Jenny Tobin

Integrating transcriptional and metabolite profiles to direct the engineering of lovastatin-producing fungal strains.

We describe a method to decipher the complex inter-relationships between metabolite production trends and gene expression events, and show how information gleaned from such studies can be applied to yield improved production strains. Genomic fragment microarrays were constructed for the Aspergillus terreus genome, and transcriptional profiles were generated from strains engineered to produce varying amounts of the medically significant natural product lovastatin. Metabolite detection methods were employed to quantify the polyketide-derived secondary metabolites lovastatin and (+)-geodin in broths from fermentations of the same strains. Association analysis of the resulting transcriptional and metabolic data sets provides mechanistic insight into the genetic and physiological control of lovastatin and (+)-geodin biosynthesis, and identifies novel components involved in the production of (+)-geodin, as well as other secondary metabolites. Furthermore, this analysis identifies specific tools, including promoters for reporter-based selection systems, that we employed to improve lovastatin production by A. terreus.

Pharmacologic Properties, Metabolism, and Disposition of Linaclotide, a Novel Therapeutic Peptide Approved for the Treatment of Irritable Bowel Syndrome with Constipation and Chronic Idiopathic Constipation

Linaclotide, a potent guanylate cyclase C agonist, is a therapeutic peptide approved in the United States for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation. We present for the first time the metabolism, degradation, and disposition of linaclotide in animals and humans. We examined the metabolic stability of linaclotide in conditions that mimic the gastrointestinal tract and characterized the metabolite MM-419447 (CCEYCCNPACTGC), which contributes to the pharmacologic effects of linaclotide. Systemic exposure to these active peptides is low in rats and humans, and the low systemic and portal vein concentrations of linaclotide and MM-419447 observed in the rat confirmed both peptides are minimally absorbed after oral administration. Linaclotide is stable in the acidic environment of the stomach and is converted to MM-419447 in the small intestine. The disulfide bonds of both peptides are reduced in the small intestine, where they are subsequently proteolyzed and degraded. After oral administration of linaclotide, <1% of the dose was excreted as active peptide in rat feces and a mean of 3–5% in human feces; in both cases MM-419447 was the predominant peptide recovered. MM-419447 exhibits high-affinity binding in vitro to T84 cells, resulting in a significant, concentration-dependent accumulation of intracellular cyclic guanosine-3′,5′-monophosphate (cGMP). In rat models of gastrointestinal function, orally dosed MM-419447 significantly increased fluid secretion into small intestinal loops, increased intraluminal cGMP, and caused a dose-dependent acceleration in gastrointestinal transit. These results demonstrate the importance of the active metabolite in contributing to linaclotide’s pharmacology.

Discovery and Characterization of a Potent Interleukin-6 Binding Peptide with Neutralizing Activity In Vivo

Interleukin-6 (IL-6) is an important member of the cytokine superfamily, exerting pleiotropic actions on many physiological processes. Over-production of IL-6 is a hallmark of immune-mediated inflammatory diseases such as Castleman’s Disease (CD) and rheumatoid arthritis (RA). Antagonism of the interleukin IL-6/IL-6 receptor (IL-6R)/gp130 signaling complex continues to show promise as a therapeutic target. Monoclonal antibodies (mAbs) directed against components of this complex have been approved as therapeutics for both CD and RA. To potentially provide an additional modality to antagonize IL-6 induced pathophysiology, a peptide-based antagonist approach was undertaken. Using a combination of molecular design, phage-display, and medicinal chemistry, disulfide-rich peptides (DRPs) directed against IL-6 were developed with low nanomolar potency in inhibiting IL-6-induced pSTAT3 in U937 monocytic cells. Targeted PEGylation of IL-6 binding peptides resulted in molecules that retained their potency against IL-6 and had a prolongation of their pharmacokinetic (PK) profiles in rodents and monkeys. One such peptide, PN-2921, contained a 40 kDa polyethylene glycol (PEG) moiety and inhibited IL-6-induced pSTAT3 in U937 cells with sub-nM potency and possessed 23, 36, and 59 h PK half-life values in mice, rats, and cynomolgus monkeys, respectively. Parenteral administration of PN-2921 to mice and cynomolgus monkeys potently inhibited IL-6-induced biomarker responses, with significant reductions in the acute inflammatory phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP). This potent, PEGylated IL-6 binding peptide offers a new approach to antagonize IL-6-induced signaling and associated pathophysiology.

MRP4 Modulation of the Guanylate Cyclase-C/cGMP Pathway: Effects on Linaclotide-Induced Electrolyte Secretion and cGMP Efflux

MRP4 mediates the efflux of cGMP and cAMP and acts as an important regulator of these secondary messengers, thereby affecting signaling events mediated by cGMP and cAMP. Immunofluorescence staining showed high MRP4 expression localized predominantly in the apical membrane of rat colonic epithelium. In vitro studies were performed using a rat colonic mucosal layer mounted in an Ussing chamber. Linaclotide activation of the guanylate cyclase-C (GC-C)/cGMP pathway induced a concentration-dependent increase in transepithelial ion current [short-circuit current (Isc)] across rat colonic mucosa (EC50: 9.2 nM). Pretreatment of colonic mucosa with the specific MRP4 inhibitor MK571 potentiated linaclotide-induced electrolyte secretion and augmented linaclotide-stimulated intracellular cGMP accumulation. Notably, pretreatment with the phosphodiesterase 5 inhibitor sildenafil increased basal Isc, but had no amplifying effect on linaclotide-induced Isc. MRP4 inhibition selectively affected the activation phase, but not the deactivation phase, of linaclotide. In contrast, incubation with a GC-C/Fc chimera binding to linaclotide abrogated linaclotide-induced Isc, returning to baseline. Furthermore, linaclotide activation of GC-C induced cGMP secretion from the apical and basolateral membranes of colonic epithelium. MRP4 inhibition blocked cGMP efflux from the apical membrane, but not the basolateral membrane. These data reveal a novel, previously unrecognized mechanism that functionally couples GC-C-induced luminal electrolyte transport and cGMP secretion to spatially restricted, compartmentalized regulation by MRP4 at the apical membrane of intestinal epithelium. These findings have important implications for gastrointestinal disorders with symptoms associated with dysregulated fluid homeostasis, such as irritable bowel syndrome with constipation, chronic idiopathic constipation, and secretory diarrhea.

Discovery of IWP-051, a Novel Orally Bioavailable sGC Stimulator with Once-Daily Dosing Potential in Humans

In recent years, soluble guanylate cyclase (sGC, EC 4.6.1.2) has emerged as an attractive therapeutic target for treating cardiovascular diseases and diseases associated with fibrosis and end-organ failure. Herein, we describe our design and synthesis of a series of 4-hydroxypyrimidine sGC stimulators starting with an internally discovered lead. Our efforts have led to the discovery of IWP-051, a molecule that achieves good alignment of potency, stability, selectivity, and pharmacodynamic effects while maintaining favorable pharmacokinetic properties with once-daily dosing potential in humans.

Pharmacological Characterization of IW-1973, a Novel Soluble Guanylate Cyclase Stimulator with Extensive Tissue Distribution, Antihypertensive, Anti-Inflammatory, and Antifibrotic Effects in Preclinical Models of Disease

Soluble guanylate cyclase (sGC), a key signal-transduction enzyme, increases the conversion of guanosine-5′-triphosphate to cGMP upon binding of nitric oxide (NO). Endothelial dysfunction and/or reduced NO signaling have been implicated in cardiovascular disease pathogenesis and complications of diabetes and have been associated with other disease states and aging. Soluble guanylate cyclase (sGC) stimulators are small-molecule drugs that bind sGC and enhance NO-mediated cGMP signaling. The pharmacological characterization of IW-1973 [1,1,1,3,3,3-hexafluoro-2-(((5-fluoro-2-(1-(2-fluorobenzyl)-5-(isoxazol-3-yl)-1H-pyrazol-3-yl) pyrimidin-4-yl)amino)methyl)propan-2-ol], a novel clinical-stage sGC stimulator under clinical investigation for treatment of heart failure with preserved ejection fraction and diabetic nephropathy, is described. In the presence of NO, IW-1973 stimulated sGC in a human purified enzyme assay and a HEK-293 whole cell assay. sGC stimulation by IW-1973 in cells was associated with increased phosphorylation of vasodilator-stimulated phosphoprotein. IW-1973, at doses of 1–10 mg/kg, significantly lowered blood pressure in normotensive and spontaneously hypertensive rats. In a Dahl salt-sensitive hypertension model, IW-1973 significantly reduced blood pressure, inflammatory cytokine levels, and renal disease markers, including proteinuria and renal fibrotic gene expression. The results were affirmed in mouse lipopolysaccharide-induced inflammation and rat unilateral ureteral obstruction renal fibrosis models. A quantitative whole-body autoradiography study of IW-1973 revealed extensive tissue distribution and pharmacokinetic studies showed a large volume of distribution and a profile consistent with predicted once-a-day dosing in humans. In summary, IW-1973 is a potent, orally available sGC stimulator that exhibits renoprotective, anti-inflammatory, and antifibrotic effects in nonclinical models.

Discovery of IWP-051, a novel orally bioavailable soluble guanylate cyclase stimulator with sustained and dose-dependent hemodynamic effects.

Discovery of IWP-051, a novel orally bioavailable soluble guanylate cyclase stimulator with sustained and dose-dependent hemodynamic effects Takashi Nakai, Nicholas R Perl, Rajesh R Iyengar, Ara Mermerian, G-Yoon J Im, Thomas W-H Lee, Glen R Rennie, James Jia, Paul A Renhowe, Timothy C Barden, James E Sheppeck II, Karthik Iyer, Joon Jung, G Todd Milne, Chrissie Segal, Kimberly Long, Joy Miyashiro, Sylvie Bernier, Sarah Jacobson, Jenny Tobin, Courtney Shea, Peter Germano, Yueh-tyng Chien, Daniel Zimmer

Concomitant administration of sGC stimulators with common classes of anti-hypertensive agents results in increased efficacy in spontaneously hypertensive rats

Background Soluble guanylate cyclase (sGC) stimulators demonstrate smooth muscle relaxation and vasodilation via the nitric oxide (NO)-sGC-cyclic guanosine monophosphate (cGMP) pathway. A novel class of sGC stimulators, the pyrazole-pyrimidines, was synthesized with the objective of creating a potent, once-a-day (QD) oral treatment for cardiovascular diseases. Several compounds from this class were identified as potent stimulators of sGC in vitro (EC50 = 40-287 nM). These compounds were evaluated in pharmacokinetic (PK) and blood pressure pharmacodynamics (PD) in vivo rat and dog models and were shown to exhibit sustained compound exposure (Thalf = >7 hours in preclinical species) after oral dosing, predicting QD dosing in humans. Further, they significantly decreased mean arterial blood pressure (MAP (≥ 10mmHg) after oral dosing. The potential for sGC stimulators to work in combination with reference antihypertensive therapies was assessed in an in vivo PD assay in a spontaneous hypertensive rat (SHR) model. Doses of losartan, atenolol, amlodipine, and our sGC stimulators that induced an effect (< 30mmHg) on MAP were chosen. IWP-121, a representative sGC stimulator, was shown to provide additional MAP lowering effects when combined with losartan, atenolol, or amlodipine, resulting in an increase in overall blood pressure effects between 5-50%. By linking compound concentration to blood pressure change for each compound alone and in combination, we were able to assess the PK/PD relationships for the individual and combined effects.

IWP-121: a novel sGC stimulator that reduces blood pressure and exhibits anti-fibrotic and anti-inflammatory activities in the Dahl Salt-Sensitive rat model

Background Soluble guanylate cyclase (sGC) is an intracellular receptor that can be activated by nitric oxide (NO) and sGC stimulators to produce cyclic guanosine monophosphate (cGMP), thereby modulating a number of downstream cellular and physiological responses including phosphorylation of VASP and vasodilation. In the Dahl Salt-Sensitive (DSS) rat model of hypertension, cGMP production by sGC is decreased, most likely due to reactive oxygen species (ROS) converting NO to peroxynitrite, resulting in depleted pools of NO available to bind to sGC. In this study we evaluated the efficacy of a novel sGC stimulator (IWP-121) in the DSS model. Male DSS rats (230-270 grams) received high-salt diet (8% NaCl) for 2 weeks followed by high salt plus compound for 6 additional weeks. IWP-121 was administered at doses of 1, 3, and 10 mg/kg/ day in the chow (n=8/group). Losartan (30 mg/kg/day in the water) was used as a positive control, in addition to both High Salt (HS) and Normal Salt (NS). All groups were compared to HS control group for analyses. IWP-121 dose dependently decreased mean blood pressure (MAP) throughout the study. Additionally, IWP-121 (at all doses tested) and losartan had statistically significant effects on decreasing heart hypertrophy and plasma NT-proBNP but only IWP-121 had an effect on attenuating liver hypertrophy. IWP-121 decreased microalbuminuria (an indicator of kidney end organ damage) as well as attenuated serum biomarkers known to be involved in inflammatory and fibrotic processes. Conclusion In the rat DSS model of hypertension, there is a decrease in cGMP levels most likely due to the inactivation of endogenous NO by ROS. The sGC stimulator IWP-121, when administered in the diet exhibited sustained dosedependent reduction in blood pressure. Additionally, IWP-121 attenuated heart and liver hypertrophy and reduced NT-proBNP, a biomarker of heart failure. The compound reduced levels of biomarkers for inflammation and fibrosis, and demonstrated renal end organ protection. sGC stimulators, like IWP-121 may have broad therapeutic application by modulating multiple relevant therapeutic endpoints including blood pressure, hypertrophy, inflammation, and fibrosis.

Tu1863 Linaclotide Induces Secretion of cGMP Into the Colonic Submucosal Layer: An In Vivo Microdialysis Study in Rats

Figure 1: Physiologic response to intraluminal BAC{BR}A) Fecal output decreased significantly in the days after BAC exposure but then recovered toward baseline output. Output was significantly more depressed in mice treated with 10% BAC compared to 5% BAC on postexposure days 2 and 3. B) Body weight also decreased in a similar fashion but then trended towards recovery. (Note: the N of mice treated with 10% BAC changed due to illness or excessive weight loss). *: p<0.05 when compared to PBS group. ‡: p<0.05 when compared to BAC 5% group. Error bars are standard error.