GB2460086A - Treatment of paediatric cancers - Google Patents

Abstract

Guanosine-rich oligonucleotides (GROs), which comprise one or more GGT motifs, are capable of forming guanosine quartets and which bind to nucleolin, are used for the treatment of paediatric cancers. The preferred GRO is AS1411 (AGRO100). The paediatric cancer may be neuroblastoma, rhabdomyosarcoma, osteosarcoma, acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), medulloblastoma, craniopharyngioma, retinoblastoma, Ewing's sarcoma, non-Hodgkins lymphoma or Hodgkin's lymphoma.

Description

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BIOLOGICAL MATERIALS AND USES THEREOF

The present invention relates to materials and methods for the treatment of cancer. In particular, the invention relates to a therapy comprising the administration of a G rich oligonucleotide for the treatment of paediatric cancers.

Oligonucleotides have the potential to recognize unique sequences of DNA or RNA with a remarkable degree of specificity. For this reason they have been considered as promising candidates to realize gene specific therapies for the treatment of malignant, viral and inflammatory diseases. Two major strategies io of oligonucleotide-mediated therapeutic intervention have been developed, namely, the antisense and antigene approaches.

The antisense strategy aims to down-regulate expression of a specific gene by hybridization of the oligonucleotide to the specific mRNA, resulting in inhibition of translation. Gewirtz et at. (1998) Blood 92,712-736; Crooke (1998) Antisense Nucleic Acid Drug Dev. 8,115-122; Branch (1998) Trends Biochem. Sci. 23, 45- 50; Agrawal et at. (1998) Antisense Nucleic Acid Drug Dev. 8,135-1 39.

The antigene strategy proposes to inhibit transcription of a target gene by means of triple helix formation between the oligonucleotide and specific sequences in the double-stranded genomic DNA. Helene et at. (1997) Ciba Found. Symp. 209,94-102.

Whereas both the antisense and antigene strategies have met with some success, it has become clear in recent years that the interactions of oligonucleotides with the components of a living organism go far beyond sequence-specific hybridization with the target nucleic acid. Recent studies and re-examination of early antisense data have suggested that some of the observed biological effects of antisense oligonucleotides cannot be due entirely to Watson-Crick hybridization with the target mRNA. In some cases, the expected biological effect (e. g. inhibition of cell growth or apoptosis) was achieved, but this was not accompanied by a down regulation of the target protein and was thus unlikely to be a true antisense effect. White et al. (1996) Biochem. Biophys. Res. Commun.

227,118-124; Dryden et at. (1998)J. Endocrinol. 157,169-1 75.

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In many cases, it was demonstrated that other non sequence specific oligonucleotides could exert biological effects that equalled or exceeded the antisense sequence. Barton et at. (1995) Br.J. Cancer 71,429437; Burgess et at.

(1995) Proc. NatI. Acad. Sci. U. S. A. 92,4051-4055; Benimetskaya et at. (1997) Nucleic Acids Res. 25,2648-2656.

Though there is currently a high awareness among antisense investigators of the importance of appropriate control oligonucleotides, and the necessity of demonstrating inhibition of target protein production (Stein (1998) Antisense io Nucleic Acid Drug Dev. 6,129-132), the mechanism of non-antisense effects is poorly understood.

In particular, phosphodiester and phosphorothioate oligodeoxynucteotides containing contiguous guanosines (G) have been repeatedly found to have non-antisense effects on the growth of cells in culture. Burgess et at. (1995) Proc. Natl. Acad. Sci. U. S. A. 92,4051-4055; Benimetskaya et al. (1997) Nucleic Acids Res. 25,2648-2656; Saijo et al. (1997) Jpn. J. Cancer Res. 88,26-33. There is evidence that this activity is related to the ability of these oligonucleotides to form stable structures involving intramolecular or intermolecular G-quartets. Burgess et at. (1995) Proc. Nati. Acad. Sci. U. S. A. 92,4051-4055; Benimetskaya et at.

(1997) Nucleic Acids Res. 25,2648-2656. G-quartets are square planar arrangements of four hydrogen-bonded guanines that are stabilized by monovalent cations.

Such structures are thought to play an important rote in vivo and putative quartet forming sequences have been identified in telomeric DNA (Sundquist et at. (1989) Nature 342,825-829), immunogtobulin switch region sequences (Sen et at. (1988) Nature 334,364-366), HtV1 RNA (Sundquist et at. (1993) Proc. Nat!. Acad. Sci U. S. A. 90,3393-3397), the fragile X repeat sequences (Fry et at (1994) Proc. Nat!.

Acad. Sci. U. S. A. 91,4950-4954) and the retinoblastoma gene (Murchie et al. (1992) Nucleic Acids Res. 20,49-53) Applicants have previously described G-rich oligonucteotides (GROs) that have potent growth inhibitory effects that are unrelated to any expected antisense or antigene activity. The antiprotiferative effects of these oligonucleotides have been identified by the applicants as being related to their ability to bind to a specific cellular protein. Because the GRO binding protein is also recognized by antinucleolin antibodies, Applicants have concluded that this protein is either nucleolin itself, or a protein of a similar size that shares immunogenic similarities with nucleolin.

s Nucleolin is an abundant multifunctional 110 kDa phosphoprotein thought to be located predominantly in the nucleolus of proliferating cells (for reviews, see Tuteja et al. (1998) Crit. Rev. Biochem. Mol. Biol. 33,407-436; Ginisty et al. (1999)J. Cell Sci. 112,761-772). Nucleolin has been implicated in many aspects of ribosome biogenesis including the control of rDNA transcription, pre-ribosome packaging and organization of nucleolar chromatin.

Tuteja et al. (1998) Crit. Rev. Biochem. Mol. Biol. 33,407-436; Ginisty et al. (1999) J. CellSci. 112,761-772; Ginisty et al. (1998) EMBO J. 17,1476-1486.

Another role for nucleolin is as a shuttle protein that transports viral and cellular proteins between the cytoplasm and nucleus/nucleolus of the cell.

Kibbey et al. (1995) J. Neurosci. Res. 42,314-322; Lee et al. (1998) J. Biol.

Chem. 2737650-7656; Waggoner et al. (1998) J. Virol. 72,6699-6709.

Nucleolin is also implicated, directly or indirectly, in other roles including nuclear matrix structure (Gotzmann et al. (1997) Electrophoresis 18,26452653), cytokinesis and nuclear division(Leger-Silvestre et al. (1997) Chromosoma 105,542-52), and as an RNA and DNA helicase (Tuteja et al. (1995) Gene 160,143-148).

The multifunctional nature of nucleolin is reflected in its multidomain structure consisting of a histone-like N-terminus, a central domain containing RNA recognition motifs, and a glycine/arginine rich C-terminus. Lapeyre et al. (1987) Proc. Natl.Acad. Sci. U. S. A. 84,1472-1 476.

Levels of nucleolin are known to relate to the rate of cellular proliferation (Derenzini et al. (1995) Lab. Invest. 73,497-502; Roussel et al. (1994) Exp. Cell Res. 214,465-472.), being elevated in rapidly proliferating cells, such as malignant cells, and lower in more slowly dividing cells.

Paediatric cancers occur in approximately 1 in every 600 children under 15 years of age and are widely recognised as exhibiting different characteristics from cancers affecting adults. Paediatric cancers tend to occur in different parts of the body, have different histology and respond differently to treatment (see, for example, Vakkila, et al, (2006) Clinical Cancer Research 12:2049-2054). Most paediatric cancers are treated using treatment regimes established for adult cancers. As such, there is a need to identify effective treatments for paediatric cancers that have been identified as effective against those paediatric conditions and not just used as an extrapolation of a related adult condition (Boklan, (2006) Mol Cancer Ther 5(8):1905-8; Balis, (2000),Oncologist, 5;2-3).

The search for anti-cancer agents and methods of treatment with improved io efficacy and reduced toxicity is ongoing and intense. The present invention seeks to provide further agents and methods for the treatment of cancers.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Summary of the invention

The inventor has identified that a surprising anti-Cancer effect can be obtained by means of treatment of paediatric cancers with a G rich oligonucleotide.

In a first aspect of the invention there is provided a use of a G-rich oligonucleotide having the sequence of one of SEQ IDs Nos. 1 to 18 in the manufacture of a medicament for treating a paediatric cancer.

In a second aspect of the invention there is provided a G-rich oligonucleotide having the sequence of one of SEQ lDs Nos. I to 18 for use in the treatment of a disease characterised by malignant, dysplastic, and/or hyperproliferative cells.

Examples of oligonucleotides of the present invention have the following nucleotide sequences: AS14I1 -5'-GGTGGTGGTGGTTGTGGTGGTGc3TGG-3' (SEQ ID No: 1) (also known as GRO26B and AGRO100) GRO14A -5' GTTGTTTGGGGTGG-3' (SEQ ID No: 2) GRO15A -5'-GTTGTTTGGGGTGGT-3' (SEQ ID No: 3) GRO25A -5'-GGTTGGGGTGGGTGGGGTGGGTG3G-3' (SEQ ID No: 4) GRO28A -5'-TTTGGTGGTGGTGGTTGTGGTGGTGGTG-3' (SEQ ID No: 5) GRO29A -5'-TUGGTGGTGGTGGTTGTGGTGGTGGTGG-3'SEQ ID No: 6) GR029-2 -5'-TTTGGTGGTGGTGG I Ti I GGTGGTGGTGG-3' (SEQ ID No: 7) GR029-3 -5'-TTTGGTGGTGGTGGTGGTGGTGGTGGTGG-3' (SEQ ID No: 8) GR029-5 -5'-TTTGGTGGTGGTGGTTTGGGTGGTGG TGG-3 (SEQ ID No: 9) GR029-13 -5'-TGGTGGTGGTGGT-3' (SEQ ID No: 10) GRO1 1A -5'-GGTGGTGGTGG-3' (SEQ ID No: 11) GRO14C 5'-GGTGGTTGTGGTGG-3' (SEQ ID No: 12) GRO56A -5'-GGTGGTGGTGGTTGTGGTGGTGGTGGTTGTGGTGGTGGTG GTTGTGGTGGTGGTGG-3' (SEQ ID No: 13) GRO32A -5'-GGTGGUGTGGTGGTTGTGGTGGTTGTGGTGG-3' (SEQ ID No: 14) GRO32B -5'-TTTGGTGGTGGTGGTTGTGGTGGTGGTGGTTT-3' (SEQ ID No: 15) GR029-6 -5'-GGTGGTGGTGGTTGTGGTGGTGGTGGTTT-3' (SEQ ID No: 16) GRO28B -5'-TTTGGTGGTGGTGGTGTGGTGGTGGTGG-3' (SEQ ID No: 17) GRO13A -5'-TGGTGGTGGT-3' (SEQ ID No: 18).

Other oligonucleotides having the same activity are also contemplated.

By G-rich oligonucleotide (GRO) it is meant that the oligonucleotides consist of 4-nucleotides (preferably 10-30 nucleotides) with DNA, RNA,2'-O-methyl, phosphorothioate or other chemically similar backbones. Their sequences contain one or more GGT motifs. The oligonucleotides have anti-proliferative activity against cells and bind to GRO binding protein and/or nucleolin. These properties can be demonstrated using techniques well known in the art such as an MU assay or the EMSA technique (see WO 2000/61597).

The oligonucleotides of the present invention are rich in guanosine and are capable of forming G-quartet structures. Specifically, the oligonucleotides of the present invention are primarily comprised of thymidine and guanosine with at east one contiguous guanosine repeat in the sequence of each oligonucleotide.

As used herein, the term "oligonucleotide" is defined as a molecule comprising two or more deoxyribonucleotides or ribonucleotides. The exact size depends on a number of factors including the specificity and binding affinity to target ligands.

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In referring to "bases" or "nucleotides" the terms include both deoxyribonucleic acids and ribonucleic acids.

In either of the first and second aspects of the invention, preferably the G-rich oligonucleotide has the sequence of SEQ ID 1.

Further preferably, the G-rich oligonucleotide has a 3' end and a 5' end, and one or both of the 3' and 5' ends have been modified to alter a property of the G-rich oligonucleotide.

The oligonucleotides can be modified at their 3' end in order to alter a specific property of the oligonucleotide. For example, the 3'-terminus of the oligonucleotide can be modified by the addition of a propylamine group which has been found to increase the stability of the oligonucleotide to serum nucleases.

Other modifications that are well known in the art include 3' and 5' modifications, for example, the binding of cholesterol, and backbone modifications, for example, phosphorothioate substitution and/or 2'-O-methyl RNA.

Advantageously, in either of the first and second aspects of the invention the paediatric cancer is selected from the following disorders: neuroblastoma; rhabdomyosarcoma; osteosarcoma; acute myeloid leukaemia (AML); acute lymphoblastic Ieukaemia (ALL); medulloblastoma; craniopharyngioma; retinoblastoma; Ewing's sarcoma; lymphomas; non-Hodgkin's lymphoma; and Hodgkin's lymphoma.

In certain embodiments of the first and second aspects of the invention, the paediatric cancer is selected from the following disorders: neuroblastoma; rhabdomyosarcoma; osteosarcoma; medulloblastoma; craniopharyngioma; retinoblastoma; Ewing's sarcoma; lymphomas; non-Hodgkin's lymphoma; and Hodgkin's lymphoma.

It will be understood that the term "paediatric" includes conditions and science associated with non-adult individuals (such as children). Thus, by "paediatric cancer" we mean a cancer found in a paediatric individual (such as a human paediatric individual, e.g. a child) or in a population of paediatric individuals (such as a population of human paediatric individuals, e.g. children).

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The "paediatric individual" may be a human or any animal (such as a cow, dog, cat, goat, sheep, and pig). Those skilled in the art are easily able to identify paediatric individuals (such as human paediatric individuals) and/or patients having a paediatric cancer condition.

By a "human paediatric individuai" we mean a human individual at any age between the day of its birth (i.e. zero (0) years of age) and 21 years of age.

"Human paediatric individual" therefore includes a "neonate" or a "newborn," which is a human individual at any age between the day of its birth (Le. zero (0) io years of age) and 30 days of age; an "infant" which is a human individual at any age between 31 days of age and two years of age; a "child", which is an individual at any age between two years of age and 12 years of age; an "adolescent", which is an individual at any age between 12 years of age and 21 years of age.

In a third aspect of the invention there is provided a method for inhibiting the proliferation of malignant, dysplastic, and/or hyperproliferative cells, said method comprising administering to the subject a therapeutically effective amount of a G-rich oligonucleotide having the sequence of one of SEQ lDs Nos. 1 to 18; wherein the malignant, dysplastic, and/or hyperproliferative cells are associated with a paediatric cancer.

The inhibition may be an in vitro or an in vivo method.

The term "inhibiting the proliferation of malignant, dysplastic, and/or hyperplastic cells" includes any partial or total growth inhibition and includes decreases in the rate of proliferation or growth of the cells.

As used herein, the term "neoplastic" includes the new, abnormal growth of tissues and/or cells, such as a cancer or tumour. The term "neoplastic" also includes malignant cells which can invade and destroy adjacent structures and/or metastasize.

As used herein, the term "dysplastic" includes any abnormal growth of cells, tissues, or structures.

The term "subject" means all animals including humans, for example a paediatric individual. Examples of subjects include humans, cows, dogs, cats, goats, sheep, and pigs. The term "patient" means a subject having a disorder in need of treatment.

By "therapeutically effective amount" we mean an amount of an oligonucleotide of the present invention or chemotherapeutic agent such as doxorubicin, that when administered to the subject either alone or in combination with another agent, ameliorates a symptom of the disease, disorder, or condition, such as by inhibiting or reducing the proliferation of dysplastic, hyperproliferative, or malignant cells.

Preferably, the G-rich oligonucleotide has the sequence of SEQ ID 1.

In one embodiment, the G-rich oligonucleotide has a 3' end and a 5' end, and one or both of the 3' and 5' ends have been modified to alter a property of the G-rich oligonucleotide.

Advantageously the malignant, dysplastic, and/or hyperproliferative cells are associated with a paediatric cancer selected from the following disorders: neuroblatoma; rhabdomyosarcoma; osteosarcoma; acute myeloid leukaemia (AML); acute lymphoblastic leukaemia (ALL); medulloblastoma; craniopharyngioma; retinoblastoma; Ewing's sarcoma; lymphomas; non-Hodgkin's lymphoma; and Hodgkin's lymphoma.

In a fourth aspect of the invention there is provided a method for treating a paediatric cancer comprising exposing paediatric cancer cells to a therapeutically effective amount of G-rich oligonucleotide having the sequence of one of SEQ JDs Nos. I to 18.

By "treatment" we include the meanings that the number of malignant, dysplastic, and/or hyperproliferative cells is reduced and/or further malignant, dysplastic, and/or hyperproliferative cell growth is retarded and/or prevented and/or the malignant, dysplastic, and/or hyperproliferative cells are killed, Malignant, dysplastic, and/or hyperproliferative cells are characteristic of tumours and of Cancers.

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Preferably the G-rich oligonucleotide has the sequence of SEQ ID 1.

In one embodiment, the G-rich oligonucleotide has a 3' end and a 5' end, and one or both of the 3' and 5' ends have been modified to alter a property of the G-rich oligonucleotide.

Advantageously, the paediatric cancer is selected from the following disorders: neuroblastoma; rhabdomyosarcoma; osteosarcoma; acute myeloid leukaemia (AM L); acute lymphoblastic leukaemia (ALL); medulloblastoma; craniopharyngioma; retinoblastoma; Ewing's sarcoma; Iymphomas; non-Hodgkin's Iymphoma; and Hodgkin's Iymphoma.

The GROs of the present invention can be administered to a patient or subject either alone or as part of a pharmaceutical composition. The GROs can be administered to patients either orally, rectally, parenterally (intravenously, intramuscularly, or subcutaneously), intracisternally, intravaginally, intraperitonally, intravesically, locally (powders, ointments, or drops), or as a buccal or nasal spray.

In a fifth aspect of the invention there is provided a kit of parts comprising: (i) a G-rich oligonucleotide having the sequence of one of SEQ IDs Nos. 1 to 18 in a therapeutically effective amount; (ii) instructions for their use in paediatric cancer patient.

Preferably the kit also comprises (iii) means for administering the G-rich oligonucleotide to a paediatric cancer patient.

Preferably the G-rich oligonucleotide has the sequence of SEQ ID 1.

In one embodiment, the G-rich oligonucleotide has a 3' end and a 5' end, and one or both of the 3' and 5' ends have been modified to alter a property of the G-rich oligonucleotide.

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Advantageously, the paediatric cancer is selected from the following disorders: neuroblastoma; rhabdomyosarcoma; osteosarcoma; acute myeloid leukaemia (AM L); acute lymphoblastic leukaemia (ALL); medulloblastoma; craniopharyngioma; retinoblastoma; Ewing's sarcoma; lymphomas; non-Hodgkin's lymphoma; and Hodgkin's lymphoma.

Examples embodying an aspect of the invention will now be described with reference to the following figures in which: Figure 1: SRB assay SRB assay showing example data from paediatric cell line assays. Paediatric cell lines exhibit similar IC50 values when exposed to AS1 411 for a 6-day assay Figure 2. Cytostatic effects Cell growth of MV4-1 1 (AML) and SUP-B15 (ALL) cells exposed to AS141 I Figure 3: Cytotoxic effects Viability of MV4-1 1 (AML) and SUP-Bi 5 (ALL) cells exposed to AS1 411 Figure 4: Western blot analysis Western blot analysis showing increased levels of Bax protein upon AS141 1 exposure Figure 5: Baxter FOLFusor LVIO device Line representation of an ambulatory device for administration.

Example I -G rich o!igonucleotide effect on paediatr!c cancer cell lines Methods Cell Culture: Cells were cultured in 175 flasks and cell counts performed using the trypan blue dye exclusion method (whereby sterile Trypan blue solution 0.4% (e.g. Sigma T-8154) is added to cell cultures and non-viable cells are unable to exclude the dye and hence appear blue).

Sulphorhodamine B assay Cells were typically seeded in wells of a 96-well plate as follows for each cell line: R-1059-D 500 A204 1000 SK-N-AS 4000 MC/CAR 10,000 SUP-B15 10,000 MV4-11 5000 AS1411 (G rich oligonucleotide of sequence ID No. 1) was added at a concentration selected from 0, 0.1, 1, 5 or 20 M and cells were incubated for 6 days.

is Cells were then washed, fixed to the 96-well plate and exposed to the dye Sulphorhodamine B (SRB; available from Sigma-Aldrich, Dorset, UK; catalogue number S-1402. The optical density of the remaining cell mass after exposure to AS141 I was measured in a microplate spectrophotometer and IC50 determined.

For the time course experiments, AS141 1 was washed off cells at the stated time-point, and then fresh medium applied to the cells which were left to grow for the full 6 days of the assay.

The experiments were run in duplicate and the mean optical density calculated.

Western Blottjp Cells were incubated with AS1411 for 4 days, after which cell lysates were analysed by non-reducing SDS-PAGE analysis using 4-12% NuPAGE Bis-Tris Gels (Invitrogen).

pg of total protein (whole cell lysate) was loaded per well as assessed by the Lowry assay (Lowry reagent available from Sigma-Aldrich, catalogue number L3540) and detected using ECL Advance western blotting kit (GE Healthcare).

Anti-nucleolin and bax antibodies were obtained from Santa Cruz and the 13-actin antibody from QED.

Results -C4otox!city Cytotoxicity and cytostatic test results are shown in figures 1, 2 and 3 and Table 1 below.

Table 1: Sensitivity of paediatric cancer cell lines to ASI4II.

Average IC50 values are shown from at least two experiments for each cell line.

Cell line Tumour type IC50 (pM) MV4-11 Acute myelogenous ieukaemia (AML) 2.1 SUP-B 15 Acute lymphoblastic leukaemia (ALL) 2.3 R-1059-D Osteosarcoma 4.7 A204 Rhabdomyosarcoma 2.6 SK-N-AS Neuroblastoma 3.2 From these results it can be seen that AS1 411 (SEQ ID No. 1) shows activity against (i.e. reduces the cell numbers of) many paediatric cancer cell lines Western blotting Nucleolin appears as several bands: these forms are expected from the literature; no effect is observed on total cell lysate nucleolin levels, Bax is observed as both a monomer or dimer. Up-regulation of Bax is observed upon exposure to ASI41 1 in both cell lines; levels of 13-actin were used to normalise protein concentrations.

Bax is a pro-apoptotic protein involved in pore formation in mitochondrial membranes, leading to apoptosis.

Example 2 -Administration of combination therapy in cancer treatment using an intravenous in fusion AS14I 1 is given to patients via intravenous infusion over a period of 7 days. The daily amount to be administered to the patient is calculated based on dose in mg/kg and the patient weight.

Fresh solutions are prepared on each infusion day, by diluting AS1411 drug product into 5% dextrose within an infusion bag (alternatives to dextrose include any known infusion system such as saline). Appropriate infusion bags are known to those skilled in the art. A fresh infusion bag is preferably prepared at the start

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of each 24-hour period. After calculation of the required dose of AS1 411, an equivalent volume of dextrose should be removed from the bag, and the required dose of AS1 411 added directly to the bag for a total final volume of 500mL.

Once prepared, infusion bags containing AS1411 can be stored at +2°C to +5°C until administration. Drug can be prepared up to 6 hours prior to dosing.

Reconstituted AS1411 in 5% dextrose is administered at room temperature as soon as possible following reconstitution. The appropriate dose of AS141 1 is ia administered as a 500m1 intravenous infusion. Infusion of AS141 1 is as close to 24 hours as possible, accounting for changing of infusion bags, or clotting of infusion lines.

Example 3 -Administration of GRO in cancer treatment using an ambulatory device Administration of AS1411 is performed using an ambulatory device, which allows improved patient mobility. Such an administration route is useful for, for example, treatment of a patient with renal cancer.

Ambulatory devices are well-known in the art of pharmacy and medicine and a skilled person would be able to select an appropriate device. A preferred device is the Baxter FOLFusor LV1O (Baxter Parkway, Deerfield, IL 60015-4625, USA; Figure 9) which been used extensively in chemotherapy treatment, is non-allergenic, and supplies product at a rate of 10 mI/hour from a 240 ml reservoir.

The FOLFusor is supplied in a "bum bag" to improve patient freedom and is replaced with a fresh, filled FOLFusor each day during the treatment cycle.

ao In the FOLFusor, product is introduced into a central elastomeric balloon via a syringe connected to a Fill Port located on the top of the device. The balloon is filled with 240 ml of AS1411. Having filled the device, the internal pressure within the balloon then drives the flow of product from the balloon through the delivery tubing via a luer-lock connector to the catheter. The flow rate is controlled by a restriction caused by a flow restrictor in the delivery tubing.

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The flow rate accuracy is +1-10% and has been calibrated by Baxter using 5% dextrose. The FOLFusor must be filled to the nominal volume (240 ml) or the flow rate is reduced. A 5 micron in-line filter removes any particulates. There is no risk of air ingress as the FOLFusor is a closed system. If the FOLFusor dispenses all product and empties, there is some risk of blood tracking back up the tubing and causing a blockage. This can be removed with a heparin flush.

Details of the administration materials are: * AS1411 Drug Product concentrate, 20 mg/mI in 20 ml vials * Baxter FOLFusor LV1 0 (Baxter, catalogue no. 2C4063K) * Sterile syringe with Luer Lock Fitting, 100 ml capacity (e.g. Becton-Dickinson Plastipak) * Sterile Hypodermic needle * 5% dextrose solution (Viaflex Container, Baxter, e.g. catalogue no. 2B0089) * Sterile Mixing vessel (preferably around 500 ml) (I) AS14II dose calculation AS1411 is delivered to the clinic as a concentrate in 20 ml vials at 20mg/mi.

AS1411 is first diluted into 5% dextrose at the clinic to give a final volume of 240 ml, the ratio of 5% dextrose to AS1 411 is dependent on patient weight (see Table 1, below).

(ii) AS 1411 solution preparation Using Table 2 as a guide, remove the required number of AS141I vials from the refrigerator and allow to stand at room temperature for 1 hour. Using a sterile 100 ml syringe fitted with a hypodermic needle, withdraw the required volume of AS1 411 concentrate from vials and add to the sterile mixing vessel. Using the same syringe, now withdraw the required volume of 5% dextrose from the Viaflex containers and add to the AS1411 concentrate in the mixing vessel. Swirl the container contents gently to mix. Note that Steps (ii) and (iii) must be carried out in a safety cabinet.

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Table 2: Preparation Guidelines for AS1411 at varying patient weight for 40 mg/kg dose Patient Total g ASI4II Volume Volume 5% weight (kg) per 24 hours at ASI4II dextrose 40mg/kg (ml) (ml) 2.4 120 120 2.6 130 110 2.8 140 100 3.0 150 90 3.2 160 80 3.4 170 70 3.6 180 60 3.8 190 50 4.0 200 40 4.2 210 30 4.4 220 20 4.6 230 10 4.8 240 0 (iii) Addition of drug to the FOLFusor.

The AS1 411/dextrose solution is added to the FOLFusor using the 100 ml syringe screwed onto the Fill Port at the top of the device. Remove the hypodermic needle from the syringe and unscrew the cap from the Fill Port on the FOLFusor and retain in the cabinet. Remove the blue cap from the end of the delivery tube

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attached to the FOLFusor and retain in the cabinet (removal of the blue cap will allow air to be expelled from the device during priming). Fill the syringe with 100 ml of the AS1 411 dextrose solution from the container and screw the syringe onto the Fill Port; slowly push the syringe plunder to transfer the solution into the device (the central balloon will inflate). Continue this process with additional syringe filling until 240 ml of the AS1411 dextrose solution is transferred to the FOLFusor (the balloon will now be fully inflated). Allow the drug solution to drip from the end of the delivery tube before replacing the blue cap.

(iv) Connecting to the catheter and patient.

Now remove the filled FOLFusor from the safety cabinet. Using aseptic technique, remove the blue cap from the end of the delivery tube and attach to the catheter via the luer lock fitting. Allow drug solution to drip from the catheter before attaching to the patient.

(v) Guidelines on use.

The FOLFusor is then placed in a "bum bag" attached to the patient's waist. The FOLFusor should be kept at roughly the same height as the entry port into the patient. The flow rate decreases by 0.5% per 2.5 cm below this level, and increases by 0.5% per 2.5 cm above this level. Temperature and viscosity also impact the flow rate. A reduced temperature increases the viscosity and decreases the flow rate. A higher temp reduces the viscosity and increases the flow rate. 33.3°C is the assumed temperature in the bum bag.

Example 4 -Preferred pharmaceutical formulations and modes and doses of administration.

The polynucleotides of the present invention may be delivered using an injectable sustained-release drug delivery system. These are designed specifically to reduce the frequency of injections. An example of such a system is Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.

The polynucleotides of the present invention can be administered by a surgically implanted device that releases the drug directly to the required site. For example, Vitrasert releases ganciclovir directly into the eye to treat CMV retinitis. The direct application of this toxic agent to the site of disease achieves effective therapy without the drug's significant systemic side-effects.

Electroporation therapy (EPT) systems can also be employed for administration.

A device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drug, resulting in a significant enhancement of io intracellular drug delivery.

Polynucleotides of the invention can also be delivered by electroincorporation (El). El occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. In El, these particles are driven through the stratum corneum and into deeper layers of the skin. The particles can be loaded or coated with drugs or genes or can simply act as "bullets" that generate pores in the skin through which the drugs can enter.

An alternative method of administration is the ReGel injectable system that is thermosensitive. Below body temperature, ReGel is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active drug is delivered over time as the biopolymers dissolve.

Polynucleotides of the invention can be introduced to cells by "Trojan peptides".

These are a class of polypeptides called penetratins which have translocating properties and are capable of carrying hydrophilic compounds across the plasma membrane. This system allows direct targeting of oligopeptides to the cytoplasm and nucleus, and may be non-cell type specific and highly efficient (Derossi et a!., 1998, Trends Cell Biol., 8, 84-87).

Preferably, the pharmaceutical formulation of the present invention i a unit dosage containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of the active ingredient.

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The polynucleotides of the invention can be administered by any parenteral route, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses.

In human therapy, the polynucleotides of the invention can be administered alone but will generally be administered in admixture with a suitable pharmaceutical exipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

The polynucleotides of the invention can also be administered parenterally, for example, intravenously, intra-arterially, intraperitoneally, intra-thecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Generally, in humans, continuous intravenous administration of the polynucleotides of the invention is the preferred route.

For veterinary use, the polynucleotides of the invention are administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.

The formulations of the pharmaceutical compositions of the invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes to one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question.

Example 5-Exemplary pharmaceutical formulations Whilst it is possible for polynucleotides of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be "acceptable" in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyroge n-free.

The following examples illustrate pharmaceutical formulations according to the invention in which the active ingredient is a polynucleotide and/or chemotherapeutic of the invention.

I

Example 5A: Ophthalmic Solution Active ingredient 0.5 g Sodium chloride, analytical gradeo.9 g Thiomersal 0.001 g Purified water to 100 ml pH adjusted to 7.5 Example 5B: Capsule Formulations Formulation A A capsule formulation is prepared by admixing the ingredients of Formulation D in Example C above and filling into a two-part hard gelatin capsule. Formulation B (infra) is prepared in a similar manner.

Formulation B mg/capsule Active ingredient 250 Lactose B.P. 143 Sodium Starch Glycolate 25 Magnesium Stearate 2 Formulation C mg/capsule Active ingredient 250 Macrogol 4000 BP 350 Capsules are prepared by melting the Macrogol 4000 BP, dispersing the active ingredient in the melt and filling the melt into a two-part hard gelatin capsule.

Formulation 0 mg/capsule Active ingredient 250 Lecithin 100 Arachis Oil 100 Capsules are prepared by dispersing the active ingredient in the lecithin and arachis oil and filling the dispersion into soft, elastic gelatin capsules.

Formulation E (Controlled Release Capsule) The following controlled release capsule formulation is prepared by extruding ingredients a, b, and c using an extruder, followed by spheronisation of the extrudate and drying. The dried pellets are then coated with release-controlling membrane (d) and filled into a two-piece, hard gelatin capsule.

mg/capsule Active ingredient 250 Microcrystalline Cellulose 125 LactoseBP 125 Ethyl Cellulose 13 513 Example 50: Injectable Formulation Active ingredient 0.200 g Sterile, pyrogen free phosphate buffer (pH7.0)to 10 ml The active ingredient is dissolved in most of the phosphate buffer (35-40C), then made up to volume and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1) and sealed with sterile closures and overseals.

Alternatively, the formulation may contain the following: -Potassium phosphate dibasic USP Quality (EMD Chemicals mc, New Jersey 08027, USA) to pH 7.4; -Potassium phosphate monobasic USP Quality (EMD Chemicals mc) to pH 7.4; -Water for Injection to 20 ml; -ASl4ll400mg The weights of these materials used in each batch will depend on batch size. For example, the following could be used to give a batch size yielding approximately 1370 vials containing 20 ml at 20 mg/mI AS1 411: -AS1411 528.5g; -Potassium phosphate dibasic 39.8 g; -Potassium phosphate monobasic 8.2 g; -Water for Injection to 28339.8g; -the formulation is mixed with 5% dextrose (Baxter) at the clinic.

Example 5D: Intramuscular injection Active ingredient 0.20 g Benzyl Alcohol 0.10 g Glucofurol75� 1.45g Water for Injection q.s. to 3.00 ml The active ingredient is dissolved in the glycofurol. The benzyl alcohol is then added and dissolved, and water added to 3 ml. The mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml glass vials (type 1).

Example SE: Syrup Suspension Active ingredient 0.2500 g Sorbitol Solution 1.5000 g Glycerol 2.0000 g Dispersible Cellulose 0.0750 g Sodium Benzoate 0.0050 g Flavour, Peach 17.42.3169 0.0125 ml Purified Waterq.s. to 5.0000 ml The sodium benzoate is dissolved in a portion of the purified water and the sorbitol solution added. The active ingredient is added and dispersed. In the glycerol is dispersed the thickener (dispersible cellulose). The two dispersions are mixed and made up to the required volume with the purified water. Further thickening is achieved as required by extra shearing of the suspension.

Example 5F: Suppository mqLsuppository Active ingredient (63 pm)* 250 Hard Fat, BP (Witepsol H15 -Dynamit Nobel) 1770 *The active ingredient is used as a powder wherein at least 90% of the particles are of 63 pm diameter or less.

One fifth of the Witepsol H15 is melted in a steam-jacketed pan at 45C maximum.

The active ingredient is sifted through a 200 pm sieve and added to the molten base with mixing, using a silverson fitted with a cutting head, until a smooth dispersion is achieved. Maintaining the mixture at 45C, the remaining Witepsol H15 is added to the suspension and stirred to ensure a homogenous mix. The entire suspension is passed through a 250 pm stainless steel screen and, with continuous stirring, is allowed to cool to 40C. At a temperature of 38C to 40CC 202 g of the mixture is filled into suitable plastic moulds. The suppositories are allowed to cool to room temperature.

Example 5G: Pessaries mq/pessary Active ingredient 250 Anhydrate Dextrose 380 Potato Starch 363 Magnesium Stearate 7 The above ingredients are mixed directly and pessaries prepared by direct compression of the resulting mixture.

Example 5H: Creams and ointments Described in Remington, The Science and Practise of Pharmacy, 19th ed., The Philadelphia College of Pharmacy and Science, ISBN 0-912734-04-3.

Example 51: Microsphere formulations The compounds of the invention may also be delivered using microsphere formulations, such as those described in Cleland (1997, Pharm. Biotechnol. 10:1- 43; and 2001, J. Control. Release 72:13-24).

Claims (18)

1. CLAIMS1. Use of a G-rich oligonucleotide having the sequence of one of SEQ IDs Nos. 1 to 18 in the manufacture of a medicament for treating a paediatric cancer.
2. 2. A G-rich oligonucleotide having the sequence of one of SEQ IDs Nos. 1 to 18 for use in the treatment of a paediatric cancer.
3. 3. A use as claimed in any previous claim wherein the G-rich oligonucleotide has the sequence of SEQ ID 1.
4. 4. A use as claimed in any previous claim wherein the G-rich oligonucleotide has a 3' end and a 5' end, and one or both of the 3' and 5' ends have been modified to alter a property of the G-rich oligonucleotide.
5. 5. A use as claimed in any previous claim wherein the paediatric cancer is selected from the following disorders: neuroblastoma; rhabdomyosarcoma; osteosarcoma; acute myeloid leukaemia (AML); acute lymphoblastic Ieukaemia (ALL); medulloblastoma; craniopharyngioma; retinoblastoma; Ewing's sarcoma; lymphomas; non-Hodgkin's lymphoma; and Hodgkin's lymphoma.
6. 6. A method for inhibiting the proliferation of malignant, dysplastic, and/or hyperproliferative cells in a subject, said method comprising administering to the subject a therapeutically effective amount of a G-rich oligonucleotide having the sequence of one of SEQ lDs Nos. I to 18; wherein the malignant, dysplastic, and/or hyperproliferative cells are associated with a paediatric cancer.
7. 7. A method for treating a paediatric cancer comprising exposing paediatric cancer cells to a G-rich oligonucleotide having the sequence of one of SEQ lDs Nos. 1 to 18.
8. 8. A method as claimed in Claims 6 or 7 wherein the G-rich oligonucleotide has the sequence of SEQ ID 1.
9. 9. A method as claimed in Claims 6 to 8 wherein the G-rich oligonucleotide has a 3' end and a 5' end, and one or both of the 3' and 5' ends have been modified to alter a property of the G-rich oligonucleotide.
10. 10. A method as claimed in Claims 6 to 9 wherein the paediatric cancer is selected from the following disorders: neuroblastoma; rhabdomyosarcoma; osteosarcoma; acute myeloid leukaemia (AML); acute lymphoblastic leukaemia (ALL); medulloblastoma; craniopharyngioma; retinoblastoma; Ewing's sarcoma; lymphomas; non-io Hodgkin's lymphoma; and Hodgkin's lymphoma.
11. 11. A kit of parts comprising: (i) a G-rich oligonucleotide having the sequence of one of SEQ IDs Nos. 1 to 18 in a therapeutically effective amount; (ii) instructions for their use in paediatric cancer patient.
12. 12. A kit as claimed in claim 11 further comprising: (iii) means for administering the C-rich oligonucleotide to a paediatric cancer patient
13. 13. A kit as claimed in either of claims 11 or 12 wherein the C-rich oligonucleotide has the sequence of SEQ ID 1.
14. 14. A kit as claimed in Claim 11 to 13 wherein the C-rich oligonucleotide has a 3' end and a 5' end, and one or both of the 3' and 5' ends have been modified to alter a property of the G-rich oligonucleotide.
15. 15. A kit as claimed in Claims 11 to 14 wherein the paediatric cancer is selected from the following disorders: neuroblastoma; rhabdomyosarcoma; osteosarcoma; acute myeloid leukaemia (AML); acute Iyrnphoblastic Ieukaemia (ALL); medulloblastoma; craniopharyngioma; retinoblastoma; Ewing's sarcoma; lymphomas; non-Hodgkin's lymphoma; and Hodgkin's lymphoma.
16. 16. A method substantially as described herein with reference to the Examples and Figures.
17. 17. A use substantially as described herein with reference to the Examples and FIgures.
18. 18. A kit substantially as described herein with reference to the Examples and Figures.