Poly (Alkyl Cyanoacrylate) Nanoparticles for Use in Treatment of Cancer

The invention is related to the field nanoparticles and medical use. In particular, it relates to cabazitaxel (CBZ) as active ingredient encapsulated into poly (alkyl cyanoacrylate) nanoparticles for use in treatment of cancer.

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Description
FIELD OF INVENTION

The invention is related to the field nanoparticles and medical use. In particular, it relates to an active ingredient encapsulated into poly (alkyl cyanoacrylate) nanoparticles for use in treatment of cancer.

BACKGROUND

The use of nanotechnology in medicine offers many exciting possibilities with potential in a number of medicinal applications envisaged. In particular, nanomedicine is expected to lead to big improvements in the treatment of complex diseases. Two areas in which the use of nanoparticles has begun to demonstrate particular value are drug delivery and molecular imaging.

Poly(alkyl cyanoacrylate) (PACA) was first developed and approved as surgical glue. PACA nanoparticles (NPs) have later demonstrated promising abilities as a drug carrier, being biodegradable and allowing high drug loading capacity.

WO2014191502 A1 discloses a one-step polymerization process for preparing stealth NPs of PACA homopolymer or copolymer comprising anionic polymerization of an oil-in-water miniemulsion. As disclosed, by utilizing a miniemulsion in combination with a particular class of polyalkylene glycol derivatives, it is possible to covalently attach targeting moieties to polyalkylene glycols, thereby enabling the simultaneous introduction of a targeting group and formation of a stealth corona. It is described that the miniemulsion may contain active agents, and a list of therapeutic agents are disclosed. However, none of the examples include encapsulation of any of these agents, and neither in vitro nor in vivo data are disclosed.

Although new, targeted treatment options and immunotherapy are being developed, chemotherapy is still the main therapeutic option for patients with advanced cancer. However, the therapeutic effect is not sufficient for certain cancer types and the treatment also results in severe side effects. Several products of drug-loaded NPs have reached the market, and many new product candidates are in clinical trials. These aspects, including the challenges and opportunities of using nanoparticles in cancer drug delivery, have been discussed in multiple reviews and commentaries including Shi et al (2017) and Torchilin (2014). In addition to improving efficacy by benefiting from the enhanced permeability and retention (EPR) effect (Matsumura et Maeda, 1986), NP encapsulated drug delivery may demonstrate reduced toxicity. The main advantage of the drug-loaded NPs in the market is that they give less adverse effects than free drug, while the therapeutic efficacy is rather similar, as described in Parahbakar et al 2013.

In Snipstad et al (2017), it is disclosed medical use of PEGylated PEBCA NPs in combination with microbubbles (MBs) and ultrasound. The drug delivery system as described consists of microbubbles stabilized by polymeric nanoparticles (NPMBs), which enables ultrasound-mediated drug delivery. The NPs are synthesized by miniemulsion polymerization. It is disclosed NPs containing cabazitaxel (CBZ), and in vitro toxicity of these NPs in triple-negative human breast adenocarcinoma cells, MDA-MB-231. The in vivo data of the drug delivery system disclosed in Snipstad et al (2017) described the therapeutic effect achieved by NP-stabilized MBs on localized, solid tumors, and how an improved effect is achieved by applying focused ultrasound.

Breast cancer is the most common non-cutaneous malignancy in women and second only to lung carcinoma in cancer mortality. There are several types of breast cancer, and in the last decade it has been possible to preform molecular classification of breast cancer based on gene expression profiles. Analyses of human breast tumors have revealed remarkably robust molecular subtypes with distinctive gene signatures and clinical outcome (Toft and Cryns, Mol Endocrinol. 2011 February; 25(2): 199-211.). Basal-like breast cancer (BLBC) is a particularly aggressive molecular subtype defined by a robust cluster of genes expressed by epithelial cells in the basal or outer layer of the adult mammary gland. BLBC is a major clinical challenge because these tumors are prevalent in young women, often relapsing rapidly. Additionally, most (but not all) basal-like tumors lack expression of steroid hormone receptors (estrogen receptor and progesterone receptor) and human epidermal growth factor receptor 2, limiting targeted therapeutic options for these predominantly triple-negative breast cancers. As described in Engebraaten et al (2013), for basal-like tumors no targeted therapies are available, and the patients would therefore benefit from improved chemotherapy regimens.

The male counterpart to breast cancer is prostate cancer. It is the most common cancer among men, and develops in the prostate, a gland in the male reproductive system. Most prostate cancers are slow growing; however, some grow relatively quickly. The cancer cells may spread from the prostate to other area of the body, particularly the bones and lymph nodes. Prostate cancer can often be treated successfully if found in the early stages.

Taxanes are important chemotherapeutic agents with proven efficacy in human cancers. Taxanes include paclitaxel, docetaxel, cabazitaxel and their pharmaceutically acceptable salts. Paclitaxel was originally derived from the Pacific yew tree. Docetaxel is a semi-synthetic analogue of paclitaxel. CBZ, which has been characterized by Vrignaud et al (2013), is a relatively novel semi-synthetic taxane derivative. CBZ has a potent cytostatic effect by microtubule stabilization but its use has been limited due to its toxicity. CBZ has been included in several clinical trials investigating efficacy against several types of cancer. It has been approved by the US Food and Drug Administration (FDA) for treatment of refractory prostate cancer as a second line drug after docetaxel chemotherapy. Taxanes present difficulties in formulation as medicines because they are poorly soluble in water.

It is therefore desirable, and hence an object of the present invention, to develop a new drug delivery system which is capable of effectively delivering a therapeutic agent to a specific location. In particular, a drug delivery system which demonstrate efficacy in addition to fewer adverse side effects would be desirable.

It is further desired if the new drug delivery system is capable of delivering hydrophobic and/or poorly soluble therapeutic agents.

Ultimately, it is desired if the drug delivery system is suitable for treatment of tumors where no targeted therapies are available.

SUMMARY OF INVENTION

In a first aspect of the present invention, it is provided herein a drug delivery system comprising PEGylated poly (alkyl cyanoacrylate) (PACA) nanoparticles (NPs) loaded with cabazitaxel (CBZ), or a pharmaceutically acceptable salt thereof, for use in treatment of cancer, provided that the drug delivery system does not comprise NP-stabilized microbubbles (MBs).

In one embodiment of this aspect, the drug delivery system does not comprise NPs that stabilize the MBs or NPs that are used to stabilize gas-filled MBs. In another embodiment, the drug delivery system does not comprise NPs that are associated with the MBs. In yet another embodiment, the drug delivery system does not comprise gas-filled MBs. In a further embodiment, the drug delivery system does not comprise MBs.

In a further embodiment, the PACA NPs are produced according to a miniemulsion anionic polymerization process.

In another embodiment, the NPs are further surface modified by a targeting moiety.

According to different embodiments of the first aspect, the PACA NP is below 800 nm, such as in a range selected from 1-800 nm or 10-500 nm or 70-150 nm.

In yet other embodiments, the CBZ comprises 1-90 wt % of the NP, preferentially 5-50 wt % of the NP, more preferentially 5-20 wt % or most preferentially 5-15 wt % of the NP. In a particular embodiment, CBZ comprises from 6-13 wt % of the NP, more particularly about 6, 7, 8, 9, 10, 11, 12 or 13 wt % of the NP.

In other embodiments of the first aspect, the drug delivery system is administered parenterally and may further comprising pharmaceutically acceptable excipients.

In yet another embodiment of the first aspect, the cancer is a tumor in a vascular phase.

In a further embodiment, the tumor belongs to a type of cancer selected from the group consisting of prostate cancer, breast cancer, glioma, lung cancer, adrenocortical carcinoma, testicular cancer, urothelium transitional cell carcinoma and ovarian cancer. In yet another embodiment, the drug delivery system is for use in prophylactic treatment of cancer to prevent metastasis through the lymph node.

In yet a further embodiment, the drug delivery system is for use as an immune modulator and/or as a vehicle to enhance the therapeutic effect of encapsulated drugs.

In a second aspect of the invention, it is also provided a method for treating cancer comprising administering a drug delivery system according to the first aspect to a patient in need thereof.

In a third aspect, it is provided a composition or solution comprising the drug delivery system according to the first aspect of the invention. The composition or solution may be a pharmaceutical formulation comprising pharmaceutically acceptable excipients and diluents.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Size distribution of the batches used in the MAS98.12 efficacy study. The size distributions for PEBCA-CBZ (the batch with size z-average of 215 nm in Table 1) is shown in dark blue and for PEBCA (without drug; the batch with z-average of 156 nm in Table 1) is shown in light blue. The size distribution of non-encapsulated CBZ, solubilized in a polysorbate 80 solution, is shown in red. Intensity (%) on the y-axis means percent intensity of total scattering.

FIG. 2. Treatment efficacy and toxicity studied in mice bearing MAS98.12 patient derived xenograft (PDX) breast tumor models. Tumor growth inhibition (A) and body weight change (B) after treatment. PEBCA-CBZ and CBZ were injected 2×15 mg CBZ/kg body weight at day 33 and 36, indicated by the red arrows. Empty PEBCA NPs (same dose as PEBCA-CBZ) and saline were used as negative controls (mean±SEM; n=8-9 tumors/5-6 mice). Tumor size is relative to the size measured at time of randomization. Non-palpable tumors at day 57 after implantation are indicated as complete remissions. The statistical p-value of Welch t-test of areas under the curves is indicated (p=0.02).

FIG. 3. Biodistribution of PEBCA particles containing the fluorescent dye NR668 measured in MAS98.12 tumor-bearing mice. Whole body images were obtained with IVIS 1, 4, 24 and 96 h after intravenous administration of the NPs; color scale on the right indicates radian efficiency×109(A). Ex vivo fluorescence images of isolated organs were obtained 24 h after injection (B). Quantification of fluorescence intensity as relative radiant efficiency per region of interest pixel data of tissues collected 24 h after injection (C). Mean values obtained for 2 animals; error bars show estimated SD values. PEBCA-CBZ: PEBCA containing CBZ and NR668; PEBCA: PEBCA containing NR668, but not CBZ. LNs: lymph nodes.

FIG. 4. CBZ concentrations in plasma and organs measured with mass spectrometry after administration of 15 mg CBZ/kg. Plasma concentration measured as function of time (A). CBZ concentrations in tumors and organs measured after 24 h (B) and 96 h (C). LNs: lymph nodes. Data shown are mean values±SD (n=3). Note that logarithmic scales are used on all y-axes.

FIG. 5. Macrophage infiltration in treated MAS98.12 tumors. The macrophage infiltration was measured in the MAS98.12 tumors 96 h after injection of saline (control), PEBCA NPs (without drug), non-encapsulated CBZ, and PEBCA-CBZ. (A): The total population of infiltrated macrophages was quantified using an antibody to CD68. (B): The population of anti-tumorigenic (pro-inflammatory) macrophages was quantified using an antibody to iNOS. (C): The population of pro-tumorigenic (anti-inflammatory) macrophages was quantified with an antibody to CD206. Data shown as mean±SEM (n=3 for control samples; n=4 for PEBCA and n=5 for CBZ and PEBCA-CBZ). Asterisks indicate statistical significance obtained by unpaired parametric t-test, where p<0.0005 is marked with *** and p<0.0001 is marked with ****.

FIG. 6. Treatment efficacy in the MAS98.12 PDX model. Each bar represents the mean area under the curve (AUC) of individual MAS98.12 tumors shown in FIG. 2. Data shown are mean±SEM (n=9 or 10 tumors in each group). The statistical p-values have been calculated using Welch's unequal variance t-test comparing the indicated groups.

FIG. 7. Treatment efficacy in mice bearing MDA-MB-231 tumors. Tumor growth inhibition was measured following two injections of PEBCA-CBZ, non-encapsulated CBZ, PEBCA NPs without drug and saline. The red arrows indicate days for injections. PEBCA-CBZ and CBZ were injected at 2×15 mg CBZ/kg; PEBCA particles not containing drug were injected 2×175 mg (similar to the amount of NPs in the PEBCA-CBZ group); 2×0.1 ml per 10 g body weight of saline were injected as a control.

FIG. 8. Ex vivo fluorescence images of isolated organs obtained after injection of PEBCA particles containing NR668. Images are shown for organs taken 1 h, 4 h and 96 h after injection of the particles (images taken 24 h after injection are shown in FIG. 3). LNs: lymph nodes.

FIG. 9. Immunohistochemical staining of macrophage infiltration in treated MAS98.12 tumors. The macrophage infiltration was measured in the tumors 96 h after injection of saline (control), PEBCA NPs (without drug), non-encapsulated CBZ, and PEBCA-CBZ (left to right columns). Immunohistochemical staining with (A) anti-CD68, (B) anti-iNOS, and (C) anti-CD206. Scale bar: 100 μm.

FIG. 10. In vitro toxicity measured as cell viability and cell proliferation in three breast cancer cell lines. PEBCA-CBZ with 100 nM CBZ contains 4.5 μg/ml PEBCA materials; equivalent amount of empty PEBCA NPs were given for comparison. Left column: Cell viability measured with the MTT assay after incubation for 72 h. Right column: Cell proliferation measured as [3H]thymidine incorporation after incubation for 24 h. The following cell lines were used: (A): MDA-MB-231, (B): MDA-MB-468, and (C): MCF-7. Data shown as mean±SD; n=3.

FIG. 11: Treatment effects of a prostate carcinoma tumor model (PC3) with CBZ formulated as Jevtana®, CBZ in PEBCA NPs and control. Plots of mean tumor size in the three groups. Arrows show treatment days, error bars show standard deviation and *** indicates p>0.001, t-test.

 DEFINITIONS

The term ‘nanoparticle, (NP)’ is used herein to describe particles or capsules with linear dimensions less than 800 nm.

The term “PEGylation” is used herein to describe the process of both covalent and non-covalent attachment or amalgamation of polyethylene glycol (PEG) polymer chains to nanoparticles, which is then described as PEGylated (pegylated). As will be known to the skilled person, the association of PEG to the NP surface can “mask” the NP from the host's immune system by creating a water corona around the NP. This can reduce the immunogenicity and antigenicity of the NP, and prolong its circulatory time by reducing renal clearance. Depending on the density of PEG on the surface, the PEG is classified as being in a brush or mushroom conformation. The PEGylation can be performed either during or after synthesis of the NPs, by either a covalent or noncovalent bond, resulting in varying properties of the PEGylation.

The term “targeting moiety” is used herein to describe any molecule that can be bound to the surface of the NP and result in selective binding to specific cells or biological surfaces.

The term “passive targeting” is used herein to describe the accumulation and/or retention of nanoparticles in inflamed and malignant tissue that occurs due to leaky blood vessels and impaired lymphatic drainage. Passive targeting is independent of targeting moieties on the surface of NPs.

The term “active targeting” is used herein to describe the accumulation and/or retention of the nanoparticle on specific cells or biological surfaces due to the specific interaction between the targeting moiety and the cell surface or the biological surface.

The term “enhanced permeability and retention (EPR)” effect is used herein to describe the phenomenon where molecules of certain sizes (typically liposomes, nanoparticles, and macromolecular drugs) tend to accumulate in tumor tissue much more than they do in normal tissues. The NPs as described herein are typically of a size from about 1-800 nm, such as about 10-500, preferably about 70-150 nm. Accordingly, the EPR effect will allow the NPs as described herein to selectively extravasate and accumulate in tumors.

The terms “parenteral administration” and “administered parenterally” are art recognized terms, and include modes of administration other than enteral and topical administration, such as injections, and include without limitation intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.

The term “pharmaceutically acceptable” as used herein denotes that the system or composition is suitable for administration to a subject, including a human patient, to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

The terms “therapy”, “treat,” “treating,” and “treatment” are used synonymously to refer to any action providing a benefit to a patient at risk for or afflicted with a disease, including improvement in the condition through lessening, inhibition, suppression or elimination of at least one symptom, delay in progression of the disease, prevention, delay in or inhibition of the likelihood of the onset of the disease, etc.

The terms “microbubble associated with nanoparticles” or “nanoparticles associated with microbubbles” are used herein to describe in what way nanoparticles can interact with the microbubble interface. The term “associated with” as used in connection with this include association by any type of chemical bonding, such as covalent bonding, non-covalent bonding, hydrogen bonding, ionic bonding or any other surface-surface interactions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a drug delivery system comprising PEGylated poly (alkyl cyanoacrylate) (PACA) nanoparticles (NPs) loaded with cabazitaxel (CBZ) for use in treatment of cancer, provided that the drug delivery system does not comprise microbubbles (MBs).

The effect of PACA NPs loaded with the cytotoxic drug CBZ is demonstrated in several in vitro and in vivo studies. As disclosed herein, the studies include demonstration of effects in three breast cancer cell lines, one basal-like patient-derived xenograft model grown in the mammary fat pad of immunodeficient mice and one prostate carcinoma tumor model. It is demonstrated that NP-encapsulated CBZ has similar or even better efficacy than similar concentrations of non-encapsulated drug. As demonstrated in the basal-like patient-derived xenograft, the results show complete remission of 6 out of 8 tumors. The different drug concentrations obtained with NP-encapsulated versus non-encapsulated CBZ was investigated with mass spectrometry analyses of CBZ, which was performed using blood and selected tissue samples. The results show that the nanoparticle-encapsulated drug has a longer circulation time in blood and a higher content in tumor tissue. The tissue biodistribution, which is obtained after 24 h using mass spectrometry analyses, correlates well with biodistribution data obtained using IVIS® Spectrum in vivo imaging of nanoparticles labeled with the fluorescent substance NR668. This is a clear indication that these data also are representative for the nanoparticle distribution. Furthermore, immunohistochemistry was used to estimate infiltration of macrophages into the tumor tissue following injection of NP-encapsulated CBZ and injection of non-encapsulated CBZ. It was also demonstrated a higher content of anti-tumorigenic versus pro-tumorigenic macrophages in tumors treated with the NPs. Without being bound by theory, this may further contribute to the improved effect obtained with the NP-encapsulated drug. In summary, encapsulation of CBZ in PACA NPs is a promising alternative to the clinically available formulation of the drug.

As will be understood by a person skilled in the art, the invention as disclosed herein is different form the drug delivery system as described in Snipstad et al (2017). As described herein, the drug delivery system of the invention does not comprise NP-stabilized MBs, as is described by Snipstad et al, 2017. In different embodiments, the drug delivery system according to the invention does not comprise NPs that stabilize the MBs nor NPs that are used to stabilize gas-filled MBs. Accordingly, the drug delivery system of the invention is not dependent on ultrasound to achieve treatments effects, in contrast to the delivery system described in Snipstad et al 2017, which is ultrasound-mediated.

Accordingly, one embodiment of the invention as provided herein is a drug delivery system comprising PEGylated PACA NPs loaded with CBZ, or a pharmaceutically acceptable salt thereof, for use in treatment of cancer, provided that the drug delivery system is not mediated by an acoustic field, such as ultrasound or focused ultrasound.

In a further embodiment of the invention, the drug delivery system does not comprise NPs that are associated with the MB. It is also disclosed a drug delivery system that does not comprise gas-filled MBs. In yet a further embodiment, the drug delivery system does not comprise MBs.

Degradation rate of PACA NPs can be controlled by the choice of the alkyl chain of the cyanoacrylate monomer, as demonstrated by Sulheim et al. (2016). It has also been demonstrated, using a panel of cell lines, that the cytotoxicity is dependent on the monomers used, i.e. n-butyl-, 2-ethyl-butyl-, or octyl cyanoacrylate (BCA, EBCA and OCA, respectively), see Sulheim et al (2017).

In different embodiments of the invention, the alkyl chain of the cyanoacrylate monomer is a linear or branched C4-C10 alkyl chain. In preferred embodiments the monomer used is selected from the group consisting of n-butyl-(BCA), 2-ethyl butyl (EBCA), polyisohexyl (IHCA) and octyl cyanoacrylate (OCA). Accordingly, in different embodiments, the drug delivery system comprises NPs selected from the group consisting of PBCA, PEBCA, PIHCA and POCA.

As described herein, the NPs are PEGylated, i.e. coated with a hydrophilic polymer such as polyethylene glycol (PEG).

The rationale of PEGylation in drug delivery is to obtain increased circulation time after parenteral administration, such as intravenous (i.v.) injection. This is well demonstrated and is known to the skilled person. Äslund et al. (2017) has studied the quantitative and qualitative effects of different types of PEG-comprising molecules on PACA nanoparticles. For example, it has been demonstrated that longer PEG on the surface will increase blood circulation time and diffusion of PACA NPs in collagen gels.

Accordingly, by varying the type of PEG one can achieve NPs with different surface modifications. This will influence the zeta potential, the protein adsorption, diffusion, cellular interaction and blood circulation half-life of the NPs described herein.

In different embodiments of the invention, the NPs are PEGylated with PEG-comprising molecules selected from the group consisting of Jeffamine, Brij, Kolliphor, Pluronic or combinations thereof.

According to an embodiment, the NPs are PEGylated with the PEG-comprising molecules selected from Pluronic and Kolliphor.

According to another embodiment, the NPs are PEGylated with the PEG-comprising molecules selected from Brij and Kolliphor.

In an embodiment of the invention, the PACA NPs is produced by a miniemulsion anionic polymerization process, in particular a one-step process as described in WO2014/191502, both with or without targeting moieties.

By using NPs that is further surface modified with targeting moieties, for example by using NPs prepared by miniemulsion anionic polymerization technique with polyalkylene glycols that is covalently attached to a targeting moiety, one can enable active targeting and potentially enhanced retention at specific locations, such as in tumors or diseased tissue. Also, this can facilitate uptake in cancer cells that is dependent upon specific ligand-receptor interactions.

The targeting moiety may be any suitable moiety that causes the NPs to bind specifically at targeted locations.

Preferably, the targeting moiety has a molecular weight in the range 100 to 200 000 Da, more preferably 200 to 50000 Da, even more preferably 300 to 15000 Da.

It should be appreciated that a single targeting moiety or a mixture of different targeting moieties may be used.

Example targeting moieties are selected from the group consisting of an amino acid, protein, peptide, antibody, antibody fragment, saccharide, carbohydrate, glycan, cytokine, chemokine, nucleotide, lectin, lipid, receptor, steroid, neurotransmitter, cell surface marker, cancer antigen, glycoprotein antigen, aptamer or mixtures thereof. Particularly preferred targeting moieties include linear and cyclic peptides. In one embodiment, the targeting moiety does not belong to the group consisting of amino acids and lipids

It is previously known that the size of nanoparticles influences the targeting effects of the nanoparticles when they are administrated systemically into the blood, as they accumulate in the areas around tumors with leaky vasculature. This is known as ‘enhanced permeability and retention’ (EPR) effect in tumor tissue. The EPR effect is as a type of targeting, commonly referred to as “passive targeting”.

Traditionally, tumor targeting approaches are classified into ‘passive targeting’ and ‘active targeting’.

The EPR effect will be known to the skilled person as a form of passive targeting. The introduction of targeting moieties on the surface of the NP will be known to the skilled person as a type of active targeting.

Angiogenesis is a biological process by which new capillaries are formed. It is essential in many physiological conditions, such as embryo development, ovulation and wound repair, and pathological conditions, such as arthritis, diabetic retinopathy, and tumors.

Tumors can grow to a size of approximately 1-2 mm3 before their metabolic demands are restricted due to the diffusion limit of oxygen and nutrients. In order to grow beyond this size, the tumor switches to an angiogenic phenotype and initiates the formation of neovasculature from surrounding blood vessels. Accordingly, tumors are endowed with angiogenic capability and their growth, invasion and metastasis are angiogenesis-dependent. Apart from some exemptions, in most cases, neoplastic cell populations will form a clinically observable tumor only after angiogenic capability has been acquired and a vascular network sufficient to sustain their growth is produced. Furthermore, new blood vessels provide them with a gateway through which they enter the circulation and metastasize to distant sites. Tumor angiogenesis is essentially mediated by angiogenic molecules elaborated by tumor cells.

Without being bound by theory, the phenomenon of the EPR effect is caused by the tumors ability to stimulate the production of blood vessels in order for tumor cells to proliferate. Vascular endothelial growth factor (VEGF) and other growth factors, known to the skilled person, are involved in cancer angiogenesis. Tumor cell aggregates as small as 150-200 μm, will start to become dependent on blood supply carried out by neovasculature for their nutritional and oxygen supply. However, these newly formed tumor vessels are usually abnormal in form and architecture. They have poorly aligned defective endothelial cells with fenestrations, lacking a smooth muscle layer, or innervation with a wider lumen, and impaired functional receptors for angiotensin II. Furthermore, tumor tissues usually lack effective lymphatic drainage. All of these factors lead to abnormal molecular and fluid transport dynamics, especially suitable for NPs as disclosed herein.

Accordingly, the EPR effect will results in passive accumulation of NPs in tumors due to the hyperpermeability of the vasculature and the lack of lymphatic drainage. This is contrary to what is seen in normal tissue where the NPs are constrained to the blood vessels. This makes the NPs as described herein attractive for tumor targeting. To increase the fraction of NPs reaching the tumor, the systemic circulation time of the NPs are increased. By extending the circulation time, an enhanced number of NPs will be able to accumulate in the tumors, as it increases the probability for the NPs to diffuse through openings in the blood vessels.

Accordingly, in preferred embodiments of the invention, the drug delivery system is for treatment of cancer, such as tumors, including but not limited to tumors of the colon, lung, breast, cervix, bladder, prostate and pancreas.

Further, it has been demonstrated that the NPs according to the invention partly accumulate in the lymph nodes. This may be utilized to treat metastasizing cancer cells in lymph nodes. Accordingly, in one embodiment of the invention, the drug delivery system is for treating metastasizing cancer cells in lymph nodes. In yet another embodiment, the drug delivery system is for prophylactic treatment of cancer by preventing metastasis through the lymph nodes. In a further embodiment, the drug delivery system is for treatment of tumors and for prophylactic treatment of metastasis.

In another embodiment of the invention, the tumor has vasculature that is hyperpermeable and/or lack lymphatic drainage.

Tumor growth consists of an avascular and a subsequent vascular phase. In one embodiment of the invention, the tumor is in a vascular phase.

The NPs used in the examples contain the cytotoxic drug cabazitaxel (CBZ). CBZ is a semi-synthetic taxane derivative that inhibits microtubule disassembly. CBZ has a very low water solubility, which complicates the administration of the free, non-encapsulated drug.

However, as demonstrated in the examples, due to excellent compatibility and solubility of CBZ in alkyl cyanoacrylate monomers, high concentrations of the drug can be dissolved in alkyl cyanoacrylate monomer solution and thus become encapsulated in PACAs.

According to different embodiments of the invention the loading capacity of CBZ in NPs can be 1-90 wt % of the NP, preferentially 5-50 wt % of the NP. In particularly preferred embodiments, the loading capacity of CBZ is from 5-15 wt % of the NP, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 wt % of the NP.

Accordingly, the drug delivery system according to the invention has a high loading capacity, which is shown to influence the treatment effects of the invention.

As CBZ is insoluble in water, the conventional formulation is CBZ solubilized in a polysorbate 80 solution. As used herein, non-encapsulated or free CBZ refers to the conventional formulation.

CBZ has been included in several clinical trials that study the effects on different types of cancer including several types of prostate cancer, adrenocortical carcinoma, testicular cancer, urothelium transitional cell carcinoma and ovarian cancer. The inventors have also demonstrated therapeutic effects of CBZ in glioma and lung cancer.

In the clinical studies, it has been demonstrated that the efficacy of CBZ is accompanied by serious side effects and toxic deaths. The toxicity rates observed in clinical trials has been assumed to pose an obstacle to use and management of CBZ, a drug that, on the other hand, has demonstrated great activity. In the transition from clinical trial to clinical practice, it has been speculated that CBZ will not be used much because of the risk of side effects, as well as high cost and discomfort derived from the administration regimes and the lack of patient compliance with the administration regimes previously proposed for CBZ-treatments. Thus, limiting the administration regimes, for example from tree-weekly to weekly has been proposed in treatment of for example prostate cancer, to improve hematologic tolerance along with a better therapeutic range to be able to increase the dose intensity and activity without increasing the associated toxicity.

Accordingly, the advantage that drug-loaded NPs give less adverse effects than free drug makes the drug delivery system as described by the inventors highly relevant for CBZ. Encapsulating CBZ in NPs offers a more sustained release profile of the drug, which can ameliorate parts of the toxicity and allows for administration of higher doses. The reduction of adverse effects allows for administration of increased doses of drugs. Accordingly, encapsulation of drug in the NPs will further improve the treatment effects. Accordingly, the inventors propose the idea that the drug delivery system as described herein will enhance treatments effects and/or reduce side effects when used in treatment of cancer.

In different embodiments, the invention provides a drug delivery system comprising PEGylated PACA NPs loaded with CBZ, or a pharmaceutically acceptable salt thereof, for use in treatment cancer, wherein the tumors belong to a type of cancer selected from the group consisting of prostate cancer, breast cancer, glioma, lung cancer, adrenocortical carcinoma, testicular cancer, urothelium transitional cell carcinoma and ovarian cancer.

In one particular embodiment of the invention, the tumor belongs to a type of prostate cancer, such as prostate carcinoma, hormone refractory prostate cancer, prostatic neoplasms or bone metastatic prostate cancer.

In another particular embodiment of the invention, the tumor belongs to a type of breast cancer, including luminal-like and basal-like breast cancer.

Breast cancer can be classified into major subgroups based on the gene expression pattern. Tumors belonging to the most aggressive subtypes are commonly treated with antracyclin and taxane-based chemotherapy regimens. In the examples, the growth inhibitory effect of CBZ encapsulated into PEBCA NPs is demonstrated in breast cancer models in vitro and in vivo, and in a prostate carcinoma tumor model in vivo. Three breast cancer cell lines representing the two main types of breast cancer, the luminal and basal-like subgroups was used. One of the cell lines is also injected and grown in the mammary fat pad of immunodeficient mice. Additionally, one patient-derived xenograft (PDX) previously demonstrated to be highly representative for aggressive basal like breast cancer is included in the examples.

One finding presented here is the surprising demonstration of improved therapeutic effect of PEBCA-CBZ compared to the non-encapsulated CBZ demonstrated in the basal-like PDX model. Analysis with mass spectrometry indicated that this could be due to the increased delivery of CBZ to the tumor following from encapsulation of the drug in NPs. Also, immunohistochemical staining revealed a lower content of pro-tumorigenic macrophages in the PEBCA-CBZ treated tumors than in tumors treated with non-encapsulated CBZ. Without being bound by theory, this could contribute to the improved efficacy. To elucidate the difference in efficacy of

PEBCA-CBZ and non-encapsulated CBZ, the examples demonstrate in vivo biodistribution of particles loaded with the lipophilic and near-infrared fluorescent substance NR668. Quantitative mass spectrometry analyses were used to describe the biodistribution of CBZ in tissues and the kinetics of CBZ in blood plasma after injection of both PEBCA-CBZ and non-encapsulated CBZ.

The example with prostate carcinoma demonstrates that the CBZ encapsulated into PEBCA NPs had similar growth inhibition as the clinically approved formulation. With the enhanced retention of drug loaded NPs in cancer cells compared to normal tissue, the drug delivery system of the present invention allows for administration of higher dosage with lower adverse effects.

Delivery of drug to lymph nodes has been discussed for treatment of metastasizing tumors. Since local lymph nodes are a significant metastatic site in many cancer types, accumulation of PEBCA-CBZ to lymphatic tissue may contribute additionally to both therapeutic and prophylactic treatment.

In summary, the PEBCA-CBZ NPs demonstrate promising results for treatment of prostate cancer and breast cancer, supporting the therapeutic effect of a drug delivery system for use in treatment of tumors as disclosed herein.

Furthermore, the observation that the PACA NPs seems to enhance intratumoral presence of anti-tumorigenic macrophages might be of more general value, and support that the drug delivery system of the invention can be used as an immune modulator and/or as a vehicle able to enhance the therapeutic effect of encapsulated drugs.

According to an embodiment of the invention, the drug delivery system is provided in a composition to be administered systemically, such as parenterally.

A last aspect of the invention includes a method of treating cancer comprising administering a drug delivery system according to the first aspect of the invention to a patient in need thereof.

EXAMPLES Example 1 Materials and Methods

Synthesis and characterization of nanoparticles. PEGylated PEBCA NPs were synthesized by miniemulsion polymerization. An oil phase consisting of 2.5 g 2-ethylbutyl cyanoacrylate (monomer, Cuantum Medical Cosmetics, Spain) containing 0.2% (w/w) butylated hydroxytoluene (Fluka, Switzerland) and 2% (w/w) Miglyol 812 (Cremer, USA) was prepared. Fluorescent particles for optical imaging were prepared by adding NR668 (modified Nile Red), custom synthesis, 0.2% (w/w) to the oil phase. Particles containing cytostatic drug for treatment were prepared by adding CBZ (10% (w/w), Biochempartner Co. Ltd., China, product item number BCP02404) to the oil phase.

An aqueous phase consisting of 0.1 M HCl (20 ml) containing Pluronic F68 (2 mM, Sigma, USA) and Kolliphor HS15 (6 mM, Sigma, Germany) was added to the oil phase and immediately sonicated for 3 min on ice (6×30 sec intervals, 60% amplitude, Branson Ultrasonics digital sonifier 450, USA). The solution was rotated (15 rpm, SB3 rotator, Stuart, UK) at room temperature overnight before adjusting the pH to 5 using 1 M NaOH. The polymerization was continued for 5 h at room temperature on rotation. The dispersion was dialyzed (Spectra/Por dialysis membrane MWCO 100,000 Da, Spectrum Labs, USA) against 1mM HCl to remove unreacted PEG. The size, polydispersity index (PDI) and the zeta potential of the NPs were measured by dynamic light scattering and laser Doppler Micro-electrophoresis using a Zetasizer Nano ZS (Malvern Instruments, UK). To calculate the amount of encapsulated drug, the drug was extracted from the particles by dissolving them in acetone (1:10), and quantified by liquid chromatography coupled to mass spectrometry (LC-MS/MS) as described below.

CBZ quantification by LC-MS/MS. CBZ, as the pure chemical or part of NPs, was quantified by LC-MS/MS, using an Agilent 1290 HPLC system coupled to an Agilent 6490 triple quadrupole mass spectrometer. The HPLC column was an Ascentis Express C8, 75×2.1 mm, 2.7 μm particles size with a 5×2.1 mm guard column of the same material (Sigma), run at 40° C. Eluent A was 25 mM formic acid in water and eluent B was 100% methanol, and flow rate was 0.5 ml/min. The mobile phase gradient was isocratic at 55% B for 1.5 min, then from 55% to 80% B over 1 min, followed by 1 min washout time and subsequently column re-equilibration. Injection volume was 5.00 μl. MS detection was in positive ESI mode (Agilent Jetstream) quantified in multiple reaction monitoring (MRM) mode using the transition m/z 858.3→577.2. The parent ion was chosen to be the Na adduct as this gave the best sensitivity. Similarly, the hexadeuterated internal standard was detected on the 864.4→583.2 transition. Both analytes were run at 380 V fragmentor and 20 V collision energy.

Reference standards were used for accurate quantification. The unlabeled CBZ standard was the same as used for synthesis (see above) at >98% purity. Hexadeuterated CBZ internal standard was purchased from Toronto Research Chemicals (Toronto, Canada; catalogue number C046502 at 99.6% isotopic purity). Standards were dissolved in acetone and were used to build an unlabeled standard series spanning at least five concentration points.

The limit of quantification (LOQ) was calculated from six replicate quantifications of the lowest concentration point in the standard curves (0.1 ng/ml), specifically as the average plus six standard deviations; this amounted to an LOQ of 0.19 ng/ml (signal/noise ratio >20). Accuracy based on the same standard sample set was 8.8% and precision was 18.0%.

Processing of tissue samples before LC-MS/MS analyses. In order to process the tissue samples such that their CBZ content could be quantified, we developed a protocol for enzymatic digestion of tissue followed by extraction and quantification of CBZ using the LC-MS/MS method described above. The enzyme buffer consisted of Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific, USA, 41965039) with 1% (v/v) penicillin-streptomycin stock solution (Sigma-Aldrich, P0781) to a final concentration of 100 U/ml penicillin and 100 μg/ml streptomycin, 0.125 mg/ml papain (Merck, F275644), 2.5 mg/ml trypsin (Sigma-Aldrich, T7409), 0.8 mg/ml collagenase (Sigma-Aldrich, C7926), 0.69 mg/ml hyaluronidase (Sigma-Aldrich, H3506) and 1% (v/v) Triton X-100 (Sigma-Aldrich, T-8787). To determine the biodistribution of CBZ, frozen organs (liver, spleen, lymph nodes, kidneys, tumors) were thawed and freshly prepared enzyme buffer was added at 1 ml per 50 mg tissue; the entire organs were digested. The samples were heated to 37° C. for 72 h with vortexing once a day, until the tissue was completely dissolved. The tissue digests, as well as the plasma samples from the animals, were diluted 10× in acetone before centrifugation; this has the dual effect of both precipitating proteins and other macromolecules, thus cleaning up the sample, and making sure all CBZ is solubilized. Internal standard (hexadeuterated CBZ) dissolved in acetone was added to a final concentration of 10 ng/ml during the acetone dilution to correct for possible matrix effects.

Cell lines. Three breast cancer cell lines were used in this study. The MDA-MB-231 (triple negative; Claudin low), was cultured in RPMI 1640; the MDA-MB-468 (triple negative; basal) and the MCF-7 (luminal A) cell lines were cultured in DMEM. All medium was fortified with 10% (w/v) fetal calf serum albumin (Sigma) and 100 units/ml penicillin/streptomycin (PenStrep®, Sigma). All cell lines were obtained from ATCC and were routinely tested for mycoplasma. Cells growing in 24- or 96-well plates were incubated with serial dilutions of PEBCA-CBZ, CBZ (non-encapsulated CBZ) dissolved in Tween-80 (Fluka)), and PEBCA without CBZ for 24, 48 or 72 h at 37° C. in an atmosphere of 5% CO2. The toxicity was assessed either by the commonly used MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay, by measuring cell proliferation based on [3H]thymidine incorporation, by measuring protein synthesis by incorporation of [3H]leucine and by measuring ATP levels using CellTiter Glo®.

MTT cell viability assay. The cells were incubated for 24, 48 and 72 h with the different NPs/substances. The cell medium was then aspirated and exchanged with 100 μl of medium containing a final concentration of 250 μg MTT/ml. The incubation was continued for 3 h at 37° C. for formation of the formazan-particles, which were dissolved in DMSO with 1% (v/v) NH4Cl. The absorbance was read in a plate reader (Biosys Ltd, Essex, UK) at 570 nm, and background from absorbance at 650 nm was subtracted.

Cell proliferation measured by [3H]thymidine incorporation. Incorporation of [3H]thymidine into DNA was used to estimate cell proliferation. The cells were incubated for 24 h with the different NPs/substances. The cell medium was then aspirated and substituted with serum free cell medium containing [3H]thymidine (3 μg/ml; 75 μCi/ml). The incubation was continued for 30 min at 37° C. The medium was removed and 5% (w/v) trichloroacetic acid (TCA) was added. After 5 min the cells were washed twice with TCA and solubilized with 200 μl of 0.1 M KOH, before mixing with 3 ml scintillation fluid (Perkin Elmer, USA). The radioactivity was counted for 1 min in a scintillation counter (Tri-Carb 2100TR, Packard Bioscience, USA).

Protein synthesis measured by [3H]leucine incorporation. To determine the impact of PEBCA-CBZ and CBZ on protein synthesis, the cells were incubated with these substances for 24 h. The cell medium was then aspirated, the cells washed once with leucine-free HEPES medium (28 mM HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) in MEM) and further incubated with leucine-free HEPES medium containing [3H]leucine (2 μCi/ml) for 30 min at 37° C. The medium was removed and 5% (w/v) TCA was added to precipitate proteins. After 5 min the cells were washed again with 5% (w/v) TCA and solubilized with 200 μl of 0.1 M KOH, before mixing with 3 ml scintillation fluid and counting the radioactivity as described above.

Cell viability estimated by measuring ATP. Viability of the cells was tested measuring the ATP levels by using the CellTiter-Glo® (Promega, Wis., USA) assay, as described by the supplier. Cells were incubated with PEBCA-CBZ or CBZ for 72 h, thereafter one half of the volume was removed, replaced with an equal volume of the ATP reagent and gently mixed. After incubation for 10 min the cell lysate was transferred to a light-protected 96-well plate and luminescence measured in a plate reader (Biosys Ltd, Essex, UK).

Treatment efficacy evaluation in nude mice. All animal experiments were approved and performed according to the Norwegian Animal Research Authority (Permit number 15-136041) and were conducted according to the regulations of the Federation of European Laboratory Animal Science Association (FELASA). The mice were kept under pathogen-free conditions, at constant temperature (21.5±0.5° C.) and humidity (55±5%); 15 air changes/h and a 12 h light/dark cycle. They had access to distilled water ad libitum, which was supplemented with 17-β-estradiol at a concentration of 4 mg/l. All mice used in this study were female athymic nude foxn1nu mice (age 5-6 weeks and body weights of 18-20 g), locally bred at the Department of Comparative Medicine, Oslo University Hospital, Norway.

The ortothopically growing basal-like xenograft model MAS98.12 has been established in house and was used as previously described in Lindholm et al (2012). When using the MDA-MB-231 cell line, 1.5 million cells were injected into the mammary fat pad and growing tumors were used for sequential implantation. As for the MAS98.12 model, 1-2 mm3 pieces of healthy tumor tissue were implanted bilaterally into the mammary fat pad of female athymic mice. After the tumors reached approximately 5 mm in diameter, the mice were randomly assigned to the different treatment groups (the average volume of each group was 49-57 mm3).

The PEBCA-CBZ NPs were given twice (day 1 and day 4 after randomization) as i.v. tail vein injections with a dose of CBZ 15 mg/kg and NPs 175 mg/kg. A comparable amount of empty PEBCA NPs were given as control. Non-encapsulated CBZ was prepared as a stock solution in Polysorbate 80 (40 mg/ml) and further diluted with 13% (v/v) ethanol to a working solution of 10 mg/ml CBZ. The injection solution was prepared directly before the administration by dilution of the working solution with 0.9% (w/v) NaCl. The same amount of ethanol (1.2-1.4% (v/v)) was used as vehicle control; injection volumes were in the range 200-290 μl. From the first day of treatment, tumor diameter and body weight were measured twice weekly. Mice were monitored daily for health status and were killed by cervical dislocation if they became moribund or if tumor reached 1500 mm3. The tumor was measured by calipers and the tumor volume was calculated according to the formula 0.5×length×width and related to the mean tumor volume at start of treatment.

In vivo imaging. PEBCA NPs labeled with the lipophilic and fluorescent dye NR668 were used to study the biodistribution in MA98.12 bearing mice using an IVIS® Spectrum in vivo imaging system (Perkin Elmer). Mice were intravenously injected the same dose PEBCA-CBZ or PEBCA without drug as in the efficacy study. The batch containing CBZ has somewhat larger particles than the batch not containing CBZ (Table 51). The excitation/emission wavelength pair of 535/640 nm was found to give the best signal-to-noise ratio and was thus used for imaging of the NPs. Whole body images were obtained 1, 4, 24 and 96 h after injection; the animals were then sacrificed by cervical dislocation and organs were harvested. The organs were imaged ex vivo with the IVIS scanner using the same settings as above. Relative signal intensity in the organs was calculated, using Living Image software (Perkin Elmer), as radiant efficiency (Emission light [photons/sec/cm2/str]/Excitation light [μW/cm2]×109) per pixel of the region of interest, which was drawn around the respective organ. Fluorescent measurements of the PEBCA NPs used for in vivo imaging showed that the particles without CBZ had a fluorescent intensity of 1.17 times that of the PEBCA NPs containing CBZ and the data shown in FIG. 3C are corrected for this difference.

Biodistribution and pharmacokinetics in blood. Blood and tissue samples were obtained following a single i.v. injection of PEBCA-CBZ and non-encapsulated CBZ (15 mg/kg) into the tail vein of mice bearing the MAS98.12 tumor (n=3). Empty particles (PEBCA) and saline were used as negative controls. The blood samples were taken either per tail vein puncture (approximately after 2 min and thereafter at 1, 4 and 24 h after injection) or by terminal cardiac puncture (96 h after injection) in Vacutainer tubes containing EDTA (BD Biosciences, San Jose, Calif., USA) and kept on ice. The 0-24 h samples were collected consecutively from the same animals (n=3) while samples after 96 h were obtained from separate mice (n=3). The blood was centrifuged for 15 min at 4° C. and 3400×g, and the supernatant plasma was collected and stored at −80° C. until LC-MS/MS analysis. The animals were killed after 24 or 96 h and tissue samples (tumors, livers, spleens, lymph nodes and kidneys) were harvested. The organs were gently washed with saline and then snap frozen in liquid nitrogen and stored at −80° C. until further processing and LC-MS/MS analyses as described above. Statistical analyses were performed using the t-test.

Immunohistochemistry. Tumors from MAS98.12 bearing mice were collected 96 h after a single injection of the same substances as were injected in the MAS98.12 efficacy study. The tumors were preserved in 4% (v/v) formalin and then paraffinized and sliced to prepare consecutive slides (3 μm thick). The deparaffinization agent Neo-clear and mounting agent Neo-mount were obtained from VWR (Radnor, Pa., USA). Heat induced epitope retrieval was performed by placing deparaffinized slides with 10 mM sodium citrate buffer (pH 6.0) in water bath for 20 min at 100° C. Endogenous peroxidase activity was blocked by incubating slides with 3% (v/v) hydrogen peroxide in Tris-buffered saline (TBS; 50 mM Tris-Cl, 150 mM NaCl, pH 7.6). Sections were washed and blocking of non-specific binding was performed with 3% (w/v) bovine serum albumin (Roche diagnostics GmbH, Mannheim, Germany) in TBS for 30 min. Sections were then incubated for 60 min with the primary antibodies. Three different antibodies, i.e. anti-CD68 (1 mg/ml; ab125212, Abcam, Cambridge, UK), anti-CD206 (0.1 μg/ml; ab64693, Abcam, Cambridge, UK), and anti-iNOS (0.5 μg/ml; ab15323, Abcam, Cambridge, UK) were used to detect different population of macrophages. CD68 is a commonly used marker for the whole macrophage population, iNOS (inducible nitric oxide synthase) is a marker for M1 macrophages (anti-tumourigenic and pro-inflammatory macrophages), and CD206 is a marker for M2 macrophages (pro-tumourigenic and anti-inflammatory macrophages). TBS was used for washing the slides between steps. Detection of primary antibodies was performed using MACH 3 rabbit HRP-polymer detection kit according to the manufacturer's protocol (Biocare Medical, Concord, Calif., USA). Signals were developed by incubation with the Chromogen solutions provided with the Betazoid DAB Chromogen kit (Biocare Medical). For counter staining a haematoxylin and 37 mM ammonium hydroxide containing solution (Sigma-Aldrich, St. Louis, Mo., USA) were used.

Stained tissue sections were scanned (NanoZoomer HT, Hamamatsu Photonics, Hamamatsu, Japan) using a 40× objective. The extent of CD68, iNOS and CD206 was automatically scored using the ImmunoPath software (Room4 Ltd., Crowborough, UK). The tumor areas were marked manually, excluding necrotic areas as well as blood vessels to avoid unspecific or false positive staining. The annotated areas were then broken down to smaller frames per image of interest for efficient processing. The annotated representative areas were analyzed further by computerized image analysis. The image analysis protocols were set up on randomly selected images from different tumors to educate the software to differentiate between haematoxylin stained blue negative staining and brown positive staining. The output of the analysis provides the number of the positive pixel fraction and the negative pixel fraction of total annotated area.

Statistical analyses. To calculate the significance in the efficacy studies, the area under the curve was calculated for each tumor and the mean values of the groups were compared using the Welch unequal variance. To determine whether there is a significant difference between the frequencies of the tumor regression in the different treatment groups, we used the Fischer's exact test. If not stated otherwise, an unpaired two-sided Student t-test without Welch correction was used. The statistical analyses were performed using either GraphPad Prism (version 7.00 for Windows, GraphPad Software, La Jolla, Calif., US) or Microsoft Excel.

Results

Characterization of PEBCA particles. The particle size, polydispersity index (PDI) and zeta potential for the batches used were in the range of 148-227 nm (z-average), 0.04-0.19 and −(0.6-2.4) mV, respectively. The drug content in the final particles was 6.0-8.6% (w/w), giving 2.0-3.4 mg CBZ/ml in the NP stock solutions (Table S1). The size of PEBCA NPs increased when adding the drug CBZ or the fluorescent label NR668 (Table S1). The size distribution curves for the two batches used for efficacy studies in the MAS98.12 tumor model and that of CBZ (forming clusters in solution) are shown in FIG. 1.

PEBCA-CBZ inhibits tumor growth more efficiently than free CBZ in the MAS98.12 mice model. To test the efficacy of the PEBCA NPs with incorporated CBZ in a PDX mode, MAS98.12 tumors were implanted into the mammary fat pad of nude mice and treated with the drug-loaded particles (PEBCA-CBZ), empty particles (PEBCA), non-encapsulated (free) CBZ and saline as control (FIG. 2A). The injected dose was 2×15 mg CBZ/kg body weight, which corresponds to a particle dose of 2×175 mg/kg (FIG. 2A). Tumor growth was not affected by empty PEBCA NPs. CBZ treatment markedly inhibited tumor growth, and the effect was more pronounced with PEBCA-CBZ treatment, which induced reduction in tumor size (FIG. 2A). In the PEBCA-CBZ treated group 6 out of 8 tumors went into complete remission while this was the case in only 2 out of 9 CBZ-treated tumors and none in the negative control groups (Fisher's exact test (1-sided): p=0.04; comparing PEBCA-CBZ with CBZ).

To further evaluate differences in tumor growth between the four groups, we calculated the area under the curve (AUC) for each individual tumor and compared the mean values of each treatment group. The efficacy of PEBCA-CBZ is significantly better than treatment with non-encapsulated CBZ (p=0.02; Figure S1). The toxicity of the different treatments measured as body weight relative to the weight at treatment start is shown in FIG. 2B. The two treatments with CBZ (non-encapsulated or encapsulated as PEBCA-CBZ) caused a decline in body weight by approximately 15%, but one week after the last injection the toxicity was reversed. This tolerable body weight loss and the recovery time were comparable for PEBCA-CBZ and CBZ. Administration of empty PEBCA NPs did not cause any toxic effect as estimated from the body weights.

The efficacy of PEBCA-CBZ and non-encapsulated CBZ was also studied in MDA-MB-231 tumor-bearing mice. In this model we did not detect a significant difference between the two CBZ formulations, but a delay in tumor growth was observed (Figure S2). When compared to MAS98.12, the CBZ (free and encapsulated drug) was less effective in the MDA-MB-231 tumors. It was not possible to improve the efficacy by increasing the dose of non-encapsulated drug, due to toxicity of the free formulation of the drug.

In vivo biodistribution of PEBCA particles. The biodistribution of the PEBCA NPs in MAS98.12 bearing mice was studied by fluorescence imaging up to 96 h after injection of particles containing the fluorescent dye NR668. The mice were imaged using the IVIS® Spectrum scanner after 1, 4, 24 and 96 h (FIG. 3A), and then sacrificed such that organs could be harvested and visualized ex vivo. The images of all organs harvested 24 h after injections are shown in FIG. 3B, and the mean radiant efficiency relative to the pixel size of the region of interest per organ is plotted in FIG. 3C. The images of organs obtained at 1, 4 and 96 h are shown in Figure S3. Injection of the free NR668 dye did not give any detectable fluorescence with the wavelengths used (data not shown), thus indicating that the detected signals are from PEBCA-bound NR668. The images shown in FIG. 3 and Figure S3 demonstrate a rapid uptake in all tissues shown; 24 h after injection the strongest signals were observed in liver, spleen and lymph nodes, although fluorescence also was easily detectable in tumors, kidneys, hearts and lungs (FIG. 3C).

Pharmacokinetics and biodistribution of encapsulated and non-encapsulated CBZ. PEBCA-CBZ and non-encapsulated CBZ (15 mg/kg) were intravenously injected into the tail vein of mice bearing the MAS98.12 tumor. Blood samples were taken approximately 2 min after injection, and 1, 4, 24 and 96 h after injection, and the plasma samples were analyzed for CBZ using an LC-MS/MS method. The CBZ concentrations at almost all time points were at least 10-fold higher in mice receiving PEBCA-CBZ compared to mice receiving free CBZ (FIG. 4A). The plasma concentration/time curves for both PEBCA-CBZ and CBZ indicate an initial distribution phase, followed by a terminal elimination phase. The low number of data points did not allow determination of distribution or elimination half-life through non-linear regression. Interpolation based on the two first data points (up to 1 hr) indicate distribution half-lives in the range of approximately 50 min and 30 min for PEBCA-CBZ and CBZ, respectively. Similarly, calculation of the elimination half-lives based upon the mean values of the two last time points (24 and 96 h) indicates half-lives of this phase of about 60 h for both compounds. The higher plasma concentration observed after administration of PEBCA-CBZ compared to CBZ suggests a lower distribution volume and lower total clearance for PEBCA-CBZ compared to CBZ. This is consistent with the NP formulation being less able to escape the vascular compartment, and elimination of NPs primarily through the reticuloendothelial system.

The CBZ concentrations were measured in tumor, liver, spleen, lymph nodes, and kidney following a single injection of PEBCA-CBZ and CBZ (15 mg/kg). The results obtained with samples taken 24 and 96 h after injections are shown in FIGS. 4B and C, respectively. The highest amount of drug per mg tissue was obtained in spleen. However, when assuming that the mass of liver is approximate 13 times that of spleen in mice15 the data in FIG. 4 indicates that the liver/spleen ratio of PEBCA-CBZ is 2.1 times 24 h after injection and 4.4 times 96 h after injection demonstrating that liver is taking up the largest part of these NPs. The amounts of CBZ in the tumor samples measured as ng CBZ/mg tissue were 20% (24 h) and 1.4% (96 h) of that in liver after injection of PEBCA-CBZ.

The concentration of CBZ was significantly higher (t-test; p-value<0.01) in all tissues analyzed 24 h following injection of particle bound (PEBCA-CBZ) as compared to non-encapsulated drug (CBZ), with the largest differences observed in liver and spleen which contained the highest amounts of PEBCA-CBZ. Following injection of non-encapsulated CBZ, the highest concentration of CBZ was found in the tumor, although this level was only about ⅓ of that obtained in the PEBCA-CBZ group. In the samples obtained 96 h after injection, CBZ could be detected (i.e. being above the LOQ of the LC-MS/MS method) in all tissues analyzed after injection of PEBCA-CBZ, but only in the tumor tissue following injection of free CBZ (FIG. 4C). At this time point the concentration of CBZ in tumors following injection of free CBZ was approximately 50% of that obtained following injection of PEBCA-CBZ, but due to high variance in the PEBCA-CBZ samples this difference was not statistically significant (p=0.18). When comparing the CBZ concentrations in the tissue samples obtained 24 and 96 h after injection of PEBCA-CBZ, there is a decrease in tumor, spleen and kidney, and an increase in liver and lymph nodes from 24 to 96 h. The only significant difference is the decrease in the MAS98.12 tumor tissue (p=0.02). Also, the CBZ concentration after injection of non-encapsulated CBZ was significantly lower in tumors after 96 h when compared to 24 h (p<0.001). Plasma and tissue samples were also analyzed for CBZ following injection of PEBCA NPs which did not contain CBZ; all these samples (similar to those samples analyzed after injection of PEBCA-CBZ) were below the LOQ of the analytical method.

Macrophage infiltration in treated MAS98.12 tumors. The infiltration of macrophages into the MAS98.12 tumors during treatment was estimated by immunohistochemistry. The extent of total population of infiltrating macrophages was quantified using an antibody against CD68 and automatic quantification of scanned slides. An increased level of tumor infiltrating macrophages was observed in mice injected PEBCA-CBZ or PEBCA without drug compared to mice receiving free CBZ or saline, but the differences did not reach statistical significance (FIG. 5A). Also the marker of pro-inflammatory M1 macrophages (iNOS) demonstrated increased macrophage infiltration into tumors of mice receiving PEBCA-CBZ or PEBCA (FIG. 5B). However, the anti-inflammatory M2 macrophages subset, measured as CD206 positive cells, demonstrated increased infiltration compared to the saline control only in tumors receiving PEBCA (FIG. 5C). Furthermore, the tumors of mice receiving PEBCA-CBZ showed significant lower levels of this pro-tumorigenic macrophage population than tumors receiving PEBCA alone (p<0.001; FIG. 5C).

In vitro cell studies. We tested cellular toxicity of PEBCA-CBZ, non-encapsulated CBZ and PEBCA without drug in three cell lines, i.e. MDA-MB-231, MDA-MB-468 and MSF-7 using two different test systems, i.e. measuring cell proliferation by incorporation of [3H]thymidine after 24 h and cell viability after 72 h using the MTT assay. PEBCA-CBZ and CBZ were significantly more toxic than PEBCA without drug for all cell lines (range 130-350 fold), but there was no difference in the toxic effect of PEBCA-CBZ and CBZ in any of these test systems in any cell line (FIG. 10). It was also apparent that a small fraction (10-20%) of the cells in all cell lines tested survived even very high CBZ concentrations when using the MTT assay after incubation for 72 h. The toxicity of PEBCA-CBZ and non-encapsulated CBZ were also tested in all three cell lines using two other test systems. The effect on protein synthesis (incorporation of [3H]leucine) was measured following 24 h of incubation and the ATP levels (CellTiter-Glo®) were measured after 72 h. The results obtained with these test systems (data not shown) were very similar to those shown in Figure S5. The MTT assay was also performed on MDA-MB-231 cells after 24 and 48 h of incubation; the data obtained were similar to those shown after 72 h of incubation (FIG. 10), although the toxic effect was smaller after these shorter incubation times (data not shown).

Discussion

The main finding of the present study was the remarkably good therapeutic effect observed with PEBCA NPs containing CBZ in the basal-like PDX mice model, where complete remission was obtained in 6 out of 8 tumors following two injections of 15 mg CBZ/kg (FIG. 2A). Several possible explanations for the advantageous effect of PEBCA-CBZ versus non-encapsulated CBZ can be envisioned, and the obtained data point at least to the following factors: The longer circulation in blood of the NP-CBZ and higher concentration of the drug in the tumor (FIG. 4) and the higher ratio of anti-iNOS labeled macrophages to anti-CD206 labeled macrophages 16-17 in the treated tumors (FIG. 5).

The amount of CBZ was quantified in blood plasma and in several tissues using an LC-MS/MS method. The plasma data clearly show CBZ to be circulating for a longer time when incorporated in the NPs, and the CBZ concentration was at least 10-fold higher at nearly all time points in mice receiving PEBCA-CBZ compared to those receiving free CBZ (FIG. 4A). When evaluating such data, it is important to remember that the CBZ incorporated in NPs does not have a therapeutic effect before being released from the NPs. Thus, biodegradation of the NPs is necessary to obtain a good therapeutic effect, as exemplified in the MAS98.12 model.

The in vivo fluorescence imaging data obtained with PEBCA NPs labeled with NR668 demonstrates accumulation of the fluorescence in the same tissues as those where CBZ was found to be present. As expected these data show that most of the NPs end up in the liver. The liver/spleen ratio of the fluorescence per pixel measured 24 h after injection was estimated to be 2.9 for NPs without CBZ and 2.4 for NPs with CBZ, whereas the ratio of total CBZ content in liver to spleen was calculated to 2.1 based on the MS analyses. Although one should be careful in interpreting the quantitative data from the fluorescent imaging (based upon per pixel measurements) these biodistribution data obtained with two different methods showed similar results. The observation herein that injection of free NR668 did not give any measurable in vivo signal, the fact that NR668 did not leak from NPs with a similar composition (as demonstrated in Snipstad et al. 2017), and the rapid elimination of non-encapsulated CBZ from blood indicate that most of CBZ and NR668 are enclosed within the NPs 24 h after injection. Therefore, the biodistribution of these low molecular substances seem to represent well the distribution of the PEBCA NPs at this time point. In the study disclosed in Snipstad et al. 2017, with somewhat similar NPs, but using another PEGylation and fluorescent marker, a liver/spleen ratio of 5.3 was reported 6 h after injection. In this study, the mean fluorescent signal obtained in the tumors 24 h after injection was 12% of that in liver for the NPs not containing CBZ and 3% for the NPs containing CBZ. The IVIS data (FIGS. 3A, B) indicate higher fluorescence following injection of PEBCA NPs without drug than for NPs containing CBZ. Perhaps the somewhat larger size of the NPs with drug (Table S1) contributes to this difference.

Macrophages are the most abundant immune cells in mammary tumors. Tumor associated macrophages (TAMs) were originally thought to exert anti-tumor activities, but increasing clinical and experimental evidence show that TAMs also may promote tumor progression and influence anticancer drug responses. The pro-tumorigenic macrophages are known as alternatively activated and referred to as anti-inflammatory (M2-type), whereas the classically activated pro-inflammatory (M1-type) macrophages exhibit anti-tumorigenic properties. Plasticity is a hallmark of the macrophage population and dynamic changes in their phenotype define the different subtypes. Markers of M1 and M2 are commonly used to recognize the main phenotypes or functions, but a set of markers is recommended for a more comprehensive characterization of the whole population. In the present examples, the well accepted nitric oxide synthase (iNOS) have been used for detection of the M1 phenotype and mannose receptor (CD206) to define the M2-type.

The TAMs are influenced by the context, e.g. by factors in the microenvironment or externally added anticancer drugs. Interestingly, the effect of docetaxel has been shown to partly depend upon depletion of M2 macrophages and expansion of M1 macrophages in models of breast cancer. In contrast, no response in the macrophage populations upon treatment with free CBZ, another taxane, was observed, despite efficient growth retardation. However, treatment with PEBCA encapsulated CBZ, resulted in significant improved anti-tumor efficacy, and complete remission in 75% of the tumors. Even though the number of tumors used for immunohistochemical quantification is small, the data suggest two possible mechanisms that may explain this good effect. First, a trend towards elevated inflammation in tumors upon treatment with PEBCA NPs (with or without drug) was observed. This may imply a role of PEBCA NPs in homing anti-tumorigenic M1 macrophages into the tumors and thus further support the effect of CBZ. Secondly, we also showed that PEBCA-CBZ treatment significantly reduced CD206 expression in tumors compared to treatment with NPs without drug, which may point towards depletion of pro-tumorigenic macrophages.

Recently it was published that M2 macrophages show a vigorous endocytic uptake by macropinocytosis, whereas this uptake was virtually inactive in the M1 macrophages. Furthermore, another study showed that the M2 macrophages use endocytosis to degrade collagen and promote tumor growth in solid tumors. Without being bound by theory, the decrease in M2 macrophages observed after treatment with PEBCA-CBZ and the strong effect on the tumor growth is related to the macropinocytic uptake of PEBCA-CBZ and subsequent killing of these M2 macrophages. Thus, the inherent properties of M1 and M2 macrophages and selective toxic effect of drug containing particles on M2 macrophages may increase the efficacy of the treatment. It has earlier been published that driving TAMs toward M1 polarization (increasing the ratio of M1/M2 macrophages) has shown promising therapeutic effects in mice cancer models.

The effect of PEBCA-CBZ and non-encapsulated CBZ was also examined in orthotopically growing MDA-MB-231 tumors without showing the same efficacy as in the MAS98.12 PDX model. Even though the data demonstrate a therapeutic effect of PEBCA-CBZ in MDA-MB-231 tumors compared to empty PEBCAs, the effect is not equal to what is seen with non-encapsulated CBZ.

Since the outcome of these NPs are based on the EPR effect, the tumor vasculature is a critical factor for the accumulation of NPs in tumor. It is shown earlier that the basal-like MAS98.12 tumor has a higher vascularization than the luminal-like MAS98.06 tumor. Although angiogenesis also has been visualized in MDA-MB-231 tumors in mice, the difference in vascularization of the MAS98.12 and MDA-MB-231 tumors is less well characterized. However, in two previously published studies, the blood volume constituted 2.4% of the tumor volume 5 weeks after inoculation of MDA-MB-231 and 5.9% at 5 weeks after transplantation of MAS98.12, suggesting more efficient vascularization in the latter model. Accordingly, the degree of vascularization may influence the efficacy.

Both the fluorescence in vivo imaging data and the quantification of CBZ in tissue samples clearly demonstrate accumulation of the PEBCA NPs in lymph nodes (FIGS. 3B, C and FIG. 4B). Accumulation of drugs or imaging agents in lymph nodes was recently reviewed, suggesting a benefit of injecting very small NPs for the best accumulation. A study reported accumulation into lymph nodes of particles similar to those used in the present study, i.e. poly(butylcyanoacrylate) modified with Pluronic F127 and loaded with vincristine. As these NPs were shown to rapidly release vincristine and vincristine also accumulated in lymph nodes following injection of the non-encapsulated drug, it is difficult to evaluate to which extent these NPs actually accumulated in the lymph nodes. Delivery of drug to lymph nodes has been discussed for treatment of metastasizing tumors. Since local lymph nodes are the first site of locoregional breast cancer metastasis, and also a significant metastatic site in the aggressive triple negative basal like breast cancer, accumulation of PEBCA-CBZ to lymphatic tissue may contribute additionally to such treatment.

In summary, the PEBCA-CBZ NPs demonstrate promising results for treatment of breast cancer, supporting the therapeutic effect of a drug delivery system for use in treatment of solid tumors as disclosed herein.

Furthermore, the observation that the PACA NPs seems to enhance intratumoral presence of anti-tumorigenic macrophages might be of more general value, and support that the drug delivery system of the invention can be used as an immune modulator and/or as a vehicle able to enhance the therapeutic effect of encapsulated drugs.

TABLE 1 Description of size, PDI, zetapotential, NP and drug content of the batches used in this study. Size z-avg. Size number- Zeta-potential CBZ content in CBZ content in NP content in Study NP description (nm) avg. (nm) PDI (mV) NPs (% w/w) stock sol. (mg/ml) stock sol. (mg/ml) MAS.98.12 PEBCA-NR668 156 86 0.19 −2.2 — — 39 MAS.98.12 PEBCA-NR668- 215 161 0.17 −2.4 8.6 2.1 24 CBZ MDA-MB-231 PEBCA 148 118 0.09 −0.6 — — 74 and biodistribution MDA-MB-231 PEBCA-CBZ 214 196 0.07 −1.1 7.0 3.4 49 and biodistribution IVIS imaging PEBCA-NR668 172 152 0.04 −0.8 — — 57 IVIS imaging PEBCA-NR668- 227 186 0.15 −1.1 6.0 3.4 56 CBZ

Example 2 Treatment of a Prostate Carcinoma Tumor model with CBZ formulated as Jevtana®, or Encapsulated into PEBCA NPs Methods:

Jevtana was formulated as described in the treatment study on breast cancer. Briefly, CBZ was dissolved to 40 mg/ml in Tween, 80, then diluted 1:4 in 13% EtOH. CBZ-loaded PEBCA NPs was prepared and characterized as described in the breast cancer treatment study. Briefly, CBZ was added to the monomer phase at 10% w/v and the pegylated PEBCA NPs were made in a one step miniemulsion polymerization. The NPs were characterized for size, size distribution and zeta-potential with DLS.

Human PC3 prostate adenocarcinoma cells were grown in DMEM with 10% FBS and 1% Pencillin/streptavidin. 3 million cells in 50 μl cell medium were injected subcutaneously on the hind leg of the mouse. When the tumor reached a volume of 200 mm3, treatment was started and the continued with one treatment weekly for three weeks. The animals were randomly distributed into three groups receiving: 1; control—no treatment, 2; 10 mg/kg CBZ-PEBCA, 3; 10 mg/kg Jevtana. The drugs were administered through a catheter in the tail vein. The tumors were measured twice weekly using a caliper and the animals were euthanized when tumors reached 1000 mm3.

Results:

The NPs were characterized with dynamic light scattering, and had a diameter of 180 nm, PDI of 0.18 and zeta-potential of −1.7 mV. CBZ-encapsulation was measured with mass spectrometry and was found to be 8.5% w/w of the NP mass.

It was found that CBZ had a similar growth inhibition when formulated either as the clinical approved formulation or in PEBCA NPs (FIG. 1). Both treatments were significantly different (p<0.001, t-test) from the untreated control 14 days after the first treatment.

In FIG. 11, mean tumor size in the three groups are plotted. Arrows show treatment days, error bars show standard deviation and *** indicates p>0.001, t-test. As can be seen from the figure, the cytostatic effects of CBZ formulated in PEBCA NPs was similar to the effects of the clinical formulation of the drug in the prostate cancer model PC3.

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Claims

1. A drug delivery system comprising PEGylated poly (alkyl cyanoacrylate) (PACA) nanoparticles (NPs) loaded with cabazitaxel (CBZ), or a pharmaceutically acceptable salt thereof, for use in treatment of cancer, wherein the CBZ comprises 1-50 wt % of the NP, provided that the drug delivery system does not comprise NP-stabilized microbubbles (MBs).

2. The drug delivery system according to claim 1, wherein the PACA NPs are produced according to a miniemulsion anionic polymerization process.

3. The drug delivery system according to any of claim 1, wherein the NPs further are surface modified by a targeting moiety.

4. The drug delivery system according to claim 1, wherein the PACA NP is below 800 nm.

5. A method of administering the drug delivery system according to claim 1, wherein the drug delivery system is administered parenterally.

6. The method according to claim 5, wherein the drug delivery system is administered into the blood.

7. The drug delivery system according to claim 1, further comprising pharmaceutically acceptable excipients.

8. The method according to claim 5, wherein the cancer is a tumor in a vascular phase.

9. The drug delivery system according to claim 1, wherein the CBZ comprises 5-20 wt % of the NPs.

10. The drug delivery system according to claim 1, wherein the PACA NP is in a range of 1-800 nm.

11. The drug delivery system according to claim 1, wherein the PACA NP is in a range of 10-500 nm.

12. The drug delivery system according to claim 1, wherein the PACA NP is in a range of 70-150 nm.

13. The drug delivery system according to claim 1, wherein the drug delivery system is in a form for parenteral administration.

Patent History
Publication number: 20210113482
Type: Application
Filed: Mar 27, 2019
Publication Date: Apr 22, 2021
Inventors: Yrr Mørch (Trondheim), Ruth Schmid (Tiller), Einar Sulheim (Trondheim), Per Stenstad (Trondheim), Heidi Johnsen (Trondheim), Kirsten Sandvig (Nittedal), Gunhild Mælandsmo (Oslo), Tore Skotland (Nittedal), Kjersti Flatmark (Oslo)
Application Number: 17/041,094
Classifications
International Classification: A61K 9/51 (20060101); A61K 31/337 (20060101); A61P 35/04 (20060101);