RO138087A2 - Polymer conjugates based on poly(2-isopropenyl-2-oxazoline) and doxorubicin for immuno-chemotherapy of cancer and process for preparing the same - Google Patents

Abstract

The invention relates to a process for preparing degradable polymer conjugates based on poly(2-isopropenyl-s-oxazoline) (PiPOx) and doxorubicin (DOX) and to the use thereof in immuno-chemotherapy of cancer. According to the invention, the process comprises the follwing steps: I. synthesis of PiPOx polymers with various polymerization degrees, by anionic polymerization, II. modifying the same by a PiPOx analogue polymer reaction with Ω -mercapto-carboxylic acid esters, to result in copolymers containing ester groups in their side chain, III. hydrazinolysis of ester groups from the side chain to introduce hydrazide groups and IV. treatment with DOX hydochloride to obtain the polymeric conjugate PiPOx-DOX to be used in the treatment of tumor cells, in order to induce immunogenic cell death.

Description

1. The technical field in which the invention can be used

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The present invention refers to the development of degradable polymer conjugates based on poly(2-isopropenyl-2-oxazoline) (PiPOx), as an advanced doxorubicin (DOX) release system, which can be used in cancer immuno-chemo-therapy. The principle of DOX release, after internalization of the polymer conjugate in the affected cells, is based on the selective hydrolysis of hydrazone bonds under relevant physiological conditions of temperature and pH specific to the tumor site. PiPOx-DOX polymer conjugates are advanced chemotherapeutic agent delivery systems because they are designed to maintain their chemical stability in the systemic circulation for a prolonged period and release DOX at the target site after internalization into tumor cells, having an effect minimally cytotoxic to healthy cells. This type of system for the controlled release of the chemotherapeutic agent presents different endocytosis pathways, in contrast to the passive diffusion through cell membranes characteristic of free DOX, being able to ameliorate some of the resistance mechanisms found in known drugs, and induces immunogenic cell death.

2. Description of the current state

Globally, cancer is the second leading cause of death that seriously affects human health, being responsible for approximately 9.6 million deaths in 2018. [1] Conventional cancer treatments include surgery, chemotherapy, immunotherapy, and radiation, while modern approaches to fighting cancer are based on stem cell therapy, hyperthermia, photodynamic therapy, laser treatment, and others. [2-5] Of these, chemotherapy, alone or in combination with other treatments such as surgery or radiation, is the most widely used therapeutic method. Neoadjuvant chemotherapy is used to shrink tumors before surgery [5], while adjuvant chemotherapy is used after surgery or radiation to kill remaining cancer cells. Even so, conventional chemotherapy is often associated with a low therapeutic window 1 | 1 3 due to the poor pharmacokinetic properties of the drugs used. [5] At the same time, the chemotherapeutic agents used do not show selectivity for tumor cells and in most cases also affect healthy cells with high mitotic rates. These can lead to side effects that raise the risk of death in cancer patients. Severity of side effects and disadvantages associated with conventional dosage forms of chemotherapeutic agents, including rapid renal clearance, enzymatic degradation, etc. can be improved by conjugation with different types of polymers. [6, 7] currently, the most successful conjugation strategy involves attaching the therapeutic agent to poly(ethylene glycol) (PEG) polymer chains, known as the "PEG-ylation" method. [8] PEG-ylation has been shown to improve therapeutic efficacy by protecting (chemo)therapeutic agents from the immune system and their degradation in the systemic circulation. [9] Thus, various commercial applications have been developed based on conjugates based on PEG with enzymes, cytokines, antibodies, and liposomes. [10, 11] However, the risks associated with the use of PEG, such as the formation of antibodies against PEG, hypersensitivity to PEG, and vacuolation, raise some reservations in the further use of PEG-ylation. [12,13] Also, due to its chemical structure, PEG only allows the introduction of a maximum of two therapeutic agent units per polymer chain, which leads to a very low loading of therapeutic agent conjugates.

consequently, new strategies have been developed for the preparation of polymer conjugates based on block copolymers or functional statistical copolymers containing reactive units in the side chain that allow the incorporation of a larger amount of therapeutic agent. [14-16] Thus, using free or controlled radical polymerization, polymer conjugates based on poly(N-2-hydroxypropyl methacrylamide), poly(methacryloyloxyethyl phosphorylcholine) or polypeptides were synthesized. [7, 17-19] However, controlled polymerization reactions do not provide a sufficiently precise control over the molecular mass distribution, or terminal groups, which are essential factors in the effective release of the drug.

Poly(2-alkyl/aryl-2-oxazolines) (PAOx) are a class of polymers recently used in the synthesis of polymer conjugates, [20] which present better biocompatibility and hydrophilicity in body fluids, and allow multiple possibilities of orthogonal functionalization in compared to PEG. [21-23] However, the synthesis of controlled high molecular weight PAOx is very difficult and even impossible for certain monomers due to the intrinsic chain transfer reactions that occur during the polymerization reaction, thus limiting in vivo applications where there is a need of relatively high molecular weight polymers for extended systemic circulation over a prolonged period. [24] | 1 3

3. The technical problem in the last 30 years, polymer conjugates based on water-soluble polymers have been reported and studied in which the drug with an anticancer effect was linked to the polymer through non-cleavable covalent bonds, hydrolytically unstable ionic bonds or covalent bonds susceptible to enzymatic hydrolysis or simple chemistry. However, very few reported polymer conjugates have reached the clinical stage, due to the high degree of difficulty of the synthesis process and problems related to the efficiency of the obtaining process on a (semi)industrial scale. Also, the influence of some vital parameters, such as: (i) the amount of cytostatic agent, (ii) the selectivity in the recognition of the affected biological material, (iii) the development of a stable bond in the systemic circulation, but susceptible to hydrolytic/enzymatic degradation at the site tumors, (iv) conjugation site identification, and (v) conjugation technology, has not been sufficiently studied. At the same time, models and quantitative correlations between these parameters and the efficiency and mode of action of the obtained conjugates have not been established. [5] Therefore, there is a great demand for the development of an alternative polymer conjugate platform that is suitable and able to overcome the current challenges and limitations.

DOX is a well-known drug used in chemotherapy to treat breast cancer, bladder cancer, Kaposi's sarcoma, lymphoma, and acute lymphocytic leukemia, etc. Recent studies have demonstrated that although systemic administration of chemotherapeutic agents is generally immunosuppressive, in situ treatments with anthracyclines such as DOX and mitoxantrone can induce immunogenic tumor cell death and activate the antitumor immune response. [25] The immunogenicity of tumor cells is a critical factor for the effectiveness of cancer therapy, the success of certain antitumor therapies being closely dependent on the ability to induce immunogenic cell death which involves the release/exposure of endogenous danger signals such as "damage- associated molecular pattem molecules' (DAMP), such as HSP70, HSP90, HMGB1, calreticulin, ATP, which leads to the triggering of an effective anti-tumor immune response and the establishment of immunological memory. [26] For most anticancer therapies based on immuno-chemo-therapy, the use of drugs in the form of free formulations, shows limited efficiency by their rapid metabolism and excretion from the body, as well as by the poor distribution of drugs within the tumor due to the chaotic arrangement of vessels of blood in the tumor tissue and high interstitial pressure, caused by the lack of efficient lymphatic drainage of the infiltrated serous fluid from the surrounding vessels. Moreover, the high systemic toxicity of cytotoxic drugs limits their clinical benefits. [27] Different studies have shown that polymer conjugates with appropriate hydrodynamic diameters can ensure the delivery of carrier therapeutic agents, preferentially in tumor tissues, | 1 3

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due to the increased permeability and retention effect of vascular endothelium in tumors. The polymers prevent the rapid release of the anti-tumor agent by glomerular filtration and allow a prolonged circulation in the blood and thus a high bioavailability of the agent. Moreover, by chemically binding the therapeutic agent to a water-soluble polymer, an increase in the solubility of the insoluble or slightly soluble therapeutic agent in aqueous media is obtained, their toxicity being significantly reduced. Also, the nature of the chemical bond between the therapeutic agent and the polymer chain can ensure the release of the therapeutic agent in a targeted and controlled manner in the presence of certain stimuli such as pH, temperature, or enzymes. [6, 7, 16, 19]

4. The technical solution

The proposed technical solution is based on the use of poly(2-isopropenyl-2-oxazoline) (PiPOx) as a water-soluble carrier polymer and the conjugation of doxorubicin (DOX) in its side chain by means of a degradable hydrazone type bond at acidic pH specific to the tumor site, to obtain an injectable polymer conjugate drug release system used in cancer immuno-chemo-therapy.

In the following, the roles of the components of the proposed release system are presented in detail:

a) PiPOx polymer: It is a functional, biocompatible and hydrophilic polymer as demonstrated in various recent literature articles. [28-34] It can be synthesized with controlled high molecular weight by anionic polymerization appropriate for the target application, [28] it is chemically inert to moisture, water, and oxygen during storage as a powder or solution [34] , and can be easily modified under simple reaction conditions by ring-opening reactions of the 2-oxazoline groups in the side chain.

b) Degradable linking group: The hydrazone group is one of the most investigated chemical links sensitive to acidic pH, with demonstrated chemical stability in the systemic circulation (pH 7.4). This grouping has the advantage of hydrolysis in the acidic lysosomal (pH 4.8) and endosomal (pH 5-6) compartments as a result of the internalization process in the target cells. [35] Covalent binding of DOX to the side chain of the PiPOx polymer via a hydrazone bond will lead to PiPOx-DOX conjugates with increased efficacy and targeted delivery in tumor tissue.

c) Chemo-therapeutic agent: chemically bound DOX, through the polymer degradable linking group, will have a significantly reduced cytotoxic effect on its way to the affected tissues of interest, due to the controlled release of the chemo-therapeutic agent in the targeted tumor sites, characterized of a slightly acidic pH compared to healthy tissues. Furthermore, the internalization, cellular trafficking, accumulation, and site of release of the cytostatic may improve chemotherapeutic efficacy.

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d) High solubility in body fluids: Due to its high hydrophilicity and the possibility of partial modification, PiPOx allows precise control of the solubility of conjugates in body fluids by varying the ratio between 2-oxazoline and DOX groups.

e) Stability vs. The degradability of the PiPOx-DOX conjugates is due to the presence of the hydrazone bond connecting DOX and the base chain of the PiPOx polymer. These conjugates are able to release the cancerostatic from the carrier in its active form, either in the tumor or, more specifically, directly in the tumor cell. The hydrazone bond ensures chemical stability of the conjugate in body fluids during systemic circulation, and after internalization, it is cleaved due to the more acidic pH in the tumor tissue, releasing the therapeutic agent, DOX.

f) Immunogenic cell death induced by PiPOx-DOX conjugates, with mechanism and kinetics of internalization and accumulation in cells, different from free DOX, but which can favor the antitumor immune response.

5. The advantages of the invention in relation to the state of the art below are presented the main advantages of the polymer conjugates, proposed as controlled release systems:

a) Versatility in the composition of the polymer conjugates developed according to this invention to address a suitable dosage range for treatment.

b) Low systemic toxicity by conjugation of DOX in the side chain of the PiPOx polymer, offering in addition the advantage of efficient activity by using lower therapeutic doses.

c) Reproducibility in the synthesis protocol that ensures reproducibility from the initial composition to the final material properties: the desired product specifications.

d) The high water solubility allows the storage of polymer conjugates in powder form and their preparation in injectable form at the time of administration.

e) Biological efficiency and controlled biodistribution due to the possibility of the PiPOx-DOX conjugate to bypass some resistance mechanisms found in known drugs.

f) Future applicability for the system developed according to this invention and for the conjugation of other therapeutic agents used in the treatment of cancer or other conditions, where controlled/targeted transport and/or release is an advantage.

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6. Description

1. in order to obtain PiPOx-DOX polymer conjugates used in the treatment of cancer according to this invention, multiple compositions based on PiPOx with a degree of polymerization between 100+350 and DOX were made which are synthesized in 4 stages based on the process to obtain as follows:

in the first synthesis step, poly(2-isopropenyl-2-oxazoline) polymers with various molecular masses were obtained by anionic polymerization. in the second stage, their modification was carried out, through an analogous polymer reaction, with esters of ω-mercapto carboxylic acids, obtaining copolymers containing ester groups in the side chain (Figure la). in the third step, the ester groups were subjected to hydrazinolysis by reaction with hydrazine hydrate, to be transformed into acyl hydrazide groups (Figure 1b). in the last step, polymer conjugates with doxorubicin were obtained by acid-catalyzed reaction of doxorubicin hydrochloride with polymers containing acyl hydrazide functional groups (Figure Ic). The content of doxorubicin was optimized to finally obtain water-soluble polymer conjugates. The final polymer conjugate is purified by precipitation and column chromatography.

Example of obtaining a PiPOx-DOX polymer conjugate composition Step 1. Synthesis of poly(2-isopropenyl-2-oxazoline)

PiPOx was synthesized by the process of anionic polymerization in solution. All reactants were purified and dried using classical procedures. [28, 32] All reactants were handled under argon flow. Thus, under argon flow, a solution was prepared containing 10 g (95.4 mmol) of iPOx dissolved in 50 mL of tetrahydrofuran. The solution was cooled to -40°C and 0.254 mL n-butyl lithium (0.636 mmol) was injected. The reaction mixture was maintained for 10 min at this temperature, and then 5 mL of methanol was added to terminate the reaction. finally the reaction mixture is allowed to warm to room temperature, precipitated in ethyl ether and dried in a vacuum oven at 50 °C overnight. By varying the iPOx: n-butyl lithium molar ratio, a series of polymers with degrees of polymerization between 100 and 350 were obtained.

Step 2. Synthesis of poly(2-isopropenyl-2-oxazoline) copolymers containing methyl ester groups (PiPOx-MMP)

The PiPOx-MMP copolymer was prepared by the analogous polymer modification reaction of PiPOx with methyl 3-mercapto propionate. 200 mg of PiPOx was dissolved in 3.4 mL of N,N'dimethylformamide. 0.2 mL of methyl 3-mercapto propionate was added to the obtained solution.

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The reaction mixture was heated at 100 °C for 1 h. if it is desired to obtain copolymers with a higher degree of modification, the duration of the reaction can be extended to 2 or 3 hours. After the appropriate time the reaction mixture was cooled to room temperature, diluted with 2 mL of methylene chloride and precipitated into 35 mL of ethyl ether. The precipitate obtained was filtered and washed on the filter with ethyl ether. The copolymer was then dried in a vacuum oven at 50°C overnight.

Step 3. Synthesis of poly(2-isopropenyl-2-oxazoline) copolymers containing hydrazine groups (PiPOx-H) in a glass vial equipped with a magnetic stirrer, 160 mg of PiPOx-MMP were dissolved in 2.3 mL of methanol. After 10 minutes, 0.5 mL of hydrazine hydrate was added. The solution thus obtained was left to stir at room temperature overnight. The next day the solvent was removed using a rotary evaporator and the obtained white powders were further used in the next reaction step.

Step 4. Synthesis of poly(2-isopropenyl-2-oxazoline)-doxorubicin (PiPOxDOX) conjugates

A solution prepared from 120 mg PiPOx-H, 10.5 mg doxorubicin hydrochloride, and 0.03 mL acetic acid in 2 mL methanol was stirred at room temperature in the dark for 3 days. The copolymer obtained was isolated by column chromatography using Sephadex LH-20 as the stationary phase and methanol as the eluent. The PiPOx-DOX polymer conjugate was obtained as a water-soluble red solid.

2. in order to test the biological efficiency and visualize the biodistributability, in vitro studies were performed on three tumor cell lines of interest, namely murine RAW267.4, human Jurkat and murine B16F10, to evaluate the cellular interaction and toxicity of PiPOx-DOX conjugates with tumor cells, as well as internalization and intracellular accumulation mechanisms at the time of induction of immune cell death. ELISA, flow cytometry, microscopy and MTT immunoenzymatic technique were used.

Example of testing the interaction of PiPOx-DOX conjugates with tumor cells

The dynamics of PiPOx-DOX polymeric conjugate accumulation in melanoma-derived tumor cells was evaluated in live cell imaging experiments. Bl6 FIO cells were grown in Lab-Tek II imaging chambers (Nune) and incubated under physiological conditions 7 | 1 3 (temperature 37 °C, humidity > 95%) throughout the experiment, in a stage-top incubator (Tokai Hit) mounted on a motorized Nikon TE-2000 microscope. Microscopy images were acquired every 30 min at 4 positions for each experimental condition in phase contrast to observe cell morphology, and in epifluorescence to monitor accumulated doxorubicin. The mean fluorescence intensity measured on the doxorubicin channel in the non-cell-covered spaces (segmented based on the phase-contrast images) was subtracted from each frame to compensate for non-specific fluorescent signal and that of doxorubicin in the medium. The mean fluorescence intensity of doxorubicin measured in the cell-covered areas, obtained after subtraction of the nonspecific fluorescent signal, was used to monitor the dynamics of the accumulation of the antitumor agent in the cells.

Free doxorubicin rapidly accumulates in cells, with predominantly nuclear localization, reaching a maximum 20-24 hours after the start of incubation (Figure 2, bottom - DOX 10). Subsequently, the fluorescent signal corresponding to doxorubicin begins to decrease, due to high cell death. in contrast, treatment of B16F10 cells with a polymeric PiPOx-DOX conjugate at a concentration of 0.29 mg/mL DOX leads to a slower but long-lasting accumulation of doxorubicin in the cells (Figure 2, bottom - PiPOx-DOX 10). Much of the fluorescent signal is located in the perinuclear region and not in the nucleus, suggesting a different mechanism of cell entry compared to DOX, based on endocytosis and active transport to membrane organelles. The results suggest that PiPOx-DOX accumulates in cells in the conjugated form, by a different mechanism than free doxorubicin.

in addition, the conjugates did not reach 50% cytotoxic effect within 72h on the three different cell lines, even at concentrations more than 10 times higher than the equivalent of free DOX.

Example protocol for induction of immunogenic tumor cell death

The Jurkat human T lymphoblast cell line was used to induce immunogenic cell death. The cells were treated for 24 - 72 hours with free DOX or conjugated PiPOxDOX with a DOX concentration of 0.29 mg/mL, after which the percentage of apoptotic cells (Annexin V+) was determined, respectively the expression of calreticulin (CRT) on the surface of apoptotic cells as well as the level of HSP70 in culture supernatants. Treatment of Jurkat cells with 10 pg/mL PiPOx-DOX conjugate induced significant increase in the percentage of apoptotic cells expressing surface calreticulin as well as HSP70 secretion similar to free DOX treatment Ipg/mL suggesting that PiPOx-DOX treatment induces immunogenic cell death (Figure 3).

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Claims (5)

1. The degradable polymeric PiPOx-DOX conjugates developed for cancer immunochemotherapy, characterized by the fact that: they present (chemo)therapeutic properties and can be used in the form of injectable solutions having as main ingredient·, (i) poly(2-isopropenyl2-oxazoline) with degree of polymerization between 100-350, chemically modified with (ii) 1-5 molar% DOX by means of a degradable hydrazone-type chemical linking group. 2. The process for obtaining degradable PiPOx-DOX polymer conjugates according to claim 1, characterized in that they can be obtained in 4 synthesis steps, and the proposed methodology offers versatility in dosage and reproducibility in chemical composition. The different compositions of the polymer conjugates allow a controlled dosing of the antitumor agent at the tumor site. 3. The degradable PiPOx-DOX polymer conjugates developed according to claim 1, characterized in that they can release the DOX chemotherapeutic agent by hydrolysis at acidic pH in contact with cellular fluids following the internalization process. 4. The degradable PiPOx-DOX polymeric conjugates developed according to claim 1, characterized in that they present a different mechanism and kinetics of internalization and accumulation in cells compared to free doxorubicin. Accumulation of the conjugate takes place in the perinuclear region, compared to the accumulation of free DOX in the nucleus. 5. The degradable PiPOx-DOX polymer conjugates developed according to claim 1, characterized in that they have a diminished cytotoxic effect compared to free DOX, follow a different internalization mechanism compared to free DOX and can induce immunogenic cell death after internalization.