Classifications

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Definitions

• the present disclosure relates to the delivery of nucleoside analogue-based oncology agents by means of drug-dendrimer conjugates.
• the drug-dendrimer conjugates comprise a dendrimer including a core and building units, with the outermost generation of building units including one or more nucleoside analogue oncology agents attached via a cleavable linker group.
• the present disclosure also relates to pharmaceutical compositions and methods of treatment comprising the drug-dendrimer conjugates, and to processes and synthetic intermediates for producing the drug-dendrimer conjugates comprising nucleoside analogue- based oncology agents.
• Oncology agents are an important class of pharmaceuticals, and there have been significant advances in the chemotherapeutic treatment of cancer in recent decades.
• therapeutic application of oncology agents is often hampered by difficulties associated with their formulation and delivery, including poor pharmacokinetics properties such as rapid metabolism and/or excretion, and/or lack of targeting to the site of action.
• a number of oncology agents are associated with severe side effects, providing a narrow therapeutic window, limiting the dosage regimen that can be used, and potentially reducing the efficacy of the treatment.
• Nucleoside analogues such as gemcitabine have been shown to be effective in the therapy of some cancers, including ovarian, breast pancreatic and lung cancers.
• Gemcitabine acts by being incorporated into the DNA and RNA of rapidly dividing cells, such as cancer cells, and interfering with their growth and repair.
• gemcitabine also has drawbacks as a pharmaceutical agent, in that it is rapidly metabolised in vivo by cytidine deaminase, and it also undergoes renal clearance (Ciccolini et al, Cancer Chemother Pharmacol, 2016, 78, pl- 12), requiring high doses.
• Gemcitabine therapy is also associated with a number of side effects, including pulmonary toxicity and respiratory failure, haemolytic uremic syndrome, renal impairment, severe hepatic toxicity, capillary leak syndrome, and posterior reversible encephalopathy syndrome (see for example the prescribing information for Gemzar ® ).
• oncology agents may be specially formulated to try and counter the limitations of the drug substance itself.
• approaches investigated include encapsulation of the pharmaceutically active agent in liposomes and micelles. Nonetheless, there remains a need for alternative and/or improved oncology therapies.
• nucleoside analogue-dendrimer conjugates have surprising properties such that the amount of nucleoside analogue required (as part of the conjugate) to provide effective therapy is significantly reduced compared to free drug.
• the conjugates of the invention facilitate controlled release of the nucleoside analogue, and use of the conjugates is expected to have reduced side effects and/or enhanced efficacy.
• a dendrimer comprising: i) a core unit (C); and ii) building units (BU), each building unit being a lysine residue or an analogue thereof; wherein the core unit is covalently attached to two building units via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and the carbon atom of an acyl group present in a building unit; the dendrimer being a five generation building unit dendrimer; wherein building units of different generations are covalently attached to one another via amide linkages formed between a nitrogen atom present in one building unit and the carbon atom of an acyl group present in another building unit; the dendrimer further comprising: iii) a plurality of first terminal groups (T1) each comprising a residue of a nucleoside analogue, which nucleoside analogue has a hydroxyl group, covalently attached to a diacyl linker group of formula: , where
• the nucleoside analogue is selected from the group consisting of gemcitabine, cytarabine, and azacitadine. In some embodiments, the nucleoside analogue is gemcitabine.
• the core unit is formed from a core unit precursor comprising two amino groups. In some embodiments, the core unit is:
• the building units are each: wherein the acyl group of each building unit provides a covalent attachment point for attachment to the core or to a previous generation building unit; and wherein each nitrogen atom provides a covalent attachment point for covalent attachment to a subsequent generation building unit, a first terminal group or a second terminal group.
• the diacyl linker is selected from the group consisting of: In some embodiments, the diacyl linker is:
• the diacyl linker is:
• the nucleoside analogue is gemcitabine and is covalently attached to the diacyl linker group as shown below:
• the nucleoside analogue is gemcitabine and is covalently attached to the diacyl linker group as shown below:
• the first terminal group is:
• the first terminal group is:
• the hydrophilic polymeric group is selected from the group consisting of polyethylene glycol (PEG), polyethyloxazoline (PEOX), and polysarcosine.
• the second terminal groups comprise PEG groups having a mean molecular weight of at least 500 Daltons. In some embodiments, the second terminal groups comprise PEG groups having an average molecular weight in the range of from 1900 to 2300 Daltons.
• the second terminal groups each comprise a PEG group covalently attached to a PEG linking group (L1) via an ether linkage formed between a carbon atom present in the PEG group and an oxygen atom present in the PEG linking group, and each second terminal group is covalently attached to a building unit via an amide linkage formed between a nitrogen atom present in a building unit and the carbon atom of an acyl group present in the PEG linking group.
• the second terminal groups are each: and wherein the PEG group is a methoxy -terminated PEG having an average molecular weight in the range of from 500 to 2500 Daltons.
• the dendrimer comprises surface units comprising an outer building unit attached to a first terminal group and a second terminal group, the surface units having the structure: and wherein the PEG group is a methoxy -terminated PEG having an average molecular weight in the range of from 500 to 2500 Daltons.
• the dendrimer has from 28 to 32 surface units. In some embodiments, at least 40% of the nitrogen atoms present in the outer building units are each covalently attached to a first terminal group; and at least 40% of the nitrogen atoms present in the outer building units are each covalently attached to a second terminal group. In some embodiments, the five generations of building units are complete generations, and wherein the outer generation of building units provides 64 nitrogen atoms for covalent attachment to a first terminal group or a second terminal group, wherein from 24 to 32 first terminal groups are covalently attached to one of said nitrogen atoms, and wherein from 24 to 32 second terminal groups are each covalently attached to one of said nitrogen atoms.
• the dendrimer when the dendrimer is exposed to PBS at pH 7.4 and 37 °C, between about 20% and about 90% of the residue of the nucleoside analogue is released from the dendrimer after 24 hours.
• the dendrimer is: in which T1' represents a first terminal group which is or ;
• T2' represents a second terminal group which is: wherein the PEG group is a methoxy-terminated PEG having an average molecular weight in the range of from 500 to 2500 Daltons, or T2' represents H, and wherein less than 5 of T2' are H.
• a pharmaceutical composition comprising: i) a dendrimer as described herein, or a pharmaceutically acceptable salt thereof; and ii) a pharmaceutically acceptable excipient.
• a dendrimer as described herein, or a pharmaceutical composition as described herein, for use in the treatment of cancer in another aspect, there is provided a dendrimer as described herein, or a pharmaceutical composition as described herein, for use in the treatment of cancer.
• a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a dendrimer as described herein, or a pharmaceutical composition as described herein.
• the cancer is selected from the group consisting of breast cancer, ovarian cancer, non-small cell lung cancer, an upper gastrointestinal cancer (e.g. pancreatic cancer), and bladder cancer.
• the amount of dendrimer administered is sufficient to deliver an amount of active agent in the range of from 5 mg to 200 mg of nucleoside analogue/m 2 .
• the dendrimer is administered in combination with a further anticancer agent.
• the further anticancer agent is selected from the group consisting of platinum-containing pharmaceutical agents, taxanes, immunooncology agents, PARP inhibitors, topoisomerase I inhibitors, antibodies, antifolates, tyrosine kinase inhibitors, anthracyclines, and vinca alkaloids.
• the anticancer agent is selected from the group consisting of capecitabine, Nab-paclitaxel (e.g.
• Abraxane ® docetaxel, cabazitaxel, doxorubicin, vindesine, irinotecan, folinic acid, 5-fluorouracil, methotrexate, pemetrexed, lapatinib, nintedanib, sunitinib, olaparib, niraparib, carboplatin, paclitaxel, SN38, cisplatin, oxaliplatin, paclitaxel, erlotinib, and irinotecan.
• the dendrimer is administered in combination with a second dendrimer, and wherein the second dendrimer comprises: i) a core unit (C); and ii) building units (BU), each building unit being a lysine residue or an analogue thereof; wherein the core unit is covalently attached to two building units via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and the carbon atom of an acyl group present in a building unit; the dendrimer being a five generation building unit dendrimer; wherein building units of different generations are covalently attached to one another via amide linkages formed between a nitrogen atom present in one building unit and the carbon atom of an acyl group present in another building unit; the dendrimer further comprising: iii) a plurality of first terminal groups (T1) each comprising a residue of an oncology agent, which oncology agent has a hydroxyl group, covalently attached to a diacyl
• T1
• the oncology agent is a taxane.
• the taxane is selected from the group consisting of docetaxel, paclitaxel and cabazitaxel.
• the taxane is docetaxel.
• the oncology agent is a topoisomerase I inhibitor. In some embodiments, the topoisomerase I inhibitor is SN-38.
• administration of the dendrimer provides enhanced clinical efficacy in comparison to administration of an equivalent dose of free nucleoside analogue. In some embodiments, administration of the dendrimer provides reduced side effects and/or toxicity in comparison to administration of an equivalent dose of free nucleoside analogue. In some embodiments, administration of the dendrimer provides therapeutically effective plasma concentration levels of gemcitabine for an extended period of time following administration, in comparison to administration of an equivalent dose of free nucleoside analogue.
• Figure 1 shows the amount of gemcitabine released (%) from the dendrimer in PBS at pH 7.4 and 37 °C.
• Figure 2 shows the amount of gemcitabine released (%) from the dendrimer in 1% citrate buffer at pH 4.5 and 37 °C.
• Figure 3 shows the results of the plasma release study, demonstrating the release rates of gemcitabine (%) from the dendrimers following administration.
• Figure 4 shows the anti-tumour efficacy (measured in mean tumour volume) of the dendrimers against the CAPAN-1 tumour xenografts.
• Figure 5 shows the anti-tumour efficacy (measured in mean tumour volume) of the dendrimers against the CAPAN-1 tumour xenografts, based on once- weekly and twice-weekly dosing intervals.
• Figure 6 shows the anti-tumour efficacy of the dendrimers against the CAPAN-1 tumour xenografts, alone or in combination with Abraxane ® or DEP-DTX.
• Figure 7 shows the anti-tumour efficacy (measured in mean tumour volume) of the dendrimers against the CAPAN-1 tumour xenografts.
• Figure 8 shows the anti-tumour efficacy (measured in mean tumour volume) of the dendrimer against the A549 tumour xenografts.
• Figure 9 shows the anti-tumour efficacy (measured in mean tumour volume) of the dendrimers against the OVCAR-3 tumour xenografts, alone or in combination with Carboplatin.
• the term “subject” refers to any organism susceptible to a disease or condition.
• the disease or condition is cancer.
• the subject can be a mammal, primate, livestock (e.g., sheep, cow, horse, pig), companion animal (e.g., dog, cat), or laboratory animal (e.g., mouse, rabbit, rat, guinea pig, hamster).
• livestock e.g., sheep, cow, horse, pig
• companion animal e.g., dog, cat
• laboratory animal e.g., mouse, rabbit, rat, guinea pig, hamster
• the subject is a mammal.
• the subject is human.
• the term “treating” includes alleviation of the symptoms associated with a specific disorder or condition and eliminating said symptoms.
• the term “treating cancer” refers to alleviating the symptoms associated with cancer and eliminating said symptoms.
• the term “treating cancer” refers to a reduction in cancerous tumour size.
• the term “treating cancer” refers to an increase in progression-free survival.
• progression-free survival refers to the length of time during and after the treatment of cancer that a patient lives with the disease, i.e., cancer, but does not have a recurrence or increase in symptoms of the disease.
• a dendrimer would be administered in a therapeutically effective amount.
• the term “therapeutically effective amount” refers to a dendrimer being administered in an amount sufficient to result in a reduction in cancerous tumour size.
• the term “therapeutically effective amount” refers to a dendrimer being administered in an amount sufficient to result in an increase in progression-free survival.
• an “effective amount”, as used herein, refers to an amount of a dendrimer effective to achieve a desired pharmacologic effect or therapeutic improvement without undue adverse side effects or to achieve a desired pharmacologic effect or therapeutic improvement with a reduced side effect profile.
• therapeutically effective amounts may be determined by routine experimentation, including but not limited to a dose escalation clinical trial.
• therapeutically effective amount includes, for example, a prophylactically effective amount.
• a prophylactically effective amount is an amount sufficient to prevent metastasis.
• an effective amount” or “a therapeutically effective amount” can vary from subject to subject, due to variation in metabolism of the compound and any of age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. Thus, it is not always possible to specify an exact “effective amount”. However, an appropriate “effective amount” in any individual case may be determined by one of ordinary skill in the art using routine experimentation. Where more than one therapeutic agent is used in combination, a “therapeutically effective amount” of each therapeutic agent can refer to an amount of the therapeutic agent that would be therapeutically effective when used on its own, or may refer to a reduced amount that is therapeutically effective by virtue of its combination with one or more additional therapeutic agents.
• Suitable salts of the dendrimers include those formed with organic or inorganic acids or bases.
• pharmaceutically acceptable salt refers to pharmaceutically acceptable organic or inorganic salts.
• Exemplary acid addition salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.
• Exemplary base addition salts include, but are not limited to, ammonium salts, alkali metal salts, for example those of potassium and sodium, alkaline earth metal salts, for example those of calcium and magnesium, and salts with organic bases, for example dicyclohexylamine, N-methyl-D-glucomine, morpholine, thiomorpholine, piperidine, pyrrolidine, a mono-, di- or tri-lower alkylamine, for example ethyl-, tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethyl -propylamine, or a mono-, di- or trihydroxy lower alkylamine, for example mono-, di- or triethanolamine.
• organic bases for example dicyclohexylamine, N-methyl-D-glucomine, morpholine, thiomorpholine, piperidine, pyrrolidine, a mono-, di-
• a pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion.
• the counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound.
• a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion. It will also be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present disclosure since these may be useful as intermediates in the preparation of pharmaceutically acceptable salts or may be useful during storage or transport.
• solvates complexes with solvents in which they are reacted or from which they are precipitated or crystallized.
• solvates a complex with water
• hydrate a complex with water
• pharmaceutically acceptable solvate refers to an association of one or more solvent molecules and a compound of the present disclosure.
• solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.
• dendrimer refers to a molecule containing a core and dendrons attached to the core. Each dendron is made up of generations of branched building units resulting in a branched structure with increasing number of branches with each generation of building units.
• a “dendrimer”, including a drug-dendrimer conjugate, may include pharmaceutically acceptable salts or solvates as defined supra.
• building unit refers to a branched molecule which is a lysine residue or an analogue thereof having three functional groups, one for attachment to the core or a previous generation of building units and at least two functional groups for attachment to the next generation of building units or forming the surface of the dendrimer molecule.
• the term “attached” refers to a connection between chemical components by way of covalent bonding.
• covalent bonding refers to a chemical bond formed by the sharing of one or more electrons, especially pairs of electrons, between atoms.
• covalent bonding is used interchangeable with the term “covalent attachment”.
• a dendrimer comprising: i) a core unit (C); and ii) building units (BU), each building unit being a lysine residue or an analogue thereof; wherein the core unit is covalently attached to two building units via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and the carbon atom of an acyl group present in a building unit; the dendrimer being a five generation building unit dendrimer; wherein building units of different generations are covalently attached to one another via amide linkages formed between a nitrogen atom present in one building unit and the carbon atom of an acyl group present in another building unit; the dendrimer further comprising: iii) a plurality of first terminal groups (T1) each comprising a residue of a nucleoside analogue, which nucleoside analogue has a hydroxyl group, covalently attached to a diacyl linker group of formula: , wherein
• the core unit (C) of the dendrimer is covalently attached to two building units via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and the carbon atom of an acyl group present in a building unit.
• the core unit may for example be formed from a core unit precursor comprising two amino groups. Any suitable diamino-containing molecule may be used as the core unit precursor.
• the core unit is: and may, for example, be formed from a core unit precursor: having two reactive (amino) nitrogens. Building units
• the building units are lysine residues or analogues thereof, and may be formed from suitable building unit precursors, e.g. lysine or lysine analogues containing appropriate protecting groups.
• Lysine analogues have two amino nitrogen atoms for bonding to a subsequent generation of building units and an acyl group for bonding to a previous generation of building units or a core.
• suitable building units include: wherein the acyl group of each building unit provides a covalent attachment point for attachment to the core or to a previous generation building unit; and wherein each nitrogen atom provides a covalent attachment point for covalent attachment to a subsequent generation building unit, a first terminal group or a second terminal group.
• the building units are each: wherein the acyl group of each building unit provides a covalent attachment point for attachment to the core or to a previous generation building unit; and wherein each nitrogen atom provides a covalent attachment point for covalent attachment to a subsequent generation building unit, a first terminal group or a second terminal group.
• the building units are each: wherein the acyl group of each building unit provides a covalent attachment point for attachment to the core or to a previous generation building unit; and wherein each nitrogen atom provides a covalent attachment point for covalent attachment to a subsequent generation building unit, a first terminal group or a second terminal group.
• the outermost generation of building units may be formed by lysine or lysine analogue building units as used in the other generations of building units (BU) as described above.
• the outermost generation of building units (BU outer ) is the generation of building units that is outermost from the core of the dendrimer, i.e., no further generations of building units are attached to the outermost generation of building units (BU outer ).
• the dendrons of the dendrimer may for example be synthesised to the required number of generations through the attachment of building units (BU) accordingly.
• each generation of building units (BU) may be formed of the same building unit, for example all of the generations of building units may be lysine building units.
• one or more generations of building units may be formed of different building units to other generations of building units.
• the dendrimer is a five generation building unit dendrimer.
• a five generation building unit dendrimer is a dendrimer having a structure which includes five building units which are covalently linked to another, for example in the case where the building units are lysines, it may comprise the substructure:
• the dendrimer has five complete generations of building units. With a core having two reactive amine groups, such a dendrimer will comprise 62 building units (i.e. core unit + 2 BU + 4 BU + 8 BU + 16 BU + 32 BU). However, it will be appreciated that, due to the nature of the synthetic process for producing the dendrimers, one or more reactions carried out to produce the dendrimers may not go fully to completion. Accordingly, in some embodiments, the dendrimer may comprise an incomplete generations of building units. For example, a population of dendrimers may be obtained, in which the dendrimers have a distribution of numbers of building units per dendrimer.
• a population of dendrimers is obtained which has a mean number of building units per dendrimer of at least 55, or at least 56, or at least 57, or at least 58, or at least 59, or at least 60. In some embodiments, a population of dendrimers is obtained in which at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the dendrimers have 55 or more building units. In some embodiments, a population of dendrimers is obtained in which at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the dendrimers have 60 or more building units.
• Each reactive (amino) group of the core represents a conjugation site for a dendron comprising one or more generations of building units.
• the core has two reactive (amino) groups, and two dendrons, for the generations of building units to be attached.
• each generation of building units in each dendron (X) may be represented by the formula [BU] 2(b-1) , wherein b is the generation number.
• a dendron (X) having five complete generations of building units is represented as [BU] 1 -[BU] 2 -[BU] 4 -[BU] 8 - [BU] 16 .
• the dendrimer comprises a residue of a nucleoside analogue.
• Nucleoside analogues are a group of therapeutic agents which find use in applications such as cancer therapy.
• the nucleoside analogues contain one or more hydroxyl groups which can be utilised for linking of the nucleoside analogue to the dendrimer via the linker.
• the nucleoside analogues may, in addition to the hydroxyl group, contain other groups which can be utilised for linking to the dendrimer.
• the nucleoside analogue may contain one or more amino groups which can be utilised for linking of the nucleoside analogue to the linker (e.g., a diacyl linker).
• nucleoside analogues examples include pyrimidine nucleoside analogues, e.g. gemcitabine, deoxycytidine, cytarabine, 5 -aza-cytidine (also known as azacitidine), capecitabine and decitabine.
• pyrimidine nucleoside analogues e.g. gemcitabine, deoxycytidine, cytarabine, 5 -aza-cytidine (also known as azacitidine), capecitabine and decitabine.
• purine nucleoside analogues such as cladribine, clofarabine, fludarabine, doxifluridine, forodesine, nelarabine and pentostatin.
• nucleoside analogue is a pyrimidine nucleoside analogue. In some embodiments, the nucleoside analogue is an anticancer nucleoside analogue. In some embodiments, the nucleoside analogue is gemcitabine, azacitidine or cytarabine.
• the nucleoside analogue is gemcitabine.
• Gemcitabine is an oncology agent having the structure:
• the residue of the nucleoside analogue active is attached to the diacyl linker through the 3 - or 5'-position of the nucleoside analogue.
• the residue of a nucleoside analogue active has the substructure: 3' -attached pyrimidine nucleoside analogue.
• the residue of a nucleoside analogue active has the substructure: 5'-attached pyrimidine nucleoside analogue.
• the residue of a nucleoside analogue active has the substructure: 3' -attached purine nucleoside analogue.
• the residue of a nucleoside analogue active has the substructure: 5'- attached purine nucleoside analogue.
• the nucleoside analogue is an anticancer nucleoside analogue which is attached via the 5’ hydroxyl group.
• the nucleoside analogue is gemcitabine, azacytidine, or cytarabine, which are attached via the 5’ hydroxyl group.
• the nucleoside analogue is gemcitabine which is attached via the 5’ hydroxyl group.
• the nucleoside analogue is covalently attached as shown below:
• the residue of a nucleoside analogue active has an amino group, and the residue of a nucleoside analogue active is attached to the diacyl linker through an amino group.
• the nucleoside analogue is gemcitabine which is attached via the amino group.
• the nucleoside analogue is covalently attached as shown below:
• the dendrimer Upon in vivo administration, typically the dendrimer releases the nucleoside analogue (e.g., gemcitabine).
• nucleoside analogue e.g., gemcitabine
• A is a C 2 -C 10 alkylene group which is interrupted by at least one O, S, NH, or N(Me), or in which A is a heterocycle selected from the group consisting of tetrahydrofuran, tetrahydrothiophene, pyrrolidine, and N-methylpyrrolidine.
• alkyl refers to straight (i.e., linear) or branched chain hydrocarbons ranging in size from one to 10 carbon atoms (i.e. C 1-10 alkyl).
• alkyl moieties include, unless explicitly limited to smaller groups, moieties ranging in size, for example, from about one to about six carbon atoms or greater, such as, methyl, ethyl, n-propyl, iso-propyl and/or butyl, pentyl, hexyl, and higher isomers.
• the alkyl moiety is of one to 10 carbon atoms (i.e. C 1-10 alkyl).
• the alkyl moiety is of 2 to 4 carbon atoms, preferably 4 carbon atoms.
• alkylene refers to straight (i.e. linear) or branched chain hydrocarbons ranging in size from 1 to 10 carbon atoms (i.e. C 1-10 alkylene).
• alkylene moieties include, for example, -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH(CH 3 )-, - CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH(CH 3 )CH 2 -, and the like.
• the diacyl linker is: wherein A is a C 2 -C 10 alkylene group (e.g straight chain or branched) which is interrupted by at least one O, S, NH, or N(Me).
• the diacyl linker is: wherein A is a C 2 -C 6 alkylene group (e.g. straight chain or branched) which is interrupted by at least one O, S, NH, or N(Me).
• A is a C 2 -C 6 alkylene group (e.g. straight chain or branched) which is interrupted by at least one O, S, NH, or N(Me).
• the diacyl linker is selected from the group consisting of: and
• the diacyl linker is:
• the diacyl linker is:
• the residue of a nucleoside analogue active is typically covalently attached to the diacyl linker via a linkage formed between an oxygen atom present as part of the nucleoside analogue active side-chain and a carbon atom of an acyl group present as part of the diacyl linker.
• the other acyl group of the diacyl linker forms an amide linkage with a nitrogen atom present in an outer building unit.
• the nucleoside analogue is gemcitabine and is covalently attached to the diacyl linker group as shown below: and wherein the diacyl linker is: wherein A is a C 2 -C 10 alkylene group which is interrupted by O, S, NH, or N(Me), or in which A is a heterocycle selected from the group consisting of tetrahydrofuran, tetrahydrothiophene, pyrrolidine and N-methylpyrrolidine .
• the first terminal group is:
• the first terminal group is:
• the inventors have found that, by the combination of particular cleavable linker groups, with specific hydroxyl groups present in the nucleoside analogue structure, that controlled and consistent release of nucleoside analogue active can be achieved, leading to good biological activity and good pharmacokinetic properties.
• the 3' site of a nucleoside analogue is typically more sterically hindered than the 5' position.
• linker groups have been identified which release drug from the dendrimer at a desirable rate, which can be conjugated with the nucleoside analogue active in high yield, and which can also be used to achieve good levels of loading of nucleoside analogue active onto the dendrimer.
• nucleoside analogue active Whilst the residue of a nucleoside analogue active is typically covalently attached to the diacyl linker via a linkage formed between an oxygen atom present as part of the nucleoside analogue active, the linkage may also be formed via another suitable atom, e g. where the nucleoside analogue active contains an amino group, the linkage may be formed via a nitrogen atom present in the amino group.
• the dendrimer comprises a plurality of second terminal groups (T2) each comprising a hydrophilic polymeric group.
• the second terminal group T2 is a pharmacokinetic modifying agent.
• a pharmacokinetic modifying agent is an agent that can modify or modulate the pharmacokinetic profile of the dendrimer or the pharmaceutically active agent (i.e. gemcitabine) that the dendrimer is delivering.
• the pharmacokinetic modifying agent may modulate the absorption, distribution, metabolism, excretion and/or toxicity of the dendrimer of the pharmaceutically active agent.
• the pharmacokinetic modifying agent (T2) may influence the rate of release of the pharmaceutically active agent, either by slowing or increasing the rate in which the active agent is released from the dendrimer by either chemical (e.g., hydrolysis) or enzymatic degradation pathways.
• the pharmacokinetic modifying agent (T2) may change the solubility profile of the dendrimer, either increasing or decreasing the solubility of the dendrimer in a pharmaceutically acceptable carrier.
• the pharmacokinetic modifying agent (T2) may assist the dendrimer in delivering the pharmaceutically active agent to specific tissues (e.g., tumours).
• the pharmacokinetic modifying agent (T2) may extend the pharmaceutically active agent half-life by reducing clearance of the dendrimer.
• hydrophilic polymeric group typically refers to a polymeric group that has a solubility in water at 25 °C of at least 25 mg/ml, more preferably at least 50 mg/ml, and still more preferably at least 100 mg/ml.
• the hydrophilic polymeric group comprises repeating units of amino acids, alkyloxy, or alkyl(acyl)amino groups.
• the hydrophilic polymeric group comprises repeating units of amino acids, such as sarcosine.
• the hydrophilic polymeric group comprises repeating units of alkyloxy groups (e.g. the hydrophilic polymer is a PEG group).
• the hydrophilic polymer comprises repeating units of alkyl(acyl)amino groups (e.g. the hydrophilic polymer is a PEOX group).
• the hydrophilic polymeric group is a PEG group.
• the hydrophilic polymeric group is a PEOX group.
• the hydrophilic polymeric group is a polysarcosine group.
• the hydrophilic polymeric group comprises at least 10 monomer units. In some embodiments, the hydrophilic polymeric group comprises up to 100 monomer units. In some embodiments, the hydrophilic polymeric group comprises from 10 to 100, or from 10 to 50 monomer units.
• the second terminal group comprises a PEG group (i.e., the hydrophilic polymeric group is a PEG group).
• a PEG group is a polyethylene glycol group, i.e. a group comprising repeat units of the formula -CH 2 CH 2 O-.
• PEG materials used to produce the dendrimer of the present disclosure typically contain a mixture of PEGs having some variance in molecular weight (i.e., ⁇ 10%), and therefore the molecular weight specified is typically an approximation of the average molecular weight of the PEG composition.
• the term “PEG ⁇ 2100 ” refers to polyethylene glycol having an average molecular weight of approximately 2100 Daltons, i.e.
• PEG ⁇ 2300 refers to polyethylene glycol having an average molecular weight of approximately 2300 Daltons, i.e. ⁇ approximately 10% (PEG 2070 to PEG 2530 ). Three methods are commonly used to calculate MW averages: number average, weight average, and z-average molecular weights.
• molecular weight is intended to refer to the weight-average molecular weight which can be measured using techniques well-known in the art including, but not limited to, NMR, mass spectrometry, matrix-assisted laser desorption ionization time of flight (MALDI-TOF), gel permeation chromatography or other liquid chromatography techniques, light scattering techniques, ultracentrifugation and viscometry.
• NMR nuclear magnetic resonance
• MALDI-TOF matrix-assisted laser desorption ionization time of flight
• the second terminal groups comprise PEG groups having an average molecular weight of between about 200 and 5000 Daltons. In some embodiments, the second terminal groups comprise PEG groups having an average molecular weight of at least 500 Daltons. In some embodiments, the second terminal groups comprise PEG groups having an average molecular weight of at least 750 Daltons. In some embodiments, the second terminal groups comprise PEG groups having an average molecular weight in the range of from 500 to 2500 Daltons.
• the second terminal groups comprise PEG groups having an average molecular weight in the range of from 1900 to 2300 Daltons. In some embodiments, the second terminal groups comprise PEG groups having an average molecular weight in the range of from 2000 to 2200 Daltons. In some embodiments, the second terminal groups comprise PEG groups having an average molecular weight of about 2100 Daltons. In some embodiments, the second terminal groups comprise PEG groups having an average molecular weight of about 1900, about 2000, about 2100, about 2200, about 2300, about 2400 or about 2500 Daltons.
• the second terminal groups comprise PEG groups having an average molecular weight in the range of from 1000 to 1200 Daltons. In some embodiments, the second terminal groups comprise PEG groups having an average molecular weight of about 1100 Daltons. In some embodiments, the second terminal groups comprise PEG groups having an average molecular weight of about 1000, about 1100, or about 1200 Daltons.
• the second terminal groups comprise PEG groups having an average molecular weight in the range of from 500 to 650 Daltons. In some embodiments, the second terminal groups comprise PEG groups having an average molecular weight of about 570 Daltons. In some embodiments, the second terminal groups comprise PEG groups having an average molecular weight of about 500, about 530, about 550, about 570, about 590, about 610, about 630, or about 650 Daltons.
• the PEG group has a polydispersity index (PDI) of between about 1.00 and about 1.50, between about 1.00 and about 1.25, or between about 1.00 and about 1.10. In some embodiments, the PEG group has a polydispersity index (PDI) of about 1.05.
• the term “polydispersity index” refers to a measure of the distribution of molecular mass in a given polymer sample.
• the polydispersity index (PDI) is equal to the weight average molecular weight (Mw) divided by the number average molecular weight (M n ) and indicates the distribution of individual molecular masses in a batch of polymers.
• the polydispersity index (PDI) has a value equal to or greater than one, but as the polymer approaches uniform chain length and average molecular weight, the polydispersity index (PDI) will be closer to one.
• the PEG groups may be linear or branched. If desired, an end-capped PEG group may be used. In some embodiments, the PEG group is a methoxy-terminated PEG.
• the second terminal group comprises a PEOX group (i.e., the hydrophilic polymeric group is a PEOX group).
• a PEOX group is a polyethyloxazoline group, i.e. a group comprising repeat units of the formula:
• PEOX groups are so named since they can be produced by polymerisation of ethyloxazoline.
• PEOX materials used to produce the dendrimer of the present disclosure typically contain a mixture of PEOXs having some variance in molecular weight (i.e., ⁇ 10%), and therefore, where a molecular weight is specified, it is typically an approximation of the average molecular weight of the PEOX composition.
• the second terminal groups comprise PEOX groups having an average molecular weight of at least 750 Daltons, at least 1000 Daltons, or at least 1500 Daltons.
• the second terminal groups comprise PEOX groups having an average molecular weight in the range of from 750 Daltons to 2500 Daltons, or from 1000 Daltons to 2000 Daltons. If desired, an end- capped PEOX group may be used.
• the PEOX group is a methoxy- terminated PEOX.
• the hydrophilic polymeric group comprises a polysarcosine group.
• the polysarcosine groups have an average molecular weight of at least 750 Daltons, at least 1000 Daltons, or at least 1500 Daltons.
• the hydrophilic polymeric groups comprise polysarcosine groups having an average molecular weight in the range of from 750 Daltons to 2500 Daltons, or from 1000 Daltons to 2000 Daltons.
• the hydrophilic polymeric group may be attached to the outer building unit via any suitable means.
• a linking group is used to attach the hydrophilic polymeric group to the outer building unit.
• the second terminal group may be attached to the outer building unit via any suitable means.
• a linking group is used to attach the hydrophilic polymeric group (e.g., PEG group, PEOX group, or polysarcosine group) to the outer building unit.
• the second terminal groups are typically attached via use of a second terminal group precursor which contains a reactive group that is reactive with an amine group, such as a reactive acyl group (which can form an amide bond), or an aldehyde (which can form an amine group under reductive amination conditions).
• a reactive group that is reactive with an amine group such as a reactive acyl group (which can form an amide bond), or an aldehyde (which can form an amine group under reductive amination conditions).
• the second terminal groups each comprise a PEG group covalently attached to a PEG linking group (L1) via an ether linkage formed between a carbon atom present in the PEG group and an oxygen atom present in the PEG linking group, and each second terminal group is covalently attached to a building unit via an amide linkage formed between a nitrogen atom present in a building unit and the carbon atom of an acyl group present in the PEG linking group.
• the second terminal groups are each: and wherein the PEG group is a methoxy-terminated PEG having an average molecular weight in the range of from about 500 to 2500 Daltons.
• the second terminal groups each comprise a PEOX group covalently attached to a PEOX linking group ( L1') via a linkage formed between a nitrogen atom present in the PEOX group and a carbon atom present in the PEOX linking group, and each second terminal group is covalently attached to a building unit via an amide linkage formed between a nitrogen atom present in a building unit and the carbon atom of an acyl group present in the PEOX linking group.
• the second terminal groups are each:
• the second terminal groups each comprise a polysarcosine group, i.e. a group comprising repeat units of the formula:
• the polysarcosine groups are attached to a building unit via an amide linkage formed between a nitrogen atom present in a building unit and the carbon atom of an acyl group present in the polysarcosine group.
• the hydrophilic polymeric groups comprise polysarcosine groups having an average molecular weight of at least 750 Daltons, at least 1000 Daltons, or at least 1500 Daltons.
• the second terminal groups comprise polysarcosine groups having an average molecular weight in the range of from 750 Daltons to 2500 Daltons, or from 1000 Daltons to 2000 Daltons.
• the dendrimers have controlled stoichiometry and/or topology.
• the dendrimers are typically produced using synthetic processes that allow for a high degree of control over the number and arrangement of first and second terminal groups present on the dendrimers.
• each functionalised outer building unit contains one first terminal group and one second terminal group.
• the dendrimer comprises surface units comprising an outer building unit attached to a first terminal group and a second terminal group, the surface units having the structure: and wherein the PEG group is a methoxy -terminated PEG having an average molecular weight in the range of from about 500 to 2500 Daltons (e.g. about 2000 to 2400 Daltons).
• the dendrimer comprises surface units comprising an outer building unit attached to a first terminal group and a second terminal group, the surface units having the structure: and wherein the PEG group is a methoxy -terminated PEG having an average molecular weight in the range of from about 500 to 2500 Daltons (e.g. about 2000 to 2400 Daltons).
• the dendrimer has from 28 to 32 surface units. In some embodiments, the dendrimer has from 30 to 32 surface units.
• At least 40% of the nitrogen atoms present in the outer building units are each covalently attached to a first terminal group. In some embodiments, at least 45% of the nitrogen atoms present in the outer building units are each covalently attached to a first terminal group. In some embodiments, about 50% of the nitrogen atoms present in the outer building units are each covalently attached to a first terminal group.
• At least 40% of the nitrogen atoms present in the outer building units are each covalently attached to a second terminal group. In some embodiments, at least 45% of the nitrogen atoms present in the outer building units are each covalently attached to a second terminal group. In some embodiments, about 50% of the nitrogen atoms present in the outer building units are each covalently attached to a second terminal group.
• At least 40% of the nitrogen atoms present in the outer building units are each covalently attached to a first terminal group; and at least 40% of the nitrogen atoms present in the outer building units are each covalently attached to a second terminal group.
• at least 45% of the nitrogen atoms present in the outer building units are each covalently attached to a first terminal group; and at least 45% of the nitrogen atoms present in the outer building units are each covalently attached to a second terminal group.
• about 50% of the nitrogen atoms present in the outer building units are each covalently attached to a first terminal group; and about 50% of the nitrogen atoms present in the outer building units are each covalently attached to a second terminal group.
• the five generations of building units are complete generations, and wherein the outer generation of building units provides 64 nitrogen atoms for covalent attachment to a first terminal group or a second terminal, wherein from 24 to 32 first terminal groups are covalently attached to one of said nitrogen atoms, and wherein from 24 to 32 second terminal groups are each covalently attached to one of said nitrogen atoms.
• first terminal groups are each covalently attached to one of said nitrogen atoms.
• from 29 to 31 first terminal groups are each covalently attached to one of said nitrogen atoms.
• from 26 to 32, or from 28 to 32 second terminal groups are each covalently attached to one of said nitrogen atoms.
• from 29 to 31 second terminal groups are each covalently attached to one of said nitrogen atoms
• the first terminal group comprises less than 20% w/w of the dendrimer, or less than 15% w/w of the dendrimer, or less than 10% w/w of the dendrimer, or between 5 and 30% w/w of the dendrimer, or between 8 and 15% w/w of the dendrimer, e.g. as measured by 1 H NMR.
• no more than one quarter of the nitrogen atoms present in the outer generation of building units are unsubstituted. In some embodiments, no more than one fifth of the nitrogen atoms present in said outer generation of building units are unsubstituted. In some embodiments, no more than one sixth of the nitrogen atoms present in said outer generation of building units are unsubstituted. In some embodiments, no more than one eighth of the nitrogen atoms present in said outer generation of building units are unsubstituted In some embodiments, no more than one tenth of the nitrogen atoms present in said outer generation of building units are unsubstituted.
• no more than 20 nitrogen atoms present in the outer generation of building units are unsubstituted. In some embodiments, no more than 10 nitrogen atoms present in the outer generation of building units are unsubstituted. In some embodiments, no more than 5 nitrogen atoms present in the outer generation of building units are unsubstituted In some embodiments, no more than 3 nitrogen atoms present in the outer generation of building units are unsubstituted. In some embodiments, no more than 2 nitrogen atoms present in the outer generation of building units are unsubstituted. In some embodiments, no more than 1 nitrogen atom present in the outer generation of building units are unsubstituted.
• the dendrimer is: in which T1' represents a first terminal group which is: T2' represents a second terminal group which is: wherein the PEG group is a methoxy-terminated PEG having an average molecular weight in the range of from 500 to 2500 Daltons, or T2' represents H, and wherein less than 5 of T2' are H.
• the dendrimer has a molecular weight in the range of from 25 to 300 kDa, or from 40 to 300 kDa, or from 75 to 200 kDa, or from 90 to 150 kDa. In some embodiments, the dendrimer has a molecular weight in the range of from 35 to 100 kDa. In one example, the dendrimer has a molecular weight in the range of from 35 to 45 kDa, or in the range of from 50 to 60 kDa, or in the range of from 85 to 95 kDa.
• the in vitro half-life is the point at which 50% of drug is released from the dendrimer. In some embodiments, the in vitro half-life may be determined by using a least squares fitting to a 1 st order release model.
• the in vitro half-life for nucleoside analogues release from the dendrimer in PBS (phosphate-buffer saline) at pH 7.4 and at 37 °C is in the range of from 2 to 50 hours. In some embodiments, the in vitro half-life for nucleoside analogue (e.g. gemcitabine) release from the dendrimer in PBS at pH 7.4 and at 37 °C is in the range of from 5 to 50 hours, or 10 to 50 hours, or 5 to 40 hours, or 5 to 30 hours, or 10 to 30 hours. In some embodiments, the in vitro half-life for nucleoside analogue (e.g.
• the in vitro half-life for nucleoside analogue (e.g. gemcitabine) release from the dendrimer in PBS at pH 7.4 and at 37 °C is in the range of from 2 to 10 hours.
• the in vitro half-life for nucleoside analogue (e.g. gemcitabine) release from the dendrimer in PBS at pH 74 and at 37 °C is in the range of from 20 to 40 hours.
• the percentage nucleoside analogue (e.g. gemcitabine) released from the dendrimer after 24 hours in PBS (phosphate-buffer saline) at pH 7.4 and at 37 °C is in the range of from 20 to 100% of total gemcitabine. In some embodiments, the percentage nucleoside analogue (e.g. gemcitabine) released from the dendrimer in PBS at pH 7.4 and at 37 °C is in the range of from 40 to 80%. In some embodiments, the percentage nucleoside analogue (e.g. gemcitabine) released from the dendrimer in PBS at pH 7.4 and at 37 °C is in the range of from 40 to 60%. In some embodiments, the percentage nucleoside analogue (e.g. gemcitabine) released from the dendrimer in PBS at pH 7.4 and at 37 °C is in the range of from 70 to 90%.
• the in vitro half-life for nucleoside analogue (e.g. gemcitabine) release from the dendrimer in citrate buffer (0.1 M, pH 4.5) at 37 °C is in the range of from 1 to 20 days. In some embodiments, the in vitro half-life for nucleoside analogue (e.g. gemcitabine) release from the dendrimer in citrate buffer (0.1 M, pH 4.5) at 37 °C is in the range of from 5 to 10 days. In some embodiments, the in vitro half-life for nucleoside analogue (e.g. gemcitabine) release from the dendrimer in citrate buffer (0.1 M, pH 4.5) at 37 °C is in the range of from 8 to 15 days.
• nucleoside analogue (e.g. gemcitabine) release from the dendrimer in citrate buffer (0.1 M, pH 4.5) at 37 °C is in the range of from 8 to 15 days.
• the in vitro half-life for nucleoside analogue e.g. gemcitabine
• the in vitro half-life for nucleoside analogue (e.g. gemcitabine) release from the dendrimer in citrate buffer (0.1 M, pH 4.5) at 37 °C is in the range of from 10 to 20 days.
• the percentage nucleoside analogue (e.g. gemcitabine) released from the dendrimer after 24 hours in citrate buffer (0.1 M, pH 4.5) at 37 °C is in the range of from 1 to 30% of total gemcitabine. In some embodiments, the percentage nucleoside analogue (e.g. gemcitabine) released from the dendrimer in citrate buffer (0.1 M, pH 4.5) at 37 °C is in the range of from 1 to 20%. In some embodiments, the percentage nucleoside analogue (e.g. gemcitabine) released from the dendrimer in citrate buffer (0.1 M, pH 4.5) at 37 °C is in the range of from 10 to 20%. In some embodiments, the percentage nucleoside analogue (e.g. gemcitabine) released from the dendrimer in citrate buffer (0.1 M, pH 4.5) at 37 °C is in the range of from 1 to 5%.
• the dendrimer is any one of the dendrimers described in the Examples below (excluding any comparative Examples).
• the dendrimer is presented as a composition, preferably a pharmaceutical composition.
• a composition comprising a plurality of dendrimers or pharmaceutically acceptable salts thereof, wherein the dendrimers are as defined herein, the mean number of first terminal groups per dendrimer in the composition is in the range of from 24 to 32, and the mean number of second terminal groups per dendrimer in the composition is in the range of from 24 to 32.
• the mean number of first terminal groups per dendrimer is in the range of from 28 to 32, and wherein the mean number of second terminal groups per dendrimer is in the range of from 28 to 32. In some embodiments, the mean number of first terminal groups per dendrimer is in the range of from 29 to 32, and wherein the mean number of second terminal groups per dendrimer is in the range of from 29 to 32. In some embodiments, the mean number of first terminal groups per dendrimer is in the range of from 30 to 32, and wherein the mean number of second terminal groups per dendrimer is in the range of from 30 to 32. In some embodiments, the composition is a pharmaceutical composition, and the composition comprises a pharmaceutically acceptable excipient.
• At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers contain at least 24 first terminal groups. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers contain at least 26 first terminal groups. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers contain at least 28 first terminal groups.
• At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers contain at least 28 second terminal groups. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers contain at least 29 second terminal groups.
• At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers contain at least 24 first terminal groups and at least 28 second terminal groups. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers contain at least 26 first terminal groups and at least 29 second terminal groups.
• compositions both for veterinary and for human medical use, which comprise the dendrimers of the present disclosure or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, and optionally any other therapeutic ingredients, stabilisers, or the like.
• the carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof.
• the composition is a pharmaceutical composition, and wherein the composition comprises a pharmaceutically acceptable excipient.
• the composition may for example contain a solvent, such as water (e.g. water for injection) or a pharmaceutically acceptable organic solvent.
• compositions may further include diluents, buffers, citrate, trehalose, binders, disintegrants, thickeners, lubricants, preservatives (including antioxidants), inorganic salts (e.g., sodium chloride), antimicrobial agents (e.g., benzalkonium chloride), sweeteners, antistatic agents, sorbitan esters, lipids (e.g., phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, fatty acids and fatty esters, steroids (e.g., cholesterol)), and chelating agents (e.g., EDTA, zinc and other such suitable cations).
• diluents e.g., buffers, citrate, trehalose, binders, disintegrants, thickeners, lubricants, preservatives (including antioxidants), inorganic salts (e.g., sodium chloride), antimicrobial agents (e.g
• compositions of the present disclosure may also include polymeric excipients/additives or carriers, e.g., polyvinylpyrrolidones, derivatised celluloses such as hydroxymethylcellulose, hydroxy ethylcellulose, and hydroxypropylmethylcellulose, Ficolls (a polymeric sugar), hydroxy ethyl starch (HES), dextrates (e.g., cyclodextrins, such as 2- hydroxy propyl-b-cyclodextrin and sulfobutylether-b-cyclodextrin), polyethylene glycols, and pectin.
• polymeric excipients/additives or carriers e.g., polyvinylpyrrolidones, derivatised celluloses such as hydroxymethylcellulose, hydroxy ethylcellulose, and hydroxypropylmethylcellulose, Ficolls (a polymeric sugar), hydroxy ethyl starch (HES), dextrates (e
• compositions according to the present disclosure are listed in “Remington: The Science & Practice of Pharmacy", 19.sup.th ed., Williams & Williams, (1995), and in the “Physician's Desk Reference", 52.sup.nd ed., Medical Economics, Montvale, N.J. (1998), and in “Handbook of Pharmaceutical Excipients", Third Ed., Ed. A. H. Kibbe, Pharmaceutical Press, 2000.
• dendrimers of the present disclosure may be formulated in compositions including those suitable for inhalation to the lung, by aerosol, or parenteral (including intraperitoneal, intravenous, subcutaneous, or intramuscular injection) administration.
• compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the dendrimer into association with a carrier that constitutes one or more accessory ingredients.
• compositions are prepared by bringing the dendrimer into association with a liquid carrier to form a solution or a suspension, or alternatively, bring the dendrimer into association with formulation components suitable for forming a solid, optionally a particulate product, and then, if warranted, shaping the product into a desired delivery form.
• Solid formulations of the present disclosure when particulate, will typically comprise particles with sizes ranging from about 1 nanometer to about 500 microns. In general, for solid formulations intended for intravenous administration, particles will typically range from about 1 nm to about 10 microns in diameter.
• the composition may contain dendrimer of the present disclosure that are nanoparticulate having a particulate diameter of below 1000 nm, for example, between 5 and 1000 nm, especially 5 and 500 nm, more especially 5 to 400 nm, such as 5 to 50 nm and especially between 5 and 20 nm. In one example, the composition contains dendrimers with a mean size of between 5 and 20nm.
• the dendrimer is polydispersed in the composition, with PDI of between 1.01 and 1.8, especially between 1.01 and 1.5, and more especially between 1.01 and 1.2. In one example, the dendrimer is monodispersed in the composition.
• the composition is formulated for parenteral delivery.
• the composition is formulated for intravenous delivery.
• the formulation may be a sterile, lyophilized composition that is suitable for reconstitution in an aqueous vehicle prior to injection.
• a composition suitable for parenteral administration conveniently comprises a sterile aqueous preparation of the dendrimer, which may for example be formulated to be isotonic with the blood of the recipient.
• compositions are also provided which are suitable for administration as an aerosol, by inhalation. These formulations comprise a solution or suspension of the desired dendrimer or a salt thereof.
• the desired formulation may be placed in a small chamber and nebulized. Nebulization may be accomplished by compressed air or by ultrasonic energy to form a plurality of liquid droplets or solid particles comprising the dendrimers or salts thereof.
• the dendrimers of the present disclosure may for example be administered in combination with one or more additional pharmaceutically active agents.
• the composition comprises a dendrimer as defined herein, or a pharmaceutically acceptable salt thereof, one or more pharmaceutically acceptable carriers, and one or more additional pharmaceutically active agents, e.g. an additional oncology agent or oncology drug.
• the additional active ingredient is an anti-cancer agent for therapy of prostate cancer or breast cancer
• the additional pharmaceutically active agent is a chemotherapeutic agent, a cytotoxic agent, or an antibody therapy. In some embodiments, the additional pharmaceutically active agent is a MAPK/ERK signalling pathway inhibitor.
• the additional pharmaceutically active agent is a further anticancer agent.
• further anticancer agents include, but are not limited to, platinum-containing pharmaceutical agents, taxanes, immunooncology agents, PARP inhibitors, topoisomerase I inhibitors, antibodies, antifolates, tyrosine kinase inhibitors, anthracyclines, and vinca alkaloids.
• the anticancer agent is selected from the group consisting of capecitabine, Nab-paclitaxel (e.g.
• Abraxane ® docetaxel, cabazitaxel, doxorubicin, vindesine, irinotecan, folinic acid, 5-fluorouracil, methotrexate, pemetrexed, lapatinib, nintedanib, sunitinib, olaparib, niraparib, carboplatin, paclitaxel, SN38, cisplatin, oxaliplatin, paclitaxel, erlotinib, and irinotecan.
• additional pharmaceutically active agents include anti-CD20 agents, for example, antibodies such as rituximab (Rituxan ® ).
• additional pharmaceutically active agents include immunooncology agents, for example, PD-1 inhibitors, PD-L1 inhibitors, or CTLA4 inhibitors, such as pembrolizumab (Keytruda ® ), nivolumab (Opdivo ® ), durvalumab (Imfinzi ® ), atezolizumab (Tecentriq ® ), avelumab (Bavencio ® ), and ipilimumab (Yervoy ® ).
• immunooncology agents for example, PD-1 inhibitors, PD-L1 inhibitors, or CTLA4 inhibitors, such as pembrolizumab (Keytruda ® ), nivolumab (Opdivo ® ), durvalumab (Imfinzi ® ), atezolizumab (Tecentriq ® ), avelumab (Bavencio ® ), and ipilimumab
• the composition is formulated for parenteral infusion as part of a chemotherapy regimen.
• the dendrimers of the present disclosure may be used to treat or prevent any disease, disorder or symptom that the unmodified pharmaceutically active agent can be used to treat or prevent. Accordingly, there is also provided a dendrimer or pharmaceutical composition as described herein for use in therapy.
• the dendrimer, or pharmaceutical composition comprising the dendrimer is used in a method of treating or preventing cancer, for example for suppressing the growth of a tumour.
• the dendrimer is for use in the treatment of cancer.
• a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the dendrimer, or of a pharmaceutical composition comprising the dendrimer.
• the cancer is a solid tumour.
• the cancer may be a primary or metastatic tumour.
• the cancer is a primary tumour.
• the cancer is a metastatic tumour.
• the cancer is selected from the group consisting of breast cancer, ovarian cancer, non-small cell lung cancer, pancreatic cancer, bladder cancer, upper gastrointestinal cancer, oesophageal cancer, stomach cancer, small bowel cancer, liver cancer, cancer of the biliary system, sarcoma, ovarian cancer, gall bladder cancer, biliary tract cancer, prostate and nasopharyngeal cancer.
• the cancer is breast cancer.
• the cancer is ovarian cancer.
• the cancer is non-small cell lung cancer.
• the cancer is pancreatic cancer.
• the cancer is bladder cancer.
• the cancer is upper gastrointestinal cancer.
• the cancer is oesophageal cancer. In some embodiments, the cancer is stomach cancer. In some embodiments, the cancer is small bowel cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is cancer of the biliary system. In some embodiments, the cancer is sarcoma. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is gall bladder cancer. In some embodiments, the cancer is biliary tract cancer. In some embodiments, the cancer is nasopharyngeal cancer.
• the dendrimer is administered in combination with one or more further pharmaceutically active agents, for example one or more further anti-cancer agents/oncology agents.
• the dendrimer and the one or more further pharmaceutically active agents may be administered simultaneously, subsequently or separately. For example, they may be administered as part of the same composition, or by administration of separate compositions.
• the one or more further pharmaceutically active agents may for example be anti-cancer agents for therapy of prostate cancer or breast cancer.
• further pharmaceutically active agents include, but are not limited to, chemotherapeutic and cytotoxic agents, antibody therapies, and MAPK/ERK signalling pathway inhibitors.
• Examples of further pharmaceutically active agents include, but are not limited to, platinum-containing pharmaceutical agents, taxanes, immunooncology agents, PARP inhibitors, topoisomerase I inhibitors, antibodies, antifolates, tyrosine kinase inhibitors, anthracyclines, and vinca alkaloids.
• the dendrimer is administered in combination with a further anticancer agent selected from the group consisting of capecitabine, Nab-paclitaxel (e.g. Abraxane ® ), docetaxel, cabazitaxel, doxorubicin, vindesine, irinotecan, folinic acid, 5- fluorouracil, methotrexate, pemetrexed, lapatinib, nintedanib, sunitinib, olaparib, niraparib, carboplatin, paclitaxel, SN38, cisplatin, oxaliplatin, paclitaxel, erlotinib, and irinotecan.
• a further anticancer agent selected from the group consisting of capecitabine, Nab-paclitaxel (e.g. Abraxane ® ), docetaxel, cabazitaxel, doxorubicin, vindesine, iri
• the further pharmaceutically active agent is an anti-CD20 agent, for example, an antibody such as rituximab (Rituxan ® ).
• the further pharmaceutically active agent is an immunooncology agent, for example, a PD-1 inhibitor, PD-L1 inhibitor, or CTLA4 inhibitor, such as pembrolizumab (Keytruda ® ), nivolumab (Opdivo ® ), durvalumab (Imfinzi ® ), atezolizumab (Tecentriq ® ), avelumab (Bavencio ® ), and ipilimumab (Yervoy ® ).
• the dendrimers of the present disclosure are effective when administered in combination with an oncology drug-containing dendrimer (e.g., taxane- containing dendrimer).
• the dendrimer is administered in combination with a second dendrimer, which second dendrimer comprises: i) a core unit (C); and ii) building units (BU), each building unit being a lysine residue or an analogue thereof; wherein the core unit is covalently attached to two building units via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and the carbon atom of an acyl group present in a building unit; the dendrimer being a five generation building unit dendrimer; wherein building units of different generations are covalently attached to one another via amide linkages formed between a nitrogen atom present in one building unit and the carbon atom of an acyl group present in another building unit; the dendrimer further comprising: ii
• the core unit (C) of the second dendrimer is as described above for the first dendrimer.
• the building units (BU) of the second dendrimer are as described above for the first dendrimer.
• the diacyl linker of the second dendrimer is as described above for the first dendrimer.
• the second terminal group of the second dendrimer is as described above for the first dendrimer.
• the oncology agent is a taxane.
• Taxane-containing dendrimers are described in, for example, WO2012/167309, US2018/0326081 and WO2020/014750A1, the contents of which are incorporated herein by reference.
• the taxane of the second dendrimer may be selected from any taxane that exhibits chemotherapeutic activity.
• the taxane is selected from the group consisting of docetaxel, paclitaxel, and cabazitaxel.
• the taxane is docetaxel.
• the taxane is paclitaxel.
• the taxane is cabazitaxel.
• the taxane may be covalently attached to a diglycolyl or thiodiglycolyl linker via an ester linkage formed between an oxygen atom present as part of the taxane and a carbon atom of an acyl group present as part of the linker.
• the second dendrimer comprises: i) a core unit (C); and ii) building units (BU), each building unit being a lysine residue; wherein the core unit is covalently attached to two building units via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and the carbon atom of an acyl group present in a building unit; the dendrimer being a five generation building unit dendrimer; wherein building units of different generations are covalently attached to one another via amide linkages formed between a nitrogen atom present in one building unit and the carbon atom of an acyl group present in another building unit; the dendrimer further comprising: iii) a plurality of first terminal groups (T1) each comprising a residue of docetaxel, which docetaxel has a hydroxyl group, covalently attached to a diacyl linker group of formula: iv) a plurality of second terminal groups (T2) each comprising a PEG
• the second dendrimer comprises: i) a core unit (C); and ii) building units (BU), each building unit being a lysine residue; wherein the core unit is covalently attached to two building units via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and the carbon atom of an acyl group present in a building unit; the dendrimer being a five generation building unit dendrimer; wherein building units of different generations are covalently attached to one another via amide linkages formed between a nitrogen atom present in one building unit and the carbon atom of an acyl group present in another building unit; the dendrimer further comprising: iii) a plurality of first terminal groups (T1) each comprising a residue of docetaxel, which docetaxel has a hydroxyl group, covalently attached to a diacyl linker group of formula: iv) a plurality of second terminal groups (T2) each comprising a PEG
• the second dendrimer comprises: i) a core unit (C); and ii) building units (BU), each building unit being a lysine residue; wherein the core unit is covalently attached to two building units via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and the carbon atom of an acyl group present in a building unit; the dendrimer being a five generation building unit dendrimer; wherein building units of different generations are covalently attached to one another via amide linkages formed between a nitrogen atom present in one building unit and the carbon atom of an acyl group present in another building unit; the dendrimer further comprising: iii) a plurality of first terminal groups (T1) each comprising a residue of cabazitaxel, which cabazitaxel has a hydroxyl group, covalently attached to a diacyl linker group of formula: iv) a plurality of second terminal groups (T2) each
• the second dendrimer comprises: i) a core unit (C); and ii) building units (BU), each building unit being a lysine residue; wherein the core unit is covalently attached to two building units via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and the carbon atom of an acyl group present in a building unit; the dendrimer being a five generation building unit dendrimer; wherein building units of different generations are covalently attached to one another via amide linkages formed between a nitrogen atom present in one building unit and the carbon atom of an acyl group present in another building unit; the dendrimer further comprising: iii) a plurality of first terminal groups (T1) each comprising a residue of cabazitaxel, which cabazitaxel has a hydroxyl group, covalently attached to a diacyl linker group of formula: iv) a plurality of second terminal groups (T2) each
• the oncology agent is a topoisomerase I inhibitor, for example, a camptothecin active.
• Camptothecin active-containing dendrimers are described in, for example, WO2020/102852A1, the contents of which are incorporated herein by reference.
• the topoisomerase I inhibitor is a camptothecin active, for example, SN38.
• the camptothecin active (e.g., SN38) may, for example, be covalently attached to a diglycolyl or thiodiglycolyl linker via an ester linkage formed between an oxygen atom present as part of the camptothecin active and a carbon atom of an acyl group present as part of the linker.
• the second dendrimer comprises: i) a core unit (C); and ii) building units (BU), each building unit being a lysine residue; wherein the core unit is covalently attached to two building units via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and the carbon atom of an acyl group present in a building unit; the dendrimer being a five generation building unit dendrimer; wherein building units of different generations are covalently attached to one another via amide linkages formed between a nitrogen atom present in one building unit and the carbon atom of an acyl group present in another building unit; the dendrimer further comprising: iii) a plurality of first terminal groups (T1) each comprising a residue of SN38, which SN38 has a hydroxyl group, covalently attached to a diacyl linker group of formula: iv) a plurality of second terminal groups (T2) each comprising a PEG; or
• the second dendrimer comprises: i) a core unit (C); and ii) building units (BU), each building unit being a lysine residue; wherein the core unit is covalently attached to two building units via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and the carbon atom of an acyl group present in a building unit; the dendrimer being a five generation building unit dendrimer; wherein building units of different generations are covalently attached to one another via amide linkages formed between a nitrogen atom present in one building unit and the carbon atom of an acyl group present in another building unit; the dendrimer further comprising: iii) a plurality of first terminal groups (T1) each comprising a residue of a camptothecin active (e.g., SN38), which camptothecin active has a hydroxyl group, covalently attached to a diacyl linker group of formula: iv) a plurality of first terminal
• a therapeutically effective amount refers to a dendrimer being administered in an amount sufficient to alleviate or prevent to some extent one or more of the symptoms of the disorder or condition being treated.
• a therapeutically effective amount of dendrimer may be referred to based on, for example, the amount of dendrimer administered. Alternatively, it may be determined based on the amount of nucleoside analogue active (e.g. gemcitabine) which the dendrimer is theoretically capable of delivering, e.g. based on the loading of nucleoside analogue active on the dendrimer.
• nucleoside analogue active e.g. gemcitabine
• a therapeutically effective amount of dendrimer-drug conjugate may be referred to on the basis of, for example, the amount of dendrimer-drug conjugate administered. Alternatively, it may be determined based on the amount of active agent (drug moiety comprising gemcitabine nucleoside) which the dendrimer-drug conjugate is theoretically capable of delivering, e.g. based on the loading of drug moiety on the dendrimer.
• the terms “unconjugated” and “released” refer to a drug moiety which has dissociated or been cleaved from a dendrimer. This dissociation or cleaving may occur in vivo following administration of the drug-dendrimer conjugate.
• the dendrimer may be administered by any suitable route.
• the route of administration may for example be targeted to the disease or disorder which the subject has.
• the subject is typically a human, although it will be understood that the dendrimer can also be used to treat conditions in non-human animals.
• the dendrimer is administered intravenously.
• Gemcitabine itself is typically administered as an IV infusion over a 30 minute period.
• the dendrimer is delivered as an IV bolus.
• the dendrimer is administered IV over a time a period in the range of from 0.5 to 15 minutes, or in the range of from 0.5 to 5 minutes.
• the dendrimer may be administered intraperitoneally.
• the disease or disorder may be an intra-abdominal malignancy such as a gynecological or gastrointestinal cancer, and the dendrimer may be administered intraperitoneally
• the dendrimer may be for treatment of a cancer of the peritoneal cavity, such as a malignant epithelial tumor (e.g., ovarian cancer) or peritoneal carcinomatosis (eg gastrointestinal especially colorectal, gastric, gynecologic cancers, and primary peritoneal neoplasms), and the dendrimer is administered intraperitoneally.
• a malignant epithelial tumor e.g., ovarian cancer
• peritoneal carcinomatosis eg gastrointestinal especially colorectal, gastric, gynecologic cancers, and primary peritoneal neoplasms
• Gemcitabine itself is typically administered at 7-day intervals, e.g., on days 1 and 8, or days 1, 8, and 15 of a dosing cycle, or once weekly.
• the dendrimers are administered in at least 10-day dosing intervals, at least 14-day dosing intervals, at least 21 -day dosing intervals, or at least 28-day dosing intervals.
• a typical dose is 1000 mg/m 2 or 1250 mg/m 2 .
• the amount of dendrimer administered is sufficient to deliver an amount of released active agent in the range of from 1 to 1500 mg of nucleoside analogue (e.g. gemcitabine)/m 2 , in the range of from 1 to 1250 mg of nucleoside analogue (e.g. gemcitabine)/m 2 , in the range of from 1 to 500 mg, in the range of from 1 to 400 mg, in the range of from 1 to 300 mg, in the range of from 5 to 200 mg, in the range of from 500 to 1250 mg of nucleoside analogue (e.g. gemcitabine)/m 2 , in the range of from 2 to 1000 mg of nucleoside analogue (e.g.
• nucleoside analogue e.g. gemcitabine
• nucleoside analogue e.g. gemcitabine
• range of from 5 to 500 mg of nucleoside analogue (e.g. gemcitabine)/m 2 in the range of from 5 to 100 mg of nucleoside analogue (e.g. gemcitabine)/m 2
• range of from 20 to 500 mg of nucleoside analogue (e.g. gemcitabine)/m 2 in the range of from 20 to 200 mg of nucleoside analogue (e.g. gemcitabine)/m 2
• range of from 50 to 200 mg of nucleoside analogue (e.g. gemcitabine)/m 2 in the range of from 50 to 100 mg of nucleoside analogue (e.g.
• gemcitabine /m 2 , in the range of from 75 to 125 mg of nucleoside analogue (e.g. gemcitabine)/m 2 , or in the range of from 50 to 75 mg of nucleoside analogue (e.g. gemcitabine)/m 2 .
• a therapeutically effective amount of the dendrimer is administered to a subject in need thereof at a predetermined frequency. In some embodiments, the dendrimer is administered to a subject in need thereof according to a dosage regimen in which the dendrimer is administered once per one to four weeks. In some embodiments, the dendrimer is administered to a subject in need thereof according to a dosage regimen in which the dendrimer is administered once per three to four weeks.
• the dendrimer or pharmaceutical composition is administered as a fast infusion or as a bolus.
• the infusion time is less than 1 hour, less than 30 minutes or less than 20 minutes, or the infusion time is 20 minutes, 15 minutes or 10 minutes.
• the administration may be as a bolus, for example, in 5 seconds to 5 minutes.
• dendrimers of the present disclosure may provide for controlled release of drug moiety comprising a residue of a nucleoside analogue in vivo, and may allow for administration of a large quantity of conjugated drug moiety in a single dose, which is then released gradually over time.
• the dendrimer will provide therapeutic levels of the residue of a nucleoside analogue for prolonged periods, and so can be administered less frequently than the free nucleoside analogues.
• the dendrimer may be administered once every two days, or once every three days, or once every seven days, or once every ten days, or once every two weeks, or once every three weeks, or one every four weeks, or once per month.
• a single dose of dendrimer provides a therapeutically effective amount of the drug moiety comprising a residue of a nucleoside analogue over a period of at least 6 hours, at least 12 hours, at least 24 hours, at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days.
• a single dose of dendrimer provides a therapeutically effective amount of a residue of a nucleoside analogue over a period of about one day, about two days, about three days, about four days, about five days, about six days, about seven days, about eight days, about nine days, about ten days, or about 14 days.
• the dendrimer when exposed to PBS at pH 7.4 and 37 °C, releases between about 5% and 90%, between about 10% and about 90%, between about 5% and 80%, between about 10% and 80%, between about 20% and 80%, between about 20% and 70%, or between about 30 and 60% of the residue of the nucleoside analogue after 24 hours. In some embodiments, when exposed to PBS at pH 7.4 and 37 °C, the dendrimer releases at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the residue of the nucleoside analogue after 24 hours.
• the dendrimer when exposed to 1% citrate buffer at pH 4.5 and 37 °C, releases between about 5% and 30%, between about 5% and 25%, between about 5% and 20%, between about 5% and 10%, of the residue of the nucleoside analogue after 24 hours. In some embodiments, when exposed to 1% citrate buffer at pH 4.5 and 37 °C, the dendrimer releases less than about 5%, less than about 10%, less than about 20%, less than about 30%, of the residue of the nucleoside analogue after 24 hours. Pharmacokinetics, Efficacy and Side Effects
• dendrimers of the present disclosure provide for good therapeutic and pharmacokinetic properties in vivo.
• free refers to a drug, e.g., gemcitabine, which has not been previously conjugated to a dendrimer.
• the direct administration of free gemcitabine refers to the direct administration of gemcitabine molecules that are not administered as being conjugated to a dendrimer.
• An example of such a therapy is Gemzar ® .
• the terms “unconjugated” and “released” refer to a drug, e.g. gemcitabine, which has dissociated or been cleaved from a dendrimer. This dissociation or cleaving may occur in vivo following administration of the drug-dendrimer conjugate. Comparisons may for example be made following administration of a dendrimer comprising gemcitabine as the nucleoside analogue, and following administration of an equivalent amount of unconjugated drug (e.g., of Gemzar ® ).
• equivalent amount or “equivalent dose” in this context refers to administration of a dose of dendrimer which, if all nucleoside analogue present as part of the dendrimer were released, would provide the same number of moles of nucleoside analogue active as in a dose of unconjugated drug being administered.
• administration of the dendrimer provides enhanced clinical efficacy in comparison to administration of an equivalent dose of free nucleoside analogue (e.g. gemcitabine).
• Enhanced clinical efficacy may include any one or more of the following: improved survival rate, reduced mortality rate, improved life expectancy, and slower progression rate of cancer.
• the dendrimers of the present disclosure provide a lower maximal concentration (Cmax), increased duration of therapeutically effective plasma concentration of nucleoside analogue (e.g. gemcitabine), and/or reduced toxicity, in comparison to administration of an equivalent amount of the unconjugated drug.
• Cmax maximal concentration
• nucleoside analogue e.g. gemcitabine
• administration of the dendrimer provides a lower maximal concentration (Cmax) of nucleoside analogue (e.g., gemcitabine), in comparison to administration of an equivalent amount of the unconjugated drug.
• Cmax of nucleoside analogue (e.g., gemcitabine) achieved following administration of the dendrimer is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 2% of the Cmax achieved following administration of an equivalent amount of the unconjugated drug.
• administration of the dendrimer provides therapeutically effective plasma concentration levels of nucleoside analogue (e.g., gemcitabine) for an extended period of time following administration, in comparison to administration of an equivalent dose of free gemcitabine.
• administration of the dendrimer provides therapeutically effective plasma concentration levels of nucleoside analogue (e.g., gemcitabine) for at least twice as long, or at least three times as long, or at least four times as long, or at least five times as long as the period of time over which therapeutically effective plasma concentrations of nucleoside analogue are achieved following administration of an equivalent dose of free nucleoside analogue.
• Oncology drugs often have significant side effects that are due to off-target toxicity.
• nucleoside analogues such as gemcitabine
• known side effects include pulmonary toxicity and respiratory failure, haemolytic uremic syndrome, renal impairment, severe hepatic toxicity, capillary leak syndrome, and posterior reversible encephalopathy syndrome.
• the toxicity of a drug refers to the degree to which damage is caused to the organism, and is measured by its effect off target. In oncology, one such measurement of toxicity in animal models is weight loss, which determines the maximum tolerated dose (MTD). In humans toxicity is commonly determined by specified adverse events (AE), which typically identify the dose limiting toxicity. It will be appreciated that usually in oncology, there is a narrow therapeutic window and off-target toxicities are considered a normal side effect of killing tumour cells. In some embodiments, administration of the dendrimer provides reduced toxicity and/or side effects in comparison to administration of an equivalent dose of free nucleoside analogue (e.g. gemcitabine).
• an equivalent dose of free nucleoside analogue e.g. gemcitabine
• the dendrimers of the present disclosure may be prepared by any suitable method, for example, by reacting a nucleoside analogue-containing precursor with a dendrimeric intermediate already containing a PEG group to introduce the pharmaceutically active agent; by reacting a PEG-containing precursor with a dendrimeric intermediate already containing a nucleoside analogue residue; or by reacting an intermediate comprising the residue of a lysine group, a nucleoside analogue residue, and a PEG group, with a dendrimeric intermediate. Accordingly, there is provided a process for producing a dendrimer as defined herein, comprising: a) reacting a nucleoside analogue intermediate which is:
• A is O or S
• X is -OH or a leaving group (e.g. an active ester), or wherein X together with the C(O) group to which it is attached forms a carboxylate salt
• a dendrimeric intermediate which comprises: i) a core unit (C); and ii) building units (BU), each building unit being a lysine residue or an analogue thereof; wherein the core unit is covalently attached to two building units via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and the carbon atom of an acyl group present in a building unit; the dendrimer being a five generation building unit dendrimer; wherein building units of different generations are covalently attached to one another via amide linkages formed between a nitrogen atom present in one building unit and the carbon atom of an acyl group present in another building unit; the dendrimer further comprising: a plurality of second terminal groups (T2) each comprising a hydrophilic
• X is -OH or a leaving group (e.g. active ester), or wherein X together with the C(O) group to which it is attached forms a carboxylate salt; with a dendrimeric intermediate which comprises: i) a core unit (C); and ii) building units (BU), each building unit being a lysine residue or an analogue thereof; wherein the core unit is covalently attached to two building units via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and the carbon atom of an acyl group present in a building unit; the dendrimer being a five generation building unit dendrimer; wherein building units of different generations are covalently attached to one another via amide linkages formed between a nitrogen atom present in one building unit and the carbon atom of an acyl group present in another building unit; the dendrimer further comprising: a plurality of first terminal groups (T1) each comprising a nucleoside analogue residue covalently
• X is -OH or a leaving group (e.g. an active ester), or wherein X together with the C(O) group to which it is attached forms a carboxylate salt; with a dendrimeric intermediate comprising: i) a core unit (C); and ii) building units (BU), each building unit being a lysine residue or an analogue thereof; wherein the core unit is covalently attached to two building units via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and the carbon atom of an acyl group present in a building unit; the dendrimeric intermediate being a four generation building unit dendrimeric intermediate; wherein building units of different generations are covalently attached to one another via amide linkages formed between a nitrogen atom present in one building unit and the carbon atom of an acyl group present in another building unit; and wherein nitrogen atoms present in the outer building units of the dendrimeric intermediate are unsubstituted; or
• a dendrimeric intermediate which comprises: i) a core unit (C); and ii) building units (BU), each building unit being a lysine residue or an analogue thereof; wherein the core unit is covalently attached to two building units via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and the carbon atom of an acyl group present in a building unit; the dendrimer being a five generation building unit dendrimer; wherein building units of different generations are covalently attached to one another via amide linkages formed between a nitrogen atom present in one building unit and the carbon atom of an acyl group present in another building unit; the dendrimer further comprising: a plurality of second terminal groups (T2) each comprising a hydrophilic polymeric group (e.g.
• a PEG, PEOX or polysarcosine group a salt thereof; under amide coupling conditions; and deprotecting PG to form the product as a salt; and optionally converting the product from the salt to another salt or to the free base.
• Process variants a), b), c), and d) involve formation of amide bonds by reaction of - C(O)X groups with amine groups present in the dendrimeric intermediates.
• Any suitable amide formation conditions also referred to as amide coupling conditions, may be used. Examples of typical conditions include the use of a suitable solvent (for example dimethylformamide) optionally a suitable base, and at a suitable temperature (for example ambient temperature, e.g. in the range of from 15 to 30 °C).
• a suitable solvent for example dimethylformamide
• a suitable base for example ambient temperature, e.g. in the range of from 15 to 30 °C.
• X is a leaving group
• any suitable leaving group may be used, for example an activated ester.
• X is an -OH group or where X together with the C(O) group to which it is attached forms a carboxylate salt, the group will typically be converted to a suitable leaving group prior to reaction with a dendrimeric intermediate, for example by use of a suitable amide coupling reagent such as PyBOP.
• the dendrimer may be obtained by dissolution in a suitable solvent (e.g. THF) and precipitation by addition into an antisolvent (e.g. MTBE).
• a suitable solvent e.g. THF
• an antisolvent e.g. MTBE
• nucleoside analogue intermediate used in variant a) may itself be obtained, for example, by reaction of nucleoside analogue or protected form thereof (e.g., gemcitabine) with diglycolic anhydride or thiodiglycolic anhydride, for example in the presence of a suitable solvent such as dichloromethane and a suitable base such as triethylamine.
• a suitable solvent such as dichloromethane
• a suitable base such as triethylamine
• the surface unit intermediate used in variant c) may itself be obtained, for example, by: i) reacting a PEG intermediate which is: wherein PEG Group is a PEG-containing group, and
• X is -OH or a leaving group, or wherein X together with the C(O) group to which it is attached forms a carboxylate salt; with: wherein PG1 is an amine protecting group (such as a Boc or Cbz group), and PG2 is either absent, or is an acid protecting group (such as a methyl or benzyl ester); ii) deprotecting PG1; iii) reacting the product of step ii) with a nucleoside analogue intermediate which is: wherein A is O or S, X is -OH or a leaving group (e.g. an active ester), or wherein X together with the C(O) group to which it is attached forms a carboxylate salt; and iv) deprotecting PG2, if present.
• PG1 is an amine protecting group (such as a Boc or Cbz group)
• PG2 is either absent, or is an acid protecting group (such as a methyl or benzyl ester
• the dendrimeric intermediate used in variant a) may itself be obtained by, for example, a sequential process involving: i) reaction of a core unit (C) containing amino groups, with building units which are protected lysines or analogues thereof, which contain a -C(O)X group, wherein X is -OH or a leaving group (e.g.
• an active ester) or -CO(X) forms a carboxylate salt, and in which the amino groups present in the lysines or analogues thereof are protected, to form amide linkages between the core unit and building units; ii) deprotecting protecting groups present on the building units; iii) reacting free amino groups present on the building units with further building units which are protected lysines or analogues thereof, which contain a - C(O)X group, wherein X is -OH or a leaving group (e.g.
• an active ester) or -CO(X) forms a carboxylate salt, and in which the amino groups present in the lysines or analogues thereof are protected, to form amide linkages between the different generations of building units; iv) deprotecting protecting groups present on the building units; v) repeating steps iii) and iv) until a four generation building unit is produced; vi) reacting free amino groups present on the building units with:
• PG is a protecting group, and wherein X is -OH or a leaving group (e.g. an active ester), or wherein X together with the C(O) group to which it is attached forms a carboxylate salt, to form amide linkages therebetween; and vii) deprotecting the protecting groups PG.
• X is -OH or a leaving group (e.g. an active ester), or wherein X together with the C(O) group to which it is attached forms a carboxylate salt, to form amide linkages therebetween; and vii) deprotecting the protecting groups PG.
• the dendrimeric intermediate used in variant a) may be obtained, for example, by carrying out steps i) to v) as described above, and: vi) reacting free amino groups present on the building units with further building units which are protected lysines or analogues thereof, which contain a -C(O)X group, wherein X is -OH or a leaving group (e.g.
• an active ester) or -CO(X) forms a carboxylate salt, and in which the amino groups present in the lysines or analogues thereof are orthogonally protected, to form amide linkages between the different generations of building units; vii) deprotecting a first set of amino protecting groups; viii) reacting free amino groups present on the building units with: wherein PEG Group is a PEG-containing group, and X is -OH or a leaving group (e.g. an active ester), or wherein X together with the C(O) group to which it is attached forms a carboxylate salt; ix) deprotecting a second set of amino protecting groups.
• the first and second sets of amino protecting groups may, for example, be Fmoc and Boc groups.
• step vii) comprises deprotecting a first set of amino protecting groups which are Fmoc groups, e.g. which protect lysine e-amino groups
• step ix) comprises deprotecting a second set of amino protecting groups which are Boc groups, e.g. which protect lysine building unit a-amino groups.
• the dendrimeric intermediate used in variant b) may itself be obtained, for example, by carrying out steps i) to v) as described above in relation to variant a), and: vi) reacting free amino groups present on the building units with:
• A is O or S
• PG is a protecting group
• X is -OH or a leaving group (e.g. an active ester), or wherein X together with the C(O) group to which it is attached forms a carboxylate salt, to form amide linkages therebetween; and vii) deprotecting the protecting groups PG.
• the dendrimeric intermediate used in variant b) may be obtained, for example, by carrying out steps i) to v) as described above, and: vi) reacting free amino groups present on the building units with further building units which are protected lysines or analogues thereof, which contain a -C(O)X group, wherein X is -OH or a leaving group (e.g. an active ester) or -CO(X) forms a carboxylate salt, and in which the amino groups present in the lysines or analogues thereof are orthogonally protected, to form amide linkages between the different generations of building units; vii) deprotecting a first set of amino protecting groups; viii) reacting free amino groups present on the building units with:
• A is O or S
• X is -OH or a leaving group (e.g. an active ester), or wherein X together with the C(O) group to which it is attached forms a carboxylate salt; vii) deprotecting a second set of amino protecting groups.
• the dendrimeric intermediate used in variant c) may itself be obtained, for example, by carrying out steps i) to v) as described above in relation to variant a).
• the present disclosure also provides synthetic intermediates useful in producing the dendrimers. Accordingly, there is also provided an intermediate for producing a dendrimer which is: wherein A is O or S, X is -OH or a leaving group (e.g. an active ester), or wherein X together with the C(O) group to which it is attached forms a carboxylate salt. Such an intermediate may be produced, for example, as described above.
• an intermediate for producing a dendrimer which is: wherein PEG Group is a PEG-containing group, A is O or S, and
• X is -OH or a leaving group (e.g. active ester), or wherein X together with the C(O) group to which it is attached forms a carboxylate salt.
• a leaving group e.g. active ester
• Such an intermediate may be produced, for example, as described above.
• Deprotection of the amino groups is undertaken utilising suitable reagents and conditions known in the art.
• deprotection of hoc protecting groups is undertaken utilising acidic conditions.
• deprotection of hoc protecting groups is undertaken utilising trifluoroacetic acid (TFA) conditions.
• TFA trifluoroacetic acid
• the resulting product of the deprotection reaction may be present in salt form.
• the resulting product of the deprotection reaction may be present as the trifluoroacetic acid salt
• a process for producing a dendrimer as defined herein comprising i) obtaining the dendrimer in salt form; ii) contacting the dendrimer with an ion exchange resin and separating the salt from the conjugate, and iii) separating the dendrimer from the ion exchange resin.
• the product of the deprotection reaction is converted from the salt to the free base. In some embodiments, the product of the deprotection reaction is converted from one particular salt to another particular salt. In some embodiments, the product of the deprotection reaction is converted from the TFA salt to the free base. In some embodiments, the product of the deprotection reaction is converted from the TFA salt to the HC1 salt.
• An ion exchange resin is an insoluble matrix of resin or polymer that acts as a medium for ion exchange. Ion exchange resins may find application in, for example, separation, purification, and decontamination processes.
• ion exchange resins include, but are not limited to, AmberLyst ® (styrene-divinylbenzene matrix with sulfonic acid functionality), and AmberLite ® (styrene-divinylbenzene matrix with sulfonic acid functionality).
• the ion exchange resin is AmberLyst ® .
• the ion exchange resin is AmberLite ® .
• the ion exchange resin is AmberLyst ® A21.
• a process for converting a dendrimer salt to its free base form comprising: i) contacting the dendrimer salt with an ion exchange resin for a period of time; and ii) separating the ion-exchange resin from the dendrimer to obtain the dendrimer in free base form.
• the product of the deprotection reaction is contacted with the ion exchange resin by passing the product through a column comprising the ion exchange resin.
• the product of the deprotection reaction is contacted with the ion exchange resin by stirring the product together with the ion exchange resin in solvent for a period of time.
• suitable solvents including, for example, dichloromethane (DCM).
• DCM dichloromethane
• the product may be stirred together with the ion exchange resin for an amount of time sufficient so as to form the free base or salt-exchanged product.
• the product is stirred together with the ion exchange resin for about 10 minutes, about 30 minutes, about 1 hour, about 12 hours, or about 24 hours.
• the ion exchange resin may be filtered so as to provide the free base or salt-exchanged product. From here, the dendrimer product may be obtained via any number of methods known in the art.
• the dendrimer is obtained as the salt. In some embodiments, the dendrimer is obtained as the TFA salt. In some embodiments, the dendrimer is obtained as the free base. In some embodiments, the dendrimer is obtained as the hydrochloric acid (HC1) salt.
• HC1 hydrochloric acid
• a dendrimer comprising: i) a core unit (C); and ii) building units (BU), each building unit being a lysine residue or an analogue thereof; wherein the core unit is covalently attached to two building units via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and the carbon atom of an acyl group present in a building unit; the dendrimer being a five generation building unit dendrimer; wherein building units of different generations are covalently attached to one another via amide linkages formed between a nitrogen atom present in one building unit and the carbon atom of an acyl group present in another building unit; the dendrimer further comprising: iii) a plurality of first terminal groups (T1) each comprising a residue of a nucleoside analogue, which nucleoside analogue has a hydroxyl group, covalently attached to a diacyl linker group of formula: , wherein A is a C 2 -
• nucleoside analogue is selected from the group consisting of gemcitabine and cytarabine.
• a dendrimer according to clause 2 wherein the nucleoside analogue is gemcitabine. 4. A dendrimer according to any one of clauses 1 to 3, wherein the core unit is formed from a core unit precursor comprising two amino groups. 5. A dendrimer according to any one of clauses 1 to 4, wherein the core unit is:
• a dendrimer according to clause 12, wherein the first terminal group is: 14. A dendrimer according to clause 12, wherein the first terminal group is:
• a dendrimer according to clause 15, wherein the second terminal groups comprise PEG groups having an average molecular weight in the range of from 500 to 2500 Daltons.
• LI PEG linking group
• 27. A dendrimer according to any one of clauses 1 to 26, wherein the five generations of building units are complete generations, and wherein the outer generation of building units provides 64 nitrogen atoms for covalent attachment to a first terminal group or a second terminal group, wherein from 24 to 32 first terminal groups are covalently attached to one of said nitrogen atoms, and wherein from 24 to 32 second terminal groups are each covalently attached to one of said nitrogen atoms.
• T2' represents a second terminal group which is: wherein the PEG group is a methoxy-terminated PEG having an average molecular weight in the range of from 500 to 2500 Daltons, or T2' represents H, and wherein less than 5 of T2' are H.
• a composition comprising a plurality of dendrimers or pharmaceutically acceptable salts thereof, wherein the dendrimers are as defined according to any one of clauses 1 to 29, the mean number of first terminal groups per dendrimer in the composition is in the range of from 24 to 32, and the mean number of second terminal groups per dendrimer in the composition is in the range of from 24 to 32.
• a pharmaceutical composition comprising: i) a dendrimer are as defined according to any one of clauses 1 to 29, or a pharmaceutically acceptable salt thereof; and ii) a pharmaceutically acceptable excipient.
• composition according to clause 31, wherein the composition is formulated for parenteral delivery.
• a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a dendrimer according to any one of clauses 1 to 29 or a pharmaceutical composition according to any one of clauses 31 to 33.
• cancer is selected from the group consisting of breast cancer, ovarian cancer, non-small cell lung cancer, an upper gastrointestinal cancer (e.g. pancreatic cancer) and bladder cancer.
• the amount of dendrimer administered is sufficient to deliver an amount of active agent in the range of from 1 to 1250 mg of nucleoside analogue/m 2 .
• a further anticancer drug selected from the group consisting of a platinum-containing pharmaceutical agent, a taxane, a PARP inhibitor, a vinca alkaloid, a topoisomerase I inhibitor, and an anthracycline.
• a further anticancer drug selected from the group consisting of carboplatin, cisplatin, oxaliplatin, paclitaxel, Nab-paclitaxel, vindesine, doxorubicin, irinotecan, SN38 and erlotinib.
• the dendrimers represented in the examples below include reference to the core and the building units in the outermost generation of the dendrimer.
• the subsurface generations are not depicted.
• the dendrimer BHALys[Lys] 32 is representative of a 5 generation dendrimer having the formula BHALys[Lys] 2 [Lys] 4 [Lys] 8 [Lys] 16 [Lys] 32 .
• the actual mean number of PEG ⁇ 2100 groups attached to the BHALys[Lys] 32 was determined experimentally by 'H NMR.
• BHALys[Lys] 32 [a-NH 2 TFA] 32 [e-PEG ⁇ 2100 ] was prepared by analogous methods to those described in, for example, WO2020/014750A1 and WO2020/102852A1, the entire contents of which are incorporated herein by reference.
• reaction mixture was stirred for 10 min then warmed to RT and stirred for 2 h.
• DCM was added (100 mL) and the organics washed with pH 3 buffer solution (3 x 100 mL) or until the aqueous phase remained at pH 3.
• the organics were washed with brine (100 mL), dried (MgSO 4 ) and concentrated in vacuo to afford the product as a pale yellow solid (0.53 g, 98%).
• Example dendrimers of the present disclosure are summarised in the table below:
• reaction mixture was monitored by HPLC analysis and left to stir overnight at RT.
• the reaction mixture was then concentrated in vacuo and the residue dissolved in TFA:DCM (10 mL, 1:1 v/v) and stirred overnight at RT.
• Example 3A [BHA Lys][Lys] 30 [Lys] 32 [(a-NH-TDA-Gemcitabine) 32 (e-NHPEG ⁇ 2100 ) 32 ] (Compound 3) Scheme: i) PyBOP, NMM, DMF, RT; ii) TFA:DCM (1 : 1 v/v), RT.
• Example 4A [BHA Lys][Lys] 30 [Lys] 32 [(a-NH-DGA-Gemcitabine) 32 (e-NHPEG ⁇ 2100 ) 32 ] (Compound 6)
• Example 5B [BHA Lys][Lys] 30 [Lys] 32 [(a-NH-TDA-Gemcitabine) 32 (e-NHPEG ⁇ 2100 ) 32 ] (Compound 3)
• Example 9 Comparative linker release rates in PBS, citrate buffer, and plasma
• Human plasma was thawed in an ice bath then centrifuged for 10 min at 13.2K rpm at 4 °C and the supernatant fdtered through a 0.2 mm filter (leaving the protein/enzyme pellet in the micro centrifuge tube).
• the plasma was placed in a CO 2 incubator at 3% CO 2 at 42% relative humidity to reach a pH of 7.4.
• Gemcitabine HC1 (28.5 mg) was dissolved in Milli Q water and the resulting stock solution transferred to a 5 mL volumetric flask and diluted to provide working standard solutions.
• the working standard solutions were centrifuged for 10 min at 13.2K rpm at 4 °C.
• the plasma release study was conducted by adding 1 mL of plasma (centrifuged and filtered) to 0.2 mL of dendrimer solution (approximately 2 mg/mL gemcitabine equivalent in miliQ water) for example dendrimers and a comparator. The mixtures were vortexed (30 sec) then incubated at 37 °C 5% CO 2 .
• GI 50 is the concentration required to inhibit cell growth by 50%.
• the cytotoxicity of compounds toward MDA-MB-231, A549, H460, BxPC3 and CAP AN-1 cells were evaluated for viability.
• Cells were seeded at a density of 1.2 - 5 x 10 3 cells, dependent on cell growth rate, in 96 well plates and incubated overnight. Cells were then treated with serial dilutions of test compositions for 72 h (MDA-MB-231, A549 and H460 lines) or 144 h (BxPC3 and CAP AN-1 lines).
• Example 11 In vivo efficacy of gemcitabine-dendrimer conjugates A CAPAN-1 (human pancreatic adenocarcinoma cell line) mouse xenograft pancreatic cancer model study was carried out to assess the anti-tumour efficacy properties of example dendrimers compared to gemcitabine.
• CAPAN-1 human pancreatic adenocarcinoma cell line
• Compounds 1, 4, 2 and 5 were prepared by dissolving in sterile saline solution immediately prior to dosing and. Gemcitabine ((DBLTM Gemcitabine injection, Hospira Pty Ltd.) was further diluted in saline solution prior to dosing.
• mice Female NOD-SCID Interleukin-2 receptor gamma chain null mice (aged 9 weeks) were inoculated subcutaneously on the flank with 5 x 10 6 CAPAN-1 cells in PBS:Matrigel (1:1). Mice were weighed and tumours measured twice weekly using electronic callipers. Tumour volume (mm 3 ) was calculated as length (mm)/2 x width (mm) 2 . On day seventeen after implantation, mice with similar sized tumours (mean tumour volume 100 mm 3 ) were randomised into 6 groups of 10 animals.
• Tumour growth inhibition was analysed at day 29 (where all vehicle animals remained in the study) using one-way ANOVA followed by Dunnett's multiple comparison test. Tumour growth curves of gemcitabine and DEP-gemcitabine were compared over 47 days (when all treatment group animals remained in the study for tumour-related endpoints) using a mixed- effects model followed by Tukey's multiple comparison test. Two mice were lost prior to day 47 due to non-tumour related endpoints (one from gemcitabine and one from SPL-9138 treatment groups). All statistical analysis was performed using GraphPad Prism 8.1.
• Figure 4 shows the anti -tumour efficacy of the treatments against the CAP AN- 1 tumour xenografts. Tumour volumes were determined twice weekly and are expressed as mean tumour volume ( ⁇ SEM). As shown in Figure 4, 4, 2, 5, induced complete stasis of CAPAN-1 tumours over the dosing study and significantly extended survival beyond that of Gemcitabine. P ⁇ 0.0001 Mantel Cox regression analysis of Kaplan-Meier survival curves).
• a CAPAN-1 (human pancreatic adenocarcinoma cell line) mouse xenograft pancreatic cancer model was established to assess the effects of weekly compared to biweekly administration of an example dendrimer.
• Compound 1 was prepared as described above. Mice were inoculated and tumours sized as described above. On day seventeen after implantation, mice with similar sized tumours (mean tumour volume 140 mm 3 ) were randomised into 2 groups of 5 animals:
• Figure 5 shows the anti -tumour efficacy of the treatments against the CAPAN- 1 tumour xenografts. Tumour volumes were determined twice weekly and are presented as mean tumour volume ( ⁇ SEM). Compound 1, given weekly at 5mg/kg was more effective at suppressing tumour growth than 1, given biweekly at 2 mg/kg.
• Example 13a In vivo efficacy study of xenograft of gemcitabine-dendrimer conjugate with Abraxane ®
• a CAPAN-1 (human pancreatic adenocarcinoma cell line) mouse xenograft pancreatic cancer model was used to assess the anti-tumour efficacy properties of 3 and 6 alone or in combination with Abraxane® (albumin-bound paclitaxel), compared to Gemcitabine, alone or in combination with Abraxane®.
• Mice were inoculated and tumours sized as described above. On day nineteen after implantation, mice with similar sized tumours (mean tumour volume 100 mm 3 ) were randomised into 7 groups of 10 animals. Gemcitabine, 3 and 6 were prepared as described above.
• Abraxane ® was dissolved in saline solution immediately prior to dosing.
• Abraxane ® , gemcitabine and dendrimers were administered at days 1, 10, and 17 via tail vein i.v. injection, at 0.1 ml/10g body weight.
• Example 13b In vivo efficacy study of xenograft of gemcitabine-dendrimer conjugate with docetaxel- dendrimer conjugate
• Example 13 a In an extension of Example 13 a, two additional groups of mice were prepared and dosed to assess the anti -tumour efficacy properties of a taxane-dendrimer combined with a nucleoside analogue-dendrimer (e.g. 6). Mice were inoculated and tumours sized as described above.
• DEP- DTX a docetaxel-dendrimer conjugate, was prepared as described in Example 19 of WO2012167309, and has the structure [BHA Lys][Lys] 30 [Lys] 32 [(a-TD-DTX) 32 (e- polyPEG ⁇ 2100 ) 32 ]
• Dendrimers were administered weekly via tail vein i.v. injection, at 0.1 ml/lOg body weight for 3 weeks. * Compound 6 was administered at 5 mg/kg on day 1 and 4 mg/kg on days 10 and 17
• Figure 6 shows the anti -tumour efficacy of the treatments against the CAP AN- 1 tumour xenografts out to day 82 when the experiment was ended. Tumour volumes were determined twice weekly and are expressed as mean tumour volume ( ⁇ SEM). Graphs are shown until the first mouse in each group reached tumour endpoint.
• Example 14 In vivo efficacy study of xenograft of two gemcitabine-dendrimer conjugates in a pancreatic adenocarcinoma mouse xenograft model.
• CAPAN-1 human pancreatic adenocarcinoma cell line
• mouse xenograft pancreatic cancer model was used to assess the anti-tumour efficacy properties of 3 and 6.
• NSG NOD- SCID Interleukin-2 receptor gamma chain null mice were inoculated and tumours sized as described above. On day 24 after implantation, mice with similar sized tumours (mean tumour volume 100 mm 3 ) were randomised into four groups of 10 animals for each tumour type. Gemcitabine, 3 and 6 were prepared as described above.
• Gemcitabine was administered twice weekly and dendrimers were administered weekly for 3 weeks via tail vein i.v. injection, at 0.1 ml/10 g body weight.
• Figure 7 shows the anti-tumour efficacy of the treatments against the CAPAN-1 tumour xenografts. Tumour volumes were determined twice weekly and are expressed as mean tumour volume ( ⁇ SEM). As shown in Figure 7, 3 was significantly more effective than Gemcitabine or 6 in CAPAN-1 tumours over the study.
• Example 15 In vivo efficacy study of xenograft of two gemcitabine-dendrimer conjugates in a lung adenocarcinoma mouse xenograft model.
• a A549 (human adenocarcinomic human alveolar basal epithelial) cell line was used to assess the anti-tumour efficacy properties of 3.
• NSG mice were inoculated and tumours sized as described above. On day 30 after implantation, mice with similar sized tumours (mean tumour volume 100 mm 3 ) were randomised into four groups of 10 animals for each tumour type.
• Gemcitabine and 3 were prepared as described above. Gemcitabine was administered twice weekly and dendrimer was administered weekly for 3 weeks via tail vein i.v. injection, at 0.1 ml/10 g body weight.
• Figure 8 shows the anti-tumour efficacy of the treatments against the A549 tumour xenografts. Tumour volumes were determined twice weekly and are expressed as mean tumour volume ( ⁇ SEM). As shown in Figure 8, 3 was more effective than Gemcitabine in A549 tumours over the study.
• Example 16 In vivo efficacy study of xenograft of gemcitabine-dendrimer conjugate with Carboplatin An OVCAR-3 (human Ovarian Carcinoma cell line) mouse xenograft cancer model was used to assess the anti -turn our efficacy properties of 3 alone or in combination with Carboplatin, compared to Gemcitabine, alone or in combination with Carboplatin. NSG Mice were inoculated and tumours sized as described above. On day twenty nine after implantation, mice with similar sized tumours (mean tumour volume 100 mm 3 ) were randomised into 10 groups of 10 animals. Gemcitabine and 3 were prepared as described above. DBLCarboplatin (Hospira) was dissolved in saline solution and further diluted in 5% glucose immediately prior to dosing.
• OVCAR-3 human Ovarian Carcinoma cell line
• Gemcitabine was administered twice weekly and dendrimers and carboplatin were administered weekly for 3 weeks via tail vein i.v. injection, at 0.1 ml/10 g body weight.
• Figure 9 shows the anti-tumour efficacy of the treatments against the OVCAR-1 tumour xenografts. Tumour volumes were determined twice weekly and are expressed as mean tumour volume ( ⁇ SEM). As shown in Figure 9, in OVCAR-1 tumours (i) 3 at 4 mg/kg and 6 mg/kg was more effective than Gemcitabine, (ii) 3 and carboplatin was as or more effective than Gemcitabine or carboplatin alone.

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