throbber
and rat uroguanylin peptide sequences. Second,a skilled artisan would have known
`
`that replacing third-position aspartic acid with glutamic acid would at least
`
`maintain, if not broaden, the intestinal surface susceptible to acid-enhanced
`
`uroguanylin signaling—thereby increasing potency. Third, a skilled artisan would
`
`have recognized that a glutamic acid substitution would reduce undesirable
`
`aspartimide formation during peptide synthesis.
`
`124. My analysis below identifies exemplary disclosures of the cited
`
`referencesrelative to the corresponding claim elements and is not meant to be
`
`exhaustive. As I set forth in my analysis below, a skilled artisan would have
`
`understood claim | to have been obvious in view of the combined teachings of
`
`Currie and Li.
`
`1. A peptide
`
`consisting of
`
`
`
`
`
`
` °786 Patent
` Currie and Li
`
`Claim
`
`
`Currie teaches:
`
`
`
`A “novel peptide...which has the following amino acid
`
`
`
`sequence. (NDDCELCVNVACTGCL)
` the amino acid
`
`
`
`
`Asn-Asp-Asp-Cys-Glu-Leu-Cys-Val-Asn-Val-Ala-Cys-Thr-
`sequence of
`
` This peptide, also referred to herein as human uroguanylin,
`
`
`has been isolated from humanurine and has been chemically
`
`Gly-Cys-Leu
`
`SEQ ID
`
`NO:20.
`
`MSN Exhibit 1002 - Page 65 of 129
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`
`-6]-
`
`

`

`synthesized by solid phase peptide synthesis. In its oxidized
`
`active biologic form, the novel peptide has two disulfide
`
`bridges, one between cysteine residues at positions 4 and 12
`
`and the other between cysteine residues at positions 7 and 15.”
`
`EX1005, 1:45-63 (emphasis added).
`
`Li teaches:
`
`“Fig. 6. Amino acid ... sequences ofrat [and human]
`
`uroguanylin. (a) Alignment of the amino acid sequence of the
`
`purified duodenal peptide (top) with published sequencesof
`
`guanylin, uroguanylin, and STa. Aminoacididentities are
`
`indicated by shading. The arrowheads denote structural
`
`features described in the text.” EX1006, 52 (emphasis added).
`
`VV
`T/E 1A THRE
`QED
`N BD
`PNT
`S HT
`Pg
`PNT
`NTFYC
`
`E Coli STa
`
`rat uroguany|! in
`opossum uroguany! in
`human uroguany|! in
`rat guanylin
`opossum guany! in
`human guany! in
`mouse guany! in
`
`EX1006, 52, FIG. 6(a) (annotated).
`
`“Alignmentof the sequence[s]...with the appropriate regions of
`
`rat guanylin and [human] uroguanylin (Fig. 6a) reveals that” rat
`
`uroguanylin “is more closely related to [human] uroguanylin
`
`(80% identity when comparedacross species, with nearly all
`
`differences representing conservative amino acid
`
`substitutions).” EX1006, 53 (emphasis added).
`
`-62-
`
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`
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`

`

`“Thus, features that are found in uroguanylin, but not in
`
`guanylin, offer information aboutstructural elements that
`
`specify the strength of the ligand/receptor interaction. Of
`
`
`
`
`
`
`
`
`
`
`
`
`
`particular interest are two residues that are basic or uncharged
`
`in guanylin but acidic in uroguanylin (stippled arrowheads).”
`
`EX1006, 54 (emphasis added).
`
`125. Claim 1 recites only [Glu*]-human uroguanylin, nothing more. Currie
`
`identified human uroguanylin. EX 1005, 1:45-63. Li further identified the glutamic
`
`acid substitution in aligning rat and human uroguanylin. EX1006, 52-54, FIG. 6(a).
`
`Thus, Currie and Li together disclose each element of [Glu*]-human uroguanylin.
`
`A. Currie Suggests Uroguanylins For Treating Constipation
`
`126. Well before 2002, Currie expressly suggested that human uroguanylin
`
`treated clinical constipation. EX1005, 2:20-24. Currie empirically validatesits
`
`suggestion in three ways. First, it confirms that human uroguanylin bindsto the
`
`intestinal guanylate cyclase. EX1005, 2:53-65, FIG. 3(b). Second, Currie confirms
`
`that human uroguanylin hasactivity upon intestinal guanylate cyclase in that it
`
`“stimulate[s] increases in cyclic GMP levels in a manner similar to guanylin and
`
`the STs.” /d., 2:8-9. Third, Currie confirms that “[h]uman uroguanylin added to the
`
`mucosal reservoir of rat colon mounted in an Ussing chamberalso caused a
`
`sustainedrise in Isc,” or Short-Circuit Current. /d., 6:29-31.
`
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`

`127. Currie noted that human uroguanylin is an “endogenous stimulator of
`
`intestinal guanylate cyclase.” EX 1005, 2:6-8. Intestinal guanylate cyclaseis a
`
`transmembrane enzyme expressed onthe surface ofcells lining the intestinal
`
`lumen. /d., 1:23-30 (“This form of the enzyme is predominantly found in the
`
`intestinal epithelial cells with the largest number of receptors oriented towards the
`
`lumen. Recently, the intestinal form of guanylate cyclase has been cloned and
`
`expressed from rat small intestinal mucosa. This enzymeis characterized by an
`
`extracellular receptor binding region, a transmembraneregion, an intracellular
`
`protein kinase-like region and a cyclase catalytic domain.”).
`
`128. Human uroguanylin binds to intestinal guanylate cyclase, thereby
`
`increasing cGMP. Following further downstream events, cGMPcauses the
`
`physiological effects associated with treating constipation or, when presentin too
`
`great an amount, the pathology of diarrhea. EX1005, 1:34-39. As Currie describes,
`
`this effect is mediated by, amongotherthings, the downstream effect of chloride
`
`secretion from intestinal endothelial cells. /d., 2:18-20. Chloride secretion
`
`decreases (and reverses) water absorption. /d. Human uroguanylin was thus known
`
`by 2002 to provide therapeutic benefits for clinical constipation.
`
`129. Giventhe above, a skilled artisan would have had good reason to
`
`modify human uroguanylin for developing a treatment for clinical constipation.
`
`130. Moreover, though human uroguanylin was knownto be naturally
`
`MSNExhibit 1002 - Page 68 of 129
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`
`-64-
`
`

`

`occurring, in view of Currie, a skilled artisan would have had good reason to turn
`
`to chemical synthesis over isolation from a natural source. For example, Currie
`
`teaches that human uroguanylin was “both isolated and chemically synthesized in a
`
`homogeneously purified form which did not exist in humanurine from whichit
`
`wasinitially obtained. That is, it has been prepared in a form whichis essentially
`
`free of other low molecular weight peptides, and free from higher molecular
`
`weight material and other cellular components and tissue matter.” EX1005, 1:64-
`
`23,
`
`131. Currie also teaches more specifically that human uroguanylin “can be
`
`prepared by knownsolution and solid phase peptide synthesis methods.” EX1005,
`
`3:7-9; see also id., 5:17-20 (teaching how “[u]roguanylin was synthesized by the
`
`solid-phase method on an Applied Biosystems Model 430A peptide synthesizer
`
`and purified by reverse-phase Cig chromatography”). This synthetic approach
`
`works by adding one aminoacid to another. See id., 3:25-28 (“This procedure,
`
`though using many of the same chemical reactions and blocking groupsofclassical
`
`peptide synthesis, provides a growing peptide chain anchoredbyits carboxy
`
`terminus to a solid support.”). As noted in Section VII, skilled artisans enjoyed a
`
`reasonable expectation of success in preparing synthetic analogs of high activity
`
`using these routine and conventional solid-state peptide synthesis techniques.
`
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`-65-
`
`

`

`B. Reason to Look to Synthetic Analogs of Human Uroguanylin
`
`132. By 2002, a skilled artisan would have had good reason to make a
`
`synthetic analog of the human uroguanylin taught by Currie for developing a
`
`therapeutic for treating constipation. As described above in Section VII, this
`
`approach wasroutine well before 2002. For example,as illustrated in my
`
`discussion of GnRH, skilled artisans routinely looked to synthetic analogs to
`
`improvethe activity of peptide hormonesandto convert them into drugs. See
`
`Section VII.E, above.
`
`133. Currie demonstrates the conventional nature of evaluating the potency
`
`of peptide analogs when discussing the differences between human uroguanylin
`
`and guanylin that made uroguanylin more potent than guanylin. EX1005, 6:13-16
`
`(confirming “[h]uman uroguanylin appeared to be more potent than human
`
`guanylin ... for activation of GC-C in T84 cells”). From Currie, a skilled artisan
`
`would have knownthat uroguanylin and guanylin comprise conserved and
`
`divergent portions. See id., 5:66-6:2 (noting that “human uroguanylin shares
`
`homology with guanylin”). From here, a skilled artisan would have had good
`
`reason to compareand contrast uroguanylin andits paralog guanylin to identify
`
`mechanistic insights for achieving enhancedactivity in a synthetic analog.
`
`134. This analysis was performed by Li, which not only compared human
`
`uroguanylin with guanylin, but also compared other related sequencesin animals.
`
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`

`For example, Li aligns and comparesthe peptide sequence of human uroguanylin
`
`with related sequences including rat uroguanylin and human guanylin in Figure
`
`6(a), reproduced below.
`
` vv
`
`L
`
`G>
`mtg
`meee Y
`
`T/E 1
`
`A THE
`QE ODO
`N B} 0
`PN T
`SHIT
`PGT
`PNT
`NTFYC
`
`rat uroguany! in
`opossum uroguany! in
`human uroguany! in
`rat guany!in
`opossum guany! in
`human guany! in
`mouse guany! in
`E Coli STa
`
`EX1006, 52, FIG. 6(a).
`
`135.
`
`In so doing, Li teachesthat “[t]he affinity of GCC for uroguanylin
`
`(opossum or human)is about 10-fold higher than its affinity for guanylin (rat or
`
`human). Thus, features that are found in uroguanylin, but not in guanylin, offer
`
`information about structural elements that specify the strength of the
`
`ligand/receptor interaction.” /d., 54. In particular, Li notes that “[o]f particular
`
`interest are two residuesthat are basic or uncharged in guanylin but acidic in
`
`uroguanylin (stippled [7.e., grey] arrowheads)” in Figure 6(a), above. /d. Thus, a
`
`skilled artisan would have had good reasonto investigate synthetic analogs of
`
`human uroguanylin with a particular focus on optimizing the residues identified by
`
`Li as being ofparticular interest given their apparent involvement in
`
`ligand/receptorinteractions.
`
`136. Skilled artisans routinely made and screened large numbers of
`
`MSNExhibit 1002 - Page 71 of 129
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`
`-67-
`
`

`

`synthetic analogs of naturally-occurring peptides, in part because of the wide
`
`availability and ease of peptide synthesis methods. As discussed above in Sections
`
`VII.D-E, multiple synthetic strategies were known androutinely used by skilled
`
`artisans for the development of synthetic analogs. For example, skilled artisans
`
`routinely sought to stabilize electrostatic pairing between hormone andreceptorto
`
`strengthen or increase binding potential. See Section VII.E.3, above. Skilled
`
`artisans also recognized naturally occurring homologs as promising avenuesfor
`
`activity- or stability-enhancing substitutions. See Section VII.E.2, above.
`
`137. When preparing synthetic analogs to human uroguanylin, a skilled
`
`artisan would have had good reasonto look first to making conservative changes to
`
`the peptide. The target receptor, as Currie discloses, is part of a “group of proteins
`
`that share structural characteristics relative to the enzymatic function of producing
`
`cyclic GMP, but differ quite remarkably in their selective activation by ligands.”
`
`EX1005, 1:7-11. Thus, as Currie notes, the guanylate cyclases are only selectively
`
`activated by their ligands. Moreover, as described above in Section VII, skilled
`
`artisans routinely began analog synthesis with conservative substitutions to avoid
`
`causing immunogenicity or ablating activity altogether.
`
`138. By 2002, a skilled artisan would have had other good reasonsto look
`
`first to making conservative changes to the human uroguanylin peptide because the
`
`potential therapeutic acts as an agonist, not an antagonist. See EX1050, 68 (“if you
`
`MSN Exhibit 1002 - Page 72 of 129
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`
`-62-
`
`

`

`wish to design a drug to effect a certain response, an agonist would be desired; if
`
`you wish to design a drug to prevent a particular response ..., an antagonist would
`
`be required”).** Thestructural requirements for agonists are more stringent than
`
`those for antagonists because agonists need to “interact[] with [the receptor] in the
`
`specific way required to elicit a response,” while antagonists only need to “block{|
`
`a receptorsite.” /d., 69-70. “In general, there are great structural similarities among
`
`a series of agonists, butlittle structural similarity exists in a series of competitive
`
`antagonists.” Jd., 69. Making conservative substitutions, especially conservative
`
`substitutions consistent with homologous sequencesalready existing in nature, thus
`
`would have been particularly attractive to a skilled artisan as the relevant time.
`
`C. Currie and Li Suggest a Glu’ Substitution as in Rat
`Uroguanylin
`
`139. As noted above, in view of Currie and Li, a skilled artisan would have
`
`had good reason to seek out information aboutthe structure-function relationship
`
`of human and rat uroguanylin with respect to their activity on the humanreceptor,
`
`intestinal guanylate cyclase.
`
`140. Currie also gave good reasonto align rat uroguanylin to human
`
`uroguanylin. For example, Currie teaches that human uroguanylin acts on the rat
`
`“4 Silverman, R. B., Chapter 3: Receptors, THE ORGANIC CHEMISTRY OF DRUG
`DESIGN AND DRUG ACTION, (Academic Press, Inc.) 1992, 52-97 (“Silverman,”
`EX1050).
`
`MSN Exhibit 1002 - Page 73 of 129
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`
`69
`
`

`

`receptor, which is displayed on the endothelialcells of rat tissue. EX1005, 2:16-21
`
`(“Human uroguanylin has been further demonstrated to act in an isolated intestinal
`
`rat preparation to stimulate an increase in short circuit current.”).
`
`141. Lialso provided good reason to look to the alignment of human and
`
`rat uroguanylin for promising substitutions for human uroguanylin. Li discloses the
`
`amino acid sequences of various mammalian uroguanylins and aligns their
`
`sequencesto illustrate their homology. EX1006, FIG. 6(a). As can be seen below,
`
`Li teaches that rat and human uroguanylin have substantial overlap:
`
`T/E
`
`QP
`
`v
`
`<zoxrzanB<4OxAnaAAnAnoCOM<” = =i
`NTVVHAVSO+
`
`rat uroguany! in
`opossum uroguany! in
`human uroguany! in
`rat guany!in
`opossum guany! in
`human guany! in
`mouse guany!in
`E Coli STa
`
`EX1006, FIG. 6(a) (annotated with yellow highlight for ease of comparing therat
`
`and human uroguanylin sequences).
`
`142.
`
`In view of teachings of Currie and Li, a skilled artisan would have had
`
`good reason to look to the amino acidsthat differ between rat and human
`
`uroguanylin sequencesin identifying promising, conservative aminoacid
`
`substitution in designing a synthetic human uroguanylin analog. Indeed, a skilled
`
`artisan would have knownthat rat uroguanylin was highly likely to stimulate the
`
`humanreceptor. As discussed above in Section VII, well before 2002, skilled
`MSN Exhibit 1002 - Page 74 of 129
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`

`artisans understood that conformation fit determined receptor-ligand binding, and
`
`that such fit may depend on pairing of hydrophobic groups betweenthe ligand and
`
`receptor and/or pairing of charged groups with opposite charge. Because human
`
`uroguanylin bindsto the rat receptor, as Currie teaches, a skilled artisan would
`
`have reasonably expected that human uroguanylin andthe rat receptor have a good
`
`conformationalfit. See EX1005, 2:17-19. So too for the binding of the human
`
`ligand-receptor and the rat ligand-receptor.
`
`143. Moreover, Li demonstrates that rat uroguanylin activates cyclic GMP
`
`synthesis in human T84 cells, providing further evidencefor the activity of the rat
`
`peptide on the human receptor. EX1006, 46-47, 54. Because human uroguanylin
`
`binds to both the human and rat receptor, and the rat receptor binds to both human
`
`and rat uroguanylin, a skilled artisan would have had goodreason to reasonably
`
`expect conformational fit between rat uroguanylin and the humanreceptor.
`
`144. Applying the principle of conservative substitutionsfirst, a skilled
`
`artisan would have read Li’s homology as a map. This homology showsthat
`
`uroguanylin orthologs generally do not vary much among mammals, with the third
`
`position being a notable and rare exception that a skilled artisan would have readily
`
`focused on. See EX1006, FIG. 6(a). Indeed, Li expressly notes that “[o]f particular
`
`interest are two residuesthat are basic or uncharged in guanylin but acidic in
`
`uroguanylin (stippled arrowheads)”in Figure 6(a), which I have annotated again
`
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`71.
`
`

`

`below, highlighting in blue the stippled arrowheadsindicating the residues of
`
`interest identified by L1. Jd.
`
`T/E I A
`
`L
`
`FNT
`<zorzenB<oAaAaAa3ng3OMm|t
`
`Q N P S P
`
`rat uroguany!in
`opossum uroguany! in
`human uroguany! in
`rat guany!lin
`opossum guany! in
`human guany! in
`p
`mouse guany!in
`ra
`Pp
`
`meerY€E Coli STa
`
`EX1006, FIG. 6(a) (annotated).
`
`145. As can be seen above,the residues “of particular interest” are those
`
`that are aligned with the second andthird positions of the human uroguanylin
`
`sequence. While the second position (left blue arrow)is an aspartic acid that is
`
`conserved betweenthe rat and human uroguanylin sequence, the third position
`
`(right blue arrow)is not. The two sequences instead demonstrate a Glu’
`
`substitution, in which the aspartic acid appearing at the third position in human
`
`guanylin, highlighted in red above,is instead a glutamic acid (E/Glu)in the aligned
`
`rat uroguanylin sequence, also highlighted in red. Thus, the Glu’ substitution of
`
`human uroguanylin wasa natural, expected, and conventional substitution that a
`
`skilled artisan would have readily identified in view of the priorart.
`
`146. Because a skilled artisan would have had good reasonto believe that
`
`[Glu*]-human uroguanylin would be an effective analog of human uroguanylin and
`MSNExhibit 1002 - Page 76 of 129
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`-72-
`
`

`

`that there was sufficient tolerance for this substitution at the third amino acid
`
`position, they would have had good reason to replace the aspartic acid at the third
`
`amino acid position of human uroguanylin with glutamic acid, as disclosed in Li.
`
`EX1006, 54, FIG. 6(a).
`
`147. A skilled artisan would have had a reasonable expectation of success
`
`in making the [Glu*]-human uroguanylin analog. Andas | noted previously in this
`
`declaration, as a general principle, conservative substitutions, such as changing
`
`aspartic acid to glutamic acid, are typically favored to maintain or even increase
`
`activity without undue toxicity.
`
`148. Thus, for the reasons discussed above, a skilled artisan’s attention
`
`immediately would have been drawnto the only three aminoacid positions on
`
`uroguanylin that were available for substitution while preserving the mammalian
`
`consensus sequencefor uroguanylin. The three conservative mammalian
`
`substitutions disclosed by Li were Asp*-to-Glu’ (human > opossum), Asp*-to-Glu*
`
`(human > rat), and Val®-to-Ile* (human > opossumorrat). See EX1006, FIG. 6(a).
`
`Prominent amongthese three, Li specifically discloses the Glu’ substitution thatis
`
`the only difference between the human uroguanylin disclosed in Currie and the
`
`claimed peptide in Shailubhai. Li suggests that this substitution would result in a
`
`functional human uroguanylin analog, noting that rat uroguanylin “follows the
`
`consensus sequence of uroguanylin rather than that of guanylin, and thus we would
`
`MSNExhibit 1002 - Page 77 of 129
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`73
`
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`

`expectits affinity to be comparable to that of opossum or human uroguanylin.” /d.,
`
`54.
`
`149. A skilled artisan also would have reasonably expected that applying
`
`this substitution would maintain or improvethe efficacy of human uroguanylin. As
`
`described above, skilled artisans knew that human and rat uroguanylin hadactivity
`
`against the human receptor. As Li demonstrates, rat and human uroguanylin are
`
`homologs. See EX 1006, FIG. 6(a). And a skilled artisan would reasonably expectat
`
`least some recombinants to exceed the activity of human uroguanylin. The Glu*
`
`substitution is one such recombinant, but because of the high homology between
`
`humanandrat uroguanylin, possible recombinantsare finite. A skilled artisan
`
`would haveapplied the well-known assays Currie recites to confirm the therapeutic
`
`activity of [Glu*]-human uroguanylin with a reasonable expectation of success.
`
`These long-knownassays are summarized above in Section IX.A, and discussed in
`
`even moredetail in Section VII.
`
`150.
`
`I also note that by 2002, skilled artisans also had identified that mouse
`
`uroguanylin sequencesalso have the same Glu’ substitution found in rat
`
`uroguanylin. EX1051, Fic. 1B, 1001-02.A skilled artisan would have understood
`
`4 Whitaker, T. L., et al., Uroguanylin and Guanylin: Distinct but Overlapping
`Patterns ofMessenger RNA Expression in Mouse Intestine, GASTROENTEROLOGY,
`113, 1997, 1000-1006 (“Whitaker,” EX1051).
`
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`7
`
`-74-
`
`

`

`this ubiquity as showing broad functionality for that particular modification with
`
`respect to the intended target receptor.
`
`151.
`
`Ialso note that while Li also aligns a sequence of opossum
`
`uroguanylin in Figure 6(a), there are several reasons whya skilled artisan would
`
`have focusedfirst on comparing human uroguanylin with rat (and mouse)
`
`uroguanylin in designing analogs. Among them, humans were known to be more
`
`closely related to mice and rats than to opossums, which are marsupials. See, e.g.,
`
`EX1052, 616, Fic. 1.*° Figure 1 of Murphy, reproduced below, showsa
`
`phylogenetic mapping of mammals. Both human (“Primates”) and rats
`
`(“Rodentia”) are in the sameclade(blue text, highlighted with blue outline).
`
`Possum (“Marsupialia’’) are as far as possible from humanswhilestill being
`
`mammals, in a separate clade (black text, highlighted with red outline).
`
`“© Murphy, W.J., et al., Molecular Phylogenetics and the Origins ofPlacental
`Mammals, NATURE, 409, 2001, 614-618 (“Murphy,” EX1052).
`
`MSN Exhibit 1002 - Page 79 of 129
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`
`-75-
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`

`

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`
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`Cavia *
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`Myocastor
`Dinamys
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`Muscardinus
`
`IS
`1
`'
`
`e
`
`88
`
`EX1052, 616, Fic. 1 (annotated).
`
`152. Moreover, uroguanylin signaling was known to beespecially similar
`
`in humansand rats. EX1053, 1331-32.4” For example, Forte 1999 confirmsthat the
`
`“GC-C receptors ofthe rat are more closely related to human and pig GC-C in the
`
`ligand-binding domainswith these proteins sharing ~70% identity in this region.”
`
`47 Forte, L. R., et al., Guanylin Peptides: Cyclic GMP Signaling Mechanisms,
`Braz. J. MED. BIOL. RES., 32, 1999, 1329-1336 (“Forte 1999,” EX1053).
`
`MSNExhibit 1002 - Page 80 of 129
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`
`-76-
`
`

`

`Id., 1332.
`
`D. The Known Chemical Properties of Uroguanylins and Amino
`Acids Would Have Further Provided a Skilled Artisan with a
`Reasonable Expectation of Improving Constipation-Related
`Activity through Glu? Substitution
`
`153. Currie teaches that natural human uroguanylin outclasses natural
`
`human guanylin for constipation-related biochemical activity. EX1005, 6:12-22
`
`(“Human uroguanylin appeared to be more potent than human guanylin.”); see also
`
`id., 2:6-24. One yearlater, Li provided mechanistic and biochemicalinsight as to
`
`why.
`
`154. Li’s mechanistic insight has to do with the different kinds of ionizable
`
`groupspresent on the aminoacids of the guanylin and uroguanylin orthologs that
`
`align with the second and third positions of human uroguanylin. This position of
`
`interest was expressly identified by Li andis indicated by the two stippled arrows
`
`present in Figure 6(a). /d., 54. I have reproducedthis figure below for convenience,
`
`highlighting the stippled arrowsin blue. I have highlighted the uroguanylin
`
`orthologs in yellow and the guanylin orthologs in purple.
`
`T/E
`
`OAAAAOOMG = —
`<zoxrzonb
`>NTDVGUMOV]ZO—4
`
`
`ey
`BY
`
`L
`
`rat uroguany! in
`opossum uroguany! in
`human uroguany! in
`rat guanylin
`opossum guany! in
`human guany! in
`mouse guany! in
`jE Coli STa
`
`MSNExhibit 1002 - Page 81 of 129
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`
`

`

`EX1006, 52, FIG. 6(a) (annotated).
`
`155.
`
`In the uroguanylin orthologs (yellow highlighting), these positions are
`
`filled only by either aspartic acid (D) or glutamic acid (E). /d., 54. For example,
`
`human uroguanylin has aspartic acid (D) at both the second and third positions. /d.,
`
`FIG. 6(a). Rat uroguanylin has an aspartic acid (D) followed by a glutamicacid (E).
`
`Id. Opossum uroguanylin also displays only aspartic or glutamic acid residues at
`
`these positions. /d.
`
`156. Aspartic and glutamic acid are the only two aminoacidsthat possess
`
`an acidic side chain. That is, both of these amino acids possess carboxylic acid
`
`groups that become deprotonated under certain physiological conditions. For
`
`convenience, andto assist in visualizing these aminoacids, I again provide the
`
`chemical structures of these residues below, shownin their deprotonatedstate.
`
`i
`H
`NL
`
`H
`NO AL
`
`co,"
`
`CO
`
`Glu
`
`Asp
`
`157. Fundamental organic chemistry teaches that carboxylic acid groups (-
`
`COH) are neutral whenthey are protonated (i.e., have an H as in -COoH). This
`
`happens underacidic conditions, predominantly where the pH of the environment
`
`is lower than the acid strength (pKa) of the -CO2H in a given carboxylic acid.
`
`:
`
`-78-
`
`MSNExhibit 1002 - Page 82 of 129
`MSNv. Bausch - IPR2023-00016
`
`

`

`Whena carboxylic acid’s pKa equals the pH, it will be 50% protonated, i.e., half of
`
`the molecules will have that carboxylic acid protonated (-CO2H) and half will be
`
`deprotonated (-CO>’), just as the glutamate and aspartate residuesare in the
`
`structures I provided above. When the pH becomeshigher(i.e., more basic) than
`
`the carboxylic acid’s pKa, the deprotonated, negatively charged state (-CO2") will
`
`dominate. EX1012, 97-99; see also Section VII.
`
`158. On the other hand, the guanylin orthologs, which are highlighted in
`
`the annotated Figure 6(a) above in purple, bear aminoacidsat these positions that
`
`have either neutral (uncharged) or positively charged (cationic) chemical groups
`
`under physiological conditions. EX1006, 52, FIG. 6(a); id., 54.
`
`159. Li expressly identifies this difference, noting, “[o]f particular interest
`
`are two residuesthat are basic or uncharged in guanylin but acidic in uroguanylin
`
`(stippled arrowheads).” /d., 54. Li further expressly notes that “affinity of GCC for
`
`uroguanylin (opossum or human),” which have only aspartic or glutamic acid at
`
`these important positions, “is about 10-fold higher than its affinity for guanylin (rat
`
`or human),” which donot. /d. (noting these “features that are found in uroguanylin,
`
`but not in guanylin, offer information aboutstructural elements that specify the
`
`strength of the ligand/receptor interaction”). Thus, in view ofLi, a skilled artisan
`
`would have understood that the acidity and ionizability of the amino acids at these
`
`positions of interest were important to uroguanylin’s increased activity as
`
`MSN Exhibit 1002 - Page 83 of 129
`MSNv. Bausch - IPR2023-00016
`
`79
`
`

`

`comparedto guanylin.
`
`160. As discussed above,in view of the teachings of Currie and Li, a skilled
`
`artisan would have had goodreason to make [Glu*]-human uroguanylin, and would
`
`have done so with a reasonable expectation of success in maintaining or improving
`
`the efficacy of human uroguanylin. This substitution would have been further
`
`supported by these known chemical properties of aspartic acid (Asp/D) and glutamic
`
`acid (Glu/E), as well as what was knownaboutthe structure/activity relationship in
`
`uroguanylins.
`
`161. More specifically, in view of the teachings of Li, a skilled artisan
`
`would avoid substituting substitute an aminoacid that did not bear an acidic side
`
`chain for the aspartic acid at the 3-position. However, a skilled artisan would have
`
`reasonably expected that a Glu’ substitution, which preserves the acidic amino acid
`
`that Li indicated was responsible for the increased activity of uroguanylin over
`
`guanylin, would maintain or even enhanceactivity.
`
`162. Hamra 1997 confirms that this is especially so in relatively acidic
`
`environments (pH 6-7) in the human large intestine as well as more acidic regions
`
`of the microenvironmentofintestinal mucosa (pH 5), both target tissues for
`
`treating clinical constipation using the receptor at issue. For example, Hamra 1997
`
`teaches that uroguanylin shows 10-fold greater affinity for T84 cells at a pH of 5.0
`
`comparedto a higher pH of 8.0. EX1021, 2707, Fic. 1; id., 2709. In contrast,
`
`MSNExhibit 1002 - Page 84 of 129
`MSNv. Bausch - IPR2023-00016
`
`-80-
`
`

`

`guanylin was knownto be 10-fold more potent at pH 8.0 than pH 5.0. /d. Thus,
`
`“Ta]t an acidic mucosal pH of 5.0, uroguanylin is 100-fold more potent than
`
`guanylin.” /d., 2705.
`
`163. Like Li, Hamra 1997 related this activity difference to the “striking
`
`difference” of “the appearance of two acidic aminoacids at the N terminus of
`
`uroguanylin,” (7.e., positions 2 and 3 of human uroguanylin) comparedto the lack
`
`of acidic residuesat this position in guanylin. EX1021, 2709. Hamra 1997 also
`
`notes that “[d]eletion of the N-terminal acidic aminoacids in uroguanylin,” i.e.,
`
`positions 2 and 3, “demonstrated that these residues are responsible for the increase
`
`in binding affinities that are observed for uroguanylin at an acidic pH.” /d., 2705;
`
`see also id., 2709 (similar). Hamra 1997 thus confirms the skilled artisan’s
`
`understanding that the acidic amino acidsat positions 2 and 3 in human
`
`uroguanylin were directly involved in the increased bindingaffinity of this peptide
`
`to receptors in the tissues relevant to treating conditions such as constipation.
`
`164. Hamra 1997 explainsthat “[iJt is likely that acidic conditions
`
`influence the ionization and/or conformationalstate of the uroguanylin molecule as
`
`a molecular mechanism for the increased biological activity of uroguanylin in this
`
`circumstance.” EX1021, 2709. A skilled artisan would have understood this
`
`commenton ionization to refer to the degree of protonation of the acidic amino
`
`acids of interest. A skilled artisan also easily would have recognizedthat, at the
`
`81
`
`MSNExhibit 1002 - Page 85 of 129
`MSNv. Bausch - IPR2023-00016
`
`

`

`acidic mucosal pH of 5.0, the acidic side-chain of Asp, which has a pKaof3.65 is
`
`mostly deprotonated, while the acidic side-chain of Glu, which has a pKaof4.25,
`
`would be comparably more protonated. See EX1012, 118, Table 5-1 (providing
`
`pKas of amino acids). Given the increased activity of uroguanylins at acidic pHs,
`
`in which a higher percentage of these acidic residues would be protonated, a
`
`skilled artisan would have reasonably suspected that making a substitution that
`
`allowed for a higher degree of protonation at these acidic residues would yield a
`
`further improvementin activity.
`
`165. More specifically, a skilled artisan would have recognizedthat the
`
`[Glu*]-human uroguanylin analog, made obvious by Currie and Li, would be
`
`comparably more protonated than native human uroguanylin in acidic
`
`environments in the large intestine and intestinal mucosa comparedto native
`
`human uroguanylin. Thus, a skilled artisan would have reasonably expected [Glu*]-
`
`human uroguanylin to have improved activity compared to native human
`
`uroguanylin in these environments.
`
`166. Moreover, a skilled artisan also would have reasonably expected that
`
`the comparably moreacidic native human uroguanylin leaves many receptors
`
`under-stimulated in less acidic sections of the intestine. For example, Fan teaches
`
`that uroguanylin is “a more effective agonist for regulating receptor-GC activity”
`
`whenthe “lumen of the intestine and the mucosal (microclimate) surface is
`
`MSNExhibit 1002 - Page 86 of 129
`MSNv. Bausch - IPR2023-00016
`
`39
`
`

`

`acidified when chyme containing HCI enters the duodenum.” EX 1016, E963; see
`
`also EX1019, G714. Increasing the degree to which a uroguanylin analog may be
`
`protonated at comparably less acidic pHs merely involves swapping the more
`
`acidic Asp residue for the more easily protonated Glu residue. The Glu*
`
`substitution thus broadensthe intestinal zone of high, or protonated, uroguanylin
`
`activity. This more permissive protonation allows for protonated [Glu*]-human
`
`uroguanylin to maximally activate receptors further away from human
`
`uroguanylin’s native maximal zone wherethe intestinal mucosalis acidified by
`
`chyme. EX1016, E963.
`
`167. A skilled artisan would have recognized the desirability of enhanced
`
`affinity of [Glu*]-human uroguanylinin these areas ofthe large intestine and
`
`intestinal mucosa because it was knownthat the large intestine is the primary target
`
`for relieving clinical constipation using human uroguanylin an

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