WO2013127885A1 - A conjugate of methotrexate and hydroxyethyl starch for use in the treatment cancer - Google Patents

Abstract

Conjugate composed of a carrier and a drug covalently bound thereto, for use in the treatment of cancer, wherein the effective dose of anticancer drug used in the form of said conjugate is lower than effective dose of said anticancer drug in unconjugated form.

Description

A CONJUGATE OF METHOTREXATE AND HYDROXYETHYL STARCH FOR

USE IN THE TREATMENTCANCER

Subject invention concerns conjugates for use in the treatment of cancer.

Most currently used medicines belong to the group of low molecular compounds [1 ]. The disadvantages of such kinds of therapeutics include: fast metabolism and excretion from an organism, unprofitable bio-distribution and low selectivity [2]. One way to solve these problems is to combine low molecular therapeutic substances with high molecular carriers [3]. This kind of approach enables an improvement in the pharmacokinetic properties and bio-distribution of both known and innovative drugs. Synthesis of such kinds of hybrid systems is one of the ways to realize the "magic bullet" concept propounded at the beginning of the 20th century by Paul Ehrlich, and which was aimed at achieving the selective deposition and release of therapeutic substances in target tissues [1 ]. This idea was developed and specified in 1975 by Helmut Ringsdorf. Synthetic and natural polymers, including polyvinyl pyrrolidone) (PVP) [4] polyglutamic acid (PGA) [5], poly(malic acid) [6, 7], poly(ethylene glycol) (PEG) [8], N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers [9], proteins [10] and polysaccharides [1 1 ], may be used as macromolecular carriers.

Conjugates of polymers with bioactive compounds may be realized for numerous reasons. One of them is the application of the most popular scheme including hydrophilic polymers with covalently bound bioactive molecules via biodegradable linkers. This kind of approach increases drug solubility, but most importantly modifies its pharmacokinetics in an organism, also on a cellular level, often increasing the therapeutic properties of a drug [12-14]. Bio-distribution of conjugates is to a high degree determined by the properties of a carrier and is realized mainly in an active way (a carrier or its fragment is a structure which potentially may selectively interact with receptors or antigens of target cells) or passive one (enhanced vascular permeability and retention - EPR) [15-19].

The EPR effect leads to preferential extravasation and accumulation of macromolecular drug-carrier conjugates (of a hydrodynamic diameter of > 3.5 nm [20, 21 ]) in the tissue of a solid tumor. The drug may be released from the conjugate in an intra- or extracellular space [1 ]. Since macromolecules cannot diffuse freely through the cell membrane, the main mechanism of their infiltration inside the cell is endocytosis [22]. In the design of high molecular drug-carrier conjugates, attention is focused inter alia on elongated half life time in the blood circulation system and accumulation in tumor tissue, at least as an effect of EPR [23]. However, elimination of a carrier/conjugate needs to take place in order to prevent the occurrence over time of negative results from polymers accumulation [23]. As in many biological systems, it is essential that an equilibrium is achieved assuring such a degree of carrier decomposition that allows gratifying therapeutic effects to be obtained with minimum side effects.

Pharmacokinetic properties and conjugate bio-distribution are also affected by the charge, conformation, hydrophobicity and immunogenicity of the carrier [22].

Drugs attached to a high molecular carrier may exhibit activity in a form attached to a carrier, as a result of carrier degradation [2] and/or dissociation of a bond between a drug and a carrier [22]. The last two processes may occur in an enzymatic or non- enzymatic way as determined by physicochemical conditions in the target site, such as temperature, pH or ionic strength.

The value of pH in affected tissues, especially cancerous ones, may reach the value of 6.5, i.e. as much as one unit below the pH of blood. This is an effect of hypoxia and intensified necrotic cell death [24, 25]. In the case of endocytosis, pH in endosomes may reach the value of 5.0 [26]. Conjugates with a pH-sensitive bond between a drug and a carrier are used for the therapeutic application of the above facts. An example may be a hydrazone bond subject to hydrolysis in pH between 6 and 5 used in conjugates of HPMA and doxorubicin [27]. These conjugates maintain their stability in blood, while with lower pH values accelerated drug release is observed. The data presented found a therapeutic reflection in this case - in the effectiveness of preparation activity in vivo in an animal model [27].

Hydroxyethyl starch is a modified polymer for use in plasma volume replacement [28]. The substrate for obtaining HES is natural maize or potato starch which is a polymer of glucose with a-1 -4 glycosidic bonds and numerous branching a-1 -6 glycosidic bonds[29]. The degree of branching, i.e. rate of a-1 -6 to a-1 -4 bonds, is usually about 1 :20 [29]. The physicochemical properties of HES are determined by its average molecular weight (MW), degree of substitution with hydroxyethyl groups (MS) and the ratio of the degree of substitution of particular sites in the glucose unit determined as the ratio of C2 C6 [30]. HES accumulation in an organism is positively correlated with the degree of substitution (MS) [31 , 32]. The detailed fate of HES in an organism depends first of all on its chemical structure [28]. HES polymers, especially those of higher molecular weight and higher degrees of substitution (MS), before they are subject to metabolism, are subject to accumulation in numerous tissues (liver, skin, spleen, lungs, kidneys) [33-37]. Polymers differing slightly in terms of structure may demonstrate significantly different accumulation properties [38]. A reducing influence was observed of HES polymers on blood platelets and the process of blood coagulation [39-42], the activity of metalloproteinase-9 (MMP-9) [43], and the expression of proinflammatory cytokines [44].

There are literature reports concerning HES conjugates with polypeptides and peptides, especially with erythropoietin (EPO) [45] [46-48], deferoxamine [49, 50]. Also, the application of HES for enzymatic synthesis of polymer-drug and polymer- protein nanoconjugates has been described in the literature [51 ].

MTX belongs to the group of antifoliates - derivatives of folic acid used in medicine [52]. MTX directly inhibits the activity of DHFR, an enzyme which contributes to the initial reduction of foliates [52, 53], and directly or indirectly affects a range of other molecular targets influencing DNA replication and cell proliferation [54]. MTX has found a wide application in cancer disease treatment and is still used both independently and combined with other chemotherapeutic. It is used in the therapy of, inter alia, acute lymphatic leukemia, osteogenic sarcoma, chorionic epithelioma, and in cases of breast, head and neck cancer [55, 56], as well as in the combined therapy of bladder cancer [57].

There have been numerous scientific reports concerning high molecular conjugates containing MTX such as MTX conjugates with albumins [58-63], fibrinogen [64] and glycated fibrinogen [10], mannan [65], dextranes [1 1 , 66] [67-69], hyaluronic acid [70, 71 ] dendrimers [72, 73], and nanotubes [74].

Nanoconjugates are a new class of therapeutic compounds, which may lead to a new therapeutic quality due to the combination of drugs already used in therapy, as well as new innovative substances with high molecular carriers . One way to verify such a thesis may be the application of a model drug, well described in terms of therapeutic activity, as an active substance. The conjugate and hybrid particle method has significant potential and may enable the elimination of numerous negative features of low molecular drugs.

The subject of the invention is a conjugate composed of a carrier and a drug covalently bound thereto, defined by the general formula:

where:

m is an integer from 60 to 60000,

R1 denotes a substituent selected from a group encompassing: hydrogen, - (CH₂)_nOH, -Drug, -(CH₂)_nO-Drug,

R2 denotes a substituent selected from a group encompassing: hydrogen, - (CH₂)_nOH, -Drug, -(CH₂)_nO-Drug, glucose unit,

wherein as the carrier, it contains starch with a molecular mass from 10 to 1000 kDa and a degree of substitution with hydroxyalkyl, preferably hydroxyethyl, groups from 0.1 to 0.9 mol/glucose unit, and as the drug it contains a molecule of a known antiinflammatory or anticancer drug which possesses a free carboxyl group, particularly methotrexate,

for use in the treatment of cancer, wherein the effective dose of anticancer drug used in the form of said conjugate is lower than effective dose of said anticancer drug in unconiugated form.

In particular embodiments of the invention, coniugate according to invention may be used in the treatment of cancer, wherein a greater inhibition of tumour growth is attained than when the same amount of said anticancer drug is used in unconiugated form.

For the purposes of the present invention, the effective dose should be understood as a dose of the anticancer drug which in a given patient can elicit the desirable effect, in particular the retardation or inhibition of tumour cell growth.

It is expected that in the case of the present invention, the effective conjugate dose will be at least twofold smaller than the dose of the unconjugated drug.

This effect may be observed as a retardation of tumour growth, which may be determined by observing changes in its volume and/or mass.

Subject invention discloses application of starch derivatives as effective, high molecular carriers for anticancer, therapeutic substances (e.g. antifoliates).

Nanoparticles composed of a starch derivative with a covalently bound anticancer drug (ester bond) were obtained. As a result of such a procedure, nanoparticles were obtained of physicochemical properties differing with respect to the carrier. These nanoparticles offered valuable therapeutic features and were composed of two medical substances, which are allowed and currently widely used in medicine. Physicochemical parameters characterizing the changes with respect to the initial polymer were analyzed, as were the biological implications. Biological examinations were performed in vitro with the application of mouse and human cancer cell lines. The anticancer effect in vivo was assessed by testing the activity on animals of lowered immunity enabling examinations with the application of human cancer line cells. The applied solution caused a considerable potentiation (a 10-fold increase in effectiveness when mean volumes of tumors were compared) of the anticancer activity of the conjugates after one intravenous administration of a preparation during a period of advanced cancer progression. The present invention opens the way for the application of modified starch as a drug carrier, especially for anticancer treatments.

LIST OF FIGURES

Figure 1 . Visual scheme of the HES-MTX conjugate.

Figure 2. Scheme of HES modification using MTX. R¹ = H, -(CH₂)_nOH, drug or - (CH₂)_nO-MTX; R² = H, -(CH₂)_nOH, drug, -(CH₂)_nO-drug or glucose unit; m=60 - 60000; n=1 - 20

Figure 3. LC-ESI-MS analysis of: A - total ion chromatogram of HES-MTX 130/0.4/48, B - total ion chromatogram of MTX pharmaceutical standard, C - ESI-MS spectrum of main peak from A.

Figure 4. Characterization of HES 130/0.4 and HES-MTX 130/0.4/59 conjugates by dynamic light scattering technique. Size distributions are shown according to intensity. Conjugates concentration - 5.5mM, solution - phosphate buffer 10mM pH=7.20.

Figure 5. Debye plot of HES 130/0.4 and HES-MTX conjugates.

Figure 6. Antiproliferative activity in vitro of HES-MTX conjugates in comparison with free MTX. All concentrations were based on the total contents of the MTX.

Figure 7. Antiproliferative activity in vitro of HES-MTX conjugates in comparison with free MTX. All concentrations were based on the total contents of the MTX.

Figure 8. Tumor growth kinetics of MV-4-1 1 bearing NOD/CDID mice. The mice were treated either with HES-MTX 130/0.4/59 conjugate (red line) or MTX (green line) alone. The control group received saline (black). Data are presented as mean tumor volume[nnnn³] ± standard deviation. The HES-MTX 130/0.4/59 conjugate diminishes the MV-4-1 1 tumor growth significantly when compared to control or MTX treated group.

Figure 9. Tumor growth kinetics of MV-4-1 1 bearing NOD/CDID mice, treated either with HES-MTX 130/0.4/59 (red), HES-MTX 130/0.4/25 (gray-red) or HES-MTX 130/0.4/1 1 (blue line) conjugate . Data are presented as mean tumor volume [mm³] ± standard deviation.

Figure 10. Tumor growth kinetics of MV-4-1 1 bearing NOD/CDID mice. The mice were treated either with HES-MTX 130/0.4/59 (red line), HES-MTX 130/0.4/25 (gray- red line) or HES-MTX 130/0.4/1 1 (blue line) conjugate, or MTX (green line) alone. The control group received saline (black). Data are presented as mean tumor volume[mm³] ± standard deviation. The HES-MTX 130/0.4/59 conjugate diminishes the MV-4-1 1 tumor growth significantly when compared to control or MTX treated group.

Figure 1 1 . The influence of HES-MTX 130/0.4/52 conjugate on the tumor growth kinetics of human leukemia MV-4-1 1 bearing mice. The tumor volumes are presented as % of control.

Figure 12. The influence of HES-MTX conjugates on the tumor growth kinetics of human leukemia MV-4-1 1 bearing mice. The tumor volumes are presented as % of control.

Figure 13. Body weight changes of MV-4-1 1 bearing NOD/CDID mice. The mice were treated either with HES-MTX 130/0.4/59, HES-MTX 130/0.4/48, HES-MTX 200/0.5/23, HES-MTX 130/0.4/25 or HES-MTX 130/0.4/1 1 conjugates, or MTX and 3% HES alone. The control group received saline (blue line). Data are presented as mean body weight [g] ± standard deviation. No body weight loss was observed during the experiment.

MATERIALS AND METHODS

Compounds

Methotrexate (EBEWE Pharma, Austria) and HES (Fresenius Kabi, Germany) were certified synthetic materials. DCC and DMF (Sigma-Aldrich) was used without further purification. Samples of the compounds were stored according to the manufacturer indications. In biological experiments, the compounds (HES, MTX and HES-MTX conjugates), prior to usage, were diluted in 0.9% saline solution to reach the required concentrations and administered to mice in a volume 0,01 ml/1 g of body weight.

Conjugates preparation (HES-MTX)

HES in aqueous solutions (6% w/w) was cooled to 4°C in ice bath and pH was adjusted to 10.5 using 1 M solution of Na2CO3. Afterwards cold solution was reacted with MTX anhydride according to the method previously described [1 1]. During conjugation reaction pH was still maintained to 10.5. After adding the total amount of MTX anhydride the pH was immediately adjusted to 7.0 (HCI 1 M) and obtained conjugate were dialyzed against ultrapure water to remove free MTX (dialysis membrane Visking 12kDA (Serva GmbH) for 96 hours or Pellicon® XL (Millipore Corp.) for 3 hours with flow rate 15ml/min). Conjugates with different levels of substitution MTX were synthesized by the same procedure using only a different (HES:MTX anhydride) stoichiometric ratio.

The stability of the prepared conjugates was assessed under different conditions and the compounds were stored at -20°C.

The conjugates were coded as:

HES-MTX 130/0.4/52

HES-MTX → conjugate of hydroxyethyl starch with covalently bound methotrexate

130 → Mean Molecular Weight of HES polymer [kDa]

0.4 → MS - molar substitution by hydroxyethyl groups on glucose units

52 → SL - molar substitution level by MTX on glucose units of the polymer x10^"3

MS and SL is expressed as number of moles of MTX/hydroxyethyl groups per mole of the glucose unit.

Analytical procedures

The analysis of total MTX in preparations was based on absorption spectrophotometry in 100 mM sodium bicarbonate at 372 nm by absorption coefficient 8571 M^"1cm^"1.

Determination of unbound MTX was based on size exclusion chromatography with UV-VIS detection at a wavelength of 302 nm. A Superdex® Peptide column (150 x 4.6 mm) and mobile phase 100 mM sodium bicarbonate with a flow rate of 400μΙ/ηηίη was applied [75]. The total glucose contents were determined by the phenol-sulphuric acid method[76] with slight modifications.

Conjugates stability in aqueous media

Conjugates were dissolved in a series of various inorganic buffers at pH from 4 to 10 and ionic strength from 10 to 600 mM to a final concentration of 0.8mM (based on the contents of the MTX in the conjugate). The buffer solutions were incubated at 4°C, 20°C and 37°C. At selected time intervals each reaction solution was diluted with NaHCO₃ 0.1 M to final concentration 0.2mM (total MTX) and analyzed of unbound MTX[75].

Conjugates stability in human plasma

Conjugates were dissolved in human plasma to a final concentration of 0.8mM (based on the contents of the MTX in the conjugate). The solutions were incubated at 37°C. At selected time intervals each reaction solution was diluted with NaHCO3 0.1 M to final concentration 0.2mM (total MTX) and analyzed of unbound MTX[75]. DLS and SLS measurements

HES and HES-MTX conjugates were characterized by dynamic light scattering (DLS) and static light scattering (SLS) to obtain hydrodynamic parameters, polydispersity information and ζ potential distribution. The sample solution was illuminated by a 633 nm laser, and the intensity of light scattered at an angle of 173° was measured. The experimental result consists of the overall average size, size distributions by intensity, overall polydispersity index - PDI, ζ potential distribution and Debye plot. All samples were measured using a Zetasizer Nano ZS (Malvern Instruments) in a low volume quartz cuvette (12 μί). All solutions were clear without evidence of insoluble material. The following parameters were used: protein refractive index (1 .450), temperature (25°C), HES and HES-MTX conjugates (5.5mM) were based on the contents of the glucose.

Cell lines

The human biphenotypic B myelomonocytic leukemia MV-4-1 1 cells, human chronic myelogenous leukemia K562 cells, human erythroleukemia HEL92 and Human T lymphoblast CCRF-CEM were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were maintained in RPMI-1460 GlutaMAX adjusted to contain 2 mM glutamine and 1 .0 mM sodium pyruvate with fetal bovine serum (10%). Murine leukemia P388 were obtained from the American Type Culture Collection (Rockville, MD, USA) and were maintained in culture or were frozen in the Cell Culture Collection of the Institute of Immunology and Experimental Therapy, Wroclaw, Poland. Twenty-four hours before addition of the tested preparations, the cells were plated in 96-well plates (Sarstedt, Germany) at a density of 0.5x10⁴ cells per well and were cultured in a mixture of RPMI 1640 and Opti-MEM (1 :1 ) medium, supplemented with 2 mM glutamine (Sigma-Aldrich Chemie GmbH, Germany), 100 mg/ml streptomycin (Polfa, Tarchomin, Poland) and 100 U/ml penicillin (Polfa), 5% fetal bovine serum (Sigma-Aldrich Chemie GmbH, Germany). The cells were cultured at 37°C in a humid atmosphere saturated with 5% CO₂. Passages of P388 leukemia cells in BDF mice were carried out according to the NIH/NCI standard screening protocols for in vivo assessment [77, 78]

An anti-proliferative assay in vitro.

The in vitro cytotoxic effect was studied after 72-h exposure of the cultured cells to different concentrations of the test preparations (based on the total contents of the MTX). The cells were placed in 96-well flat bottom plates (Sarstedt, Inc., Newton, NC, USA) at a density of 1 x10⁴ cells per well, 24 hours before addition of the tested compounds. The cells were exposed for 72 hours to various concentrations (0.1 ; 1 , 10 and 100 g/ml of MTX and HES-MTX conjugates. The MTT assay for evaluation of the cytostatic effect was performed. The results were calculated as a percent of proliferation inhibition by the tested compounds. The average values were counted using the data from 3-7 repetitions.

Experimental animals:

Male B6D2F1 mice, aged 16-20 weeks were used. The mice were supplied from the Animal Breeding Centre of the Medical Academy, Wroclaw, Poland, and were maintained in standard laboratory conditions. Experiments were performed according to the Guide for the Care and Use of Laboratory Animals (National Academy of Science, National Academy Press, Washington, D.C.) and were approved by the First Local Ethical Committee for the use of Laboratory Animals, Wroclaw, Poland. Male NOD/SCID mice, aged 8- 12 weeks old were used, weighing 20-25 g. The mice were supplied from the JU Children's University Hospital Krakow, Poland, were maintained in standard laboratory conditions. All experiments were performed according to Interdisciplinary Principles and Guidelines for the Use of Animals in Research, (National Academy of Science, National Academy Press, Washington, D.C.). Ad Hoc Committee on Animal Research and were approved by the 1 st Local Committee for Experiments with the Use of Laboratory Animals, Wroclaw, Poland. Design of the in vivo experiments.

Experiment 1 (P388)

Mice were injected with 10⁶ leukemia (P388) cells i.p. (day 0), and 24 h later (day 1 ) each mouse was injected once i.p. with the appropriate agent. All doses (4(Vmol/kg) were based on the contents of the MTX in the conjugate. Body weight and survival data were collected on a daily basis throughout the duration of the experiment.

Experiment 2 and 3

In two subsequent experiments, NOD/SCID male mice were subcutaneously inoculated in the right flank of the abdomen with 7.5-8x10⁶ viable MV-4-1 1 tumor cells per mouse in 0.2 ml Hanks solution (Sigma) and when the tumors become measurable (60-100mm³), mice were randomly divided into three (2^nd experiment) or eight (3^rd experiment) groups receiving different HES-MTX conjugates (HES-MTX 130/0.4/52 in 2^nd experiment and all other conjugates in 3^rd experiment) or HES, MTX or 0.9% saline solution alone. All doses (40μηηοΙ^) were based on the contents of the MTX in the conjugate. Body weight data were collected on a daily basis throughout the duration of the experiment. At the end of the experiment blood was harvested before subsequent sacrifice of the mice and tumors were collected during autopsy for further analysis.

Data handling

The in vitro results were presented in terms of IC50 values. The IC50 is the concentration of a tested agent which inhibits the proliferation of 50% of the cancer cell population. Average IC50 values for each preparation were calculated using data from at least three independent experiments.

Experiment 1 : The antileukemic effect in vivo was evaluated as the increase in lifespan (ILS) of treated mice over the control, calculated from the following formula: (MSTT/MSTC) x 100-100, where MSTT is the median survival time of treated animals and MSTC is the median survival time of untreated control mice.

Experiment 2 and 3: The tumor volume was calculated using the formula (a2 x b)/2, where a = shorter tumor diameter in mm and b=longer tumor diameter in mm. The inhibition of tumor growth was calculated from the following formula: TGI [%] = (WT/WC) x 100-100%, where WT was the median tumor volume of treated mice and WC that of the untreated control animals. Statistical evaluation:

Experiment 1 : Survival data in experimental in vivo groups were compared using Cox's F test with the Bonferroni correction for multiple comparisons (p adjusted = p counted χ N, where N = number of pairwise comparisons). P values less than 0.05 were considered significant.

Statistical evaluation.

Experiment 2 and 3: Statistical analysis was performed using STATISTICA version 7.1/10 (StatSoft Inc., USA). The data were analyzed by Kruskal-Wallis ANOVA. Multiple Comparisons p values 2-tailed) and Mann-Whitney test were used in analysis. P values below 0.05 were considered as significant.

RESULTS

Synthesis of conjugates

Synthesis of the HES-MTX conjugate runs according to the S_N2 nucleophilic substitution mechanism. Therefore, the highest probability of modification concerns the hydroxyl group by an atom of C2 carbon. However, taking into account the fact that the polymer is primarily modified by hydroxyethyl groups (which may react with MTX as well), it may be assumed that there is a statistical possibility of a reaction with all the hydroxyl groups of the polymer. A visual scheme of the conjugate is presented in Fig. 1 , while the scheme of HES modification using MTX is shown in Fig. 2. Conjugates, according the protocol described in materials and method section, were obtained in two independent syntheses. Conjugates were purified from an excess of free drug and classified in terms of the substitution level of MTX (SL) and the kind of HES polymer applied (Table 1)

Table 1. Physicochemical properties of HES-MTX conjugates. C_g [M] - glucose unit content, C^⁹ _TX - MTX total content in preparations, C¾- - MTX covalently bound to HES, SL - molar substitution level by MTX on glucose units, MTX-(COOH)₂ - unbound (free) MTX [%]

Analysis of MTX released from conjugates (Fig. 3)

LC-MS analysis of MTX released from HES-MTX 130/0.4/48 conjugates was performed. The results were compared with an analysis of MTX standard performed in identical conditions. The identity of both samples was demonstrated by comparing the courses of total ion chromatograms and ESI-MS spectra of the main chromatographic peak.

Hydrodynamic characteristics of conjugates.

It may be concluded from the data presented that both the hydrodynamic diameter of conjugates and their polydispersity differ between particular degrees of MTX polymer substitution. All HES-MTX conjugates were characterized by a slightly higher hydrodynamic diameter towards an initial polymer. The polydispersity of conjugates is also characterized by higher values when compared to initial (non-modified) polymers. The increase in hydrodynamic diameter is higher, when the degree of substitution of a given MTX conjugate is higher. This is accompanied by an increase in conjugates polydispersity (Table 2, Fig. 4).

Table 2. Hydrodynamic parameters of HES-MTX conjugates. d(H) - hydrodynamic diameter, Pd - polydispersity. Conjugates concentration - 5.5mM, solution - phosphate buffer 10mM pH=7.20.

The zeta potential of all examined HES-MTX conjugates in water solution receives negative values. The zeta potential value decreases with an increase in the degree of SL substitution, and its distribution is subject to narrowing. The higher the degree of substitution of a given MTX conjugate, the more negative is the zeta potential of conjugates (Table 3). Changes in electric properties of these molecules may be of key significance with respect to their biological properties. Table 3. Hydrodynamic parameters of HES-MTX conjugates. ZP - Zeta potential, Conjugates concentration - 5.5mM, solution - phosphate buffer 10mM pH=7.20.

Measurements of static light dispersion demonstrate drastic changes in conjugate properties in solutions when compared to non-modified polymers. The differences are mainly concerned with the interaction of conjugate molecules with their surroundings (ie solvents). The change in character of the second viral coefficient points to the different character of interactions of free HES and HES-MTX conjugates with the environment, and that the differences are enhanced by the degree of MTX polymer substitution (Table 4, Fig. 5).

Table 4. Hydrodynamic parameters of HES-MTX conjugates. A2 - second virial coefficient, MW - estimated molecular weight, R² - correlation coefficient of Debye plot. Conjugates concentration - 0.55 - 5.5mM, solution - NaCI 154mM.

Analysis of in vitro stability.

Analyses of stability were performed for HES-MTX 130/0.4/59 conjugate. Table 5 contains the data concerning the stability of the conjugate depending on solution pH, ionic strength and temperature. The cited data demonstrate the properties of bonds between HES and MTX in a manner typical for esters. Hydrolysis of these bonds is catalyzed by bases (fast decomposition in alkaline pH) and strongly depends on temperature.

The stability of the conjugates (half life time) in serum reaches 65 hours (Table 6) and is almost twice as short as the half life time in phosphate buffer of a similar pH. This fact points to the catalytic influence of serum components on the decomposition of HES-MTX conjugate.

Table 5. Conjugate (HES-MTX 130/0.4/59) stability in aqueous media. I - ionic strength, to.5 - half-life after which the release will be half of the total content of MTX in the conjugate.

Table 6. Conjugate (HES-MTX 130/0.4/59) stability in human plasma. I - ionic strength, to.5 - half-life after which the release will be half of the total content of MTX in the conjugate. pH T [°C] to.5 [h]

Human

7.24±0.05 37 65.4714.962

plasma

Phosphate

7.20±0.05 37 121.515.198

buffer 50mM BIOLOGICAL ACTIVITY ANALYSIS

In vitro examinations.

In vitro examinations of the antiproliferative activity of HES-MTX conjugates were performed on the cells of human leukemia lines: MV4-11 , K562, HEL92, CCRF-CEM. IC₅₀ (inhibitory concentration 50%) values were determined in comparison with the reference compound - MTX - which was a positive control. The experimental data are presented in Table 7. Graphic presentation of an activity as mean values of IC50 for the MV4-11 line in Fig. 6 and Fig. 7.

IC50 values for the examined conjugates were about 100 nM (10 nM for free MTX) in the case of MV4-11 line, and within the range of 65-124 nM (7.7 nM for free MTX) in the case of the K562 line. All HES-MTX preparations demonstrated lower in vitro activity when compared to free MTX.

Table 7. Antiproliferative activity in vitro of HES-MTX conjugates in comparison with free MTX. All concentrations were based on the total contents of the MTX.

In vivo examinations.

Model of mouse leukemia P388 (experiment 1).

The experimental data are presented in Table 8. HES-MTX 130/0.4/52 preparation elongated the life time of females by 66% when compared to the life time of control mice, while the life time of males was elongated by 45.2% when compared to males in the control group. The life time length of mice in groups treated with the HES-MTX 130/0.4/52 preparation exceeded the life time of mice treated with MTX. Table 8. Survival data of leukemia-bearing mice (P388) in control group (untreated, n = 8), and mice treated with either free MTX (n = 7) or HES-MTX 130/0.4/52 conjugate (n = 8). All doses were based on the total contents of the MTX.

Model of human leukemia MV-4-11 (experiment 2).

HES-MTX 130/0.4/52 conjugate was characterized by a higher than free MTX effectiveness in the inhibition of the growth of experimental MV4- 1 tumors (

Table 9).

Table 9. The influence of HES-MTX 130/0.4/52 conjugate on the tumor growth kinetics of human leukemia MV-4-11 bearing mice. Mice were treated with HES-MTX conjugates, or MTX and 3% HEST alone. The control group received saline. The tumor volumes are presented as mean ± standard deviation, % tumor growth inhibition TGI, N- number of mice per group.

Model of human leukemia MV-4-11 (experiment 3).

The experiment aimed at:

• confirmation of previous results obtained for the HES-MTX 130/0.4/52 preparation, • examination of the relationship between the therapeutic activity of conjugate and the degree of MTX substitution (SL),

• determination of perspectives for the application of polymers differentiated in terms of MW and MS HES as MTX carriers.

All the examined HES-MTX conjugates were characterized by a higher than free MTX effectiveness in the inhibition of the growth of experimental MV4-11 tumors (Table 10).

Table 10. The influence of HES-MTX conjugates on the tumor growth kinetics of human leukemia MV-4-11 bearing mice. Mice were treated with HES-MTX conjugates, or MTX and 3% HEST alone. The control group received saline. The tumor volumes are presented as mean ± standard deviation, % tumor growth inhibition TGI, N- number of mice per group.

HES-MTX 130/0.4/59 and HES-MTX 130/0.4/48 conjugates demonstrated the highest activity among all the examined conjugates (Fig. 8). Both conjugates reduce the volume of MV4-11 tumors to a considerable degree (p<0.05) when compared to the control starting from day 13 up to day 29. Comparing the size of tumors in the group which was administered the HES-MTX 130/0.4/59 preparation in one dose, to the size of tumors in groups receiving MTX, significant differences in the size of tumors in days 15 to 29 were noted, while in the HES-MTX 130/0.4/48 group on days 23 to 29 (p<0.05) when compared to MTX.

Preparations HES-MTX 200/0.5/23, 130/0.4/25 and 130/0.4/11 demonstrated a poorer effect in terms of the inhibition of tumor growth when compared to HES-MTX 130/0.4/59 and 130/0.4/48 conjugates (Fig. 9, Fig. 10); however, their activity was higher than those of free MTX (Fig. 1 1 , Fig. 12).

Analyzing the TGI parameter (tumor growth inhibition) on the 29th day of the experiment, the highest inhibition in tumor growth was 83.3% in the HES-MTX 130/0.4/59 group, then TGI values were 74.8% for HES-MTX 130/0.4/48, 61.6% for HES-MTX 200/0.5/23, 45.8% and 41.8% for HES-MTX 130/0.4/11 and HES-MTX 130/0.4/25, respectively. For a comparison, the TGI value for free MTX on day 29 was 28.97%.

Based on the analysis of changes in the body weight of mice, no toxicity was observed in the accepted scheme of preparations administration. The average body weight of mice during preparation administration was not lower than on the first day of treatment in any of the groups observed (Fig. 13).

DISCUSSION AND CONCLUSIONS

Physicochemical properties of HES-MTX conjugates and their biological activity. The obtained HES-MTX conjugates contained hydroxyethyl starch as a carrier substance (HES 130/0.4 and HES 200/0.5). Modifications to HES using MTX were conducted using the anhydride method. The linker between the drug and the polymer was thus glutamic acid (an integral part of the MTX particle) and the connecting bond was an ester bond. Consequently, the following facts should be taken into consideration:

• HES-MTX conjugates are composed of two medical substances, certified and currently widely used in medicine,

• a linker in a conjugate is an integral part of the drug particle - there is a lack of additional linking substances or groups,

• esterification is an equilibrium reaction, and a by- product of this reaction is water - during drug release from a conjugate (hydrolysis of esters) we deal with the reconstruction of the substrates of the reaction, i.e. we obtain free MTX and non-modified HES,

• identity of the MTX released from the conjugate with the pharmaceutical MTX standard was demonstrated,

• the kinetics of MTX release from conjugates strongly depends on environmental pH and temperature. It should thus be expected that drug release would occur with a differentiated rate depending on the pH observed in a given tissue (blood, cancer tissue, endosome). There is a possibility that the described feature may lead to conjugate deposition in sites of lowered pH value.

An additional mechanism of MTX release from a conjugate is HES-MTX hydrolysis by a-amylases. Conjugate subjected to partial enzymatic degradation may exhibit profitable properties. Moreover, digestion of the conjugate may limit MTX release in sites of lowered pH.

Enzymatic HES hydrolysis may also be caused by the fact that no toxicity of HES- MTX preparations was observed in in vivo examinations. They are subject, as in the case of HES application in volume therapy, to gradual degradation and removal from an organism.

The examinations of hydrodynamic parameters revealed the following facts:

• HES-MTX conjugates are characterized by insignificantly higher hydrodynamic diameters when compared to initial HES,

• HES-MTX conjugates are characterized by insignificantly higher polydispersity when compared to initial HES,

• when compared to initial HES, conjugates are characterized by negative zeta potential of a value and distribution dependent on the degree of MTX polymer substitution,

• HES-MTX conjugates are characterized by a quite different Debye curve course in comparison to initial HES. This may prove the fact that intermolecular conjugate-conjugate and conjugate-environment interactions are of a different character from interactions between non-modified HES, which may influence the adhesion and distribution of conjugates in an organism.

In summary, hybrid HES-MTX molecules demonstrate differences in hydrodynamic and electric parameters when compared to non-modified HES polymers. The differences centre on hydrodynamic diameter, polydispersity, charge of molecules and interaction with other molecules of the environment.

An antiproliferation study in vitro of HES-MTX conjugates performed on the control (MTX) revealed an expected effect - antiproliferation activity was an order lower than that for the free drug. A lack of the presence of the enzymatic apparatus of an organism in this type of experiment means that mainly MTX released as a result of chemical hydrolysis is responsible for an antiproliferative effect.

Biological study in vivo provided the following information:

• anticancer activity in in vivo experiments was the most effective when cancer developed in the form of a tumor,

• all synthesized conjugates profitably influenced the kinetics of the growth of experimental cancer tumors in mice, and all were more effective than free MTX (in an accepted administration scheme),

the best effects were obtained for conjugates of the highest degree of MTX substitution, obtaining over 90% reduction in tumor mass, and in individual cases their temporary atrophy was noted,

• conjugates of the lowest degree of substitution - HES-MTX 130/0,4/11 - demonstrated the lowest activity among the examined conjugates, but this activity was higher than that for free MTX.

Comparison of the effectiveness of two HES types as carriers did not reveal any considerable differences - it only gave a suggestion that HES with higher values of MW, MS and ratio of C₂/C₆ (when compared to HES 130/0,4) may be more effective carriers of therapeutic substances. There is a probability that 1st and 2nd generation HES used years ago in medicine will return to use drug carriers, since their negative features in volume therapy, such as increased accumulation in some tissues, may appear positive in the case of oncological drug administration.

Perspectives for HES application as high molecular carriers of anticancer drugs.

The term "polymer therapeutics" includes natural and synthetic macromolecules used in interdisciplinary studies combining polymer chemistry, biology, pharmacy and medicine for the design of biologically active polymer-drug, polymer-protein conjugates or polymeric micelles [79]. Many polymers with attractive physicochemical features have no possibility to become effective drug carriers due to their biological properties, such as toxicity or immunogenicity [80]. Therefore, selection or design of carrier polymers is a key issue, limiting the process of conjugate formation. Similarly, a significant element is the suitable selection of the linker connecting a polymer with a drug. If it is too stable or not subject to a biodegradation bond connecting the drug with a carrier, it would result in limited drug release and its insufficient concentration in a target site. In turn, quick degradation of a connecting bond may lead to excessive removal of a dissociated drug by the kidneys [13] Attention should be paid to the fact that bio-distribution of compounds in an organism never occurs entirely on one selected path. In fact, we observe an effect as being a result of a range of physiological, physicochemical factors and overlapping interactions. Thus, when designing drug-carrier systems, attention should be paid to all factors which may affect the realization of the assumed effect.

The application of natural polymers as drug carriers seems to the safest; however, modifications of these polymers enabling therapeutic settlement are not biological property neutral. [81-83].

Ringsdorfs idealistic concept [84], and elements connected to the influence of endocytosis on conjugate behavior in an organism [85] may be useful in the design of polymeric drug-carrier conjugates.

Combining low molecular drugs with high molecular carriers can inter alia:

• regulate drug transport in an organism and cellular transport determining preferential transport to target sites, limiting concurrently passive transport to healthy tissues [86],

• cause accumulation of conjugates in serum or/and tissues making use of the EPR effect [18]

• give the possibility of the application of targeted therapy, for example by modification of polymers with antibodies [87],

Desirable properties of polymers used for the design of therapeutic conjugates, according to the current opinions, are as follows [13, 79, 88]:

• polymers should be non-toxic and non-immunogenic,

• mean molar mass of polymers should be large enough to ensure a suitably long period of conjugate circulation in an organism; however, for polymers not subject to biodegradation they must be optimized in such a manner as to ensure gradual elimination from an organism,

• polymers should ensure the possibility of the attachment of a suitable amount of the drug,

• linkers connecting drugs with polymers should be stable in conditions of conjugate transport and ensure effectiveness in a target site,

The application of HES polymers as anticancer drug carriers creates large possibilities. Depending on its origin, an initial polymer (natural starch) is characterized by different properties (mean molecular weight, number of a-1 ,6- glucoside bonds). These polymers may be designed according to established parameters critical for biological properties, such as MW, MS and ratio of C₂/C6, with respect to a specified therapeutic substance. The degree of substitution (MS) and its localization may be regulated precisely in the process of modification with hydroxyethyl groups. The whole process may be led in a way enabling the acquisition of a polymer of designed properties adequate to the specified biological targets. These facts mean that HES is a very interesting polymer with medical applications, and currently its utilization in volume therapy may be considered as only the beginning of its "medical career".

HES demonstrates practically all the above-presented desirable properties of polymers used for the design of therapeutic conjugates, and additionally:

• drugs subjected to conjugation may be any biologically active particle possessing a free carboxyl group (in particular MTX), even if this group is essential for the biological activity of this particle,

• conjugates may be composed of two or more biologically active substances (including diagnostic substances) revealing new physicochemical and biological properties after conjugation.

Finally, examination and then control of structural and functional differences of high molecular polymers in connection with continuous examinations of molecular mechanisms of various complex diseases, including cancers, has the chance to lead to a new generation of polymer drugs based on HES.

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Claims

Claims

1 . A conjugate composed of a carrier and a drug covalently bound thereto, defined by the general formula:

where:

m is an integer from 60 to 60000,

R1 denotes a substituent selected from a group encompassing: hydrogen, -(CH₂)_nOH, -Drug, -(CH₂)_nO-Drug,

R2 denotes a substituent selected from a group encompassing: hydrogen, -(CH₂)_nOH, -Drug, -(CH₂)_nO-Drug, glucose unit,

wherein as the carrier, it contains starch with a molecular mass from 10 to 1000 kDa and a degree of substitution with hydroxyalkyl, preferably hydroxyethyl, groups from 0.1 to 0.9 mol/glucose unit, and as the drug it contains a molecule of a known antiinflammatory or anticancer drug which possesses a free carboxyl group, particularly methotrexate,

for use in the treatment of cancer, wherein the effective dose of anticancer drug used in the form of said conjugate is lower than effective dose of said anticancer drug in unconjugated form.

2. A conjugate according to claim 1 for use in the treatment of cancer, wherein a greater inhibition of tumour growth is attained than when the same amount of said anticancer drug is used in unconjugated form.