WO2010/065751
`
`PCT/US2009/066600
`
`are inhibitors of neprilysin are useful analgesic agents which can be administered with
`
`the GCC agonists described herein or covalently linked to a GCC agonist to form a
`
`therapeutic conjugate. Sialorphin and related polypeptides are described in U.S. Patent
`
`6,589,750; U.S. 20030078200 Al; and WO 02/051435 A2.
`
`[197]
`In another embodiment, a GCC agonist formulation of the invention is
`administered as part of a regimen of combination therapy with an opioid receptor
`antagonist or agonist. In one embodiment, the GCC agonist and the opioid receptor
`antagonist or agonist are linked via a covalent bond. Non-limiting examplesof opioid
`
`receptor antagonists include naloxone, naltrexone, methyl nalozone, nalmefene,
`cypridime, beta funaltrexamine, naloxonazine,naltrindole, nor-binaltorphimine,
`enkephalin pentapeptide (HOE825; Tyr-D-Lys-Gly-Phe-L-homoserine; SEQ ID
`NO:258), trimebutine, vasoactive intestinal polypeptide, gastrin, glucagons. Non-
`limiting examples of opioid receptor agonists include fedotozine, asimadoline, and
`ketocyclazocine, the compounds described in WO03/09705 1! and WO05/007626,
`morphine, diphenyloxylate, frakefamide (H-Tyr-D-Ala-Phe(F)-Phe-NH 23 SEQ ID
`NO:259; WO 01/019849 Al), and loperamide.
`
`Further non-limiting examples of analgesic agents that can be used ina
`[198]
`regimen of combination therapy along with the GCCagonist formulations ofthe
`invention include the dipeptide Tyr-Arg (kyotorphin); the chromogranin-derived
`‘polypeptide (CgA 47-66; See, e.2., Ghia et al. 2004 Regulatory polypeptides 119:199);
`CCKreceptor agonists such as caerulein; conotoxin polypeptides; peptide analogs of
`thymulin (FR Application 2830451); CCK (CCKa or CCKb) receptor antagonists,
`including loxiglumide and dexloxiglumide (the R- isomer of loxighumide) (WO
`
`88/05774); 5-HT4 agonists such as tegaserod (Zelnorm®), mosapride, metoclopramide,
`
`zacopride, cisapride, renzapride, benzimidazolone derivatives such as BIMU 1 and
`
`BIMU8,andlirexapride; calcium channel blockers such as ziconotide and related
`
`compoundsdescribedin, for example, EP625162B1, US 5,364,842, US 5,587,454, US
`5,824,645, US 5,859,186, US 5,994,305, US 6087,091, US 6,136,786, WO 93/13128 Al,
`
`88
`
`RECTIFIED SHEET (RULE 91) ISA/EP
`MYLAN - EXHIBIT 1022 Part 12 of 16
`3776
`
`3776
`
`MYLAN - EXHIBIT 1022 Part 12 of 16
`
`

`

`WO2010/065751
`
`PCT/US2009/066600
`
`EP 1336409 Al, EP 835126 Al, EP 835126 Bl, US 5,795,864, US 5,891,849, US
`6,054,429, WO 97/01351 Al; NK-I, receptor antagonists such as aprepitant (Merck & Co
`Inc), vofopitant, ezlopitant (Pfizer, Inc.), R-673 (Hoffmann-La Roche Ltd), SR-48968
`(Sanofi Synthelabo), CP-122,721 (Pfizer, Inc.), GW679769 (Glaxo Smith Kline), TAK-
`637 (Takeda/Abbot), SR-14033,
`
`88a
`
`RECTIFIED SHEET (RULE 91) ISA/EP
`
`3777
`
`3777
`
`

`

`WO 2010/065751
`
`PCT/US2009/066600
`
`and related compounds describedin, for example, EP 873753 Al, US 20010006972 Al, US
`
`20030109417 Al, WO 01/52844 Al (for a review see Giardinaet al. 2003.Drugs 6:758); NK-2
`
`receptor antagonists such as nepadutant (Menarini Ricerche SpA), saredutant (Sanofi-
`
`Synthelabo), GW597599 (Glaxo Smith Kline), SR-144190 (Sanofi-Synthelabo) and UK-290795
`
`(Pfizer Inc); NK3 receptor antagonists such as osanetant (SR-142801; Sanofi-Synthelabo), SSR-
`
`241586, talnetant and related compounds described in, for example, WO 02/094187 A2, EP
`
`876347 Al, WO 97/21680 Al, US 6,277,862, WO 98/1 1090, WO 95/28418, WO 97/19927, and
`
`Bodenet al. (J Med Chem. 39:1664-75, 1996); norepinephrine-serotonin reuptake inhibitors
`
`C(NSRI) such as milnacipran and related compounds described in WO 03/077897; and vanilloid
`
`receptor antagonists such as arvanil and related compouds described in WO 01/64212 AL.
`
`[199]
`
`In addition to sialorphin-related polypeptides, analgesic polypeptides include:
`
`AspPhe, endomorphin-1, endomorphin-2, nocistatin, dalargin, lupron, ziconotide, and substance
`
`P.
`
`EXAMPLES
`
`Example 1: Synthesis and Purification of GCC Agonist Peptides
`
`[200]
`
`The GCC agonist peptides were synthesized using standard methods for solid-
`
`phase peptide synthesis. Either a Boc/Bzl or Fmoc/tBu protecting group strategy was seleceted
`
`depending upon the scale of the peptide to be produced. In the case of smaller quantities, it is
`
`possible to get the desired product using an Fmoc/tBu protocol, but for larger quantitics (1 g or
`
`more), Boc/Bzl is superior.
`
`[201]
`
`In each case the GCC agonist peptide was started by either using a pre-loaded
`
`Wang (Fmoc) or Merrifield (Boc) or Pam (Boc)resin. For products with C-terminal Leu, Fmoc-
`
`Leu-Wang (D-1115) or Boc-Leu-Pam resin (D-1230) or Boc-Leu-Merrifield (D-1030) Thus, for
`
`peptides containing the C-terminal d-Leu, the resin was Fmoc-dLeu-Wang Resin (D-2535) and
`
`Boc-dLeu-Merrifield, Boc-dLeu-Pam-Resin (Bachem Product D-1230 and D-1590, respectively)
`
`(SP-332 and rclated analogs). For peptides produced as C-terminal amides, a resin with Ramage
`
`89
`
`3778
`
`3778
`
`

`

`WO 2010/065751
`
`PCT/US2009/066600
`
`linker (Bachem Product D-2200) (Fmoc) or mBHA (Boc) (Bachem Product D-1210 was used
`
`and loaded with the C-terminal residue as the first synthetic step.
`
`Fmoc-tBu Overview
`
`[202]
`
`Each synthetic cycle consisted deprotection with 20% piperidine in DMF. Resin
`
`washes were accomplished with alternating DMF and IpOHto swell and shrink the resin,
`
`respectively. Peptide synthesis elongated the chain from the C-terminus to the N-terminus.
`
`Activation chemistry for cach amino acid was with HBTU/DIEAin a 4 fold cxcess for 45
`
`minutes. In automated chemistries, each amino acid was double coupled to maximize the
`
`coupling efficiency. To insure the correct position of disulfide bonds, the Cys residues were
`
`introduced as Cys (Acm)at positions 15 and 7. Cys (Trt) was positioned at Cys4 and Cys12.
`
`This protecting group strategy yields the correct topoisomer as the dominant product (75:25).
`
`(For enterotoxin analogs, a third disulfide bond protecting group (Mob) wasutilized).
`
`[203]
`
`For peptides containing C-terminal Acea (aminoethyloxyethyloxyacetyl) groups,
`
`these were coupled to a Ramage amide linker using the same activation chemistry above by
`
`using an Fmoc-protected Acea derivative. The Cys numbering in these cases remains the same
`
`and the positioning of the protecting groups as well. For the peptides containing the N-terminal
`
`extension of Acea, the Cys residue numbering will be increased by three Cys4 becomes Cys7,
`
`Cys12 becomes Cys15; Cys7 becomes Cys10 and Cys 15 becomes Cys18. The latter pair is
`
`protected with Acm and the former pair keeps the Trt groups.
`
`[204]
`
`For analogs containing D-aminoacid substitutions, these were introduced directly
`
`by incorporating the correctly protected derivative at the desired position using the same
`
`activation chemistry described in this document. For Fmocstrategics, Fmoc-dAsn(Trt)-OH,
`
`Fmoc-dAsn(Xan)-OH, Fmoc-dAsp(tBu)-OH, Fmoc-dGlu(tBu)-OH and for Boc strategies, Boc-
`
`dAsn(Xan)-OH, Boc-dAsn(Trt)-OH, Boc-dAsp(Chx), Boc-dAsp(Bzl)-OH, Boc-dGlu(Chx)-OH
`
`and Boc-dGlu(Bzl)-OH would be utilized.
`
`[205]
`
`Each peptide is cleaved from the solid-phase support using a cleavage cocktail of
`
`TFA:H20:Trisisopropylsilane (8.5:0.75:0.75) ml/g of resin for 2 hr at RT. The crude deprotected
`
`peptideis filtered to remove the spent resin beads and precipitated into ice-cold diethylether.
`
`90
`
`3779
`
`3779
`
`

`

`WO 2010/065751
`
`PCT/US2009/066600
`
`[206]
`
`Each disulfide bonds was introduced orthogonally. Briefly, the crude synthetic
`
`product wasdissolved in water containing NH4OHto increase the pH to 9. Following complete
`
`solubilization of the product, the disulfide bond was made between the Trt deprotected Cys
`
`residues bytitration with H2O2. The monocyclic product was purified by RP-HPLC. The purified
`
`mono-cyclic product was subsequently treated with a solution of iodine to simultaneously
`
`remove the Acm protecting groups and introduce the second disulfide bond.
`
`[207]
`
`For cntcrotoxin analogs, the Mob group was removed via treatment of the dicyclic
`
`product with TFA 85% containing 10% DMSO and 5% thicanisole for 2 hr at RT.
`
`[208]
`
`Each product was then purified by RP-HPLC using a combination buffer system
`
`of TEAP in H20 versus MeCN,followed by TFA in H20 versus MeCN. Highly purefractions
`
`were combined and lyophilized. The final product was converted to an Acetate salt using either
`
`ion exchange with Acetate loaded Dow-Exresin or using RP-HPLC using a base-wash step with
`
`NH,4OAc followed by 1% AcOH in water versus MeCN.
`
`[209]
`
`It is also possible to prepare enterotoxin analogs using a random oxidation
`
`methodology using Cys(Trt) in Fmoc or Cys(MeB)in Boc. Following cleavage, the disulfide
`
`bonds can be formed using disulfide interchange redox pairs such as glutathione (red/ox) and/or
`
`cysteine/cystine. This process will yield a folded product that the disulfide pairs must be
`
`determined as there would be no way of knowing their position directly.
`
`Boc-Bil Process
`
`[210]
`
`Peptide synthesis is initiated on a Merrifield or Pam pre-loaded resin or with
`
`mBHAfor peptides produced as C-terminal amides. Each synthetic cycle consists of a
`
`deprotection step with 50% TFA in MeCL2. The resin is washed repetitively with MeCl2 and
`
`MeOH. The TFAsalt formed is neutralized with a base wash of 10% TEA in MeCl2. Theresin is
`
`washed with MeCl2 and MeOHandlastly with DMFprior to coupling steps. A colorimetric test
`
`is conducted to ensure deprotection. Each coupling is mediated with diisopropyl carbodiimide
`
`with HOBTto form the active ester. Each coupling is allowed to continue for 2 hr at RT or
`
`overnight on difficult couplings. Recouplings are conducted with either Uronium or
`
`Phosphoniumreagents until a negative colorimetric test is obtained for free primary amines. The
`
`91
`
`3780
`
`3780
`
`

`

`WO 2010/065751
`
`PCT/US2009/066600
`
`resin is then washed with DMF, MeCl2 and MeOHandprepared for the next solid-phase step.
`
`Cysprotection utilizes Cys(Acm) at positions 7 and 15, and Cys(MeB)at Cys 4 and Cys12.
`
`[211]
`
`Cleavage and simultaneous deprotection is accomplished by treatment with HF
`
`using anisole as a scavenger (9:1:1) ml:ml:g (resin) at 0°C for 60 min. The peptide is
`
`subsequently extracted from the resin and precipitated in ice cold ether. The introduction of
`
`disulfide bonds and purification follows the exact same protocol described above for the Fmoc-
`
`produced product.
`
`Example 2: Jn vitro Biological and Chemical Stability of SP-304 after Incubation in
`
`Simulated Gastric Fluid (SGF)
`
`[212]
`
`Thestability of SP-304 in the presence of simulated gastric fluid (SGF) was
`
`determined by biological activity measurements and HPLC analyses (Figs. 1A & 1B). SP-304
`
`(final concentration of 8.5 mg/ml) was incubated in SGF (Proteose peptone (8.3 g/liter; Difco),
`
`D-Glucose (3.5 g/liter; Sigma), NaCl (2.05 g/liter; Sigma), KH 2PO, (0.6 g/liter; Sigma), CaCl,
`
`(0.11 g/liter), KCI (0.37 g/liter; Sigma), Porcine bile (final 1 X concentration 0.05 g/liter; Sigma)
`
`in PBS, Lysozyme(final 1 X concentration 0.10 g/liter; Sigma) in PBS, Pepsin (final 1 X
`
`concentration 0.0133 g/liter; Sigma) in PBS). SGF was made on the day of the experiment and
`
`the pH wasadjusted to 2.0 + 0.1 using HCl or NaOHas necessary. After the pH adjustment,
`
`SGFis filter sterilized with 0.22 um membranefilters. SP-304 (final concentration of 8.5
`
`mg/ml) was incubated in SGF at 37°C for 0, 15, 30, 45, 60 and 120 min, respectively, in
`
`triplicate aliquots. Following incubations, samples were snap frozen in dry ice then stored in a -
`
`80°C freezer until assayed in duplicate.
`
`[213]
`
`Figure 1A showsa bar chart showing the biological activity of SP-304 after
`
`incubation with SGF for times as indicated. The activity at 0 min was taken as 100%. The data
`
`are an average oftriplicates + SD for each data point. The data demonstrate that SP-304 is
`
`resistant to breakdown in SGFfor incubationslasting as long as two hours. In addition, the data
`
`also suggest that the activity of SP-304 is unaltcred by exposure to the acidic pH of the SGF.
`
`[214]
`
`The HPLC chromatograms of samples of SP-304 incubated in SGF for 0 and 120
`
`min are shown in Fig. 1B. Here, aliquots of the two samples were analyzed by HPLC using a
`
`92
`
`3781
`
`3781
`
`

`

`WO 2010/065751
`
`PCT/US2009/066600
`
`previously developed method for analyzing SP-304 peptide. Samples from the SGF incubations
`
`were diluted to give a final concentration 0.17 mg/mL of SP-304. The major peak of SP-304 did
`
`not change following incubation with SGF, indicating that the peptide was resistant to SGF
`
`treatment.
`
`Example 3: én vitro Biological and Chemical Stability of SP-304 after Incubation in
`
`Simulated Intestinal Fluid (SLF)
`
`[215]
`
`The stability of SP-304 was also evaluated after incubation with simulated
`
`intestinal fluid (SIF) by measuring its biological activity and by HPLC analyses (Figs. 2A & 2B).
`
`SIF solution was prepared by the method as described in the United States Pharmacopoeia, 24th
`
`edition, p2236. The recipe to prepare SIF solution was as described below. The SIF solution
`
`contained NaCl (2.05 g/liter; Sigma), KH 2PO4 (0.6 g/liter; Sigma), CaCl, (0.11 g/liter), KCl
`
`(0.37 g/liter; Sigma), and Pacreatin 10 mg/ml. The pH wasadjusted to 6 and the solution was
`
`filter sterilized. A solution of SP-304 (8.5 mg/ml) was incubated in SGF at 37°C for 0, 30, 60,
`
`90, 120, 150 and 300 min respectively, in triplicate aliquots. Following incubations, samples
`
`were removed and snap frozen with dry ice and stored in a -80°C freezer until assayed in
`
`duplicate. Figure 2A is a bar chart showing the ability of SP-304, after incubation in SIF for
`
`times as indicated, to stimulate cGMP synthesis in T84 cells. The cGMPstimulation activity at 0
`
`min was taken as 100%. The data are an average of 3 triplicates + SD. The data indicated that
`
`the biological activity of SP-304 is reduced by about 30% following incubation in SIFfor 300
`
`min.
`
`[216]
`
`The physical stability of SP-304 peptide exposed to SIF was evaluated by HPLC
`
`using the method described for SGF digestion. Figure 2B shows HPLC chromatograms for SP-
`
`304 after incubation with heat-inactivated SIF for 300 min, and SIF for 120 min, respectively.
`
`SP-304 treated with heat-inactivated SIF remained intact (Note: the major peak of SP-304
`
`eluting at 16.2 min), whereas SP-304 treated with SIF for 120 min was completely converted
`
`into another peak eluting at 9.4 min plus a few minor additional peaks.
`
`[217]
`
`Figure 3 is a schematic representation of the possible metabolites of SP-304. The
`
`major degradation products involve Asn and Aspclipped from the N-terminus and Leu from the
`
`93
`
`3782
`
`3782
`
`

`

`WO 2010/065751
`
`PCT/US2009/066600
`
`C-terminus of SP304. The fact that only 30% reduction in biological activity was observed even
`
`after 2 hours incubation in SIF implies that one or more of the degradation products observed in
`
`Fig. 2B are also biologically active. To address this possibility, several truncated peptides were
`
`synthesized and evaluated for their abilities to stimulate cGMPsynthesis in T84 cells (Figure 4).
`
`[218]
`
`Figure 4 showsdata from the analyses of various peptides in the T&84 cell cGMP
`
`stimulation assay (essentially as described in Shailubhai, et a/., Cancer Research 60, 5151-5157
`
`(2000) . Bricfly, conflucnt monolayers of T-84 cells in 24-well plates were washed twice with
`
`250 wl of DMEM containing 50 mM HEPES (pH 7.4) and pre-incubated at 37°C for 10 minutes
`
`with 250 ul of DMEM containing 50 mM HEPES (pH 7.4) and 1 mM isobutyl methylxanthine
`
`(BMX). Monolayers of T&4 cells were then incubated with 250 ul of DMEM containing 50 mM
`
`HEPES(pH 7.4) containing one of the peptides shown in the Figure 4 at a concentration of 1.0
`
`uM for 30 min. After the 30 min incubation, the medium was aspirated and the reaction was
`
`terminated by the addition of 3% perchloric acid. Following centrifugation and the addition of
`
`NaOH (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, MI). Peptide incubations
`
`were run in duplicate, and samples taken from each incubation were run as duplicates in the
`
`ELISA test.
`
`[219]
`
`The data indicate that SP-338, the 15-mer peptide missing the leucine (L) residue
`
`at the C-terminus of SP-304, retains about 80% ofthe biological activity of the full length 16-
`
`mer SP-304 peptide. Thus, the C-terminal Leu clearly does make some contribution to the
`
`biological potency of the peptide. Similarly, peptides SP-327, SP-329 and SP-331, whichareall
`
`missing their C-terminal Leu, also showed a 20-25% reduction in biological potency relative to
`
`their counterpart parent peptides SP-326, SP-328 and SP-330, respectively. In addition, the data
`
`also suggest that amino acid residues at the N-terminus may also contribute to the stability and/or
`
`potency of the peptides. Several additional peptides were synthesized with D-forms of amino
`
`acids replacing the corresponding L-forms at the C- and N-termini of the peptides. These
`
`peptides were evaluated for their abilities to stimulate cGMP synthesis in T84 cells as shown in
`
`Figure 5.
`
`94
`
`3783
`
`3783
`
`

`

`WO 2010/065751
`
`PCT/US2009/066600
`
`[220]
`
`The results presented in Figure 5 indicate that substitution of L-aminoacids with
`
`D-aminoacids at the C- and N-termini did not significantly alter their potency relative to SP-304.
`
`Peptides SP-332, SP-333 and SP-335 all showed comparable ability to stimulate cGMP synthesis
`
`in T84 cells. These results suggest that the amino acid residues Asn, Asp and Glu at the N-
`
`terminus and Leuat the C-terminus can be replaced with their respective D-aminoacid forms.
`Onthe other hand, substitution of L-leucine with D-leucineat the 6"position (SP-337) resulted
`
`in virtually complete loss of biological activity.
`
`[221]
`
`Figure 7 (A-F) showsthe stabilities of peptides SP-332, SP-333 and SP-304 when
`
`incubated in SIF for two hours. The results demonstrate that SP-333, which has D-Asnat the N-
`
`terminus and D-Leu at the C-terminus, remained virtually 100% biologically active after a two
`
`hour incubation in SIF (Figure 7A), and remained virtually intact to digestion with SIF after two
`
`hours (Figs. 7F-1 & 7F-2). Subsequent incubation studies with SP-333 performed in SIF for up
`
`to 24 hours indicate that there is very little degradation even after 24 hours in SIF (Fig. 7G). The
`
`data indicated that SP-333 is stable against digestion with SIF for up to 24 hours. Peptide SP-
`
`332 with D-Leu at the C-terminus showed a minor reduction in potency following the 120 min
`
`incubation with SIF (Figure 7B). Interestingly, the HPLC analyses of SP-332 did not reveal any
`
`clear-cut degradation of the peptide (Figure 7E-1 & 7E2), also suggesting that this peptide is also
`
`almost completely resistant to proteoysis by SIF during the 2-hr incubation. On the other hand,
`
`peptide SP-304 lost about 30% of its potency following digestion with SIF for just one hour
`
`(Figure 7C). The HPLC analysis of SP-304 following SIF incubation confirmedits degradation
`
`(Figure 7D-1 & 7D-2). These results suggest that SP-304 undergoes substantial proteolysis
`
`following incubation with SIF within one hour.
`
`Example 4: Cyclic GMP Stimulation Assays
`
`[222]
`
`Theability of the GCC agonist peptide to bind to and activate the intestinal GC-C
`
`receptor wastested usingT 84 human colon carcinomacell line. Human T84 colon carcinoma
`
`cells were obtained from the American Type Culture Collection. 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 Lg/ml streptomycin.
`
`The cells were fed fresh medium every third day andsplit at a confluence of approximately 80%.
`
`95
`
`3784
`
`3784
`
`

`

`WO 2010/065751
`
`PCT/US2009/066600
`
`[223]
`
`Biological activity of the GCC agonist peptides was assayed as previously
`
`reported (Shailubhai, et a/., Cancer Research 60, 5151-5157 (2000)). Briefly, the confluent
`
`monolayers of T-84 cells in 24-well plates were washed twice with 250 ul of DMEMcontaining
`
`50 mM HEPES(pH 7.4), pre-incubated at 37°C for 10 min with 250 wl of DMEM containing 50
`
`mM HEPES(pH 7.4) and 1 mM isobutylmcthylxanthinc (BMX), followed by incubation with
`
`GCC agonist peptides (0.1 nM to 10 .mu.M) for 30 min. The medium wasaspirated, and the
`
`reaction was terminated by the addition of 3% perchloric acid. Following centrifugation, and
`
`neutralization with 0.1 N NaOH, the supernatant was used directly for measurements of cGMP
`
`using an ELISA kit (Caymen Chemical, Ann Arbor, Mich.).
`
`[224]
`
`Figure 6 showsresults from experiments evaluating the potency of peptides (via
`
`cGMPstimulation assay) having structures similar to the 14-mer peptide SP-339, also referred to
`
`as linaclotide. SP-339 is a truncated analog of the E. coli enterotoxin ST peptide. SP-354 was
`
`foundto be virtually identical to SP-339 in biological activity. Notably, peptide SP-353, which
`has a Serresidue at the 6" position, was found to be more potent than SP-339, and was the most
`
`potent of all the peptides tested. Peptide SP-355 which has a D-Tyrat the C-terminus showed
`
`considerably less potency than the other peptidestested.
`
`Example 5: Peggylated Peptides
`
`[225]
`
`An additional strategy to render peptides moreresistant towards digestion by
`
`digestive proteases is to peggylate them at the N- and C-terminus. The peptide SP-333 was
`
`peggylated with the aminoethyloxy-ethyloxy-acetic acid (Acea) group at the C-terminus (SP-
`
`347) or at the N-terminus (SP-350) or at both termini (SP-343). Cyclic GMP synthesis in T84
`
`cells was measured by the method as described above.
`
`[226]
`
`The peptides SP-347 and SP-350 showed potencies comparable to SP-333 in their
`
`abilities to stimulate cGMPsynthesis in T84 cells. However, peptide SP-343 was considerably
`
`less potent as compared to the other peptides tested. The poor activity of SP-343 might be due to
`
`the considerable steric hindrance afforded by the large Aeea groups at both termini.
`
`96
`
`3785
`
`3785
`
`

`

`WO 2010/065751
`
`PCT/US2009/066600
`
`Example 6: Combination of Guanylate Cyclase Receptor Agonists with
`Phosphodiesterase Inhibitors
`
`[227]
`
`Regulation of intracellular concentrations of cyclic nucleotides (7.e., cAMP and
`
`cGMP)and thus, signaling via these second messengers, has been generally considered to be
`
`governedbytheir rates of production versus their rates of destruction within cells. Thus, levels of
`
`cGMPintissues and organs can also be regulated by the levels of expression of cGMP-specific
`
`phosphodiesterases (CGMP-PDE), which are generally overexpressed in cancer and
`
`inflammatory diseases. Therefore, a combination consisting of an agonist of GC-C with an
`
`inhibitor of cGMP-PDE might produce synergistic effect on levels of cGMPin the target tissues
`
`and organs.
`
`[228]
`
`Sulindac Sulfone (SS) and Zaprinast (ZAP) are two of the knowninhibitors of
`
`cGMP-PDEand have been shownto induce apoptosis in cancer cells via a cGMP-dependent
`
`mechanism. SS and ZAP in combination with SP-304 or SP-333 were evaluated to see if these
`
`PDEinhibitors had any synergistic effects on intracellular accumulation of cGMP(Fig. 9-12).
`
`As the data show, SS at a concentration of 100 uM did not enhance intracellular accumulation of
`
`cGMP. However, the combination of SS with SP-304 stimulated cGMP production several-fold
`
`more then stimulation by SP-304 alone. This synergistic effect on cGMP levels was more
`
`pronounced when SP-304 were used at a 0.1 uM concentration (Fig 10). Similar observations
`
`were made when SP-304 or SP-333 were used in combination with ZAP (Fig 10, Fig 11 and Fig
`
`12). These results suggest that the intracellular levels of cGMPare stabilized because SS inhibits
`
`cGMP-PDEthat might be responsible for depletion of intracellular cGMP. Thus, the approach to
`
`use a combination of GCC agonist with a cGMP-PDEinhibitoris attractive.
`
`[229]
`
`For the results shown in Figure 9, cyclic GMP synthesis in T84 cclls was assessed
`
`essentially as described in Shailubhai et al., Cancer Research 60, 5151-5157 (2000). Briefly,
`
`confluent monolayers of T-84 cells in 24-well plates were washed twice with 250 ul of DMEM
`
`containing 50 mM HEPES(pH 7.4) and pre-incubated at 37°C for 10 minutes with 250 ul of
`
`DMEMcontaining 50 mM HEPES (pH 7.4) and 1 mM isobutyl methylxanthine (BMX).
`
`Monolayers of T84 cells were then incubated with 250 ul of DMEM containing 50 mM HEPES
`
`(pH 7.4) containing SP-304 or PDE inhibitors either alone or in combinations, as indicated below
`
`in the following experimental sets: 1) Control; 2) SP-304 (0.1 uM); 3) Sulindac Sulfone (100
`
`97
`
`3786
`
`3786
`
`

`

`WO 2010/065751
`
`PCT/US2009/066600
`
`uM); 4) Zaprinast (100 uM); 5) SP-304 (0.1 uM) + Sulindac Sulfone (100 uM); and 6) SP-304
`
`(0.1 uM) + Zaprinast (100 uM). After the 30 min incubation, the medium wasaspirated and the
`
`reaction was terminated by the addition of 3% perchloric acid. Following centrifugation and the
`
`addition of NaOH (0.1 N) to neutralize the pH,intracellular cGMP levels were determined in
`
`lysates using a cGMPELISAkit (Cat. No. 581021 ; Cayman Chemical, Ann Arbor, MI).
`
`Incubations were performed in duplicate, and cach sample was run in duplicate using the ELISA
`
`test.
`
`[230]
`
`For the results shown in Figure 10, the method used was same as the one used for
`
`Fig. 9 except that the monolayers of T84 cells were incubated with 500 wl of DMEM containing
`
`50 mM HEPES(pH 7.4) containing SP-304 (0.1 or 1.0 uM)or increasing concentrations of PDE
`
`inhibitors (0 to 750 uM) either alone or in combination with SP-304. After the 30 min
`
`incubation, the medium wasaspirated and the reaction was terminated by the addition of 3%
`
`perchloric acid. Following centrifugation and the addition of NaOH (0.1 N) to neutralize the pH,
`
`intracellular cGMPlevels were determined in lysates using a cGMP ELISAkit (Cat. No.
`
`581021; Cayman Chemical, Ann Arbor, MI). Samples were runintriplicate using the ELISA
`
`test.
`
`[231]
`
`For the results shown in Figure 11,
`
`the method used was sameas the one used for
`
`Fig. 10 except that the monolayers of T84 cells were incubated with 500 ul of DMEM containing
`
`50 mM HEPES(pH 7.4) containing SP-3333 (0.1 or 1.0 uM) or increasing concentrations of
`
`ZAP (0 to 500 uM) either alone or in combination with SP-333. After the 30 min incubation, the
`
`medium wasaspirated and the reaction was terminated by the addition of 3% perchloric acid.
`
`Following centrifugation and the addition of NaOH (0.1 N) to neutralize the pH, intracellular
`
`cGMPlevels were determined in lysates using a cGMP ELISAkit (Cat. No. 581021 ; Cayman
`
`Chemical, Ann Arbor, MI). Samples were run in triplicate using the ELISAtest.
`
`[232]
`
`For the results shown in Figure 12, the method used was sameas the one usedfor
`
`Fig. 10 except that the monolayers of T84 cells were incubated with 500 Ul of DMEM containing
`
`50 mM HEPES(pH 7.4) containing SP-333 (0.1 LM) or increasing concentrations of Sulindac
`
`Sulfone (0 to 500 UM) either alone or in combination with SP-333. After the 30 min incubation,
`
`the medium was aspirated and the reaction was terminated by the addition of 3% perchloric acid.
`
`98
`
`3787
`
`3787
`
`

`

`WO 2010/065751
`
`PCT/US2009/066600
`
`Following centrifugation and the addition of NaOH (0.1 N) to neutralize the pH, intracellular
`
`cGMPlevels were determined in lysates using a cGMP ELISA kit (Cat. No. 581021 ; Cayman
`
`Chemical, Ann Arbor, MI). Samples were run in triplicate using the ELISAtest.
`
`Example 7: A Repeated Oral Dose Toxicity Study of SP-304 in Cynomolgus Monkeys.
`
`[233]
`
`The primary purpose ofthis experiment was to evaluate the toxicity and
`
`pharmacokinetics of a repeated oral dose of SP-304 in cynomolgus monkeys. Treatment with a
`
`daily dose of 250 mg of SP-304 for 14 consecutive days was well tolerated by all of the
`
`monkeys, however the treatment consistently produced liquid feces and watery diarrhea (Figure
`
`14). Monkeys returned to normal stool consistency within 24-48 hours following the last dose of
`
`SP-304.
`
`Example 8:
`
`SP-304 Treatment Improves Stool Consistency and Clears TNBS-induced
`Intestinal Blockage in a TNBS-induced Murine Modelof Colitis.
`
`[234]
`
`SP-304 is a superior analog of uroguanylin and an agonist of GC-C. The anal
`
`administration of trinitrobenzene sulphonic acid (TNBS)is typically used to produce intestinal
`
`blockage, resulting in poor stool consistency. As shown in Figure 13, oral administration of SP-
`
`304 considcrably improved stool consistency in mice treated with TNBS. Treatment with SP-304
`
`at a dose between 0.05 to 0.5 mg/kg body weight was sufficient to completely restore the
`
`consistency score to the level observed in mice treated with phosphate buffer instead of TNBS
`
`(minus TNBS control). Sulfasalazine, a FDA approved drug used as a positive control, also
`
`restored normal stool consistency.
`
`Example 9: A Randomized, Double-blind, Placebo-Controlled, Single-, Ascending-, Oral-
`Dose Safety, Tolerability, and Pharmacokinetic Study of SP-304 in Healthy
`Adult Human Male and Female Volunteers
`
`[235]
`
`The objectives of this study were to assess the safety and pharmacokinetics of a
`
`single oral dose of SP-304 in healthy subjects. This was a phase 1, single-site, randomized,
`
`double-blind, placebo-controlled, single-, ascending-, oral-dose, sequential dose escalation study
`
`of SP-304 in fasted, healthy male and female subjects. A total of 9 cohorts utilizing 8 subjects
`
`per cohort (6 SP-304; 2 placebo) were utilized, totaling 71 volunteers administered drug (one
`
`99
`
`3788
`
`3788
`
`

`

`WO 2010/065751
`
`PCT/US2009/066600
`
`volunteer dropping out). Each cohort was administered a single, oral dose or matching placebo
`
`administered in 10-fold diluted phosphate buffered saline (PBS) (240 mL). Subjects were only
`
`administered one dose of study treatment and could not be enrolled in subsequent cohorts. The
`
`nine cohort doses included 0.1, 0.3, 0.9, 2.7, 5.4, 8.1, 16.2, 24.3 mg and 48.6 mg SP-304.
`
`Doses of SP-304
`
`0.1 mg (6 active, 2 placebo)
`0.3 mg (6 active, 2 placebo)
`0.9 mg (6 active, 2 placcbo)
`2.7 mg (6 active, 2 placebo)
`5.4 mg (6 active, 2 placebo)
`8.1 mg (6 active, 2 placebo)
`16.2 mg (5 active, 2 placcbo)
`24.3 mg(6 active, 2 placebo)
`48.6 mg(6 active, 2 placebo)
`
`[236]
`
`The decision to proceed to the next cohort was madeby the study sponsor and
`
`principal investigator after reviewing the preliminary blinded, safety information from the
`
`cohort. All safety data collected through the 48 hours after dosing were reviewedto assess
`
`tolerability of the dose level. A minimum of 3 evaluable subjects were required for the
`
`determination of safety and tolerability at each dose level.
`
`[237]
`
`The stopping criteria were: 1) clinically significant adverse events [including
`
`clinically significant changes in laboratory or electrocardiogram (ECG) parameters] in >4
`
`subjects (collectively within a cohort), or 2) 1 drug related, serious adverse event (SAE). No
`
`higher doses were to be administered if one of these criteria was met. Otherwise, the study could
`
`proceed to the next higher dose cohort.
`
`[238]
`
`Safety was monitored by physical examinations, vital signs, clinical laboratory
`
`tests (hematology, chemistry, urinalysis, fecal occult blood), ECG, and adverse experience
`
`assessments). Serial blood samples were collected 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 16, 24, 36, and
`
`48 hours after dosing. Plasma samples were assayed by a validated method for SP-304, and
`
`pharmacokinetic parameters calculated. Pharmacodynamic endpoints that were evaluated
`
`includedtimeto first stool, stool frequency (48-hour period), and stool consistency (48-hour
`
`period) using the Bristol Stool Form Scale (BSFS).
`
`100
`
`3789
`
`3789
`
`

`

`WO 2010/065751
`
`PCT/US2009/066600
`
`[239]
`
`The phase | study (Protocol No. SP-SP304101-08) used an oral solution prepared
`
`by a registered licensed pharmacist at the investigation site not more than one hour before
`
`administration of dose.
`
`[240]
`
`The primary objectives of this clinical evaluation were to determine safety,
`
`toxicity and systemic absorption of a single oral dose of SP-304. The data indicated that SP-304
`
`was well-tolerated at all dosage levels and there were no severe adverse events (SAEs). The
`
`most prevalent adverse event (AE) observed during this study was grade | diarrhea (12.7%), as
`
`defined using the Common Terminology Criteria for Adverse Events (CTCAE), which is an
`
`increase in the number of bowel movements from 1 and <4 in a 24-hour period. Notably, SP-
`
`304 was expected to promote bowel movement, thus the increase in number of bowel
`
`movements was considered to be related to the pharmacodynamic (PD) action of SP-304.
`
`[241]
`
`The effect of a single oral dose of SP-304 on stool consistency, as judged by the
`
`Bristol form stool scale (BSFS), was also examined in volunteers. The BSFS sco