Case IPR2022-00722
`U.S. Patent No. 7,041,786
`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`__________________
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`__________________
`
`MYLAN PHARMACEUTICALS INC.,
`
`Petitioner,
`
`v.
`
`BAUSCH HEALTH IRELAND LIMITED,
`
`Patent Owner.
`
`__________________
`
`Case IPR2022-00722
`U.S. Patent No. 7,041,786
`__________________
`
`DECLARATION OF KUNWAR SHAILUBHAI
`
`Bausch Health Ireland Exhibit 2023, Page 1 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
`U.S. Patent No. 7,041,786
`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`__________________
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`__________________
`
`MYLAN PHARMACEUTICALS INC.,
`
`Petitioner,
`
`v.
`
`BAUSCH HEALTH IRELAND LIMITED,
`
`Patent Owner.
`
`__________________
`
`Case IPR2022-00722
`U.S. Patent No. 7,041,786
`__________________
`
`DECLARATION OF KUNWAR SHAILUBHAI
`
`Bausch Health Ireland Exhibit 2023, Page 1 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
`
`Case IPR2022-00722
`U.S. Patent No. 7,041,786
`
`
`I, Kunwar Shailubhai, under penalty of perjury, declare as follows:
`INTRODUCTION
`1.
`I am of legal age and otherwise competent to make this declaration.
`
`I.
`
`2.
`
`I am the first named inventor on U.S. Patent No. 7,041,786 (Ex. 1001,
`
`“the ’786 patent”). I have been asked to submit a declaration attesting to how the
`
`data disclosed in Table 4 in the specification of the ’786 patent and other relevant
`
`data related to the peptides disclosed in the ’786 patent were generated.
`
`II. EDUCATION AND WORK EXPERIENCE
`3.
`I graduated from the Maharaja Sayajirao University of Baroda with a
`
`Ph.D. in Microbiology in 1984 and from the University of Missouri-Saint Louis
`
`with an M.B.A. in 2002.
`
`4.
`
`I am currently employed by the Pennsylvania Biotechnology Center
`
`and Baruch S. Blumberg Institute as a Senior Advisor and Professor.
`
`III. BIOLOGICAL ACTIVITY DATA IN TABLE 4 OF THE ’786 PATENT
`5.
`I was involved in synthesizing and testing of guanylate cyclase (“GC-
`
`C”) receptor agonists that enhance intracellular production of cyclic guanosine
`
`monophosphate (“cGMP”), including the peptides of the experimental examples
`
`reported in the ’786 patent. As such, I have first-hand knowledge of how the
`
`peptides reported below were made and tested.
`
`1
`
`Bausch Health Ireland Exhibit 2023, Page 2 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
`
`
`I directed the synthesis and testing of the GC-C receptor agonist
`
`Case IPR2022-00722
`U.S. Patent No. 7,041,786
`
`6.
`
`peptides in order to examine biological activity as disclosed in Table 4 of the ’786
`
`patent.
`
`7.
`
`Human T84 colon carcinoma cells were obtained from the American
`
`Type Culture Collection. (Id. at 15:27-29). Cells were grown in a 1:1 mixture of
`
`Ham’s F-12 medium and Dulbecco’s modified Eagle’s medium (“DMEM”)
`
`supplemented with 10% fetal bovine serum, 100 U penicillin/ml, and 100 μg/ml
`
`streptomycin. (Id. at 15:29-32). The cells were fed fresh medium every third day
`
`and split at a confluence of approximately 80%. (Id. at 15:32-34).
`
`8.
`
`Peptides were custom synthesized by Multiple Peptide Systems, San
`
`Diego, California, and by Princeton Biomolecules, Langhorne, Pennsylvania. (Id.
`
`at 15:36-38). Biological activity of the synthetic peptides was assayed. (Id. at
`
`15:38-39). The confluent monolayers of T84 cells in 24-well plates were washed
`
`twice with 250 μl of DMEM containing 50 mM HEPES (pH 7.4), pre-incubated at
`
`37°C for 10 minutes with 250 μl DMEM containing 50 mM HEPES (pH 7.4) and 1
`
`mM isobutylmethylxanthine (“IBMX”), followed by incubation with peptides (0.1
`
`nM to 10 μM) for 30 minutes. (Id. at 15:40-46). The medium was aspirated, and
`
`the reaction was terminated by the addition of 3% perchloric acid. (Id. at 15:46-
`
`47). Following centrifugation, and neutralization with 0.1 N NaOH, the
`
`2
`
`Bausch Health Ireland Exhibit 2023, Page 3 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
`
`
`supernatant was used directly for measurements of cGMP using an ELISA kit
`
`Case IPR2022-00722
`U.S. Patent No. 7,041,786
`
`(Caymen Chemical, Ann Arbor, Michigan). (Id. at 15:47-50).
`
`9.
`
`As indicated in the following table, the peptides were custom
`
`synthesized and purified (>95% purity) using a published procedure (procedure
`
`from Klodt, et al., J. Peptide Res. 50:222-230 (1997)). (Id. at 15:53-54, 18:32).
`
`Peptides were evaluated in the T84 cell-based assay for their ability to enhance
`
`intracellular levels of cGMP. (Id. at 15:55-56). The results of this test are shown
`
`in Table 4 below.
`
`(Id. at 16:1-19). I note that the p value below Table 4 contains a typographical
`
`
`
`error and should say p < 0.05. (Id. at 16:19).
`3
`
`Bausch Health Ireland Exhibit 2023, Page 4 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
`
`
`IV. DATA IN STUDY NUMBER SP-PH-001
`10.
`I directed the preparation and testing of the ability of SP-304
`
`Case IPR2022-00722
`U.S. Patent No. 7,041,786
`
`(plecanatide) to stimulate cGMP production in T84 human colon carcinoma cells
`
`in vitro and evaluated the effects of SP-304, uroguanylin, and other peptides. (Ex.
`
`2027 at TRUL00018209).
`
`11. SP-304, uroguanylin, and two other peptides (SP-302 and SP-303)
`
`were tested at concentrations ranging from 10-9 M to 10-5 M. (Id.). Cell culture
`
`supernatants were prepared following 30-minute incubation, and cGMP levels
`
`were measured using a commercial ELISA assay. (Id.).
`
`12. 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. (Ex. 2027 at TRUL00018210). The cells were cultured in 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. (Id.). Cells were split every 5-6
`
`days by trypsinization. (Id.). Frozen stocks of cells were stored in liquid nitrogen.
`
`(Id.).
`
`13. The following test peptides were synthesized by BACHEM
`
`Biosciences, Inc. (King of Prussia, PA) as the trifluoroacetic acid salt form:
`
`4
`
`Bausch Health Ireland Exhibit 2023, Page 5 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
`
`Case IPR2022-00722
`U.S. Patent No. 7,041,786
`
`
`
`
`
`(Id. at TRUL00018210, TRUL00018218).
`
`14. All peptides were prepared as stock solutions at a concentration of 1
`
`mM in deionized water. (Id. at TRUL00018210). For each peptide, cGMP levels
`
`were tested at concentrations ranging from 10-9 M (0.001 μM) to 10-5 M (10 μM).
`
`(Id.). Control wells contained no peptide. (Id.).
`
`15. The potency of test peptides to stimulate cGMP synthesis in T84 cells
`
`was assessed using a published procedure. (Ex. 2027 at TRUL00018211).
`
`Confluent monolayers of T84 cells in 24-well plates were washed twice with 250
`
`μl of DMEM and containing 50 mM N-(2-hydroxyethyl)piperazine-N’-(2-
`
`ethanesulfonic acid) (“HEPES”) (pH 7.4) and pre-incubated at 37°C for 10 minutes
`
`with 250 μl of DMEM containing 50 mM HEPES (pH 7.4) and 1 mM IBMX.
`
`(Id.). The test peptides were then incubated for 30 min. (Id.). The medium was
`
`then aspirated and the reaction was terminated by the addition of 3% perchloric
`
`5
`
`Bausch Health Ireland Exhibit 2023, Page 6 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
`
`
`acid. (Id.). Following centrifugation and the addition of NaOH (0.1 N) to
`
`Case IPR2022-00722
`U.S. Patent No. 7,041,786
`
`neutralize the pH, intracellular cGMP levels were determined in lysates using a
`
`cGMP ELISA kit (Cat No. 581021, Cayman Chemical, Ann Arbor, MI.). (Id.).
`
`Samples were run in duplicate incubations and each sample was run as duplicates
`
`in ELISA test. (Id.).
`
`16. As illustrated by Table 1 and Figure 1 below, the results of this study
`
`demonstrated that SP-304 stimulated cGMP production in T84 colon carcinoma
`
`cells in a concentration-related manner, with an EC50 of 1.1 x 10-7 M (185 ng/mL).
`
`(Ex. 2027 at TRUL0001822-TRUL00018224). Uroguanylin showed an EC50 of
`
`2.3 x 10-7, SP-302 showed an EC50 of 3.5 x 10-7, and SP-303 showed an EC50 of 2.4
`
`x 10-7. (Id.).
`
`6
`
`Bausch Health Ireland Exhibit 2023, Page 7 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
`
`Case IPR2022-00722
`U.S. Patent No. 7,041,786
`
`
`
`(Ex. 2027 at TRUL00018222).
`
`
`
`7
`
`Bausch Health Ireland Exhibit 2023, Page 8 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
`
`Case IPR2022-00722
`U.S. Patent No. 7,041,786
`
`
`
`
`
`(Ex. 2027 at TRUL00018223).
`
`V. DATA IN STUDY NUMBER SP-PH-004
`A. Uroguanylin and SP-304 Topoisomeric Properties
`17.
`I directed the preparation and testing of topoisomeric conformations
`
`of SP-304 (plecanatide) and uroguanylin monitored by HPLC analysis and further
`
`evaluated by NMR to characterize their three-dimensional structures. (Ex. 2028 at
`
`TRUL00018269).
`
`18. Sample SP-304 (10 μM) and uroguanylin (10 μM) were incubated for
`
`16 hours at 37°C in a pH 3.0 solution comprised of 25 mM sodium acetate, 150
`
`mM NaCl, and then analyzed by HPLC. (Id.).
`
`19. Peptide solutions were subjected to reverse phase chromatography on
`
`an HPLC system attached with a VYDAC C18 column using a 20-40% gradient of
`
`8
`
`Bausch Health Ireland Exhibit 2023, Page 9 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
`
`
`solvent A (0.1% TFA in water) and solvent B (0.1 % TFA in Acetonitrile). The
`
`Case IPR2022-00722
`U.S. Patent No. 7,041,786
`
`column flow rate was adjusted to 1 ml/min. Under these conditions, the
`
`biologically active and inactive fractions were eluted at 21.4 and 23.7 minutes,
`
`respectively. (Ex. 2028 at TRUL00018268).
`
`20. As shown in Figure 1 below, after the 16-hour incubation,
`
`uroguanylin showed two major peaks and two minor peaks. (Ex. 2028 at
`
`TRUL00018269). In contrast, SP-304 showed only a single peak. (Id.).
`
`
`
`(Id.).
`
`B.
`
`21.
`
`pH Dependency of cGMP Stimulation by SP-304, Uroguanylin,
`SP-302, and SP-303
`I directed the preparation and testing of SP-304, uroguanylin, and two
`
`other peptides (SP-302 and SP-303) in T84 cells to determine if cGMP stimulation
`
`9
`
`Bausch Health Ireland Exhibit 2023, Page 10 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
`
`
`by 0.1 μM concentrations was affected by the pH of culture medium. (Ex. 2028 at
`
`Case IPR2022-00722
`U.S. Patent No. 7,041,786
`
`TRUL00018269). I tested a range of pH values (pH 5.5-7.0). (Id.).
`
`22. Human colon carcinoma T84 cells (ATCC Number CCL-248),
`
`provided by Dr. Lenard Forte, University of Missouri at Columbia, MO, were used
`
`in these assays. (Ex. 2028 at TRUL00018267). The cells were cultured in 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. (Id.). Cells were split every 5-6
`
`days by trypsinization. (Id.). Frozen stocks of cells were stored in liquid nitrogen.
`
`(Id.).
`
`23. Confluent monolayers of cultured T84 colon carcinoma cells were
`
`washed and pre-incubated with Hanks Balanced Salt Solution containing IBMX at
`
`pH 5.5, 6.0, 6.5, or 7.0 for 10 minutes, and were then incubated with test peptides
`
`(uroguanylin, SP-302, SP-303, or SP-304) for 30 minutes at a concentration of 0.1
`
`μM at pH 5.5, 6.0, 6.5, or 7.0, respectively. (Ex. 2028 at TRUL00018277; see
`
`infra Section V.C (discussing procedure)). The reactions were then terminated,
`
`cells were lysed, and the resulting lysates were assayed for intracellular cGMP
`
`content using ELISA. (Id.).
`
`24. The results of the study are shown in Figure 2 below.
`
`10
`
`Bausch Health Ireland Exhibit 2023, Page 11 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
`
`Case IPR2022-00722
`U.S. Patent No. 7,041,786
`
`
`
`
`
`
`
`(Ex. 2028 at TRUL00018270). The raw data reported in Figure 2 are presented in
`
`Table 2 below.
`
`(Ex. 2028 at TRUL00018273).
`
`C. Thermostability of SP-304, Uroguanylin, SP-302, and SP-303
`25.
`I directed the preparation and testing of SP-304, uroguanylin, SP-302,
`
`and SP-303 to stimulate cGMP after incubation at 95°C for various lengths of time
`
`11
`
`Bausch Health Ireland Exhibit 2023, Page 12 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
`
`
`to compare their thermostability to high temperature. (Ex. 2028 at
`
`Case IPR2022-00722
`U.S. Patent No. 7,041,786
`
`TRUL00018270).
`
`26. Human colon carcinoma T84 cells (ATCC Number CCL-248),
`
`provided by Dr. Lenard Forte, University of Missouri at Columbia, MO, were used
`
`in these assays. (Ex. 2028 at TRUL00018267). The cells were cultured in 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. (Id.). Cells were split every 5-6
`
`days by trypsinization. (Id.). Frozen stocks of cells were stored in liquid nitrogen.
`
`(Id.).
`
`27. Peptides for analysis were prepared as 1 mM stock solutions in
`
`deionized water. (Ex. 2028 at TRUL00018277). The potency of test peptides to
`
`stimulate cGMP synthesis in T84 cells was assessed using a published procedure
`
`(procedure from Shailubhai, K., et al., Uroguanylin treatment suppresses polyps
`
`formation in APCmin/+ mouse and induces apoptosis in human colon
`
`adenocarcinoma cells by a cGMP dependent mechanism, Cancer Res. 60:5151-
`
`5157 (2000)). (Id.). Confluent monolayers of T84 cells in 24-well plates were
`
`washed twice with 250 μl of DMEM containing 50 mM HEPES (pH 7.4) and pre-
`
`incubated at 37°C for 10 minutes with 250 μl of DMEM containing 50 mM
`
`HEPES (pH 7.4) and 1 mM IBMX. (Id.). Monolayers of T84 cells were then
`
`incubated with 250 μl of a solution of DMEM, 40 mM HEPES (pH 7.4) plus
`12
`
`Bausch Health Ireland Exhibit 2023, Page 13 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
`
`
`peptide for 30 minutes. (Id.). Control cells were incubated in the above solution
`
`Case IPR2022-00722
`U.S. Patent No. 7,041,786
`
`minus peptide. (Id.). After 30 minutes, the medium was aspirated and the reaction
`
`was terminated by the addition of 3% perchloric acid to cells. (Id.). Following
`
`centrifugation and the addition of NaOH (0.1 N) to neutralize the pH, cell lysates
`
`were made and intracellular cGMP levels measured using a cGMP ELISA kit (Cat.
`
`No. 581021, Cayman Chemical, Ann Arbor, MI.). (Id.). All incubations were
`
`performed in duplicate. Cell lysates subjected to ELISA determinations were also
`
`performed in duplicate. (Id.).
`
`28. Test peptides (uroguanylin, SP-302, SP-303, or SP-304) were
`
`dissolved in phosphate buffered saline solution (pH 7.4) at a concentration of
`
`0.1 μM and were incubated at 95°C for times ranging from 0-90 minutes. (Ex.
`
`2028 at TRUL00018268). After the incubation, the peptide solutions were rapidly
`
`cooled to 4°C and then assayed for stimulation of cGMP production in T84 cells
`
`using the standard cGMP T84 cell assay. (Id.). This involved taking confluent
`
`monolayers of cultured T84 colon carcinoma cells, washed and pre-incubated with
`
`DMEM and IBMX for 10 minutes, and then incubating them for 30 minutes with
`
`the test peptides (1 μM) obtained from the earlier 95°C incubations. (Id.). The
`
`reactions were terminated, cells were lysed, and the resulting lysates were assayed
`
`for intracellular cGMP levels using ELISA. (Id.).
`
`29. Figure 3 below shows the results from the study.
`13
`
`Bausch Health Ireland Exhibit 2023, Page 14 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
`
`Case IPR2022-00722
`U.S. Patent No. 7,041,786
`
`
`
`(Ex. 2028 at TRUL00018271). The raw data for Figure 3 are presented in Table 3
`
`below.
`
`
`
`
`
`(Ex. 2028 at TRUL00018273).
`
`14
`
`Bausch Health Ireland Exhibit 2023, Page 15 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
`
`Case IPR2022-00722
`U.S. Patent No. 7,041,786
`
`
`VI. DATA DISCLOSED IN TABLE 4 OF THE ’786 PATENT, STUDY NO.
`SP-PH-001, AND STUDY NO. SP-PH-004
`30. Based on my involvement with the synthesis and testing of GC-C
`
`receptor agonist peptides in order to examine their isomerization, biological
`
`activity, and heat stability as disclosed in Table 4 of the ’786 patent, Study No. SP-
`
`PH-001, and Study No. SP-PH-004, I have first-hand knowledge of how these test
`
`data disclosed were generated.
`
`31.
`
`I hereby certify that each of the records for Study No. SP-PH-001 and
`
`Study No. SP-PH-004 (i.e., Ex. 2027 and Ex. 2028):
`
`(A) were made or obtained at or near the time of occurrence of the matters
`
`set forth in the records, by, or from information transmitted by, a person
`
`with personal knowledge of those matters;
`
`(B) were kept in the course of regularly conducted business activity;
`
`(C) were created or obtained as a regular business practice; and
`
`(D) are the types of information created, obtained, and used in the ordinary
`
`course of business.
`
`32.
`
`I declare that all statements made herein of my knowledge are true
`
`and that all statements made on information and belief are believed to be true; and
`
`further, that these statements were made with knowledge that willful false
`
`15
`
`Bausch Health Ireland Exhibit 2023, Page 16 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
`
`
`statements and the like so made are punishable by fine or imprisonment, or both,
`
`Case IPR2022-00722
`U.S. Patent No. 7,041,786
`
`under 18 U.S.C. § 1001.
`
`
`
`
`
`
`
`
`
`16
`
`Bausch Health Ireland Exhibit 2023, Page 17 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Bausch Health Ireland Exhibit 2023, Page 18 of 18
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`
Accessing this document will incur an additional charge of $.
After purchase, you can access this document again without charge.
Accept $ Charge
Still Working On It
This document is taking longer than usual to download. This can happen if we need to contact the court directly to obtain the document and their servers are running slowly.
Give it another minute or two to complete, and then try the refresh button.
A few More Minutes ... Still Working
It can take up to 5 minutes for us to download a document if the court servers are running slowly.
Thank you for your continued patience.
This document could not be displayed.
We could not find this document within its docket. Please go back to the docket page and check the link. If that does not work, go back to the docket and refresh it to pull the newest information.
Your account does not support viewing this document.
You need a Paid Account to view this document. Click here to change your account type.
Your account does not support viewing this document.
Set your membership
status to view this document.
With a Docket Alarm membership, you'll
get a whole lot more, including:
- Up-to-date information for this case.
- Email alerts whenever there is an update.
- Full text search for other cases.
- Get email alerts whenever a new case matches your search.
One Moment Please
The filing “” is large (MB) and is being downloaded.
Please refresh this page in a few minutes to see if the filing has been downloaded. The filing will also be emailed to you when the download completes.
Your document is on its way!
If you do not receive the document in five minutes, contact support at support@docketalarm.com.
Sealed Document
We are unable to display this document, it may be under a court ordered seal.
If you have proper credentials to access the file, you may proceed directly to the court's system using your government issued username and password.
Access Government Site
