(i9) United States
`(12) Patent Application Publication
`Chegini et al.
`
`US20080300147A1
`
`(io) Pub. No.: US 2008/0300147 Al
`(43) Pub. Date: Dec. 4, 2008
`
`(54) DETECTION AND TREATMENT OF
`FIBROTIC DISORDERS
`
`(76) Inventors:
`
`Nasser Chegini, Gainesville, FL
`(US); Xiaoping Luo, Gainesville,
`FL (US); Li Ding, Gainesville, FL
`(US); R. Stan Williams,
`Gainesville, FL (US)
`
`Correspondence Address:
`SALIWANCHIK LLOYD & SALIWANCHIK
`A PROFESSIONAL ASSOCIATION
`PO BOX 142950
`GAINESVILLE, FL 32614-2950 (US)
`
`(21) Appl.No.:
`
`10/590,675
`
`(22) PCX Filed:
`
`Mar. 28, 2005
`
`(86) PCX No.:
`
`PCT/US05/10257
`
`§ 371 (c)(1),
`(2), (4) Date:
`
`Oct. 18, 2007
`
`Related U.S. Application Data
`
`(60) Provisional application No. 60/556,546, filed on Mar.
`26, 2004, provisional application No. 60/620,444,
`filed on Oct. 19, 2004, provisional application No.
`60/636,240, filed on Dec. 15, 2004.
`
`Publication Classification
`
`(51) Int. CI.
`C40B 40/06
`C12Q 1/68
`
`(52) U.S. CI
`
`(2006.01)
`(2006.01)
`
`506/16; 435/6
`
`(57)
`
`ABSTRACT
`
`Xhe present invention provides a method for detecting a
`fibrotic disorder in a subject by: (a) providing a biological
`sample obtained from the subject (such as endometrium,
`peritoneal fluid, and/or smooth muscle cells); (b) analyzing
`the expression of at least one gene that is differentially
`expressed in the fibrotic disorder of interest; and (c) correlat-
`ing the expression of the gene(s) with the presence or absence
`of the fibrotic disorder in the subject. Xhe present invention
`also provides a method and compositions for modulating the
`expression of genes that are differentially expressed in
`fibrotic tissues, compared to normal tissues. Restoration of
`gene expression to levels associated with normal tissue is
`expected to ameliorate at least some of the symptoms of the
`fibrotic disorder. Xhis method includes the step of contacting
`the tissue with an agent that modulates expression of one or
`more differentially expressed genes in the tissue. Xhe present
`invention also includes arrays, such as microfluidic cards, for
`detecting differential gene expression in samples of fibrotic
`tissue.
`
`Lassen - Exhibit 1065, p. 1
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 1 of 53 US 2008/0300147 Al
`
`-BDNF
`
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`
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`
`Lassen - Exhibit 1065, p. 2
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 2 of 53 US 2008/0300147 Al
`
`•—cAMP-
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`Lassen - Exhibit 1065, p. 3
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 3 of 53 US 2008/0300147 Al
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`-•—BHLHB2
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`
`Lassen - Exhibit 1065, p. 4
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 4 of 53 US 2008/0300147 Al
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`c
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`
`Lassen - Exhibit 1065, p. 5
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 5 of 53 US 2008/0300147 Al
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`GnRHa Treatment (hrs)
`
`FIG. II
`
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`GnRHa Treatment (hrs)
`
`FIG. U
`
`Lassen - Exhibit 1065, p. 6
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 6 of 53 US 2008/0300147 Al
`
`FIG. 2A
`
`FIG. 2B
`
`FIG. 2C
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`Lassen - Exhibit 1065, p. 7
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 7 of 53 US 2008/0300147 Al
`
`1.5
`
`TGIF
`
`-+—Realtime PCR
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`
`FIG. 2D
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`
`Lassen - Exhibit 1065, p. 8
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 8 of 53 US 2008/0300147 Al
`
`FIG. 2G
`
`FIG. 2H
`
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`Lassen - Exhibit 1065, p. 9
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 9 of 53 US 2008/0300147 Al
`
`1.5 i
`
`c
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`GnRHa Treatment (hrs)
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`
`FIG. 3B
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`GnRHa Treatment (hrs)
`
`Lassen - Exhibit 1065, p. 10
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 10 of 53 US 2008/0300147 Al
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`c
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`Lassen - Exhibit 1065, p. 11
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 11 of 53 US 2008/0300147 Al
`
`FIG. 3F
`
`FIG. 3G
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`at «
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`Lassen - Exhibit 1065, p. 12
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 12 of 53 US 2008/0300147 Al
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`FIG. 31
`
`FIG. 3J
`
`FIG. 3K
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`Lassen - Exhibit 1065, p. 13
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 13 of 53 US 2008/0300147 Al
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`MSMC
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`FIG. 3L
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`FIG. 3M
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`Lassen - Exhibit 1065, p. 14
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`

`

`Patent Application Publication Dec. 4,2008 Sheet 14 of 53 US 2008/0300147 Al
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`c
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`FIG. 3Q
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`Lassen - Exhibit 1065, p. 15
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 15 of 53 US 2008/0300147 Al
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`MSMC
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`FIG. 3R
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`12
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`FIG. 3S
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`Lassen - Exhibit 1065, p. 16
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 16 of 53 US 2008/0300147 Al
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`Patent Application Publication Dec. 4, 2008 Sheet 17 of 53 US 2008/0300147 Al
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`Patent Application Publication Dec. 4, 2008 Sheet 18 of 53 US 2008/0300147 Al
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`Patent Application Publication Dec. 4, 2008 Sheet 19 of 53 US 2008/0300147 Al
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`Lassen - Exhibit 1065, p. 20
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`Patent Application Publication Dec. 4, 2008 Sheet 20 of 53 US 2008/0300147 Al
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`Patent Application Publication Dec. 4, 2008 Sheet 21 of 53 US 2008/0300147 Al
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`EGR3
`
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`
`Ctrl 2 6 12
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`FIG. 5A
`
`14
`
`12
`
`10
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`FIG. 5B
`
`Lassen - Exhibit 1065, p. 22
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 22 of 53 US 2008/0300147 Al
`
`-•— BM P-6
`
`-•—CAR
`
`EPHA2
`
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`
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`
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`
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`
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`
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`
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`
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`
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`
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`
`FIG. 5C
`
`Ctrl 2 6 12
`TGF-p Treatment (hrs)
`
`FIG. 5D
`
`Lassen - Exhibit 1065, p. 23
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 23 of 53 US 2008/0300147 Al
`
`-•—GEM
`
`-•—LIMK2
`
`MAP2K3
`
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`
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`
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`
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`
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`
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`
` S100A5
`
`SM AD 6
`
`SMAD 7
`
`Ctrl 2 6 12
`TGF-0 Treatment (hrs)
`
`FIG. 5E
`
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`
`TGF-p Treatment (hrs)
`
`FIG. 5F
`
`Lassen - Exhibit 1065, p. 24
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 24 of 53 US 2008/0300147 Al
`
`1.5
`
`1
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`FIG. 5G
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`
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`TGF-p Treatment (hrs)
`
`FIG. 5H
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`-•—ADCY9
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`
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`
`——ROR1
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`——SM AD3
`
`Lassen - Exhibit 1065, p. 25
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 25 of 53 US 2008/0300147 Al
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`c
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`TGF-0 Treatment (hrs)
`
`FIG. 51
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`Ctrl 2 6 12
`
`TGF-p Treatment (hrs)
`
`FIG. 5J
`
`Lassen - Exhibit 1065, p. 26
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 26 of 53 US 2008/0300147 Al
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`10
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`FIG. 5K
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`
`FIG. 5L
`
`Lassen - Exhibit 1065, p. 27
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 27 of 53 US 2008/0300147 Al
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`c
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`FIG. 5M
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`
`FIG. 5N
`
`Lassen - Exhibit 1065, p. 28
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 28 of 53 US 2008/0300147 Al
`
`FIG. 6A
`
`FIG. 6B
`
`FIG. 6C
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`Ctrl 2 6
`
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`
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`
`Lassen - Exhibit 1065, p. 29
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 29 of 53 US 2008/0300147 Al
`
`MSMC
`
`EGR3
`
`RealTimePCR
`-Microarray
`
`Ctrl 2 6
`
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`
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`LSMC
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`MSMC
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`FIG. 6F
`
`Ctrl 2 6
`
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`
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`
`Lassen - Exhibit 1065, p. 30
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 30 of 53 US 2008/0300147 Al
`
`TGIF
`
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`
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`FIG. 6H
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`FIG. 61
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`1
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`
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`
`MSMC
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`
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`Ctrl 2 6
`
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`LSMC
`
`1.5
`
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`
`Ctrl
`
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`
`12
`
`TGF-p Treatment (hrs)
`
`Lassen - Exhibit 1065, p. 31
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 31 of 53 US 2008/0300147 Al
`
`1.5
`
`CITED2
`
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`
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`
`FIG. 6J
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`FIG. 6K
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`
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`
`300
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`FIG. 6L
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`
`12
`
`Lassen - Exhibit 1065, p. 32
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 32 of 53 US 2008/0300147 Al
`
`FIG. 6M
`
`FIG. 6N
`
`FIG. 60
`
`LSMC
`
`RealTimePCR
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`
`Ctrl 2 6 12
`
`TGF-p Treatment (hrs)
`
`MSMC
`
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`Ctrl 2 6
`
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`
`TGF-p Treatment (hrs)
`
`LSMC
`
`p57
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`1.5
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`Lassen - Exhibit 1065, p. 33
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 33 of 53 US 2008/0300147 Al
`
`FIG. 6P
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`
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`
`LSMC
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`Ctrl 2 6
`
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`
`1.5
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`
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`
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`-Microarray
`
`Ctrl 2 6
`
`12
`
`TGF-p Treatment (hrs)
`
`Lassen - Exhibit 1065, p. 34
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 34 of 53 US 2008/0300147 Al
`
`-•—Runxl-Realtime PCR
`-•— Runx1-M icroarray
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`
`FIG. 7A
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`o
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`1.5
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`FIG. 7C
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`
`-•— Runxl-RealtimePCR
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`
`MSMC
`
`Ctrl 2 6 12
`
`GnRHa Treatment (hrs)
`
`Lassen - Exhibit 1065, p. 35
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 35 of 53 US 2008/0300147 Al
`
`FIG. 7D
`
`FIG. 7E
`
`-•— Runxl-Realtime PCR
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`
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`
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`
`Lassen - Exhibit 1065, p. 36
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 36 of 53 US 2008/0300147 Al
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`Lassen - Exhibit 1065, p. 37
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`Patent Application Publication Dec. 4, 2008 Sheet 37 of 53 US 2008/0300147 Al
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`Lassen - Exhibit 1065, p. 38
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`

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`Patent Application Publication Dec. 4, 2008 Sheet 38 of 53 US 2008/0300147 Al
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`Lassen - Exhibit 1065, p. 39
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 39 of 53 US 2008/0300147 Al
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`FIG. 10
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`Lassen - Exhibit 1065, p. 40
`
`

`

`Patent Application Publication Dec. 4, 2008 Sheet 40 of 53 US 2008/0300147 Al
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`Lassen - Exhibit 1065, p. 41
`
`

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`Patent Application Publication Dec. 4, 2008 Sheet 41 of 53 US 2008/0300147 Al
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`Lassen - Exhibit 1065, p. 42
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`Patent Application Publication Dec. 4, 2008 Sheet 42 of 53 US 2008/0300147 Al
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`Lassen - Exhibit 1065, p. 43
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`Patent Application Publication Dec. 4, 2008 Sheet 43 of 53 US 2008/0300147 Al
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`Lassen - Exhibit 1065, p. 44
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`Patent Application Publication Dec. 4, 2008 Sheet 44 of 53 US 2008/0300147 Al
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`Lassen - Exhibit 1065, p. 45
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`Patent Application Publication Dec. 4, 2008 Sheet 45 of 53 US 2008/0300147 Al
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`

`

`US 2008/0300147 Al
`
`Dec. 4, 2008
`
`DETECTION AND TREATMENT OF
`FIBROTIC DISORDERS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`[0001] The present application claims the benefit of U.S.
`Application Ser. Nos. 60/556,546, filed Mar. 26, 2004,
`60/620,444, filed Oct. 19, 2004, and 60/636,240, filed Dec.
`15, 2004, each of which is hereby incorporated by reference
`herein in its entirety, including any figures, tables, nucleic
`acid sequences, amino acid sequences, and drawings.
`
`GOVERNMENT SUPPORT
`
`[0002] The subject invention was made with government
`support under a research project supported by the National
`Institutes of Health Grant No. HD37432.
`[0003] The Sequence Listing for this application is four
`compact discs labeled "Copy 1", "Copy 2", "Copy 3", and
`"CRF". Each copy contains only one file named "03-28-05.
`ST25.txt" which was created on Mar. 28, 2005. The file is
`9,994 KB. The entire contents of each of the computer discs
`are incorporated herein by reference in their entireties.
`
`BACKGROUND OF INVENTION
`
`[0004] Leiomyomas are benign uterine smooth muscle
`tumors, accounting for more than 30% of hysterectomies
`performed in the United States annually. Leiomyomas consist
`mainly of smooth muscle cells of myometrial origin and a
`network of connective tissue (Anderson, Semin. Reprod.
`Endocrinol, 1996, 14:269-282; Chegini, Cytokines and
`Reproduction, 1999, 133-162).
`[0005] Abnormal vaginal bleeding, pelvic pain and pelvic
`masses are among the major symptoms associated with lei-
`omyomas. Leiomyomas are considered to originate from cel-
`lular transformation of myometrial smooth muscle cells and/
`or connective tissue fibroblasts during the reproductive years.
`The identity of factors that initiate such cellular transforma-
`tion is not known; however, ovarian steroids are essential for
`leiomyoma growth, and GnRH anolog (GnRHa) therapy, cre-
`ating a hypoestrogenic condition, is often used for their medi-
`cal management (Chegini, N "Implication of growth factor
`and cytokine networks in leiomyomas" In Cytokines in
`human reproduction. J. Hill ed. New York, Wiley & Sons
`Publisher, 2000, 133-162; Maruo, T et al. Hum Reprod
`Update, 2004, 10:207-20; Takeuchi, H et al.JObstet Gynae-
`col Res, 2000,26:325-331; Steinauer, J et al. Obstet Gynecol,
`2004, 103:1331-6; Palomba, S et al. Hum Reprod, 2002,
`17:3213-3219; DeManno, D et al. Steroids, 2003, 68:1019-
`32; Carr, B R et al. J Clin Endocrinol Metab, 1993, 76:1217-
`1223).
`[0006] Hypoestrogenic conditions created by GnRHa
`therapy affect both leiomyoma and myometrium; however,
`clinical observations indicate a difference in their response to
`changes in the hormonal environment (Carr, B R et al. J Clin
`Endocrinol Metab, 1993, 76:1217-1223). In addition to
`GnRHa therapy, clinical and preclinical assessments of selec-
`tive estrogen and progesterone receptor modulators, either
`alone or in combination with GnRHa therapy, have shown
`efficacy in leiomyoma regression (Steinauer, J et al. Obstet
`Gynecol, 2004, 103:1331-6; Palomba, S et al. Hum Reprod,
`2002, 17:3213-3219; DeManno, D et al. Steroids, 2003,
`68:1019-32).
`
`[0007] GnRHa-induced leiomyoma regression is accompa-
`nied by alterations in uterine arteriole size, blood flow, and
`cellular content as well as changes in the expression of several
`growth factors, cytokines, extracellular matrix, proteases, and
`protease inhibitors (reviewed in Chegini, Cytokines in
`Human Reproduction, 2000, 133-162; Nowak, Bailliere Best
`PractRes. Clin Obstet. Gynaecol, 1999, 13:223-238). Dif-
`ferential expression and autocrine/paracrine action of many
`of these molecules are considered to play a central role in
`leiomyoma growth and GnRHa-induced regression (Chegini,
`Cytokines in Human Reproduction, 2000, 133-162; Nowak,
`Bailliere Best Pract Res. Clin Obstet. Gynaecol, 1999,
`13:223-238).
`[0008] At the cellular level, a combination of mitotic activ-
`ity, cellular hypertrophy, and accumulation of extracellular
`matrix (ECM) are considered to participate in leiomyoma
`growth (Anderson, Semin. Reprod. Endocrinol, 1996,
`14:269-282; Chegini, Cytokines and Reproduction, 1999,
`133-162; Stewart et al., J. Clin. Endocrinal Metab, 1994,
`79:900-906; Wolanska et al., Mol Cell Biochem., 1998, 189:
`145-152). Compared to myometrium, leiomyomas are
`reported to overexpress estrogen and progesterone receptors,
`and GnRHa therapy lowers their content in both tissues
`(Stewart et al., Semin, Reprod. Endocrinol., 1995, 10:344-
`357; Englund et al., J. Clin. Endocrinol Metab, 1998,
`83:4092-4092). Clinical and basic science research shows
`that GnRHa acting through suppression of the pituitary-go-
`nadal axis cause leiomyoma to regress by affecting uterine
`arteriole size, blood flow at the tumor level. But its effect at
`cellular and molecular levels in leiomyoma has not been
`investigated.
`[0009] With respect to the leiomyoma molecular environ-
`ment, several genome-wide allel-typing studies have evalu-
`ated the association between genomic instability and the
`pathogenesis of leiomyoma (for review; Ligon, A H and
`Morton, CCHumReprod Update, 2001,7:8-14). These stud-
`ies have led to the identification of several candidate genes,
`however in the majority of cases evidence of genomic insta-
`bility is either lacking or inconsistent (Ligon, A H and Mor-
`ton, C C Hum Reprod Update, 2001, 7:8-14), implying the
`existence of unrecognized pathways that can lead to the
`development of leiomyoma. Further studies have provided
`support for various autocrine/paracrine regulators in the
`pathogenesis of leiomyoma including local estrogen produc-
`tion, growth factors, cytokines, chemokines and their recep-
`tors, whose expression are regulated by ovarian steroids
`(Chegini, N "Implication of growth factor and cytokine net-
`works in leiomyomas" In Cytokines in human reproduction.
`J. Hill ed. New York, Wiley & Sons Publisher, 2000,133-162;
`Maruo, T et al. Hum Reprod Update, 2004,10:207-20). These
`studies in many instances demonstrated altered expression of
`these factors and/or their receptors in leiomyoma compared
`to normal myometrium. In recent years cDNA microarray has
`been utilized as a high throughput method to identify a large
`number of differentially expressed and regulated genes in
`various tissues and cells. Using this approach, several recent
`studies have further assisted in fingerprinting the gene expres-
`sion profile of leiomyoma and myometrium during the men-
`strual cycle (Tsibris, J C M et al. Fertil Steril, 2002, 78:114-
`121; Chegini, N et al. JSoc Gynecol Investig, 2003, 10:161-
`71; Wang, Hetal.FertilSteril, 2003, 80:266-76; Weston, Get
`al. Mol Hum Reprod, 2003, 9:541-9; Ahn, W S et al. IntJExp
`Pathol, 2003, 84:267-79; Quade, B J et al. Genes Chromo-
`somes Cancer, 2004, 40:97-108). However, only the expres-
`
`Lassen - Exhibit 1065, p. 55
`
`

`

`US 2008/0300147 Al
`
`Dec. 4, 2008
`
`sion of a few of these newly identified genes has been vali-
`dated, and their regulation and correlation with pathogenesis
`of leiomyoma remains to be investigated.
`[0010] With respect to GnRHa therapeutic action, it is tra-
`di