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`111111111111110111111010 1111111111111Ji111101°1!141141911)1111111111111111111111110111111
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`(19) United States
`(12) Patent Application Publication
`Prakash et al.
`
`(10) Pub. No.: US 2010/0028449 Al
`Feb. 4, 2010
`(43) Pub. Date:
`
`(54) FERMENTED MILK PRODUCT AND USE
`THEREOF
`
`(76)
`
`Inventors:
`
`Satya Prakash, Brossard (CA);
`Aleksandra Malgorzata
`Urbanska, Montreal (CA)
`
`Correspondence Address:
`BERESKIN AND PARR LLP/S.E.N.C.R.L., s.r.l.
`40 KING STREET WEST, BOX 401
`TORONTO, ON M5H 3Y2 (CA)
`
`(21)
`
`Appl. No.:
`
`12/303,758
`
`(22)
`
`PCT Filed:
`
`Jun. 6, 2007
`
`(86)
`
`PCT No.:
`
`PCT/CA2007/001010
`
`§ 371 (c)(1),
`(2), (4) Date:
`
`Apr. 1, 2009
`
`Related U.S. Application Data
`
`(60) Provisional application No. 60/804,008, filed on Jun.
`6, 2006.
`
`Publication Classification
`
`(51) Int. Cl.
`A61K 9/14
`A61K 35/74
`A61P 29/00
`A61P 13/00
`A61P 35/04
`A61P 1/00
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`
`(52) U.S. Cl.
`
`424/490; 424/93.4; 424/93.45
`
`(57)
`
`ABSTRACT
`
`The present invention relates to an oral formulation compris-
`ing a microcapsule containing bacteria and a fermented milk
`carrier. There is also provided a method of medical treatment
`of an inflammatory gastrointestinal disease or disorder in a
`subject in need thereof, comprising detecting the presence of
`inflammatory gastrointestinal disease or disorder in the sub-
`ject, wherein if inflammatory gastrointestinal disease or dis-
`order is detected, then administering the formulation of the
`present invention to the subject.
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`'f.;
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`*St
`x
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`Figure. 2
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`F isiure
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`Feb. 4, 2010 Sheet 2 of 22
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`SGF 3h
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`SGF3h+SIF3h
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`SGF3h+SIF12h
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`SGF3h+SIF24h
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`Fluid and Incubation Time
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`Figure 4
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`APA microcapsules undamaged (%)
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`Figure 5
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`Feb. 4, 2010 Sheet 4 of 22
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`A)
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`B)
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`12
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`10 •
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`6,
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`4 •
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`2.
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`0
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`3
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`a.
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`2.5
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`1—e— L. eciaophlius cells)
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`0.5
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`1
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`2.5
`2
`1.5
`Storage Time (weeks)
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`3
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`3.5
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`% live L. acidophilus cells
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`g
`I t r -
`A "5 1.5 ..
`J 4- es o o
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`10 10
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`2
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`1
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`0.5
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`0'
`0
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`0:5
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`1.5
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`2.5
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`3
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`3.5
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`Storage lime (weeks)
`Figure 6
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`Feb. 4, 2010 Sheet 5 of 22
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`10 7
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`p1-12 #
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`pH3 -* pH4
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`0-16
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`pH8
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`a 6
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`= 4
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`0
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`03
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`3
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`2
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`10
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`7
`e
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`3 •
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`2 •
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`1•
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`0
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`2
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`500
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`1000
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`1500
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`2000
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`2500
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`3000
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`3500
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`4000
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`4500
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`Time (mins)
`Figure 7
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`--o- L. acidophilus APA in SGF + 2% yogurt
`2% yogurt in SGF
`-a- L. acidophilus APA in SGF
`-411- control - SGF
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` I- I
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`10
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`20
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`30
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`40
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`50
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`60
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`70
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`80
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`90
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`100
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`110
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`120
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`Time (mins)
`Figure 8
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`Feb. 4, 2010 Sheet 6 of 22
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`Figure 9
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`Figure 10
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`f.:
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`FigUte 11
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`2
`3
`Storage time (weeks)
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`Figure 12
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`4
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`•
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`-r- L. acidophilus AC in SGF + 2% M.F. yogurt
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`is L. acidophilus AC in SGF
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`2% M.F. yogurt in SGF
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`Control - SGF
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`20
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`40
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`60
`Time (mins)
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`80
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`100
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`120
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`Figure 13
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`120 -
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`100 -
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`80 -
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`60 -
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`40 -
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`20 -
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`AC microencapsules undamaged %
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`L. acidophilus cells log (cfu/g)
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`4
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`1•
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`L. acidophilus AC in SIF + 2% M.F. yogurt
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`a L. acidophilus AC in SIF
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`2% M.F. yogurt in SIF
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`Control - SIF
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`20 40
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`60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360
`Time (mins)
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`Figure 14
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`-j 2_
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`1
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`o-
`0
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`12 -
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`10
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`E
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`—4— LA AC (0.5%4 0) in 2% yogurt at 100rpm/4C
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`5 LA AC (0.5%/1 0) in 0.85% P.S. at 100rpm/4C
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`2% yogurt at 100rpm/4C
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`2% yogurt at 4C
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`0
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`0.5
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`1
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`1.5
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`2
`Time (weeks)
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`2.5
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`3
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`3.5
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`Figure 15
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`LA AC (0.5%/10) in 0.85% PS. at 4C
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`* LA AC (0.25%/10) in 0.85% P.S. at 4C
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`LA AC (0.1%710) in 0,85% P.S. at 4C
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`L. aciclophdus in 0.85% at 4C
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`A
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`-r
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`12
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`10 -
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`L. acidophilus cells log(cfu/g)
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`0.5
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`1
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`1.5
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`2
`2.5
`Time (weeks)
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`Figure 16
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`3
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`3.5
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`4
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`4.5
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`Patent Application Publication
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`Feb. 4, 2010 Sheet 10 of 22
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`• pH2
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`a pH3
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`pH4
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`pH6
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`phIB
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`500
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`1000
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`1500
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`•
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`2500
`2000
`Time (mins)
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`Figure 17
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`3000
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`3500
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`4000
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`4500
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`pH3
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`pH4
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`pH6
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`pF18
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`500
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`1000
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`1500
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`2000
`2500
`Time (mins)
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`Figure 18
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`3000
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`3500
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`4000
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`4500
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`Iglus cells Iog(cfulml)
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`acidophilus cells log(cfu/m1)
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`Patent Application Publication
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`a)
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`Figure 19
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`22
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`21
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`20
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`-.- Control - empty APA microcapsules + 085% saline
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`- a- Treatment 1 - L. acidophilus in APA microcapsules + 2% M.F. yogurt
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`Treatment 2 - L. acidophilus in APA microcapsules + 0.85% saline
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`9
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`10
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`11
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`12
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`13
`14
`15
`Animal age (weeks)
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`Figure 20
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`16
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`17
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`18
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`19
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`20
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`25
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`20
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`0
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`-b-- Control - empty APA microcapsules + 0.85% saline
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`- Treatment 1 - L. acidophilus in APA microcapsules + 2% M.F. yogurt
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`Treatment 2 - L. acidophilus in APA microcapsules + 0.85% saline
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`9
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`10
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`11
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`12
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`13
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`14
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`15
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`16
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`17
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`18
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`19
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`20
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`'
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`Animal Age (weeks)
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`Figure 21
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`Q. 350 -
`E
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`To
`6.) w
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`300 -
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`250 -
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`150 -
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`a) 100
`Ed
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`50 -
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`a>
`LL
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`D Control - empty APA microcapsules + 0.85% same
`• Treatment i - L. ac iclaphlus in APA microcapsules + 2% M.F. yogurt
`Treatment 2 - L. ecidophius in APA microcapsules + 0.85% saline
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`4
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`6
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`8
`9
`Treatment lime (weeks)
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`10
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`11
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`12
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`Figure 22
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`Feb. 4, 2010 Sheet 15 of 22
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`1.2 -
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`a)
`4:4, 0.8 -
`Co
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`E 0.6 -
`0
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`• 1:1 Co 0.4
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`0
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`E 0.2 -
`=
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`0
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`Control
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`Treatment 1
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`Treatment 2
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`Figure 23 A
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`z
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`to
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`0.0
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`75
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`to
`aL i =
`. w
`c
`co En
`c eo
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`—
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`c
`2
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`cr)
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`0.6 -
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`0.5 -
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`0.4 -
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`0.3
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`0.2
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`.1
`o
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`0
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`Control
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`Treatment 1
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`Treatment 2
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`(b)
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`Figure 23 B
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`I
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`Control
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`Treatment 1
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`Treatment 2
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`Figure 24 A
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`35 -
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`30
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`25 -
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`20 -
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`15
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`10 -
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`5
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`0
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`Number of adenoma in small intestine
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`z
`N
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`co
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`0
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`Tu.
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`a)
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`w
`c
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`10 -
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`9 -
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`8-
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`7
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`6-
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`5 -
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`a
`a) cl)
`c
`c —
`Tts E 4 -
`c
`c 3 -
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`rn
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`0
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`Control
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`Treatment 1
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`Treatment 2
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`Figure 24 B
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`Feb. 4, 2010 Sheet 19 of 22
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`Figure 25 A
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`Figure 25 B
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`Patent Application Publication
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`Figure 25 C
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`Patent Application Publication
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`Figure 25 D
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`US 2010/0028449 Al
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`Feb. 4, 2010
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`1
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`FERMENTED MILK PRODUCT AND USE
`THEREOF
`
`BACKGROUND OF THE INVENTION
`
`(a) Field of the Invention
`[0001]
`[0002] The present invention relates to a novel method for
`encapsulating live bacteria; an encapsulated live bacteria; an
`oral formulation for probiotic therapy and method of treat-
`ment thereof
`[0003]
`(b) Description of Prior Art
`[0004] A well balanced gut microflora is known to contrib-
`ute to the maintenance of a healthy intestinal mucosa. The
`density of gastrointestinal (GI) microflora increases from the
`stomach to the large intestine reaching 1010-1012 cfu/g in the
`colon. One of the most important groups of bacteria for intes-
`tinal health is lactic acid bacteria (LAB) (Adolfsson, 0. et al.,
`(2004), American Journal of Clinical Nutrition 80:245-256).
`LAB are considered probiotic; live microorganisms that
`remain in the GI tract to benefit the host (Adolfsson, 0. et al.,
`(2004), American Journal of Clinical Nutrition 80:245-256;
`Roberfroid, 2000). Although their mechanism of action is not
`known, it is believed that LAB, like other probiotic microor-
`ganisms, compete and suppress the growth of undesirable
`microorganisms in the colon and intestines leading to the
`stabilization of the digestive system (Adhikari, K. et al.,
`(2000), Journal of Dairy Science 83:1946-1951).
`[0005] There are several reports that probiotic yogurt has
`significant clinical benefits (Donaldson, M. S., (2004), Nutri-
`tion Journal 3:19). It is estimated that a decrease of at least
`60-70 percent in breast, colorectal, and prostate cancers and
`40-50 percent in lung cancer would occur when a diet is
`complied with (according to the anti-cancer diet guidelines)
`which includes probiotic yogurt products. In order to be
`labeled probiotic, yogurt must contain a cell load of at least
`107 cfu/g at the time of manufacture (Chandan, R. C. et al.,
`(1993), Ed Hui, Y H, VCH Publishers, Inc, New York 1-56).
`However, it has been found that this level of live bacterial cells
`in probiotic yogurt is not adequate to provide the maximum
`benefit, especially considering that many bacteria do not sur-
`vive storage (Donaldson, M. S., (2004), Nutrition Journal
`3:19), (Dave, R. I. et al., (1998), Journal of Dairy Science
`81:2804-2816), (Shah, N. P., et al., (1995), International
`Dairy Journal 5:515-521) or passage through the stomach.
`[0006] Therefore several attempts have been made to
`deliver a greater number of live bacterial cells. One strategy to
`deliver more live bacteria to the intestines is bioencapsula-
`tion. This technology has developed over the last 20 years,
`(Orive, G. et al., (2003b), International Journal of Pharma-
`ceutics 259:57-68; Prakash, S. et al., (1996b), Nature Medi-
`cine 2:883-887; Sun et al., 1987, Chang, T. M. S. et al.,
`(1998), Molecular Medicine Today 4:221-227; Prakash, S. et
`al., (1998), Artificial Cells Blood Substitutes and Immobili-
`zation Biotechnology 26:35-51; Jones, M. L. et al, (2004),
`Journal of Biomedicine and Biotechnology 1:61-69). How-
`ever, the use of this technology in probiotic yogurt formula-
`tion containing live bacteria has not yet been investigated.
`[0007] The transit of free bacteria through the gastrointes-
`tinal tract is often problematic because of low pH conditions,
`enzymatic digestion and very few probiotic cells finally reach
`their targeted site. The challenge here consist in producing a
`support allowing successful storage and transport of bacteria
`which could, if added to one's diet, constitute an alternative
`
`but effective treatment to various medical issues caused by an
`imbalance between desirable and undesirable microorgan-
`isms in the GI microflora.
`[0008] Therefore, it would be highly desirable to be pro-
`vided with a novel method to encapsulate live bacteria and
`which would be safe for oral administration as a probiotic
`formulation to improve gastrointestinal microflora's condi-
`tion.
`
`SUMMARY OF THE INVENTION
`
`[0009] Herein we report the potential of microcapsules as a
`platform for probiotic live bacterial cell oral delivery. In vitro
`data suggests that capsules containing live Lactobacillus aci-
`dophilus cells showed superior mechanical stability and dem-
`onstrated significantly higher bacterial cell survivals com-
`pared to free bacterial cells over a period of 4 weeks. Using an
`in vitro simulation human stomach model, we monitored the
`survival rates of free and alginate-poly-L-lysine (PLL)-algi-
`nate (APA) membrane microencapsulated L. acidophilus
`cells at 37° C. over two hours, the approximate time it takes
`food to pass through the stomach. Results show that 7.10 log
`cfu/g of microencapsulated L. acidophilus cells were found
`alive compared to only 5.51 log cfu/g of free L. acidophilus
`cells in the presence of simulated gastric fluid (SGF) and 2%
`milk fat M.F. yogurt. In addition, data shows that only 6.66
`log cfu/g of microencapsulated L. acidophilus cells survived
`in SGF fluid in the absence of yogurt. The high survival rates
`of encapsulated L. acidophilus cells strongly suggest the use
`of microcapsules and yogurt for probiotic bacterial cell deliv-
`ery.
`In accordance with the present invention there is
`[0010]
`provided an oral formulation to improve a patient gastrointes-
`tinal microflora, which comprises coated microcapsule con-
`taining bacteria in suspension in a probiotic acceptable car-
`rier, wherein said coated microcapsule comprises an
`encapsulated bacteria in a semipermeable capsule coated
`with poly-L-Lysine (PLL) and alginate and is also resistant in
`gastrointestinal conditions.
`[0011] The bacteria may be chosen from Lactobacilli cells,
`Bifidobacterium cells, Lactobacillus plantarum 80, Lactoba-
`cllus delbrueckii subsp. Lactis, Lactobacillus Rhamnosus,
`Lactobacillus, more particularly from Lactobacillus acido-
`philus, Lactobacillus casei, Bifidobacterium, Lactobacillus
`plantarum 80, Lactobacillus delbrueckii subsp. Lactis, Lac-
`tobacillus Rhamnosus, Lactobacillus GG.
`[0012]
`In accordance with a preferred embodiment of the
`oral formulation of the present invention, the bacteria is live.
`[0013]
`In accordance with another embodiment of the oral
`formulation of the present invention, the microcapsule is
`made of a material chosen from alginate-poly-L-Lysine-algi-
`nate (APA), alginate-chitosan (AC), alginate pectinate polyl-
`ysine pectinate alginate (APPPA), alginate polyethylene gly-
`col alginate (APEGA), alginate chitosan genipin alginate
`(ACGA).
`[0014]
`In accordance with another embodiment of the oral
`formulation of the present invention, the probiotic acceptable
`carrier is at a substantially basic pH to further protect from
`gastrointestinal fluids.
`[0015]
`In accordance with another embodiment of the oral
`formulation of the present invention, the probiotic acceptable
`carrier is chosen from a food supplement or food.
`[0016]
`In accordance with another embodiment of the oral
`formulation of the present invention, the food carrier is cho-
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`sen from yogurt, ice cream, cheese, chocolate, nutritional
`bars, cereal, milk, infant formulation, fruit juices.
`[0017]
`In accordance with the present invention there is
`provided a method for probiotic therapy of a patient for
`improving gastrointestinal microflora, which comprises
`orally administering the oral formulation of the present inven-
`tion.
`In accordance with another embodiment of the
`[0018]
`method of the present invention, the patient is suffering from
`a disease or disorder chosen from breast cancer, colorectal
`cancer, prostate cancer, lung cancer, urinary tract infections,
`yeast infections and inflammatory bowel diseases (IBD),
`Crone's diseases (CD).
`[0019]
`In accordance with another embodiment of the
`present invention, there is provided an oral formulation com-
`prising:
`[0020] a microcapsule containing bacteria; and
`[0021] a fermented milk carrier.
`[0022] The microcapsule may comprise a semipermeable
`capsule comprising poly-L-Lysine (PLL) and alginate and
`wherein the microcapsule is resistant to degradation in gas-
`trointestinal conditions.
`[0023] The bacteria may be Lactobacilli bacteria or Bifido-
`bacterium bacteria. The Lactobacilli bacteria are selected
`from the group consisting of Lactobacillus acidophilus, Lac-
`tobacillus casei; Lactobacillus plantarum 80, Lactobacillus
`delbrueckii subsp. Lactis, Lactobacillus Rhamnosus,
`[0024]
`In accordance with another embodiment of the
`present invention, there is provided a method for treatment or
`prevention of a disease or disorder in a subject in need thereof
`or for nutritional supplementation of a subject, comprising
`orally administering to the subject the oral formulation of the
`present invention.
`[0025]
`In accordance with another embodiment of the
`present invention, there is provided the use of the oral formu-
`lation of the present invention for the preparation of a medi-
`cament for the treatment or prevention of a disease or disorder
`or for the preparation of a nutritional supplement.
`[0026]
`In accordance with another embodiment of the
`present invention, there is provided a fermented milk carrier
`i) for use as a prebiotic carrier in increasing the efficacy of
`microencapsulated bacteria in the treatment of a disease or
`disorder in a subject or ii) for preparation of a medicament for
`the treatment of a disease or disorder in a subject; wherein
`optionally the carrier is used in the oral formulation of the
`present invention.
`[0027] The subject may be a mammal, optionally a human.
`[0028] The disease or disorder includes a gastrointestinal
`disease or disorder.
`[0029] The gastrointestinal disease or disorder includes an
`inflammation gastrointestinal disease or disorder such as
`Inflammatory Bowel Disease (IBD), Crohn's Disease, colitis,
`enteroinvasive colitis, C. d cile colitis, Ulcerative Colitis
`(UC), Inflammatory Bowel Syndrome (IBS), pouchitis,
`diverticulitis, gastroenteritis, colic, appendicitis, appendici-
`tis, ascending colangitis, esophagitis, gastritis, or enteritis.
`[0030] The disease or disorder includes cancer, such as
`breast cancer, colorectal cancer, prostate cancer, lung cancer,
`colon cancer and inflammation-related colon cancer, includ-
`ing adenoma, carcinoma, leiomyosarcoma, carcinoid tumor,
`or squamous cell carcinoma. Lactobacillus GG, Bifidobacte-
`rium infantis, Bifidobacterium breve, Bifidobacterium ion-
`gum, Bifidobacterium bifidum.
`
`[0031] The bacteria may be live.
`[0032] The bacteria may be present in a range from 109 to
`1012 colony forming units (CFU).
`[0033] The microcapsule may comprise a material selected
`from the group consisting of alginate-poly-L-Lysine-alginate
`(APA), alginate-chitosan (AC), alginate pectinate polylysine
`pectinate alginate (APPPA), alginate polyethylene glycol
`alginate (APEGA) and alginate chitosan genipin alginate
`(ACGA).
`[0034] The fermented milk carrier may comprise a basic
`pH buffer and protects the bacteria and/or the microcapsule
`from gastrointestinal fluids. The basic pH buffer may be
`between pH 7-9.
`[0035] The fermented milk carrier may comprise a food
`supplement or food, such as yogurt, cheese, milk, powdered
`milk, kefer or a fermented milk formulation.
`[0036] The yogurt may be selected from the group consist-
`ing of plain yogurt, flavored yogurt, yogurt beverage, Dahi,
`Dadiah, Labneh, Bulgarian Yogurt, Tarator, Cacik, Lassi and
`Kefir.
`[0037] The yogurt may comprise 1-10 grams of microen-
`capsulated bacteria per 100 grams of yogurt, optionally 5-10
`grams of microencapsulated bacteria per 100 grams of
`yogurt, optionally 8-10 grams of microencapsulated bacteria
`per 100 grams of yogurt.
`[0038] The yogurt may comprise 4.2 grams of harvested
`bacteria in 100 mL of 1.65% alginate solution.
`[0039] The oral formulation of the present invention may
`be use in nutritional supplementation of a subject or for use in
`preventing or treating a disease or disorder in a subject.
`[0040] The disease or disorder includes inflammation of
`tissue in bowel, colon, sigmoid colon, rectum, appendix,
`anus, esophagus, stomach, mouth, liver, billiary, tract or pan-
`creas, including inflammation of colon.
`[0041] The inflamed tissue or colon comprises increased
`interleukins and cytokines compared to non-inflamed tissue
`or colon, such as up-regulated inflammatory response and
`markers compared to non-inflamed tissue or colon, such as
`tumor necrosis factor-a[TNF-a], interleukin-1 [IL-1], IL-6,
`IL-12, and y-interferon in macrophages.
`[0042] The disease or disorder includes a urinary tract
`related disease or disorder.
`[0043] The urinary tract related disease or disorder includes
`a urinary tract infection or a yeast infection.
`[0044]
`In accordance with another embodiment of the
`present invention, there is provided a method of medical
`treatment of an inflammatory gastrointestinal disease or dis-
`order in a subject in need thereof, comprising detecting the
`presence of inflammatory gastrointestinal disease or disorder
`in the subject, wherein if inflammatory gastrointestinal dis-
`ease or disorder is detected, then administering the formula-
`tion of any one of claims 1 to 13 to the subject.
`[0045] The detecting step may comprise determining the
`presence of inflammatory gastrointestinal disease or disorder
`in the subject with a biopsy of the subject's tissue or a blood
`test of the subject, such as detection of: elevated C Reactive
`Protein (CRP), increased Erythrocyte Sedimentation Rate
`(ESR), elevated neutrophil count, elevated eosinophil count,
`elevated monocyte count, elevated white blood cell count
`(WBC), elevated immunoglobulin count or elevated IgA,
`compared to a subject not having inflammation.
`[0046]
`In accordance with another embodiment of the
`present invention, there is provided a method of medical
`treatment of inflammation-related colon cancer in a subject in
`
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`need thereof, comprising detecting the presence of inflamma-
`tion-related colon cancer in the subject, wherein if cancer is
`detected, next administering the formulation of the present
`invention.
`[0047] The detecting step may comprise determining the
`presence of cancer in the subject using fecal occult blood
`(FOB), visible protrusion adenomatous polyps from the
`mucosal surface, digital rectal exam, colonoscopy, sigmoi-
`discopy, abdominal series radiograph with contrast, double
`contrast enema abdominal radiograph or abdominal CT scan.
`[0048] The detecting step may comprise determining the
`presence of cancer in the subject with a blood test of the
`subject comprising detection of elevated carcinoembryonic
`antigen (CEA) compared to a subject not having cancer.
`[0049] The detecting step may comprise determining the
`presence of cancer in the subject with a biopsy of the subject's
`tissue or a blood test of the subject, such as detection of:
`elevated C Reactive Protein (CRP), increased Erythrocyte
`Sedimentation Rate (ESR), elevated neutrophil count,
`elevated eosinophil count, elevated monocyte count, elevated
`white blood cell count (WBC), elevated immunoglobulin
`count or elevated IgA, compared to a subject not having
`cancer, such as adenoma or carcinoma.
`[0050]
`In accordance with another embodiment of the
`present invention, there is provided an oral formulation for
`the treatment and/or prevention of a disease and/or disorder,
`which comprises coated microcapsule containing bacteria in
`suspension in a fermented milk probiotic acceptable carrier,
`wherein said coated microcapsule comprises encapsulated
`bacteria in a semipermeable capsule coated with poly-L-
`Lysine (PLL) and alginate and is also resistant in gastrointes-
`tinal conditions.
`[0051] For the purpose of the present invention the follow-
`ing terms are defined below.
`[0052] The expression "mechanically resistant" is referring
`to an intrinsic construction's capacity of a microcapsule
`which allows to maintain its original structure and shape
`against physical and/or mechanical stresses in a particular
`environment.
`[0053] The expression "gastrointestinal conditions" is
`referring to the various mechanical stresses of the gastrointes-
`tinal tract and to the different acidity levels of the gastrointes-
`tinal fluids which ingested substances undergo.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0054] FIG. 1, left, illustrates freshly prepared empty APA
`microcapsules whereas FIG. 1, right, illustrates freshly pre-
`pared APA microcapsules loaded with L. acidophilus cells.
`[0055] FIG. 2 is a photomicrograph of four different stages
`of APA microcapsules. In photomicrograph (a), freshly pre-
`pared empty APA microcapsules are shown whereas in pho-
`tomicrograph (b), they were loaded with L. acidophilus. Pho-
`tomicrograph (c) is an illustration APA microcapsules loaded
`withL. acidophilus cells after 76 hours of incubation in MRS
`broth and 370 rpm in vitro shaking at 37° C.
`[0056] FIG. 3
`illustrates empty APA microcapsules
`exposed to shaking at 150 rpm at 37° C. in three different
`conditions. In (a), the microcapsules were introduced in SGF
`(pH 1.98) for 3 hrs. In b), they were incorporated in SGF (pH
`1.98) for 12 hrs and in (c), they were incorporated in SGF (pH
`1.98) for 3 hrs and in SIF (pH 6.5) for 24 hrs.
`[0057] FIG. 4 is a graph of the mechanical stability of
`empty APA microcapsules at various exposure times in simu-
`
`lated gastric fluid (SGF) (pH 1.98) and simulated intestinal
`fluid (SIF) (pH 6.5) after shaking at 150 rpm at 37° C.
`[0058] FIG. 5 is a photomicrograph of various APA micro-
`capsules loaded with L. acidophilus cells, exposed to
`mechanical shaking of 100 rpm at 4° C. and stored in various
`conditions. InY1) storage was in 2% M.F. yogurt for 1 week.
`In P1), storage was in 0.85% physiological solution for 1
`week. In Y2) storage was in 2% M.F. yogurt for 2 weeks. In
`P2) storage was in 0.85% physiological solution for 2 weeks.
`In Y3) storage was in 2% M.F. yogurt for 3 weeks. In P3),
`storage was in 0.85% physiological solution for 3 weeks. In
`Y4) storage was in 2% M.F. yogurt for 4 weeks and in P4),
`storage was in 0.85% physiological solution for 4 weeks.
`[0059] FIG. 6 A) is a graph of the viability of live L. acido-
`philus cells in 2% M.F. yogurt during 4 weeks of mechanical
`shaking at 100 rpm at 4° C. FIG. 6 B) is a graph illustrating the
`retention capacity of APA microcapsules. The number of
`viable L. acidophilus bacteria in the supernatant of storage
`media gives an indication of how many L. acidophilus bacte-
`ria have leaked from the microcapsules. The APA microcap-
`sules loaded with L. acidophilus cells were stored in 0.85%
`physiological solution for 4 weeks at 4° C. No mechanical
`stress was applied.
`[0060] FIG. 7 is a graph evaluating the survival of APA
`encapsulated L. acidophilus cells in pH 2, 3, 4, 6 and 8 in
`presence of 2% M. F. yogurt at 37° C.
`[0061] FIG. 8 is a graph effectuating a comparison of the
`survival of APA encapsulated and free L. acidophilus cells in
`conditions simulating the stomach supplemented with 2%
`M.F. yogurt at 37° C.
`[0062] FIG. 9 displays photomicrographs of freshly encap-
`sulated empty capsules and capsules loaded withL. acidophi-
`lus cells of 550±26 nm in size and magnification of 2.5x using
`light microscopy. Left: Photomicrograph of freshly prepared
`empty AC microcapsules (size 550±96 nm, magnification:
`2.5x). Right: Photomicrograph of freshly prepared AC micro -
`capsules loaded with L. acidophilus cells.
`[0063] FIG. 10 shows three comparative photomicrographs
`of freshly prepared microcapsules. FIG. 10 (a) Photomicro-
`graph of freshly prepared empty AC microcapsules. (b) Pho-
`tomicrograph of freshly prepared AC microcapsules loaded
`with L. acidophilus. (c) Photomicrograph of AC microcap-
`sules loaded with L. acidophilus cells after 76 hours of incu-
`bation in MRS broth and 150 rpm in-vitro shaking at 37° C.
`(Magnification: 2.5x).
`[0064] FIG. 11 displays three photomicrographs of AC
`microcapsules exposed to simulated gastrointestinal fluid
`(SGF) (pH 1.98) for 3 hours (11a), to SGF for 12 hours (11b)
`and to simulated intestinal fluid (SIF) (pH6.5) for 24 hours.
`FIG. 11. Photomicrographs ofAC microcapsules loaded with
`L. acidophilus cells exposed to shaking at 150 rpm at 37° C.:
`(a) in SGF (pH 1.98) for 3 hrs. (b) in SGF (pH 1.98) for 12 hrs.
`(c) in SGF (pH 1.98) for 3 hrs and in SIF (pH 6.5) for 24 hrs.
`(Magnification: 6.3x).
`[0065] FIG. 12 further demonstrates physical property of
`exposed microcapsules to a combination of simulated fluids.
`Mechanical stability of AC microcapsules loaded with L.
`acidophilus cells at various exposure times in simulated gas-
`tric fluid (SGF) (pH 1.98) and simulated intestinal fluid (SIF)
`(pH 6.5) after shaking at 150 rpm at 37° C.
`[0066] FIG. 13 illustrates the survival of encapsulated bac-
`terial cells in SGF with and without addition of 2% M.F.
`yogurt as well as the survival of free bacteria contained in the
`yogurt. Comparison of the survival of AC (chitosan 10)
`
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`encapsulated and free L. acidophilus cells using Simulated
`Human Intestinal Microbial Ecosystem—conditions simulat-
`ing the stomach supplemented with 2% M.F. yogurt at 37° C.
`[0067] FIG. 14 displays survival of AC encapsulated and
`free bacterial cells obtained by exposure to simulated intes-
`tinal fluid conditions. Comparison of the survival of AC (chi-
`tosan 10) encapsulated and free L. acidophilus cells using
`Simulated Human Intestinal Microbial Ecosystem condi-
`tion simulating the intestines supplemented with 2% M.F.
`yogurt at 37° C.
`survival of AC 10
`[0068] FIG. 15 is a comparative study
`encapsulated L. acidophilus in presence and of 2% M.F.
`yogurt at 4° C. and mechanical shaking of 100 rpm.
`[0069] FIG. 16 illustrates comparative study of microen-
`capsulated L. acidophilus bacterial cells viability in various
`chitosan concentrations and polymers (0.5%/10, 0.25%/10,
`0.1%/10) in 2% M.F. yogurt with free L. acidophilus bacterial
`cells in 0.85% saline during 4 weeks of mechanical shaking at
`100 rpm at 4° C.
`[0070] FIG. 17 Viability of free L. acidophilus cells in 2%
`M.F. plain yogurt in buffers: pH2, pH3, pH4, pH6 and pH8.
`[0071] FIG. 18 Survival of AC encapsulated live L. acido-
`philus cells in buffers: pH2, pH3, pH4, pH6 and pHB supple-
`mented with 2% M.F. plain yogurt.
`[0072] FIG. 19 is a photomicrograph ofAPA microcapsules
`loaded with Lactobacillus acidophilus bacterial cells at 77x
`magnification and (b) at 112x magnification. (size 433
`um±67)
`[0073] FIG. 20 illustrates the effect of the treatment on
`animal body weights in the Min mouse. Data represent the
`mean±SEM per group.
`[0074] FIG. 21 illustrates the changes in the expression
`levels of anti-inflammatory interleukin-6 examined during
`treatment at different time intervals. The data represent the
`mean±SEM of expression levels per group.
`[0075] FIG. 22 illustrates the effect of the treatment on total
`fecal bile acid levels. The data represent the mean±SEM of
`expression levels per group.
`[0076] FIG. 23 illustrates the number of adenoma (a) and
`Gastrointestinal Intraepithelial Neoplasias (b) for three
`groups: Control gavaged empty APA microcapsules+0.
`85% saline, Treatment 1 gavaged L. acidophilus bacterial
`cells in APA microcapsules+2% M.F. yogurt and Treatment
`2 gavaged L. acidophilus bacterial cells in APA microcap-
`sules+0.85% saline found in the large intestines. Data repre-
`sent the mean±SEM per group.
`[0077] FIG. 24 illustrates the number of adenoma (a) and
`Gastrointestinal Intraepithelial Neoplasias (b) for three
`groups: Control gavaged empty APA microcapsules+0.
`85% saline, Treatment 1 gavaged L. acidophilus bacterial
`cells in APA microcapsules+2% M.F. yogurt and Treatment
`2 gavaged L. acidophilus bacterial cells in APA microcap-
`sules+0.85% saline found in the small intestines. Data repre-
`sent the mean±SEM per group.
`[0078] FIG. 25 illustrates histological sections showing
`intestinal changes in C57BL/6J-Apcm'"/+ mice. FIG. 25 (a)
`consists of a representative tumor of the colon found in a
`control untreated mouse shows pedunculated (polypoid)
`adenoma with high grade of dysplasia. Original magnifica-
`tion 40x. FIG. 25 (b) consists of gastrointestinal intraepithe-
`lial neoplasia (microadenoma) of the small intestine found in
`a mouse gavaged with L. acidophilus bacterial cells in APA
`microcapsules+2% M.F. yogurt. Note the increased Nuclear/
`Cytoplasmic ratio, the nuclear crowding and the hy