115
`United States Patent
`5,489,670
`[11] Patent Number:
`
`[45] Date of Patent: Feb. 6, 1996
`Currie et al.
`
`CNOA ATAAAT
`
`US005489670A
`
`[54] HUMAN UROGUANYLIN
`
`[75]
`
`Inventors: Mark G. Currie, St. Charles;
`Toshihiro Kita, Creve Coeur; Kam F.
`Fok,St. Louis; Christine E. Smith,
`Manchester, all of Mo.
`
`[73] Assignee: G. D. Searle & Co., Chicago,Ill.
`
`de Sauvage et al., Proc. Natl. Acad. Sci. 89: 9089-9093
`(1992).
`Kuhnet al., FEBS Lett. 318: 205-209 (1993).
`Wiegand et al., FEBS Lett. 311: 150-154 (1992).
`Savarino et al., Proc. Natl. Acad. Sci. 90: 3093-3097 (1993).
`Wiegand et al., Biochem. Biophys. Res. Commun. 185:
`812-817 (1992).
`Schulz et al., J. Biol. Chem. 267: 16019-16021 (1992).
`
`[21] Appl. No.: 145,940
`
`[22]
`
`Filed:
`
`Oct. 29, 1993
`
`TS1]
`
` Eint, C1o oneeeccccecessesseceee A61K 38/00; CO7K 5/00;
`CO7K 7/00; CO7K 4/00
`assess 530/326
`
`[52] U.S. Che
`seseseseesssecessseessssessnsssenseeseraneesss 530/326
`[58] Field of Search
`
`Primary Examiner—Jill A. Warden
`Assistant Examiner—Sheela J. Huff
`Attorney, Agent, or Firm—Dennis A. Bennett
`
`[57]
`
`ABSTRACT
`
`A novel peptide is disclosed which is useful for the control
`of intestinal fluid absorption and that has the following
`amino acid sequence
`References Cited
` [SEQ ID NO:1]
`U.S. PATENT DOCUMENTS
`
`Asn— Asp—~ Asp—Cys—Glu—Leu—Cys—Val—Asn— Val
`1
`5
`10
`5,140,102—8/1992 Currie aveccccsessceecssectsenenssreens 530/36
`Ala—Cys—~Thr —Gly—Cys —Leu
`15
`
`[56]
`
`OTHER PUBLICATIONS
`
`Schulz et al., Cell 63: 941-948 (1985).
`Yoshimura et al., FEBS Lett. 18]: 138-142 (1985).
`Currie et al., Proc. Natl. Acad. Sci. 89: 947~—951 (1992).
`
`2 Claims, 4 Drawing Sheets
`
`MSN Exhibit 1005 - Page 1 of 9
`MSNv. Bausch - IPR2023-00016
`
`

`

`U.S. Patent
`
`Feb.6, 1996
`
`Sheet 1 of 4
`
`5,489,670
`
`i
`insulin
`
`APIII
`
`
`
`
`
`
`
`
`guanylin
`
`|
`
`|
`
`(lartatnanatllanttana:
`
`20
`
`30
`
`40
`Fraction number
`
`50
`
`60
`
`Fig.
`
`1
`
`MSNExhibit 1005 - Page 2 of 9
`MSNv. Bausch - IPR2023-00016
`
`
`Vo
`
`1000
`
`@=
`
`= 600
`
`400
`
`2000
`
`E o
`
`=3
`
`— 800 |
`
`

`

`(fmol/well)
`10000
`
`0.04
`
`0.03
`
`0.01
`
`15000
`
`5000
`
`)
`
`Absorbanceat220nm(----
`cGMP
`
`US. Patent
`
`Feb. 6, 1996
`
`Sheet 2 of 4
`
`5,489,670
`
`
`
`%CHaCN(---
`
`90
`
`100
`
`120
`
`130
`
`110
`Time (min)
`Fig. 2
`
`MSN Exhibit 1005 - Page 3 of 9
`MSNv. Bausch - IPR2023-00016
`
`

`

`US.
`
`Patent
`
`Feb. 6, 1996
`
`Sheet 3 of 4
`
`5,489,670
`
`106
`
`TTTTN
`
`110 +
`
`90 +
`
`704
`
`50 4
`
`x0.
`
`
`
`cGMP(fmoles/well)
`
`
`
`125|-STaBound,%ofBo
`
`——t— Human Guanylin
`
`0
`
`-10
`
`-9
`
`-8
`
`-7
`
`-6
`
`TTTTT
`—-oO— §$Ta TTt ——®— Human Uroguanylin
`— o aoTtTItttit1a
`10 5
`
`-10SS
`0
`-10
`-9
`-8
`-7
`-6
`-5
`[Peptide],
`log (M)
`
`Fig. 3B
`
`MSN Exhibit 1005 - Page 4 of 9
`MSNv. Bausch - IPR2023-00016
`
`

`

`U.S. Patent
`
`Feb. 6, 1996
`
`Sheet 4 of 4
`
`5,489,670
`
`5 minutes
`
`Uroguanylin [0.2 pM]
`
`Fig. 4
`
`MSNExhibit 1005 - Page 5 of 9
`MSNv.Bausch - IPR2023-00016
`
`
`
` ,
`
`50 pA/cm2
`
`

`

`5,489,670
`
`1
`HUMAN UROGUANYLIN
`
`BACKGROUNDOF THE INVENTION
`
`This invention relates to a novel peptide and, more
`particularly, to a sedecapeptide that is an endogenousregu-
`lator of intestinal guanylate cyclase.
`Guanylate cyclase is composedof a group ofproteinsthat
`share structural characteristics relative to the enzymatic
`function of producing cyclic GMP, but differ quite remark-
`ably in their selective activation by ligands. The three major
`forms of guanylate cyclase are the soluble, particulate, and
`intestinal (cytoskeletal-associated particulate or STa-sensi-
`tive) with each of these forms regulated by different ligands
`(1,2). Activation of the soluble guanylate cyclase occursin
`response to nitric oxide (EDRF), while activation of the
`particulate enzyme occurs in response to the natriuretic
`peptides(atrial natriuretic peptide, brain natriuretic peptide,
`and C-type natriuretic peptide) (1,2). An endogenousacti-
`vator of the intestinal guanylate cyclase only recently been
`identified. The first known endogenousactivator was termed
`guanylin (3). However, the heat stable enterotoxin from E.
`coli has been knownto selectively activate this form of the
`enzyme (4,5). This form of the enzyme is predominantly
`foundin the intestinal epithelial cells with the largest num-
`ber of receptors oriented towards the lumen(1,2). Recently,
`the intestinal form of guanylate cyclase has been cloned and
`expressed from rat small intestinal mucosa (6). This enzyme
`is characterized by an extracellular receptor binding region,
`a transmembraneregion, an intracellular protein kinase-like
`region and a cyclase catalytic domain (6).
`Pathogenic strains of E. coli and other bacteria produce a
`family of heat stable entertoxins (STs) thatactivate intestinal
`guanylate cyclase. STs are acidic peptides 18~19 amino
`acids in length with six cysteines and three disulfide bridges
`that are required for full expression of bioactivity (7). The
`increase of intestinal epithelial cyclic GMPelicited by STs
`is thought to cause a decrease in water and sodium absorb-
`tion and an increase in chloride secretion (8,9). These
`changes in intestinal fluid and electrolyte transport then act
`to cause secretory diarrhea. In developing countries,
`the
`diarrhea due to STs is the cause of many deaths, particularly
`in the infant population (10). STs are also considered to be
`a major cause of traveler’s diarrhea in developed countries
`(11). STs have also been reported to be a leading cause of
`morbidity in domestic animals (12).
`
`BRIEF DESCRIPTION OF THE INVENTION
`
`In accordance with the present invention a novel peptide
`is provided which has the following amino acid sequence.
`(NDDCELCVNVACTGCL)
`
`[SEQ ID NO:1]
`Asn— Asp— Asp —Cys—Glu—Leu—Cys — Val—Asn—Val—
`1
`5
`10
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`Ala—Cys —Thr--Gly —Cys—Leu
`15
`
`35
`
`This peptide, also referred to herein as human uroguany-
`lin, has been isolated from human urine and has been
`chemically 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.
`
`The peptide of this invention has been both isolated and
`chemically synthesized in a homogeneously purified form
`which did not exist in human urine from which it was
`initially obtained. That is, it has been prepared in a form
`
`60
`
`65
`
`2
`which is essentially free of other low molecular weight
`peptides, and free from higher molecular weight material
`and other cellular components and tissue matter. This novel
`peptide has physiological characteristics which suggest that
`it is important to medical sciencein the study of regulators
`of guanylate cyclase. In particular, the novel peptide of this
`invention is an endogenousstimulator of intestinal guanylate
`cyclase. It has been found to stimulate increases in cyclic
`GMPlevels in a manner similar to guanylin and the STs. As
`such regulator,
`it
`is useful for the control of intestinal
`absorption. It has potential to regulate fluid and electrolyte
`transport. Human uroguanylin also has been foundto dis-
`place heat stable enterotoxin binding to cultured T84 human
`colon carcinomacells. This cell line is knownto selectively
`respond to the toxin in a very sensitive manner with an
`increase in intracellular cyclic GMP.
`Human uroguanylin has been further demonstratedto act
`in an isolated intestinal rat preparation to stimulate an
`increase in short circuit current. This action is believed to be
`the physiologic driving force foreliciting chloride secretion
`and ultimately decreased water absorption. The human
`uroguanylin may thus act as a laxative and be useful in
`patients suffering from constipation, e.g. cystic fibrosis
`patients who suffer with severe intestinal complications
`from constipation.
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`While the specification concludes with claimsparticularly
`pointing out and specifically claiming the subject matter
`regarded as forming the present invention,it is believed that
`the invention will be better understood from the following
`detailed description of preferred embodiments taken in
`conjunction with the accompanying drawings in which:
`FIG. 1. Purification of uroguanylin from human urine by
`gel filtration chromatography. The extract of 5 liters of
`human urine was applied to 2.6x94 cm sephadex G-25
`(superfine) gel filtration column. Isocratic SO mM ammo-
`nium acetate was used to elute peptides at a rate of 0.5
`ml/min and 5 ml of fractions were collected after 100 ml of
`initial elution. Molecular weight standards were separately
`assessed (Vo: blue dextran 200, insulin (MW 5750), atrio-
`peptin TI (AP II, MW 2550), rat guanylin (MW 1516)). All
`fractions were assessed in T84 cell cyclic GMP accumula-
`tion bioassay.
`FIG. 2. Purification of uroguanylin from human urine by
`reverse-phase HPLC.Fiveliters of human urine extract was
`purified through the semipreparative reverse-phase HPLC
`and active fraction was fractionated on an analytical C,,
`column (Vydac). A linear gradient of 10% to 40% acetoni-
`torile, 0.1% TFA was developed at 1.0 ml/min over 3 hrs.
`One min fractions were collected and assayed foractivity in
`T84 cell cyclic GMP bioassay. This figure shows the bio-
`logical active region with two peaks associated with changes
`in UV absorbance.
`
`FIG. 3(a) and (b). FIG. 3(a) Concentration-response
`effect of synthetic human uroguanylin, human guanylin and
`E. coli ST;.13 (STa) on cyclic GMPlevels in T84 cells. The
`cells were incubated with various concentrations of ligands
`for 30 min. Values represent mean+SE (n=4). FIG. 3(b)
`Displacementof **°I-STa specific binding from T84 cells by
`human uroguanylin, human guanylin and STa. Cells were
`incubated for 30 min at 37° C. with labeled STa and
`indicated concentrations of ligands. Specific binding
`(%)
`was determined by dividing the specifically bound ‘*°I-STa
`at each ligand concentration by the specifically bound 1?°]-
`STa in the absence of ligands. Each determination represents
`the mean of four wells examined.
`
`FIG. 4. Effect of synthetic human uroguanylin on short-
`circuit current (Isc) of rat colon. Effect of human urogua-
`MSNExhibit 1005 - Page 6 of 9
`MSNv.Bausch - IPR2023-00016
`
`

`

`5,489,670
`
`3
`nylin (0.2 uM) on Isc across rat proximal colon after a
`mucosal addition. The response is characteristic of results
`from 3 other experiments.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`The novel peptide of this invention can be prepared by
`knownsolution and solid phase peptide synthesis methods.
`In conventional solution phase peptide synthesis,
`the
`peptide chain can be prepared by a series of coupling
`reactions in which the constituent amino acids are added to
`the growing peptide chain in the desired sequence. The use
`of various N-protecting groups, e.g. the carbobenzyloxy
`grouporthe t-butyloxycarbony] group (BOC), various cou-
`pling reagents, e.g., dicyclohexylcarbodiimide or carbon-
`yldimidazole, various active esters, e.g., esters of N-hydrox-
`yphthalirnide or N-hydroxy-succinimide, and the various
`cleavage reagents, e.g., trifluoroacetic acid (TFA), HC1 in
`dioxane, borontris-(trifluoracetate) and cyanogen bromide,
`arid reaction in solution with isolation and purification of
`intermediates is well-knownclassical peptide methodology.
`The preferred peptide synthesis method follows conven-
`tional Merrifield solid-phase procedures. See Merrifield, J.
`Amer. Chem. Soc. 85, 2149-54 (1963) and Science 150,
`178-85 (1965). This procedure, though using many of the
`same chemical reactions and blocking groups of classical
`peptide synthesis, provides
`a growing peptide chain
`anchored by its carboxy terminusto a solid support, usually
`cross-linked polystyrene, styrenedivinylbenzene copolymer
`or, preferably, p-methylbenzhydrylamine polymer for syn-
`thesizing peptide amides. This method conveniently simpli-
`fies the number of procedural manipulations since removal
`of the excess reagents at each step is effected simply by
`washing the polymer.
`The acyl group on the N-terminus is conveniently intro-
`duced byreaction of an alkanoic anhydride with the peptide
`on the solid support after deprotection with TFA.
`Further background information on the established solid
`phase synthesis procedure can be had by reference to the
`treatise by Stewart and Young, “Solid Phase Peptide Syn-
`thesis,” W. H. Freeman & Co., San Francisco, 1969, and the
`review chapter by Merrifield in Advances in Enzymology,
`32, pp. 221-296, F. P. Nold, Ed., Interscience Publishers,
`New York, 1969; and Erickson and Merrifield, The Proteins,
`1 Vol. 2, p. 255 et seq. (ed. Neurath and Hill), Academic
`Press, New York, 1976.
`All references, patents or applications. U.S. or foreign,
`cited in the application are hereby incorporated by reference
`as if written herein.
`
`In order to further illustrate the invention, the following
`exemplary laboratory preparative work was carried out.
`However, it will be appreciated that the invention is not
`limited to these examples or the details described therein.
`
`EXAMPLE1
`
`Materials and Methods
`Cell Culture. A cultured human colon carcinomacell line
`(T84) was obtained from the American Type Culture Col-
`lection (Rockville, Maryland)
`(ATCC No. CCL 248) at
`passage 52. Cells were grown to confluency in 24-well
`culture plates with a 1:1 mixture of Ham’s F12 medium and
`Dulbecco’s modified Eagle’s medium (DMEM), supple-
`mented with 10% fetal calf serum, 100 IU/mlpenicillin, and
`100 pg/ml streptomycin. Cells were used at passages 54-60.
`Cyclic GMP determination. Monolayers of T84 cells in
`24-well plates were washed twice with 1 ml/well DMEM,
`then incubated at 37° C. for 10 min with 0.5 mi DMEM
`
`10
`
`15
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4
`containing 1 mM isobutylmethylxanthine, a phosphodi-
`esterase inhibitor. Agents and fractions were then added for
`the indicated time as described in theresults section, below.
`The media wasthen aspirated and the reaction terminated by
`the addition of ice cold 0.5 ml of 0.1N HC1. Aliquots were
`then evaporated to dryness under nitrogen and then resus-
`pended in 5 mM sodium acetate buffer, pH 6.4. The samples
`were then measured for cyclic GMP by conventional RIA as
`described by Steiner et al. (13).
`Purification of Uroquanylin. Five separate batches of
`adult male human urine samples, 5 liters each, were col-
`lected and immediately placed on ice. The urine samples
`were applied to C,, Sap-Yak columns (Waters). The col-
`umns were washed with 10% acetonitrile/0.1% trifluoroace-
`tic acid (TFA)/H,O and eluted with 40% acetonitrile/0.1%
`TFA/H,O. The eluted peptide fraction was lyophilized and
`resuspended in 7 ml of distilled H,O and centrifuged at
`20,000xg for 20 min at 4° C. The resulting supernatant was
`separated by gel filtration chromatography (Sephadex G-25,
`superfine, 2.6x94 cm). The fractions were bioassayed and
`the active fraction was lyophilized. The sample was resus-
`pended in 1 mi of 10% acetonitrile/0.1% TFA/H,O and
`applied to a C,, semipreparative HPLC column (Vydac,
`Hasparia, Calif.). The column was developed with the
`following linear gradient: 10% acetonitrile/0.1% TFA/H,O
`to 40% acetonitrile/0.1% TFA/H,O in 120 minat a flow rate
`of 3 ml/min. The active fraction was lyophilized and resus-
`pended in 1 m! of 10% acetonitrile/0.1% TFA/H20. The
`sample was applied to a a C,, analytical HPLC column
`(Vydac) and active peptides were eluted using the above
`gradient over 180 min at a flow rate of 1 ml/min. Two active
`fractions were separately lyophilized and reconstituted in
`0.05 ml of 0.1% TFA/H,O. The samples were then sepa-
`rately applied to a C, microbore column (Applied Biosys-
`tems, Foster City, Calif.), eluted with an increasing gradient
`of 0.323% acetonitrile/min in 0.1% TFA/H,O. Two sepa-
`rately purified peptides of each batch were then subjected to
`sequence analyses.
`N-Terminal Protein Sequence Analysis. Automated
`Edman degradation chemistry was used to determine the
`N-terminal protein sequence. An applied Biosystems, Model
`470A gas-phase sequencer was employed for the degrada-
`tions (14) using the standard sequencer cycle, O3RPTH. The
`respective phenylthiohydantoin (PTH)-amino acid deriva-
`tives were identified by reverse-phase HPLC analysis in an
`on-line fashion employing an Applied Biosystems, Model
`120A PTH Analyzer fitted with a Brownlee PTH-C,, col-
`umn. Reduction and pyridylethylation for cysteine residue
`identification was performed directly on the filter (15).
`Electrospray Mass Spectrometry. The molecular weights
`of uroguanylin samples were determined by a previously
`described technique (3,16). Briefly, a triple quadrupole mass
`spectrometer equipped with an atmospheric pressure ion
`source was used to sample positive ions produced from an
`electrospray interface. Mass analysis of sample ions was
`accomplished by scanning the first quadrupole in 1 atomic
`mass unit increments from 1000 to 2400 atomic mass units
`in =3 s and passing mass-selected ions through the second
`and third quadrupoles operated in the rf-only mode to the
`multiplier. For maximum sensitivity, the mass resolution of
`the quadrupole mass analyzer was set so that ion signals
`were =2 atomic mass units wide at half peak height, but the
`centroid ofthe ion signal, still represented the correct mass
`of the ion.
`Radioligand Binding Assay. '**I-labeled ST;., (17°I-ST)
`was prepared by the Iodo-Gen method as previously
`described (3). T84 cell monolayers were washed two times
`with 1.0 ml per well of ice-cold binding assay buffer (Earle’s
`medium containing 25 mM 2{N-Morpholino] ethanesulfonic
`acid (MES), pH 5.5). The cells were incubated for 30 minat
`MSN Exhibit 1005 - Page 7 of 9
`MSNv.Bausch - IPR2023-00016
`
`

`

`5,489,670
`
`5
`37° C. in 0.5 ml per well of binding assay buffer with !7*1-ST
`(100,000 cpm per well) and unlabeled peptides. Then the
`cells were washed four times with 1 ml of ice-cold binding
`assay buffer and solubilized with 0.5 ml of 1M NaOH per
`well. This volume was transferred to tubes and assayed for
`radioactivity by a multigamma counter. Results
`are
`expressed as the percentage specifically bound.
`Measurementof Short-Circuit Current ([Sc) in Rat Colon.
`Rat proximal colon tissue, consisting of only mucosa and
`submucosa, was mounted between two Ussing half-cham-
`bers and bathed on both sides similar to previously reported
`(3). Electrical measurements were monitored with an auto-
`matic voltage clamp, and direct connecting voltage-and
`current-passing electrodes were used to measuretrans epi-
`thelial potential difference and Isc. Tissues were equilibrated
`under short-circuit conditions until Isc had stabilized.
`Chemical Synthesis of Uroquanylin. Uroguanylin was
`synthesized by the solid-phase method (17,18) on an
`Applied Biosystems Model 430A peptide synthesizer and
`purified by reverse-phase C,, chromatography. The purity
`and the structure were verified by analytical HPLC, amino
`acid composition analysis, mass spectroscopy, and sequence
`analysis.
`
`RESULTS
`
`In initial experiments, the peptide fraction of haman urine
`samples resulting from C,, Sep-Pak extraction was assayed
`for activity to increase cyclic GMPlevels in T84cells. These
`preliminary experiments strongly suggested the presence of
`guanylate cyclase stimulatory activity. The urine extract was
`subjected to fractionation by gel-filtration and a series of
`reverse-phase HPLC steps in order to producea sufficiently
`pure preparation for the purpose of structural determination.
`Fractionation by G-25gelfiltration chromatography yielded
`a single major bioactive fraction that migrated on the
`column with an apparent size of 5,000 daltons (FIG. 1).
`Subsequently, this active fraction was further purified by
`reverse-phase HPLC using a semipreparative C,, column
`and the bioactivity was determined to reside in only one
`fraction eluting at 27.8% acetonitrile/0.1% TPA/H,O (data
`not shown). Further purification by reverse-phase HPLC
`using a C,, analytical column yielded two active fractions
`that appeared to elute with peaks of substances that absorbed
`at 220 nm (FIG. 2). These two fractions were separately
`subjected to further characterization by microbore HPLC
`(Cg, column) and each fraction exhibited a single bioactive
`peak that absorbed at 220 nm (data not shown). The amino
`acid sequences of the two peaks were independently deter-
`mined by the Edman degradation procedure. The sequences
`are NDDCELCVNVACTGCL [SEQ ID NO:1]
`and
`DDCELCVNVACTGCL [SEQ ID NO:2], respectively for
`peaks 1 and 2. These two peptides are identical except that
`the peptide contained in peak one possesses an additional
`amino acid (asparagine) at the N-terminus. It is likely that
`peak two is a degradation product of peak 1, probably a
`result of aminopeptidase action. Electrospray massspectro-
`metric analysis of the two fractions yielded observed
`molecular weights of 1666.6 and 1552.6 atomic massunits,
`respectively for the peptides contained in peaks 1 and 2,
`respectively. These molecular weights correspond to the
`theoretical molecular weights derived from the sequencesif
`two disulfide bonds link the four cysteines, and therefore
`indicate that the full sequences of these peptides were
`determined by N-terminal protein sequence analysis.
`Comparison of the sequence of peak 1 with other proteins
`in the GenBank, National Biomedical Research Foundation,
`and SwissProt databases by computer-basedsearch indicates
`that this sequence is a unique sequence. This search did
`reveal that human uroguanylin shares homology with gua-
`
`20
`
`25
`
`35
`
`40
`
`45
`
`50
`
`355
`
`60
`
`65
`
`6
`nylin and ST. Thus, human uroguanylin appears to be a
`member of the guanylin/ST family of peptides.
`Chemical synthesis of bioactive human uroguanylin (the
`sedecapeptide) was accomplished by directed folding of the
`peptide. The synthetic bioactive peptide possessesdisulfide-
`linked bridges between the 4-12 and 7-15 amino acid
`positions. Analysis of the biological activity of human
`uroguanylin was assessed by determining its effect on T84
`cyclic GHP levels, competition binding studies with '5[-ST
`as the radioligand in T84 cells, and stimulation of Cl-
`secretion as reflected by increasesin Isc rat colon.
`Synthetic human uroguanylin caused a concentration-
`dependent
`increase in T84 cell cyclic GMP (FIG. 4a).
`Human uroguanylin appeared to be more potent than human
`guanylin, but less potent than ST foractivation of GC-C in
`T84 cells. A different profile of relative affinity was obtained
`using the competitive binding assay with '*5I-ST..,, as the
`radioligand. ST and human uroguanylin hadsimilar affinities
`for the receptors on T84 cells and human guanylin had a
`much loweraffinity (FIG. 4b). The data indicate that these
`peptides all possess the ability to stimulate GC-C and share
`similar binding sites with varying degrees ofrelative affini-
`ties for the receptors in T84 cells.
`To assess the effect of human uroguanylin on well char-
`acterized ST- and guanylin-sensitive transport functions, we
`assessed the effects of the peptide on Isc of proximal rat
`colon. In these experiments, the measurementofIsc is used
`as an indicatorof transepithelial chloride secretion. Previous
`studies in these preparations have indicated that the charge
`in Isc elicited by ST and guanylin is mostly accounted for by
`an increase in chloride secretion. Human uroguanyglin
`added to the mucosal reservoir of rat colon mounted in an
`Ussing chamber also caused a sustained rise in Isc (FIG. 5).
`Various other examples will be apparent to the person
`skilled in the art after reading the present disclosure without
`departing from the spirit and scope of the invention.It is
`intendedthat all such other examples be included within the
`scope of the appended claims.
`REFERENCES
`
`1. Singh, S. Lowe, K. G., Thorpe, D. S. Rodriquez, H.,
`Kuang, W.-J., Dangott, L. J., Chinkers, H., Goeddel, D. B.,
`and Garbers, D. L. {1988) Nature 334, 708-712.
`2. Waldman, S. A., and Murad, F, (1987) Pharmacologi-
`cal Reviews 39, 163-196.
`3. Currie, H. G., Fok, K. F. , Karo, J., Moore, R. J., Hamra,
`F. K. Duffin, K. L., and Smith, C. E. {1992) Proc. Nail.
`Acad. Sci. USA 89,947-951,
`4. Field, H., Graf, L. H., Laird, W. J., and Smith, P. L.
`(1978) Proc. Natl Acad. Sci. USA 75, 2800-2804.
`5. Guerrant, R. L., Hughes, J. M., Chang, B., Robertson,
`D.C., and Hurad, F. (1980) J. Infect Dis. 142, 220-228.
`6. Schulz, S., Green, C. K., Yuen, P. S. T., and Garbers,D.
`L. (1990) Cell 63,941-948.
`7. Yoshimura, S., Ikemura, H., Watanabe, H., Aimoto, S.,
`Shimonishi, Y., Hara, S., Takeda, T., Miwatani, T., and
`Takeda, Y. (1985) FEBS Letters 181, 138-142.
`8. Field, H., Rao, C. H., and Chang, E.B. (1980) New
`England J. Med. 321, 879-883.
`9. Guarino, A., Cohen, M., Thompson, M., Dharm-
`sathaphorn, K., and Giannella, R. (1987) Am. I. Physiol.
`253, G775G 780.
`10. Robins-Browne, R. M. (1987) Rev. Infect. Dis. 4, 28-
`53.
`
`11. Levine, M. M. (1987) J. Infect. Dis, 155, 377-389.
`12. Burgess, M. N., Bywater, R. J., Cowley, C. M.,
`Mullan, N. A., and Newsome, D. M. Infect. Immun. 21,
`526-531.
`MSNExhibit 1005 - Page 8 of 9
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`

`

`8
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`5,489,670
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`7
`13, Steiner, A. L., Paghara, A. S., Chase, L. R., and
`Kipnis, D. M. (1972) J. Biol. Chem. 247, 1114-1120.
`14. Hunkapiller, M. W., Hewick, R. M., Dreyer, R. J., and
`Hood, L. E. (1983) Methods Enzymol, 91, 399-413.
`15. Kruft, V., Ulrike, K., and Wittmann-Liebold, B.
`(1991) Anal. Biochem. 193, 306-309.
`16. Bruins, A. P., Covey, T. R., Henion, J. D. (1987) Anal.
`Chem. 59, 2642-2651.
`17. Merrifield, R. B.
`2149-2154.
`18. Tam, J. P, Wu, C.-R., Liu, W., and Zhang, J.-W.
`(1991) Twelfth American Peptide Symposium, Abstract
`LWS.
`
`(1963) J. Am, Chem. Soc. 85,
`
`5
`
`10
`
`SEQUENCE LISTING
`
`(
`
`1
`
`) GENERAL INFORMATION:
`
`(i i
`
`t
`
`) NUMBER OF SEQUENCES: 2
`
`( 2 ) INFORMATION FOR SEQ ID NO:1:
`
`(
`
`i
`
`) SEQUENCE CHARACTERISTICS:
`( A ) LENGTH: 16 amino acids
`( B ) TYPE: amino acid
`( D ) TOPOLOGY:lincar
`
`(i i
`
`) MOLECULE TYPE:peptide
`
`(x i
`
`) SEQUENCE DESCRIPTION: SEQ ID NO:1:
`
`Asn Asp Asp Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu
`1
`5
`10
`15
`
`( 2 ) INFORMATION FOR SEQ ID NO:2;
`
`(
`
`i
`
`) SEQUENCE CHARACTERISTICS:
`( A ) LENGTH:15 amino acids
`( B ) TYPE: amino acid
`(.D ) TOPOLOGY:linear
`
`(i i
`
`) MOLECULE TYPE:peptide
`
`(x i
`
`} SEQUENCE DESCRIPTION: SEQ ID NO:2:
`
`Asp Asp Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu
`1
`5
`190
`15
`
`Whatis claimedis:
`1. A purified peptide having the following amino acid
`sequence
`
`350
`
`(SEQ ID NO: 1]
`Asn— Asp—Asp—Cys—Glu—Leu—Cys — Val ~ Asn—Val—
`1
`5
`10
`
`Ala—Cys—Thr Gly Cys —~ Leu
`15
`
`35
`
`2. The peptide of claim 1 in oxidized form having two
`disulfide bridges, one between cysteine residues 4 and 12
`and the other between cysteine residues 7 and 15.
`
`+e +e F
`
`60
`
`65
`
`MSN Exhibit 1005 - Page 9 of 9
`MSNv. Bausch - IPR2023-00016
`
`