Suppression of Chemotherapy-Induced Cancer Metastasis and Cognitive Dysfunction by Cationic Polymeric Nanocarriers

Abstract

PAMAM-based nanocarriers with high filling efficiency and high cfNA binding ability of chemotherapeutic drugs enable continuous delivery of chemotherapeutic drugs while suppressing systemic inflammation caused by chemotherapy. This is useful for treating primary and metastatic tumors along with attenuation of cognitive impairment.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the invention disclosed in U.S. Provisional Application No. 63 / 349,492, filed on June 6, 2022, with the title "Cationic Polymeric Nanocarriers to Inhibit Chemotherapy - Induced Cancer Metastasis and Cognitive Impairment". The benefit under 35 U.S.C. § 119(e) of the U.S. provisional application is hereby claimed, and the above - mentioned application is hereby incorporated herein by reference.

[0002] Government Use License This invention was made with government support under grant number AR073935 awarded by the National Institutes of Health (NIH) of the United States and grant number W81XWH1910463 awarded by the Department of Defense (DoD). The government has certain rights in this invention.

Background Art

[0003] Cancer is the leading cause of death in most countries. In women, breast cancer is the most frequently diagnosed cancer and has the second - highest mortality rate. Many early - stage non - metastatic breast cancer patients can be cured with a combination of surgery, chemotherapy, and radiotherapy. However, approximately 50% of patients develop distant organ metastases, which are generally not curable. Chemotherapeutic drugs such as paclitaxel and doxorubicin are used in the treatment of primary breast cancer in combination with surgery. Evidence is increasing to suggest that these drugs, while suppressing the growth of primary tumors, can promote metastasis by increasing the levels of inflammatory cell - free nucleic acids (cfNA) released from damaged cells into the tumor microenvironment. New research is beginning to focus on attenuating the metastasis - promoting effects of chemotherapy.

[0004] Cognitive impairment is also a side effect that cancer patients undergoing chemotherapy may face. These cognitive impairments are also known as chemo brain, chemo fog, i.e., chemotherapy-induced cognitive impairment. Chemo brain was first cited in the 1980s when the cognitive function assessment of a cohort of cancer patients was significantly lower during and after chemotherapy. Psychiatrists were advised to recognize chemotherapy as a possible cause for behavioral changes. Qualitative studies reported problems with concentration, working memory, and multitasking in breast cancer survivors, and changes in executive function and learning / memory were pointed out. Cohort studies have shown that chemotherapy-induced cognitive impairment can persist for about 4 to 10 years in subjects showing hyporeactivity in the parahippocampal gyrus during an associative task for evaluating episodic memory. Therefore, there is a strong desire to develop new nanomedicines to reduce chemo brain.

[0005] High levels of cfNA - single-stranded and double-stranded DNA and RNA - in the blood circulation around tumors after chemotherapy are associated with cancer metastasis and demobrain. 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, inducing the secretion of inflammatory cytokines, and TLR-induced systemic inflammation develops, promoting tumor metastasis and cognitive impairment. cfNA is being explored as a biomarker for cancer diagnosis and prognosis but is hardly recognized as a therapeutic target for preventing metastasis and chemo brain.

[0006] Recently, we discovered that cationic polyamidoamine (PAMAM) dendrimers can remove nucleic acids via electrostatic interactions. When used to treat inflammatory and autoimmune diseases, PAMAM removes cfNA and suppresses cfNA-induced TLR activation and TLR-induced inflammation. In pancreatic and breast cancer models, PAMAM dendrimers suppress metastasis by removing cfNA. Through careful molecular design based on PAMAM dendrimers, cationic polymeric nanocarriers for chemotherapeutic drugs can suppress both cancer metastasis and cognitive dysfunction induced by chemotherapy.

Summary of the Invention

[0007] We developed cationic polymer-based nanoparticles that can encapsulate chemotherapy and bind to inflammatory circulating cell-free DNA molecules. This nanoparticle technology has been shown to reduce chemotherapy-induced systemic inflammation and cancer metastasis in vivo. Thereby, these nanoparticles can reduce potential neuroinflammation by reducing chemotherapy-related systemic inflammation, thereby alleviating the symptoms of chemobrain. Cognitive dysfunction is one of the side effects that cancer patients receiving chemotherapy may face. These cognitive dysfunctions are also referred to as chemobrain, chemo fog, i.e., chemotherapy-induced cognitive dysfunction. Qualitative studies have reported problems with concentration, indeterminacy, work area, and multitasking in cancer survivors, and changes in executive function and learning / memory have been pointed out.

[0008] Chemotherapy promotes chronic inflammation through necrotic cancer cell death and the release of their cellular contents, known as damage-associated molecular patterns (DAMPs), into the bloodstream. DAMPs include cell-free nucleic acids (such as cell-free DNA and miRNA) that can activate immune Toll-like receptors (TLRs). The persistence of DAMPs in the blood induces a state of chronic inflammation with elevated levels of inflammatory cytokines in the blood circulation, which can then invade 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 chemobrain and depression. Since chemotherapy is a mainstay of cancer treatment, it is essential to find ways to minimize off-target inflammation and incidental cognitive dysfunction. Our nanomaterials can reduce systemic inflammation induced as a byproduct of chemotherapy treatment, and by reducing systemic inflammation, they can also reduce neuroinflammation and thereby mitigate the impact on cognitive function resulting from chemotherapy treatment.

[0009] In some aspects, the present disclosure relates to a polymeric nanocarrier for the delivery of chemotherapeutic agents, the polymeric nanocarrier comprising a cationic polyamidoamine (PAMAM) dendrimer modified with cholesterol. Some polymeric nanocarriers further comprise a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is one or both of paclitaxel or doxorubicin.

[0010] In one embodiment, a cholesterol-modified PAMAM (polyamidoamine) dendrimer comprises at least one cholesterol residue at a terminal position of the PAMAM. In one embodiment, the cholesterol residue is attached to an external amine functional group of the PAMAM.

[0011] The polymeric nanocarrier may have a spherical shape.

[0012] Other aspects of the present disclosure relate to a method of treating a patient's cancer by administering to the patient the polymeric nanocarrier disclosed herein. In some embodiments, the cancer is one or both of a primary tumor and a metastatic tumor.

[0013] In certain embodiments, the method prevents or reduces chemotherapy-induced cognitive dysfunction in a patient. In some embodiments, the method prevents or reduces the metastasis-promoting effect of chemotherapy in a patient. It has also been found that cholesterol-modified cationic PAMAM (polyamidoamine) dendrimers reduce the levels of cell-free nucleic acids (cfNA) within a patient.

[0014] In certain embodiments, the nanocarriers and methods disclosed herein are used for the treatment of cancer. In some embodiments, the nanocarriers and methods are utilized to prevent cancer recurrence after surgery, chemotherapy, or radiation therapy.

[0015] In certain embodiments, a polymeric nanocarrier delivers at least one immune checkpoint inhibitor.

[0016] In yet other aspects, the disclosure includes methods of making polymeric nanocarriers for the delivery of chemotherapeutic agents described herein. Some methods comprise generating a nanocarrier comprising a cholesterol-modified cationic polyamidoamine (PAMAM) dendrimer and a chemotherapeutic agent using a water-in-oil emulsion process. BRIEF DESCRIPTION OF THE DRAWINGS

[0017]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 1E

Figure 1F

Figure 2A

Figure 2B

Figure 2C

Figure 2D

Figure 2E

Figure 2F

Figure 2G

Figure 2H

Figure 2I

Figure 2J

Figure 2K

Figure 3A

Figure 3B

Figure 3C

Mode for Carrying Out the Invention

[0018] Therefore, it should be understood that the embodiments of the present invention described herein are merely exemplary examples of the application of the principles of the invention. The reference herein to the details of the described embodiments does not limit the scope of the claims, which describe the features that are considered essential to the invention itself.

[0019] PAMAM-based nanocarriers having a high loading efficiency of chemotherapeutic agents and a high cfNA binding ability can deliver chemotherapeutic agents while suppressing the systemic inflammation caused by chemotherapy. These nanocarriers can enhance the antitumor efficacy by continuously and locally delivering chemodrugs to improve the pharmacokinetics. Therefore, a chemotherapeutic agent-loaded nanoparticle system can be developed to treat both primary and metastatic tumors with attenuation of cognitive impairment.

[0020] Cationic polymer nanocarriers are synthesized by modifying PAMAM dendrimers with cholesterol. The nanoparticles can be prepared through a water-in-oil single emulsion by drug loading. During the emulsion process, cholesterol-modified PAMAM dendrimers can self-assemble based on their amphiphilicity to form nanoparticles. The hydrophilic PAMAM dendrimers are on the outer surface of the nanoparticles, providing a positively charged environment for cfNA binding. The hydrophobic cholesterol segments form the core of the nanoparticles and encapsulate the chemotherapeutic agent, which forms nanoparticles with a size of ~160 nm and a zeta potential of +50 mV or more. The cfNA binding ability and drug loading efficiency of the nanocarriers can be controlled by the difference in the conjugation number of cholesterol.

[0021] Several chemotherapeutic agents have the potential to be encapsulated within the polymer nanocarriers. Non-limiting examples of chemotherapeutic agents 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, azacitidine, capecitabine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, methotrexate, pemetrexed, pentostatin, pralatrexate, or trifluridine and tipiracil; plant alkaloids such as vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, etoposide, teniposide, irinotecan, or topotecan; and antitumor antibiotics such as daunorubicin, doxorubicin, epirubicin, idarubicin, or valrubicin.

[0022] In some embodiments, the chemotherapeutic agent is one or both of paclitaxel or doxorubicin.

[0023] In certain embodiments, a cholesterol-modified PAMAM (polyamidoamine) dendrimer comprises at least one cholesterol residue at a terminal position of the PAMAM. In certain embodiments, the cholesterol residue is attached to an external amine functional group of the PAMAM.

[0024] In some embodiments, the nanoparticles may additionally comprise one or more immune checkpoint inhibitors. Immune checkpoint inhibitors inhibit proteins referred to as checkpoints that are formed by immune system cells such as certain types of T cells and some cancer cells. Such checkpoints sometimes help prevent the immune response from becoming too strong. An overly strong response can stop T cells from killing cancer cells. When these checkpoints are inhibited, T cells can more efficiently kill cancer cells. 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.

[0025] The anti-cancer efficacy of the nanoparticles was evaluated in an NSG mouse model bearing MDA-MB-231 human breast cancer with lung metastases. Paclitaxel-loaded nanoparticles showed higher primary tumor suppression efficacy than the free drug, which was attributed to improved stability and tumor targeting efficiency. A thorough characterization of lung metastases was performed by combining IVIS imaging, H&E staining, and machine learning. The results showed that drugs without paclitaxel suppressed the primary tumor but showed no therapeutic effect on metastases and even induced metastases in some cases. In contrast, paclitaxel-loaded nanoparticles significantly suppressed tumor metastasis. The anti-metastatic effect correlated with serum cfDNA levels, suggesting the importance of cfNA binding in cancer metastasis.

[0026] A Balb / c mouse model with chemotherapy-induced cognitive dysfunction was evaluated using drugs and drug-loaded nanoparticles that do not contain doxorubicin. To examine whether the mice underwent any behavioral changes during treatment, an open field test and a pain sensitivity behavioral test were conducted. The results of the open field test show a significant decrease in the distance traveled by the doxorubicin-treated mice compared to the saline control mice and the doxorubicin-loaded nanoparticle-treated mice. These results suggest that doxorubicin can induce cognitive dysfunction while the nanocarrier reduces this effect. Our findings are presented below in the drawings.

[0027] Some non-limiting application examples of the disclosed method are shown below.

[0028] The first application example is to deliver chemotherapeutic drugs using this system to suppress untreated early-stage cancer and reduce the risk of recurrence after surgery / chemotherapy / radiation therapy. In cancer patients, cfNA levels are evaluated in the blood when compared to healthy controls. The levels of cfNA further increase after surgery, chemotherapy, or radiation therapy and are elevated in patients with metastatic cancer.

[0029] The second application example is to deliver chemotherapeutic drugs using this system to suppress untreated metastatic cancer. Chemotherapeutic drugs can suppress the growth of primary tumors but may increase the risk of cancer metastasis and cognitive dysfunction. Cationic polymer nanocarriers remove inflammatory cfNA and suppress these side effects.

[0030] The third application example is to use this system to treat metastatic cancer that is resistant to other treatments.

[0031] The fourth application example is to use this system after surgery / chemotherapy / radiation therapy to prevent recurrence.

[0032] The fifth application example uses this system to deliver immune checkpoint inhibitors and combine immune checkpoint inhibition therapy and cfNA capture agents. Delivery of immune checkpoint inhibitors to cancer reduces anti-cancer immunosuppression, and the cfNA capture agent attenuates metastasis-promoting inflammation.

[0033] This technology can benefit at least the following three groups of patients: 1) patients with high-risk early cancers such as breast cancer who require adjuvant chemotherapy to reduce the risk of recurrence after surgery; 2) patients with metastatic cancers such as metastatic breast cancer who require systemic chemotherapy for life extension; 3) patients with metastatic cancers such as metastatic breast cancer for whom other treatments are ineffective. All groups can benefit from our nanomedicine's ability to reduce the side effects of cytotoxic agents and prevent the development of further tumor metastases from circulating tumor cells. Since some of the materials we used have already been approved for pharmacological use in human patients, the approach here has the potential to be quickly translated to the clinical setting.

[0034] We concluded in vivo studies using Balb / c mice with 4T1 breast tumors. We demonstrated that the drug-loaded capture agent nanoparticles can suppress both primary tumor growth and cancer metastasis. We are examining how these nanoparticles modify the tumor microenvironment and identifying the effects of the nanoparticles on the levels of neuroinflammation, mouse behavior (specifically anxiety), and nerve damage. We are also attempting to evaluate the side effects, biodistribution, and pharmacokinetics of the nanoparticles.

[0035] Detailed embodiments of the present invention are disclosed herein as needed. It should be understood that the disclosed embodiments are merely examples of the present invention, which can be realized in various forms. Therefore, the specific structures and basic details disclosed herein should not be construed as limitations, but merely as a basis for teaching those skilled in the art to adopt the present invention. The following specific examples will make the present invention better understood. However, they are provided merely as a guide and do not imply any limitation.

[0036] The present invention can be more easily understood with reference to the following detailed description, which is presented in the context of the accompanying drawings and examples that form a part of this 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 illustrated herein, and that the terms used herein are for the purpose of describing particular embodiments by way of example and are not intended to limit the claimed invention. As used herein, the term "a plurality of" means "more than one." When a range of values is expressed, other embodiments include from and / or to one particular value and / or the other particular value. Similarly, when a value is expressed as an approximation by use of the preceding "about," it should be understood that the particular value forms other embodiments. All ranges are inclusive and combinable.

[0037] It should be understood that certain features of the present invention described herein in the context of separate embodiments may be provided in combination in a single embodiment for purposes of clarity. Conversely, various features of the present invention described in the context of a single embodiment may be provided separately or in any subset for purposes of brevity. Further reference to values described in ranges includes each value and combination of values within that range.

[0038] The following definitions are provided to assist in understanding the present invention.

[0039] As used herein, the singular forms "a," "an," and "the" include the plural, and reference to a particular numerical value includes at least that particular value unless expressly stated otherwise.

[0040] When a range of values is expressed, other embodiments include from and / or to one particular value and / or the other particular value. Similarly, when a value is expressed as an approximation by use of the preceding "about," it should be understood that the particular value forms other embodiments. All ranges are inclusive and combinable.

[0041] Throughout the detailed description and the entire scope of the claims of this specification, the terms "comprise / include (base form)" and "contain (base form)" and variations of those terms, such as "comprising / including (present progressive)" and "comprises / includes (third-person singular present tense)", mean "including but not limited to" and are not intended to exclude other elements (do not exclude).

[0042] The term "spherical" means an object similar to a sphere. In some embodiments, the object is such that each point on the surface of the object is at approximately the same difference from the center of the object. The approximately the same difference may vary by 5%, 10%, 15% or 20% from the average difference.

Claims

1. A polymeric nanocarrier for the delivery of chemotherapeutic agents, comprising a cationic polyamidoamine (PAMAM) dendrimer modified with cholesterol.

2. The polymeric nanocarrier according to claim 1, further comprising at least one chemotherapeutic agent.

3. The polymeric nanocarrier according to claim 2, wherein the at least one chemotherapeutic agent comprises at least one of paclitaxel or doxorubicin.

4. The polymeric 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 polymeric nanocarrier according to claim 4, wherein the cholesterol residue is bonded to the external amine functional group of the PAMAM.

6. The polymeric nanocarrier according to any one of claims 1 to 5, having a spherical shape.

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

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

9. The method according to claim 7 or 8, wherein the method prevents or reduces chemotherapy-induced cognitive dysfunction in the patient.

10. The method according to any one of claims 7 to 9, wherein the method prevents or reduces the metastasis-promoting effect of chemotherapy in the patient.

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

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

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

14. A method for producing a polymeric nanocarrier for the delivery of chemotherapeutic agents, comprising the step of generating a nanocarrier comprising a cationic polyamidoamine (PAMAM) dendrimer modified with cholesterol and at least one chemotherapeutic agent using a water-in-oil emulsion process.

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

Images