"NANODELIVERY TISSUE TARGETING AT LOW SYSTEMIC EXPOSURES

AND METHODS THEREOF"

Technical field

This application relates to the therapy based on surface modified nanoparticles encapsulating agents with high volume of distribution and associated high systemic exposure, enabling relevant decrease of the latter, while providing targeted delivery of agents to tumour cells and increased therapeutic efficacy.

Background art

Significant efforts have been taken over the last two decades on the development of nanotechnology-based systems, with approximately two-thirds of its applications focusing on cancer treatment (1) . Despite the envisioned potential of nanotechnology (1), the therapeutic benefit of the first approvals of nanomedicine-based strategies resulted from enhanced safety profiles, with only a modest benefit on patient survival (2) . The accessibility of nanomedicines to cancer cells and their ability to diffuse and penetrate the neoplastic mass is compromised by, among other barriers (3), the limited extent of the enhanced permeability and retention (EPR) effect in patient tumors, resulting in limited drug bioavailability (4, 5) . As the overall efficacy will ultimately rely on drug bioavailability at the tumor and/or at the tumor cell level, there is the need to engineer novel mechanisms of drug delivery to overcome the EPR dependency associated with existing clinical-approved nanomedicines (6, 7) This limitation has been recently evidenced with the HER2 antibody-targeted liposomal doxorubicin (DXR) (codenamed MM- 302) . Notwithstanding preclinical studies demonstrated its superior antitumor activity relative to non-targeted liposomal DXR (8), in a phase II study with HER2-positive metastatic breast cancer patients (NCT02213744 ) , MM-302 did not show any benefit over the control arms, which included non-encapsulated chemotherapy. Being the HER2 antigen markedly overexpressed on the surface of cancer cells, MM- 302 still depends, in a first level of tumor targeting, on a relevant component of the EPR effect, as the extravasation from tumor leaky vasculature to access its target receptor (9) . Successful exploration of the EPR effect relies on the use of prolonged blood circulating, high systemic exposure, nanosystems. While these may alter the toxicokinetics of the encapsulated agents, relative to the unencapsulated ones, the referred dose-dependent high systemic exposure is associated with a toxicological profile of its own, as for example, Palmar-Plantar erythrodysesthesia or stomatitis (10) . In fact, such prolonged blood circulating nanosystems, encapsulating doxorubicin, present a system exposure 300-fold higher relative to the free drug, at a dosing of 50mg/m ² , that correlates with above side effects (11) . Alternatively, positively charged liposomes, without surface modifications, namely with ligands, provide low improvement over free drug in terms of systemic exposure (below, 50 pg.mL ^_1 .h (12) as compared to >1500 pg.mL ^_1 .h of long circulating nanosystems (13), in mice), compromising efficacy and safety.

To address those problems, targeting readily accessible overexpressed specific proteins within the tumor microenvironment that promote cell internalization, combined with efficient intracellular drug release, using platforms presenting lower systemic exposures, than the ones provided by prolonged circulating nanosystems, may enable increased efficacy and safety of drugs with high volume of distribution, which is associated with systemic toxicity (6) . Accordingly, herein cell surface nucleolin is used as an example of a specific protein of the tumor microenvironment .

Nucleolin is a nucleolar protein involved in various cellular functions that control RNA and DNA metabolism, playing a central role in cell cycle, microtubule nucleation and nucleolus structure (14-16) . Its overexpression has been identified in several human neoplasia and has been increasingly suggested as an unfavorable prognostic factor, associated with a high risk of relapse and low overall survival (17-21) . In fact, under pathological conditions, this protein is responsible for the development of malignant traits, contributing to tumorigenesis , and promoting invasion and angiogenesis ( 22 ) . Interestingly, an increased localization of nucleolin at the cell membrane has been identified in both cancer cells and endothelial cells of tumor angiogenic vessels, where the protein modulates the internalization of different ligands as part of the nucleus- cytoplasm-membrane shuttling (23-25) .

The prior art teaches the use of prolonged blood circulating nanoparticles, namely liposomes. The document US4920016A teaches that prolonged blood circulating liposomes would benefit from the prolonged blood residency as a mean to promote its accumulation in tissues as solid tumors, through EPR effect. However, this document does not teach any strategy favoring the decrease of the systemic exposure of the nanoparticle (ex. liposomes) while maintaining tissue targeting effectiveness, as into solid tumors, and therapeutic efficacy.

The document "Antibody targeting of long-circulating lipidic nanoparticles does not increase tumor localization but does increase internalization in animal models." (doi: 10.1158/0008-5472. CAN-05-4199) teaches a surface modification of long circulating liposomes enabling targeting of a specific protein in cancer cells, that does not affect the systemic exposure relative to an unmodified liposome. However, this document does not provide a surface modification that simultaneously enables, on one hand, targeting of a specific protein at the tumor site and, on the other hand, decrease of the systemic exposure, relative to the non-functionalized liposome, as a means to improve safety without compromising activity, such as antitumor activity .

The document "Doxil (R) -the first FDA-approved nano-drug: lessons learned." (doi: 10.1016/j . j conrel .2012.03.020 ) teaches that liposomes with high systemic exposures enable a better tolerability of the encapsulated drug relative to its free form. However, the document does not discuss the modification of liposomes that enable low systemic exposures and promote the targeting towards a specific protein and subsequent increase of therapeutic efficacy.

The documents WO2009142525 and "Targeted and intracellular triggered delivery of therapeutics to cancer cells and the tumor microenvironment: impact on the treatment of breast cancer." (doi: 10.1007/sl 0549-011-1688-7 ) discuss the use of a peptide, on the surface of liposomes, targeting a specific protein as a mean to promote the targeted delivery of agents to solid tumors. However, these documents do not discuss the properties of the ligand in the context of promoting the low systemic exposure of the modified nanoparticle, and thus safety, while retaining the tumor targeting and efficacy relative to the unmodified nanoparticle.

Summary The present invention describes the modification of a nanoparticle's surface with a targeting moiety that simultaneously lessens the dependence of high in vivo systemic exposures for successful tumor targeting, favoring safety of the encapsulated drug, namely those with high distribution volumes and subsequent systemic toxicity, while enabling efficient and specific intracellular delivery to tumor cells. The present invention discloses the characteristics of said chemical modification.

The present invention describes the chemical modification as the functionalization of the surface of the nanoparticle with a ligand that promotes the shift of the nanoparticle's surface towards a positive zeta potential.

The present invention discusses a relative residue composition, as a peptide, with a certain ratio of positive residues, at neutral pH, that shifts the nanoparticle's surface towards a positive zeta potential.

The present invention provides, as an example, a liposomal formulation with said surface modification encapsulating doxorubicin as a drug model characterized by a high volume of distribution and associated systemic toxicity.

It was discovered that said modification of the liposome decreased the systemic exposure of the encapsulated drug model, relative to a non-functionalized equivalent liposomal formulation .

It was surprisingly discovered that the ligand, targeting a readily accessible target in the tumor microenvironment, as nucleolin, improved the intracellular tumor bioavailability of doxorubicin, at low systemic exposure, with an associated safer toxicological profile compared to non-functionalized liposomal formulation encapsulating doxorubicin, an observation that could not be anticipated from prior art, thus overcoming the EPR dependence for tumor drug delivery. The functionalized liposomes containing doxorubicin, with low systemic exposure profile, enable a therapeutic response that depend on the level of expression of the target protein (as nucleolin) tumor cells.

Disclosure/Detailed Description

The present invention in one of its aspects provides the description of a chemical modification that can be introduced onto the surface of a nanosystem carrying an agent or combination of agents. Said chemical modification provides a mean to decrease the systemic exposure of encapsulated agent or a combination of agents with a high volume of distribution (triggering systemic toxicity) , following intravenous administration in mammals, to safer levels, compared to a non-functionalized nanosystem. Said chemical modification simultaneously provides a mean to bind the functionalized nanosystem to a specific protein on the target tumor cell. Said chemical modification further promotes the internalization of the modified nanosystem by the target tumor cell, upon binding to specific protein at the cell surface. The chemically modified nanosystem, according to the above, is termed "TarSSE-NP" (standing for Targeted Short Systemic Exposure Nanoparticle)".

The term "chemical modification" designates the non-covalent or covalent coupling of a ligand to the surface of the nanosystem, with or without the need of a neutral spacer. Said coupling can be accomplished by, but not limited to, conjugation with a PEG modified lipid, for instance DSPE- PEG, bearing a suitable functional group at the terminus of the PEG chain. This modified PEG-lipid can either be an integrant part of the nanosystem or may be coupled to the ligand apart from the nanosystem, aiming at further insertion. Other spacers include, but not limited to, carbon spacers typically between 0 and 20 carbons long, more typically between 1 and 10 carbons which can be represented by R-(CH2)n-R', wherein "n" can be, independently for each of R and R' , any "n" length (e.g., n= 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, etc.); 2- iminothiolane-derived ligands linked by thioether bond to PEG-maleimide ; pyridylditiopropionoylamino (PDP) -PEG, hydrazide (Hz) -PEG, or p-nitrophenylcarbonyl (pNP) -PEG dependent chemistry can also be used for spacing ligands and PEG.

The term "ligand" designates a positively charged peptide, at neutral pH, that enables said functions of the chemical modification. Said peptide is short in length, specifically shorter than 70 residues, preferentially shorter than 40 residues. The term "peptide" is broadly used to describe peptides, protein fragments, peptide-like molecules, peptides or protein fragments containing non-naturally occurring aminoacids, peptoids and similar structures, that may be chemically modified, which bind preferentially to a specific protein at target cell.

The term "nanosystem" designates the physical, chemical or biological material to which the ligand is linked to, preferentially a nanostructure enclosing an agent or a combination of agents to be targeted to a specific cell. Examples include "nanostructures" such as, but are not restricted to, quantum dots, magnetic particles, gold nanoparticles, nanoshells, carbon nanotubes, liposomes, virus containing an agent or a nucleic acid, polymeric particles such as microcapsules, biodegradable microdevices, agarose, gelatin, or other biological or inert material, and the like. The preferred nanosystem is a liposome composed of but not limited to fully hydrogenated soy phosphatidylcholine (HSPC) , methoxy-polyethylene glycol phosphatidylethanolamine (DSPE-mPEG, maleimide-polyethylene glycol phosphatidylethanolamine (DSPE-PEGmaleimide) , N- methylpalmitoyloleoylphosphatidylcholine (MPOPC) , phosphatidylserine (PS), phosphatidylcholine (PC), palmitoyloleoylphosphatidylcholine (POPC) , dipalmitoylphosphatidylcholine (PPC) , distearoylphosphatidylcholine (DSPC) , diphytanoylphosphatidylcholine (DPhPC) , sphingomyelin (SM) , phosphatidylglycerol (PG) , dioleoylphosphatidylethanolamine (DOPE) , N-acetoyl-D-erythro-sphingosine (C2-Ceramide) , N- butyroyl-D-erythro-sphingosine (C4-Ceramide) , N-hexanoyl-D- erythro-sphingosine (C6-Ceramide) , N-octanoyl-D-erythro- sphingosine (C8-Ceramide) , N-decanoyl-D-erythro-sphingosine (ClO-Ceramide) , N-lauroyl-D-erythro-sphingosine (C12- Ceramide) , N-myristoyl-D-erythro-sphingosine (C14- Ceramide) , N-palmitoyl-D-erythro-sphingosine (C16- Ceramide) , 3b-hydroxy-5-cholestene-3-hemisuccinate (CHEMS), cholesterol (CHOL) or a combination thereof. The term "enclosing" and "carrying" refer to the entrapment and/or protection of the agents within the nanosystem as follows, but not limited to: adsorbed, associated or anchored at the surface of the nanosystem; integrated in the membrane of a lipidic system; and entrapped in the inner core of the nanosystem.

The term "specific protein" designates a molecular structure that is expressed at the cell surface of the target cells, comprising, but not limited to, proteins, glycoproteins, or modifications thereof.

The term "target cell" designates tumor cells that express a specific protein in a reasonably superior amount compared to a normal somatic cell outside the tumor, without excluding tumor metastasis, preferentially at tumor cell surface. Such difference enables the TarSSE-NP to preferentially target the tumor cells. Such specific protein may be, but not limited to CD19, sigma receptor, HER2 (human epidermal growth factor receptor 2), epidermal growth factor receptor, Nucleolin, PSMA (prostate specific membrane antigen) , SSEA1 (stage-specific embryonic antigen 1), SSEA3/4 (stage- specific embryonic antigen 3/4), TRA-1-60 (tumor-related antigen 1-60), TRA-1-81 (tumor-related antigen 1-81), GCTM2, GCT343, CD9, Thyl (a.k.a. CD90), HLA1 (human leucocyte antigen 1), CD24, CD44, CD133, EpCAM (epithelial cell adhesion molecule), CD34, CD38, epithelial membrane protein, and VEGFR, pituitary adenylate cyclase-activating peptide receptors, lymphatic vessel endothelial hyaluronan receptors, thrombin receptor protease-activated receptor type 1, Notch, integrin receptors, lectin receptor, lactoferrin receptor, annexin 1, platelet-derived growth factor receptors, ephrin receptors as, but not limited to, the ephrinA4 receptor, C-kit, glycoprotein 60, aminopeptidase A, aminopeptidase N, CD13, endosialin, plectin-1, p32/gClq receptor, fibronectin ED-B, fibrin- fibronectin complexes, interleukin 11 receptor alpha, protease -cleaved collagen IV, hyaluronic binding protein, NG2 proteoglycan, prohibitin, heat shock protein 90, neuropilin 1, neuropilin 2, matrix metalloproteinase 2, matrix metalloproteinase 9. In the preferred embodiments, the ligand binds to nucleolin. In the embodiments, the ligand is a positively charged peptide (at neutral pH) shorter than 70 residues, preferentially shorter than 40 residues, that may be administered intravenously coupled to a nanosystem. The term "tumor cells" designates cells that are present in the tumor and include, but not limited to, tumor endothelial cells, cancer cells, cancer stem cells and immune system cells . The term "neutral pH" encompasses any pH values between 7.0 and 7.6.

The term "systemic exposure" designates the amount of agent or combination of agents in the blood stream after intravenous administration, and is defined as the area under the curve of a concentration in blood stream as a function of time.

The "agent" designates a drug, preferentially a cytotoxic molecule with high volume of distribution, a feature that is associated with systemic toxicity. In the embodiments, the agent is one or more selected from the group of alkylating drugs; cytotoxic antibiotics; antimetabolites; vinca alkaloids; amsacrine; altetarmine; crisantaspase ; dacarbazine; temozolomide ; hydroxycarbamide (hydroxyurea) ; pentostatin; platinum compounds; porfimer sodium; procarbazine; razoxane; taxanes; topoisomerase I inhibitors; tretinoin; SN-38; ET-743; TLK 286; anti-inflammatory agents; antiangiogenic agents or angiolytic agents; ABT-627; Bay 12- 9566; Benefin; BMS-275291; cartilage-derived inhibitor (CDI ) ; CAI ; CD59 complement fragment; CEP-7055; Col 3; Combretastatin A-4; Endostatin (collagen XVIII fragment); Fibronectin fragment; Gro-beta; Halofuginone ; Heparinases; Heparin hexasaccharide fragment; HMV833; Human chorionic gonadotropin (hCG) ; IM-862; Interferon alpha/beta/gamma; Interferon inducible protein (IP-10); Interleukin-12; Kringle 5 (plasminogen fragment) ; Marimastat; Metalloproteinase inhibitors (TIMPs); 2-Methoxyestradiol ; MMI 270 ( CGS 27023A); MoAbIMC- 1C11 ; Neovastat; NM-3; Panzem; PI-88; Placental ribonuclease inhibitor; Plasminogen activator inhibitor; Platelet factor-4 (PF4); Prinomastat; Prolactinl 6kD fragment; Proliferin-related protein (PRP) ; PTK 787/ZK 222594; Retinoids; Solimastat; Squalamine; SS 3304; SU 5416; 5U6668; SU11248; Tetrahydrocortisol-S ; tetrathiomolybdate ; thalidomide; Thrombospondin-1 (TSP-1 ) ; TNP-470; Transforming growth factor-beta (TGF-b) ; Vasculostatin; Vasostatin ( calreticulin fragment) ; ZD6126; ZD 6474; farnesyl transferase inhibitors (FTI); bisphosphonates ; and porphyrins. The cytotoxic agent also may be one or more quinolones (including, but not limited to, ciprofloxacin and trovafloxacin) ; and/or tyrosine kinase inhibitors (TKI), (including, but not limited to, imatinib, dasatinib, nilotinib, bosutinib, lapatinib, gefitinib, erlotinib, vandetanib, vemurafenib, crizotinib, sorafenib, sunitinib, pazopanib, regorafenib, cabozantinib) . In the preferred embodiments, anthracyclines are used, preferentially doxorubicin.

The term "dosing" or "effective dosing" describes the treatment regimen in terms of dose of the agent or combination of agents and the schedule of administration as well as the route of administration. The exact amount required will vary from subject to subject, depending on age, general condition of the subject, the severity of the condition being treated, and the particular active agent administered, and the like. Thus, it is not possible to specify an exact "effective amount" However, an appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation .

In another aspect of the invention, a method for killing tumor cells is provided. The method involves contacting a tumor cell with any one of the TarSSE-NP described above, wherein the agent is a cytotoxic drug. The TarSSE-NP are administered in amounts effective to kill the tumor cell. In embodiments, the contacting occurs as a result of administering the TarSSE-NP to a subject, wherein the subject is a mammal, including, but not limited to, a human.

Brief description of drawings

For an easier understanding of this application, figures are attached in the annex that represent the preferred forms of implementation which nevertheless are not intended to limit the technique disclosed herein.

Figure 1A illustrates the plasma clearance profile of doxorubicin [DXR] (and corresponding areas under the curve, AUC or systemic exposure) following intravenous administration of a TarSSE-NP or a non-TarSSE-NP_#l (at indicated DXR doses), encapsulating said agent, to nucleolin ^high tumor-bearing female BALB/c ^nu/nu mice. Figure IB illustrates the plasma clearance profile of doxorubicin [DXR] (and corresponding AUC) following intravenous (i.v.) administration of a TarSSE-NP (at indicated DXR doses) to female BALB/c mice.

Figure 2A illustrates the doxorubicin [DXR] tumor (bulk) accumulation, 24 h after intravenous administration of a TarSSE-NP or non-TarSSE-NP_#l , encapsulating said agent. Bulk accumulation includes both tumor parenchymal DXR and liposomal DXR circulating through the tumor vasculature. Bars and points represent the mean of DXR levels and individual values, respectively (p-value calculated using Dunn's test, n=4-6) . Figure 2B illustrates the number of doxorubicin positive (DXR ⁺ ) cells, drawn from confocal image analysis, 24 h after the administration of indicated treatments and doses. Bars and dots represent the mean and individual values, respectively (p-value calculated using Dunn's test, n=102-140) . Figure 3A illustrates the cell surface nucleolin density of live MDA-MB-435S and 4T1 cells measured by flow cytometry and compared with a standard curve generated with bead- Alexa488 (p-value calculated using t test, n=3-4) . As an example, Figure 3B illustrates the impact of a TarSSE-NP (q7dx5) , encapsulating doxorubicin, on MDA-MB-435S (nucleolin ^high ) and 4T1 (nucleolin ^low ) tumor burden. Bars and dots represent the mean and individual tumor burden values, respectively (p-values calculated using Mann-Whitney non- parametric test, n=4-12) .

Figure 4 illustrates the area of nucleolin ⁺ vessels, after the indicated treatments (q7dx5) of female BALB/c nude mice bearing mammary nucleolin ^hi 9 ^h (MDA-MB-435S ) tumors. Bars and points represent the mean and individual values, respectively (p-value calculated using Dunn's test, n=3-4) . Figure 5 represents the MM473:Luc and MM487:Luc mesothelioma tumor burden after the indicated treatments at the end of the experiment. Individual tumor BLI values were normalized to the mean tumor BLI of non-treated mice. Bars and dots represent the mean and individual tumor burden values, respectively (p-values were calculated by Dunn's test).

Figure 6A illustrates the relative body weight variation of dogs after multi-administration (q7dx4) of the indicated treatments (p-value calculated with Dunn's test at week 3, n=2-5) . Figure 6B illustrates the survival of dogs administered with a TarSSE-NP or a non-TarSSE-NP_#l , at 1 mg of doxorubicin/kg (q7dx4) (n=6-10) .

Figure 7 illustrates doxorubicin (DXR) plasma profile in dogs upon single administration of a TarSSE-NP and non- TarSSE-NP_#l , encapsulating said agent. Each timepoint represent the mean of DXR plasma concentration (±SEM, n=2- 3) . Insert tables refer to the corresponding area under curve (AUC, or systemic exposure) . Examples

Example 1

Characterization of the nanoparticles

The modified liposomes (TarSSE-NP) and non-modified liposomes (non-TarSSE-NP_#l ) liposomes were prepared from lipid mixes as described (26, 27) . The functionalization of liposomes with the ligand (into a TarSSE-NP) was adapted from the literature (28) . The ligand consisted in a peptide shorter than 40 residues, preferentially with 31 residues, containing less than 70% of positively charged residues (at pH 7.4), preferentially less than 62% but higher than 40%. Within the peptide, 25-50% of the residues on the amino terminal side belonged to a target recognition and binding motif, in this example, towards cell surface nucleolin. Data demonstrated that the functionalization of liposomes with the described ligand (TarSSE-NP) enabled an increase in the zeta potential, as compared to the liposomes without functionalization (non-TarSSE-NP_#2 ) (Table 1) . Similarly, a non-functionalized commercially available formulation of doxorubicin described in the literature (29) , but qualitatively identical in terms of the nature of the lipids used, presented a negative zeta potential (Table 1), was further used in subsequent examples.

Table 1. Nanoparticle characterization.

* Diluted 10 times in water for injection.

** Diluted 100 times in HBS pH 7.4. Example 2

Pharmacokinetics and biodistribution of doxorubicin- containing TarSSE-NP compared to a non-TarSSE-NP

Doxorubicin (DXR) blood clearance profile was performed in both female BALB/c and female MDA-MB-435S tumor-bearing BALB/c nude mice (once tumors have reached 100-150 mm ³ ) . TarSSE-NP was intravenously administered, in the tail vein, at 5, 6 or 7 mg of DXR per kg of body weight. As a control, non-modified liposome encapsulating doxorubicin (non-TarSSE- NP_#1) was administered by the same route, at 5 mg of DXR/Kg. When appropriate, animals were euthanized and MDA-MB-435S- derived tumors were collected and frozen.

Systemic exposure (AUC5 min-48 h) of DXR delivered by non- TarSSE-NP_#l was 8.7-fold higher than the one enabled by TarSSE-NP, at the dose of 5 mg DXR/kg (Fig. 1A) . Higher doses of TarSSE-NP were then tested to address the question on whether an increase in the systemic exposure of DXR delivered by TarSSE-NP would increase its bulk tumor accumulation. Although DXR delivered by TarSSE-NP presented a dose- dependent systemic exposure (AUC5 _min- 48 _h at 6 and 7 mg/kg was 3.7- and 4.4-fold higher, respectively, than at 5 mg/kg) (Fig. IB), it did not influence the corresponding bulk tumor accumulation 24 h after intravenous administration (within the range of 1 - 2 pg DXR/g of tumor and, at least, 4.3-fold lower than non-TarSSE-NP_#l , at 5 mg/kg), as assessed by LC- MS/MS (Fig. 2A) . These results demonstrated that bulk tumor accumulation of DXR delivered by TarSSE-NP was neither dependent on the administered dose nor proportional to the systemic exposure, within the range of tested doses.

Example 3

Intratumoral localization of doxorubicin by laser confocal microscopy Previously frozen MDA-MB-435S-derived breast (nucleolin ^hi 9 ^h ) tumors (approximately 150 mm ³ , 4 tumors per group) , recovered 24 h after single intravenous administration of TarSSE-NP at 6 and 7 mg of DXR/kg or non-TarSSE-NP_#l at 5 mg of DXR/kg, were thawed, sliced and mounted using Fluoroshield® mounting medium with DAPI (Abeam, UK) . Each tumor slice area was fully inspected for DXR fluorescence using LSM 710 AxioObserver confocal microscope and image acquisition was performed blindly by the operators to minimize bias. Image analysis was performed blindly using Zen v2.0 software enabling one to count DXR positive cells. Analysis of tumor sections by confocal microscopy (Fig. 2B) evidenced that the intracellular delivery of DXR enabled by the TarSSE-NP (assessed by the number of DXR ⁺ cells per area) , was dose- dependent and at 7 mg/kg, it was 2.3-fold higher than the one enabled by the non-TarSSE-NP_#l at 5 mg/kg (Fig. 2B) . This supported the enhanced intracellular delivery and improved intratumor bioavailability of the payload delivered by the TarSSE-NP, relative to the unmodified formulation and was in clear contrast with the assessment based on the bulk drug tumor accumulation performed by LC-MS/MS.

Example 4

Impact of nucleolin levels in the antitumor activity of

TarSSE-NP against mammary implanted tumors

Estimation of nucleolin cell surface density in MDA-MB-435 and 4T1 cells was performed upon comparing the cell- associated fluorescence values of anti-nucleolin-Alexa488 antibody, with standard curves prepared with the Quantum™ MESF Alexa488-labeled microspheres (Bangs Laboratories, USA) . This procedure has enabled to determine the number of nucleolin molecules per cell. The antitumor activity of the TarSSE-NP was evaluated against breast tumors from both cell types. Once NCL-overexpressing MDA-MB-435S-derived tumors reached a tumor volume between 100-150 mm ³ , the animals were treated with TarSSE-NP at 6 and 7 mg of DXR/kg, once a week, for 5 weeks. Additionally, as control, animals were treated with non-TarSSE-NP_#l at 5 mg/Kg, once a week, for 5 weeks. Alternatively, antitumor activity of TarSSE-NP was evaluated in a triple-negative murine breast cancer model. Treatment started once 4T1 triple-negative breast tumors reached 50- 90 mm ³ . Animals were treated with 7 or 9 mg of DXR/kg, once a week, for 5 consecutive weeks. Animals treated with saline solution were used as controls.

MDA-MB-435S cells presented a cell surface nucleolin density that was 2.3-fold higher than the one in 4T1 cells, as determined by flow cytometry at 4°C (Fig. 3A) .

Accordingly, the nucleolin-targeted TarSSE-NP inhibited MDA- MB-435S tumor growth (TG) , containing the tumor volume below the 2-fold threshold in a dose-dependent manner. This supported a marked reduction of tumor burden of, approximately, 2.3-fold (p=0.0043 or 0.0095) at the end of the treatment (Fig. 3B) . In contrast, all 4T1 tumors treated with TarSSE-NP at 7 or 9 mg/kg evidenced a tumor growth above the 2-fold threshold, after the third administration. This translated into a residual effect on tumor burden (p=0.08 or 0.142) (Fig. 3B) .

Further histological analysis of nucleolin ^hi 9 ^h MDA-MB-435S tumors was performed to assess the biological impact of the improved intratumoral DXR bioavailability provided by the TarSSE-NP (Fig. 4), upon comparison with the non-TarSSE- NP_#1 at 5 mg/kg. This value was close to the maximum tolerated dose and enabled half of the TarSSE-NP' s survival at the end of experiment with mice bearing nucleolin ^hi 9 ^h MDA- MB-435S tumors. TarSSE-NP at 7 mg/kg (q7dx5) decreased by 95% the density of nucleolin ⁺ vasculature, relative to the non-TarSSE-NP_#l (Fig. 4) . The impairment of the tumor vasculature, particularly nucleolin positive one, supported a component of nucleolin- mediated targeting associated with the nucleolin-targeted TarSSE-NP that further explained its antitumor activity. Importantly, the efficacy (Fig. 3B) , subsequent to the increased intratumor bioavailability of doxorubicin enabled by TarSSE-NP, as assessed by confocal microscopy (Fig. 2B) , could not be anticipated in light of prior art and given the higher systemic exposure and bulk tumor accumulation of the encapsulated payload (as assessed by mass spectrometry (Fig. 1A, IB and 2A) delivered by liposomes devoid of ligand- mediated targeting and presenting high systemic exposure (non-TarSSE-NP_#l ) .

Example 5

TarSSE-NP enabled significant mesothelioma growth

inhibition relative to the standard-of-care

Therapeutic activity of TarSSE-NP, encapsulating doxorubicin, was evaluated against human mesothelioma orthotopic animal models established in female CD-l ^nu/nu mice : the highest incidence epithelioid subtype (derived from MM473:Luc cells), and the most aggressive biphasic subtype (derived from MM487:Luc cells) . Cancer cells expressed luciferase and tumor growth was assessed by In Vivo Imaging System (IVIS) .

Mice were treated for 5 weeks with TarSSE-NP (once a week at 7 mg/kg) or the standard-of-care combination of pemetrexed (100 mg/kg, q2dx3x5) and cisplatin (4 mg/kg, q7dx5) . Untreated animals were used as control. A combination of the TarSSE-NP and cisplatin was also considered to assess the benefits of the combination over single treatments. In the combined treatment, TarSSE-NP was administered at weekly doses of 5.6 mg of doxorubicin/kg and cisplatin was maintained at 4.0 mg/kg/week, over five weeks. Data demonstrated that the overall tumor growth inhibition enabled by TarSSE-NP at 7 mg/kg, translated into a significant reduction of tumor burden in the epithelioid and biphasic animal models, compared to the standard-of-care (79- or 1223-fold for MM473:Luc or MM487:Luc, respectively)

( Fig . 5 ) .

Overall, treatment with the standard-of-care did not show any effect on tumor burden across in the different models. Moreover, no significant differences in the tumor burden between TarSSE-NP in monotherapy (both 5.6 and 7.0 mg/kg doses) or in combination with cisplatin (Fig. 5) have been observed, in any of the animal models tested.

Example 6

Toxicokinetic assessment of TarSSE-NP in beagle dogs

In another set of studies, TarSSE-NP was intravenously administered at 1 mg of DXR/kg (equivalent to 7 mg of DXR/kg in mice (30)) to male and female beagle dogs (n=3 per sex gender), once every week for 4 consecutive weeks. Control consisted of a non-TarSSE-NP_#l at 1 mg of DXR/kg. The blood clearance profile of DXR was established after first intravenous administration of nucleolin-targeted TarSSE-NP. As a general indication of toxicity, the TarSSE-NP demonstrated a lower impact on body weight variation as compared to a non-modified liposome (non-TarSSE-NP_#l ) (Fig. 6A) . The latter has caused a decrease of the mean relative body weight that was equal or higher than 10% in male or female dogs, respectively (Fig. 6A) . Moreover, the absence of significant impact on body weight evidenced in TarSSE-NP- treated group (Fig. 6A) , correlated with a better survival of animals, as compared to non-TarSSE-NP_#l-treated group at equimolar dose (Fig. 6B) . This correlated with the higher systemic exposure to DXR enabled by the non-TarSSE-NP_#l , as assessed by the area under the curve (AUC) of plasma concentrations, that was more than 4.5-fold higher than the one enabled by modified liposomes (TarSSE-NP) (Fig. 7), in accordance to Example 2.

Thus, delivery of DXR by a nanoparticle modified with a small, positively charged, targeting ligand (TarSSE-NP) presented fewer debilitating effects than a strategy based on the delivery of the same drug through an unmodified formulation (non-TarSSE-NP_#l ) .

Prior art teaches that a drug delivery system's efficacy relies on long circulating properties, enabling high systemic exposures, exploring the EPR effect to promote tumor accumulation and said efficacy. The prior art also teaches us that even targeted drug delivery systems rely in part on the EPR effect to accumulate at the tumor site, to later take advantage of the targeting moiety to target cancer cells .

Herein, the described data teaches that the modification of nanosystems with a positively charged moiety that simultaneously enables the binding to an overexpressed target while lowering the nanosystem systemic exposure (along with the one of the encapsulated agent) , retains the antitumor efficacy (dependent on the target expression level) with a safer profile, relative to unmodified longer circulating nanosystems.

Description of the embodiments

Now, preferred embodiments of the present application will be described in detail with reference to the annexed drawings. However, they are not intended to limit the scope of this application. According to the main embodiment of the present patent application, it is disclosed herein a nanosystem functionalized with a ligand for use in the treatment of tumors ,

Wherein said ligand is a positively charged peptide, at neutral pH, and the nanosystem is a nanoparticle carrying an agent or a combination of agents with high volume of distribution, associated with significant systemic toxicity, wherein said agent is a therapeutic molecule;

Wherein the nanosystem functionalized with the said ligand has a positive zeta potential between +0.1 and +10 mV at neutral pH;

Wherein the non-covalent or covalent attachment, at the surface of the nanosystem, of said ligand decreases the plasma concentration-time under the curve below 700 pg.mL- l.h in mammals of said nanosystem and encapsulated agent or combination of said agents at an effective dosing,

Wherein such decrease enables a safer toxic profile with less than 10% of body weight loss and less than 10% non human mammal death at effective dosing,

Wherein said ligand simultaneously enables the targeting to an overexpressed and accessible protein in tumor cells, reducing the tumor burden to less than 50% of the tumor burden of an untreated individual when administered, at an effective dosing.

In one preferential embodiment the positive zeta potential of the nanosystem functionalized with the said ligand is between +0.1 and +5 mV at neutral pH.

In one preferential embodiment, the non-covalent or covalent attachment, at the surface of the nanosystem, of said ligand decreases the plasma concentration-time under the curve to between 40 and 600 pg.mL-l.h in mammals, of said nanosystem and encapsulated agent or combination of said agents at an effective dosing.

In one embodiment the decrease in the plasma concentration time area under the curve of the said nanosystem and the encapsulated agent or combination of agents ameliorates the toxicological profile of the unmodified nanosystem encapsulating an agent or a combination of agents, characterized by high volume of distribution and associated with systemic toxicity.

In one embodiment, the plasma concentration-time area under the curve at the effective dosing induces less than 10% of mammal body weight loss and no more than 10% of mammal death.

In one embodiment, ligand is a peptide shorter than 70 residues, preferentially shorter than 40 residues. In one preferred embodiment the peptide presents, at physiological pH, less than 70% of positively charged residues, between 25% and 65%, preferentially between 40% and 62%.; wherein "physiological pH" is a pH between 7 and 8, preferentially 7.4.

In one preferential embodiment, effective dosing of said nanosystem reduces the tumor burden to 10% of the tumor burden of untreated mammals.

In one embodiment, 25-50% of the residues on the amino terminal of the ligand peptide side belong to a target recognition and binding motif towards a protein overexpressed at the surface of tumor cells, wherein said proteins are epidermal growth factor receptors, integrin receptors, VEGF receptors, nucleolin and neuropilin proteins. In one preferred embodiment the target recognition and binding motif is towards nucleolin.

In one embodiment at least one active agent or combination of agents are typically associated with a high volume of distribution and systemic toxicity.

In one embodiment, the preclinical systemic exposure of the encapsulated agent or combination of agents is preferentially below 700 pg.mL ^_1 .h, or more preferentially below 600 pg.mL ^_1 .h, within 48 h after administration.

In one preferred embodiment, the nanosystem as described herein is for use in the treatment of tumor and cancer cells. While various embodiments of the invention have been described, it will be apparent to those of ordinary skill in the art that many more embodiments and implementations are possible within the scope of this invention. Accordingly, the invention is not to be restricted except in light of the attached claims and their equivalents.

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