ES2350978T3 - TREATMENT OF MELANOMA THROUGH THE REDUCTION OF CLUSTERINE LEVELS. - Google Patents

Abstract

Use of a composition comprising a therapeutic agent in the form of an antisense oligodeoxynucleotide or siRNA oligonucleotide directed to the clusterin gene or an antibody directed to the clusterin and effective to reduce the effective amount of clusterin in melanoma cells for the formulation of a Pharmaceutical composition for the treatment of melanoma.

Description

This application claims the benefit and priority of U.S. Provisional Applications No. 60 / 405,193 filed on August 21, 2002, 60 / 408,152 filed on 3 September 2002, 60 / 319,748 filed December 2 of 2002 and 60 / 472,387 filed on May 20, 2003.

Background of the invention

This application refers to treatments for melanoma by inhibiting clusterin, also known as Testosterone inhibited prostate message 2 (TRPM-2), by example by administering oligonucleotides specific antisense for clusterin.

Clusterin or TRPM-2 is a ubiquitous protein, with a Diverse range of proposed activities. In the cells epithelial prostate, clusterin expression increases immediately after castration, reaching maximum levels in rat prostate cells at 3 to 4 days after castration, coincident with the onset of massive cell death. These results have led some researchers to the conclusion that clusterin is a marker for death cell, and a promoter of apoptosis. Moreover, the observation that Sertoli cells and some cells epithelials express high levels of clusterin without increasing cell death levels raises questions as to whether This conclusion is correct. Sensibar et al., Cancer Research

55: 2431-2437 (1995) reported in in vitro experiments made to clarify more clearly the role of the clusterin in prostate cell death. They used cells LNCaP transfected with a gene that codes for clusterin and they observed if the expression of this protein altered the effects

of tumor necrosis factor  (TNF), to which LNCaP cells are very sensitive, with cell death normally occurring within approximately 12 hours. The treatment of LNCaP cells transfected with TNF was shown to result in a transient increase in clusterin levels over a period of a few hours, but these levels had dissipated by the time the DNA fragmentation was observed. preceded cell death. The use of an antisense molecule corresponding to bases 1-21 of the clusterin sequence, but not other clusterin antisense oligonucleotides, resulted in a substantial reduction in clusterin expression, and an increase in the

Apoptotic cell death in LNCaP cells exposed to TNF. This led to Sensibar et al. to the hypothesis that clusterin overexpression could protect cells from the cytotoxic effect of TNF, and that the depletion responsible for the onset of cell death, although the mechanism of action remains unclear.

PCT publication WO00 / 049937 describes the use of therapy antisense that reduces clusterin expression for provide therapeutic benefits in the treatment of prostate cancer, renal cell cancer and some breast cancers In addition, the combined use of the antisense clusterin plus cytotoxic chemotherapy (for example, taxanes) synergistically powers the chemosensitivity in prostate cancer resistant to hormones The radiation sensitivity is also enhanced when cells expressing clusterin are treated with oligodeoxynucleotides (ODN) of antisense clusterin.

Summary of the invention

The present application refers to the treatment of melanoma by reducing the effective amount of clusterin. By therefore, according to one aspect of the invention, a method for treating melanoma in a mammalian subject, preferably a human being, comprising the stage of administer to the subject an effective therapeutic agent to reduce the effective amount of clusterin in melanoma cells. He

therapeutic agent can be, for example, an ODN compound antisense or small inhibitory RNA (siRNA) targeting clusterina.

The present invention also provides a method for regulate bcl-xL expression in a subject or cell line which comprises administering to the subject or the cell line an agent effective to modulate the amount of clusterin expression. In particular, in cells expressing clusterin, the expression of bcl-xL is regulated by decrease when the amount is reduced effective clusterin. Such inhibition is significant because it knows that bcl-xL acts as an apoptosis inhibitor. See, for example, U.S. Patent No. 6,172,216.

Brief description of the drawings

Figure 1 shows the results when treated 607B melanoma cells with either the oligonucleotide antisense at concentrations of 100, 250 or 500 nM, or a mating control randomized to a concentration 100 nM on two consecutive days.

Figure 2 provides graphical representations of the Clusterin expression in 518A2 cells after treatment with cisplatin and either an antisense oligonucleotide or a mating control erroneous, randomized.

Figure 3 shows cell survival of cells from Mel Juso melanoma stably transfected with either a empty control vector (Neo) or a vector that directs the overexpression of clusterin, which were grown in medium that

It contained 10 M cisplatin.

Description of the invention

As used in the specification and the claims of this application, the term “clusterina” is refers to the testicularly derived glycoprotein rat, and homologous proteins derived from other species of mammals, including humans, whether called clusterina as with an alternative name. The

sequences of numerous species of clusterin. For example, it reports the sequence of the human clusterin in Wong et al., Eur. J. Biochem. 221 (3), 917-925 (1994), and in the number of NCBI sequence record NM_001831 and is shown in the sequence listing as SEQ ID NO: 1. In this sequence, the coding sequence covers bases 48 to 1397.

The present invention provides a composition therapeutic, and methods for using such a composition for the treatment of melanoma, particularly in beings humans. The therapeutic compositions and methods of invention achieve a reduction in the effective amount of Clusterin present in the individual being treated. Such as used in this application, the “effective amount of clusterina ”is the amount of clusterina that is present in a way that is functional to provide protection antiapoptotic The effective amount of clusterin can be reduced decreasing the clusterin expression rate, increasing the degradation rate of clusterin, or modifying clusterin (for example, by binding with an antibody) in such a way It becomes inactive.

Therapeutic agents of antisense ODN

In one embodiment of the invention, the reduction of the effective amount of clusterin by administration of antisense ODN, particularly ODN antisense that are complementary to a region of the mRNA of clusterina that covers either the initiation site of the translation or termination site. The sequences by way example that can be used as antisense molecules in the method of the invention are disclosed in the publication of PCT patent WO 00/49937, patent publication US-2002-0128220-A1, and the US patent No. 6,383,808. The specific antisense sequences are show in this application as SEQ ID NO: 2 to 12.

The ODNs used to increase the ODN stability in vivo. For example, the ODN as phosphorothioate derivatives (substitution of an atom of

phosphoryl oxygen that does not form bridges by an atom of sulfur) that have increased resistance to digestion with nucleases. Modification with MOE (2’-O- (2methoxyethyl) (ISIS main structure) is also effective. Modified ODN is described in detail in the patent application U.S. 10,080,794. A composition particularly preferred is a 21-mer oligonucleotide (cagcagcagagtcttcatcat; SEQ ID NO: 4) directed to the codon of translation initiation and the following 6 codons of the human clusterin sequence (Genbank registration no .: NM_001831) with a modification with 2’-MOE. This oligonucleotide It has a main structure of phosphorothioate in its entirety. The sugar residues of nucleotides 1-4 and 18-21 (the "wings") carry modifications with 2’-O-methoxyethyl and nucleotides remaining (nucleotides 5-17; the "deoxy hole") are 2'-deoxynucleotides. The cytosines on the wings (i.e. nucleotides 1, 4 and 19) are 5-methylcytosines.

Antisense ODN administration can be carried out using the various mechanisms known in the art, including vehicleless administration and administration in Pharmaceutically acceptable lipid vehicles. For example, U.S. Patent Nos. 5,855,911 and 5,417,978 are given to know lipid vehicles for agent administration antisense In general, the antisense agent is administered through intravenous, intraperitoneal, subcutaneous or oral routes, or direct local tumor injection.

The amount of antisense ODN administered is an effective to inhibit the expression of clusterin in the cells of melanoma. It will be appreciated that this amount will vary so much with the effectiveness of the antisense ODN employed, as with the nature of Any used vehicle. The determination of quantities appropriate for any given composition is within the skill in the art, using conventional series of tests designed to assess therapeutic levels appropriate.

IRNA therapeutic agents

The reduction of the effective amount of clusterin also It can be achieved using iRNA therapy. RNA interference

or "iRNA" is a term initially coined by Fire and collaborators to describe the observation that RNA double stranded (cRNA) can block gene expression when introduced into worms (Fire et al. (1998) Nature 391, 806-811). The tRNA directs posttranscriptional, specific silencing of genes, in many organisms, including vertebrates, and has provided a new tool to study the function gene. The iRNA implies the degradation of mRNA, however it they don't know many of the biochemical mechanisms that underlie This interference The use of the RNAi has been described additionally in Carthew et al. (2001) Current Opinions in Cell Biology 13, 244-248, and Elbashir et al. (2001) Nature 411, 494

498.

In the present invention, isolated RNA molecules they mediate the iRNA. That is, the RNA molecules isolated from the present invention mediate degradation or block expression of the mRNA that is the transcriptional product of the gene, which also It is called the target gene. For convenience, such mRNA can also be referred to herein as mRNA to be degraded. The expressions RNA, RNA molecule (s), RNA segment (s) and RNA fragment (s) can be used interchangeably to refer to the RNA that mediates RNA interference. These expressions include double stranded RNA, single stranded RNA, isolated RNA (RNA partially purified, essentially pure RNA, synthetic RNA, Recombinantly produced RNA), as well as altered RNA that it differs from the RNA that occurs naturally by the addition, deletion, substitution and / or alteration of one or more nucleotides Such alterations may include the addition of non-nucleotide material, such as at the end (s) of the RNA

or internally (in one or more RNA nucleotides). The nucleotides in the RNA molecules of the present invention they can also comprise unconventional nucleotides, including nucleotides that do not occur naturally or deoxyribonucleotides. Together, all iRNA molecules altered of this type are called analogs or RNA analogs

They occur naturally. The RNA of this invention just needs to be similar enough to RNA natural to have the ability to mediate the iRNA. Such as is used herein, the phrase "mediate the iRNA" is refers to, and indicates, the ability to distinguish which mRNA are going to be affected by the machinery or process of the iRNA. He RNA mediating the iRNA interacts with the iRNA machinery of such that it directs the machinery to degrade mRNA particular or otherwise to reduce the expression of the target protein In one embodiment, the present invention is refers to RNA molecules that direct the direct cleavage of Specific mRNA to which its sequence corresponds. It is not necessary that there is perfect correspondence of the sequences, but the correspondence must be sufficient to allow the RNA direct inhibition by iRNA by excision or blocking of the expression of the target mRNA.

As noted above, the RNA molecules of the present invention generally comprise a portion of RNA and some additional part, for example a part of deoxyribonucleotide. The total number of nucleotides in the RNA molecule is suitably less than 49 in order to be effective mediators of iRNA. In preferred RNA molecules, the number of nucleotides is 16 to 29, more preferably of 18 to 23, and most preferably 21-23. At the moment Application suitable sequences are shown as SEQ ID NO: 20 a

43

The siRNA molecules of the invention are used in therapy. to treat patients, including human patients, who have cancers or other diseases of a type in which you get a therapeutic benefit by inhibiting the expression of the selected protein as target. SiRNA molecules of the invention is administered to patients by one or more daily injections (intravenous, subcutaneous or intrathecal) or by continuous intravenous or intrathecal administration during one or more treatment cycles to achieve plasma and tissue concentrations suitable for regulation of the mRNA and protein selected as the target.

Additional therapeutic agents

The method of treating melanoma according to the invention can also include the administration of chemotherapy agents or other agents useful in the therapy of melanoma and / or ODN additional antisense aimed at different targets in combination with therapeutic efficacy to reduce the amount of active clusterin. For example, the clusterin ODN antisense increases sensitivity to chemotherapy agents conventional such as taxanes (paclitaxel or docetaxel), mitoxantrone and gemcitabine. Other agents that probably show synergistic activity include other cytotoxic agents (for example, cyclophosphamide, dacarbazine, inhibitors of topoisomerase), angiogenesis inhibitors, agents of differentiation and inhibitors of signal transduction. From similarly, the antisense agent combinations of clusterin with other species of antisense agents such as Antisense ODN of Bcl-2, Bcl-xl and c-myc provide greater effectiveness.

Bcl-xL expression regulation method

Although the chaperone type function has been proposed for clusterin protein, the molecular mechanism is still not understood specific responsible for the role of clusterin in the apoptosis In the human melanoma cell line that expressed clusterin at a very low level, clusterin overexpression through stable transfection not only led to an increase marked resistance against cytotoxic treatment (figure 3), but also led to regulation by increased member of the antiapoptotic bcl-2 family, bcl-xL, as shown by immunoblot type Western. On the contrary, the treatment of melanoma cells which express clusterin led to a regulation by decline marked bcl-xL thereby providing a possible mechanism for clusteria antiapoptotic potency. Not even overexpression of clusterin by transfection or Clusterin antisense agent treatment altered the

expression of other members of the Bcl-2 family undergoing Human melanoma cell test. Therefore, the clusterina regulates the member of the antiapoptotic bcl-2 family, bcl-xL. Such inhibition is significant because it is known that bcl-xL acts as an apoptosis inhibitor (see patent U.S. 6,182,216).

The invention will be described below by making reference to the following non-limiting examples.

Example 1

Clusterin expression in two different batches of normal human melanocytes (NHEM 6083 and 2489) and four lines Human melanoma cells (518A2, SKMEL-28, Mel-Juso and 607B). Cells were grown in 6 cm plates and collected when They were 80-90% confluent. 30: g of protein were applied per lane on a 10% SDS-PAGE gel and treated with probes of goat anti-clusterin polyclonal antibody. They were used

Panceau's red stain and an antibody directed against -actin as a loading control. In each case, the clusterin antisense inhibitor used was based on the advanced antisense chemistry of 2'MOE as described in US Patent Application 10 / 080,794 and has the sequence of SEQ ID NO: 4.

Figure 1 shows the results when treated 607B melanoma cells with either the oligonucleotide antisense at concentrations of 100, 250 or 500 nM, or a randomized control at a concentration of 100 nM in two days consecutive. Lipofectin ™ (lip) without oligonucleotide was used as a control. Cell numbers were measured on 96 plates wells photometrically using MTS (Cell Titer 96 ™, Pierce). As shown, they were significantly reduced cell counts in the presence of wells treated with antisense agent at 250 and 500 nM.

Figure 2 provides a graphical representation of the Clusterin expression in 518A2 cells after treatment with cisplatin and either an antisense oligonucleotide or a randomized control. Lip is a Lipofectin control without

oligonucleotide Detection was performed using an antibody directed against clusterina.

The results in showed that in melanoma cells human, clusterin is expressed at significantly levels greater than in human melanocytes in all cell lines tested less in one. The antisense inhibitor (modification with MOE of SEQ ID NO: 4) led to regulation by dose dependent clusterin decrease such as It is shown by RT-PCR at mRNA level and by Western blots at the protein level in comparison with the randomized mating control. This regulation by decrease led to an increase in the apoptotic cell death by agent treatment antisense alone. In a melanoma cell line (607B), this it was only enough to lead to complete cell death (Figure 1). In another melanoma cell line, the cells survivors showed increased sensitivity to a consecutive treatment with the cytotoxic drug cisplatin in comparison with cells treated with an oligonucleotide of wrong control mating (figure 2).

Example 2

Mel Juso melanoma cells transfected from stable manner with either an empty control vector (Neo) or well a vector that directed clusterin overexpression, in

medium containing 10 M cisplatin. Cell survival was measured using Promega Cell-titer 96 kits. The results are summarized in Figure 3. As shown, clusterin overexpression dramatically enhanced cell survival, or put it differently, reduced the effectiveness of the chemotherapy agent.

SEQUENCE LIST

<110> THE UNIVERSITY OF BRITISH COLUMBIA GLEAVE, Martin JANSEN, Burkhard

<120> TREATMENT OF MELANOMA THROUGH REDUCTION OF CLUSTERINE LEVELS

<130> 49101-4

<140> NOT ALREADY ASSIGNED

<141> 08-21-2003

<150> US 60 / 405,193

<151> 08-21-2002

<150> US 60 / 319,748

<151> 02-12-2002

<150> US 60 / 408,152

<151> 03-09-2002

<150> US 60 / 473,387

<151> 05-20-2003

<160> 43

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Claims (12)

one.

Use of a composition comprising a therapeutic agent in the form of an antisense oligodeoxynucleotide or siRNA oligonucleotide directed to the clusterin gene or an antibody directed to clusterin and effective for reduce the effective amount of clusterin in cells melanoma for the formulation of a composition Pharmaceutical for the treatment of melanoma.

2.

Use according to claim 1, wherein the agent Therapeutic is an antisense oligodeoxynucleotide.

3.

Use according to claim 2, wherein the antisense oligodeoxynucleotide covers either the site of translation initiation or termination site.

Four.

Use according to claim 2 or 3, wherein it is modified the antisense oligodeoxynucleotide to enhance your in vivo stability in relation to a unmodified oligodeoxynucleotide of the same sequence.

5.

Use according to claim 4, wherein the modification it is a modification with (2’-O- (2-methoxyethyl).

6.

Use according to any of claims 2-5, in the that the antisense oligodeoxynucleotide consists essentially in an oligodeoxynucleotide selected from group consisting of SEQ ID NO: 2 to 12.

7.

Use according to claim 6, wherein the antisense oligodeoxynucleotide consists of SEQ ID NO:

Four.

8.

Use according to claim 7, wherein the oligodeoxynucleotide has a main structure of Phosphorothioate in its entirety, the sugar residues of nucleotides 1-4 and 18-21, the "wings", carry modifications with 2’-O-methoxyethyl and nucleotides remaining are 2’-deoxynucleotides.

9.

Use according to claim 1, wherein the agent therapeutic is an effective RNA molecule to reduce the effective amount of clusterin in melanoma cells through an iRNA mechanism.

10.

Use according to claim 9, wherein the molecule of

RNA comprises an oligonucleotide selected from the group that

consists of SEQ ID NO: 20

to 43 or a RNA molecule

double stranded comprising a selected oligonucleotide

5

of the group consisting of SEQ ID NO: 20 to 43 as a

chain.

eleven.

Use according to claim 9, wherein the molecule of

RNA consists of an oligonucleotide selected from the group

consisting of SEQ ID NO: 20 to 43 or an RNA molecule

10

double stranded containing an oligonucleotide consisting of

an oligonucleotide selected from the group consisting of

SEQ ID NO: 20 to 43 as a string.