ITMI20101064A1 - CARRIER OF CELL TYPE FOR THE SIGHTED TRANSPORT OF AT LEAST ONE MOLECULE AND / OR AT LEAST A MOLECULAR COMPOUND AT LEAST ONE TARGET CELL IN A HUMAN OR NON-HUMAN MAMMALY - Google Patents

Description

Description of a patent application for an industrial invention

DESCRIPTION

The present invention refers to a cellular type carrier, to a complex product comprising in combination at least one molecule and / or a molecular compound and the cellular type carrier for the targeted transport of said at least one molecule and / or said at least one compound molecular to at least one target cell in a human or non-human mammal, and to a process for the preparation of said complex product usable for diagnostic, prognostic and therapeutic purposes.

It is known that classical pharmacological therapy, despite the considerable progress, still has important limitations which concern first and foremost the ability to be selective and therefore not to cause side effects. Pharmacokinetic data also indicate that often the quantities of drug capable of reaching the target in effective concentrations can only be obtained at the expense of systemic or organ-specific toxicity, sometimes even considerable. In some cases the pathological tissue does not receive adequate therapeutic concentrations due to anatomophysiological barriers. This aspect is particularly relevant in the field of cancer chemotherapy, where the chemotherapy drug used is often extremely toxic and therefore produces significant harmful side effects to the cancer patient.

As it is known, to increase the selectivity / specificity of pharmacological therapies, the parenteral administration of vectors capable of transporting molecules has been used for some time, such as systems based on the use of antibodies, nanoparticles of different materials, liposomes or, by genetically manipulating cells by means of viral vectors capable of encoding the production of directly cytotoxic substances or capable of transforming prodrugs into active drugs. Disadvantageously, all these solutions, which use molecules or cells as â € œcarrierâ € are not, however, free from collateral toxicity. In particular, those obtained with viral transfections can present serious safety problems. The technical task of the present invention is therefore to provide a cell-type carrier, a complex product comprising in combination at least one molecule and / or a molecular compound and the cell-type carrier for the targeted transport of said at least one molecule and / or said at least one molecular compound to at least one target cell in a human or non-human mammal, and a process for the preparation of said complex product which allows to eliminate the drawbacks complained of by the known art.

In the context of this technical task, an aim of the invention is to produce a complex product of the type referred to above in which the carrier is able to gradually release a high concentration of a molecule or molecular compound.

Another object of the invention is to provide a complex product of the type referred to above in which the carrier is able to gradually release a concentration of a cytotoxic molecule and / or molecular compound mainly in the pathological target cell in a localized manner. and / or pharmacologically active.

Yet another purpose of the invention is to provide a complex product of the type referred to above in which the carrier allows the delivery of a pharmacologically active molecule and / or molecular compound in such a way as to increase its therapeutic specificity and at the same time reduce or even eliminate it. any side effects.

Another purpose of the invention is to produce a complex product of the type referred to above for use with diagnostic, prognostic and therapeutic purposes.

The technical task, as well as these and other purposes, according to the present invention are achieved by providing a cell-type carrier for the targeted transport of at least one molecule and / or at least one molecular compound to at least one target cell in a human or non-human mammal , characterized in that it comprises at least one autologous or heterologous stromal cell isolated from a human or non-human mammal.

In the carrier, the stromal cell preferably consists of a mesenchymal stem cell.

The present invention also discloses a complex product comprising in combination at least one molecule and / or a molecular compound and a cell-type carrier for the targeted transport of said at least one molecule and / or said at least one molecular compound to at least one target cell in a human or non-human mammal, characterized in that said carrier comprises at least one autologous or heterologous stromal cell isolated from a human or non-human mammal.

In the complex product, the stromal cell consists of a mesenchymal stem cell.

Preferably the molecule and / or the molecular compound are cytotoxic.

Preferably the molecule and / or the molecular compound are pharmacologically active.

Preferably the molecule and / or the molecular compound are anticancer drugs.

Preferably the molecule and / or the molecular compound are anti-angiogenic drugs.

Preferably the drugs include Paclitaxel (PTX).

The present invention also discloses a process for the preparation of a complex product characterized in that it comprises a step of isolating stromal cells from a human or non-human mammal for the production of a cell-type carrier for the targeted transport of at least one molecule. and / or at least one molecular compound to at least one target cell in a human or non-human mammal, an in vitro expansion step of said stromal cells, and a step of exposing said stromal cells to high concentrations of said at least one molecule and / or of said at least one molecular compound.

The process preferably comprises a cryopreservation step of said stromal cells before or after said exposure step.

In the process, stromal cells are mesenchymal stem cells.

The present invention also discloses a method of targeted and controlled release of a therapeutically effective dose of at least one cytotoxic molecule and / or at least one cytotoxic compound, characterized by the fact of delivering to pathological target cells a population of mesenchymal stem cells internalizing a high concentration of said at least one cytotoxic molecule and / or said at least one cytotoxic compound for their gradual and targeted release.

The present invention also discloses a mouse model treated with the complex product.

The present invention also discloses a use of the complex product for diagnosis, prognosis, therapy and therapeutic monitoring.

Further characteristics and advantages of the invention will become more evident from the following description, illustrated by way of non-limiting example with reference to the attached figures 1 to 17:

Figure 1 shows the phenotypic characterization of mesenchymal stem cells (MSCs): the photos show a freshly observed MSC culture under the microscope and their multipotency expressed by the ability to differentiate into osteoblasts, chondroblasts and adipocytes. The figure confirms the mesenchymal character evaluated on the basis of the phenotypic expression of CD analyzed by FACS.

Figure 2 shows the resistance of MSCs to the cytotoxic activity of Paclitaxel: the histogram shows the cell viability determined in MTT test after 24 hours of treatment with PTX. Each point represents the mean / - ds of 5 experiments. The% is calculated on the D.O. of untreated cells (considered as 100% viability).

Figure 3 shows the time-dependent release kinetics of Paclitaxel by MSCs-PTX: the presence of PTX in the conditioned medium was evaluated by assaying its antitumor activity on Molt-4 leukemia cells. The histogram shows the release at different hours of culture (with total change of the cultural medium at each time studied). The curve indicates the accumulation of PTX in the conditioned medium. Each value reports the mean / - ds of 5 experiments.

Figure 4 shows the incorporation of PTX by MSCs: the incorporation is evaluated using PTX conjugated with Fluorescein. The cells are observed both under the microscope (A) and analyzed by FACS (B).

Figure 5 shows the analysis by HPLC: the figure shows the chromatograms of pure Paclitaxel (high) and of conditioned medium obtained from cells treated with PTX, trypsinized and recultivated 24 hours (low). The chromatogram confirms the presence of PTX in the conditioned medium.

Figure 6 shows the antiproliferative activity of the conditioned medium by MSCs-PTX on Molt-4, DU145 and T98G tumor cells: the activity of the conditioned medium (B) was evaluated at serial dilutions 1: 2 up to 1: 128 by 7-day MTT test. The activity of pure PTX was evaluated at serial dilutions 1: 2 starting from the concentration of 50 ng / ml. The figure demonstrates that the conditioned medium is active on neoplastic growth with a kinetics similar to that of pure taxol. The PTX Equivalent Concentration (CEP) was calculated as described in 3.5. The grid shows the IC5O values of the PTX for the three tumors.

Figure 7 shows the effect of growth in adhesion / suspension and freezing on the release of PTX by MSCs-PTX: the figure shows the quantities of PTX released by each single cell in the different experimental conditions:

CTRL = Standard culture with treatment and release evaluated on attached cells Co / Sc = Treated cells attached subsequently frozen. Cultivate adese after thawing. A / S = Adhesive treated cells, PTX release evaluated in cell suspension cultures.

Figures 8-9-10 show the direct anti-tumor activity of MSCs-PTX on the proliferation of human tumors in vitro: the histogram shows the mean / - ds of tumor proliferation determined in vitro in the presence of normal MSCs and MSCs-PTX in different ratios. Proliferation is expressed as% of the proliferation of tumor cells that did not receive MSCs in a 7-day MTT test. Figure 8 reports the results on MOLT-4 leukemia cells. Figure 9 on T98G glioblastoma cells. Figure 10 on DU145 prostate cancer cells.

Figure 11 shows how PTX inhibits the proliferation of human endothelial cells (ECs): HUVECs and HMECs (2 x 10 <5>) were cultured for 72 hrs in the presence of different PTX concentrations. The numbers in the figure are means ± SD of three experiments in triplicate. Note that PTX at a concentration of 10 ng / ml completely blocks the growth of both HUVECs and HMECs, at higher doses it is toxic. ** p <0.01 PTX treated vs controls.

Figure 12 shows the growth inhibition of ECs co-cultured with hMSCsPTX: HUVECs (panel A) and HMECs (panel B) were mixed with hMSCsPTX or control hMSCs and cultured for 72 hours. As the figure shows, adding MSCsPTX to ECs at ratios 1: 1 and 1: 5 is cytotoxic for both HUVECs and HMECs. At 1:10 ratios hMSCsPTX continue to inhibit the proliferation of ECs. Mixed co-cultures of HUVECs or HMECs with untreated control hMSCs do not inhibit the proliferation of ECs. Panel C shows images of (20x magnification) HMECs mixed 5: 1 with hMSCsPTX fixed and stained at different incubation times after seed. HMSCsMTX kill HMECs already at an early time (24 hours). The white arrows in the images show the endothelial islands that are still present in the mixed culture. Note that at 72 hours virtually all seeded HMECs were killed by the hMSCsPTXs, which are the only ones left in culture. The numbers in (A) and (B) are the means ± SD of three separate experiments * p <0.05, ** p <0.01 vs untreated control hMSCs.

Figure 13 shows that the conditioned medium (CM) of hMSCsPTX (hMSCsPTX-CM) is as effective as the free PTX at inhibiting the growth of both HUVECs and HMECs: HUVECs (panel A and C) and HMECs (panel B and D) were cultured for 72 hours in the presence of different dilutions of CM deriving from both cultures of hMSCsPTX (hMSCsPTX-CM) and untreated control MSCs (hMSCs-CM). The addition of hMSCsPTX-CM induces a strong inhibition of the growth of ECs at all dilutions tested. In particular hMSCsPTX-CM at 1: 2 dilution is even cytotoxic for ECs, at 1: 4, it is as effective as PTX at 5ng / ml. Panels C and D show photographic images of cultures of HUVECs and HMECs exposed to different dilutions of hMSCsPTX-CM. Note that the photographic image of the cultures in the 1: 2 presence of hMSCsPTX-CM shows how only a few ECs survive the treatment, confirming the numerical data of the graph. The arrows indicate the few surviving ECs that remain anchored to the plastic. (10x photo magnification). The values in graphs A and B are means ± SD from three different experiments. * p <0.05, ** p <0.01 vs CTRL control medium or hMSCs-CM at the same dilution.

Figure 14 shows that HMSCsPTX-CM inhibits the growth of microvessels from the aorta rings and facilitates their regression: the rat aorta ring test was used to test the effectiveness of hMSCsPTX-CM in inhibiting the growth of vessels. in vitro. Aorta rings were placed in a collagen gel and then exposed to different dilutions of hMSCsPTX-CM or control hMSCs-CM. VEGFa at 20ng / ml has been added in some rings cultures as an angiogenic stimulus and as a positive control to evaluate the efficiency of the prepared aorta rings. Twelve days later the newly formed capillaries from the rings are counted under the microscope. As shown in panel A in the figure, adding HMSCsPTX-CM at 1.2 and 1: 4 produces a strong reduction in the number of out-of-ring vessels when compared to control rings or those treated with hMSCs-CM. Panel B shows the photos of the effect of hMSCsPTX-CM and hMSCs-CM added to the aorta rings after 7 days of culture, ie when the capillaries are already present. The photos (20x magnification) were taken on day 10. Note that adding hMSCsPTX-CM at 1: 2 or 1: 4 induces regression of already formed capillaries while control medium CTRL or adding hMSCs-CM is not effective. The arrows indicate the capillaries in an advanced phase of regression with evident areas of necrosis. The numerical values in (A) are the means ± SD of two separate experiments. ** p <0.01 vs hMSCs-CM or CTRL medium alone.

Figure 15 shows that the in vivo co-inoculation of hMSCsPTX with DU145 human prostate cancer cells retards their growth in Nod-Scid mice: to evaluate the efficacy of hMSCsPTX in vivo on tumor growth, approximately 2x10 <6> DU145 are state inoculate s.c. (in the right flank) in 8-week-old male Nod / Scid mice, alone or mixed in a 5: 1 ratio (tumor cells / hMSCs) with hMSCsPTX (0.4x10 <6>) or with control hMSCs The appearance of tumors à It was assessed by palpation, while the tumor volumes were calculated by measuring the diameters of the tumors taken with a caliper at various times after inoculation. The Figure shows that co-injection of DU145 with hMSCsPTX significantly reduces tumor volume as well as delays its initial onset. Co-injection of DU145 and control hMSCs has no effect when compared with the growth of inoculated DU145 alone. Even mice that received DU145 mixed with free PTX (100 times more concentrated) did not differ from the control. The mice were sacrificed 40 days after inoculation and the tumors removed and weighed. In the inset the image of a control tumor DU145, treated with hMSCsPTX and hMSCs after removal from the mouse flank. * p <0.05 and ** p <0.01 vs DU145 alone or treated with hMSCs Figure 16 shows that the co-inoculation of hMSCsPTX with B16 murine melanoma cells reduces its growth in Nude mice: the ability of hMSCsPTX to inhibit growth of an in vivo tumor was also evaluated using the experimental murine tumor B16 melanoma. 8-week-old male nude mice were inoculated s.c. with 2x10 <6> B16 cells alone or mixed in a 5: 1 ratio (tumor cells / MSCs) with hMSCsPTX (0.4x10 <6>) or with control hMSCs. The volumes of the tumors were calculated by measuring the diameter taken with a caliper at different times from the inoculation. As the Figure shows, adding hMSCsPTX to B16 cells significantly reduces B16 growth when compared with both B16 alone and with B16 mixed with control hMSCs. On day 17, the animals were sacrificed and the tumors removed and weighed. In the frame of the figure the photographic image of a control B16 tumor, treated with hMSCsPTX or with hMSCs. * p <0.05 vs control B16 alone ** p <0.01 vs control B16 alone or treated with hMSCs.

Figure 17 shows that the co-inoculation of murine mesenchymal MMSCsPTX with B16 melanoma inhibits its growth in syngenic C57Bl6 mice: the ability of murine MSCs cells to inhibit the growth of B16 in syngenic mice was tested. The figure shows the effect of 0.4 x 10 <5> MMSCs isolated from BM of PTX-treated or untreated C57Bl6 mice and mixed with 2x10 <5> B16 s.c. in a 5: 1 ratio (tumor B16 / MSCs) and then inoculated into C57BL6 male mice aged 8 weeks. Tumor volumes were calculated by measuring the diameters with a caliper at various times from inoculation. The Figure shows that treatment with MMSCsPTX inhibits the growth of B16 melanoma even in the syngenic mouse strain. In this case, however, control MMSCs inoculated with B16 also reduce the growth of B16 when compared to mice transplanted with control B16 alone. The inset in the Figure shows a photo of a control B16 tumor treated with MMSCsPTX or MMSCs. * p <0.05 and ** p <0.01 vs control B16.

Figure 18 shows Table 1 which shows the antitumor activity of hMSCsPTX and MMSCsPTX.

At the basis of this invention there is the experimental evidence that in the mammalian organism there are cells contained in the connective / stroma of various tissues and organs that are able to incorporate molecules and drugs and subsequently release them both in the original form and in the form of metabolites.

In previous studies it has been shown that in the adult organism there is a cell population, (preferentially contained in the mesenchymal and connective tissues of all organs, including blood), within a larger population called by the generic term of â € œstromal cellsâ € or, with the more specific term â € œ mesenchymal stem cellsâ € (MSCs), which is particularly resistant to high concentrations of cytotoxic drugs. In fact, these resistant MSCs (MSCs-R) may not suffer biological damage even in the presence of drug concentrations even 100-1000 times higher than those which are capable of killing many other cells in our organism. This property of theirs is initially expressed through the ability to internalize toxic drugs, thus providing for an acute detoxification phenomenon (removing / sequestering drugs from the microenvironment of the organ or tissue and thus preventing them from acting on more sensitive cells) . Subsequently, however, these MSCs-Rs can release into the microenvironment, thanks to an active secretion mechanism (probably regulated by particular proteins called â € œP glycoproteinsâ €), more or less rapidly the internalized toxic drug, both in its original form and under form of metabolites. Obviously, the secretion of the drug in its original form can still cause toxicity phenomena ("acute delayed toxicity" or chronic if the cells slowly release the drug "slow release") on adjacent and drug-sensitive cells. In any case, the cells most sensitive to the drug avoid direct damage that could be fatal, but would still suffer damage, which is precisely dependent on the quantities and temporal modalities with which the MSCs-R release the original active drug. In the event that the MSCs-R no longer release the original drug but its â € œmetabolitesâ €, both the condition of complete neutralization of toxicity (ineffective metabolites) as well as increased toxicity (more toxic metabolites) of the original drug. These observations were published and discussed by Pessina et al. (1999).

The principle on which this invention is based is therefore the experimental evidence that in our organism there are MSCs-R capable of internalizing exogenous molecules and subsequently releasing them. The invention consists in the practical application of this mechanism and in the use of these cells for therapeutic, diagnostic and prognostic purposes, precisely defining a new way of using drugs in man. In particular, for example, the application of our invention can lead to an increase in the effectiveness of chemotherapy drugs in the field of oncological pathologies, thanks to the ability of the MSCs-R to transport and concentrate the chemotherapy almost exclusively in the neoplastic tissue , or where the application of chemotherapy is recommended. For this purpose, having verified the veracity of the biological principle and deepened the phenotype of the MSCs-R, a procedure has been standardized that involves the production of an MSCs-Drug (MSCs-F) product and its therapeutic use which is defined by the type. drug used.

The procedure consists of several phases: 1) isolation of MSCs from tissues, in particular from the bone marrow and adipose tissue which appear to be the richest; 2) their in vitro expansion according to standard culture methods; 3) selection of the MSCs-R by exposure to high concentrations of the drug that is to be transported / internalized by the MSCs-R; 4) evaluation of the internalization of the drug in cells with formation of the MSCs-F product and evaluation, through biological tests (in vitro and in vivo), of the therapeutic efficacy of the MSCs-F product; 5) cryopreservation of the MSCs-F product for subsequent therapeutic use.

Thanks to our procedure, the MSCs of our organism (but also of other mammalian species) therefore become â € œcarrier â € œ of drugs (or molecules) which subsequently, thanks to their innate expulsion capacity, can be released into the tissue where you want the drug to work.

To do this, the MSCs-F can be either inoculated directly into the diseased tissue of interest, or administered systemically (by blood), also exploiting their ability (homing) to concentrate mainly in the diseased pathological tissue. This new form of drug therapy should therefore contribute to increasing the therapeutic specificity of the drug and at the same time to reduce, or even eliminate, any unwanted side effects.

The procedure has been standardized for the pharmacological application of a chemotherapy called Paclitaxel (PTX) already used in anti-tumor therapy in humans. The protocol described is however also applicable to other chemotherapeutic drugs, and potentially expandable to molecules with activities other than chemotherapeutic drugs. The procedure described in detail below leads to the packaging of a pharmaceutical product composed of MSCs and PTX (MSCs-PTX) which can be used immediately at the end of its preparation, or can be stored (by freezing in liquid nitrogen -196 ° C) for a possible subsequent use. In fact, the MSCs-PTX product thus obtained from this procedure does not lose its effectiveness after defrosting after some time (months / years) from its initial preparation.

The preparation of the MSCs-PTX product was carried out with MSCs isolated from bone marrow and from human and murine adipose tissue, but it can also be reproduced using MSCs / stromal cells isolated from other organs and tissues, provided they have similar morphological and phenotypic characteristics. to those used in this study.

However, since both the bone marrow, and in particular the adipose tissue, are easily accessible tissues for sampling in humans, it is foreseeable that this procedure can be performed mainly with MSCs isolated from these two tissue sources. The easy accessibility of these tissues will also allow the successful preparation of MSCs from virtually any donor. This technical aspect is important to successfully prepare an autologous type MSCs-F product. The procedure is therefore applied to both samples of MSCs of heterologous origin (patient-donor other than the recipient) and autologous (MSCs of the same patient-donor). This makes it possible to develop an MSCs-F product to implement personalized drug therapy.

It is possible to foresee the possible cryopreservation of the MSCs of donors in bio-banks without prior manipulation. This does not prevent a donor's MSCs from being used, in case of need, in the production of the MSCs-F product. subsequently after their defrosting.

The procedure is generally safe because it does not involve any â € œgenetic manipulation â € œ of the MSCs that are used. The procedure exploits an innate physiological behavior of MSCs and therefore does not pose safety problems related to the introduction of new genes (such as gene therapy) or of nanoparticles which are synthetic elements foreign to the cell. However, some security issues can arise with MSCs-Fs. First, MSCs per se have their own spontaneous secretory activity and then are able to differentiate (being immature cells) once inoculated into various cell phenotypes. However, their therapeutic use is already in clinical practice and currently no particular â € œtoxic â € œ activities are documented in their use. The safety issue of the MSCs-F product is potentially more related to the type of drug you want to carry. For example, if you want to use MSCs for the transport of drugs that act directly or indirectly on the DNA of a cell, these drugs, when incorporated, can also induce modifications on the DNA of the transporter MSCs themselves, potentially inducing changes in the genome themselves.

Materials and methods

Isolation, expansion and phenotypic characterization of MSCs.

The in vitro isolation and expansion procedure of MSCs applied to human and murine bone marrow and human adipose tissue is that described by Pittenger et al, (1999). The stabilized SR-4987 line generated and maintained in our laboratory by serial passages was also used as a model of murine MSCs (Pessina et al, 1992). Both murine and human MSCs exhibit similar morphology in vitro. MSCs are cells that grow in adhesion on the culture plastic and appear with a morphology similar to fibroblasts. When grown in their NHEM growth medium (Miltenyi, Germany) in general MSCs double in number in about 72-96 hours and their cell cycle appears quite slow with a high percentage of cells in the G0 / G1 phase (70-90% ), a modest fraction in the S phase (8-25%) and only a small fraction in the G2 / M phase (2-4%). As shown in figure 1, the phenotypic characterization reveals that the MSCs used show the classic markers present on mesenchymal cells such as Cd13, Cd90, Cd105, Cd29, Cd44, Cd73 and Cd166, and endothelial and hematopoietic mouse markers Cd31, Cd34, Cd45 are absent. , Cd133. There are scarcely any markers such as Cd54, Cd146. Cd117 and Stro-1. Finally, the isocompatibility antigen of class I (HLA-I) is present while the one of class II (HLA-II) is absent.

Preparation of the drug.

In this study, as previously illustrated, a chemotherapy drug called Paclitaxel (PTX), already known for its anti-tumor activity in humans, was used to make the final product MSCs-PTX. The stock solution of PTX (Serva, Germany) is prepared by dissolving 5 mg / ml in DMSO and stored in aliquots of 10ul at -20 ° C. The working solution is freshly prepared by diluting 1:25 in DMEM medium (Lonza, Usa) so as to obtain a PTX solution of 200 micrograms / ml.

MSCs-PTX product training procedure and verification of PTX internalization in MSCs

Confluent cultures of mesenchymal MSCs prepared and characterized as described above, are treated with trypsin-edta (0.05% / 0.02%), washed in PBS and counted in a Burker chamber.

10 <5> cells are seeded in plastic bottles (Costar) of 25 cmq in 5 ml of NHEM medium and incubated at 37 ° C in an atmosphere of 5% CO2 air After 96-120 hours. Upon reaching confluence (3-4 x 10 <5> cells), 50 microlitres of a freshly prepared 200 ug / ml PTX solution (as described in 3.2) are added to the culture medium to obtain a final concentration of 2ug / ml of PTX. To generate the MSCs-R-PTXs, the MSCs are kept at 37 ° C in 5% CO2 air for at least 24 hours. At the end of the incubation, the supernatant of the cell culture is aspirated and discarded, the adherent cells washed twice with 5 ml of HBSS, and then detached by adding 1 ml of a solution of trypsinâ € "edta (0.05% 0.02% weight / vol) for 5â € ™. When about 80% of the cells in the flask are detached from the plastic, 2 ml of DMEM medium complete with 2 mM L-glutamine containing 20% of FCS are added.

After vigorous shaking (to favor the detachment of all cells), the detached cells are aspirated and transferred into a 15 ml conical tube. The flask is further washed with 10ml of HBSS and the wash added to the conical tube containing the cells initially recovered. Centrifugation is then performed at 400xG for 10â € ™ at room temperature to recover the cells. At the end of the centrifugation, the supernatant is discarded and the MSCs-PTX resuspended in 2.0 ml of HBSS. A second centrifugation is performed at 400xG, 10â € ™ to completely eliminate any non-incorporated PTX from the MSCs. Finally the MSCs-PTX are resuspended 1 ml of NHEM and counted in the presence of vital dye (trypan blue) to evaluate their viability. At this point the MSCs-PTX are ready to be used directly in a possible therapeutic application (for example as an anticancer) or frozen and stored until use. The possible verification of the PTX incorporation in the MSCs can be confirmed, in the specific case of the PTX, through the parallel use of a PTX labeled with fluorescent substance (FITC) and subsequently analyzed by flow cytometry (FACS). One or more aliquots of MSCs-PTX produced are used to establish the temporal release of PTX from the MSCs and to establish its therapeutic efficacy after release.

Evaluation of the in vitro anti-tumor activity of conditioned mediums (CM) deriving from MSCs-PTX cultures.

The release of PTX by the MSCs-PTX produced can be analyzed by evaluating the anti-proliferative and cytotoxic activity of the MSCs-PTX. This evaluation was performed by initially testing their conditioned medium (CM). To prepare it, about 3-5 x10 <4> MSCsPTX / ml (viability> 90%) are sown in 5 ml of NHEM in a new 25 cmq flask.

After 24 hours of cell culture (37 ° C in air + 5% CO2) as described above, their CM is collected and replaced with fresh medium. To study the release kinetics, this operation is repeated at different times (48, 72, 96 and 144 hours) from sowing. The collected CMs are centrifuged at 2,500xG for 15 minutes, to eliminate any dead cells. The CMs are then divided into aliquots and can be tested immediately or frozen at -70 ° C until the moment of use.

The release in the CM of PTX of part of the MSCs-PTX was verified by evaluating the anti-proliferative or cytotoxic activity on a Molt-4 reference tumor model (acute human lymphoblastic leukemia; Minowada et al 1972) which is highly sensitive to the PTX. The proliferative test on Molt-4 is performed by MTT test as previously described (Mossman 1983; Pessina et al., 1994). The antitumor activity in CM of MSCs-PTX was evaluated by making various dilutions in order to establish an IC50 and IC90 dose. The same type of procedure has also been applied on other human tumor lines such as glioblastoma T98G (Stain et al, 1979) and prostate cancer DU-145 (Mickey et al., 1977) and also on a murine melanoma B16 line ( Riley, 1963). Molt 4, DU145 and B16 were cultured and expanded in vitro according to previously published culture modalities (Pessina et al 1994, Mickey et al 1977, Riley et al 1963). CM from MSCs grown under the same conditions but not treated with PTX was used as the conditioned reference medium (negative control). The value of IC50 and IC90 for each line is determined according to the formula of Reed and Muench (1938). Evaluation of the anti-tumor activity of MSCs-PTX co-cultured with tumor cells.

The evaluation of the anti-tumor activity of MSCs-PTX in vitro has also been studied through mixed cultures with tumor cells. MSCs-PTX were co-cultured with Molt-4 reference tumors. The MSCs-PTX obtained as described in 3.3 were mixed at different ratios with the Molt-4s. (MSCs-PTX / Molt-4 1: 1, 1: 5, 1:10 etc ..) to define the IC50 and IC90 of the Molt-4. The proliferative test was performed as described in 4.1 according to the MTT test. The anti-tumor activity of MSCs-PTX was also evaluated by making mixed cultures with other tumor lines described in the previous paragraph. Also in this case the control cells were the MSCs not treated with PTX and mixed with the tumors in the same ratios used for the MSCs-PTX.

Evaluation of the in vitro anti-angiogenic activity of MSCs-PTX

PTX is also considered a chemotherapy with anti-angiogenic capacity, therefore able to inhibit the growth of capillaries. Endothelial cell cultures (ECs) were used to evaluate the in vitro anti-angiogenic action of MSCs-PTX. Two different types of ECs have been used - HUVECs which are ECs derived from human umbilical cord veins and ECs derived from human dermal microvessels (HMECs). HUVECs and HMECs were isolated and cultured as described by Alessandri et al (2005). The anti-proliferative effect of the CM of the MSCs-PTX on the ECs was performed, with the same modalities already described for the tumor lines described above. Similarly, also the co-culture between MSCs-PTX and ECs took place with the same modalities described in the previous paragraph. The anti-angiogenic activity of MSCs-PTX was also evaluated with the rat aorta ring test. With this test described by Nicosia et al (1990) it is possible to study the growth of microvessels in vitro. The CM derived from the MSCs-PTX was tested, at various dilutions. The count of microvessels originating from the rings was performed according to the methods described by Nicosia et al (1990). The controls of this whole trial were either the CMs deriving from the MSCs not treated with PTX, or the normal MSCs in the co-culture tests with ECs.

Biological assay of PTX release in vitro

The quantity of PTX released by the MSCs-PTX was calculated by biological assay of the antitumor activity of their CM. The dosage is calculated on the activity of a known quantity of pure PTX. The concentration is therefore expressed as equivalent concentration of paclitaxel (CEP) according to the following algorithm: CEP (ng / ml) = FD50mc x PTXc / DF50PTX

where FD50mc and FD50ptx are the dilution factors at which CM and pure PTX respectively cause 50% inhibition of the proliferation of Molt-4 and PTXc the starting concentration of pure PTX. The release of PTX referred to each single cell (Rptx) expressed in pg / cell is calculated with the formula Rptx = CEP (ng / ml) x Volume of CM (ml) / number of cells seeded. The CM assay of the released PTX of the MSCs-PTX was also evaluated with HPLC according to the method suggested by Gianni et al., 1995.

Evaluation of the anti-tumor activity of MSCs-PTX in vivo.

The ability to inhibit tunoral growth in vivo was performed with both human (hMSCs-PTX) and murine (MMSCs-PTX) MSCs-PTX. Control cells were the MSCs of the two species not treated with PTX. The preparation of the hMSCs-PTX and MMSCs-PTX took place according to the previously described procedure. The tumor cells used for the in vivo study were the human DU145 line and the murine B16 line. The two tumor lines were cultured in vitro according to standard culture conditions described in the paragraph relating to the evaluation of the in vitro anti-tumor activity of conditioned mediums deriving from MSCs-PTX cultures. 60-day-old male NOD / SCID mice were used to evaluate the antitumor activity of hMSCs-PTX on DU145 (human prostate cancer). The experimental groups were composed as follows: 1) control mice that each received only DU145 at a dose of 2x10 <6> /0.2ml of physiological mouse inoculated subcutaneously (s.c.) in the right flank; 2) mice treated each SC with 2x10 <6> DU145 and 0.4x 10 <6> hMSCs-PTX (ratio 1: 5) in 0.2 ml. mix 5â € ™ before inoculation; 3) mice treated each SC with 2 x10 <6> DU145 and 0.4 x10 <6> hMSCs (ratio 1: 5) control (i.e. not treated with PTX) in 0.2 ml mixed 5â € ™ before inoculation: 4) mice treated each s.c. with 2 x 10 <6> DU145 resuspended in 0.2 ml of saline containing 100ug / ml of pure PTX 5â € ™ before inoculation. To evaluate the anti-tumor activity of hMSCs-PTX on B16 murine melanoma, 60-day-old male nude mice were used. The experimental groups were composed as follows: 1) control mice inoculated each s.c. with 2x10 <6> B16 in 0.2 ml; 2) mice inoculated each SC with 2x10 <6> B16 and 0.4 x10 <6> hMSCs-PTX (ratio 1: 5) in 0.2 ml mixed 5â € ™ before inoculation; 3) mice treated s.c. each with 2x10 <6> B16 and 0.4x10 <6> hMSCs (ratio 1: 5) control mixed 5â € ™ before inoculation; 4) mice treated s.c. each with 2x10 <6> B16 resuspended 5â € ™ before inoculation in 0.2 ml of saline containing 100ug / ml of pure PTX. To evaluate the anti-tumor activity of MMSCs-PTX on B16 murine melanoma, 45-day-old male C57 / Bl6 syngeneic mice were used for both cell preparations. The experimental groups were composed as follows: 1) control mice inoculated each s.c. with 2x10 <5> B16 melanoma cells in 0.2 ml; 2) mice treated each SC with 2x10 <5> B16 and 0.4 x10 <5> MMSCs-PTX (ratio 1: 5) in 0.2 ml mixed 5â € ™ before inoculation; 3) mice treated each SC with 2x10 <5> B16 and 0.4 x10 <5> MMSCs control (ratio 1: 5) in 0.2ml mix 5â € ™ before inoculation; 4) mice treated each SC with 2 x10 <5> B16 resuspended 5â € ™ before inoculation in 0.2 ml of saline containing 100ug / ml pure PTX. The appearance of the tumor was monitored by palpation of the flank of the inoculated mouse SC. with the cells, while the growth rate of the tumors was obtained by measuring, with a caliper, the diameter of the tumors at various times after transplantation. At the sacrifice of the mice the tumors were removed from the subcutis and weighed.

Synthesis of parallel procedures for the verification of some experimental conditions

1. The passive release of PTX (not internalized by the cells, therefore bound specifically to the cells) adsorbed on the cell membrane was evaluated by treating the pre-fixed confluent MSCs with 10% formalin for 30 minutes and washed three times with PTX for 24 hours. times with double distilled water before adding the drug. The rest of the procedure is as described above.

2. The ability to internalize PTX was also evaluated with MSCs treated with the drug under conditions of forced growth in suspension. For this purpose 3x 10 <5> StCs / resuspended in a conical tube with 5 ml in their culture medium containing 2ug / ml of PTX. The cells in the tube were incubated for 24 hours with continuous shaking to prevent their deposition. At the end of the incubation the MSCs-PTX were recovered by centrifugation and washed twice, again by centrifugation, to eliminate any non-incorporated PTX. The evaluation of the subsequent release of the internalized PTX from the MSCs-PTX was done by evaluating the cytotoxic activity of their CM after the MSCs-PTX were cultured for 24 hours adhesion in a new culture flask. In another series of experiments we also evaluated the ability of MSCs-PTX, treated in adhesion, to release PTX if I was forcibly suspended. Briefly, the procedure for incorporating PTX into MSCs was done as previously described. After detachment with trypsin the MSCs-PTX were incubated for 24 hours in suspension in their medium. At the end of the incubation the CM was recovered after centrifugation and tested for its cytotoxic activity as already described.

Release after freezing is verified with cells treated as previously described and, after trypsinization, frozen in liquid nitrogen (-196 ° C). The cells are thawed after one month and sown in the bottle. Cytotoxic activity was evaluated as previously described.

Experimental results

As Figure 2 shows, high dosages of PTX (up to 10,000 ng / ml) do not affect the viability of MSCs demonstrating their particular resistance. The PTX resistance analysis of MSCs was also confirmed by analyzing their cell cycle which is not substantially modified by PTX treatment (data not shown).

As shown in Figure 3, MSCs-PTX (treated for 24 hours with PTX), trypsinized, washed and re-cultured in vitro release the drug with time dependent kinetics at pharmacologically effective concentrations on neoplastic proliferation.

As shown in Figure 4, the incorporation of PTX into MSCs was verified by treating MSCs with Fluorescein-labeled drug (Invitrogen, USA). Subsequently, according to the procedure described above, the cells were trypsinized, washed, suspended in PBS (Phosphate Buffered Saline) and then analyzed with the Fluorescence Activated Cell Sorter (FACS) and observed under the microscope (Ex: 496nm; Em: 524 nm).

This showed that most of the cells contain PTX and that subsequently it is released as shown by the fall curve over time (2, 4, 8, 24, 48 hours) of the fluorescent emission evaluated by FACS and reported in Figure 4 expressed as ratio calculated on the unmarked PTX.

The presence of PTX in the conditioned medium of the treated cells was confirmed by HPLC analysis as shown in figure 5. As shown in figure 6, the antineoplastic activity â € œin vitroâ € of the conditioned medium of the cells collected 24 hours after treatment evaluated in three different human tumor models, namely Molt-4 lymphatic leukemia, T98G glioblastoma and DU-145 prostate cancer, is similar to that of pure PTX. On the basis of this activity, the equivalent concentration in Paclitaxel (CEP) is calculated as described in the paragraph on the biological assay of PTX release in vivo.

As shown in Figure 7, MSCs-PTXs held in suspension release PTXs. The same cells after treatment can be frozen in liquid nitrogen and subsequently thawed and cultured without losing the ability to release the drug.

As shown in Figures 8,9 and 10, trypsinized and washed MSCs-PTX are effective on the in vitro proliferation of the three human tumor models. These (pharmacologically loaded) cells mixed with tumor cells (in different proportions of 1: 1 - 1:10 - 1: 100) show significantly greater antiproliferative activity than non-PTX-treated control MSCs. The same analysis of the efficacy of MSCs-PTX to inhibit tumor growth in vitro was also performed to evaluate the anti-angiogenic capacity. The efficacy of pure PTX in inhibiting the growth of ECs was initially determined. As Figure 11 shows, the addition of PTX at concentrations above 10ng / ml in both HUVECs and microvascular HMECs cultures is cytotoxic. The dose that inhibits the growth of ECs at 50% is around 4.69ng / ml of PTX.

The ability of MSCs-PTX to inhibit the growth of ECs was then evaluated. In a first series of experiments, MSCs-PTX of human origin were tested and mixed with ECs at various ratios. Co-cultivation of hMSCs-PTX with ECs at 1: 1 ratios. 1: 5 and 1:10 produces in a dose dependent manner a strong growth inhibition of both HUVECs and HMECs, as shown in figure 12. The ratio 1: 5 hMSCs-PTX / ECs at 72 hours of co-culture is sufficient to determine a complete elimination of the ECs from the culture as shown in the image in Figure 12 (panel C). The co-cultivation of control hMSCs, not treated with PTX, at the same ratios, did not result in any particular growth inhibitory effect on both HUVECs and HMECs, as shown in panel A and B of Figure 12.

The strongly inhibitory action of hMSCs-PTX on the proliferation of ECs was also confirmed by the analysis of their CM. Up to 1: 8 dilutions the hMSC-PTX-CM is able to significantly inhibit the growth of both HUVECs and HMECs as shown in figure 13. At 1: 4 dilutions, or higher, the activity of hMSCs -PTX-CM produces a cytotoxic effect on ECs even higher than the effect obtained with 5ng / ml of pure PTX, as shown in panel A and B of Figure 13. The cytotoxic action of MSCs-PTX-CM at dilutions 1 : 2 and 1: 4 is so high as to leave only a few ECs alive after a 72 h treatment as shown in the images of panel C and D of Figure 13. Also in this case the addition of control hMSCs-CM on ECs at the same dilutions it shows no efficacy.

Finally, the ability of hMSCs-PTX-CM to inhibit the growth of ex-vivo microvessels was analyzed with the aorta ring test. By applying various dilutions of hMSCs-PTX-CM to aorta ring cultures, microvessel growth inhibitions were observed in a dose dependent manner. Dilutions 1: 2 and 1: 4 of hMSCs-PTX-CM inhibit the escape of new capillaries from the rings in a very significant way, as can be seen in panel A of figure 14. Interestingly, the addition of hMSCs-PTX -CM at dilutions 1: 2 and 1: 4 after about 7-10 days of culture of rings, that is, when the vessels are already formed, it induces a vascular regression, as shown in panel B of Figure 14. The addition to the aorta rings of hMSCs-CM has no inhibitory effect, on the contrary it seems to increase their number, as shown in panel A of figure 14.

Even the addition of hMSCs-CM after seven days does not induce capillary regression but even seems to increase its vascular stability (panel B of Figure 14)

The same experiment described above on the antiangiogenic activity of hMSCs- has been confirmed using mouse mesenchymals. MMSCs-PTX both in co-cultures with ECs and with the use of their CM were as effective as in humans (data not shown)

To verify that the PTX treatment of MSCs is effective in inhibiting the growth of human tumor cells in vivo, a DU145 prostate tumor line transplanted into NOD / SCID (immunodeficient) mice was used. DU145 (at a dose of 2x10 <6> mouse) were co-transplanted subcutaneously (s.c.) alone or mixed in a 5: 1 ratio (tumor / mesenchymal) with hMSCs-PTX (0.4x10 <6>) or with hMSCs not treated used as cell control. Finally, mice with DU145 resuspended with free PTX at a dose of 10ug / ml were also treated. As shown in Figure 15, the co-injection of DU145 + hMSCs-PTX produces a significant inhibition of tumor growth (measured as tumor volume reduction at various times after transplantation). This inhibition of the growth of DU145 also resulted in a reduction in the weight of the tumor after their sacrifice occurred about 40 days after transplantation as shown in Table 1. The co-inoculation of DU145 with control hMSCs did not produce any significant effect, as well as previously treated DU145s. with pure PTX (the curve is not shown in Figure 15 because it is practically superimposable to that with DU145 only). Interestingly, the co-injection of DU145 with hMSCs-PTX also produces a delay in tumor appearance as seen in Figure 15.

The evaluation of the efficacy of hMSCs-PTX in inhibiting the growth of tumor cells in vivo was also analyzed using an experimental mouse tumor, melanoma B16. This tumor model is widely used in cancer research.

In this experimentation he was co-transplanted s.c. in nude mice melanoma B16 at a dose 2x10 <6> cells / mouse always in a 5: 1 ratio (tumor / mesenchymal) with hMSCs-PTX (0.4 x 10 <6>) or with control hMSCs and B16 in the presence of pure PTX free at 10ug / ml. Tumor B16 has a much faster growth rate than DU145, however, the co-injection of B16 hMSCs-PTX showed a significant reduction in B16 growth and tumor weight at the time of mouse sacrifice, which occurred at 14. day after inoculation as shown in Figure 16 and Table 1. Also in this case, a delay in tumor appearance was observed in animals co-inoculated with B16-hMSCs-PTX as well as the absence of effect on tumor weight at the time of sacrifice by co-transplantation of B16 with control hMSCs or of B16 inoculated in the presence of pure PTX (Table 1).

Finally, the ability of murine MSCs (MMSCs) to inhibit the growth of B16 melanoma was investigated. The aim of this experiment was to verify the ability of MMSCs-PTX to function in the syngene system of tumor presence. In fact, the MMSCs used were derived from the bone marrow of C57Bl6 mice which are the strain of mice on which B16 melanoma grows. C57Bl6 males were then co-transplanted s.c. with 2x10 <5> B16 melanoma cells alone, or mixed 1: 1 or 5: 1 with MMSCs-PTX or control MMSCs. Also in this case a group of mice was treated with B16 mixed at the moment of inoculation with pure PTX. As shown in Figure 17, MMSCs-PTX significantly inhibited the growth of B16 in C57BL6, both at 1: 1 and at dilutions greater than 5: 1 (tumor / mesenchymal), indeed, at this ratio, the effect on tumor weight B16 it seemed even more effective than the 1: 1 ratio (Table 1). In this experiment, a certain efficacy was also observed in reducing the growth of B16 also by the control MMSCs, particularly at the 1: 1 ratio (Table 1). The action of free PTX added to B16 had no effect on tumor growth (Table 1). Finally, also in the syngenic model of C57Bl6, the MMSCs-PTX delay the engraftment of B16 (data not shown) in the SC. suggesting the little influence of the immune system at least in the initial phase of tumor engraftment.

CONCLUSIONS

The present invention discloses a procedure for the preparation of a product defined with the generic name â € œMSCs-Fâ €. This product is made up of two elements, one consisting of mesenchymal cells called MSCs, the other element is made up of a specific drug. The terminal pharmaceutical product will then be named according to the drug that will be used in therapy. In our case, having used the PTX drug, the final product was named â € œMSCs-PTXâ €.

At the basis of this invention there is the experimental evidence that in the mammalian organism there are cells contained in the connective / stroma of various tissues and organs that are able to incorporate molecules and drugs and subsequently release them both in the original form and in the form of metabolites. These MSCs cells operate a detoxification process to neutralize the activity of molecules with toxic potential for our organism. This property makes these cells usable as drug carriers, thus developing an alternative approach to the current classic drug administration in humans.

The procedure described here was developed to obtain an MSCs-F product with anti-tumor and anti-angiogenic cytotoxic activity. In fact, the drug used was Paclitaxel (PTX), a well-known anti-tumor drug with proven antiangiogenic capacity. The procedure is not exclusive for the transport of chemotherapy drugs, but it can in any case also be applied to transport drugs and / or molecules other than the one described here. The demonstration of the pharmacological efficacy (with antitumor and anti-angiogenic activity) of the â € œnew drugâ € MSCs-PTX produced, has been confirmed both on tumor and endothelial cells in vitro, and in human and murine tumors in vivo. The MSCs-PTX product has therefore proved to be active in vitro, as much as free PTX, while much more effective in vivo. In fact, the addition of free PTX to tumor cells (100 times more concentrated than that incorporated in the cells) has not proved effective in inhibiting their growth in vivo, as well as the same â € œcarrier cellsâ € MSCs, not previously treated with PTX.

In conclusion, the experimental data, reported here, fully support the validity of our discovery. The procedure for obtaining a new pharmaceutical product generically called â € œMSCs-Fâ € and to be used in the therapy of human pathologies, is described in detail.

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Claims (15)

CLAIMS 1. Cell-type carrier for the targeted transport of at least one molecule and / or at least one molecular compound to at least one target cell in a human or non-human mammal, characterized in that it comprises at least one autologous or heterologous stromal cell isolated from a mammal human or non-human. Cell-type carrier according to claim 1, characterized in that said at least one stromal cell consists of a mesenchymal stem cell. 3. A complex product comprising in combination at least one molecule and / or a molecular compound and a cell-type carrier for the targeted transport of said at least one molecule and / or said at least one molecular compound to at least one target cell in a human or non-human mammal human, characterized in that said carrier comprises at least one autologous or heterologous stromal cell isolated from a human or non-human mammal. 4. Complex product according to the preceding claim, characterized in that said at least one stromal cell consists of a mesenchymal stem cell. 5. Complex product according to any one of claims 3 and 4, characterized in that said at least one molecule and / or said at least one molecular compound are cytotoxic. Complex product according to any one of claims 3 to 5, characterized in that said at least one molecule and / or said at least one molecular compound are pharmacologically active. Complex product according to any one of claims 5 and 6, characterized in that said at least one molecule and / or said at least one molecular compound are anticancer drugs. Complex product according to any one of claims from 5 to 7, characterized in that said at least one molecule and / or said at least one molecular compound are anti-angiogenic drugs. Complex product according to any one of claims 7 and 8, characterized in that said drugs comprise Paclitaxel (PTX). 10. Process for the preparation of a complex product characterized in that it comprises a step of isolation of stromal cells from a human or non-human mammal for the production of a cell-type carrier for the targeted transport of at least one molecule and / or at least a molecular compound to at least one target cell in a human or non-human mammal, an in vitro expansion step of said stromal cells, and a step of exposing said stromal cells to high concentrations of said at least one molecule and / or said at least a molecular compound. 11. Process for the preparation of a complex product according to the preceding claim, characterized in that it comprises a cryopreservation step of said stromal cells before or after said exposure step. 12. Process for the preparation of a complex product according to any one of claims 10 and 11, characterized in that said stromal cells are mesenchymal stem cells. 13. Method of targeted and controlled release of a therapeutically effective dose of at least one cytotoxic molecule and / or at least one cytotoxic compound, characterized by the fact of delivering to pathological target cells a population of mesenchymal stem cells internalizing a high concentration of said at least one molecule cytotoxic and / or said at least one cytotoxic compound for their gradual and targeted release. 14. Mouse model characterized in that it is treated with a complex product according to any one of claims 3 to 9. 15. Use of a complex product conforming to any of claims 3 to 9 for diagnosis, prognosis, therapy and therapeutic monitoring.