IT201800006278A1 - Bionanofenretinide new antitumor formulation - Google Patents

Description

DESCRIPTION

Attached to a patent application for INDUSTRIAL INVENTION entitled "Bionanofenretinide new antitumor formulation"

STATE OF THE ART

Most cancers are heterogeneous and contain a small population of cancer stem cells (CSCs) that exhibit distinctive features of self-renewal, proliferation and differentiation, which are believed to play a crucial role in tumor progression, drug resistance, recurrence and metastases in multiple neoplasms [1]. As the existence of CSCs is a primary obstacle to cancer therapy, a huge effort has been made in developing anti-CSC strategies and several potential approaches to kill therapy-resistant CSCs have been explored, including inhibition of ATP binding. -receptors, blocking the essential signaling pathways involved in the self-renewal and survival of CSCs, targeting the surface markers of CSCs and destroying the tumor microenvironment. In addition to overcoming the primary intrinsic resistance of tumor cells, CSC-directed therapies have also been shown to be effective against tumor recurrence and metastasis [2,3]. Meanwhile: an increasing number of therapeutic agents (e.g. small molecule drugs, nucleic acids and antibodies) with efficacy against CSC have been investigated or proposed in recent years. However, in consideration of the plasticity of tumor cells, it appears evident that drugs with wide toxicity against tumor cell populations with different properties and aggressiveness must be preferred to target the tumor in its heterogeneity and obtain long-term antitumor efficacy of the treatments. [4]. Another huge obstacle to the efficacy of the treatment is the limited distribution of drugs into the target cell environment due to limited water solubility, short circulation time and reduced stability of the compounds, as well as doselimiting systemic toxicity which does not allow for achievement of therapeutic concentrations within tumors [5]. Specially designed nanocarrier-based therapeutic (or nanomedicine) agents offer new possibilities to increase therapeutic drug accumulation at the tumor site by enabling the therapeutic window to be reached for drugs that can potentially efficiently kill CSCs but whose activity in vivo antitumor is prevented by low solubility or bioavailability at the tumor site, resulting in negligible accumulation of drugs at the target site, below the therapeutic window.

Fenretinide (4-HPR) is a synthetic retinoid that has emerged as a promising anticancer agent based on numerous in vitro and animal studies as well as clinical chemotherapy studies [6,7,8]. It has been reported to induce cytotoxicity through multiple mechanisms including p53-dependent apoptosis, caspase-dependent and independent and / or non-apoptotic mechanisms; generation of reactive oxygen species (ROS), modulation of ceramide metabolism associated with increased levels of potentially cytotoxic dihydroceramides and inhibition of the mTOR pathway [9,10,11,12,13,14], Significantly, fenretinide was cytotoxic for lymphoblastic leukemic cell lines in vitro, but minimally toxic to fibroblasts, peripheral blood mononuclear cells and marrow myeloid progenitors, suggesting a potential safe therapeutic use in haematological malignancies. Importantly, fenretinide has shown the ability to target cancer stem cell-associated targets, further increasing its therapeutic value [15,16,17,18]. An oral formulation of fenretinide consisting of soft gelatin capsules containing fenretinide in corn oil and polysorbate is currently available from the National Cancer Institute (NCI-FeR). However, despite the promising in vitro cytotoxicity, its antitumor activity in clinical studies (both as a therapeutic and chemo-preventive agent) remains limited, probably due to the poor bioavailability of this pharmaceutical formulation which requires the administration of too high doses of the drug. which limit both tolerability and patient compliance [19,20,21]. Numerous attempts have been made to develop formulations of fenretinide capable of increasing its bioavailability to a level that elicits a satisfactory therapeutic response in the absence of toxicity. Promising, although still unsatisfactory, therapeutic activity was achieved in a study with children with neuroblastoma, while in adult patients (lymphoma) the high doses of drug needed to target therapeutic plasma concentrations of the drug resulted in limited patient compliance. , gastrointestinal toxicity and reversible nocthalopia (reduced night vision). Other studies have reported improved efficacy of oral fenretinide when co-administered with other drugs, possibly due to the lower drug concentration required in combination with synergistic compounds or to increased fenretinide exposure induced by concomitant drug treatment. specific [19,22,23, 24,25]. Phenretinide administered intravenously can obviously increase plasma levels of the drug and a soybean oil in water emulsion is currently being tested in patients with haematological malignancies with promising results, although limited solubility in body fluids and tissues of the drug formulations. unmodified may represent an unsolved problem [26].

The development of a highly effective oral formulation opens up important therapeutic perspectives. It allows the administration of prolonged therapies over time without the patient being dependent on health facilities or specialized personnel, facilitates pediatric administration and in general improves the patient's perception of care.

The inventors of the present invention in a previous patent (Fenretinide Complexes WO2016038534) presented a formulation which provides for the complexation of fenretinide with cyclodextrin thus obtaining a higher solubility in water capable of providing a better bioavailability of the fenretinide. In this case, the bioavailability of the drug in the tumor site made it possible to reach the drug concentrations suitable to elicit a therapeutic response also thanks to the presence of adequate levels of drug solubilization. The efficacy of the drug object of the previous patent is limited to intravenous administration, making the use of fenretinide as a prescription drug not very accessible for a therapy to be combined with chemo or radio therapy.

The results obtained on athymic nude mice in the presence of subcutaneous tumors of different origin treated orally with the old formulation fenretinide-cyclodextrin, are such that they do not allow to proceed with a clinical trial, since the minimum thresholds of bioavailability of fenretinide are not reached. in plasma.

Despite the good results achieved, all these studies have suggested that it is necessary to continue research and experimentation in order to obtain formulations of fenretinide of which acceptable doses can be administered, which are highly bioavailable, tolerable and which ensure patient compilation even with significant systemic exposures, aiming for satisfactory therapeutic efficacy also for single agent treatment: for adult patients,

For these reasons, the present invention concerns the development of a nano-formulation of fenretinide, called bionanofenretinide (Bio-nFeR) designed to increase the low bioavailability of fenretinide, which limits its clinical use despite its remarkable antitumor activity. Bio-nFeR was prepared by nanoencapsulation of fenretinide in an anhydrous lipid matrix composed of phospholipids and triglycerides (derived from natural lecithins). Bio-nFeR shows very promising antitumor activity, superior to NCI-FeR, against CSCs of various origins in vitro at low concentrations, and in xenografts of CSC-derived tumors of the lung, colon and melanoma, achieving a good drug concentration in the blood and drug levels with therapeutic efficacy in xenografts, in the absence of toxicity.

Summary of the invention

An increasing number of anticancer agents have been proposed in recent years with an attempt to overcome the resistance to treatment of cancer cells. In particular, most preclinical trial efforts have been aimed at targeting the more aggressive cancer stem cell (CSC) population, with partial improvement in long-term drug response. However, in addition to the therapy resistance of cancer cells, a further obstacle to the elimination of CSCs is the ineffective administration of drugs in the environment of the target cell, due to limited water solubility, short circulation time or low stability. of compounds, which together with the limit imposed by toxicity due to systemic administration, contributes to hindering the achievement of therapeutic concentrations of drugs within tumors, ultimately leading to treatment failure. Nanocarrier-based therapeutic agents (nanomedicines) offer new possibilities to increase drug accumulation at the tumor site by enabling therapeutic doses to be achieved for drug effective against CSCs but whose antitumor activity in vivo is impeded by low solubility or low release at the tumor site. The synthetic retinoid fenretinide (4-HPR) is an excellent anticancer drug active against a wide range of cancers with excellent tolerability and practically free of limiting side effects. However, its extremely low solubility in aqueous solutions limits its therapeutic use as even the administration of high doses of the drug does not provide sufficient plasma levels to produce an adequate therapeutic effect. Promising, although still unsatisfactory, attempts have been made to develop formulations of fenretinide capable of increasing its bioavailability. Given these premises, the inventors have developed a formulation of nano-micellar fenretinide called bionanofenretinide (Bio-nFeR), based on the encapsulation of the drug in a micellar lipid matrix, designed to increase the low bioavailability of fenretinide, which limits its clinical use despite its considerable antitumor activity. Bionanofenretinide is particularly suitable for oral administration of fenretinide, as it provides a stable delivery throughout the gastrointestinal tract against the presence of enzymes and pH changes, but above all it allows the drug to be absorbed by the lymphatic route through fat uptake. mediated by the presence of bile salts. In this way the oral bioavailability of fenretinide is further increased as the solubilizing effect of the formulation is added to the lack of hepatic metabolization or 'first pass effect' which strongly limits the bioavailability of most of the drugs administered orally.

Bio-nFer showed marked antitumor activity in vitro against CSCs of the lung, colon and melanoma and against xenografts, superior to the standard capsular formulation used in clinical studies, reaching therapeutic concentrations within tumors and therapeutic activity without in vivo history as a single agent, in the absence of toxicity.

As is known, oral pharmaceutical formulations represent the majority of formulations in the pharmaceutical market since the ease and safety of use and the high patient compliance make this route the first choice among the various administration methods.

Detailed description of the images

Fig. 1 Illustration of the biochemical structure of Bio-nFeR and fluorescence microscopic image of micelles containing drugs. A) Schematic representation of micelles composed of phosphplipids in the outer layer that contain fenretinide in the nucleus. B) Solubility in water and distribution of Bio-nFeR and NCI-FeR formulations. (Left) Optical microscopy image showing the structure and size of the micelles of Bio-nFeR dissolved in water, (center) Confocal image of Bio-nFeR (the same on the left) showing the presence of autofluorescent fenretinide within the micelles. (Right) Confocal image of NCI-FeR dissolved in water forming large aggregates of insoluble material. The magnification of the images is 60X and the scale bar of 10 μιm is shown;

Fig. 2 (A) Cytotoxic activity of different doses of Bio-nFeR in CSC of lung cancer, melanoma, colon cancer, glioblastoma and sarcoma, in vitro. The indicated CSCs were exposed to drug doses of 0.1, 0.5, 1, 2, 5, 10, 20 and 30 μΜ and cell viability was assessed by Cell Titer-Glo after 72 hours and indicated as a percentage compared to control cells. (B) Antitumor activity of Bio-nFeR in CSC-derived xenografts of lung, melanoma and colon cancer. (Upper panels) Growth curves of CSC-derived xenografts in control mice or mice treated with Bio-nFeR at 100 (lung cancer and melanoma) or 150 (colon cancer) mg / kg for the indicated times. The mean ± S.D. is shown. of three independent experiments. * P <0.05 ** P <0.01. (lower panels) Table of drug-induced systemic toxicity in the three groups of mice shown as percentage of body weight loss (BWL) or number of deaths / total number of mice. C) Levels of fenretinide in plasma and tumors of the same samples as B. Phenretinide concentration in plasma (expressed in ng / ml and corresponding concentration of μΜ, as indicated) and tumors (in ng / g and extrapolated uM concentration assuming tumor density like about 1). (D). Levels of phenretinide metabolites in plasma and in the same tumor samples described in B-C. Concentration levels of Fenretinide, and its metabolites 4HPR, 0X0 and DH-4HPR in plasma and tumor specimens shown in C.

Fig. 3 Effect of Bio-nFeR (100 mg / Kg / 5 days a week) on the growth of subcutaneous tumor xenografts of different origins. Treatment was started when the tumor generated by the injected cells had reached the size of 0.05-0.1 cm3 (day 0). Treatment was administered orally. The in vitro cytotoxic effect of Bio-nFeR at different dose levels is shown in the upper left of each panel.

Fig. 4 A) Comparison of the piasmatic concentration decay curves of fenretinide with different oral dosages of Bio-nFeR. B) Main pharmacokinetic parameters of fenretinide in female CD1 mice after oral administration of different doses of Bio-nFeR. Fenretinide Cmax (C) and AUC (D) as a function of the administered dose of BionFeR.

Fig. 5 Pharmacokinetic profile in acute and chronic administration of Bio-nFeR compared to NCI-FeR. Pharmacokinetic profile of oral BionFeR compared to the phenretinide MCI formulation after acute treatment (left), and after 2 weeks of chronic treatment (right).

Fig. 6 Plasma time-concentration profiles of 4-oxo-4-HPR and MPR metabolites compared with 4-HPR, obtained in the multiple-dose study, which correspond to approximately 50% and 15% respectively to the parental drug, both in the case of Bio-nFeR and NCI-FeR.

Fig. 7 (A) Cytotoxic activity of Bio-nFeR compared with the standard formulation of fenretinide (NCI-FeR) in lung CSCs. Pulmonary CSCs were exposed at the indicated drug doses and cell viability was assessed by CellTiter-Glo after 72 hours and reported as a percentage of control cells. (B) antitumor activity of Bio-nFeR and NCI-FeR in xenografts derived from lung CSCs. (left) Growth curves of xenografts derived from lung CSCs in mice treated or control mice with Bio-nFeR or NCI-FeR at the dose of 50 mg / kg for the indicated times. Lung CSC sample # 136 was used for the generation of xenografts. The mean ± S.D. is shown. of three independent experiments. ** P <0.01. (right) the table of drug-induced systemic toxicity in the three groups of mice, shown as percentage of body weight loss (BWL) or number of deaths / total number of mice.

Fig. 8 Viability curves (obtained by MTT assay) of control or exposed lung CSCs at the different indicated doses of Bio-nFeR or NCI-FeR and corresponding IC50 values calculated for the two different formulations.

Fig. 9 Modulation of the lipid metabolism of Bio-nFeR in tumor tissues. A) Confocal images of KI-67 immunofluorescence and evaluation of TUNEL positivity of lung cancer, melanoma and colorectal cancer (CRC) xenografts obtained from control mice or treated with Bio-nFeR.

B) Quantification of the different forms of dihydroceramides present in lung, melanoma and colon tumors obtained from the same control mice or treated with BionFer as in Fig 9A. The values obtained were normalized with respect to the controls and represented as a percentage with respect to the untreated samples . Quantification of sphinganins in the same samples as in A and B, normalized with respect to the controls and represented as variation with respect to the untreated samples. Fig. 10 Quantification of the different forms of sphygomyelin-ceramides and -dihydroceramides, glucosylceramides and -dihydroceramides present in the same samples of lung, melanoma and control colon tumors and treated with Bio-nFer as in Fig 9. The values obtained were normalized with respect to controls and plotted as a percentage with respect to untreated samples.

Detailed description of the invention Bìonanofenretinide (Bio-nFeR) is an innovative nanoformulation conceived with the aim of increasing the low bioavailability of fenretinide which limits its clinical use despite its remarkable antitumor activity with negligible side effects. Bio-nFeR is based on the technological platform of drug nanoencapsulation in amphiphilic carriers that allow the formation of drug-carrier complexes with physico-chemical characteristics suitable for controlling the solubilization of the drug in the body fluids present in the administration and absorption sites. Such high systemic bioavailability can produce a better distribution of the drug in the body and increase drug levels bringing it into specific therapeutic targets. Bio-nFeR was prepared from the formation of a phenretinide complex with biocompatible and biofunctional carrier molecules consisting of phospholipids and triglycerides (derived from natural lecithins), Fig. 1A. In this formulation, for the first time, fenretinide has been encapsulated in micellar form with phospholipids to obtain nanocapsules characterized by a high percentage by weight of the drug (101 w: w), a high solubilization of the drug (up to 30 mg / ml) and stability in aqueous body fluids at the sites of administration and absorption.

In accordance with the present invention, fenretinide can be complexed with phospholipids and / or sphingolipids, or with cationic lipids, for an embodiment of the invention the fenretinide-carrier complex is a vesicular system. Another embodiment of the invention provides for the use of pegylated phospholipids or phospholipids with cationic residues.

A particular embodiment of the phenretinide-carrier complex in the form of liposomes are the complexes with phosphatidylcholine.

According to a further embodiment of the invention, when fenretinide is complexed with amphiphilic polymers and / or amphiphilic macromolecules optionally mixed with surfactants and / or cationic lipids and / or phospholipids and / or sphingolipids, the phenretinide-carrier complex is a micelle comprising fenretinide, or, if prepared by standard methods known to those skilled in the art, nanoparticles comprising fenretinide.

The analysis by fluorescence microscopy allowed to detect the fenretinide inside the micellar aggregates, exploiting the autofluorescence of the drug (Fig. 1B). NCI-FeR, on the other hand, appeared in the form of coarse aggregates of fenretinide, indicating the lack of solubilization of the drug in water (Fig. 1B).

Bio-nFeR has been specially designed to protect the drug from the destabilizing effects of gastrointestinal enzymes and pH variation and in general due to its chylomicron structure to promote the absorption of drugs through the lymphatic pathway as an additional means to further increase the 'systemic exposure of the drug via the lack of hepatic metabolism or' first pass effect '.

Compared to the standard formulation, based on soft gelatin capsules containing fenretinide dispersed in corn oil and polysorbate 80 and a more recent formulation based on fenretinide dispersed in a lipid matrix called LYM-X-SQRB, Bio-nFeR appears to have a greater piasmatic concentration after a single administration with a dose comparable to that which in other formulations is administered in multiple modalities. Furthermore, the Bio-nFeR provides a linear correlation between the doses administered orally and the corresponding piasmatic concentrations of the effect drug necessary to increase the systemic exposure compared to LYM-X-SORB which, on the contrary, undergoes a decrease in gastrointestinal absorption at increasing doses [25]. In vivo studies have shown that Bio-nFeR is completely free of toxic side effects, as indicated by the lack of weight change of the treated mice (Fig.2B) and the lack of visible signs of toxicity after single administration of doses up to 200 mg / Kg.

As shown in Fig.4A, after oral administration of multiple doses of Bio-nFeR to mice, 4-HPR reaches piasmatic Cmax between 2 and 4 hours, is rapidly distributed and eliminated with a half-life of approximately 7 hours, ensuring a plasma drug exposure for up to 48 hours at each dose studied.

Table 1

The main derived pharmacokinetic parameters are listed in Table 1, Cmax and AUC increase proportionally with doses indicating that the pharmacokinetics of Bio-nFeR are not dose-dependent over the dose range studied as shown in Fig. 4C and D which report dose correlations. vs Cmax and against AUC.

These results suggest that the drug dose could be further increased to achieve higher piasmatic exposure, possibly improving the therapeutic effect.

Following the pharmacokinetic study of different doses as acute treatment, the pharmacokinetics were explored with a large comparative study with chronic administration, at therapeutic doses of Bio-nFeR and NCI-FeR. The drugs were well tolerated in mice. No body weight loss or impairment of liver function was observed in the treated mice compared to the control mice. These controls were performed by evaluating the levels of liver enzymes (Table 2).

Table 2

The protocols and assays used are highly compatible with in vivo activity studies. 150 mg / kg of Bio-nFeR and NCl-FeR were administered as a single administration (formally on day 1) or for 2 weeks as a daily administration (12 days, two days off) and the 4-HPR vs piasmatic concentration profiles. time are shown in Fig. 5. From a visual inspection of the curves and as demonstrated by the parameters listed in Table 3, it can be seen that exposure to 4-HPR is 1.3 and 1.6 times higher with Bio-nFeR oral versus oral NCI-FeR after the first treatment and after repeated treatments, respectively.

Table 3

In detail, following the administration of Bio-nFeR, 4-HPR reaches Cmax between 2 and 4 hours, is rapidly distributed and eliminated with an HL of about 5 ~ 6 hours reaching an adequate piasmatic exposure up to 48 hours after the last administration. On the first day, the mean Cmax and AUC0-24h are 6350 ng / mL and 56881 ng / mL * h and after repeat treatment 6813 ng / mL and 78608 ng / mL * h. The last value indicates a 40% increase in 4-HPR piasmatic exposure after chronic administration of Bio-nFeR compared to the first day.

Elimination of fenretinide occurs mainly by metabolic process with formation of 4-oxo-4-HPR and MPR and by faecal excretion of 4-HPR and unmodified metabolites.

This result is shown in Fig. 6 which reports the plasma time-concentration profiles of the 4-oxo-4-HPR and MPR metabolites compared to 4-HPR, obtained in the multiple-dose study, which correspond to approximately 50% and 15 respectively. % of the parental drug, after administration of both formulations, Bio-nFeR or NCI-FeR. The main pharmacokinetic parameters obtained for the metabolites are shown in Table 4,

Table 4

A similar metabolic pattern has been observed during chronic administrations where metabolites in plasma are measurable one hour after administration and with maximum exposure at 2-4 hours. The metabolites disappear with the same half-life as the parent compound (Table 5).

In the Acute Study, the relative percentage to 4-HPR of 4-oxo-4-HPR, known to be 2-4 times more cytotoxic than 4-HPR and inactive metabolic MPR, after single and repeated administration is approximately 50% and 15%, respectively. In addition, the inventors have detected the presence of a third metabolite, DH-4-HPR, previously described by Cooper et al.

[19] this amounted to approximately 5-7% of the parental drug.

Table 5

As a final consideration on metabolite formation, the differences in piasmatic AUC between day 1 and day 14 should be noted, which are negligible compared to a hypothetical linear trend between administered dose and metabolites in plasma (Table 5). These results indicate that no saturation or induction of metabolism is present after chronic treatment.

The presence of the parental drug and its metabolites in faeces for acute multiple-dose treatment is shown in Table 6, while that for chronic treatment is shown in Table 7. The amount of dose recovered in urine is negligible (≤0.002%) given the low polarity of fenretinide and metabolites.

Table 6

Table 7

Bio-nFeR was further validated for its anticancer activity and pharmacokinetic properties.

In order to determine if Bio-nFeR had relevant antitumor activity, its ability to influence the viability of lung CSCs was evaluated against the standard oral capsule formulation of fenretinide (NCI-FeR). First in vitro tests were performed and five previously isolated and validated patient-derived CSC-lung lines (squamous cell carcinoma, adenocarcinoma and large cell carcinoma) [27] were exposed to three different doses of Bio -nFeR or in parallel with NCI-FeR at the same concentrations. Bio-nFeR exerted a substantial cytotoxic effect, against all lung CSCs examined even at low concentrations, although with varying efficacy in different samples, while at higher doses more than 95% of the cells were killed in all samples tested. Conversely, NCI-FeR resulted in a moderate (40-60%) reduction in cell viability only at higher doses (10 μΜ). More precisely, Bio-nFeR inhibited cell viability at a level comparable to that determined by a 10 times higher concentration of NCI-FeR in the dose range of the drug tested. This different efficacy is also visible in the viability curve of the MTT assay and confirmed by the IC50 values calculated through an experiment comparing the response to the administered dose performed on pulmonary GSCs. This experiment shows a difference greater than 10 times between IC5G with Bio-nFeR IC50 = 0.72 μΜ and NCI-FeR IC50 = 9 μΜ (Fig.7A and Fig.8).

An example of application with which the inventors examined the antitumor activity of Bio-nFeR in vivo was on CSC-derived lung xenografts on NSG immunodeficient mice, compared to the standard formulation of NCI-FeR. While the administration of NCI-FeR (50 mg / kg) did not substantially affect tumor growth rates compared to tumors of untreated mice, the same dose of Bio-nFeR resulted in a significant inhibition of tumor growth (Fig. 7B). It is important to note that no signs of systemic toxicity were observed (Fig. 7B right), suggesting that increasing doses of Bio-nFeR could be explored for future experiments in order to further improve efficacy.

Given the marked anticancer activity of BionFeR against lung cancer cells in vitro and in vivo and the better systemic exposure compared to the reference formulation NCI-FeR, the inventors extended the evaluation of the cytotoxic activity of Bio-nFeR over a wide range. of CSC concentrations of lung and colon cancer, melanoma, sarcoma and glioblastoma. Three days of exposure to Bio-nFeR resulted in marked inhibition of CSC viability in most of the samples of each type of tumor tested (Fig. 2A).

Generally, CSCs of lung, colon, and melanoma show a higher fraction of Bio-nFeR-sensitive lines than the more resistant CSCs of glioblastoma and sarcoma, prompting in vivo studies to evaluate the antitumor activity of the drug in these. tumors. Subcutaneous tumors were generated by CSC inoculation of lung cancer, melanoma and colon carcinoma in immunodeficient mice. For a particular embodiment of the invention the in vivo tests were performed on mice to which Bio-nFeR was administered orally for 3 weeks at 100 (lung cancer and melanoma) or 150 (colon cancer) mg / kg, based on the absence of toxicity demonstrated in the previous experiment (Fig. 7B), where lower doses of the drug were used. Tumors from treated mice showed a significant reduction in tumor growth rate compared to control tumors (Fig. 2B). In addition, the drug shows high tolerability, as no systemic toxicity is observed, except for a slight weight loss (8%) in the group of mice with higher dose (150 mg / kg), in the absence of other signs. visible toxicity (Fig.2B tables below), The lack of toxicity in mice at the doses used, together with the previous evidence of tolerability of fenretinide observed in clinical studies (when doses of the drug were administered 10 times higher suggests that the dose maximum tolerated was not reached under these experimental conditions and drug doses could be increased in order to increase drug concentration at the tumor site and therapeutic efficacy [29],

The complexes of the present invention, due to the solubility of the drug and the effective drug release just discussed, are extremely suitable for use as medicaments.

The complexes of the present invention are of particular interest for the therapeutic targeting of cancer cells, including cancer stem cells, and therefore for the treatment or prevention of solid and / or hematological tumors.

Given the large amount of literature on the efficacy of fenretinide on various tumors or cancer cells, including cancer stem cells, the skilled person will easily identify all tumors for which the complexes of the invention provide effective treatment.

A non-limiting example of these cancers is represented by: gynecological cancers, skin cancers, brain and neurological cancers, urological cancers, hepatobiliopancreatic cancers, neuroendocrine cancers, endocrine cancers, adenocarcinomas, cancers of the digestive tract, respiratory tract cancers, cancers haematological, mesothelioma, embryonic carcinoma, head and neck cancer, sarcomas.

According to the present disclosure and according to the art, the term urological cancer includes but is not limited to cancers of the bladder, kidney, prostate and testicles; the term hepatobiliopancreatic cancers includes of the liver, gallbladder, bile ducts, and pancreas.

According to the present disclosure and according to the art, cancers of the respiratory tract include but are not limited to cancers of the lung, cancers of the larynx.

According to the present disclosure and according to the art, cancers of the digestive tract include but are not limited to cancers of the pharynx and esophagus and gastrointestinal cancers such as, by way of example, cancers of the stomach, pancreas, small intestine, large intestine, colorectal, rectal, gastric, and anus and gastrointestinal stromal tumor.

According to the present disclosure and according to the art, gynecological cancers include but are not limited to breast, ovarian, uterine, cervical, vaginal and vulvar cancers.

According to the present description and according to the technique, hematological cancers include but are not limited to adult and pediatric cancers. Non-limiting examples thereof are leukemias such as acute and chronic leukemia, hairy cell leukemia, lymphomas including Hodgkin and non-Hodgkin lymphomas, mycosis fungoides, CNS lymphoma, Sezary syndrome, cutaneous lymphomas, myelomas, myelodysplastic and myeloproliferative diseases, Waldenstrom's macroglobulinemia.

According to the present disclosure and according to the art, brain and neurological cancers include but are not limited to adult and pediatric neurological tumors, non-limiting examples thereof are gliomas including glioblastoma, medulloblastomas, oligodendroglioma, neurinomas, chordomas, ependymoma, neuroblastoma and cancers of the peripheral nerves.

According to the present disclosure and according to the art skin cancers include, but are not limited to, basal cell cancer (BCC), squamous cell cancer (SCC), melanoma and Merkel's cancer.

According to the present description and according to the art, endocrine cancers include but are not limited to adrenocortical carcinoma, thyroid cancer, parathyroid cancer, pituitary tumors.

According to the present description and according to the technique, neuroendocrine cancers are cancers that affect the neuroendocrine system. The neuroendocrine system produces chemical messengers called hormones, which regulate the functioning of different organs in the body. Neuroendocrine cells are scattered throughout the body in organs such as the stomach, intestines and lungs.

Cancerous NETs are classified according to where the cancer started (where is the primary tumor) in the body. Non-limiting examples of neuroendocrine cancers according to the present disclosure include: NET of the small intestine, NET of the large intestine, NET of the appendix, Pancreatic NETs, Gastric NETs, NET of the lung.

Rarely, NETs are found in other areas, including the liver, gallbladder, bile ducts, kidneys, ovaries, or testes.

According to the present description and according to the technique, adenocarcinoma is a type of cancer which forms in the mucus-secreting glands throughout the body.

According to the present description and according to the technique, sarcomas derive from transformed cells of mesenchymal origin therefore malignant tumors made of bone, cartilage, adipose, muscle, vascular, or hematopoietic cancerous tissues, by definition, are considered sarcomas. Non-limiting examples include: osteosarcomas, chondrosarcomas, liposarcomas, leiomyosarcomas and rhabdomyosaromas.

According to the present invention, the Bio-nFeR complexes described in the present invention and claimed can be used alone in patients with cancer, or alternatively or in combination with chemotherapeutic agents commonly used for each type of tumor, and / or with small molecules commonly used for any type of tumor and / or with biological agents commonly used in biological therapies for any type of tumor (including immunotherapy and monoclonal antibodies).

Non-limiting examples of said chemotherapeutic agents are selected from the group comprising or consisting of cisplatin, gemcitabine, docetaxel, cytosine arabinoside, bleomycin, busulfan, capecitabine, carboplatin cyclophosphamide, daunorubicin, doxorubicin, epirubicin, fluoracouracourin 5 , Ifosfamide, hydroxyurea, lomustine, melphalan, mitomycin C, mitoxantrone, oxaliplatin, paclitaxel, temozolomide, topotecan, vinblastine, vincristine, vinorelbine, vindesine.

Consistent with the observed pharmacological activity, the parallel pharmacokinetic study showed that fenretinide administered orally achieved highly encouraging concentrations in plasma (Fig.2C), higher than that achieved in the plasma of patients treated with equivalent doses (300 mg / m2 is the equivalent dose corresponding to 100 mg / kg in mice) of the standard drug (NCI-FeR) or other best formulations of drugs in clinical trials (phenretinide-cyclodextrin complex), either as single or multiple administration [22, 25].

Importantly, as measured in tumor models, the drug showed a pharmacologically active intratumor concentration (1.88 ug / g to 2.23 pg / g, equivalent to 4.8-5.7 pM) at both doses. of drug analyzed (Fig. 2C). These concentrations are higher than those required for in vitro cytotoxicity in these cell lines (Fig. 2A).

In the tumors analyzed, the inventors detected the presence of the three main metabolites of 4-HPR (Fig. 2D) already observed in the plasma. The most abundant is 4-oxo-4-HPR, the active one, which reached comparable tumor concentrations in the three different tumors. These concentrations were in the same range as those of the parent drug, but since this metabolic is 2-4 times more active than 4-HPR, we can hypothesize that the antitumor activity in vivo is mainly due to its abundant presence (Table 8).

Table 8

To analyze the molecular effects of the Bio-nFeR treatment in vivo, the proliferative index and the extent of cell death induction of tumor xenografts were analyzed. In the confocal images of the tumor slides in Fig. 9Ά, the expression of KI-67 was strongly reduced and the fraction of positive TUNEL cells markedly increased in tumors treated with Bio-nFeR compared to controls. Therefore, Bio-nFeR induced a marked inhibition of tumor cell proliferation associated with the induction of apoptosis, in line with previous reports [24,28].

Previous studies have shown that fenretinide cytotoxicity is associated with lipid metabolism

[7,10, 12,14], In particular, fenretinide is able to increase the intracellular levels of dihydroceramide species by targeting dihydroceramide desaturase [10], With liquid chromatography-mass spectrometry (LC-MS), the inventors found that the Bio-nFeR treatment induced a marked increase in the dihydroceramide and dihydrosphingolipid (glucosyldihydroceramide) species (Fig. 9B and Fig, 10). This effect is more evident for the hydroceramide species with fatty acid chains 18 and 16. Conversely, the saturated ceramide levels were not affected by the Bio-nFeR treatment. In conjunction with the accumulation of long-chain dihydroceramide, the inventors observed a significant increase in intracellular levels of sphinganine in tumor xenografts treated with Bio-nFeR {Fig. 9C).

On the basis of these results, Bio-nFeR for oral administration proves to have greater efficacy and lower toxicity than all formulations known to date in the literature.

By way of example, the pharmaceutical composition of the invention can be formulated in the form of capsule, tablet, lozenge, granule, powder, solution, suspension, emulsion, syrup, elixir, hard gelatin and soft gelatin.

Examples

Preparative example

In the present example a preparation technique of Bio-nFeR is reported.

to. Fenretinide is homogeneously mixed with egg phosphatidylcholine and glyceryl tributyrate dispersed in ethanol up to a weight ratio of 1: 9: 1 w: w: w respectively.

b. Ethanol is removed from the mixture in a rotary evaporator and the dry residue stored at -18 ° C until use, c. The reconstitution of the dry residue in Bio-nFeR nanomicells was obtained by dissolving the residue in water at 30 ° C in an ultrasonic bath where a wave frequency of 40 kHz was applied for 1 hour until the dissolution phase.

To compare Bio-nFeR with soft gelatin capsules which represent the standard formulation most used in clinical trials for fenretinide, the inventors reconstituted the contents of the capsules according to the directions provided by the National Cancer Institute (fenretinide: 100 mg, corn oil: 704 mg, polysorbate 80: 60 mg). The final concentration in the reconstituted content of NCI capsules (NCI-FeR) is 11.6% w: fenretinide: components of the inner capsule, as per US instructions.

Example of cell culture, drug treatments and cell viability tests

Patient-derived tumor stem cell lines have been obtained from primary tumors, melanomas, glioblastomas, lung and colon cancers.

For drug treatments and cell viability tests, 3000 cells were triplicated in 96-well plates and left untreated or exposed for 72 hours to BionFeR (200 mg / ml stock solution freshly dissolved in water) or NCI-FeR ( 20 mg / ml dissolved in corn oil and polysorbate 80). Cell viability was detected with Cell TiterGlo (Promega).

Pharmacokinetic study example

Animals

The experiments were conducted on 7-week-old female CDl mice (body weight 25 ± 2 g) obtained from Envigo RMS SrL (Udine, Italy). They were kept in specific pathogen-free conditions at constant temperature and humidity, according to institutional guidelines regularly checked by a certified veterinarian. The experimentation on animals was conducted in accordance with the following laws, regulations and policies governing the care and use of laboratory animals: Italian government law (D. 1. 26/2014, Authorization n.19 / 2008-A issued on March 6, 2008 by the Ministry of Health). Institutional regulations and policies that provide internal authorizations for people conducting animal experiments (Quality Management System Certificate - UNI EN ISO 9001: 2008 - Reg. N 86121). The experimental protocol was examined by the Institute's Ethics Committee

and approved by the Italian Ministry of Health (Min. Aut. No 489/2016-Pr).

Example of method of administration

The pharmacokinetics of oral Bio-nFeR were studied relative to that of the reference formulation NCI-FeR, after single administration of multiple doses and after chronic administrations, every day for two weeks or twice a day for one week. Bio-nFeR was reconstituted at 20 mg / ml of fenretinide to allow administration of 200 mg / kg of fenretinide corresponding to the highest dose. Doses of 100, 50 and 10 mg / kg were administered using serial dilution of the 20 mg / ml solution.

NCI-FeR was prepared, according to NCI instructions, at 11.6% w: w fenretinide: components of the inner capsule, it was diluted with corn oil to obtain the treatment solutions at concentrations of 20, 15 and 10 mg / ml, corresponding to the administration doses of 200, 150 and 100 mg / kg respectively.

Examples of treatments

Acute study: BionFeR oral pharmacokinetics were studied in four independent groups of 24 randomized mice, to recive different treatment with the following equivalent doses of fenretinide: 10, 50, 100 and 200 mg / kg. The drug was administered for orally and after treatment, blood samples were taken at 15 and 30 minutes and at 1, 2, 4, 8/10, 24 and 48 hours from the retro-orbital plexus of mice under isoflurane anesthesia and collected in heparinized tubes. The mice were then sacrificed by cervical dislocation. Liver, kidney and brain were removed and immediately frozen on dry ice for future studies. To obtain the plasma, the blood was centrifuged at 4000 rpm for 10 minutes at 4 ° C. To evaluate the excretion of fenretinide and metabolites, urine and faeces were collected over a period of 0-24 hours and 24-48 hours. Collection was performed in three mice per treatment group, individually housed in metabolic cages for urine and faeces collection. All biological samples were stored at -20 ° C until analysis. To compare the absorption of Bio-nFeR with NCI-nFeR and to study bioavailability, another independent group of 24 mice were randomized to receive an oral dose of 200 mg / kg of F-NCI. Blood samples were taken at the times reported above. Twenty-four hour fractions of urine and faeces were collected in three mice per treatment group. Plasma and other biological samples were stored at -20 ° C until analysis.

Cranial study: The oral pharmacokinetics of Bio-nFeR in comparison with NCI-FeR was also studied after chronic administration. The mice were divided into three independent randomized groups, to receive a single treatment of 150 mg / kg or several repeat treatments of 150 mg / kg, every day for two weeks (12 days and 2 days off) b 100 mg / kg two. times a day for a week (5 days and 2 days of rest). Bio-nFeR and NCI-FeR were administered orally, taking blood samples at 0.5, 1, 2, 4, 8/10 and 24 hours, on the day of single treatment and at 0, 1, 2, 4, 8/10, 24 and 48 hours after the last dose of both chronic treatments. Procedures for blood collection, plasma preparation, mouse sacrifice, and specimen storage were reported in the acute study. Furthermore, during chronic treatment, excretions of fenretinide and metabolites in urine and faeces were determined by collecting samples in three mice per group, both at the start of treatment and at the end of repeated administration. All collected samples were stored at -20 ° C until analysis.

Pharmacokinetic processing example

The determination of the presence of fenretinide in the plasma of mice and other biological samples was determined according to a method specifically developed and validated for the quantification of phenretinide (4-HPR) based on liquid chromatography-mass spectrometry (LC-MS / MS) . The sample preparation and quantification procedure can be summarized as follows. A 30 µl aliquot of plasma was added with 3 ng of deuterated internal standard (2H4 4-HPR), deproteinized with 90 µL of acetonitrile and centrifuged 5 min at 13200 rpm at room temperature. The supernatant was recovered and 5 μL injected into the HPLC system. Detection was obtained by MS / MS, monitoring transitions 392.3> 283.2 m / z for 4-HPR and 396.3> 283.2 m / z for the deuterated internal standard. To analyze study samples, a six-point calibration curve (range: 1.0-500.00 ng / mL) was prepared in the plasma of the control mouse by evaluating the precision and accuracy of the run by simultaneously analyzing a series of freshly prepared quality control samples. The LOQ of the method was 1 ng / mL in plasma and urine and 50 ng / g in faeces.

Example of study of antitumor activity in vivo

Cell suspensions of CSCs of lung, colon and melanoma cancer were mixed 1: 1 with Matrigel containing low concentrations of growth factors (Growth Factor-Reduced Matrigel) and injected subcutaneously into both hips of 5-week-old immunocompromised mice. age; in the case of lung tumors NSG mice (non-obese diabetic mice with severe combined immunodeficiency and defective for the gamma chain of the interleukin 2 receptor) were used while for colon and melanoma NOD / SCID mice (non-obese diabetic mice with severe combined immunodeficiency) (both purchased from Charles River, Wilmington, MA, USA). For drug treatment, when tumors reached a mean diameter of 0.5 cm, groups of 6 mice were randomized into 3 groups and treated with a placebo, with Bio-nFeR (50,100 or 150 mg / kg as described) freshly dissolved. in sterile water or with the standard NCI-FeR formulation that reproduces the NCI capsule (50 mg / kg). Treatments were administered by gavage for 5 days and 2 days off for 3 weeks. Tumors were measured twice a week using a caliper, and mice were monitored for signs of drug-induced toxicity and weighed regularly. At the end of the treatments, in a study parallel to the evaluation of the antitumor activity, tumors and blood samples were collected for pharmacokinetic and molecular studies three hours after the last administration of the drug. The collection of blood, plasma, separation and storage have been described above. Immediately after blood collection the mice were sacrificed via cervical dislocation, the tumors removed and quickly frozen in liquid nitrogen and stored in small pieces at -80 ° C for subsequent fenretinide analysis, protein and lipid analysis. All procedures were performed with the approval of the Ethics Committee for Animal Testing.

Lipid study example

The tissue samples extracted by the inventors (20 μl) were injected into an ultra high performance liquid chromatography (UHPLC) system (Ultimate 3000, Thermo). The samples were loaded onto a Reprosil C18 column (2.0 mra x 150 mm, 2.5 μm - Dr Maisch, Germany) for lipid separation. The chromatographic separations were obtained at a column temperature of 30 ° C; and flow rate of 0.2 mL / min. The multi-step gradient program was used for lipids. It started with 8% solvent A (ddH20, 20 mmol L-1 ammonium formate, pH 5) at 6% solvent A for 3 minutes versus 2% solvent A for 35 minutes and finally at 100% solvent B (methanol) in 30 minutes. At the end of the gradient, the column was reconditioned with 8% solvent A for 5 minutes.

The UHPLC system was paired online with a Q-Exactive (Thermo) mass spectrometer. The tool was used with full scan (FS) and a subsequent data-dependent acquisition mode (ODA). The full scan settings were as follows: resolution, 70,000; automatic gain control target (AGC), 3e6; maximum injection time (IT), 100ms; and scan range, m / z 150-2000. The remaining settings for DDA mode were as follows: resolution, 17,500; Target AGC, 1e5; maximum IT, 50ms; isolation window, m / z1.0, HCD with step normalized collision energy (NCE), 30. Calibration was performed before each analysis according to the positive or negative ion mode [Piercenet, Thermo Fisher, Rockford, IL) to ensure the subppm error of the intact mass. Lipid identification was performed with LipidSearch (Thermo) software which processes LC-MS data to provide accurate lipid identification.

Example of immunofluorescence of xenografts

Tumor tissue samples were collected in OCT, frozen in dry ice and stored at -80 ° C until next use. 6 micron thick tissue sections were flawed with cryostat and mounted on a coverslip.

Tissue sections were fixed with 2% paraformaldehyde, permeabilized with 0.1% Triton X-100 / PBS, chopped with 1M glycine in PBS and incubated with anti-KI-67 antibodies (Dako, MIB-1 clone) followed by secondary anti-mouse antibodies coupled with Alexa 488 (Invitrogen). the TUNEL assay was performed using the fluorescent in situ cell death detection KIT (Roche): following the manufacturer's instructions, the nuclei were counterstained with DAPI (Invitrogen) and the tissue slices were permanently mounted. The images were obtained using a FV1000 confocal microscope (Olympus, Tokyo, Japan), with a 10x objective (Olympus). The excitation light was obtained from a Dapi 408 nm laser for DAPI, an argon ion laser (488 nm) for Alexa 488. DAPI emission was recorded from 415 to 485 nm, FITC emission was recorded from 495 to 550 nm.

Claims (14)

CLAIMS 1. Oral nanoformulation of a phenretinide complex called Bionanofenretinide (BionFeR) which involves the nanoencapsulation of fenretinide in an anhydrous lipid matrix, its application in the medical field, the pharmaceutical formulations comprising the same and the preparation processes of the same. 2. Formulation according to claim 1, wherein the Bio-nFeR complex is in the form of liposome, micelle, nanoparticle. 3. Formulation according to claim 1, wherein the anhydrous lipid matrix can be composed of: phospholipids, sphingolipids, pegylated phospholipids, sphingolipids with cationic residues or cationic lipids 4. Formulation according to claim 1 wherein the fenretinide is complexed with amphiphilic polymers and / or amphiphilic macromolecules optionally mixed with surfactants and / or cationic lipids and / or phospholipids and / or sphingolipids. 5. Formulation according to claim 1, wherein the anhydrous lipid matrix can be composed of triglycerides selected from: glyceryl tributyrate, glyceryl trimyristate, glyceryl tripalmitate, glyceryl tristearate, glyceryl trioleate. 6. Formulation according to claims 1 to 5 in which Bio-nFeR is used as a drug. 7. Formulation according to claim 6 wherein Bio-nFeR is used in the treatment of cancer prevention. 8. Formulation according to claim 6 in capsule form; tablet; lozenge; granule; dust; solution; Suspension; emulsion; syrup; elixir; stiff gelatin; soft jelly. 9. Formulation according to claims 6 and 7 wherein Bio-nFeR is used in the treatment or prevention of tumors in which said treatment is carried out alternatively or in combination with further chemotherapeutic agents and / or biological anticancer agents. 10. A preparation process of the Bio-nFeR complex as claimed in claims 1 to 5, comprising the following steps: to. Fenretinide is homogeneously mixed with a lipid matrix dispersed in a suitable solvent up to a weight ratio of 1: 9: 1 w: w: w respectively. b. The solvent is removed from the mixture in a rotary evaporator and the dry residue stored at -18 ° C until use. c. The reconstitution of the dry residue in Bio-nFeR nanomicells was obtained by dissolving the residue in water at 30 ° C in an ultrasonic bath where a wave frequency of 40 kHz was applied for 1 hour until the dissolution phase. 11. A process of preparation of the Bio-nFeR complex as claimed in claim 10 wherein the lipid matrix is composed of lipids which are selected from phospholipids, sphingolipids, pegylated phospholipids, sphingolipids with cationic residues or cationic lipids. 12. A preparation process of the Bio-nFeR complex as claimed in claim 10 wherein the lipid matrix is composed of amphiphilic polymers and / or amphiphilic macromolecules optionally mixed with surfactants and / or cationic and / or phospholipids and / or sphingolipids. 13. A process of preparation of the Bio-nFeR complex as claimed in claim 10 wherein the lipid matrix is composed of triglycerides which are selected from glyceryl tributyrate, glyceryl trilaurate, glyceryl trimyristate, glyceryl tripalmitate, glyceryl tristearate, glyceryl trioleate. 14. A process for preparing the Bio-nFeR complex as claimed in claim 10 wherein the solvent is selected from methanol, ethanol, chloroform, methylene chloride, acetone, ethyl ether and mixtures thereof.