WO2022010441A1 - Oxaliplatin containing therapeutic polyamidoamine (pamam) dendrimers and production method thereof - Google Patents

Oxaliplatin containing therapeutic polyamidoamine (pamam) dendrimers and production method thereof Download PDF

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WO2022010441A1
WO2022010441A1 PCT/TR2021/050633 TR2021050633W WO2022010441A1 WO 2022010441 A1 WO2022010441 A1 WO 2022010441A1 TR 2021050633 W TR2021050633 W TR 2021050633W WO 2022010441 A1 WO2022010441 A1 WO 2022010441A1
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oxaliplatin
dendrimer
solution
dendrimers
modified
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PCT/TR2021/050633
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French (fr)
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Gülşah Gedi̇k
Hakan NAZLI
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T. C Trakya Üni̇versi̇tesi̇
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Priority to EP21827812.5A priority Critical patent/EP3976024A4/en
Publication of WO2022010441A1 publication Critical patent/WO2022010441A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/595—Polyamides, e.g. nylon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines
    • C08G73/028—Polyamidoamines

Definitions

  • the invention relates to the anticancer medication containing oxaliplatin as the active substance, and to the macromolecules with dendrimer oxaliplation complexes (prepared by physical methods) and oxaliplation derivative dendrimer conjugates (prepared by chemical methods) to decrease the release time and toxic effect of the said medication, and to the production methods thereof.
  • the invention has the potential to be used in first and second-line therapy for the treatment of cancer types such as metastatic colorectal cancer, non-Hodgkin lymphoma, breast cancer, non-small cell lung cancer, head and neck cancer, mesothelioma and squamous cell carcinoma advanced ovarian cancer in which oxaliplatin is used.
  • cancer types such as metastatic colorectal cancer, non-Hodgkin lymphoma, breast cancer, non-small cell lung cancer, head and neck cancer, mesothelioma and squamous cell carcinoma advanced ovarian cancer in which oxaliplatin is used.
  • Oxaliplatin is a platinum ( ⁇ ) analogue similar to cisplatin and carboplatin.
  • New drug application (NDA) for use in metastatic colorectal cancers was first accepted by the US Food and Drug Administration (FDA) in February 1999.
  • Oxaliplatin, a DACH platinum derivative has a similar mechanism of action to other platinum derivatives.
  • its antitumour activity differs from other platinum derivatives such as cisplatin and carboplatin.
  • oxaliplatin is effective against cisplatin-resistant colon carcinoma cell lines.
  • Cisplatin, carboplatin and oxaliplatin are platinum compounds that are accepted and used worldwide. Apart from these, there are also other platinum derivatives which have been regionally approved in various countries of the world, such as nedaplatin (Japan), lobaplatin (China), heptaplatin (South Korea) and some others such as picoplatin and satraplatin (Pt* 4 ) that are still being developed and the phase studies of which still continue.
  • Particularly octahedral structured platinum (TV) compounds, also called as fourth generation platinum compounds, are of interest as they are less reactive, and are more suitable than platinum (II) derivatives for oral administration.
  • Platinum compounds that are in clinical use worldwide are also called classical platinum complexes. These are cis-structured, uncharged, square planar complexes in the +2-oxidation state (Pt +2 ) and are shown below.
  • A2 represents the nitrogen donor two monodentate or one bidentate ligand
  • X2 represents two monodentate or one bidentate anionic ligands. Alteration of the A2 moiety results in the formation of structurally different DNA-drug complexes, and this alters the anticancer activity of the formed complexes.
  • the X2 group affects the distribution of the formed complexes in the body and thereby its side effects.
  • Oxaliplatin C 8 H 14 N 2 O 4
  • Oxaliplatin is designed to overcome cellular resistance seen against cisplatin and carboplatin.
  • oxaliplatin whose chemical nomenclature is (cis- [(1R,2R)- 1 ,2-cyclohexanediamine-N,N9] oxalato(2-)-0,09] platinum, differs from that of cisplatin or carboplatin by bonding of the cyclohexane ring to its nitrogen atoms.
  • Oxaliplatin is a white/off-white crystalline structured powder with a molecular weight of 397.3 g/mol. It is solubility in water is low (6-7g/L at 20°C), in methanol is very low (0.125g/L at 20°C), and it is almost insoluble in ethanol or acetone. The pKa value is 6.1.
  • the main lesions responsible for cytotoxicity are intrastrand cross-links between active platinum compounds and specific base sequences. These crosslinks are particularly between two adjacent guanine bases or adjacent guanine-adenine bases. Interstrand cross-links can also occur. However, they constitute less than 5% of the total platinum-DNA complexes. Approximately 60-65% of the cross-links formed between DNA and oxaliplatin are intrastrand guanine-guanines and 25-30% are intrastrand adenine-guanine links.
  • Oxaliplatin turns into active platinum compound by interacting with HCO3- (bicarbonate) and H2PO4- (dihydrogen phosphate) anions in physiological environment.
  • HCO3- bicarbonate
  • H2PO4- dihydrogen phosphate
  • a chloride ion leaves this compound and causes a single aqueous chlorine complex (monoaquamonochloro complex) to form.
  • This complex rapidly reacts with guanine at the N-7 position on DNA. By one more chlorine leaving the monochloro complex, a double aqueous complex is formed.
  • the formation of the double aqueous complex from oxaliplatin is shown below.
  • the DACH-Pt complex formed by oxaliplatin forms a more voluminous hydrophobic region than the cis-diamine-Pt complex formed by both cisplatin and carboplatin.
  • oxaliplatin causes a more effective inhibition of DNA synthesis, resulting in higher cytotoxicity than the cis-diamino-Pt complex.
  • these voluminous complexes prevent the mismatch repair proteins responsible for DNA repair from binding to the region and initiating the DNA repair mechanism.
  • the development of resistance to oxaliplatin can be explained by the cellular interactions of oxaliplatin. The various mechanisms that cause oxaliplatin resistance are summarized in Table 1.
  • Oxaliplatin undergoes rapid nonenzymatic biotransformation to form various reactive platinum intermediates.
  • the intermediates formed are rapidly and extensively bound to plasma proteins and erythrocytes.
  • the active and inactive metabolites of oxaliplatin formed as a result of biotransformation are shown below.
  • Half maximum inhibitory concentrations (IC50) are in vitro data for the HT-29 cell line.
  • the pharmacokinetics of unbound platinum in plasma after oxaliplatin administration is characterized by a short initial a and ⁇ distribution phase and a long terminal ⁇ elimination phase.
  • the main route of elimination is urinary excretion (53.8 ⁇ 9.1%), and a very small amount of platinum (2.1 ⁇ 1.9%) is excreted in the faeces.
  • No platinum accumulation was observed in the plasma when administered as 85mg/m 2 every 2 weeks or 130mg/m 2 every 3 weeks, which are the routine use doses.
  • Oxaliplatin exhibits a dose-dependent pharmacokinetics in the dose range of 20-180mg/m 2 .
  • oxaliplatin is not a substrate for CYP450 in vitro.
  • the critical pharmacokinetics parameters of oxaliplatin such as peak concentration (Cmax), area under the curve (AUC), half-lives for the three elimination phases (tl/2), virtual volume of distribution (Vd), and clearance (Cl) obtained from the ultrafiltrate of blood plasma are shown in Table 2.
  • oxaliplatin in clinical use is metastatic colorectal cancer.
  • the utility of oxaliplatin as first-line and second-line therapy for the treatment of advanced ovarian cancer is under investigation by clinical trials.
  • the activity of this drug in other malignancies such as non- Hodgkin lymphoma, breast cancer, non-small cell lung cancer, head and neck cancer, mesothelioma and squamous cell carcinoma is also being investigated.
  • Oxaliplatin is used in combination with 5-FU and calcium folinate (calcium salt of leucovorin) in colorectal cancers.
  • 5-FU is a pyrimidine antagonist and is used together with oxaliplatin to achieve a synergistic effect. When calcium folinate is used with 5-FU, it has been observed that the antineoplastic activity is strengthened.
  • dilution with 250- 500mL of 5% dextrose solution should be made. 0.9% NaCl or other electrolyte solutions should not be used as a dilution solution.
  • the diluted solution is administered to the patient as intravenous infusion over 2-6 hours.
  • Acute neuropathies are characterized by paresthesia and dysesthesia in the hands, feet, and peroral region that occur during or shortly after the infusion. However, although less frequently it happens, they can also cause cumulative distal neuropathy. Events such as sensory ataxia, functional disorders, jaw pain, eye pain, ptosis, leg cramps, and changes in vision or voice may occur. Neuropathies seen in association with cumulative oxaliplatin affect all patients receiving a total dose of 540 mg/m 2 or more for four or more cycles. While reversible neuropathies regress within 4-6 months in 82% of patients, 41% recover completely within 6-8 months. The cause of sensory-motor neuropathy caused by oxaliplatin is mainly due to hyperexcitability.
  • Peripheral neuropathy due to oxaliplatin is the result of the drug's action on sensory, motor neurons, or muscles, while cumulative chronic distal neurotoxicity is due to damage to neurons in the dorsal root ganglia.
  • additional drugs such as amifostine (cytoprotective), a-lipoic acid (antioxidant), glutathione (antioxidant), calcium-magnesium ions (oxalate chelator), carbamazepine, oxcarbazepine, topiramate, and gabapentin (antiepileptic) to take advantage of their neuroprotective properties and the clinical studies were carried out.
  • additional drugs such as amifostine (cytoprotective), a-lipoic acid (antioxidant), glutathione (antioxidant), calcium-magnesium ions (oxalate chelator), carbamazepine, oxcarbazepine, topiramate, and gabapentin (antiepileptic)
  • Haematological side effects are the rarely seen low severity anaemia, thrombocytopenia, and neutropenia. As they are not severe, these are often manageable cases.
  • Nausea and vomiting are gastrointestinal side effects frequently seen with most other chemotherapeutic agents. Nausea and vomiting due to oxaliplatin are mild to moderate and can be easily overcome with 5-hydroxytryptamine (5HT3) receptor antagonists given as premedication before treatment. Allergic reactions to oxaliplatin, like in nausea and vomiting, can be prevented with antihistamines and glucocorticoids given as pre-medication.
  • 5HT3 5-hydroxytryptamine
  • Liposomal cisplatin Lipoplatin
  • liposomal oxaliplatin Lipoxal
  • polymeric conjugates and dendrimers can also be used as carrier systems.
  • Dendrimers are nano-sized, branched, three-dimensional molecules that mimic the biological structure consisting of synthetic polymers and have promising properties in the field of diagnosis and treatment. Unlike conventional polymers, they have a three-dimensional structure with low polydispersity, with long or spiral branching around the central core. Formed by branching on the functional groups in the core, these molecules can be given the desired shape and properties by altering the core structure or the functional groups added to the outer surface. The ability of dendrimers to be designed as desired has given them with a application area in the medical field.
  • linkers There are several disadvantages of using linkers.
  • the use of linkers causes an increase in the number of steps in the synthesis processes, thereby causing the need for new processes such as synthesis and purification for the additional steps. Additional purification steps often cause a decrease in the yield of the final product. Additional processes complicate the synthesis process and cause a waste of time. All these situations considered together, the cost of the product increases.
  • the structure responsible for the cytotoxic effect of the final product is the platinum group.
  • the platinum group responsible for the cytotoxic effect is released by enzymatic reactions inside the cell.
  • the rest of the molecule acts as a carrier and is not directly related to the cytotoxicity of the molecule.
  • WO2015035446A1 which is about the dendrimer with amino groups on the nucleus and surface, and macromolecules containing one or more platinum attached to the dendrimer amino groups
  • amine (-NH2) terminated dendrimers are conjugated with various cytotoxic platinum compounds through linkers.
  • the dendrimers used have -NH2 termination and in the said patent, the conjugation with the dendrimer was performed over polydentate ligands. It is said that pharmaceutical compositions containing these macromolecules can be used in cancer treatment.
  • dendrimers with -NH2 termination or more generally + charged groups when ionized have higher toxicity compared to anionic dendrimers with carboxyl groups on the surface or neutral dendrimers with hydroxyl groups on the surface.
  • the underlying reason of it is that the cationic ends interact with the negatively charged cell membrane and thus destabilize it.
  • haemolytic and aggregate activities of cationic dendrimers are also much more clear than anionic ones.
  • Cationic dendrimers are not preferred as it is not desirable for dendrimers to interact with the membranes of normal cells and disrupt their structure until they are removed from the body after being separated from their platinum structure.
  • polydentate or monodentate ligands do not have a direct effect on toxicity.
  • Cationic dendrimers used in the methods of prior art are disadvantageous due to their toxicity.
  • PAMAM type cationic dendrimers used demonstrate haemolytic activity at concentrations above lmg/mL.
  • the main aim of the invention is to develop various dendrimeric formulations with effects similar to oxaliplatin which may be candidates for use in cancer treatment, in vitro examination of their various properties, and to validate their production.
  • Dendrimers can increase the solubility of active substances by various mechanisms. In this study, G 3.5 and G 4.5 PAMAM dendrimer were used, and it was seen that both of them showed an increasing effect on oxaliplatin solubility. It was also found that this effect has increased linearly with dendrimer concentrations. Comparing between the dendrimer generations, it was seen that the G4.5 generation dendrimers increased the solubility statistically significantly more than the G3.5 generation ones.
  • Dendrimer oxaliplatin physical complexes were prepared by co-precipitation method and oxaliplatin derivative dendrimer conjugates were prepared by Steglich esterification reactions. The charge properties of the physical complexes were examined, and it was observed that they differed according to the dendrimer concentration and pH values of the medium. In general, it was observed that as the dendrimer concentration increased, the charge rate increased, and the charge efficiency decreased.
  • G4.5 dendrimers can charge 2 to 5 times more oxaliplatin per mole than G3.5 dendrimers.
  • the amount of substance conjugated to dendrimer ends in chemical conjugates was determined by 1 ⁇ -NMR. 37.9 molecules were bound to G3.5 conjugate and 26.3 molecules were bound to G4.5 conjugate per 1 mole of dendrimer.
  • Release studies were carried out with the characterized formulations in two different media with pH 5.5 and pH 7.4. Release studies were carried out by the dialysis membrane method. According to the release studies, the release time of all substances placed in the membrane took an average of 30 minutes for free oxaliplatin, an average of 60 minutes for physical complexes, and an average of 120 minutes for chemical conjugates. Pure oxaliplatin was seen to tend to be released more rapidly at pH 7.4. The release profiles were compared using the fl difference factor and f2 similarity factor.
  • the dendrimers used in the invention are 3.5 and 4.5 generation PAMAM dendrimers with carboxyl (-COOH) termination.
  • the conjugation is realised by bonding the hydroxyl (-OH) groups of the platinum (TV) complex with these -COOH groups.
  • the dendrimers used are different from the study of Sommerfeld et al. in terms of both generation and end groups. While conjugating the dendrimer and the platinum (TV) compound, Sommerfeld et al. have bound the -NH2 groups of the dendrimer with the -COOH group of the succinato group and realised the conjugation through a succinato bridge.
  • conjugation was realised over -COOH and -OH groups. Conjugation was realised directly without the use of any linker molecules.
  • linker leads to an increase in the number of steps in the synthesis processes, and the increase in the number of steps causes the need for new processes such as synthesis and purification for the additional steps. Additional purification steps often lead to a decrease in the yield of the final product. Additional processes complicate the synthesis process and cause a waste of time. All these situations considered together; it is inevitable for the cost of the product to increase.
  • the elimination of the use of additional chemical substances and processes such as additional purification due to the increase in the step plays a role in reducing both monetary and temporal costs of the formulation.
  • the dendrimers used in the invention are 3.5 and 4.5 generation PAMAM dendrimers and have carboxyl (-COOH) termination.
  • the way of conjugation with the dendrimer also differs.
  • the conjugation with the dendrimer is realised over polydentate ligands in the said patent document, in the invention the conjugation is realised over -OH groups, in other words, over the monodentate ligand. Conjugation directly over the -OH group eliminates the need to use an linker molecule between the platinum-containing structure and the dendrimer.
  • the invention solves the problem of toxic and haemolytic risks that may occur due to cationic dendrimer that could not have been solved in the prior art or that may occur, and at the same time, it meets the need for an effective and more cost-effective pharmaceutical form by providing a cost advantage by reducing the number of steps in the synthesis process.
  • FIG 1 Oxaliplatin Solubility A. Oxaliplatin solubility in various solvents (mg/ml), B. Oxaliplatin solubility at various pHs (mg/ml)
  • Figure 2 Effect of dendrimers on solubility
  • Figure 3 Process steps of obtaining dendrimer-oxaliplatin aqueous complex solution
  • Figure 4 Chromatograms of pure oxaliplatin, PAMAM-G3.5 and PAMAM-G4.5 dendrimer solutions and blank solutions prepared without active substance
  • Figure 5 Spectrums between 200-400nm
  • Figure 7 Spectra between 200-400nm A. Oxaliplatin, B. The fact that the oxaliplatin peak seen in oxaliplatin-PAMAM G3.5 Complex (F6) at 255nm was suppressed in the F6 formulation indicates that oxaliplatin forms a complex with the dendrimer.
  • Figure 9 Spectra between 200-400nm A. Oxaliplatin, B. The fact that the oxaliplatin peak seen in oxaliplatin-PAMAM G4.5 Complex (F21) at 255nm was suppressed in the F21 formulation indicates that oxaliplatin forms a complex with the dendrimer.
  • Figure 11 FTIR spectra of pure oxaliplatin and synthesized modified oxaliplatin
  • Figure 12 1 ⁇ - ⁇ results of modified oxaliplatin
  • Figure 13 13 C-NMR results of modified oxaliplatin
  • Figure 14 FT-IR spectra of modified oxaliplatin, PAMAM-G3.5 dendrimer and KK-3.5 formulation
  • Figure 15 1 ⁇ -NMR (DMSO-d6) results of KK-3.5 formulation
  • Figure 16 FT-IR spectra of modified oxaliplatin, PAMAM-G4.5 dendrimer and KK-4.5 formulation
  • Figure 17 1 ⁇ -NMR (DMSO-d6) results of KK-4.5 formulation A. 1 HNMR spectra of the conjugate, B 1 HNMR spectra of modified oxaliplatin Figure 18: Amounts of Oxaliplatin Complexed with 1 mole of Dendrimer Figure 19: Release Data A. Release data of F6 Formulation at pH 5.5 and pH 7.4, B. Release data at F21 Formulation pH 5.5 and pH 7.4
  • Figure 21 As a result of the study carried out on the HT-29 cell line, the viability data (%) of PAMAM G3.5 dendrimer, oxaliplatin and KK-3.
  • Figure 22 IC 50 values of oxaliplatin and KK-3.5 formulation A. Oxaliplatin IC 50 , B. KK-3.5 conjugate IC 50
  • the macromolecule of the invention comprises; • G3.5 or G.4.5 generation PAMAM dendrimer with at least one carboxyl structure (- COOH) anionic surface groups,
  • One or more oxaliplatin the monodentate ligand of which is modified to be the -OH group, and is characterized with -OH groups of one or more oxaliplatin modified to be -OH group of monodentate ligand directly bonding with at least one carboxyl structure (-COOH) anionic surface groups in the structure of G3.5 or G.4.5 generation PAMAM dendrimer.
  • -COOH carboxyl structure
  • the main aim of the invention is to develop various dendrimer formulations with effects similar to oxaliplatin that may be candidates for use in cancer treatment and in vitro examination of their various properties.
  • Dendrimers can increase the solubility of active substances by various mechanisms.
  • two different generations of dendrimers (G 3.5, G4.5) were used and it was observed that both had an effect on increasing oxaliplatin solubility. It was also found that this effect increased linearly with dendrimer concentrations. Comparing the dendrimer generations, it was seen that the G4.5 generation dendrimers increased the solubility statistically significantly more than the G3.5 generation ones.
  • Dendrimer-mediated solubility increase depends on several factors. These are properties like the structure of the core and surface groups, concentration and generation of the dendrimer, the pH of the solution, and the type of solvent. Increasing dendrimer generation and dendrimer concentration seems to increase oxaliplatin solubility. The reason why the solubility increases in G4.5 dendrimers is higher than that in G3.5 dendrimers under similar ambient conditions can be explained by the increase in the number of surface groups that can interact with oxaliplatin in G4.5 dendrimers.
  • oxaliplatin derivative dendrimer conjugates were prepared with dendrimer oxaliplatin physical complexes and Steglich esterification reactions by coprecipitation method. Formulations prepared using various methods have been characterized. The charge properties of the physical complexes were examined, and it was observed that they differed according to the dendrimer concentration and pH values of the medium. In general, it was observed that as the dendrimer concentration increased, the charge rate increased, and the charge efficiency decreased. It has been observed that G4.5 dendrimers can charge 2 to 5 times more oxaliplatin per mole than G3.5 dendrimers.
  • the ratio of active substance/dendrimer charged with the highest active substance per mole was found to be 25.
  • the amount of active substance is 1.26 ⁇ mol and the amount of dendrimer is O.OS ⁇ mol.
  • the amount of substance conjugated to dendrimer ends in chemical conjugates was determined by 1H-NMR. 37.9 molecules were bound to G3.5 conjugate and 26.3 molecules were bound to G4.5 conjugate per 1 mole of dendrimer. It is thought that this situation may be caused by the molecular density formed around the dendrimer ends by the increase in dendrimer generation. It is known that more active substances can be prevented from reaching the dendrimer ends and reacting due to the steric obstacles formed during the chemical reaction.
  • zeta potential values of G3.5, G4.5 dendrimers and conjugate formulations used in the invention were examined.
  • the particle sizes were small ( ⁇ 10nm) and the size distributions were in a narrow range (PDK0.25).
  • the dendrimers were dispersed in water by evaporating methanol, the particle size increased significantly (200-250nm) and the dispersion got worse (PDIX).40).
  • the dimensions of the chemical conjugates in water were found to be 179.30 ⁇ 77.30 for G3.5 and 155.93 ⁇ 13.59 for G4.5, and it was seen that they have an average polydispersity of 0.377 and 0.340, respectively.
  • G3.5 and G4.5 dendrimers were found to be negatively charged as they have carboxyl structured anionic surface groups. With the conjugation of the active substance to the dendrimers, the surface charge changed from negative to positive.
  • Release studies were carried out with the characterized formulations in two different media with pH 5.5 and pH 7.4. Release studies were carried out by the dialysis membrane method. According to the release studies, the release time of all substances placed in the membrane took an average of 30 minutes for free oxaliplatin, an average of 60 minutes for physical complexes, and an average of 120 minutes for chemical conjugates. Pure oxaliplatin was seen to tend to be released more rapidly at pH 7.4. The release profiles were compared using the fl difference factor and f2 similarity factor. Comparing the chemical conjugate formulations among themselves, it was seen that there was no significant difference between the release profiles.
  • the release profile of the F6 formulation obtained in a pH 5.5 medium was significantly faster than the others.
  • the release time of all substances is 30 minutes on average for free oxaliplatin, 60 minutes on average for physical complexes and 120 minutes on average for chemical conjugates. It is known that the release time in complex structures varies depending on the binding constants of the complexes formed by platinum ( ⁇ ) compounds with PAMAM dendrimer. While the low binding constants of the complexes formed cause the release to accelerate, the high ones cause the release to slow down.
  • the concord of the release profiles to various release kinetic models was examined and it was seen that the kinetic with the highest concord was generally Weibull.
  • Dendrimers can interact with drugs in various ways. It is possible to classify these interactions as physical and chemical. Physical interactions are the binding of the active substance to between dendrimer branches or to end groups by non-covalent bonds. Electrostatic bonds, hydrogen bonds or hydrophobic interactions play a role in the formation of such physical complexes. In chemical interactions, between the surface groups of the dendrimer and the drug molecule covalent bonds are established. These physical or covalent connections formed cause formulations to release for a longer period of time compared to the free active substance.
  • Thermo Surveyor was used as HPLC system.
  • the system consists of a pump unit (Surveyor LC Pump) that pushes the mobile phase to the column, auto sampler (Surveyor Autosampler) that automatically takes the sample and injects it into the column, and detector (Surveyor UV/Vis Detector) units that analyse the material passing through the column at the appropriate wavelength. Drawing of chromatograms and analysis of peaks were done with ChromQuest 4.2.34 program. Shimadzu UV-1601 spectrophotometer and quartz vessels were used for spectrophotometric quantification. Properties of the HPLC method used:
  • Validation of the determination methods used According to the Q2 (Rl) guideline of the International Conference on Harmonization (ICH), the aim of validation is to prove the suitability of the process for its intended use in analytical procedures. In the recognition according to the guideline, it is necessary to validate the methods used in determination of the amount of active substances within a medication or a sample in limit tests used for the determination and control of impurities. Apart from this, validation should be done again when there is a change in the synthesis of the active substance, the composition of the final product or the analytical method. Validation of the analytical method used in the determination of the amount was performed as described in the ICH guidelines.
  • the % standard deviation between measurements, relative standard deviation and confidence interval should be specified.
  • the relative standard deviation is found by dividing the standard deviation by the mean and multiplying it by one hundred.
  • Relative standard deviation should be 2% or less is to ensure desirable accuracy.
  • Three solutions were each prepared at a concentration of 4 ⁇ g/mL (80%), 5 ⁇ g/mL (100%), 6 ⁇ g/mL (120%) for HPLC method, and 40 ⁇ g/mL (80%), 50 ⁇ g/mL (100%), 60 ⁇ g/mL 120% (120%) for spectrophotometric method, and measurements were made three times in the relevant device. Relative standard deviation values between measurements were calculated. The precision study was repeated three times on different days to validate the between-days precision.
  • System suitability testing is an integral part of many analytical procedures, and the system suitability test parameters to be established for a particular procedure depend on the type of method. Values like retention time, peak area, theoretical plate number, tailing factor, their mean and relative standard deviation for HPLC are important for instance. Relative standard deviation of values such as retention time and peak area should be ⁇ 1%, tailing factor should be ⁇ 2, and theoretical plate numbershould be > 2000.
  • LOD and LOQ can be determined by different approaches.
  • LOD is mathematically calculated by dividing the residual standard deviation ( ⁇ ) of the regression line by the slope of the regression line and multiplying it by 3.3, and LOQ is calculated by multiplying it by 10.
  • Solubility Data of Oxaliplatin In various organic solvents, water and buffer solutions at various pHs used throughout the invention, the solubility of oxaliplatin was investigated. Solubility studies of oxaliplatin were carried out in organic solvents such as methanol, ethanol, dimethylformamide (DMF), and dichloromethane (DCM), distilled water and seven buffer solutions at pH 2, 4, 6, 7, 8, 10, 12 and are shown in Figure 1. Determination of solubility was realised according to the shake flask method. First, solid oxaliplatin is added to the glass vials in excess rates. Then, lmL of the medium for solubility test is added into the vials. Three vials are prepared in this way for each solvent.
  • organic solvents such as methanol, ethanol, dimethylformamide (DMF), and dichloromethane (DCM)
  • the vials are mixed at room temperature for 24 hours. At the end of the period, solid oxaliplatin that is taken into tubes and not dissolved were centrifuged at 10,000rpm for 15 minutes to precipitate. At the end of the centrifuge, the supernatant is removed and filtered through a 0.45 pm nylon or PTFE membrane filter. If necessary, the amount is determined by dilution. The results are stated below.
  • Dendrimeric formulations of oxaliplatin were prepared with two different approaches: physical complex and chemical conjugate formulations.
  • PAMAM-G3.5 and PAMAM-G4.5 dendrimers with anionic structures were used. Therefore, it was possible to examine the effects of dendrimer generation and preparation alterations on formulations. The abbreviations given to the formulations are given below.
  • dendrimeric oxaliplatin formulations to be prepared by physical complexation are expected to have a property to of immediate release
  • formulations to be prepared by chemical conjugation are expected to release the active substance more slowly and in a longer time.
  • Dendrimeric complexes of oxaliplatin FK-3.5 and FK-4.5
  • FK-3.5 and FK-4.5 Dendrimeric complexes of oxaliplatin
  • Dendrimeric conjugates of oxaliplatin were prepared by chemical reaction. Basically, it is aimed to establish an ester bond between the carboxyl groups on the surface of half-generation dendrimers and the hydroxyl groups obtained by modifying oxaliplatin. The establishment of this ester bond was accomplished by the esterification reactions shown above and described by Wolfgang Steglich. In Steglich esterification, N, N 1 - dicyclohexylcarbodiimide (DCC) was used as the binding agent, 4-(dimethylamino) pyridine (DMAP) was used as the catalyst, and dichloromethane (DCM), an aprotic solvent, was used as the reaction medium. Rl, R2, R3, R4 symbolize different chemical structures.
  • DCC N 1 - dicyclohexylcarbodiimide
  • DMAP 4-(dimethylamino) pyridine
  • DCM dichloromethane
  • the 3 and 4 generation PAMAM dendrimers are -NH2 terminated cationic dendrimers
  • the 3.5 and 4.5 generation dendrimers -COOH are terminated anionic dendrimers.
  • Cationic dendrimers are known to have higher toxicity compared to anionic dendrimers. The underlying reason of it is that the cationic ends interact with the negatively charged cell membrane and thus destabilize it. In addition to toxicity, haemolytic and aggregate activities of cationic dendrimers are also much more clear than anionic ones. Cationic dendrimers are not preferred as it is not desirable for dendrimers to interact with the membranes of normal cells and disrupt their structure until they are removed from the body after being separated from their platinum structure.
  • Cationic dendrimers used in the methods of prior art are disadvantageous due to their toxicity.
  • PAMAM type cationic dendrimers used demonstrate haemolytic activity at concentrations above lmg/mL. It has been shown that even at non-haemolytic doses as low as 10 ⁇ g/mL, they cause alterations in erythrocyte morphology and cause a transformation from disc shape to spherical shape.
  • anionic dendrimers no haemolytic activity or a alteration in erythrocyte morphology was observed even at high concentrations like 2mg/mL.
  • drug carrier systems it is desired that the structure that carries the drug is compatible with the body and non-toxic as much as possible. The remaining part not causing any toxicity in the body after performing its main duty of drug carrying will increase the benefit/harm ratio of the formulation. Therefore, anionic 3.5 and 4.5 generation dendrimers (G3.5 and G4.5) are preferred in the invention.
  • the pKa values of the primary amines on the surface of full generation PAMAM dendrimers vary between 7 and 9
  • the pKa values of the tertiary amines between the branches are between 3-6. Therefore, in mediums with different pH values, the ionization states of the functional groups of dendrimers differ.
  • the primary and tertiary amines of PAMAM dendrimers are in not protonated statuses. While only primary amines are protonated at near neutral pH, both primary and tertiary amines are protonated at low pH.
  • the pKa value of the carboxylic acid surface groups found in half-generation dendrimers is approximately between 4-5.
  • physical dendrimer complex formulations are prepared by adding different rates of oxaliplatin to different mediums.
  • formulations were prepared in in three different media (distilled water, pH 4 acetate buffer and pH 7 phosphate buffer) and five different oxaliplatin dendrimers.
  • a basic buffer was not used as a charge medium because media with high hydroxyl ions can cause stability problems in oxaliplatin.
  • Dendrimeric conjugates of oxaliplatin were prepared by chemical reaction. Basically, it is aimed to establish an ester bond between the carboxyl groups (- COOH) on the surface of half-generation dendrimers and the hydroxyl groups (-OH) obtained by modifying oxaliplatin. The establishment of this ester bond was realised by esterification reactions described by Wolfgang Steglich. In Steglich esterification, ⁇ , ⁇ '- dicyclohexylcarbodiimide (DCC) is used as the binding agent, 4-(dimethylamino)pyridine (DMAP) is used as the catalyst, and dichloromethane (DCM), an aprotic solvent, is used as the reaction medium.
  • DCC carboxyl groups
  • DMAP 4-(dimethylamino)pyridine
  • DCM dichloromethane
  • DCM N,N-dimethylformamide
  • Synthesis of physical complexes generally comprises the steps of;
  • FK-3.5 formulations In order to prepare FK-3.5 formulations, first 25mg of oxaliplatin was weighed separately for each and dissolved in 5 mL water, pH 4 and pH 7 mediums. By this way, a stock solution of oxaliplatin at a concentration of 5mg/mL in three different media was prepared. The amounts of stock solution and methanolic G3.5 dendrimer solution required to prepare 25:1, 50:1, 75:1, 150:1 and 250:1 ratios, respectively, are summarized in Table 5 below.
  • the molecular weight of oxaliplatin and G3.5 dendrimer was taken as 397.286 g/mol and 12927.69 g/mol, respectively, and the density of 10% (w/w) solution of G3.5 dendrimer was 0.810 g/mL as specified by the manufacturer, and ⁇ mol values are rounded to 2 digits after the decimal point.
  • the amovmt of stock solution and dendrimer solution indicated in the table is added to a glass vial and mixed for 24 hours on a magnetic stirrer. At the end of the period, the solvent medium is evaporated. The residue is suspended in anhydrous ethanol. It is centrifuged at 10,000rpm for 5 minutes to precipitate oxaliplatin that did not enter the branches of the dendrimers and complex. At the end of the centrifuge, the supernatant is removed and filtered through a 0.45 pm nylon or PTFE membrane filter. An aqueous complex solution is obtained by evaporating the ethanol of the filtrate and dissolving the residue in water. Measurements are made with this solution.
  • the molecular weight of oxaliplatin and G4.5 dendrimer was taken as 397.286 g/mol and 26251.86 g/mol, respectively, and the density of 10% (w/w) solution of G4.5 dendrimer was 0.791 g/mL as specified by the manufacturer, and ⁇ mol values are rounded to 2 digits after the decimal point.
  • the amount of stock solution and dendrimer solution indicated in the table is added to a glass vial and mixed for 24 hours on a magnetic stirrer. At the end of the period, the solvent medium is evaporated. The residue is suspended in anhydrous ethanol. It is centrifuged at 10,000rpm for 5 minutes to precipitate oxaliplatin that did not complex with the dendrimers.
  • KK-3.5 formulations were synthesized by a two-step reaction. In the first step, the modification of oxaliplatin was carried out, and then the binding of this compound to the G3.5 dendrimer was carried out by the Steglich esterification reaction as previously described.
  • Synthesis of chemical conjugates with G 3.5 dendrimer generally comprises the steps of:
  • oxaliplatin 20mL of acetic acid is added onto 1,192g (3mmol) of oxaliplatin and suspended on a magnetic stirrer. 250pL of 30% H2O2 solution (perhydrol) is added onto the suspension. 30% H2O2, 250pL and 750pL, is added with 30 minutes intervals, respectively. Within 1,5 hours the suspension turns into a yellowish and almost clear solution. This solution is stirred overnight. The acetic acid is evaporated and removed from the medium and the oily residue is suspended again with 40mL of distilled water to form a turbid mixture. The added water is removed by evaporation. This process (water addition and removal) is repeated 2 more times until the product becomes solid.
  • the solid product obtained is suspended again with some (10-20mL) ethyl acetate. Methanol is added to this suspension until a clear solution is formed and until it is seen adding more methanol does not change the appearance of the solution.
  • the mixture is filtered through a 0,45 pm nylon membrane filter. The solvent mixture is removed from the medium.
  • the slightly yellowish product obtained is stored at +4°C to be used in the preparation of conjugates.
  • the chemical synthesis reaction of the modification of oxaliplatin is shown below.
  • Dendrimer conjugation 250pL (l,566 ⁇ mol) is taken from the methanolic solution (10% w/w) of the G3.5 dendrimer. The methanol is evaporated under vacuum and the carboxyl ends are activated by dissolving the residue in HC1 solution with pH 3. After evaporating the water, the dried dendrimers are dissolved again in 6mL of DMF. 60mg (126,8 ⁇ mol) modified oxaliplatin, 26,2mg (126,8 ⁇ mol) DCC and 15,5mg (126,8 ⁇ mol) DMAP are added onto this solution. The mixture is stirred under vacuum for 24 hours at room temperature.
  • the precipitates resulting from DCC (for example, dicyclohexylurea-DCU) formed during the reaction are removed by filtrating through a 0,45 pm nylon membrane filter.
  • the solvent medium is evaporated.
  • the remaining residue is dissolved in distilled water and filtered again through a 0,45 pm nylon membrane filter if needed. It is extracted 5 times with 25mL DCM to remove the hydrophobic organic substances that remained from the reaction.
  • the aqueous part is purified and separated by dialysis to be lyophilized.
  • KK-4.5 formulations were also synthesized by a two-step reaction like KK-3.5 formulation.
  • the modification of oxaliplatin was done as described in the preparation of the KK-3.5 formulation.
  • 500pL (0,753 ⁇ mol) is taken from the methanolic solution (5% w/w) of G4.5 dendrimer. The methanol is evaporated under vacuum and the carboxyl ends are activated by dissolving the residue in HC1 solution with pH 3. After evaporating the water, the dried dendrimers are dissolved again in 5mL of DMF.
  • DMAP are added on to this solution.
  • the mixture is stirred under vacuum for 24 hours at room temperature.
  • the precipitates resulting from DCC for example, dicyclohexylurea-DCU
  • the solvent medium is evaporated.
  • the remaining residue is dissolved in distilled water and filtered again through a 0.45 pm nylon membrane filter if needed. It is extracted 5 times with 25mL DCM to remove the hydrophobic organic substances that remained from the reaction.
  • the aqueous part is purified by dialysis and separated to be lyophilized.
  • Lyophilization is performed by freezing the aqueous solution at -80°C for 3 hours, keeping it at -40°C for 22 hours at 0,01mbar pressure, and at -20°C for 3 hours at 0,00 lbar pressure.
  • the obtained lyophilized conjugate is stored at +4°C to be used in characterization and cell culture studies.
  • the molar ratio of the reactants to the dendrimer is approximately 140:1. Therefore, it was possible to bind all 128 surface groups of the G4.5 dendrimer with the active substance.
  • the reaction of the synthesis is shown below.
  • UV -Vis Ultraviolet- Visible Spectroscopy
  • FT-IR Fourier Transform Infrared
  • NMR Nuclear Magnetic Resonance
  • the residual standard deviation ( ⁇ ) of the regression line and the slope of the regression line were found for both methods.
  • LOD and LOQ were calculated with the formulas described in the methods section. Accordingly, the LOD values for HPLC and spectrophotometric methods were found to be 0, 1 ⁇ g/mL and 1,1 ⁇ g/mL, respectively, and the LOQ values were found to be 0,3 ⁇ g/mL and 3,3 ⁇ g/mL, respectively.
  • FT-IR Fourier transformed infrared
  • FT-IR Fourier transformed infrared
  • KK-3.5 has peaks belonging to both dendrimer and modified oxaliplatin functional groups to which it is conjugated, due to the free dendrimer ends that are not conjugated with the active substance.
  • Pt-0 stretching peak was observed in the fingerprint region 573cm -1 .
  • KK-4.5 Formulation Using the Steglich esterification reaction, the G4.5 dendrimer and modified oxaliplatin was connected by ester bonds. Reaction yield was 46,6%. To show that the desired compound was synthesized, characterization studies were conducted. i) Fourier transformed infrared (FT-IR) spectroscopy: The FT-IR spectra of the modified oxaliplatin, PAMAM-G4.5 dendrimer and KK-4.5 formulation are shown in Figure 16. The FT-IR spectrum of the KK-4.5 formulation showed that the functional groups seen in the modified oxaliplatin and G4.5 dendrimer are also present in the conjugate structure.
  • FT-IR Fourier transformed infrared
  • Particle Size-Distribution and Zeta ( ⁇ ) Potential Measurements, particle size and polydispersity index (PDI) data of G3.5 and G4.5 PAMAM dendrimers in methanol and water, and of chemical conjugate formulations in water are given in the tables below.
  • the invention macromolecule sizes can be up to lOOOnm, PDI values can be up to 0.6, and the zeta potential range can be between -lOOmV +100mV.
  • the invention macromolecule sizes are between 10- lOOOnm, PDI values are between 0.05-0.6 and zeta potential values are between -lOOmV +100mV.
  • Table 20 Average size and polydispersity index data of dendrimers.

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Abstract

The invention relates to the anticancer medication containing oxaliplatin as the active substance, and to dendrimer oxaliplation complexes (prepared by physical methods) and oxaliplation derivative dendrimer conjugates (prepared by chemical methods) to decrease the release time and toxic effect of the said medication, and to the production methods thereof.

Description

OXALIPLATIN CONTAINING THERAPEUTIC POLYAMIDOAMINE (PAMAM) DENDRIMERS AND PRODUCTION METHOD THEREOF Technical Field of the Invention
The invention relates to the anticancer medication containing oxaliplatin as the active substance, and to the macromolecules with dendrimer oxaliplation complexes (prepared by physical methods) and oxaliplation derivative dendrimer conjugates (prepared by chemical methods) to decrease the release time and toxic effect of the said medication, and to the production methods thereof.
The invention has the potential to be used in first and second-line therapy for the treatment of cancer types such as metastatic colorectal cancer, non-Hodgkin lymphoma, breast cancer, non-small cell lung cancer, head and neck cancer, mesothelioma and squamous cell carcinoma advanced ovarian cancer in which oxaliplatin is used.
State of the Art of the Invention (Prior Art) The anti-proliferative properties of platinum-derived compounds were first observed in the 1960s. Cisplatin, the first platinum compound used in cancer chemotherapy, was put into use in 1978 and became one of the most successful cancer drugs. Despite its success in routine use, its success has remained very low in patients who do not respond or are resistant to first- line treatment with cisplatin. In an effort to develop cisplatin, many different platinum analogues have been synthesized. For example, carboplatin is a second-generation platinum compound used in treatment. The main clinical success with the use of carboplatin is the observed reduction of nephrotoxicity compared to cisplatin. However, myelotoxicity and the inability of carboplatin to overcome cisplatin-induced resistance (cross-resistance) caused the quest to continue.
As one of the analogues synthesized in the continuation of the studies, oxaliplation was first obtained in the late 1970s by attaching a diaminocyclohexane (DACH) ring to the amine groups of cisplatin. Oxaliplatin is a platinum (Π) analogue similar to cisplatin and carboplatin. New drug application (NDA) for use in metastatic colorectal cancers was first accepted by the US Food and Drug Administration (FDA) in February 1999. Oxaliplatin, a DACH platinum derivative, has a similar mechanism of action to other platinum derivatives. However, its antitumour activity differs from other platinum derivatives such as cisplatin and carboplatin. For example, it has been demonstrated that oxaliplatin is effective against cisplatin-resistant colon carcinoma cell lines.
Cisplatin, carboplatin and oxaliplatin are platinum compounds that are accepted and used worldwide. Apart from these, there are also other platinum derivatives which have been regionally approved in various countries of the world, such as nedaplatin (Japan), lobaplatin (China), heptaplatin (South Korea) and some others such as picoplatin and satraplatin (Pt*4) that are still being developed and the phase studies of which still continue. Particularly octahedral structured platinum (TV) compounds, also called as fourth generation platinum compounds, are of interest as they are less reactive, and are more suitable than platinum (II) derivatives for oral administration.
Chemical and Physical Properties: Platinum compounds that are in clinical use worldwide (cisplatin, carboplatin, oxaliplatin) are also called classical platinum complexes. These are cis-structured, uncharged, square planar complexes in the +2-oxidation state (Pt+2) and are shown below.
Figure imgf000003_0001
The general formula describing these classical platinum complexes is cis- [A2PtX2], Here, A2 represents the nitrogen donor two monodentate or one bidentate ligand, and X2 represents two monodentate or one bidentate anionic ligands. Alteration of the A2 moiety results in the formation of structurally different DNA-drug complexes, and this alters the anticancer activity of the formed complexes. The X2 group, on the other hand, affects the distribution of the formed complexes in the body and thereby its side effects. Oxaliplatin (C8H14N2O4) is designed to overcome cellular resistance seen against cisplatin and carboplatin. The chemical structure of oxaliplatin, whose chemical nomenclature is (cis- [(1R,2R)- 1 ,2-cyclohexanediamine-N,N9] oxalato(2-)-0,09] platinum, differs from that of cisplatin or carboplatin by bonding of the cyclohexane ring to its nitrogen atoms. Oxaliplatin is a white/off-white crystalline structured powder with a molecular weight of 397.3 g/mol. It is solubility in water is low (6-7g/L at 20°C), in methanol is very low (0.125g/L at 20°C), and it is almost insoluble in ethanol or acetone. The pKa value is 6.1.
Effect Mechanism: Although the exact effect mechanism of oxaliplatin has not been able to be fully explained, it is thought that the cytotoxicity of platinum compounds results from the inhibition of DNA synthesis. The main lesions responsible for cytotoxicity are intrastrand cross-links between active platinum compounds and specific base sequences. These crosslinks are particularly between two adjacent guanine bases or adjacent guanine-adenine bases. Interstrand cross-links can also occur. However, they constitute less than 5% of the total platinum-DNA complexes. Approximately 60-65% of the cross-links formed between DNA and oxaliplatin are intrastrand guanine-guanines and 25-30% are intrastrand adenine-guanine links. Additionally, activation of the apoptosis pathway may contribute to this effect mechanism described. Oxaliplatin turns into active platinum compound by interacting with HCO3- (bicarbonate) and H2PO4- (dihydrogen phosphate) anions in physiological environment. When the activated platinum compound enters the cell, a chloride ion leaves this compound and causes a single aqueous chlorine complex (monoaquamonochloro complex) to form. This complex rapidly reacts with guanine at the N-7 position on DNA. By one more chlorine leaving the monochloro complex, a double aqueous complex is formed. The formation of the double aqueous complex from oxaliplatin is shown below.
Figure imgf000004_0001
With this event, as the conversion process of oxaliplatin to active platinum compounds is shown above, the transformation of complexes formed transiently and with one-point-link with DNA into more stable complexes with two-point-links occurs. Although the sites of interaction with DNA are the same for cisplatin, carboplatin, and oxaliplatin, the interaction kinetics is highly dependent on the chemistry of the ligands carrying the platinum group. Binding of oxaliplatin with DNA takes approximately 15 minutes in vitro. This time is longer than the binding duration of cisplatin. It is thought that the reason for this case is the N-Pt-N bond of the DACH complex. In terms of mobility, this bond has a more restricted structure than the bond of cisplatin and carboplatin complexes.
The DACH-Pt complex formed by oxaliplatin forms a more voluminous hydrophobic region than the cis-diamine-Pt complex formed by both cisplatin and carboplatin. As a result, oxaliplatin causes a more effective inhibition of DNA synthesis, resulting in higher cytotoxicity than the cis-diamino-Pt complex. Additionally, by creating various conformational problems, these voluminous complexes prevent the mismatch repair proteins responsible for DNA repair from binding to the region and initiating the DNA repair mechanism. The development of resistance to oxaliplatin can be explained by the cellular interactions of oxaliplatin. The various mechanisms that cause oxaliplatin resistance are summarized in Table 1.
Table 1. Various mechanisms causing oxaliplatin resistance
Figure imgf000005_0001
Figure imgf000006_0002
Biotransfonnation and Pharmacokinetic Properties: Oxaliplatin undergoes rapid nonenzymatic biotransformation to form various reactive platinum intermediates. The intermediates formed are rapidly and extensively bound to plasma proteins and erythrocytes. The active and inactive metabolites of oxaliplatin formed as a result of biotransformation are shown below. Half maximum inhibitory concentrations (IC50) are in vitro data for the HT-29 cell line.
Figure imgf000006_0001
The pharmacokinetics of unbound platinum in plasma after oxaliplatin administration is characterized by a short initial a and β distribution phase and a long terminal γ elimination phase. The main route of elimination is urinary excretion (53.8±9.1%), and a very small amount of platinum (2.1±1.9%) is excreted in the faeces. No platinum accumulation was observed in the plasma when administered as 85mg/m2 every 2 weeks or 130mg/m2 every 3 weeks, which are the routine use doses. Oxaliplatin exhibits a dose-dependent pharmacokinetics in the dose range of 20-180mg/m2. Studies to investigate oxaliplatin metabolism with human liver microsomal extracts have shown that oxaliplatin is not a substrate for CYP450 in vitro. The critical pharmacokinetics parameters of oxaliplatin such as peak concentration (Cmax), area under the curve (AUC), half-lives for the three elimination phases (tl/2), virtual volume of distribution (Vd), and clearance (Cl) obtained from the ultrafiltrate of blood plasma are shown in Table 2.
Table 2. Critical pharmacokinetics parameters of oxaliplation
Figure imgf000007_0001
Cancers where it is used and the Mode of Administration: The focus point of oxaliplatin in clinical use is metastatic colorectal cancer. However, the utility of oxaliplatin as first-line and second-line therapy for the treatment of advanced ovarian cancer is under investigation by clinical trials. In addition, the activity of this drug in other malignancies such as non- Hodgkin lymphoma, breast cancer, non-small cell lung cancer, head and neck cancer, mesothelioma and squamous cell carcinoma is also being investigated. Oxaliplatin is used in combination with 5-FU and calcium folinate (calcium salt of leucovorin) in colorectal cancers. 5-FU is a pyrimidine antagonist and is used together with oxaliplatin to achieve a synergistic effect. When calcium folinate is used with 5-FU, it has been observed that the antineoplastic activity is strengthened.
To reach a concentration between 0.2-0.7mg/mL during administration, dilution with 250- 500mL of 5% dextrose solution should be made. 0.9% NaCl or other electrolyte solutions should not be used as a dilution solution. The diluted solution is administered to the patient as intravenous infusion over 2-6 hours.
Side Effects: The clinical toxicity seen due to the use of oxaliplatin differs from other platinum group drugs. Nephrotoxicity and hematotoxicity seen in other platinum-derived drugs are minimal in oxaliplatin. Oxaliplatin rapidly causes a reversible acute neuropathy in 90% of cases. Neurotoxicity is actually a side effect specific to all platinum-containing antineoplastic agents. While this effect is generally tolerated in oxaliplatin, tetraplatin for example, one of the third-generation platinum compounds, had to be withdrawn from the market due to its severe neurotoxic effects. No significant neurotoxicity is usually observed in patients receiving carboplatin. However, the reason of this is the fact that the haematological toxicity of carboplatin has a dose-limiting effect.
Acute neuropathies are characterized by paresthesia and dysesthesia in the hands, feet, and peroral region that occur during or shortly after the infusion. However, although less frequently it happens, they can also cause cumulative distal neuropathy. Events such as sensory ataxia, functional disorders, jaw pain, eye pain, ptosis, leg cramps, and changes in vision or voice may occur. Neuropathies seen in association with cumulative oxaliplatin affect all patients receiving a total dose of 540 mg/m2 or more for four or more cycles. While reversible neuropathies regress within 4-6 months in 82% of patients, 41% recover completely within 6-8 months. The cause of sensory-motor neuropathy caused by oxaliplatin is mainly due to hyperexcitability. Peripheral neuropathy due to oxaliplatin is the result of the drug's action on sensory, motor neurons, or muscles, while cumulative chronic distal neurotoxicity is due to damage to neurons in the dorsal root ganglia. To protect patients from neuropathic side effects during oxaliplatin treatment, patients were given additional drugs such as amifostine (cytoprotective), a-lipoic acid (antioxidant), glutathione (antioxidant), calcium-magnesium ions (oxalate chelator), carbamazepine, oxcarbazepine, topiramate, and gabapentin (antiepileptic) to take advantage of their neuroprotective properties and the clinical studies were carried out. However, discussions in this matter still continue. Other side effects seen are various haematological side effects, gastrointestinal disturbances, and various allergies. Haematological side effects are the rarely seen low severity anaemia, thrombocytopenia, and neutropenia. As they are not severe, these are often manageable cases. Nausea and vomiting are gastrointestinal side effects frequently seen with most other chemotherapeutic agents. Nausea and vomiting due to oxaliplatin are mild to moderate and can be easily overcome with 5-hydroxytryptamine (5HT3) receptor antagonists given as premedication before treatment. Allergic reactions to oxaliplatin, like in nausea and vomiting, can be prevented with antihistamines and glucocorticoids given as pre-medication. Even when used with 5-FU, the low probability of alopecia is an important advantage for patients' quality of life. Pharmaceutical preparations in the Turkish Market: as of 2019 there are 32 licensed preparations in Turkey containing oxaliplatin as the active substance (Table 3). There are introduced into market as vials containing 50mg and lOOmg lyophilised powder or 50mg/10mL, 100mg/20mL, 150mg/30mL, 200mg/40mL concentrated solution (RX Media Pharma Interactive Medication Information Source (Computer Program). Version 1.94 Izmir (TUR): GEMA§ GMM A.§.:2019.).
Table 3. Pharmaceutical preparations of oxaliplation in Turkish market.
Figure imgf000009_0001
Delivery of anticancer drugs such as oxaliplatin with carrier-based systems attracts attention as it increases drug efficacy, reduces side effects and prevents the development of cellular resistance. In previous studies, it has been demonstrated that there are changes in the side effects of platinum-derived cytotoxic drugs when they are prepared in different pharmaceutical forms and in combination with different active substances. Liposomal cisplatin (Lipoplatin) and liposomal oxaliplatin (Lipoxal) are some of the promising particulate delivery systems that have come to the clinical trial stage. Apart from liposomes, polymeric conjugates and dendrimers can also be used as carrier systems. Dendrimers are nano-sized, branched, three-dimensional molecules that mimic the biological structure consisting of synthetic polymers and have promising properties in the field of diagnosis and treatment. Unlike conventional polymers, they have a three-dimensional structure with low polydispersity, with long or spiral branching around the central core. Formed by branching on the functional groups in the core, these molecules can be given the desired shape and properties by altering the core structure or the functional groups added to the outer surface. The ability of dendrimers to be designed as desired has given them with a application area in the medical field.
In the prior art, it is known that dendrimers with platinum (TV) complex form a conjugate and show less toxic effects than pure oxaliplatin and these pharmaceutical compositions could have been used in cancer treatment.
The molecules in the study of Nadine S. Sommerfeld et al. related to obtaining dendrimer- platinum conjugates from G-2 and G-4 type PAMAM dendrimers and platinum (IV) complexes with a less toxic effect than pure oxaliplatin by forming conjugate of dendrimers with platinum (IV) complex are shown below.
Figure imgf000010_0001
In the study, 2nd and 4th generation PAMAM dendrimers with amine (-NH2) termination were used. Oxaliplatin is converted to the platinum (TV) complex by asymmetric oxidation before being conjugated with these dendrimers. Then, this platinum (TV) compound was reacted with succinic anhydride to form a free carboxyl (-COOH) group. Conjugation was carried out by connecting the -NH2 groups of the dendrimer to the -COOH group on the platinum (TV) complex. Sommerfeld et al. conjugated dendrimer and platinum (TV) compound via a succinato bridge. That is, dendrimer-platinum conjugates cannot be obtained directly, without the need for any linker. There are several disadvantages of using linkers. The use of linkers causes an increase in the number of steps in the synthesis processes, thereby causing the need for new processes such as synthesis and purification for the additional steps. Additional purification steps often cause a decrease in the yield of the final product. Additional processes complicate the synthesis process and cause a waste of time. All these situations considered together, the cost of the product increases. Both in structures with and without linkers, the structure responsible for the cytotoxic effect of the final product is the platinum group. The platinum group responsible for the cytotoxic effect is released by enzymatic reactions inside the cell. The rest of the molecule acts as a carrier and is not directly related to the cytotoxicity of the molecule.
Similar to Sommerfeld et al., in the patent document no WO2015035446A1, which is about the dendrimer with amino groups on the nucleus and surface, and macromolecules containing one or more platinum attached to the dendrimer amino groups, amine (-NH2) terminated dendrimers are conjugated with various cytotoxic platinum compounds through linkers. The dendrimers used have -NH2 termination and in the said patent, the conjugation with the dendrimer was performed over polydentate ligands. It is said that pharmaceutical compositions containing these macromolecules can be used in cancer treatment. However, it is known that dendrimers with -NH2 termination or more generally + charged groups when ionized have higher toxicity compared to anionic dendrimers with carboxyl groups on the surface or neutral dendrimers with hydroxyl groups on the surface. The underlying reason of it is that the cationic ends interact with the negatively charged cell membrane and thus destabilize it. In addition to toxicity, haemolytic and aggregate activities of cationic dendrimers are also much more clear than anionic ones. Cationic dendrimers are not preferred as it is not desirable for dendrimers to interact with the membranes of normal cells and disrupt their structure until they are removed from the body after being separated from their platinum structure. Unlike the carrier dendrimer structure, polydentate or monodentate ligands do not have a direct effect on toxicity. Although there are many methods in the previous art, there is a need to develop innovative methods for modifying chemical conjugates to bind dendrimer to oxaliplatin so as to prepare dendrimers in which oxaliplatin is the active substance, and various dendrimeric pharmaceutical formulations derived from oxaliplatin. Cationic dendrimers used in the methods of prior art are disadvantageous due to their toxicity. PAMAM type cationic dendrimers used demonstrate haemolytic activity at concentrations above lmg/mL. It has been shown that even at non-haemolytic doses as low as 10μg/mL, they cause alterations in erythrocyte morphology and cause a transformation from disc shape to spherical shape. In anionic dendrimers, no haemolytic activity or a alteration in erythrocyte morphology was observed even at high concentrations like 2mg/mL. In drug carrier systems, it is desired that the structure that carries the drug is compatible with the body and non-toxic as much as possible. The remaining part not causing any toxicity in the body after performing its main duty of drug carrying will increase the benefit/harm ratio of the formulation. In addition to this advantage, there is a need for rapid and low-cost production of oxaliplatin-dendrimer complexes and conjugates that bind to the platinum carrier dendrimers without linker groups, and thereby shortening the process steps without the use of additional chemicals and additional purification processes.
Brief Description and Aims of the Invention
The main aim of the invention is to develop various dendrimeric formulations with effects similar to oxaliplatin which may be candidates for use in cancer treatment, in vitro examination of their various properties, and to validate their production. Dendrimers can increase the solubility of active substances by various mechanisms. In this study, G 3.5 and G 4.5 PAMAM dendrimer were used, and it was seen that both of them showed an increasing effect on oxaliplatin solubility. It was also found that this effect has increased linearly with dendrimer concentrations. Comparing between the dendrimer generations, it was seen that the G4.5 generation dendrimers increased the solubility statistically significantly more than the G3.5 generation ones.
Dendrimer oxaliplatin physical complexes were prepared by co-precipitation method and oxaliplatin derivative dendrimer conjugates were prepared by Steglich esterification reactions. The charge properties of the physical complexes were examined, and it was observed that they differed according to the dendrimer concentration and pH values of the medium. In general, it was observed that as the dendrimer concentration increased, the charge rate increased, and the charge efficiency decreased.
It has been observed that G4.5 dendrimers can charge 2 to 5 times more oxaliplatin per mole than G3.5 dendrimers.
The amount of substance conjugated to dendrimer ends in chemical conjugates was determined by1Η -NMR. 37.9 molecules were bound to G3.5 conjugate and 26.3 molecules were bound to G4.5 conjugate per 1 mole of dendrimer.
Particle size and distribution, and zeta potential values of the G3.5, G4.5 dendrimers used, and the conjugate formulations prepared were examined.
When measured directly with dendrimers in methanol, it was observed that the particle sizes were small (<10nm) and the size distributions were in a narrow range (PDK0.25). When the dendrimers were dispersed in water by evaporating methanol, the particle size increased significantly (200-250nm) and the dispersion got worse (PDIX).40). The dimensions of the chemical conjugates in water were found to be 179.30 ± 77.30 for G3.5 and 155.93 ± 13.59 for G4.5, and it was seen that they have an average polydispersity of 0.377 and 0.340, respectively. In the zeta potential measurements, G3.5 and G4.5 dendrimers were found to be negatively charged as they have carboxyl structured anionic surface groups. With the conjugation of the active substance to the dendrimers, the surface charge changed from negative to positive.
Release studies were carried out with the characterized formulations in two different media with pH 5.5 and pH 7.4. Release studies were carried out by the dialysis membrane method. According to the release studies, the release time of all substances placed in the membrane took an average of 30 minutes for free oxaliplatin, an average of 60 minutes for physical complexes, and an average of 120 minutes for chemical conjugates. Pure oxaliplatin was seen to tend to be released more rapidly at pH 7.4. The release profiles were compared using the fl difference factor and f2 similarity factor.
Comparing the chemical conjugate formulations among themselves, it was seen that there was no significant difference between the release profiles. In the physical complex formulations, it was seen that the release profile of the F6 formulation obtained in a pH 5.5 medium was significantly faster than the others. The concord of the release profiles to various release kinetic models was examined and it was seen that the kinetic with the highest concord was generally Weibull.
With the KK-3.5 formulation lastly selected within the scope of the invention, an efficacy study was conducted in the HT-29 colon cancer cell line. In addition to the KK-3.5 formulation, the cytotoxicities of pure oxaliplatin and empty G3.5 dendrimers were also examined. According to the study, after 48 hours, it was seen that the cells to which pure G3.5 dendrimers were applied demonstrated more than 90% viability even at the highest dose (256μΜ = 3.321mg/mL). This shows that the toxicity of G3.5 dendrimers alone is quite low. By applying non-linear regression to the data, the IC50 values of pure oxaliplatin and KK3.5 conjugates were found to be 14.03 μΜ and 0.72μΜ, respectively.
In addition, the smaller Hill slope in the KK-3.5 formulation indicates a faster response to dose increase.
With the preparation of various dendrimeric pharmaceutical formulations derived from oxaliplatin, and validation of the production method, it was aimed to help reduce the temporary acute syndrome characterized by muscle cramps and spasms that can be seen due to oxaliplatin treatment with dose reduction, and dose-limiting neuropathies such as sensory disorders that affect the patient's normal life, paresthesia in the extremities, and muscle coordination disorders as well as the cancer treatment.
Compared to the work of Nadine S. Sommerfeld et al., the dendrimers used in the invention are 3.5 and 4.5 generation PAMAM dendrimers with carboxyl (-COOH) termination. In the invention, the conjugation is realised by bonding the hydroxyl (-OH) groups of the platinum (TV) complex with these -COOH groups. The dendrimers used are different from the study of Sommerfeld et al. in terms of both generation and end groups. While conjugating the dendrimer and the platinum (TV) compound, Sommerfeld et al. have bound the -NH2 groups of the dendrimer with the -COOH group of the succinato group and realised the conjugation through a succinato bridge. In the invention, conjugation was realised over -COOH and -OH groups. Conjugation was realised directly without the use of any linker molecules. The use of linker leads to an increase in the number of steps in the synthesis processes, and the increase in the number of steps causes the need for new processes such as synthesis and purification for the additional steps. Additional purification steps often lead to a decrease in the yield of the final product. Additional processes complicate the synthesis process and cause a waste of time. All these situations considered together; it is inevitable for the cost of the product to increase. The elimination of the use of additional chemical substances and processes such as additional purification due to the increase in the step plays a role in reducing both monetary and temporal costs of the formulation.
Compared to the WO2015035446A1 patent document, the dendrimers used in the invention are 3.5 and 4.5 generation PAMAM dendrimers and have carboxyl (-COOH) termination. In addition to the end groups of the dendrimers used in the invention being different, the way of conjugation with the dendrimer also differs. Although the conjugation with the dendrimer is realised over polydentate ligands in the said patent document, in the invention the conjugation is realised over -OH groups, in other words, over the monodentate ligand. Conjugation directly over the -OH group eliminates the need to use an linker molecule between the platinum-containing structure and the dendrimer.
Briefly, the invention solves the problem of toxic and haemolytic risks that may occur due to cationic dendrimer that could not have been solved in the prior art or that may occur, and at the same time, it meets the need for an effective and more cost-effective pharmaceutical form by providing a cost advantage by reducing the number of steps in the synthesis process.
Description of Drawings Describing the Invention
Figure 1: Oxaliplatin Solubility A. Oxaliplatin solubility in various solvents (mg/ml), B. Oxaliplatin solubility at various pHs (mg/ml) Figure 2: Effect of dendrimers on solubility
Figure 3: Process steps of obtaining dendrimer-oxaliplatin aqueous complex solution Figure 4: Chromatograms of pure oxaliplatin, PAMAM-G3.5 and PAMAM-G4.5 dendrimer solutions and blank solutions prepared without active substance Figure 5: Spectrums between 200-400nm A. Oxaliplatin, B. PAMAM G3.5, C. PAMAM
G4.5
Figure 6: Oxaliplatin standard curves A. HPLC standard curve, B. Spectrophotometer standard curve
Figure 7: Spectra between 200-400nm A. Oxaliplatin, B. The fact that the oxaliplatin peak seen in oxaliplatin-PAMAM G3.5 Complex (F6) at 255nm was suppressed in the F6 formulation indicates that oxaliplatin forms a complex with the dendrimer.
Figure 8: FT-IR spectra of the F6 formulation
Figure 9: Spectra between 200-400nm A. Oxaliplatin, B. The fact that the oxaliplatin peak seen in oxaliplatin-PAMAM G4.5 Complex (F21) at 255nm was suppressed in the F21 formulation indicates that oxaliplatin forms a complex with the dendrimer.
Figure 10: FT-IR spectra of the F21 formulation
Figure 11: FTIR spectra of pure oxaliplatin and synthesized modified oxaliplatin Figure 12: 1Η-ΝΜΚ results of modified oxaliplatin Figure 13: 13C-NMR results of modified oxaliplatin Figure 14: FT-IR spectra of modified oxaliplatin, PAMAM-G3.5 dendrimer and KK-3.5 formulation
Figure 15: 1Η -NMR (DMSO-d6) results of KK-3.5 formulation A. 1HNMR spectra of the conjugate, B. 1HNMR spectra of modified oxaliplatin
Figure 16: FT-IR spectra of modified oxaliplatin, PAMAM-G4.5 dendrimer and KK-4.5 formulation
Figure 17:1Η -NMR (DMSO-d6) results of KK-4.5 formulation A. 1HNMR spectra of the conjugate, B 1HNMR spectra of modified oxaliplatin Figure 18: Amounts of Oxaliplatin Complexed with 1 mole of Dendrimer Figure 19: Release Data A. Release data of F6 Formulation at pH 5.5 and pH 7.4, B. Release data at F21 Formulation pH 5.5 and pH 7.4
Figure 20: In vitro release data of chemical conjugate formulations
Figure 21: As a result of the study carried out on the HT-29 cell line, the viability data (%) of PAMAM G3.5 dendrimer, oxaliplatin and KK-3.
Figure 22: IC50 values of oxaliplatin and KK-3.5 formulation A. Oxaliplatin IC50, B. KK-3.5 conjugate IC50
Detailed Description of the Invention The macromolecule of the invention comprises; • G3.5 or G.4.5 generation PAMAM dendrimer with at least one carboxyl structure (- COOH) anionic surface groups,
• One or more oxaliplatin, the monodentate ligand of which is modified to be the -OH group, and is characterized with -OH groups of one or more oxaliplatin modified to be -OH group of monodentate ligand directly bonding with at least one carboxyl structure (-COOH) anionic surface groups in the structure of G3.5 or G.4.5 generation PAMAM dendrimer.
The main aim of the invention is to develop various dendrimer formulations with effects similar to oxaliplatin that may be candidates for use in cancer treatment and in vitro examination of their various properties. Dendrimers can increase the solubility of active substances by various mechanisms. Within the scope of the invention, two different generations of dendrimers (G 3.5, G4.5) were used and it was observed that both had an effect on increasing oxaliplatin solubility. It was also found that this effect increased linearly with dendrimer concentrations. Comparing the dendrimer generations, it was seen that the G4.5 generation dendrimers increased the solubility statistically significantly more than the G3.5 generation ones.
Dendrimer-mediated solubility increase depends on several factors. These are properties like the structure of the core and surface groups, concentration and generation of the dendrimer, the pH of the solution, and the type of solvent. Increasing dendrimer generation and dendrimer concentration seems to increase oxaliplatin solubility. The reason why the solubility increases in G4.5 dendrimers is higher than that in G3.5 dendrimers under similar ambient conditions can be explained by the increase in the number of surface groups that can interact with oxaliplatin in G4.5 dendrimers.
Within the scope of the invention, oxaliplatin derivative dendrimer conjugates were prepared with dendrimer oxaliplatin physical complexes and Steglich esterification reactions by coprecipitation method. Formulations prepared using various methods have been characterized. The charge properties of the physical complexes were examined, and it was observed that they differed according to the dendrimer concentration and pH values of the medium. In general, it was observed that as the dendrimer concentration increased, the charge rate increased, and the charge efficiency decreased. It has been observed that G4.5 dendrimers can charge 2 to 5 times more oxaliplatin per mole than G3.5 dendrimers. In the studies carried out within the scope of the invention, the ratio of active substance/dendrimer charged with the highest active substance per mole was found to be 25. Here, the amount of active substance is 1.26μmol and the amount of dendrimer is O.OSμmol. The amount of substance conjugated to dendrimer ends in chemical conjugates was determined by 1H-NMR. 37.9 molecules were bound to G3.5 conjugate and 26.3 molecules were bound to G4.5 conjugate per 1 mole of dendrimer. It is thought that this situation may be caused by the molecular density formed around the dendrimer ends by the increase in dendrimer generation. It is known that more active substances can be prevented from reaching the dendrimer ends and reacting due to the steric obstacles formed during the chemical reaction.
Particle size and distribution, zeta potential values of G3.5, G4.5 dendrimers and conjugate formulations used in the invention were examined. When measured directly with commercially available dendrimers in methanol, it was observed that the particle sizes were small (<10nm) and the size distributions were in a narrow range (PDK0.25). When the dendrimers were dispersed in water by evaporating methanol, the particle size increased significantly (200-250nm) and the dispersion got worse (PDIX).40). The dimensions of the chemical conjugates in water were found to be 179.30 ± 77.30 for G3.5 and 155.93 ± 13.59 for G4.5, and it was seen that they have an average polydispersity of 0.377 and 0.340, respectively. In the zeta potential measurements, G3.5 and G4.5 dendrimers were found to be negatively charged as they have carboxyl structured anionic surface groups. With the conjugation of the active substance to the dendrimers, the surface charge changed from negative to positive.
Release studies were carried out with the characterized formulations in two different media with pH 5.5 and pH 7.4. Release studies were carried out by the dialysis membrane method. According to the release studies, the release time of all substances placed in the membrane took an average of 30 minutes for free oxaliplatin, an average of 60 minutes for physical complexes, and an average of 120 minutes for chemical conjugates. Pure oxaliplatin was seen to tend to be released more rapidly at pH 7.4. The release profiles were compared using the fl difference factor and f2 similarity factor. Comparing the chemical conjugate formulations among themselves, it was seen that there was no significant difference between the release profiles.
In the physical complex formulations, it was seen that the release profile of the F6 formulation obtained in a pH 5.5 medium was significantly faster than the others. In studies conducted by using dialysis membranes, the release time of all substances is 30 minutes on average for free oxaliplatin, 60 minutes on average for physical complexes and 120 minutes on average for chemical conjugates. It is known that the release time in complex structures varies depending on the binding constants of the complexes formed by platinum (Π) compounds with PAMAM dendrimer. While the low binding constants of the complexes formed cause the release to accelerate, the high ones cause the release to slow down. The concord of the release profiles to various release kinetic models was examined and it was seen that the kinetic with the highest concord was generally Weibull.
With the KK-3.5 formulation lastly selected within the scope of the invention, an efficacy study was conducted in the HT-29 colon cancer cell line. In addition to the KK-3.5 formulation, the cytotoxicities of pure oxaliplatin and empty G3.5 dendrimers were also examined. According to the study, after 48 hours, it was seen that the cells to which pure G3.5 dendrimers were applied demonstrated more than 90% viability even at the highest dose (256μΜ = 3.321mg/mL). This shows that the toxicity of G3.5 dendrimers alone is quite low. By applying non-linear regression to the data, the IC50 values of pure oxaliplatin and KK3.5 conjugates were found to be 14.03μΜ and 0.72μΜ, respectively. In addition, the smaller Hill slope in the KK-3.5 formulation indicates a faster response to dose increase.
The slowdown in the release rate of the active ingredient oxaliplatin was realised by the use of dendrimers in the formulation preparation. Dendrimers can interact with drugs in various ways. It is possible to classify these interactions as physical and chemical. Physical interactions are the binding of the active substance to between dendrimer branches or to end groups by non-covalent bonds. Electrostatic bonds, hydrogen bonds or hydrophobic interactions play a role in the formation of such physical complexes. In chemical interactions, between the surface groups of the dendrimer and the drug molecule covalent bonds are established. These physical or covalent connections formed cause formulations to release for a longer period of time compared to the free active substance. The shifts between 0-30cm-1 in the FT-IR spectra makes us think the hydrophilic interactions, and the disappearance of the amide Π band peak makes us think that H bonds play a role in the interaction between the drug and the dendrimer. In vitro studies indicate that the uptake of platinum pro-drugs with the macromolecular structure is significantly increased compared to small molecule platinum compounds. It is thought that the reason behind the dendrimeric formulation being more toxic than pure oxaliplatin in HT-29 colon cancer cells is that platinum is more actively taken up into the cell and thereby more DNA-platinum complex is formed. The increase in the cytotoxic effect makes it possible to reduce the dose used in the treatment, and therefore, the side effects of oxaliplatin are reduced.
HPLC and Spectrophotometric Method for the Determination of the Oxaliplation Amount:
Thermo Surveyor was used as HPLC system. The system consists of a pump unit (Surveyor LC Pump) that pushes the mobile phase to the column, auto sampler (Surveyor Autosampler) that automatically takes the sample and injects it into the column, and detector (Surveyor UV/Vis Detector) units that analyse the material passing through the column at the appropriate wavelength. Drawing of chromatograms and analysis of peaks were done with ChromQuest 4.2.34 program. Shimadzu UV-1601 spectrophotometer and quartz vessels were used for spectrophotometric quantification. Properties of the HPLC method used:
Column C18 Waters Symmetry 4.6x250mm 5μm, Mobile Phase Acetonitrile: Water (adjusted to pH=3 with H3PO4) (10:90) (h/h), Flow Rate 0.8mL/min, Injection Volume 50μL, Column Temperature 25°C, Column Pressure 98-100bars, Wavelength (λ ) 255nm, Analysis Time 7 minutes, Retention Time between 5.4 - 5.5 minutes, Dilution Medium Water, LOD-LOQ 0.1- 0.3μg/Ml
Validation of the determination methods used: According to the Q2 (Rl) guideline of the International Conference on Harmonization (ICH), the aim of validation is to prove the suitability of the process for its intended use in analytical procedures. In the recognition according to the guideline, it is necessary to validate the methods used in determination of the amount of active substances within a medication or a sample in limit tests used for the determination and control of impurities. Apart from this, validation should be done again when there is a change in the synthesis of the active substance, the composition of the final product or the analytical method. Validation of the analytical method used in the determination of the amount was performed as described in the ICH guidelines. Within the scope of validation, specificity, linearity, intra-day and inter-day precision, accuracy, stability, system suitability test, limit of detection (LOD) and limit of qualification (LOQ) studies were performed. a) Specificity: It is to show that the method gives results only for the desired item and that other items do not interfere with the analysis. When blank solution is injected in HPLC, the peak should not be seen when the active substance peak is seen. PAMAM-G3.5 and PAMAM-G4.5 dendrimer solutions (100 μg/mL), and blank solutions were given to HPLC, and it was examined whether they interfere with the oxaliplatin peak. Additionally, the spectra of these solutions between 200-400nm wavelengths were recorded with a spectrophotometer, and how much absorbance they provide at which wavelengths was examined. b) Linearity: It is necessary to establish a linear relationship in the determined measuring range. Linearity studies can be prepared by standard solutions, by dilution from stock solutions, or by weighing the substances in different determined amounts. There must be at least 5 concentration points for linearity to be demonstrated. The coefficient of determination (r2) of the standard curve is desired to be greater than 0.999. HPLC standard solutions were prepared at 1-lOμg/mL measuring range, in 7 different concentrations (lOμg/mL, 8 μg/mL, 6 μg/mL, 5μg/mL, 4μg/mL, 2μg/mL, lμg/mL) and was given to HPLC as triplicates (n=3). Spectrophotometer standards were prepared in the measuring range of 10-100μg/mL, in 7 different concentrations (lOOμg/mL, 80 μg/mL, 60 μg/mL, 50μg/mL, 40μg/mL, 20μg/mL, lOμg/mL) and was read in the spectrophotometer as triplicates (n=3). While preparing the standards, a stock solution of lOmg/lOOmL (lOOμg/mL) concentration was prepared by dissolving lOmg of solid oxaliplatin in lOOmL of distilled water. c) Precision: Precision is demonstrated by multiple measurements of the same solution. Precision studies can be repeated on the same day and on different days. The % standard deviation between measurements, relative standard deviation and confidence interval should be specified. The relative standard deviation is found by dividing the standard deviation by the mean and multiplying it by one hundred. Relative standard deviation should be 2% or less is to ensure desirable accuracy. Three solutions were each prepared at a concentration of 4μg/mL (80%), 5μg/mL (100%), 6μg/mL (120%) for HPLC method, and 40μg/mL (80%), 50μg/mL (100%), 60μg/mL 120% (120%) for spectrophotometric method, and measurements were made three times in the relevant device. Relative standard deviation values between measurements were calculated. The precision study was repeated three times on different days to validate the between-days precision. d) Accuracy: It is a demonstration that samples of known concentration are correctly measured. Validation of accuracy should be performed in triplicates (n=3) at a minimum of 3 different concentrations. The value taken from the device versus the known concentration is expressed as % recovery. Accuracy can be demonstrated once the precision, linearity and authenticity are established. The % recovery value should be in the range of 100% ± 2. Three solutions were each prepared at a concentration of 4μg/mL (80%), 5μg/mL (100%), 6μg/mL (120%) for HPLC method, and 40μg/mL (80%), 50μg/mL (100%), 60μg/mL 120% (120%) for spectrophotometric method for the validation of the accuracy and measurement result % recovery oxaliplatin amounts were calculated. e) Stability Studies: It is done to show that the substance to be analysed remains stable in the sample solutions during the analysis period. A solution was prepared at a concentration of 5μg/mL for the HPLC method and 50μg/mL for the spectrophotometric method, and samples were taken from the solution at 0, 1, 3, 8 and 24 hours for amount determination, and % relative standard deviation was calculated for the determined amounts. f) System Compatibility Test: System suitability testing is an integral part of many analytical procedures, and the system suitability test parameters to be established for a particular procedure depend on the type of method. Values like retention time, peak area, theoretical plate number, tailing factor, their mean and relative standard deviation for HPLC are important for instance. Relative standard deviation of values such as retention time and peak area should be < 1%, tailing factor should be < 2, and theoretical plate numbershould be > 2000. g) LOD and LOQ: According to the ICH Q2 (Rl) guideline, LOD and LOQ can be determined by different approaches. In this method, LOD is mathematically calculated by dividing the residual standard deviation (σ) of the regression line by the slope of the regression line and multiplying it by 3.3, and LOQ is calculated by multiplying it by 10.
Solubility Data of Oxaliplatin: In various organic solvents, water and buffer solutions at various pHs used throughout the invention, the solubility of oxaliplatin was investigated. Solubility studies of oxaliplatin were carried out in organic solvents such as methanol, ethanol, dimethylformamide (DMF), and dichloromethane (DCM), distilled water and seven buffer solutions at pH 2, 4, 6, 7, 8, 10, 12 and are shown in Figure 1. Determination of solubility was realised according to the shake flask method. First, solid oxaliplatin is added to the glass vials in excess rates. Then, lmL of the medium for solubility test is added into the vials. Three vials are prepared in this way for each solvent. The vials are mixed at room temperature for 24 hours. At the end of the period, solid oxaliplatin that is taken into tubes and not dissolved were centrifuged at 10,000rpm for 15 minutes to precipitate. At the end of the centrifuge, the supernatant is removed and filtered through a 0.45 pm nylon or PTFE membrane filter. If necessary, the amount is determined by dilution. The results are stated below.
Ingredients required for the preparation of lOOmL buffer solution: pH 2 HC1-KC1 Buffer 0,373g KC1 + 0, 107mL HC1 pH 4 Acetate Buffer 0,238mL CH3COOH + 0,074g CH3COONa pH 6 Phosphate Buffer 0,68 lg KH2PO4 + 0,022g NaOH pH 7 Phosphate Buffer 0,681g KH2PO4 + 0,116g NaOH pH 8 Phosphate Buffer 0,68 lg KH2PO4 + 0,184g NaOH pH 10 Borate Buffer 0,309g H3BO3 + 0,373g KC1 + 0,175g NaOH pH 12 NaOH-KCl Buffer 0,048g NaOH + 0,373g KC1
Examination of the Effect of Dendrimers on Solubility: In order to examine the effect of the presence of dendrimer in the dissolution medium on the solubility, a solubility study was performed using two different types of dendrimers and their five different concentrations. Water, pH4 acetate buffer and pH7 phosphate buffer were used as medium. As it has been shown in the literature that basic pH values can cause stability problems in oxaliplatin, a basic buffer medium was not used. The solubility study was carried out as previously described, distinctly, 0.05 μmol, 0.10μmol, 0.20μmol, 0.40μmol, 0.80μmol PAMAM-G3.5 or PAMAM-G4.5 dendrimers were added to the media. At the end of the study, the amount of oxaliplatin was determined by appropriate dilution. The results are shown in the table below and in Figure 2.
Table 4. Effect of dendrimers on solubility
Figure imgf000024_0001
Preparation of Oxaliplatin-Dendrimer Formulations:
Dendrimeric formulations of oxaliplatin were prepared with two different approaches: physical complex and chemical conjugate formulations. In the invention, PAMAM-G3.5 and PAMAM-G4.5 dendrimers with anionic structures were used. Therefore, it was possible to examine the effects of dendrimer generation and preparation alterations on formulations. The abbreviations given to the formulations are given below.
Figure imgf000024_0002
While dendrimeric oxaliplatin formulations to be prepared by physical complexation are expected to have a property to of immediate release, formulations to be prepared by chemical conjugation are expected to release the active substance more slowly and in a longer time. Dendrimeric complexes of oxaliplatin (FK-3.5 and FK-4.5) were prepared by co-precipitation method. As dendrimers have multiple (primary and tertiary amines) connection points, it is assumed that a dendrimer molecule can interact with many drug molecules. Therefore, the different drug in which the dendrimer ratio is always one: Mixtures at molar concentration ratios of dendrimer (N: 1) were prepared. The number N can take a value from 1 to the number of possible connection points of the dendrimer generation.
Figure imgf000025_0001
Dendrimeric conjugates of oxaliplatin (KK-3.5 and KK-4.5) were prepared by chemical reaction. Basically, it is aimed to establish an ester bond between the carboxyl groups on the surface of half-generation dendrimers and the hydroxyl groups obtained by modifying oxaliplatin. The establishment of this ester bond was accomplished by the esterification reactions shown above and described by Wolfgang Steglich. In Steglich esterification, N, N1- dicyclohexylcarbodiimide (DCC) was used as the binding agent, 4-(dimethylamino) pyridine (DMAP) was used as the catalyst, and dichloromethane (DCM), an aprotic solvent, was used as the reaction medium. Rl, R2, R3, R4 symbolize different chemical structures.
Figure imgf000025_0002
-J
While the 3 and 4 generation PAMAM dendrimers are -NH2 terminated cationic dendrimers, the 3.5 and 4.5 generation dendrimers -COOH are terminated anionic dendrimers. Cationic dendrimers are known to have higher toxicity compared to anionic dendrimers. The underlying reason of it is that the cationic ends interact with the negatively charged cell membrane and thus destabilize it. In addition to toxicity, haemolytic and aggregate activities of cationic dendrimers are also much more clear than anionic ones. Cationic dendrimers are not preferred as it is not desirable for dendrimers to interact with the membranes of normal cells and disrupt their structure until they are removed from the body after being separated from their platinum structure. Cationic dendrimers used in the methods of prior art are disadvantageous due to their toxicity. PAMAM type cationic dendrimers used demonstrate haemolytic activity at concentrations above lmg/mL. It has been shown that even at non-haemolytic doses as low as 10μg/mL, they cause alterations in erythrocyte morphology and cause a transformation from disc shape to spherical shape. In anionic dendrimers, no haemolytic activity or a alteration in erythrocyte morphology was observed even at high concentrations like 2mg/mL. In drug carrier systems, it is desired that the structure that carries the drug is compatible with the body and non-toxic as much as possible. The remaining part not causing any toxicity in the body after performing its main duty of drug carrying will increase the benefit/harm ratio of the formulation. Therefore, anionic 3.5 and 4.5 generation dendrimers (G3.5 and G4.5) are preferred in the invention.
While the pKa values of the primary amines on the surface of full generation PAMAM dendrimers vary between 7 and 9, the pKa values of the tertiary amines between the branches are between 3-6. Therefore, in mediums with different pH values, the ionization states of the functional groups of dendrimers differ. At high pHs, the primary and tertiary amines of PAMAM dendrimers are in not protonated statuses. While only primary amines are protonated at near neutral pH, both primary and tertiary amines are protonated at low pH.
The pKa value of the carboxylic acid surface groups found in half-generation dendrimers is approximately between 4-5. As it is believed that different medium pHs can alter the property of physical attachment of active substance to dendrimers, physical dendrimer complex formulations are prepared by adding different rates of oxaliplatin to different mediums. For G3.5 and G4.5 dendrimers, formulations were prepared in in three different media (distilled water, pH 4 acetate buffer and pH 7 phosphate buffer) and five different oxaliplatin dendrimers. A basic buffer was not used as a charge medium because media with high hydroxyl ions can cause stability problems in oxaliplatin.
The reason that the highest ratio was chosen as 250: 1 is because the sum of all points that can bind oxaliplatin is maximum 254 (in the case of oxaliplatin binding to 128 carboxylic surface groups and 126 tertiary amine groups between branches in the G4.5 dendrimer).
Dendrimeric conjugates of oxaliplatin (KK-3.5 and KK-4.5) were prepared by chemical reaction. Basically, it is aimed to establish an ester bond between the carboxyl groups (- COOH) on the surface of half-generation dendrimers and the hydroxyl groups (-OH) obtained by modifying oxaliplatin. The establishment of this ester bond was realised by esterification reactions described by Wolfgang Steglich. In Steglich esterification, Ν,Ν'- dicyclohexylcarbodiimide (DCC) is used as the binding agent, 4-(dimethylamino)pyridine (DMAP) is used as the catalyst, and dichloromethane (DCM), an aprotic solvent, is used as the reaction medium. As dendrimers and oxaliplatin are insoluble in DCM, another aprotic solvent, N,N-dimethylformamide (DMF), was used as the reaction medium in this study, in which both oxaliplatin and dendrimers, and DCC and DMAP are well soluble.
Synthesis of physical complexes generally comprises the steps of;
Preparation of 1-10 mg/mL stock solutions by dissolving oxaliplatin in water, pH 4 or pH 7 media,
• Obtaining 0.05-5 mL of oxaliplatin solution from the prepared stock solutions,
• Taking 0.65 mg of methanolic G3.5 or G4.5 dendrimer solution,
• Preparation of oxaliplatin dendrimer solutions in the range of 25-250:1 from the oxaliplatin and dendrimer solutions taken,
• Mixing the solutions with a magnetic stirrer for 24 hours,
• evaporating the solvent,
• Suspending the residue in anhydrous ethanol,
• Centrifuging at 10,000rpm for 5 minutes to precipitate oxaliplatin that did not enter between the branches of the dendrimers and complex,
• Removing the supernatant at the end of the centrifuge and filtering through a 0.45 pm nylon or PTFE membrane filter, and
• Obtaining an aqueous complex solution by evaporating the ethanol of the filtrate and dissolving the residue in water. a) Preparation of FK-3.5 formulations:
The method used in the preparation of physical complex formulations (FK-3.5 and FK-4.5) is based on simple mixing and known in the art. Appropriate mole ratios to be mixed were calculated and mixed in the determined ratios.
In order to prepare FK-3.5 formulations, first 25mg of oxaliplatin was weighed separately for each and dissolved in 5 mL water, pH 4 and pH 7 mediums. By this way, a stock solution of oxaliplatin at a concentration of 5mg/mL in three different media was prepared. The amounts of stock solution and methanolic G3.5 dendrimer solution required to prepare 25:1, 50:1, 75:1, 150:1 and 250:1 ratios, respectively, are summarized in Table 5 below. While making the calculations, the molecular weight of oxaliplatin and G3.5 dendrimer was taken as 397.286 g/mol and 12927.69 g/mol, respectively, and the density of 10% (w/w) solution of G3.5 dendrimer was 0.810 g/mL as specified by the manufacturer, and μmol values are rounded to 2 digits after the decimal point.
Table 5. The amounts of the used stock solution and methanolic G3.5 dendrimer solution Fl-
Figure imgf000028_0001
The amovmt of stock solution and dendrimer solution indicated in the table is added to a glass vial and mixed for 24 hours on a magnetic stirrer. At the end of the period, the solvent medium is evaporated. The residue is suspended in anhydrous ethanol. It is centrifuged at 10,000rpm for 5 minutes to precipitate oxaliplatin that did not enter the branches of the dendrimers and complex. At the end of the centrifuge, the supernatant is removed and filtered through a 0.45 pm nylon or PTFE membrane filter. An aqueous complex solution is obtained by evaporating the ethanol of the filtrate and dissolving the residue in water. Measurements are made with this solution.
To examine the amount of active substance that can be charged to the dendrimer while preparing the physical complex formulations, different active substance/dendrimer ratios were tried. It was desired to obtain different mole ratios by adding oxaliplatin stock solution in increasing rates to a fixed amount of dendrimer (0.05 μmol). For this reason, 25-250 more ratio was investigated by increasing the amount of active substance added while keeping the dendrimer amount constant. The selection of the molar ratio range to be examined was made considering the potential interaction points of the dendrimers. While the G3.5 dendrimers used theoretically have a total of 126 interaction points, 64 carboxyl groups on the surface and 62 tertiary amine groups in the branches, in the G4.5 dendrimers, these values are 128, 124 and 252, respectively. As oxaliplatin is added to a 250:1 oxaliplatimdendrimer ratio, it is possible for almost all possible interaction sites to interact with oxaliplatin. b) Preparation of FK-4.5 formulations: In order to prepare FK-4.5 formulations, first 25mg of oxaliplatin was weighed separately for each and dissolved in 5 mL water, pH 4 and pH 7 mediums. By this way, a stock solution of oxaliplatin at a concentration of 5mg/mL in three different media was prepared. The amounts of stock solution and methanolic G4.5 dendrimer solution required to prepare 25:1, 50:1, 75:1, 150:1 and 250:1 ratios, respectively, are summarized in Table 6 below.
While making the calculations, the molecular weight of oxaliplatin and G4.5 dendrimer was taken as 397.286 g/mol and 26251.86 g/mol, respectively, and the density of 10% (w/w) solution of G4.5 dendrimer was 0.791 g/mL as specified by the manufacturer, and μmol values are rounded to 2 digits after the decimal point.
Table 6. The amounts of the used stock solution and methanolic G4.5 dendrimer solution
F16-F30.
Figure imgf000030_0001
The amount of stock solution and dendrimer solution indicated in the table is added to a glass vial and mixed for 24 hours on a magnetic stirrer. At the end of the period, the solvent medium is evaporated. The residue is suspended in anhydrous ethanol. It is centrifuged at 10,000rpm for 5 minutes to precipitate oxaliplatin that did not complex with the dendrimers.
At the end of the centrifuge, the supernatant is removed and filtered through a 0.45 pm nylon or PTFE membrane filter. An aqueous complex solution is obtained by evaporating the ethanol of the filtrate and dissolving the residue in water. Measurements are made with this solution. The process steps are shown in Figure 3. c) Preparation of KK-3.5 formulation: KK-3.5 formulations were synthesized by a two-step reaction. In the first step, the modification of oxaliplatin was carried out, and then the binding of this compound to the G3.5 dendrimer was carried out by the Steglich esterification reaction as previously described.
Synthesis of chemical conjugates with G 3.5 dendrimer generally comprises the steps of:
Preparing a solution of oxaliplatin in acetic acid in the concentration range of 10-
1000mM,
Suspending the mixture in a magnetic stirrer,
Adding H2O2 solution to the suspension, with H2O2 being 0,1-1% by volume, Adding 30% H2O2 with 30 minutes intervals,
Stirring the solution overnight, • Removing the acetic acid by evaporation,
• Resuspending the oily residue with distilled water,
• Removing the added water from the medium by evaporation,
• Repeating the addition and removal of water until the product is solid,
• Suspending the obtained solid product with 10-20 mL of ethyl acetate,
• Adding methanol to the suspension until a clear solution is formed,
• Filtering the mixture through a 0,45 pm nylon membrane filter,
• Removing the solvent mixture from the medium,
• Storing the obtained modified oxaliplatin at +4°C,
• Removing G3.5 dendrimer from methanolic solution (10% w/w),
• Evaporation of methanol under vacuum and activating the carboxyl ends by dissolving the residue in HC1 solution with a pH of 3.
• Dissolving the dried dendrimers again in DMF after evaporation of water,
• Adding modified oxaliplatin, DCC and DMAP to this solution,
• Adjusting the modified oxaliplatin mole percentage as 25-35%, DCC mole percentage as 25-35%, DMAP mole percentage as 25-35% and G3.5 dendrimer as 0-10% in the final mixture,
• Stirring the mixture under vacuum for 24 hours at room temperature,
• At the end of 24 hours, removing the precipitates from DCC formed during the reaction by filtrating through a 0,45 pm nylon membrane filter,
• Evaporating the solvent,
• Dissolving the remaining residue in distilled water and if needed filtering again through a 0,45 pm nylon membrane filter,
• Extracting with DCM 5 times to remove the hydrophobic organic substances remained from the reaction from the medium,
• At the end of the extraction, purifying the aqueous part by dialysis and separating for lyophilization,
Freezing, primary and secondary drying of the aqueous solution in lyophilization, Obtaining lyophilized conjugates.
Modification of oxaliplatin: 20mL of acetic acid is added onto 1,192g (3mmol) of oxaliplatin and suspended on a magnetic stirrer. 250pL of 30% H2O2 solution (perhydrol) is added onto the suspension. 30% H2O2, 250pL and 750pL, is added with 30 minutes intervals, respectively. Within 1,5 hours the suspension turns into a yellowish and almost clear solution. This solution is stirred overnight. The acetic acid is evaporated and removed from the medium and the oily residue is suspended again with 40mL of distilled water to form a turbid mixture. The added water is removed by evaporation. This process (water addition and removal) is repeated 2 more times until the product becomes solid. The solid product obtained is suspended again with some (10-20mL) ethyl acetate. Methanol is added to this suspension until a clear solution is formed and until it is seen adding more methanol does not change the appearance of the solution. The mixture is filtered through a 0,45 pm nylon membrane filter. The solvent mixture is removed from the medium. The slightly yellowish product obtained is stored at +4°C to be used in the preparation of conjugates. The chemical synthesis reaction of the modification of oxaliplatin is shown below.
Figure imgf000032_0001
Dendrimer conjugation: 250pL (l,566μmol) is taken from the methanolic solution (10% w/w) of the G3.5 dendrimer. The methanol is evaporated under vacuum and the carboxyl ends are activated by dissolving the residue in HC1 solution with pH 3. After evaporating the water, the dried dendrimers are dissolved again in 6mL of DMF. 60mg (126,8μmol) modified oxaliplatin, 26,2mg (126,8μmol) DCC and 15,5mg (126,8μmol) DMAP are added onto this solution. The mixture is stirred under vacuum for 24 hours at room temperature. After 24 hours, the precipitates resulting from DCC (for example, dicyclohexylurea-DCU) formed during the reaction are removed by filtrating through a 0,45 pm nylon membrane filter. The solvent medium is evaporated. The remaining residue is dissolved in distilled water and filtered again through a 0,45 pm nylon membrane filter if needed. It is extracted 5 times with 25mL DCM to remove the hydrophobic organic substances that remained from the reaction. At the end of the extraction, the aqueous part is purified and separated by dialysis to be lyophilized. Lyophilization is performed by freezing the aqueous solution at -80°C for 3 hours, keeping it at -40°C for 22 hours at 0,01 mbar pressure, and at -20°C for 3 hours at 0,00 lbar pressure. The obtained lyophilized conjugate is stored at +4°C to be used in characterization and cell culture studies. The molar ratio of the reactants to the dendrimer is approximately 80:1. Therefore, it was possible to bind all 64 surface groups of the G3.5 dendrimer with the active substance. d) Preparation of KK-4.5 formulation: Synthesis of chemical conjugates with G 4.5 dendrimer generally comprises the steps of:
Preparing a solution of oxaliplatin in acetic acid in the concentration range of 10-
1000mM,
• Suspending the mixture in a magnetic stirrer,
• Adding H2O2 solution to the suspension with, H2O2 being 0.1-1% by volume,
• Adding 30% H2O2 with 30 minutes intervals,
• Stirring the solution overnight,
• Removing the acetic acid by evaporation,
• Resuspending the oily residue with distilled water,
• Removing the added water from the medium by evaporation,
• Repeating the addition and removal of water until the product is solid,
• Suspending the obtained solid product with 10-20 mL of ethyl acetate,
• Adding methanol to the suspension until a clear solution is formed,
• Filtering the mixture through a 0,45 pm nylon membrane filter,
• Removing the solvent mixture from the medium,
• Storing the obtained modified oxaliplatin at +4°C,
• Removing G4.5 dendrimer from methanolic solution (5% w/w),
• Evaporating the methanol under vacuum and activating the carboxyl ends by dissolving the residue in HC1 solution with pH 3,
• Dissolving the dendrimers dried after evaporation of the water again in 5mL DMF,
• Adding modified oxaliplatin, DCC and DMAP to this solution,
• Adjusting the modified oxaliplatin mole percentage as 25-35%, DCC mole percentage as 25-35%, DMAP mole percentage as 25-35% and G3.5 dendrimer mole percentage as 0- 10% in the final mixture,
• Stirring the mixture under vacuum for 24 hours at room temperature,
• At the end of 24 hours, removing the precipitates from DCC formed during the reaction by filtrating through a 0.45 pm nylon membrane filter,
• Removing the solvent from the medium, • Dissolving the remaining residue in distilled water and if needed, filtering again through a 0.45 pm nylon membrane filter,
• Extracting with DCM 5 times to remove the hydrophobic organic substances remained from the reaction from the medium,
• At the end of the extraction, purifying the aqueous part by dialysis and separating for lyophilization,
• Freezing, primary and secondary drying of the aqueous solution in lyophilization,
• Obtaining lyophilized conjugates.
KK-4.5 formulations were also synthesized by a two-step reaction like KK-3.5 formulation. In the first step, the modification of oxaliplatin was realised, and then the binding of this compound to the G4.5 dendrimer was realised. The modification of oxaliplatin was done as described in the preparation of the KK-3.5 formulation. 500pL (0,753 μmol) is taken from the methanolic solution (5% w/w) of G4.5 dendrimer. The methanol is evaporated under vacuum and the carboxyl ends are activated by dissolving the residue in HC1 solution with pH 3. After evaporating the water, the dried dendrimers are dissolved again in 5mL of DMF. 50mg (105,6μmol) modified oxaliplatin, 21,8mg (105,6μmol) DCC and 12,9mg (105,6μmol)
DMAP are added on to this solution. The mixture is stirred under vacuum for 24 hours at room temperature. After 24 hours, the precipitates resulting from DCC (for example, dicyclohexylurea-DCU) formed during the reaction are removed by filtrating through a 0,45 pm nylon membrane filter. The solvent medium is evaporated. The remaining residue is dissolved in distilled water and filtered again through a 0.45 pm nylon membrane filter if needed. It is extracted 5 times with 25mL DCM to remove the hydrophobic organic substances that remained from the reaction. At the end of the extraction, the aqueous part is purified by dialysis and separated to be lyophilized. Lyophilization is performed by freezing the aqueous solution at -80°C for 3 hours, keeping it at -40°C for 22 hours at 0,01mbar pressure, and at -20°C for 3 hours at 0,00 lbar pressure. The obtained lyophilized conjugate is stored at +4°C to be used in characterization and cell culture studies. The molar ratio of the reactants to the dendrimer is approximately 140:1. Therefore, it was possible to bind all 128 surface groups of the G4.5 dendrimer with the active substance. The reaction of the synthesis is shown below.
Figure imgf000035_0001
Characterization of Oxaliplatin-Dendrimer Formulations
The methods used in the characterization of dendrimeric formulations of oxaliplatin are Ultraviolet- Visible Spectroscopy (UV -Vis), Fourier Transform Infrared (FT-IR) Spectroscopy, and 1H and 13C Nuclear Magnetic Resonance (NMR) Spectroscopy. Additionally, particle size and distribution were examined on the basis of dynamic light scattering, and zeta (ζ) potential values were measured to get information about the surface charges of the formulations. The charge rate and charge efficiency were investigated for the FK-3.5 and FK-4.5 formulations, and the approximate conjugated oxaliplatin amounts per mole for the KK-3.5 and KK-4.5 formulations were found by proportioning the areas of the integrated characteristic peaks in 1H-NMR.
Validation of Oxaliplatin Ouantification Methods The validation of HPLC and spectrophotometric methods used for the determination of the amount of the active substance was made in accordance with the ICH Q2 (Rl) guideline and it was found that the methods were suitable for use to determine the amount of oxaliplatin.
Selectivity (Specificity): The chromatograms of PAMAM-G3.5 and PAMAM-G4.5 dendrimer solutions and (blank) solutions prepared without active ingredient are shown in Figure 4. As seen from the chromatograms, it is found that dendrimers and solutions without active substance do not interfere with the oxaliplatin peak, therefore the method is specific to oxaliplatin. Furthermore, in Figure 5, it is demonstrated as spectrum and chromatogram that PAMAM-G3.5 or PAMAM-G4.5 dendrimers do not give an absorbance to interfere with the measurement at 255nm which is the wavelength where oxaliplatin shows maximum absorbance in the spectra taken from the spectrophotometer between 200-400nm wavelengths.
Linearity: The prepared standard solutions were measured in HPLC and spectrophotometer, the peak area and absorbance values corresponding to each standard solution are shown in Table 7 below.
Figure imgf000036_0001
These data were processed with the help of Microsoft Office Excel 2016 program, oxaliplatin standard curves are shown in Figure 6 and information on regression lines are shown in Table 8. Accordingly, the regression line equation for HPLC was calculated as y=20989x-317,82, the coefficient of determination (r2) was calculated as 0,9999, and for the spectrophotometer, the line equation was calculated as y=0,0056x+0,0089, and r2 was calculated as 0,9999. Consequently, both methods show linearity in the specified concentration ranges.
Table 8. Information on oxaliplatin regression lines.
Figure imgf000037_0001
With the help of LOD and LOQ Microsoft Office Excel 2016 program, the residual standard deviation (σ) of the regression line and the slope of the regression line were found for both methods. LOD and LOQ were calculated with the formulas described in the methods section. Accordingly, the LOD values for HPLC and spectrophotometric methods were found to be 0, 1 μg/mL and 1,1 μg/mL, respectively, and the LOQ values were found to be 0,3μg/mL and 3,3 μg/mL, respectively.
Precision: The method was validated in terms of intra-day and inter-day precision, as the percent relative standard deviation calculated at each concentration in intra-day and inter-day precision studies on three separate days was below 2%. Precision data for the first, second and third days for both methods are given in the following tables (Tables 9-14), as first HPLC and then Spectrophotometric data, respectively.
Figure imgf000038_0001
Figure imgf000039_0001
Accuracy: The method was validated for accuracy as % recovery value calculated for three separate concentration for both methods remained within the % 100 ± 2 range in the accuracy studies. The data obtained from the accuracy study are given below (Table 15-16).
Table 15. HPLC method accuracy data.
Figure imgf000040_0002
Stability Studies: The results of the stability studies are shown in the tables below (Tables 17-18). The changes in oxaliplatin concentration were examined using the peak areas in HPLC and absorbance values in spectrophotometer. Additionally, it was checked whether there was a different peak formation that might stem from the degradation products in HPLC, and no new peak formation was observed. As the relative standard deviation between the concentrations did not exceed 2% at the end of 24 hours, it was determined that the oxaliplatin solution in water remained stable throughout the analysis,
Figure imgf000040_0001
Table 18. Stability data for spectrophotometer.
Figure imgf000041_0002
System Compatibility Test: Results of the system compatibility test for the HPLC method are shown below. As the number of theoretical plates is greater than 2000 and the tailing factor is less than 2, the HPLC method has passed the system compatibility test.
Figure imgf000041_0001
Characterization of FK-3.5 Formulations i) Ultraviolet-visible region (UV -Vis) spectroscopy: The spectrum of oxaliplatin and F6 formulation from the FK-3.5 formulations in the 200-400nm wavelength range was taken and is shown in Figure 7. Changes in the spectrum like the disappearance of the peak, the shift of the peak or a decrease in its intensity provide evidence of the realisation of the complex. As can be seen in Figure 7, the suppression of the oxaliplatin peak at 255nm in the F6 formulation suggests that oxaliplatin is adhered between the dendrimer branches and forms a complex.
Fourier transformed infrared (FT-IR) spectroscopy: To represent FK-3.5 formulations, the FT-IR spectra of the F6 formulation are shown in Figure 8. Peaks of O-H stretching at 3308cm-1, 2948cm-1 asymmetrical C-H stretching, 2836cm-1 symmetrical C-H stretching, 1648cm-1 C=0 stretching, and C-0 stretching at 1408cm-1 are seen in the FT-IR spectrum. The shifts observed particularly in the O-H and C=0 peaks of PAMAM G3.5 dendrimers suggest that a physical interaction between oxaliplatin and the dendrimer was established, and complex was formed.
Characterization of FK-4.5 Formulations i) Ultraviolet-visible region (UV -Vis) spectroscopy: The spectrum of the oxaliplatin and F21 formulation of the FK-4.5 formulations in the 200-400nm wavelength range was taken and shown in Figure 9. Changes in the spectrum like the disappearance of the peak, the shift of the peak or a decrease in its intensity provide evidence of the realisation of the complex. As seen in Figure 9, the decrease in intensity and suppression of the oxaliplatin peak seen at 255nm in the F21 formulation suggests that oxaliplatin is adhered between the dendrimer branches and forms a complex.
Fourier transformed infrared (FT-IR) spectroscopy: To represent FK-4.5 formulations, the FT-IR spectra of the F21 formulation are shown in Figure 10. Peaks of O-H stretching at 3296cm-1, 2949cm-1 asymmetrical C-H stretching, 2838cm-1 symmetrical C-H stretching, 1648cm-1 C=0 stretching, and C-0 stretching at 1407cm-1 are seen in the FT-IR spectrum.
The shifts observed particularly in the O-H and C=0 peaks of PAMAM G4.5 dendrimers suggest that a physical interaction between oxaliplatin and the dendrimer was established, and complex was formed.
Characterization of Modified Oxaliplatin
Chemical modifications were carried out in oxaliplatin before the KK-3,5 and KK-4,5 formulations were prepared. The chemical synthesis yield was 98,2%. To show that the desired compound was synthesized, characterization studies were conducted. i) Fourier transformed infrared (FT-IR) spectroscopy: The FTIR results of pure oxaliplatin and synthesized modified oxaliplatin are shown in Figure 11. In the FT-IR spectrum of pure oxaliplatin, peaks of NH stretching at 3210 cm-1 and 3082 cm-1, 2928cm-1 asymmetrical C-H stretching, 2863cm-1 symmetrical C-H stretching, C=0 stretching at 1699cm-1 and 1661cm-1, and C-0 stretching at 1376cm-1 are seen. In the FT-IR spectrum of modified oxaliplatin, peaks of O-H stretching at 3480cm-1, N-H stretching at 3090cm-1, 2920cm-1 asymmetrical C-H stretching, 2856cm-1 symmetrical C-H stretching, C=0 stretching at 1713cm-1 and 1662cm-1, and C-0 stretching at 1361cm-1 are seen. Also, a Pt-0 stretching peak was observed at 574cm-1 in the fingerprint region. The fact that O-H and Pt-0 stretching peaks are seen in the synthesized compound suggested that the desired compound was synthesized, and 1H-NMR and 13C-NMR spectra of the compound were taken for further characterization.
According to the 1H-NMR and 13C-NMR results shown in Figures 12 and Figure 13, the desired modification was realised in oxaliplatin.
Characterization of KK-3.5 Formulation
Using the Steglich esterification reaction, the G3.5 dendrimer and modified oxaliplatin was connected by ester bonds. The reaction yield was 50,3%. To show that the desired compound was synthesized, characterization studies were conducted. i) Fourier transformed infrared (FT-IR) spectroscopy: The FT-IR spectra of the modified oxaliplatin, PAMAM-G3.5 dendrimer and KK-3.5 formulation are shown in Figure 14. The FT-IR spectrum of the KK-3.5 formulation showed that the functional groups seen in the modified oxaliplatin and G3.5 dendrimer are also present in the conjugate structure. KK-3.5 has peaks belonging to both dendrimer and modified oxaliplatin functional groups to which it is conjugated, due to the free dendrimer ends that are not conjugated with the active substance. In the FT-IR spectrum, peaks of O-H stretching at 3476cm-1, N-H stretching at 3135cm-1, 2941cm-1 asymmetrical C-H stretching, 2880cm-1 symmetrical C-H stretching, 1718cm-1 C=0 stretching (amide I band), N-H bending at 1626cm-1 (amide Π band), and C-0 stretch at 1359cm-1 are seen. Also, Pt-0 stretching peak was observed in the fingerprint region 573cm-1. Expected functional groups were seen, 1H-NMR spectrum was taken for further characterization. ii) 1Η-NMR spectroscopy: 1Η-NMR (DMSO-d6) results of the KK-3.5 formulation are given in Figure 15. Figure 15. A shows the 1HNMR spectra of the conjugate and Figure 15. B shows of the modified oxaliplatin.
Characterization of KK-4.5 Formulation Using the Steglich esterification reaction, the G4.5 dendrimer and modified oxaliplatin was connected by ester bonds. Reaction yield was 46,6%. To show that the desired compound was synthesized, characterization studies were conducted. i) Fourier transformed infrared (FT-IR) spectroscopy: The FT-IR spectra of the modified oxaliplatin, PAMAM-G4.5 dendrimer and KK-4.5 formulation are shown in Figure 16. The FT-IR spectrum of the KK-4.5 formulation showed that the functional groups seen in the modified oxaliplatin and G4.5 dendrimer are also present in the conjugate structure. KK-4.5 has peaks belonging to both dendrimer and modified oxaliplatin functional groups to which it is conjugated, due to the free dendrimer ends that are not conjugated with the active substance. Peaks of O-H stretching at 3475cm-1, N-H stretching at 3085cm-1, 2941cm-1 asymmetric C-H stretching, 2860cm-1 symmetrical C-H stretching, 1719cm-1 C=0 stretching (amide I band), NH bending at 1625cm-1 (amide Π band), and C-0 stretch at 1355cm-1 in the FT-IR spectrum are seen in Figure 16. Also, Pt-0 stretching peak was observed in the fingerprint region 573cm-1. Expected functional groups were seen, 1HNMR spectrum was taken for further characterization. ii) 1Η -NMR spectroscopy: 1Η-ΝΜR (DMSO-d6) results of the KK-4.5 formulation are given in Figure 17. Figure 17. A shows the 1HNMR spectra of the conjugate and Figure 17. B shows of the modified oxaliplatin.
Particle Size-Distribution and Zeta (ζ) Potential Measurements, particle size and polydispersity index (PDI) data of G3.5 and G4.5 PAMAM dendrimers in methanol and water, and of chemical conjugate formulations in water are given in the tables below. The invention macromolecule sizes can be up to lOOOnm, PDI values can be up to 0.6, and the zeta potential range can be between -lOOmV +100mV. For all physical complex and chemical conjugates, the invention macromolecule sizes are between 10- lOOOnm, PDI values are between 0.05-0.6 and zeta potential values are between -lOOmV +100mV. Table 20. Average size and polydispersity index data of dendrimers.
Figure imgf000045_0001
Zeta potential values of G3.5 and G4.5 PAMAM dendrimers and chemical conjugate formulations in water are given below.
Table 21. Zeta (Q potential data of dendrimers.
Figure imgf000045_0002
Charge Rate and Efficiency, Amount of Conjugated Oxaliplatin
The % charge rate, % charge efficiency, and mole values of oxaliplatin charged per mole of dendrimer for physical complex formulations and chemical conjugates are shown in Table 22 and Figure 18.
Table 22. % charge rate, % charge efficiency, and mole values of oxaliplatin charged per
Figure imgf000046_0001
In Vitro Release Studies in Physical Complex and Chemical Conjugates:
The results of the release study performed in two different mediums, pH 5.5 and pH 7.4, with selected physical complex formulations (F6 and F21) and chemical conjugates are shown in Figure 19 and Figure 20. The release profiles of physical and chemical conjugates in pH 5.5 and pH 7.4 mediums are compared and the fl difference factor and f2 similarity factor values are shown in the tables below, respectively.
Figure imgf000047_0002
Figure imgf000047_0001
Table 25. Application of various kinetic models to physical complex (F1-F21) and chemical
Figure imgf000048_0001
Cell Culture Studies: As a result of the study conducted on the HT-29 cell line, the % viability data ofPAMAM G3.5 dendrimer, oxaliplatin and KK-3.5 formulation are given in Figure 21. The IC50 values of the oxaliplatin and KK-3.5 formulation were foimd using non-linear regression with the GraphPad Prism 8.2.0 program. The results are shown in Figure 22 and Table 26.
Table 26. IC50 values of pure Dendrimer, Oxaliplatin and KK-3.5 formulation.
Figure imgf000049_0001

Claims

1. A macromolecule, characterized in that the molecule comprises;
• G3.5 or G.4.5 generation polyamidoamine (PAMAM) dendrimer with at least one carboxyl structured (-COOH) anionic surface groups,
• At least one oxaliplatin whose monodentate ligand has been modified to be the -OH group, and in that at least one carboxyl structured (-COOH) anionic surface groups in the structure of G3.5 or G.4.5 generation PAMAM dendrimer and -OH groups of at least one oxaliplatin whose monodentate ligand has been modified to be the -OH group, are bound to each other without using linker molecule.
2. Macromolecule according to Claim 1, characterized in that the oxaliplatin compounds whose monodentate ligand has been modified to be the -OH group are platinum (IV) compounds with an octahedral structure.
3. Macromolecule according to Claim 2, characterized in that the oxaliplatin whose monodentate ligand has been modified to be the -OH group is
Figure imgf000050_0001
4. Macromolecule according to Claim 1, characterized in that the particle size is in the range of 10-1000nm.
5. Macromolecule according to Claim 4, characterized in that the particle size is 179,30 ± 77,30.
6. Macromolecule according to Claim 4, characterized in that the particle size is 155,93 ± 13,59.
7. Macromolecule according to Claim 1, characterized in that the PDI value is in the range of 0,05-0,6.
8. Macromolecule according to Claim 7, characterized in that the PDI value is 0,377.
9. Macromolecule according to Claim 7, characterized in that the PDI value is 0,340.
10. Macromolecule according to Claim 1, characterized in that the zeta potential is in the range of -lOOmV +100mV.
11. The production method of the macromolecule according to Claim 1, characterized in that in the case that the physical complexes are synthesized, it comprises the steps of
• Preparing 1-10 mg/mL stock solutions by dissolving oxaliplatin in mediums of water, pH 4 or pH 7,
• Taking 0.05-5 mL of oxaliplatin solution from the prepared stock solutions,
• Taking 0.65 mg of methanolic G3.5 or G4.5 dendrimer solution,
• Preparing oxaliplatin:dendrimer solutions in the range of 25-250:1 from the taken oxaliplatin and dendrimer solutions,
• Mixing the solutions with a magnetic stirrer for 24 hours,
• Evaporating the solvent,
• Suspending the residue in anhydrous ethanol,
• Centrifuging at 10.000rpm for 5 minutes to precipitate oxaliplatin that does not enter between the branches of the dendrimers and complex,
• At the end of the centrifuge, removing the supernatant and filtering through a 0,45 pm nylon or PTFE membrane filter,
• Obtaining an aqueous complex solution by evaporating the ethanol of the filtrate and dissolving the residue in water.
12. The method according to Claim 11, characterized in that the molar ratio of oxaliplatin: dendrimer is 25:1.
13. The method according to Claim 12, characterized in that 1,26 μmol of oxaliplatin and 0,05 μmol of dendrimer are mixed in an aqueous, pH 4 or pH 7 medium.
14. The production method of the macromolecule according to Claim 1, characterized in that in the case that chemical conjugates are synthesized with the G 3.5 dendrimer, it comprises the steps of
• Preparing a solution of oxaliplatin in acetic acid in the concentration range of 10- lOOOmM,
• Suspending the mixture in a magnetic stirrer,
• Adding H2O2 solution to the suspension, with H2O2 being 0, 1-1% by volume,
• Adding 30% H2O2 with 30 minutes intervals,
• Stirring the solution overnight,
• Removing the acetic acid by evaporation,
• Resuspending the oily residue with distilled water,
• Removing the added water from the medium by evaporation,
• Repeating the addition and removal of water until the product is solid,
• Suspending the obtained solid product with 10-20 mL of ethyl acetate,
• Adding methanol to the suspension until a clear solution is formed,
• Filtering the mixture through a 0,45 pm nylon membrane filter,
• Removing the solvent mixture from the medium,
• Storing the obtained modified oxaliplatin at +4°C,
• Removing G3.5 dendrimer from methanolic solution (10% w/w),
• Evaporating the methanol under vacuum and activating the carboxyl ends by dissolving the residue in HC1 solution with pH 3,
• Dissolving the dendrimers dried after evaporation of the water again in DMF,
• Adding modified oxaliplatin, DCC and DMAP to this solution,
• Adjusting the modified oxaliplatin mole percentage as 25-35%, DCC mole percentage as 25-35%, DMAP mole percentage as 25-35% and G3.5 dendrimer as 0-10% in the final mixture,
• Stirring the mixture under vacuum for 24 hours at room temperature,
• At the end of 24 hours, removing the precipitates from DCC formed during the reaction by filtrating through a 0,45 pm nylon membrane filter,
• Evaporating the solvent, • Dissolving the remaining residue in distilled water and if needed, filtering again through a 0,45 pm nylon membrane filter,
• Extracting with DCM 5 times to remove the hydrophobic organic substances remaining from the reaction from the medium,
• At the end of the extraction, purifying the aqueous part by dialysis and separating for lyophilization,
• Freezing, and primary and secondary drying of the aqueous solution in lyophilization,
• Obtaining lyophilized conjugates.
15. The production method of the macromolecule according to Claim 1, characterized in that in the case that chemical conjugates are synthesized with the G 4.5 dendrimer, it comprises the steps of
• Preparing a solution of oxaliplatin in acetic acid in the concentration range of 10- 1000mM,
• Suspending the mixture in a magnetic stirrer,
• Adding H2O2 solution to the suspension, with H2O2 being 0, 1 - 1 % by volume,
• Adding 30% H2O2 with 30 minutes intervals,
• Stirring the solution overnight,
• Removing the acetic acid by evaporation,
• Resuspending the oily residue with distilled water,
• Removing the added water from the medium by evaporation,
• Repeating the addition and removal of water until the product is solid,
• Suspending the obtained solid product with 10-20 mL of ethyl acetate,
• Adding methanol to the suspension until a clear solution is formed,
• Filtering the mixture through a 0,45 pm nylon membrane filter,
• Removing the the solvent mixture from the medium,
• Storing the obtained modified oxaliplatin at +4°C,
• Removing G4.5 dendrimer from methanolic solution (5% w/w),
• Evaporating the methanol under vacuum and activating the carboxyl ends by dissolving the residue in HC1 solution with pH 3,
• Dissolving the dendrimers dried after evaporation of the water again in 5mL DMF, • Adding modified oxaliplatin, DCC and DMAP to this solution,
• Adjusting the modified oxaliplatin mole percentage as 25-35%, DCC mole percentage as 25-35%, DMAP mole percentage as 25-35% and G3.5 dendrimer mole percentage as 0-10% in the final mixture,
• Stirring the mixture under vacuum for 24 hours at room temperature,
• At the end of 24 hours, removing the precipitates from DCC formed during the reaction by filtrating through a 0,45 pm nylon membrane filter,
• Removing solvent from the medium,
• Dissolving the remaining residue in distilled water and if needed filtering again through a 0,45 pm nylon membrane filter,
• Extracting with DCM 5 times to remove the hydrophobic organic substances remained from the reaction from the medium,
• At the end of the extraction, purifying the aqueous part by dialysis and separating for lyophilization,
• Freezing, primary and secondary drying of the aqueous solution in lyophilization,
• Obtaining lyophilized conjugates.
16. Macromolecule according to Claim 1, characterized in that it is used in the preparation of drugs to be used in cancer treatment.
17. Macromolecule according to Claim 16, characterized in that the said cancer is metastatic colorectal cancer, non-Hodgkin lymphoma, breast cancer, non-small cell lung cancer, head and neck cancer, mesothelioma, squamous cell carcinoma, or advanced ovarian cancer.
18. Macromolecule according to Claim 1, characterized in that it is used in the treatment of metastatic colorectal cancer, non-Hodgkin lymphoma, breast cancer, non-small cell lung cancer, head and neck cancer, mesothelioma, squamous cell carcinoma, or advanced ovarian cancer.
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