract

The nano-carrier based on PAMAM has high chemotherapeutic drug loading efficiency and high cfNA binding capacity, can realize continuous delivery of chemotherapeutic drugs, and inhibits systemic inflammation caused by chemotherapy at the same time. This contributes to the treatment of primary and metastatic tumors while mitigating cognitive impairment.

Description

[0001] Citation of Related Applications

[0002] This application claims protection to an invention disclosed in U.S. Provisional Application No. 63 / 349,492 filed on June 6, 2022, entitled “Cationic Polymeric Nanocarriers to Inhibit Chemotherapy-Induced Cancer Metastasis and Cognitive Impairment”. The benefits of 35 U.S.C. §119(e) of the U.S. Provisional Application are hereby claimed, and the aforementioned application is hereby incorporated herein by reference.

[0003] Government licensing rights

[0004] This invention was made with government support under Grant No. AR073935 awarded by the National Institutes of Health (NIH) and Grant No. W81XWH1910463 awarded by the Department of Defense (DoD). The government has certain rights in this invention. Background Art

[0005] Cancer is the leading cause of death in most countries. Among women, breast cancer is the most commonly diagnosed type of cancer and has the second highest mortality rate. Many patients with early-stage, non-metastatic breast cancer can be cured with a combination of surgery, chemotherapy, and radiation therapy, but approximately 50% of patients develop distant organ metastases, which are usually incurable. Chemotherapeutic drugs such as paclitaxel and doxorubicin are used in combination with surgery to treat primary breast cancer. Although these drugs inhibit the growth of primary tumors, there is growing evidence that they may promote metastasis by causing elevated levels of proinflammatory cell-free nucleic acids (cfNA), which are released by damaged cells into the tumor microenvironment. New research has begun to focus on attenuating the pro-metastatic effects of chemotherapy.

[0006] Cognitive impairment is another side effect that cancer patients receiving chemotherapy may face. These cognitive impairments are also called chemobrain, chemofog, or chemotherapy-induced cognitive impairment. Chemobrain was first mentioned in 1980, when a group of cancer patients scored significantly lower on cognitive function assessments during and after chemotherapy, suggesting that psychiatrists should be aware of chemotherapy as a possible source of behavioral changes. In qualitative studies, breast cancer survivors reported problems with attention, working memory, and multitasking, pointing to changes in executive function and learning / memory. Cohort studies have found that chemotherapy-induced cognitive impairment may persist for 4 to 10 years, with subjects showing hyporesponsiveness in the parahippocampal gyrus in paired associate tasks assessing episodic memory. Therefore, the development of new nanomedicines to alleviate chemobrain is highly anticipated.

[0007] Elevated levels of cfNA (single-stranded and double-stranded DNA and RNA) in the peri-tumor and post-chemotherapy circulation are associated with cancer metastasis and chemo-brain. cfNA is a damage-associated molecular pattern (DAMP) that induces chronic inflammation by activating toll-like receptors (TLRs); for example, dsRNA activates TLR3, ssRNA activates TLR8, and ssDNA activates TLR9. Activated TLRs upregulate MYD88 and NF-κB to induce the secretion of inflammatory cytokines, and TLR-induced systemic inflammation appears to promote tumor metastasis and cognitive impairment. cfNA is being explored as a biomarker for cancer diagnosis and prognosis, but is rarely recognized as a therapeutic target for the prevention of metastasis and chemo-brain.

[0008] Recently, we found that cationic polyamidoamine (PAMAM) dendrimers can clear nucleic acids through electrostatic interactions. In the treatment of inflammatory and autoimmune diseases, PAMAM clears cfNA and inhibits cfNA-induced TLR activation and TLR-induced inflammation. In pancreatic and breast cancer models, PAMAM dendrimers inhibit metastasis by clearing cfNA. Through careful molecular design based on PAMAM dendrimers, cationic polymer nanocarriers for chemotherapeutic drugs can inhibit cancer metastasis and chemotherapy-induced cognitive impairment. Summary of the invention

[0009] We have developed cationic polymer-based nanoparticles that are able to encapsulate chemotherapeutic drugs and bind to free DNA molecules of inflammatory circulating cells. This nanoparticle technology has been found to reduce chemotherapy-induced systemic inflammation and cancer metastasis in vivo. These nanoparticles may therefore reduce possible neuroinflammation by reducing chemotherapy-associated systemic inflammation, thereby alleviating chemo-brain symptoms. Cognitive impairment is a side effect that cancer patients who have undergone chemotherapy may face. These cognitive impairments are referred to as chemo-brain, chemo-fog, or chemotherapy-induced cognitive impairment. In qualitative studies, cancer survivors have reported problems with attention, anxiety, working memory, and multitasking, pointing to changes in executive function and learning / memory.

[0010] Chemotherapy promotes chronic inflammation through necrotic cancer cell death and the release of their cellular contents (called damage-associated molecular patterns, DAMPs) into the blood. DAMPs include cell-free nucleic acids (e.g., cell-free DNA and miRNAs) that can activate immune toll-like receptors (TLRs). The persistent presence of DAMPs in the blood induces a chronic inflammatory state with elevated levels of circulating inflammatory cytokines that can subsequently enter the central nervous system and induce neuroinflammation. Neuroinflammation has been identified as a risk factor in the pathophysiology of depression, and a positive correlation has been found between chemo brain and depression. Chemotherapy is the mainstay of cancer treatment, and therefore, it is critical to find methods that can minimize off-target inflammation and unintended cognitive impairment. Since our nanomaterials can reduce systemic inflammation induced as a side effect of chemotherapy treatment, they may also reduce systemic inflammation and thus neuroinflammation, thereby attenuating the cognitive effects of chemotherapy treatment.

[0011] In some aspects, the present disclosure relates to polymer nanocarriers for delivering chemotherapeutic drugs, the nanocarriers comprising cationic polyamidoamine (PAMAM) dendrimers modified with cholesterol. Some polymer nanocarriers also comprise chemotherapeutic drugs. In some embodiments, the chemotherapeutic drug is one or both of paclitaxel or doxorubicin.

[0012] In certain embodiments, the cholesterol-modified PAMAM (polyamidoamine) dendrimer comprises at least one cholesterol residue at the terminal position of the PAMAM. In certain embodiments, the cholesterol residue is attached to an external amine functional group of the PAMAM.

[0013] Some polymer nanocarriers have a spherical shape.

[0014] Other aspects of the present disclosure relate to methods of treating cancer in a patient by administering to the patient a polymeric nanocarrier disclosed herein. In some embodiments, the cancer is one or both of a primary tumor and a metastatic tumor.

[0015] In certain embodiments, the method prevents or reduces chemotherapy-induced cognitive impairment in patients. In some embodiments, the method prevents or reduces the pro-metastatic effect of chemotherapy in patients. It has also been found that cholesterol-modified cationic polyamidoamine (PAMAM) dendrimers can reduce cell-free nucleic acid (cfNA) levels in patients.

[0016] In certain embodiments, the nanocarriers and methods disclosed herein are used to treat cancer. In some embodiments, the nanocarriers and methods are used to prevent cancer recurrence after surgery, chemotherapy or radiotherapy.

[0017] In certain embodiments, polymeric nanocarriers deliver at least one immune checkpoint inhibitor to a patient.

[0018] In yet another aspect, the disclosure includes methods for preparing polymer nanocarriers described herein for delivering chemotherapeutic drugs. Some methods include utilizing an oil-in-water emulsification process to produce nanocarriers comprising cholesterol-modified cationic polyamidoamine (PAMAM) dendrimers and chemotherapeutic drugs.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figures 1A-1F The synthesis and characterization of cationic nanocarriers are shown. 1A) Synthesis route of cholesterol-modified PAMAM dendrimers; 1B) TEM image of cationic nanocarrier PAMAM-Chol (5); 1C) Basic physicochemical properties of cationic nanocarriers; 1D) 1H-NMR spectrum of polymers; 1E) DNA binding efficiency of cationic nanocarriers; 1F) Cytotoxicity of cationic nanocarriers to MDA-MB-231 cells.

[0021] Figures 2A-2K The anticancer efficacy of cationic nanoparticles is shown. 2A) Schematic diagram of NSG mouse study; 2B) Primary tumor growth curve; 2C) Tumor image; 2D) In ​​vivo imaging of tumor metastasis; 2E) Quantification of in vivo imaging signal; 2F) Ex vivo imaging of lungs showing tumor metastasis; 2G) Quantification of ex vivo imaging signal; 2H) Probability map created by machine learning based on hematoxylin and eosin (H&E) stained lung sections showing lung metastasis; 2I) Quantification of metastasis level by machine learning; 2J) Serum cell-free DNA (cfDNA) level at different time points; 2K) Serum cfDNA level at the final time point.

[0022] Figures 3A-3C Evaluation of cognitive impairment is shown. 3A) Schematic diagram of the Balb / c mouse study; 3B) Total distance traveled by mice in the open field test; 3C) Distance traveled by mice in the central area during the open field test.

[0023] It should be understood, therefore, that the embodiments of the invention described herein are merely illustrative of the application of the principles of the invention. Reference herein to details of the illustrated embodiments is not intended to limit the scope of the claims, which themselves recite those features regarded as essential to the invention. DETAILED DESCRIPTION OF THE INVENTION

[0025] PAMAM-based nanocarriers with high chemotherapeutic drug loading efficiency and high cfNA binding capacity are able to deliver chemotherapeutic drugs while suppressing chemotherapy-induced systemic inflammation. These nanocarriers are able to deliver chemotherapeutic drugs in a sustained and localized manner, improving the anti-tumor efficacy by improving the pharmacokinetics of the drugs. Therefore, a chemotherapeutic drug-loaded nanoparticle system can be developed for the treatment of primary and metastatic tumors and to alleviate cognitive impairment.

[0026] Cationic polymer nanocarriers are synthesized by modifying PAMAM dendrimers with cholesterol. Nanoparticles can be prepared by water-in-oil single emulsification and can load drugs. During the emulsification process, cholesterol-modified PAMAM dendrimers can form nanoparticles based on their amphiphilic self-assembly. The hydrophilic PAMAM dendrimers will be the outer surface of the nanoparticles, providing a positively charged environment for cfNA binding. The hydrophobic cholesterol fragments will form the core of the nanoparticles and encapsulate chemotherapeutic drugs. It forms nanoparticles with a size of about 160nm and a ζ (zeta) potential greater than +50mV. The cfNA binding ability and drug loading efficiency of the nanocarrier can be adjusted by different conjugation numbers of cholesterol.

[0027] Several chemotherapeutic drugs can potentially be encapsulated in polymeric nanocarriers. Non-limiting examples of chemotherapeutic drugs include alkylating agents such as altretamine, busulfan, carboplatin, carmustine, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, melphalan, temozolomide, or trabectedin; antimetabolites such as 5-fluorouracil, 6-mercaptopurine, azacytidine, capecitabine, clofarabine, cytarabine, floxuridine, fludarabine, gemtuzumab, cytarabine ... gemcitabine, methotrexate, pemetrexed, pentostatin, pralatrexate or trifluridine and tipiracil; plant alkaloids such as vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, etoposide, teniposide, irinotecan or topotecan; and anticancer antibiotics such as daunorubicin, doxorubicin, epirubicin, idarubicin or valrubicin.

[0028] In some embodiments, the chemotherapeutic drug is one or both of paclitaxel or doxorubicin.

[0029] In certain embodiments, the cholesterol-modified PAMAM (polyamidoamine) dendrimer comprises at least one cholesterol residue at the terminal position of the PAMAM. In certain embodiments, the cholesterol residue is attached to an external amine functional group of the PAMAM.

[0030] In some embodiments, the nanoparticles may additionally include one or more immune checkpoint inhibitors. Immune checkpoint inhibitors block proteins called checkpoints, which are composed of certain types of immune system cells (e.g., T cells) and some cancer cells. These checkpoints sometimes help prevent the immune response from being too strong. Too strong a response prevents T cells from killing cancer cells. When these checkpoints are blocked, T cells can kill cancer cells more effectively. Non-limiting examples of immune checkpoint inhibitors include PD-1 inhibitors, such as pembrolizumab, nivolumab, or cemiplimab; PD-L1 inhibitors, such as atezolizumab, avelumab, or durvalumab; CTLA-4 inhibitors, such as ipilimumab or tremelimumab; and LAG-3 inhibitors, such as relatlimab.

[0031] The anticancer efficacy of the nanoparticles has been evaluated in an NSG mouse model bearing MDA-MB-231 human breast tumors with lung metastases. Paclitaxel-loaded nanoparticles showed higher primary tumor inhibitory efficacy than the free drug, which was attributed to its improved stability and tumor targeting efficiency. A thorough characterization of lung metastases has been performed using a combination of IVIS imaging, H&E staining, and machine learning. The results showed that while paclitaxel free drug inhibited primary tumors, it had no therapeutic effect on metastases and even induced more metastases. In contrast, paclitaxel-loaded nanoparticles significantly inhibited tumor metastasis. The anti-metastatic effect was correlated with serum cfDNA levels, indicating the importance of cfNA binding in combating cancer metastasis.

[0032] Chemotherapy-induced cognitive impairment has been evaluated in a Balb / c mouse model treated with doxorubicin free drug and drug-loaded nanoparticles. To detect whether the mice showed any behavioral changes under treatment, an open field test and a behavioral test of pain sensitivity were performed. The results of the open field test showed that the distance traveled by doxorubicin-treated mice was significantly attenuated compared to saline control mice and mice treated with doxorubicin-loaded nanoparticles. These results suggest that doxorubicin may induce cognitive impairment, and the nanocarriers mitigate this effect. The results of our study are shown in the accompanying figures.

[0033] Several non-limiting applications of the disclosed methods are discussed below.

[0034] The first application is to use this system to deliver chemotherapy drugs to suppress untreated early-stage cancers and reduce the risk of recurrence after surgery / chemotherapy / radiation. In cancer patients, cfNA levels in the blood are elevated compared to healthy controls. cfNA levels will increase further after surgery, chemotherapy or radiotherapy, and are elevated in patients with metastatic cancer.

[0035] The second application is to use this system to deliver chemotherapy drugs to inhibit untreated metastatic cancer. Chemotherapeutic drugs can inhibit the growth of primary tumors, but may increase the risk of cancer metastasis and cognitive impairment. Cationic nanocarriers will scavenge pro-inflammatory cfNA to inhibit these side effects.

[0036] A third application is to use this system to treat metastatic cancers that are resistant to other treatments.

[0037] A fourth application is to use this system after surgery / chemo / radiotherapy to prevent recurrence.

[0038] The fifth application is to use this system to deliver immune checkpoint inhibitors and combine immune checkpoint blockade therapy with cfNA depletion agents. Delivery of immune checkpoint inhibitors to cancer will reduce anti-cancer immunosuppression, and cfNA depletion agents will attenuate pro-metastatic inflammation.

[0039] This technology will help at least three types of patients: 1) patients with high-risk early-stage cancers such as breast cancer who need adjuvant chemotherapy to reduce the risk of recurrence after surgery; 2) patients with metastatic cancers such as metastatic breast cancer who need systemic chemotherapy to prolong survival; 3) patients with metastatic cancers such as metastatic breast cancer who are resistant to other treatments. All of these patient populations may benefit from the ability of the nanomedicine of the present invention to reduce cytotoxic drug side effects and prevent circulating tumor cells from producing additional tumor metastases. This approach has the potential to be rapidly translated in a clinical setting because some of the materials we use have been approved for pharmacological use in human patients.

[0040] We have conducted an in vivo study in Balb / c mice bearing murine 4T1 mammary tumors. We have demonstrated that drug-loaded scavenger nanoparticles are able to inhibit primary tumor growth and cancer metastasis. We are investigating how these nanoparticles alter the tumor microenvironment and determining the effects of the nanoparticles on neuroinflammation, mouse behavior (particularly anxiety), and levels of neuronal damage. We will also evaluate the side effects, biodistribution, and pharmacokinetics of the nanoparticles.

[0041] Upon request, detailed embodiments of the present invention are disclosed herein. It should be understood that the disclosed embodiments are only examples of the present invention, and the present invention can be embodied in various forms. Therefore, the specific structural and functional details disclosed herein should not be interpreted as limitations, but only as a basis for teaching those skilled in the art how to apply the present invention. The specific examples provided below will help to better understand the present invention. However, these examples are provided only as guidance and are not meant to be any limitation.

[0042] The present invention can be more easily understood by reference to the following detailed description, in conjunction with the accompanying drawings and examples, which form a part of the present disclosure. It should be understood that the present invention is not limited to the specific materials, devices, methods, applications, conditions or parameters described and / or shown herein, and the terms used herein are only used to describe specific embodiments by way of example, and are not intended to limit the claimed invention. The term "multiple" as used herein means "more than one". When expressing the range of values, another embodiment includes from one specific value and / or to another specific value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it should be understood that the specific value constitutes another embodiment. All ranges are inclusive and combinable.

[0043] It should be understood that certain features of the invention, for the sake of clarity, are described herein separately in the context of different embodiments, but these features may also be provided in combination in a single embodiment. Conversely, various features of the invention described in a single embodiment for the sake of brevity may also be provided separately or in any subcombination. Further references to values ​​stated in ranges include every value within that range and any combination of these values.

[0044] The following definitions are intended to aid in the understanding of the present invention.

[0045] As used herein, the singular form "a," "an," and "the" include plural referents and a reference to a specific value includes at least that specific value unless the context clearly dictates otherwise.

[0046] When expressing a range of values, another embodiment includes from one specific value and / or to another specific value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it is understood that the specific value forms another embodiment. All ranges are inclusive and combinable.

[0047] Throughout the description and claims of this specification, the words “include” and “comprising” and variations of these words, such as “containing” and “having”, mean “including but not limited to”, and are not intended to (and do not) exclude other components.

[0048] The term "sphere-like" refers to an object that resembles a sphere. In some embodiments, the object is such that every point on the surface of the object has approximately the same variance from the center of the object. Approximately the same variance may differ from the average variance by 5%, 10%, 15%, or 20%.

Claims

1. A polymer nanocarrier for delivering chemotherapeutic drugs, comprising a cholesterol-modified cationic polyamidoamine (PAMAM) dendrimer.

2. The polymer nanocarrier according to claim 1, further comprising at least one chemotherapeutic drug.

3. The polymer nanocarrier of claim 2, wherein the at least one chemotherapeutic drug comprises at least one of paclitaxel or doxorubicin.

4. The polymer nanocarrier according to any one of claims 1 to 3, wherein the cholesterol-modified PAMAM (polyamidoamine) dendrimer comprises at least one cholesterol residue at the terminal position of the PAMAM.

5. The polymer nanocarrier of claim 4, wherein the cholesterol residue is attached to an external amine functional group of the PAMAM.

6. The polymer nanocarrier according to any one of claims 1 to 5, wherein the polymer nanocarrier has a spherical shape.

7. A method of treating cancer in a patient by administering to the patient the polymer nanocarrier of any one of claims 1-6.

8. The method of claim 7, wherein the cancer is one or both of a primary tumor and a metastatic tumor.

9. The method according to any one of claims 7-8, wherein the method prevents or reduces chemotherapy-induced cognitive impairment in a patient.

10. The method of any one of claims 7-9, wherein the method prevents or reduces the pro-metastatic effects of chemotherapy in a patient.

11. The method of any one of claims 7-10, wherein the cholesterol-modified cationic polyamidoamine (PAMAM) dendrimer reduces cell-free nucleic acid (cfNA) levels in the patient.

12. The method according to any one of claims 7-11, wherein the method is used after surgery, chemotherapy or radiotherapy to prevent cancer recurrence.

13. The method of any one of claims 7-12, wherein the polymer nanocarrier delivers at least one immune checkpoint inhibitor to the patient.

14. A method for preparing a polymer nanocarrier for delivering a chemotherapeutic drug, comprising producing a nanocarrier comprising a cholesterol-modified cationic polyamidoamine (PAMAM) dendrimer and at least one chemotherapeutic drug using an oil-in-water emulsification process.

15. The method of claim 14, wherein the at least one chemotherapeutic agent comprises at least one of paclitaxel or doxorubicin.

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