`
`SP-304: STIMULATION OF INTRACELLULAR cGMP SYNTHESIS IN T84 CELLS
`
`
`
`
`
`
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`Study Number:
`
`SP-PH-001
`
`Test Article:
`
`SP-304
`
`Author:
`
`Kunwar Shailubhai
`
`
`Senior Vice President, Discovery
`
`
`Synergy Pharmaceuticals, Inc.
`
`Study Dates:
`
`
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`September 2001 to November 2001
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`
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`Testing Facility: R&D Center, Synergy Pharmaceuticals, Inc., Norristown, PA
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`Final Report Date:
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`
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`February 15, 2008
`
`Source Data:
`
`Synergy Pharmaceuticals Lab Notebook# 2; Pages: 70-91
`
`
`
`
`Dr. Surendra Dheer
`
`Synergy Pharmaceuticals, Inc.
`
`
`
`420 Lexington Ave. -Suite 1609
`New York, NY 10170
`
`
`
`Confidentiality Statement
`
`This document is the property of Synergy Pham1aceuticals Inc., and is confidential and proprietary. This
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`information contained herein is believed to be accurate and complete as of the date of preparation. The contents
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`may not be used in regulatory submissions without the expressed consent of Synergy Pharmaceuticals Inc.
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018203
`
`Bausch Health Ireland Exhibit 2027, Page 1 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Synergy Pharmaceuticals Inc.
`
`Study No. SP-PH-001
`
`Page2
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`TABLE OF CONTENTS
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`SIGNATURE PAGE ...................................................................................................................... 3
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`LIST OF TABLES .......................................................................................................................... 4
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`LIST OF FIGURES ........................................................................................................................ 5
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`LIST OF ABBREVIATIONS ......................................................................................................... 6
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`l. ABSTRACT ............................................................................................................................... 7
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`2. OBJECTIVE .............................................................................................................................. 8
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`3 . MATERIALS AND METHODS ............................................................................................... 8
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`3.1 Cell Lines ......................................................................................................................... 8
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`3.2 Chemicals and Reagents ................................................................................................... 8
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`.............................. 8 ...............................................3 .3 Test Materials ......................................
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`3.3 cGMP Stimulation Assay ................................................................................................. 9
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`4. RE SUL TS .................................................................................................................................. 9
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`4.1 Stimulation of cGMP Synthesis by SP-304, Uroguanylin, and Related Peptides ................ 9
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`5. CONCLUSION ........................................................................................................................ 11
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`6. REFERENCES ........................................................................................................................ 12
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`
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`CONFIDENTIAL
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018204
`
`Bausch Health Ireland Exhibit 2027, Page 2 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Synergy Pharmaceuticals Inc.
`
`Study No. SP-PH-001
`
`Page3
`
`EFFECTS OF SP-304 IN THE T84 CELL cGMP STIMULATION BIOASSAY
`
`SIGNATURE PAGE
`
`
`
`The undersigned have reviewed the format and content ofthis report and have approved the
`
`
`
`
`
`
`
`
`
`report for final issuance.
`
`�.
`
`Kunwar Shailubhai, Ph.D.
`Sr. Vice President Discovery
`Synergy Phannaccuticals, Inc.
`
`Date
`
`CSOandCEO
`
`
`
`Inc. . Callisto Pharmaceuticals,
`
`.Date
`
`CONFIDENTIAL
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018205
`
`Bausch Health Ireland Exhibit 2027, Page 3 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Synergy Pharmaceuticals Inc.
`
`Study No. SP-PH-001
`
`Page4
`
`LIST OF TABLES
`Table l: Effects of SP-304, Uroguanylin, and Other Test Peptides in the T84 Cell cGMP
`Stimulation Bioassay .................................................................................................................... 10
`
`CONFIDENTIAL
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018206
`
`Bausch Health Ireland Exhibit 2027, Page 4 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Synergy Pharmaceuticals Inc.
`
`Study No. SP-PH-001
`
`Page5
`
`LIST OF FIGURES
`Figure 1: Dose-Response for SP-304, Uroguanylin (UroG), and Other Test Peptides in the T84
`Cell cGMP Stimulation Bioassay ................................. .................................... ....................... ..... 11
`
`CONFIDENTIAL
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018207
`
`Bausch Health Ireland Exhibit 2027, Page 5 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Synergy Pharmaceuticals Inc.
`
`Study No. SP-PH-001
`
`Page6
`
`LIST OF ABBREVIATIONS
`
`cGMP
`DMEM
`ECso
`ELISA
`IBMX
`UG orUroG
`
`cyclic guanosine monophosphate
`Dulbecco's modified Eagle medium
`median effective concentration (required to induce a 50% effect)
`enzyme-linked immunosorbent assay
`isobutylmethylxanthine
`uroguanylin
`
`CONFIDENTIAL
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018208
`
`Bausch Health Ireland Exhibit 2027, Page 6 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Synergy Pharmaceuticals Inc.
`
`Study No. SP-PH-001
`
`Page7
`
`1. ABSTRACT
`
`The objective of this study was to examine the ability of SP-304 to stimulate cyclic guanosine
`
`monophosphate (cGMP) production in T84 human colon carcinoma cells in vitro. The effects of
`SP-304 were compared to uroguanylin and to other structurally related peptides.
`
`SP-304, uroguanylin, and two related peptides (SP-302 and SP-303) were tested at
`concentrations ranging from 10-9 M to 10-5 M. Cell culture supematants were prepared following
`30-minute incubation, and cGMP levels were measured using a commercial ELISA assay.
`
`All four peptides increased cGMP production in a concentration-dependent manner. At a
`common concentration of 10-6 M (1 µM), cGMP levels were observed to be 54% higher in SP-
`304 treated cells compared to uroguanylin treated cells. SP-304 was the most potent peptide
`tested, with an EC50 of 10-7 M. SP-304 was at least 10-fold more potent than uroguanylin and
`the other test peptides. These data confirm the activity and potency of SP-304 in the cGMP
`stimulation bioassay in T84 cells.
`
`CONFIDENTIAL
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018209
`
`Bausch Health Ireland Exhibit 2027, Page 7 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Synergy Pharmaceuticals Inc.
`
`Study No. SP-PH-001
`
`Page 8
`
`2. OBJECTIVE
`
`The objective of this study was to examine the ability of SP-304 to stimulate cyclic guanosine
`
`monophosphate (cGMP) production in T84 human colon carcinoma cells in vitro. The effects of
`SP-304 were compared to uroguanylin (UG or UroG) and to other structurally related peptides.
`
`3. MATERIALS AND METHODS
`
`3.1
`
`Cell Lines
`
`Human colon carcinoma cells T84 (ATCC Number CCL-248), provided by Dr. Lenard Forte,
`University of Missouri at Columbia, MO, were used in these assays. The cells were cultured in
`Dulbecco's Modified Eagle Medium (DMEM) and Ham's F-12 medium (1:1) containing 5% fetal
`bovine serum and 60 µg of penicillin plus 100 µg of streptomycin per ml ( 1 ). Cells were split
`every 5-6 days by trypsanization. Frozen stocks of cells were stored in liquid nitrogen.
`
`3.2 Chemicals and Reagents
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`All chemicals and reagents were obtained from commercial vendors such as Sigma Chemical
`Co., St. Louis, MO.
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`3.3
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`Test Materials
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`The following test peptides were synthesized by BACHEM Biosciences, Inc. (King of Prussia,
`PA) as the trifluoroacetic acid salt form.
`
`Test Peptide
`
`Mol.
`Wei2ht
`
`Amino Acid Sequence *
`
`SP-301
`(uroguanylin)
`
`1668
`
`I
`I
`Asn 1-Asp2-Glu3-Cys4-Glu5-Leu6-Cys7-Val8-Asn9-Val10-Ala 11-Cys 12-Thr13-Gly 14-Cys 15-Leu 16
`I
`I
`
`SP-302
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`1696
`
`I
`Asn 1-Glu2-Glu3-Cys4-Glu5-Leu6-rys7-Val8-Asn9-Val10-Ala 11-Cys 12-Thr13-Gly14-Cr 15-Leu 16
`
`I
`
`SP-303
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`1682
`
`SP-304
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`1682
`
`Asn 1-Glu2-Asp3-Cys4-Glu5-Leu6-Cys7-Val8-Asn9-Vai10-Ala 11-Cys 12-Thr13-Giy14-Cys 15-Leu 16
`
`I
`
`I
`
`I
`
`f
`
`I
`Asn 1-Asp2-Glu3-Cys4-Glu5-Leu6-Cys7-Val8-Asn9-Vai10-Ala 11-Cys 12-Thr13-Gly14-Cys 15-Leu 16
`
`I
`
`f
`
`I
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`* Note: brackets indicate the location of disulfide bonds
`
`All peptides were prepared as stock solutions at a concentration of 1 mM in deionized water.
`For each peptide, cGMP levels were tested at concentrations ranging from 10-9 M (0.001 µM) to
`10-5 M (10 µM). Control wells contained no peptide.
`
`CONFIDENTIAL
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018210
`
`Bausch Health Ireland Exhibit 2027, Page 8 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Synergy Pharmaceuticals Inc.
`
`Study No. SP-PH-001
`
`Page9
`
`cGMP Stimulation Assay
`3.3
`The potency oftest peptides to stimulate cGMP synthesis in T84 cells was assessed using a
`published procedure (8). Briefly, confluent monolayers of T-84 cells in 24-well plates were
`washed twice with 250 µl ofDMEM containing 50 mM HEPES (pH 7.4) and pre-incubated at
`37°C for 10 min with 250 µl ofDMEM containing 50 mM HEPES (pH 7.4) and 1 mM isobutyl
`methylxanthine (IBMX). The test peptides were then incubated for 30 min. The medium was
`then aspirated and the reaction was terminated by the addition of 3% perchloric acid. Following
`centrifugation and the addition ofNaOH (0.1 N) to neutralize the pH, intracellular cGMP levels
`were determined in lysates using a cGMP ELISA kit (Cat. No. 581021, Cayman Chemical, Ann
`Arbor, Ml.). Samples were run in duplicates incubations and each sample was run as duplicates
`in ELISA test.
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`4. RESULTS
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`4.1 Stimulation of cGMP Synthesis by SP-304, Uroguanylin, and Related Peptides
`The results with SP-304, uroguanylin and other related peptides are presented in Table 1. At a
`common concentration of 10-6 M (1 µM), cGMP levels were observed to be 54% higher in SP-
`304 treated cells compared to uroguanylin treated cells. Cyclic GMP levels in SP-302- treated
`cells were just 10% higher than uroguanylin treated cells, while cGMP levels in SP-303-treated
`cells were 5% lower compared to uroguanylin.
`Figure 1 shows the concentration-response for all four peptides with respect to cGMP production
`in T84 cells. All four peptides increased cGMP production in a concentration-dependent
`manner. SP-304 was the most potent peptide tested, with an EC50 of 10-7 M. SP-304 was at least
`10-fold more potent than uroguanylin and the other test peptides.
`
`CONFIDENTIAL
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018211
`
`Bausch Health Ireland Exhibit 2027, Page 9 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Synergy Pharmaceuticals Inc.
`
`Study No. SP-PH-001
`
`Page 10
`
`Table 1: Effects of SP-304, Uroguanylin, and Other Test Peptides in the T84 Cell cGMP
`Stimulation Bioassay
`
`Test Material
`
`SP-301
`(uroguanylin)
`
`SP-302
`
`SP-303
`
`SP-304
`
`Concentration
`ng/mL
`Molar
`0
`0
`10-• M
`1.668
`10-•M
`16.668
`10-'M
`166.8
`10-0 M
`1668
`10-' M
`16680
`0
`0
`10-"M
`1.696
`10-8M
`16.96
`10-'M
`169.6
`10-0 M
`1696
`10-'M
`16960
`0
`0
`10-" M
`1.682
`10-•M
`16.82
`10-'M
`168.2
`10-6M
`1682
`10-' M
`16820
`0
`0
`10-"M
`1.682
`10-•M
`16.82
`10-1M
`168.2
`10-0 M
`1682
`10-' M
`16820
`* cGMP levels in T84 cells after a 30-minute incubation.
`** EC50: median effective concentration (required to induce a 50% effect)
`
`cGMP Levels
`(pmol/well) *
`0
`0
`12
`82
`205
`254
`0
`0
`8
`62
`185
`248
`0
`0
`12
`82
`195
`254
`0
`0
`17
`149
`320
`315
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`ECso * *
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`Molar
`
`ng/mL
`
`10-6M
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`1668
`
`10-6M
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`1696
`
`10-6M
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`1682
`
`10-7 M
`
`168.2
`
`CONFIDENTIAL
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018212
`
`Bausch Health Ireland Exhibit 2027, Page 10 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Synergy Pharmaceuticals Inc.
`
`Study No. SP-PH-001
`
`Page 1 1
`
`Figure 1: Dose-Response for SP-304, Uroguanylin (UroG), and Other Test Peptides in the
`T84 Cell cGMP Stimulation Bioassay
`
`350
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`300
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`250
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`200
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`150
`
`100
`
`50
`
`0
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`0
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`-9
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`-7
`-8
`-6
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`Log Peptide [M]
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`-6
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`5. CONCLUSION
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`Overall, the results of this study demonstrate that SP-304 stimulated cGMP production in T84
`colon carcinoma cells in a concentration-related manner, with an EC50 of 10-7 M (168.2 ng/mL).
`SP-304 was at least 10-fold more potent than uroguanylin and other related test peptides in this
`bioassay, and induced higher cGMP levels (>50% greater) compared to uroguanylin.
`
`CONFIDENTIAL
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018213
`
`Bausch Health Ireland Exhibit 2027, Page 11 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Synergy Pharmaceuticals Inc.
`
`Study No. SP-PH-001
`
`Page 12
`
`6. REFERENCES
`
`1. Shailubhai, K., Yu, H. H., Karunanandaa, K., Wang, J. Y., Eber, S. L., Wang, Y., Joo, N.
`S., Kim, H. D., Miedema, B. W., Abbas, Z., Boddupalli, S. B., Mark G. Currie., and
`Forte, L. R. "Uroguanylin treatment suppresses polyps formation in APC"in;+ mouse and
`induces apoptosis in human colon adenocarcinoma cells by a cGMP dependent
`mechanism". Cancer Res, 60:5151-5157, 2000.
`
`
`CONFIDENTIAL
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018214
`
`Bausch Health Ireland Exhibit 2027, Page 12 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Final Report Amendment Number 1
`
`SP-304: Stimulation of Intracellular cGMP Synthesis in T84 Cells
`
`Study Number:
`
`SP-PH-001
`
`Test Articles:
`
`SP-304
`
`Author:
`
`Kunwar Shailubhai
`
`Study Dates:
`
`September 2001 to November 2001
`
`Testing Facility:
`
`R&D Center, Synergy Pharmaceuticals, Inc., Norristown, PA
`
`Final Report Date:
`
`February 15, 2008
`
`Final Report
`Amendment Date:
`
`25 June 2015
`
`Synergy Pharmaceuticals, Inc.
`420 Lexington Ave. - Suite 1609
`New York, NY 10170
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018215
`
`Bausch Health Ireland Exhibit 2027, Page 13 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Page 2 SP-PH-001 Final Report Amendment Number 1
`
`TABLE OF CONTENTS
`
`SIGNATURE PAGE ...................................................................................................................... 3
`l. DETAILS OF THE FINAL REPORT AMENDMENT ............................................................ 4
`Item 1 .......................................................................................................................................... 4
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018216
`
`Bausch Health Ireland Exhibit 2027, Page 14 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`
`
`Page 3 SP-PH-001 Final Repo11 Amendment Number I
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`
`
`Final Report Amendment Number 1
`
`
`
`
`
`
`
`
`
`SP-304: Stimulation of Inti-accUular cGMP Synthesis in T84 Cells
`
`SIGNATURE PAGE
`
`
`
`The undersigned reviewed the format and content of this final report amendment and approved
`
`
`
`
`
`
`
`
`the amendment for final issuance.
`
`Kunwar Shailubhai
`
`Date
`
`
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018217
`
`Bausch Health Ireland Exhibit 2027, Page 15 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Page 4 SP-PH-001 Final Report Amendment Number 1
`
`1. DETAILS OF THE FINAL REPORT AMENDMENT
`Note: Text to be removed is identified by a strik&hrough and new text is presented in bold.
`
`Item 1
`Report Page Number: 8
`Report Section Number: 3 .3
`Report Section Title: Test Materials
`Section Test peptide Table: Uroguanylin amino acid sequence
`
`1
`
`:I
`
`;i
`
`4
`
`:,
`
`ti
`
`/
`
`
`
`8 1U 1 1 1:1 1 J 14 1 :, B
`
`
`
`Asn -Asp -Gil! -Cys -Glu -Leu -Cys -Val -Asn -Val -Ala -Cys -Thr -Gly -Cys -Leu
`
`1 ti
`
`Replace with
`
`1
`
`Z
`
`J
`
`4
`
`0
`
`0
`
`f
`
`Asn -Asp -Asp -Cys -Glu -Leu -Crs -Val -Asn -Val -Ala -Cys -Thr -Gly -Cr -Leu
`
`
`
`ts lU 7 7 lZ H
`
`
`
`1;:s
`
`14
`
`10 1 0
`
`Reason for Amendment: Correct error in uroguanylin amino acid sequence.
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018218
`
`Bausch Health Ireland Exhibit 2027, Page 16 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Final Report Amendment Number 2
`
`SP-304: Stimulation of Intracellular cGMP Synthesis in T84 Cells
`
`Study Number:
`
`SP-PH-001
`
`Test Articles:
`
`SP-304
`
`Author:
`
`Kunwar Shailubhai
`
`Study Dates:
`
`September 2001 to November 2001
`
`Testing Facility:
`
`R&D Center, Synergy Pharmaceuticals, Inc., Norristown, PA
`
`Final Report Date:
`
`February 15, 2008
`
`Final Report
`Amendment Date:
`
`17 September 2015
`
`Synergy Pharmaceuticals, Inc.
`420 Lexington Ave. - Suite 1609
`New York, NY 10170
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL0001821 9
`
`Bausch Health Ireland Exhibit 2027, Page 17 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Page 2 SP-PH-001 Final Report Amendment Number 2
`
`TABLE OF CONTENTS
`
`SIGNATURE PAGE ...................................................................................................................... 3
`l. DETAILS OF THE FINAL REPORT AMENDMENT ............................................................ 4
`Item 1 .......................................................................................................................................... 4
`Iten1 2 .......................................................................................................................................... 5
`Item 3 .......................................................................................................................................... 6
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018220
`
`Bausch Health Ireland Exhibit 2027, Page 18 of 22
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`

`

`
`
`
`
`Pag,e 3 SP-PH-001 Final Report A.wendment Number 2
`
`Final Report Amendment Number 2
`
`SP-304: Stimulation of Intracellular cGMP Synthesis in T84 Cells
`
`SIGNATURE PAGE
`
`
`
`The undersigned reviewed the format and content of this final report amendment and approved
`
`
`
`
`
`the amendment for final issuance.
`
`Kunwar Shailubhai
`
`Date
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018221
`
`Bausch Health Ireland Exhibit 2027, Page 19 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`
`
`Page 4 SP-PH-0 0 1 Final Report Amendment Number 2
`
`
`
`
`
`1. DETAILS OF THE FINAL REPORT AMENDMENT
`
`Note: Text to be removed is identified by a strik&hrough and new text is presented in bold.
`
`Item 1
`
`Report Page Number: 10
`Report Section Number: 4.1
`Report Section Title: Stimulation of cGMP Synthesis by SP-304, Uroguanylin, and Related
`Peptides
`
`Effects of SP-304, Uroguanylin, and Other Test Peptides in the T84 cGMP
`Table 1.
`Stimulation Bioassay
`
`Test Material
`
`ECso **
`
`Molar
`
`ng/mL
`
`Concentration
`cGMP Levels
`(pmol/well) *
`ng/mL
`Molar
`0
`0
`0
`1 0-'M
`1 .668
`0
`SP-30 1
`12
`1 0-•M
`1 6 . 668
`(uroguanylin)
`82
`1 0- M
`1 66 . 8
`205
`1 0-0 M
`1 66 8
`1 0-, M
`1 6680
`254
`0
`0
`0
`1 0-•M
`1 .696
`0
`1 6.96
`8
`1 0-"M
`62
`1 0- 'M
`1 69.6
`1 8 5
`1 0-0M
`1 696
`248
`1 0-'M
`1 6960
`0
`0
`0
`1 .682
`0
`1 0-'M
`12
`1 0-"M
`1 6.82
`82
`1 68.2
`1 0-'M
`195
`1 0-0 M
`1 682
`254
`1 0-'M
`1 6820
`0
`0
`0
`1 0-'M
`1 .682
`0
`17
`1 6.82
`1 0-"M
`1 68.2
`149
`1 0-'M
`1 0-0 M
`320
`1 682
`3 1 5
`1 0--M
`1 6820
`
`
`
`cGMP levels in T84 cells after a 3 0-minute incubation.
`
`
`
`
`
`a 5 0% effect) to induce (required EC50: median effective concentration
`
`-1-Q.-&
`-M
`2.3x10-7M
`
`-1-Q.-&-M
`3.5x10-7M
`
`-1-Q.-&
`-M
`2.4x10-7M
`
`w-+
`-M
`1.1x10-7M
`
`-Me%
`383.6
`
`-±-9%
`593.6
`
`-l-6&2-
`403.7
`
`-1-6&4
`1 85.0
`
`SP-3 02
`
`SP-303
`
`SP-304
`
`*
`
`**
`
`To update the scientific notation for the EC50 molar value for each
`Reason for Amendment:
`
`peptide to include the coefficient as well as the exponent and to update the corresponding ng/mL
`values. The new values were calculated using Prism 6 (Version 6.05) using a nonlinear
`regression curve fit (log[peptide concentration] versus cGMP level) using a least squares fit with
`no constraints)
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018222
`
`Bausch Health Ireland Exhibit 2027, Page 20 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Page 5 SP-PH-001 Final Report Amendment Number 2
`
`Item 2
`
`Report Page Number: 1 1
`
`
`
`
`Report Section Number: 4.1
`
`
`
`
`Peptides
`
`
`
`
`
`Report Section Title: Stimulation of cGMP Synthesis by SP-304, Uroguanylin, and Related
`
`
`
`Replace the original Figure l below
`
`
`
`
`
`350
`
`$()()
`
`260
`
`200
`
`• UfOG·
`* SP-302
`,, Sf'-303
`❖ SP-304
`
`150
`
`100
`
`60
`
`o ,
`
`
`
`Log Peptide l.lVl]
`
`
`
`
`
`with the following updated figure:
`
`....... SP-302
`
`350 ...... UG
`- 300
`SP-304
`---
`'ai
`� 250
`In _..,_. SP-303
`,St 200
`� 150
`
`� 100
`u 50
`
`0
`-10
`
`?_.,,...,----.
`
`,·
`-J AY· ··
`�"?'
`
`i
`
`'./
`
`..g
`
`� '"'5
`-8
`-7
`Log Peptide [M]
`
`
`
`
`
`
`Reason for Amendment:
`
`the updated analyses described in Item l .
`
`The curves in the new figure depict the dose-response curves derived from
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018223
`
`Bausch Health Ireland Exhibit 2027, Page 21 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`Page 6 SP-PH-001 Final Report Amendment Number 2
`
`Item 3
`Report Page Number: 11
`Report Section Number: 5
`Report Section Title: Conclusion
`
`Overall, the results of this study demonstrate that SP-304 stimulated cGMP production in T84
`colon carcinoma cells in a concentration-related manner, with an BGw ef-1-0-1- EC50 of 1.1 x
`10-7M (168.2 ag/ml,) (185 ng/mL). SP-304 was at least 10 2-3 fold more potent than
`uroguanylin (EC50 2.3 x 10-7M) and other related test peptides (SP-302 EC50 3.5 x 10-7M and
`SP-303 EC50 2.4 x 10-7M) in this bioassay, and induced higher cGMP levels (>50% greater)
`compared to uroguanylin
`Reason for Amendment: To incorporate the updated results into the conclusion.
`
`HIGHLY CONFIDENTIAL INFORMATION
`
`TRUL00018224
`
`Bausch Health Ireland Exhibit 2027, Page 22 of 22
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`