Targeting Anti-TGF-ß Therapy to Fibrotic Kidneys with a Dual Specificity Antibody Approach.

Targeted delivery of a therapeutic agent to a site of pathology to ameliorate disease while limiting exposure at undesired tissues is an aspirational treatment scenario. Targeting diseased kidneys for pharmacologic treatment has had limited success. We designed an approach to target an extracellular matrix protein, the fibronectin extra domain A isoform (FnEDA), which is relatively restricted in distribution to sites of tissue injury. In a mouse unilateral ureteral obstruction (UUO) model of renal fibrosis, injury induced significant upregulation of FnEDA in the obstructed kidney. Using dual variable domain Ig (DVD-Ig) technology, we constructed a molecule with a moiety to target FnEDA and a second moiety to neutralize TGF-ß After systemic injection of the bispecific TGF-ß + FnEDA DVD-Ig or an FnEDA mAb, chemiluminescent detection and imaging with whole-body single-photon emission computed tomography (SPECT) revealed significantly higher levels of each molecule in the obstructed kidney than in the nonobstructed kidney, the ipsilateral kidney of sham animals, and other tissues. In comparison, a systemically administered TGF-ß mAb accumulated at lower concentrations in the obstructed kidney and exhibited a more diffuse whole-body distribution. Systemic administration of the bispecific DVD-Ig or the TGF-ß mAb (1-10 mg/kg) but not the FnEDA mAb attenuated the injury-induced collagen deposition detected by immunohistochemistry and elevation in Col1a1, FnEDA, and TIMP1 mRNA expression in the obstructed kidney. Overall, systemic delivery of a bispecific molecule targeting an extracellular matrix protein and delivering a TGF-ß mAb resulted in a relatively focal uptake in the fibrotic kidney and reduced renal fibrosis.

Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin.

For complex diseases in which multiple mediators contribute to overall disease pathogenesis by distinct or redundant mechanisms, simultaneous blockade of multiple targets may yield better therapeutic efficacy than inhibition of a single target. However, developing two separate monoclonal antibodies for clinical use as combination therapy is impractical, owing to regulatory hurdles and cost. Multi-specific, antibody-based molecules have been investigated; however, their therapeutic use has been hampered by poor pharmacokinetics, stability and manufacturing feasibility. Here, we describe a generally applicable model of a dual-specific, tetravalent immunoglobulin G (IgG)-like molecule--termed dual-variable-domain immunoglobulin (DVD-Ig)--that can be engineered from any two monoclonal antibodies while preserving activities of the parental antibodies. This molecule can be efficiently produced from mammalian cells and exhibits good physicochemical and pharmacokinetic properties. Preclinical studies of a DVD-Ig protein in an animal disease model demonstrate its potential for therapeutic application in human diseases.

Central pituitary adenylate cyclase 1 receptors modulate nociceptive behaviors in both inflammatory and neuropathic pain states.

UNLABELLED: The pituitary adenylate cyclase-activating polypeptide type 1 receptor (PAC(1)-R) is a member of the 7-transmembrane domain, group 2 G-protein coupled receptor family. PAC(1)-Rs modulate neurotransmission and neurotrophic actions and have been implicated in both pronociception and antinociception. To better understand the role of PAC(1)-Rs in pain, PACAP 6-38, a PAC(1)-R antagonist, was evaluated in several inflammatory and neuropathic pain models after intrathecal (i.t.) administration. PACAP 6-38 potently reduced mechanical allodynia in a neuropathic spinal nerve ligation model (77% +/- 15% maximal effect at 12 nmol, P < .01) and was also effective in reducing thermal hyperalgesia in the carrageenan model of inflammatory pain (89% +/- 17% maximal effect at 12 nmol, P < .01). Although nociceptive responses were also attenuated with PACAP 6-38 in a dose-dependent manner in models of chronic inflammatory and persistent pain, no effects on motor performance were observed at analgesic doses. Taken together, these data demonstrate that blockade of the PAC(1)-R/PACAP complex by PACAP 6-38 can effectively attenuate thermal hyperalgesia and mechanical allodynia associated with inflammatory and neuropathic pain states. These results further emphasize that at the level of the spinal cord, PAC(1)-R activation is pronociceptive. PERSPECTIVE: This article presents the analgesic profile generated by the blockade, at the spinal cord level, of the PAC-1 receptor by a potent peptide antagonist. This comprehensive data set demonstrates that if small molecule PAC-1 receptor antagonists could be identified, they would potentially produce broad-spectrum analgesia in both inflammatory and neuropathic pain states.

Discovery and SAR of hydrazide antagonists of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor type 1 (PAC1-R).

Potent small molecule antagonists for the PAC(1)-R have been discovered. Previously known antagonists for the PAC(1)-R were slightly truncated peptide ligands. The hydrazides reported here are the first small molecule antagonists ever reported for this class B GPCR.

A high throughput fluorescent assay for measuring the activity of fatty acid amide hydrolase.

Fatty acid amide hydrolase (FAAH) is the enzyme responsible for the rapid degradation of fatty acid amides such as the endocannabinoid anandamide. Inhibition of FAAH activity has been suggested as a therapeutic approach for the treatment of chronic pain, depression and anxiety, through local activation of the cannabinoid receptor CB1. We have developed a high throughput screening assay for identification of FAAH inhibitors using a novel substrate, decanoyl 7-amino-4-methyl coumarin (D-AMC) that is cleaved by FAAH to release decanoic acid and the highly fluorescent molecule 7-amino-4-methyl coumarin (AMC). This assay gives an excellent signal window for measuring FAAH activity and, as a continuous assay, inherently offers improved sensitivity and accuracy over previously reported endpoint assays. The assay was validated using a panel of known FAAH inhibitors and purified recombinant human FAAH, then converted to a 384 well format and used to screen a large library of compounds (>600,000 compounds) to identify FAAH inhibitors. This screen identified numerous novel FAAH inhibitors of diverse chemotypes. These hits confirmed using a native FAAH substrate, anandamide, and had very similar rank order potency to that obtained using the D-AMC substrate. Collectively these data demonstrate that D-AMC can be successfully used to rapidly and effectively identify novel FAAH inhibitors for potential therapeutic use.

Pharmacological characterization of a 1,4-dihydropyridine analogue, 9-(3,4-dichlorophenyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydro-1,8(2H,5H)-acridinedione (A-184209) as a novelK(ATP) channel inhibitor.

1. This study reports on the identification and characterization of a 1,4-dihydropyridine analogue, 9-(3,4-dichlorophenyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydro-1,8(2H,5H)-acridinedione (A-184209) as a novel inhibitor of ATP-sensitive K(+) channels. 2. A-184209 inhibited membrane potential changes evoked by the prototypical cyanoguanidine ATP-sensitive K(+) channel opener (KCO) P1075 in both vascular (A10) and urinary bladder smooth muscle cells with IC(50) values of 1.44 and 2.24 micro M respectively. 3. P1075-evoked relaxation of 25 mM K(+) stimulated aortic strips was inhibited by A-184209 in an apparently competitive fashion with a pA(2) value of 6.34. 4. The potencies of A-184209 to inhibit P1075-evoked decreases in membrane potential responses in cardiac myocytes (IC(50)=0.53 micro M) and to inhibit 2-deoxyglucose-evoked cation efflux pancreatic RINm5F cells (IC(50)=0.52 micro M) were comparable to the values for inhibition of smooth muscle K(ATP) channels. 5. On the other hand, a structural analogue of A-184209 that lacked the gem-dimethyl substituent, 9-(3,4-dichlorophenyl)-3,4,6,7,9,10-hexahydro-1,8(2H,5H)-acridinedione (A-184208), was found to be a K(ATP) channel opener, evoking membrane potential responses in A10 smooth muscle cells (EC(50)=385 nM) and relaxing aortic smooth muscle strips (IC(50)=101 nM) in a glyburide-sensitive manner. 6. Radioligand binding studies demonstrated that A-184209 displaced SUR1 binding defined by [(3)H]glyburide binding to RINm5F cell membranes with a K(i) value of 0.11 micro M whereas A-184208 was ineffective. On the other hand, both A-184209 (K(i)=1.34 micro M) and A-184208 (K(i)=1.14 micro M) displaced binding of the KCO radioligand, [(125)I]A-312110 in guinea-pig bladder membranes with similar affinities. 7. These studies demonstrate that A-184209 is a novel and structurally distinct compound that inhibits K(ATP) channels in smooth muscle with potencies comparable to glyburide. The structural overlap between DHP openers and blockers, together with their differential interaction with ligand binding sites, support the notion that both openers and blockers bind to similar or very closely coupled sites on the sulfonylurea receptor and that subtle changes in the pharmacophore itself could switch functional properties from K(ATP) channel activation to inhibition.

In vitro and in vivo characterization of a novel naphthylamide ATP-sensitive K+ channel opener, A-151892.

1. Openers of ATP-sensitive K(+) channels are of interest in several therapeutic indications including overactive bladder and other lower urinary tract disorders. This study reports on the in vitro and in vivo characterization of a structurally novel naphthylamide N-[2-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-naphthalen-1-yl]-acetamide (A-151892), as an opener of the ATP-sensitive potassium channels. 2. A-151892 was found to be a potent and efficacious potassium channel opener (KCO) as assessed by glibenclamide-sensitive whole-cell current and fluorescence-based membrane potential responses (-log EC(50)=7.63) in guinea-pig bladder smooth muscle cells. 3. Evidence for direct interaction with KCO binding sites was derived from displacement of binding of the 1,4-dihydropyridine opener [(125)I]A-312110. A-151892 displaced [(125)I]A-312110 binding to bladder membranes with a -log Ki value of 7.45, but lacked affinity against over 70 neurotransmitter receptor and ion channel binding sites. 4. In pig bladder strips, A-151892 suppressed phasic, carbachol-evoked and electrical field stimulus-evoked contractility in a glibenclamide-reversible manner with -log IC(50) values of 8.07, 7.33 and 7.02 respectively, comparable to that of the potencies of the prototypical cyanoguanidine KCO, P1075. The potencies to suppress contractions in thoracic aorta (-log IC(50)=7.81) and portal vein (-log IC(50)=7.98) were not substantially different from those observed for suppression of phasic contractility of the bladder smooth muscle. 5. In vivo, A-151892 was found to potently suppress unstable bladder contractions in obstructed models of unstable contractions in both pigs and rats with pED(35%) values of 8.05 and 7.43, respectively. 6. These results demonstrate that naphthylamide analogs exemplified by A-151892 are novel K(ATP) channel openers and may serve as chemotypes to exploit additional analogs with potential for the treatment of overactive bladder and lower urinary tract symptoms.

Characterization of human urinary bladder KATP channels containing SUR2B splice variants expressed in L-cells.

The molecular properties of the sulfonylurea receptor 2 (SUR2) subunits of K(ATP) channels expressed in urinary bladder were assessed by polymerase chain reaction (PCR). This showed that SUR2B exon 17- mRNA (72%) was predominant over the SUR2B exon 17+ splice variant (28%). The pharmacological properties of both of these isoforms stably expressed in mouse Ltk(-)cells (L-cells) with K(IR) 6.2 were determined by measuring changes in membrane potential responses evoked by K(+) channel openers using bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC(4)(3)) fluorescence. The rank order potency of a variety of structurally distinct K(+) channel openers was found to be the same in both stable cell lines and compared well with guinea pig bladder cells. The potency of these compounds in the SUR2B exon 17- cells more closely resembled the potency measured in guinea pig bladder unlike the cell line containing the SUR2B exon 17+ subtype. Analysis of the displacement of [125I]A-312110 binding with the same K(+) channel openers to the SUR2B exon 17- cells showed excellent correlation to those measured in guinea pig bladder. This study supports the notion that K(ATP) channels containing SUR2B exon 17- represent a major splice variant expressed in urinary bladder smooth muscle.

Expression of antibodies using single open reading frame (sORF) vector design: Demonstration of manufacturing feasibility.

Efficient production of large quantities of therapeutic antibodies is becoming a major goal of the pharmaceutical industry. We developed a proprietary expression system using a polyprotein precursor-based approach to antibody expression in mammalian cells. In this approach, the coding regions for heavy and light chains are included within a single open reading frame (sORF) separated by an in-frame intein gene. A single mRNA and subsequent polypeptide are produced upon transient and stable transfection into HEK293 and CHO cells, respectively. Heavy and light chains are separated by the autocatalytic action of the intein and antibody processing proceeds to produce active, secreted antibody. Here, we report advances in sORF technology toward establishment of a viable manufacturing platform for therapeutic antibodies in CHO cells. Increasing expression levels and improving antibody processing by intein and signal peptide selection are discussed.

Solution structure and mutational analysis of pituitary adenylate cyclase-activating polypeptide binding to the extracellular domain of PAC1-RS.

The pituitary adenylate cyclase-activating polypeptide (PACAP) receptor is a class II G protein-coupled receptor that contributes to many different cellular functions including neurotransmission, neuronal survival, and synaptic plasticity. The solution structure of the potent antagonist PACAP (residues 6'-38') complexed to the N-terminal extracellular (EC) domain of the human splice variant hPAC1-R-short (hPAC1-R(S)) was determined by NMR. The PACAP peptide adopts a helical conformation when bound to hPAC1-R(S) with a bend at residue A18' and makes extensive hydrophobic and electrostatic interactions along the exposed beta-sheet and interconnecting loops of the N-terminal EC domain. Mutagenesis data on both the peptide and the receptor delineate the critical interactions between the C terminus of the peptide and the C terminus of the EC domain that define the high affinity and specificity of hormone binding to hPAC1-R(S). These results present a structural basis for hPAC1-R(S) selectivity for PACAP versus the vasoactive intestinal peptide and also differentiate PACAP residues involved in binding to the N-terminal extracellular domain versus other parts of the full-length hPAC1-R(S) receptor. The structural, mutational, and binding data are consistent with a model for peptide binding in which the C terminus of the peptide hormone interacts almost exclusively with the N-terminal EC domain, whereas the central region makes contacts to both the N-terminal and other extracellular parts of the receptor, ultimately positioning the N terminus of the peptide to contact the transmembrane region and result in receptor activation.

[125I]A-312110, a novel high-affinity 1,4-dihydropyridine ATP-sensitive K+ channel opener: characterization and pharmacology of binding.

Although ATP-sensitive K+ channels continue to be explored for their therapeutic potential, developments in high-affinity radioligands to investigate native and recombinant KATP channels have been less forthcoming. This study reports the identification and pharmacological characterization of a novel iodinated 1,4-dihydropyridine KATP channel opener, [125I]A-312110 [(9R)-9-(4-fluoro-3-125iodophenyl)-2,3,5,9-tetrahydro-4H-pyrano[3,4-b]thieno[2,3-e]pyridin-8(7H)-one-1,1-dioxide]. Binding of [125I]A-312110 to guinea pig cardiac (KD = 5.8 nM) and urinary bladder (KD = 4.9 nM) membranes were of high affinity, saturable, and to a single set of binding sites. Displacement of [125I]A-312110 by structurally diverse potassium channel openers (KCOs) indicated a similar rank order of potency in both guinea pig cardiac and bladder membranes (Ki, heart): A-312110 (4.3 nM) > N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine (P1075) > (-)-N-(2-ethoxyphenyl)-N'-(1,2,3-trimethylpropyl)-2-nitroethene-1,1-diamine (Bay X 9228) > pinacidil > (-)-cromakalim > N-(4-benzoyl phenyl)-3,3,3-trifluro-2-hydroxy-2-methylpropionamine (ZD6169) > 9-(3-cyanophenyl)-3,4,6,7,9,10-hexahydro-1,8-(2H,5H)-acridinedione (ZM244085) >> diazoxide (16.7 microM). Displacement by KATP channel blockers, the sulfonylurea glyburide, and the cyanoguanidine N-[1-(3-chlorophenyl)cyclobutyl]-N'-cyano-N"-3-pyridinyl-guanidine (PNU-99963) were biphasic in the heart but monophasic in bladder with about a 100- to 500-fold difference in Ki values between high- and low-affinity sites. Good correlations were observed between cardiac or bladder-binding affinities of KCOs with functional activation as assessed by their respective potencies to either suppress action potential duration (APD) in Purkinje fibers or to relax electrical field-stimulated bladder contractions. Collectively, these results demonstrate that [125I]A-312110 binds with high affinity and has an improved activity profile compared with other radiolabeled KCOs. [125I]A-312110 is a useful tool for investigation of the molecular and functional properties of the KATP channel complex and for the identification, in a high throughput manner, of both novel channel blockers and openers that interact with cardiac/smooth muscle-type KATP channels.

(-)-(9S)-9-(3-Bromo-4-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno[3,2-b]quinolin-8(4H)-one 1,1-dioxide (A-278637): a novel ATP-sensitive potassium channel opener efficacious in suppressing urinary bladder contractions. I. In vitro characterization.

Alterations in the myogenic activity of the bladder smooth muscle are thought to serve as a basis for the involuntary detrusor contractions associated with the overactive bladder. Activation of ATP-sensitive K(+) (K(ATP)) channels has been recognized as a potentially viable mechanism to modulate membrane excitability in bladder smooth muscle. In this study, we describe the preclinical pharmacology of (-)-(9S)-9-(3-bromo-4-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno[3,2-b]quinolin-8(4H)-one 1,1-dioxide (A-278637), a novel 1,4-dihydropyridine K(ATP) channel opener (KCO) that demonstrates enhanced bladder selectivity for the suppression of unstable bladder contractions in vivo relative to other reference KCOs. A-278637 activated K(ATP) channels in bladder smooth muscle cells in a glyburide (glibenclamide)-sensitive manner as assessed by fluorescence membrane potential assays using bis-(1,3-dibutylbarbituric acid)trimethine oxonol (EC(50) = 102 nM) and by whole cell patch clamp. Spontaneous (myogenic) phasic activity of pig bladder strips was suppressed (IC(50) = 23 nM) in a glyburide-sensitive manner by A-278637. A-278637 also inhibited carbachol- and electrical field-stimulated contractions of bladder strips, although the respective potencies were 8- and 13-fold lower compared with inhibition of spontaneous phasic activity. As shown in the accompanying article [Brune ME, Fey TA, Brioni JD, Sullivan JP, Williams M, Carroll WA, Coghlan MJ, and Gopalakrishnan M (2002) J Pharmacol Exp Ther 303:387-394], A-278637 suppressed myogenic contractions in vivo in a model of bladder instability with superior selectivity compared with other KCOs, WAY-133537 [(R)-4-[3,4-dioxo-2-(1,2,2-trimethyl-propylamino)cyclobut-1-enylamino]-3-ethyl-benzonitrile] and ZD6169 [(S)-N-(4-benzoylphenyl)3,3,3-trifluro-2hydroxy-2-methyl-priopionamide]. A-278637 did not interact with other ion channels, including L-type calcium channels or other neurotransmitter receptor systems. The pharmacological profile of A-278637 represents an attractive basis for further investigations of selective K(ATP) channel openers for the treatment of overactive bladder via myogenic etiology.