(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`11111111111111111111111111111111111111111111111111111111111111111111111111111111
`
`(43) International Publication Date
`25 October 2001 (25.10.2001)
`
`PCT
`
`(10) International Publication Number
`WO 01/79554 Al
`
`(51) International Patent Classification7:
`C07H 21/04
`
`C12Q 1/68,
`
`(21) International Application Number: PCT/USOl/12087
`
`(72) Inventor; and
`(75) Inventor/Applicant (jor US only): WOOSLEY, Ray(cid:173)
`mond, L. [US/US]; 4106 18th Road, North Arlington, VA
`22207 (US).
`
`(22) International Filing Date:
`
`13 April2001 (13.04.2001)
`
`(25) Filing Language:
`
`English
`
`(74) Agent: TESKIN, Robin, L.; Pillsbury Winthrop LLP,
`1100 New York Avenue, NW, Washington, DC 20005
`(US).
`
`(26) Publication Language:
`
`English
`
`(81) Designated States (national): CA, JP, US.
`
`(84) Designated States (regional): European patent (AT, BE,
`CH, CY, DE, DK, ES, Fl, FR, GB, GR, IE, IT, LU, MC,
`NL, PT, SE, TR).
`
`Published:
`with international search report
`
`For two-letter codes and other abbreviations .. refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`(30) Priority Data:
`60/196,916
`
`13 April :woo (13.04.2000) US
`
`(63) Related by continuation (CON) or continuation-in-part
`(CIP) to earlier application:
`us
`Filed on
`
`60/196,916 (CON)
`13 April2000 (13.04.2000)
`
`(71) Applicant
`(for all designated States except US):
`GEORGETOWN UNIVERSITY
`[US/US];
`3900
`Reservior Road, N.W., Washington, DC 20007 (US).
`
`iiiiiiiiiiii
`
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`
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`iiiiiiiiiiii -iiiiiiiiiiii
`!!!!!!! --
`--==
`iiiiiiiiiiii ---==
`
`~
`
`<-------------------------------------------------------
`~ (54) Title: GENETIC DIAGNOSIS FOR QT PROLONGATION RELATED ADVERSE DRUG REACTIONS
`ln
`ln (57) Abstract: The specification is directed to a method of diagnosing whether a subject is predisposed to an adverse reaction to
`~ one or more pharmaceutical agents which may induce a prolonged QT interval or acquired LQTS in that individual. The diagnosis is
`S risk for prolonged QT intervals or Torsades de Pointes (TdP). Genetic screening for determining the predisposition of prolonged
`-... genetic analysis of at least two polymorphisms or mutations which the individual may have, which are associated with an increased
`0 two classes or genes, wherein the genes are (1) LQT genes, (2) altered sensitivity genes (e.g., MiRP 1) or (3) increased exposure
`> genes (e.g.. MDR genes orP450 cytochrome genes). The specification is also directed to compositions and kits for determining such
`~ predispositions to adverse drug reactions.
`
`QT intervals induced by a pharmaceutical agent is performed by identifying genetic polymorphisms or mutations located in at least
`
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`Page 001
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`GENETIC DIAGNOSIS FOR QT PROLONGATION RELATED
`
`ADVERSE DRUG REACTIONS
`
`CROSS REFERENCE TO RELATED APPLICATION
`
`5
`
`This application claims benefit of priority from U.S. Provisional Patent
`
`Application Serial No. 60/196,916, filed April13, 2000, the entirety of which is
`
`incorporated herein by reference.
`
`FIELD OF THE INVENTION
`
`1 0
`
`The invention relates to methods of determining a predisposition for QT
`
`interval prolongation in a subject after the administration of a pharmaceutical agent or
`
`agents. Compositions and kits for determining said predispositions to the QT interval
`
`prolongation are also described.
`
`15
`
`BACKGROUND OF THE INVENTION
`
`The invention relates to a method of screening a subject for a predisposition to
`
`an adverse drug reaction involving prolonged QT intervals. The genetic screening of
`
`patients for said predisposition focuses on genes associated with QT interval
`
`prolongation, including LQT genes, P-glycoprotein membrane pump proteins (P-gp ),
`
`20 multidrug resistance genes and cytochrome P450-mediated drug metabolism genes.
`
`I.
`
`1.
`
`LOT and Cytochrome P450 Genes and Polymorphisms
`
`LQT Genes
`
`Genes associated with long QT (LQT) syndrome (LQTS) include KVLQTJ
`
`25
`
`(LQTJ), HERG (LQT2), SCN5A (LQT3) and MinK (LQT5). A fifth gene locus exists
`
`on human chromosome 4 (e.g., LQT4). Recently, a sixth gene (LQT6) has been
`
`identified (Wang et al., Ann. Med. 30: 58-65 (1998)). All but LQT3 encode cardiac
`potassium ion (Kl channel proteins; LQT3 encodes a cardiac sodium ion (Na l
`channel protein (Vincent, Annu. Rev. Med. 49: 263-74 (1998)). At least 180
`
`30 mutations have been identified among these genes (Abbott et al., Cell97: 175-87
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`-2-
`
`(1999); Vincent, Annu. Rev. Med. 49: 263-74 (1998); Curran et al., Cell80: 795-803
`
`(1995); Berthet et al., Circulation 99: 1464-70 (1999); Dausse et al., J. Mol. Cell
`
`Cardiol. 28: 1609-15 (1996); Chen et al., J. Biol. Chern. 274: 10113-8 (1999); and
`
`Sanguinetti et al., Proc. Nat!. Acad. Sci. US.A. 93: 2208-12 (1996)). Some ofthese
`
`5 mutations cause altered ion channel function resulting in non-drug induced prolonged
`
`QT intervals and a pro:pensity for Torsades de Paiutes (TdP) (See, e.g., Berthet et al.,
`
`Circulation 99: 1464-70 (1999)). Accordingly, genetic screening can be performed on
`
`subjects suspected of having long QT syndrome, as well as other patients (see, e.g.,
`
`Satler et al., Hum. Genet. 102: 265-72 (1998)). Larson et al., Hum. Mutat. 13: 318-27
`
`10
`
`(1999) reported a high-throughout single strand polymorphism (SSCP) analysis for
`
`detecting point mutations associated with LQTS.
`
`U.S. Patent No. 5,599,673 claims two (e.g., HERG and SCN5A) of the six LQT
`
`genes. Two BERG-related genes have also been claimed (U.S. Patent No. 5,986,081).
`
`futemational PCT Application WO 97/23598 describes a method of assessing a
`
`15
`
`patient's risk for long QT syndrome (LQTS) by screening for genetic mutations in the
`
`MinK gene. However, these patents do not disclose methods of diagnosing a patient's
`
`predisposition to an adverse drug reaction involving elongation of the QT interval due
`
`to mutations in any of the LQT genes.
`
`Drugs have been identified that cause QT interval prolongation, an:d thereby
`
`20
`
`adverse drug reactions. Certain antihistamines, such as terfenadine (e.g., Seldane®)
`
`and astemizole (e.g., Hismanal®), reportedly block potassium channels (Woosley,
`
`Annu. Rev. Pharmacal. Toxicol. 36: 233-52 (1996)) and inhibit the HERG protein,
`
`and thereby were postulated to induce Torsades de Pointes (Wang et al., 1998). All
`
`antiarrhythmic drugs that lengthen repolarization reportedly can cause Torsades de
`
`25
`
`Pointes (Drici et al., Circulation 94: 1471-4 (1996)). Additional non-cardiac and
`
`cardiac drugs capable of inducing QT prolongation including many that were
`
`identified by the inventor were released on March 27, 1998 at the following web site:
`
`www.qtdrugs.org. However, Wei et al., Circulation 92: I-125 (1995) could not
`
`identify HERG or SCN5A gene mutations that were linked to acquired LQTS in
`
`30
`
`patients treated with an anti-arrhythmic agent. To the best knowledge of the inventor,
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`no one has described diagnosing a predisposition towards an adverse drug or drug(cid:173)
`
`drug reaction whic~ causes QT interval elongation by screening patients for one or
`
`more polymorphisms in one or more LQT genes.
`
`5
`
`1.
`
`Cytochrome P450 Genes
`
`The cytochrome P450 enzymes have also been linked to adverse drug
`
`reactions. CYP2D6 was the first cytochrome P450 isoform found to be genetically
`
`polymorphic in its distribution (Eichelbaum et al., Eur. J. Clin. Pharmacal. 16: 183-7
`
`I
`
`(1979); and Mahgoub et al., Lancet 2: 584-6 (1977)), and it is now clear that-this
`
`10
`
`enzyme metabolizes a large number of drugs (fuaba et al., Can. J. Physiol.
`
`Pharmacal. 73: 331-8 (1995); and Buchert et al., Pharmacogenetics 2: 2-11 (1992)).
`
`At least 30 mutations exist which alter the activity or specificity ofCYP2D6 (Jordan
`
`et al., Endocr. Rev. 20: 253-78 (1999)). These include alleles that contain single point
`
`mutations resulting in no activity (e.g., CYP2D6*4), alleles in which the CYP2D6
`
`15
`
`gene has been deleted (e.g., CYP2D6*5) and alleles in which it has been duplicated
`
`(e.g., CYP2D6*2_n) (Aklillu et al., J. Pharmacal. Exp. Ther. 278: 441-6 (1996)).
`
`There are numerous cytochrome P450 genes which are involved in the
`
`metabolism of drugs and drug metabolites. Several of them include CYP IA2,
`
`CYP2C19, CYP2C9, CYP2D6, CYP2El, CYP3A4, CYP3A5 and CYP3A7. Allelic
`
`20
`
`variations exist amongst these genes. Certain of these allelic variations combine to
`
`produce a poor metabolizer phenotype in 7% of Caucasians, but smaller percentages
`
`of Africans and Asians and the "ultrarapid" phenotype in ~5% of Caucasian and up to
`
`30% Africans. As ethnic-specific alleles for both Asians (Y okoi et al., Pharm. Res.
`
`15: 517-24 (1998)) and Africans (Aklillu et al., J. Pharmacal. Exp. Ther. 278: 441-6
`
`25
`
`(1996); and Oscarson et al., Mol. Pharmacal. 52: 1034-40 (1997)) have been
`
`identified, that may alter the mean activity of the enzymes in these populations (see
`
`Table 1 below), it is also important to test for these alleles in studies of the
`
`relationship between genotype and phenotype.
`
`30
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`Table 1
`
`Chromosome Distribution of Cytochrome P450 Gene
`
`Chr.
`15
`
`Chr. 10
`Polymorphic
`
`Chr. 10
`Polymorphic
`
`Chr. 22
`
`Chr.lO Chr. 7
`
`3-5%
`Caucasian
`PMs
`
`1-3%
`Caucasian
`PMs
`
`15-20% Asian
`PMs
`
`Polymorphic
`
`5-10%
`Caucasian
`PMs
`
`In fact, due to the metabolic differences, methods have been reported which identify a
`
`5
`
`drug which interacts with the CYP2C19 gene product, S-mephenytoin 4'-hydroxylase
`
`(U.S. Patent No. 5,786,191).
`
`Methods for detecting the presence or absence ofmutations in certain ofthe
`
`cytochrome P450 genes have been described. For example, U.S. Patent No.
`
`5,891,633 relates to a method of identifying mutations in the cytochrome P450 genes
`
`10
`
`CYP2C9 and CYP2A6.
`
`futemational PCT Application WO 95/30772 reportedly describes a CYP2D6
`
`gene polymorphisms involving a 9 bp insertion in exon 9, which is associated with a
`
`slower than normal rate of drug metabolism in individuals bearing it and may be
`
`therefore useful diagnostically. PCR primers have been described for detecting
`
`15 mutations in drug metabolism enzymes, including detection of the debrisoquine
`
`polymorphism, mephenytoin polymorphism and the acetylation polymorphism (U.S.
`
`Patent Nos. 5,648,484 and 5,844,108). Additional mutations have been identified in
`
`CYP2D6 bufuralol-1 '-hydroxylase, including mutations at positions 271, 281, 294,
`
`and 506 whlch result in metabolizer/poor metabolizer phenotypes as described in
`
`20
`
`futemational PCT Application WO 91/10745 and U.S. Patent No. 5,981,174.
`
`Japanese Patent No. 8168400 provides a method of determining mutations in
`
`exons 6 and 7 ofthe CYP2C19 gene. Japanese Patent No. 10014585 describes
`
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`-5-
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`primers and methods of detecting a mutation in exon 5 of CYP2C19, which is related
`
`to the abnormal metabolism of diazepam, imipramine, omeprazole and propranolol.
`
`U.S. Patent No. 5,912,120 claims a method of diagnosing a patient having a
`
`deficiency in S-mephenytoin 4'-hydroxylase activity by detecting polymorphisms at
`
`5
`
`nucleotides 681 or 636.
`
`U.S. Patent 5,719,026 provides methods and primers for detecting a
`
`polymorphisms in CYP JA2 and assessing the changes in the drug activity of
`
`theophylline associated with those polymorphisms.
`
`Japanese Patent No. 10286090 reportedly describes methods and primers to
`
`10
`
`detect mutations in CYP2El. These mutations are reported as being useful for
`
`determining the safety margin for drug administration for the treatment or related
`
`diseases.
`
`Despite these teachings and to the best of the inventor's knowledge, no one has
`
`described or suggested that a combination of polymorphisms in LQT and cytochrome
`
`15
`
`P450 genes can induce acquired LQTS in a subject in response to the administration
`
`of a drug or drugs.
`
`C.
`
`P-glycoprotein Pump Genes
`
`P-Glycoprotein Pump (P-gp) in the development of drug-resistant tumor cells
`
`20
`
`has been extensively studied (Lo et al., J. Clin. Pharmacal. 39: 995-1005 (1999)). P(cid:173)
`
`gp is an ATP-dependent drug pump that extrudes a broad range of cytotoxic agents
`
`from the cells end is encoded by a gene called MDR -1, for multi drug resistance (Loo
`
`et al., Biochem. Cell. Bioi. 77: 11-23 (1999); and Robert, Eur. J. Clin. Invest. 29: 536-
`
`45 (1999)). The human P-gp sequence has been described by Chen et al., Cell47:
`
`25
`
`381-9 (1986) and has the GenBank Accession No. M14758.
`
`Its physiological role may be to protect the body from endogenous and
`
`exogenous cytotoxic agents. The protein has clinical importance because it
`
`contributes to the phenomenon of multi drug resistance during chemotherapy (Loo et
`
`al., 1999) and the development of simultaneous resistance to multiple cytotoxic drugs
`
`30
`
`in cancer cells (Ambudkar et al., Annu. Rev. Pharmacal. Toxicol. 39: 361-98 (1999)).
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`Specifically, the over expression ofthis membrane pump appears to extrude many
`
`xenobiotics out of the tumor cells (Robert, 1999). However, considerable controversy
`
`remains about the mechanism of action of this efflux pump and its function in normal
`
`cells (Ambudkar eta!., 1999).
`
`5
`
`Multidrug resistance (MDR) can be diagnosed in tumors using molecular
`
`biology techniques (e.g., gene expression at the mRNA level), by immunological
`
`techniques (e.g., quantification of the P-glycoprotein itself) or by functional
`
`approaches (e.g., measuring dye exclusion) (Robert, 1999).
`
`Drugs have been developed which reverse or modulate MDR. For example,
`
`10
`
`PSC-833 is a non-immunosuppressive cyclosporin derivative that potently and
`
`specifically inhibits P-gp (Atadja eta!., Cancer Metastasis Rev. 17: 163-8 (1998)).
`
`Also, compounds have been identified which increase or modulate the bioavailability
`
`of pharmaceutical compounds. See, e.g., U.S. Patent Nos. 6,004,927; 5,962,522;
`
`5,916,566; 5,716,928; 5,665,386; and 5,567,592. P-gp activity has been altered by
`
`15
`
`expression of antisense nucleotides specific to MDR-1 (U.S. Patent No. 6,001,991).
`
`Methods and assays have also been developed which assess whether multidrug
`
`resistance has been reversed (U.S. Patent No. 5,403,574).
`
`Mutations have also been identified which alter an agents interaction with P(cid:173)
`
`gp. For instance U.S. Patent No. 5,830,697 discloses single and double mutations
`
`20
`
`(Phe335 and/or 336) which alters the spectrum of cross-reactivity to cytotoxins and
`
`resistance to modulation by cyclosporins. Another mutation at V185G in P-gp
`
`confers increased resistence to colchicine (U.S. Patent No. 5,830,697). P-gp
`
`sensitivity to vinblastine, colchicine, VP16 and adriamycin, common
`chemotherapeutic agents, was up- and down-regulated by altering 61His to another
`
`25
`
`amino acid residue (Taguchi eta!., Biochemist1y 36: 8883-9 (1997)). Moreover,
`
`different drugs interact differently with P-gp and mutated forms ofP-gp, such that one
`
`mutation may influence the activity of one drug and not another (See, e.g., Chen et a!.,
`
`J. Biol. Chem. 272: 5974-82 (1997); Bakos eta!., Biochem. J. 323: 777-83 (1997);
`
`and Gros eta!., Proc. Nat!. Acad. Sci. USA 88: 7289-93 (1991)). However, despite the
`
`30
`
`information regarding the influence such mutations may have on drug activity, no
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`association has been made linking P-gp by itself, or in combination with another
`
`protein in influencing QT intervals or inducing Torsades de Pointes.
`
`II.
`
`Nucleic Acid Hybridization
`
`5
`
`The capacity of a nucleic acid "probe" molecule to hybridize (i.e., base pair) to
`
`a complementary nucleic acid "target" molecule forms the cornerstone for a wide
`
`array of diagnostic and therapeutic procedures. Hybridization assays are extensively
`
`used in molecular biology and medicine. Methods of performing such hybridization
`
`reactions are disclosed by, for example, SAMBROOK ET AL., MOLECULAR CLONING: A
`
`10
`
`LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
`
`N.Y. (1989)), HAYMES ET AL., NUCLEIC ACID HYBRIDIZATION: A PRACTICAL
`
`APPROACH (IRL Press, Washington, D.C. (1985)) and KELLERET AL., DNA PROBES
`
`(2nd Ed., Stockton Press, New York (1993)).
`
`Many hybridization assays require the immobilization of one component to a
`
`15
`
`solid support. Nagata et al., FEBS Letters 183: 379-82 (1985) described a method for
`
`quantifying DNA which involved binding unknown amounts of cloned DNA to
`
`microtiter wells in the presence of 0.1 M MgCh. A complementary biotinylated probe
`
`was then hybridized to the DNA in each well and the bound probe measured
`
`colorimetrically. Dahlen et al., Mol .. Cell. Probes 1: 159-168 (1987) have discussed
`
`20
`
`sandwich hybridization in microtiter wells using cloned capture DNA adsorbed to the
`
`wells. An assay for detecting HN -1 DNA using PCR amplification and capture
`
`hybridization in microtiter wells also has been reported (Keller et al., J. Clin.
`
`Microbial. 29: 638-41 (1991)). The NaCl-mediated binding of oligomers to
`
`polystyrene wells has been discussed by Cros et al. (French Patent No. 2,663,040) and
`
`25
`
`by Nikiforov et al., PCR Methods Applic. 3: 285-291 (1994). A cationic
`
`detergent-mediated binding of oligomers to polystyrene wells has been described by
`
`Nikiforov et al., Nucleic Acids Res. 22: 4167-75 (1994).
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`III. Analysis Of Single Nucleotide DNA Polymorphisms
`
`Many genetic diseases and traits (i.e. hemophilia, sickle-cell anemia, cystic
`
`fibrosis, etc.) reflect the consequences of mutations that have arisen in the genomes of
`
`some members of a species through mutation or evolution (Gusella, Ann. Rev.
`
`5
`
`Biochem. 55: 831-54 (1986)). fu. some cases, suchpolymorphisms are linked to a
`
`genetic locus responsible for the disease or trait; in other cases, the polymorphisms are
`
`the determinative characteristic of the condition .
`
`.Single nucleotide polymorphisms (SNPs) differ significantly from the variable
`
`nucleotide type polymorphisms (VNTRs ), that arise from spontaneous tandem
`
`10
`
`duplications of di- or tri-nucleotide repeated motifs of nucleotides (Weber, U.S. Pat.
`
`No. 5,075,217; Armour et al., FEBS Lett. 307: 113-5 (1992); Hom et·al., PCT
`
`Application No. WO 91/14003; Moore et al., Genomics 10: 654-60 (1991); Hillel et
`
`al., Genet. 124: 783-9 (1990)), and from the restriction fragment length
`
`polymorphisms ("RFLPs") that comprise variations which alter the lengths of the
`
`15
`
`fragments that are generated by restriction endonuclease cleavage (e.g., Fischer et al.,
`
`(PCT Application No. WO 90/13668); and Uhlen (PCT Application No. WO
`
`90/11369)).
`
`Because SNPs constitute sites of variation flanked by regions of invariant
`
`sequence, their analysis requires no more than the determination of the identity of the
`
`20
`
`single nucleotide present at the site of variation; it is unnecessary to determine a
`
`complete gene sequence for each patient. Several methods have been developed to
`
`facilitate the analysis of such single nucleotide polymorphisms.
`
`Mundy, C. R. (U.S. Pat. No. 4,656, 127), for example, discusses a method for
`
`determining the identity of the nucleotide present at a particular polymorphic site that
`
`25
`
`employs a specialized exonuclease-resistant nucleotide derivative.
`
`Cohen et al. (French Patent 2,650,840; and PCT Application No. WO
`
`91/02087) discuss a solution-based method for determining the identity of the
`
`nucleotide of a polymorphic site. As in the Mundy method ofU.S. Patent. No.
`
`4,656,127, a primer is employed that is complementary to allelic sequences
`
`30
`
`immediately 3' to a polymorphic site.
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`Additional SNP detection methods include the Genetic Bit Analysis method
`
`described by Goelet et al. (PCT Application No. 92115712). The method ofGoelet et
`
`al. uses mixtures of labeled terminators and a primer that is complementary to the
`
`sequence 3' to a polymorphic site.
`
`5
`
`Cheesman (U.S. Pat. No. 5,302,509) describes a method for sequencing a
`
`single stranded DNA molecule using fluorescently labeled 3 '-blocked nucleotide
`
`triphosphates. An apparatus for the separation, concentration and detection of a DNA
`
`molecule in a liquid sample has been described by Ritterband et al. (PCT Patent
`
`Application No. WO 95/17676).
`
`10
`
`Several primer-guided nucleotide incorporation procedures for assaying
`
`polymorphic sites in DNA have been described (Prezant et al., Hum. Mutat. 1: 159-64
`
`(1992); Ugozzoli et al., GATA 9: 107-12 (1992); and Nyren et al., Anal. Biochem.
`
`208: 171-5 (1993)).
`
`15
`
`IV. Methods of Immobilizing Nucleic Acids to a Solid-Phase
`
`Several ofthe above-described methods involve procedures in which one or
`
`more of the nucleic acid reactants are immobilized to a solid support. Currently,
`
`96-well polystyrene plates are widely used in solid-phase immunoassays, PCR
`
`product detection methods that use plates as a solid support and DNA chips have been
`
`20
`
`described. The microtiter plate method requires the immobilization of a suitable
`
`oligonucleotide probe into the microtiter wells, followed by the capture ofthe PCR
`
`product by hybridization and colorimetric detection of a suitable hapten.
`
`Covalent disulfide bonds have been previously used to immobilize both
`
`proteins and oligonucleotides. Chu et al. (Nucl. Acids Res. 16: 3671-91 (1988))
`
`25
`
`discloses a method for coupling oligonucleotides to nucleic acids or proteins via
`
`cleavable disulfide bonds.
`
`Gentalen et al., Nucl. Acids Res. 27: 1485-91 (1999) describe a cooperative
`
`hybridization method to establish physical linkage between two loci on a DNA strand
`
`by using hybridization to a new type of high-density oligonucleotide array. This same
`
`30 method can be used to determine SNP haplotypes.
`
`Roxane Labs., Inc.
`Exhibit 1014
`Page 010
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`-10-
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`Yershov et al., Proc. Nat!. Acad. Sci. USA 93: 4913-8 (1996) describe an
`
`oligonucleotide microchip which has been used to detect beta-thalassemia mutations
`
`in patients by hybridizing PCR-amplified DNA with the microchips. This technology
`
`was suggested for large scale diagnostics in gene polymorphism studies.
`
`5
`
`Guo et al., Nucl. Acids Res. 22: 5456-65 (1994) describe a simple method for
`
`the analysis of genetic polymorphisms allele-specific oligonucleotide raised bound to
`
`glass supports. This method was demonstrated in parallel analysis of 5 point
`
`mutations from exon 4 of the human tyrosinase gene.
`
`More recently, Gilles et al., Nat. Biotechnol. 17: 365-70 (1999) have described
`
`10
`
`a rapid assay for SNP detection utilizing electronics circuitry on silicon microchips.
`
`Holloway et al., Hum. Mutat. 14: 340-7 (1999) also compares methods for high(cid:173)
`
`throughput SNP typing using TaqMan® liquid phase hybridization, PCR-SSOP or
`
`ARMS-microplate array diagonal gel electrophoresis (MADGE).
`
`As the world population ages and new drugs are identified, more and more
`
`15
`
`patients will administer one or more phannaceutical compositions, such that an
`
`individual drug or drug combination can cause adverse drug reactions. Therefore, not
`
`withstanding what has been previously reported in the literature, the inventor herein
`
`describes methods and compositions for diagnosing drug interactions which involve at
`
`least one mutation in a LQT gene. Additional mutations may exist in certain
`
`20
`
`cytochrome 450 genes and P-glycoprotein pumps, which work in concert with a LQT
`
`gene mutation or other ion chrumel (e.g., K+ or Nal gene polymorphisms to produce
`
`an adverse drug or drug-drug reaction. The specification also discloses kits and
`
`compositions for diagnosing a subject's predisposition to QT interval elongation in
`
`response to the administration of one or more pharmaceutical agents.
`
`25
`
`SUMMARY OF THE INVENTION
`
`It is an object of the invention to provide novel and improved methods for
`
`determining whether a subject has a predisposition for QT interval elongation or
`
`Torsades de Pointes due to one or more pharmaceutical agents. The methods
`
`30
`
`comprise the step of screening a biological sample from the subject through a nucleic
`
`Roxane Labs., Inc.
`Exhibit 1014
`Page 011
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`PCT/USOl/12087
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`-11-
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`acid array, wherein said nucleic acid array contains probes for at least two genetic
`
`mutations or polymorphisms. These two genetic mutations or polymorphisms are
`
`located in two or more of the group of genes consisting of (1) LQT genes, (2) altered
`
`sensitivity genes, and (3) increased exposure genes. Preferred genes include LQT
`
`5
`
`genes andMDR genes (e.g., MDR-1). The nucleic acid array can be in the form of a
`
`chip, a microchip, a bead or a microsphere. The LQT gene which may contain a
`
`polymorphism which induces QT interval elongation include LQTI, LQT2, LQT3,
`
`LQT4, LQT5 and LQT6.
`
`The method may further comprise screening both LQT and increased exposure
`
`10
`
`gene (e.g., cytochrome P450 genes) mutations and polymorphisms. The P450
`
`cytochrome isoforms which may contain a mutation which can result in excessive
`
`accumulation of drugs and thereby induce QT interval elongation include: CYP IA2,
`
`CYP2C19, CYP2C9, CYP2D6, CYP2El, CYP3A, CYP3A5 and CYP3A7.
`
`A further object of the invention is to provide a method for determining
`
`15 whether a subject has a predisposition for QT interval elongation (e.g., acquired
`
`LQTS) when treated with one or more pharmaceutical agents comprising the step of
`
`screening a biological sample from the subject through a nucleic acid array, such as a
`
`DNA array. The DNA array contains probes for two or more genetic mutations or
`
`polymorphisms in at least two or more groups of genes wherein the genes are selected
`
`20
`
`from the group consisting of(1) LQT genes, (2) altered sensitivity genes (e.g., MiRP-
`
`1 genes and its related genes), and (3) increased exposure genes (e.g., multidrug
`
`resistant genes and cytochrome P450 genes). The two or more genetic mutations or
`
`polymorphisms are found in these genes as at least one or more genetic mutations or
`
`polymorphisms in each of the two or more groups of genes. The genes can be
`
`25
`
`selected from those described above.
`
`Another object of the invention is to provide a nucleic acid array comprising
`
`nucleic acids which recognize and bind to mutations of the QT syndrome genes (e.g.,
`
`LQT genes), the altered sensitivity genes (MirR-1 genes) and/or the increased
`
`exposure genes.
`
`Roxane Labs., Inc.
`Exhibit 1014
`Page 012
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`-12-
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`Another object of the invention is to provide a method of screening one or
`
`more pharmaceutical agents in vitro for its or their ability to induce prolonged cardiac
`
`repolarization of a cell comprising the steps of A) measuring IKr and IKs currents of the
`
`cell using a voltage clamp before superfusing the cell with a candidate agent or
`
`5
`
`agents; B) superfusing and incubating the cell with the candidate agent or agents; C)
`
`measuring the Ir<r and IKs currents after superfusion and incubation of the cell with the
`
`candidate. agent or agents using a voltage clamp; and D) determining whether the IKr
`
`and IKs currents are inhibited or abolished thereby indicating that the drug prolongs
`
`repolarization.
`
`10
`
`It is another object of the invention to provide a method for identifying genetic
`
`polymorphisms and mutations, which can cause QT interval prolongation in a subject
`
`comprising the steps of inserting at least two nucleic acids each encoding a
`
`polymorphism or mutation of at least two of the following genes: a LQT gene, an
`
`altered sensitivity gene, and/or an increased drug exposure gene into a cell; B)
`
`15 measuring IKr and IKs currents of the cell before administering a drug lmown to cause a
`
`change in IKr and/or IKs; C) measuring IKr and IKs currents of the cell after superfusion
`
`of the cell with the drug; D) comparing the IKr and IKs values of the cell expressing the
`
`polymorphisms and/or mutations to the IKr and IKs values of a cell expressing a wild(cid:173)
`
`type genes; and E) determining if the presence ofthe polymorphisms and/or mutations
`
`20
`
`leads to greater inhibition or blockage ofiKr and IKs currents in the cell expressing said
`
`polymorphism or polymorphisms.
`
`Roxane Labs., Inc.
`Exhibit 1014
`Page 013
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`-13-
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`BRIEF DESCRIPTION OF THE DRAWINGS
`
`Figure 1. Recordings oflKn Ito (A and B) and IK1 (C) in the same cell
`
`before and after 5 minutes superfusion with 5 flmol/L E-4031. Panel A) IKr and Ito
`
`currents before and after superfusion with E-4031. E-4031 abolished the IKr tail
`
`5
`
`current and also reduced the time-dependent IKr current without affecting the transient
`
`outward current Cito) or the holding current; Panel B) E-4031 sensitive currents
`
`obtained by digital subtraction of currents after E-4031 exposure from currents before
`
`E-4031 exposure. Note the inward rectification of the time-dependent IKr currents at
`
`10
`
`very positive potentials compared with the tail currents; Panel C) IK1 current before
`and after superfusion with E-4031. E-4031 showed little effect on the inward IK1
`current recorded at -120 mV. The outward holding currents that represent the
`
`amplitude ofk1 at -40 mV are superimposed.
`
`Figure 2. Recordings of IKr, Ito (A and B) and IK1 (C) in the same cell
`
`before and after 5 minutes superfusion with 10 flmol/L tamoxifen. Panel A) IKr
`
`15
`
`and Ito currents recorded before and after superfusion with tamoxifen. Tamoxifen
`
`abolished the IKr tail current and also reduced the time-dependent IKr current, without
`
`affecting the transient outward current Oto); Panel B) Tamoxifen-sensitive currents
`
`obtained by digital subtraction of currents before and after tamoxifen superfusion.
`
`Note the inward rectification of the time-dependent IKr currents at very positive
`
`20
`
`potentials compared with the tail currents and their similarity to the E-4031 sensitive
`
`currents; Panel C) IK1 current recorded before and after superfusion with tamoxifen.
`
`Tamoxifen showed no block ofiK1 inward current. The outward holding currents,
`which represent the an1plitude ofiK1 at -40 mV, were superimposed together.
`Figure 3. Time-dependent block of IKr by tamoxifen. IKr currents were
`
`25
`
`recorded in the same cell before drug administration, 3, 5 and 9 min. after superfusion
`
`with 1 J.Lmol/1 tamoxifen.
`
`Figure 4. Voltage- and concentration-dependent block of IKr by
`tamoxifen. IKr tail currents ~ere measured 5-7 min. after superfusion with tamoxifen.
`
`Panel A) Effect of 1 Jlmol/L I-V relationship; Panel B) Effect of3.3 J.Lmol/1
`
`Roxane Labs., Inc.
`Exhibit 1014
`Page 014
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`PCT/USOl/12087
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`-14-
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`tamoxifen on I-V (current-voltage) relationship. Data are expressed as mean± SD,
`
`n=4, *p<0.05.
`
`Figure 5. Comparison of IKr block by tamoxifen and quinidine. Panel A)
`
`IKr currents recorded from the same cell before drug administration, 5 minutes after
`
`5
`
`super:fusion with 10 )lmol/L tamoxifen and 5 minutes after washout. IKr tail currents
`
`were abolished by tamoxifen without recovery. Panel B) IKr currents recorded from
`
`another cell before dmg administration, 5 minutes after superfusion with 10 )lmol/L
`
`quinidine and 3 minutes after washout demonstrate that IKr tail currents were reduced
`
`but not abolished by quinidine, with partial recovery after 3 minutes washout. Panel
`
`10
`
`C) Inhibition ofiKr by 3.3 ~-tmol/L tamoxifen and 3.3 )lmol/L quinidine. Data are
`
`expressed as mean± SD, n=4, **p<O.Ol.
`
`Figure 6. Effect of tamoxifen on the action potential duration (APD).
`
`Action potentials were elicited by injecting 100 pA depolarizing currents of 2 ms, at a
`
`frequency of 0.45 HZ. Shown in the figure are two superimposed action potential
`
`15
`
`tracings recorded in a single ventricular myocyte before and 4 minutes after exposure
`
`to 3.3 )lmol/L tamoxifen. Th