The Novel, Clinical-Stage Soluble Guanylate Cyclase Activator BI 685509 Protects from Disease Progression in Models of Renal Injury and Disease.

Activation of soluble guanylate cyclase (sGC) to restore cyclic guanosine monophosphate (cGMP) and improve functionality of nitric oxide (NO) pathways impaired by oxidative stress is a potential treatment of diabetic and chronic kidney disease. We report the pharmacology of BI 685509, a novel, orally active small molecule sGC activator with disease-modifying potential. BI 685509 and human sGC α1/ß1 heterodimer containing a reduced heme group produced concentration-dependent increases in cGMP that were elevated modestly by NO, whereas heme-free sGC and BI 685509 greatly enhanced cGMP with no effect of NO. BI 685509 increased cGMP in human and rat platelet-rich plasma treated with the heme-oxidant ODQ; respective EC50 values were 467 nM and 304 nM. In conscious telemetry-instrumented rats, BI 685509 did not affect mean arterial pressure (MAP) or heart rate (HR) at 3 and 10 mg/kg (p.o.), whereas 30 mg/kg decreased MAP and increased HR. Ten days of BI 685509 at supratherapeutic doses (60 or 100 mg/kg p.o., daily) attenuated MAP and HR responses to a single 100 mg/kg challenge. In the ZSF1 rat model, BI 685509 (1, 3, 10, and 30 mg/kg per day, daily) coadministered with enalapril (3 mg/kg per day) dose-dependently reduced proteinuria and incidence of glomerular sclerosis; MAP was modestly reduced at the higher doses versus enalapril. In the 7-day rat unilateral ureteral obstruction model, BI 685509 dose-dependently reduced tubulointerstitial fibrosis (P < 0.05 at 30 mg/kg). In conclusion, BI 685509 is a potent, orally bioavailable sGC activator with clear renal protection and antifibrotic activity in preclinical models of kidney injury and disease. SIGNIFICANCE STATEMENT: BI 685509 is a novel small soluble guanylate cyclase (sGC) molecule activator that exhibits an in vitro profile consistent with that of an sGC activator. BI 685509 reduced proteinuria and glomerulosclerosis in the ZSF1 rat, a model of diabetic kidney disease (DKD), and reduced tubulointerstitial fibrosis in a rat 7-day unilateral ureteral obstruction model. Thus, BI 685509 is a promising new therapeutic agent and is currently in phase II clinical trials for chronic kidney disease and DKD.

Targeting endogenous kidney regeneration using anti-IL11 therapy in acute and chronic models of kidney disease.

The kidney has large regenerative capacity, but this is compromised when kidney damage is excessive and renal tubular epithelial cells (TECs) undergo SNAI1-driven growth arrest. Here we investigate the role of IL11 in TECs, kidney injury and renal repair. IL11 stimulation of TECs induces ERK- and p90RSK-mediated GSK3ß inactivation, SNAI1 upregulation and pro-inflammatory gene expression. Mice with acute kidney injury upregulate IL11 in TECs leading to SNAI1 expression and kidney dysfunction, which is not seen in Il11 deleted mice or in mice administered a neutralizing IL11 antibody in either preemptive or treatment modes. In acute kidney injury, anti-TGFß reduces renal fibrosis but exacerbates inflammation and tubule damage whereas anti-IL11 reduces all pathologies. Mice with TEC-specific deletion of Il11ra1 have reduced pathogenic signaling and are protected from renal injury-induced inflammation, fibrosis, and failure. In a model of chronic kidney disease, anti-IL11 therapy promotes TEC proliferation and parenchymal regeneration, reverses fibroinflammation and restores renal mass and function. These data highlight IL11-induced mesenchymal transition of injured TECs as an important renal pathology and suggest IL11 as a therapeutic target for restoring stalled endogenous regeneration in the diseased kidney.

The key role of NLRP3 and STING in APOL1-associated podocytopathy.

Coding variants in apolipoprotein L1 (APOL1), termed G1 and G2, can explain most excess kidney disease risk in African Americans; however, the molecular pathways of APOL1-induced kidney dysfunction remain poorly understood. Here, we report that expression of G2 APOL1 in the podocytes of Nphs1rtTA/TRE-G2APOL1 (G2APOL1) mice leads to early activation of the cytosolic nucleotide sensor, stimulator of interferon genes (STING), and the NLR family pyrin domain-containing 3 (NLRP3) inflammasome. STING and NLRP3 expression was increased in podocytes from patients with high-risk APOL1 genotypes, and expression of APOL1 correlated with caspase-1 and gasdermin D (GSDMD) levels. To demonstrate the role of NLRP3 and STING in APOL1-associated kidney disease, we generated transgenic mice with the G2 APOL1 risk variant and genetic deletion of Nlrp3 (G2APOL1/Nlrp3 KO), Gsdmd (G2APOL1/Gsdmd KO), and STING (G2APOL1/STING KO). Knockout mice displayed marked reduction in albuminuria, azotemia, and kidney fibrosis compared with G2APOL1 mice. To evaluate the therapeutic potential of targeting NLRP3, GSDMD, and STING, we treated mice with MCC950, disulfiram, and C176, potent and selective inhibitors of NLRP3, GSDMD, and STING, respectively. G2APOL1 mice treated with MCC950, disulfiram, and C176 showed lower albuminuria and improved kidney function even when inhibitor treatment was initiated after the development of albuminuria.

Molecular characterization and cell type composition deconvolution of fibrosis in NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver disease worldwide. In adults with NAFLD, fibrosis can develop and progress to liver cirrhosis and liver failure. However, the underlying molecular mechanisms of fibrosis progression are not fully understood. Using total RNA-Seq, we investigated the molecular mechanisms of NAFLD and fibrosis. We sequenced liver tissue from 143 adults across the full spectrum of fibrosis stage including those with stage 4 fibrosis (cirrhosis). We identified gene expression clusters that strongly correlate with fibrosis stage including four genes that have been found consistently across previously published transcriptomic studies on NASH i.e. COL1A2, EFEMP2, FBLN5 and THBS2. Using cell type deconvolution, we estimated the loss of hepatocytes versus gain of hepatic stellate cells, macrophages and cholangiocytes with advancing fibrosis stage. Hepatocyte-specific functional analysis indicated increase of pro-apoptotic pathways and markers of bipotent hepatocyte/cholangiocyte precursors. Regression modelling was used to derive predictors of fibrosis stage. This study elucidated molecular and cell composition changes associated with increasing fibrosis stage in NAFLD and defined informative gene signatures for the disease.

Urinary Single-Cell Profiling Captures the Cellular Diversity of the Kidney.

BACKGROUND: Microscopic analysis of urine sediment is probably the most commonly used diagnostic procedure in nephrology. The urinary cells, however, have not yet undergone careful unbiased characterization. METHODS: Single-cell transcriptomic analysis was performed on 17 urine samples obtained from five subjects at two different occasions, using both spot and 24-hour urine collection. A pooled urine sample from multiple healthy individuals served as a reference control. In total 23,082 cells were analyzed. Urinary cells were compared with human kidney and human bladder datasets to understand similarities and differences among the observed cell types. RESULTS: Almost all kidney cell types can be identified in urine, such as podocyte, proximal tubule, loop of Henle, and collecting duct, in addition to macrophages, lymphocytes, and bladder cells. The urinary cell-type composition was subject specific and reasonably stable using different collection methods and over time. Urinary cells clustered with kidney and bladder cells, such as urinary podocytes with kidney podocytes, and principal cells of the kidney and urine, indicating their similarities in gene expression. CONCLUSIONS: A reference dataset for cells in human urine was generated. Single-cell transcriptomics enables detection and quantification of almost all types of cells in the kidney and urinary tract.

Genome-Wide Association Study of Diabetic Kidney Disease Highlights Biology Involved in Glomerular Basement Membrane Collagen.

BACKGROUND: Although diabetic kidney disease demonstrates both familial clustering and single nucleotide polymorphism heritability, the specific genetic factors influencing risk remain largely unknown. METHODS: To identify genetic variants predisposing to diabetic kidney disease, we performed genome-wide association study (GWAS) analyses. Through collaboration with the Diabetes Nephropathy Collaborative Research Initiative, we assembled a large collection of type 1 diabetes cohorts with harmonized diabetic kidney disease phenotypes. We used a spectrum of ten diabetic kidney disease definitions based on albuminuria and renal function. RESULTS: Our GWAS meta-analysis included association results for up to 19,406 individuals of European descent with type 1 diabetes. We identified 16 genome-wide significant risk loci. The variant with the strongest association (rs55703767) is a common missense mutation in the collagen type IV alpha 3 chain (COL4A3) gene, which encodes a major structural component of the glomerular basement membrane (GBM). Mutations in COL4A3 are implicated in heritable nephropathies, including the progressive inherited nephropathy Alport syndrome. The rs55703767 minor allele (Asp326Tyr) is protective against several definitions of diabetic kidney disease, including albuminuria and ESKD, and demonstrated a significant association with GBM width; protective allele carriers had thinner GBM before any signs of kidney disease, and its effect was dependent on glycemia. Three other loci are in or near genes with known or suggestive involvement in this condition (BMP7) or renal biology (COLEC11 and DDR1). CONCLUSIONS: The 16 diabetic kidney disease-associated loci may provide novel insights into the pathogenesis of this condition and help identify potential biologic targets for prevention and treatment.

Changes in Albuminuria But Not GFR are Associated with Early Changes in Kidney Structure in Type 2 Diabetes.

BACKGROUND: In type 1 diabetes, changes in the GFR and urine albumin-to-creatinine ratio (ACR) are related to changes in kidney structure that reflect disease progression. However, such changes have not been studied in type 2 diabetes. METHODS: Participants were American Indians with type 2 diabetes enrolled in a clinical trial of losartan versus placebo. We followed a subset who underwent kidney biopsy at the end of the 6-year trial, with annual measurements of GFR (by urinary clearance of iothalamate) and ACR. Participants had a second kidney biopsy after a mean follow-up of 9.3 years. We used quantitative morphometric analyses to evaluate both biopsy specimens. RESULTS: Baseline measures for 48 participants (12 men and 36 women, mean age 45.6 years) who completed the study included diabetes duration (14.6 years), GFR (156 ml/min), and ACR (15 mg/g). During follow-up, glomerular basement membrane (GBM) width, mesangial fractional volume, and ACR increased, and surface density of peripheral GBM and GFR decreased. After adjustment for sex, age, ACR, and each morphometric variable at baseline, an increase in ACR during follow-up was significantly associated with increases in GBM width, mesangial fractional volume, and mean glomerular volume, and a decrease in surface density of peripheral GBM. Decline in GFR was not associated with changes in these morphometric variables after additionally adjusting for baseline GFR. CONCLUSIONS: In American Indians with type 2 diabetes and preserved GFR at baseline, increasing ACR reflects the progression of earlier structural glomerular lesions, whereas early GFR decline may not accurately reflect such lesions.

Renal compartment-specific genetic variation analyses identify new pathways in chronic kidney disease.

Chronic kidney disease (CKD), a condition in which the kidneys are unable to clear waste products, affects 700 million people globally. Genome-wide association studies (GWASs) have identified sequence variants for CKD; however, the biological basis of these GWAS results remains poorly understood. To address this issue, we created an expression quantitative trait loci (eQTL) atlas for the glomerular and tubular compartments of the human kidney. Through integrating the CKD GWAS with eQTL, single-cell RNA sequencing and regulatory region maps, we identified novel genes for CKD. Putative causal genes were enriched for proximal tubule expression and endolysosomal function, where DAB2, an adaptor protein in the TGF-ß pathway, formed a central node. Functional experiments confirmed that reducing Dab2 expression in renal tubules protected mice from CKD. In conclusion, compartment-specific eQTL analysis is an important avenue for the identification of novel genes and cellular pathways involved in CKD development and thus potential new opportunities for its treatment.

A molecular morphometric approach to diabetic kidney disease can link structure to function and outcome.

Diabetic kidney disease is the leading cause of kidney failure. However, studies of molecular mechanisms of early kidney damage are lacking. Here we examined for possible linkage between transcriptional regulation and quantitative structural damage in early diabetic kidney disease in Pima Indians with type 2 diabetes. Tissue obtained from protocol kidney biopsies underwent genome-wide compartment-specific gene expression profiling and quantitative morphometric analysis. The ultrastructural lesion most strongly associated with transcriptional regulation was cortical interstitial fractional volume (VvInt), an index of tubule-interstitial damage. Transcriptional co-expression network analysis identified 1843 transcripts that correlated significantly with VvInt. These transcripts were enriched for pathways associated with mitochondrial dysfunction, inflammation, migratory mechanisms, and tubular metabolic functions. Pathway network analysis identified IL-1ß as a key upstream regulator of the inflammatory response and five transcription factors cooperating with p53 to regulate metabolic functions. VvInt-associated transcripts showed significant correlation with the urine albumin to creatinine ratio and measured glomerular filtration rate 10 years after biopsy, establishing a link between the early molecular events and long-term disease progression. Thus, molecular mechanisms active early in diabetic kidney disease were revealed by correlating intrarenal transcripts with quantitative morphometry and long-term outcomes. This provides a starting point for identification of urgently needed therapeutic targets and non-invasive biomarkers of early diabetic kidney disease.

Transgenic expression of human APOL1 risk variants in podocytes induces kidney disease in mice.

African Americans have a heightened risk of developing chronic and end-stage kidney disease, an association that is largely attributed to two common genetic variants, termed G1 and G2, in the APOL1 gene. Direct evidence demonstrating that these APOL1 risk alleles are pathogenic is still lacking because the APOL1 gene is present in only some primates and humans; thus it has been challenging to demonstrate experimental proof of causality of these risk alleles for renal disease. Here we generated mice with podocyte-specific inducible expression of the APOL1 reference allele (termed G0) or each of the risk-conferring alleles (G1 or G2). We show that mice with podocyte-specific expression of either APOL1 risk allele, but not of the G0 allele, develop functional (albuminuria and azotemia), structural (foot-process effacement and glomerulosclerosis) and molecular (gene-expression) changes that closely resemble human kidney disease. Disease development was cell-type specific and likely reversible, and the severity correlated with the level of expression of the risk allele. We further found that expression of the risk-variant APOL1 alleles interferes with endosomal trafficking and blocks autophagic flux, which ultimately leads to inflammatory-mediated podocyte death and glomerular scarring. In summary, this is the first demonstration that the expression of APOL1 risk alleles is causal for altered podocyte function and glomerular disease in vivo.

A Soluble Guanylate Cyclase Activator Inhibits the Progression of Diabetic Nephropathy in the ZSF1 Rat.

Therapies that restore renal cGMP levels are hypothesized to slow the progression of diabetic nephropathy. We investigated the effect of BI 703704, a soluble guanylate cyclase (sGC) activator, on disease progression in obese ZSF1 rats. BI 703704 was administered at doses of 0.3, 1, 3, and 10 mg/kg/d to male ZSF1 rats for 15 weeks, during which mean arterial pressure (MAP), heart rate (HR), and urinary protein excretion (UPE) were determined. Histologic assessment of glomerular and interstitial lesions was also performed. Renal cGMP levels were quantified as an indicator of target modulation. BI 703704 resulted in sGC activation, as evidenced by dose-dependent increases in renal cGMP levels. After 15 weeks of treatment, sGC activation resulted in dose-dependent decreases in UPE (from 463 ± 58 mg/d in vehicle controls to 328 ± 55, 348 ± 23, 283 ± 45, and 108 ± 23 mg/d in BI 703704-treated rats at 0.3, 1, 3, and 10 mg/kg, respectively). These effects were accompanied by a significant reduction in the incidence of glomerulosclerosis and interstitial lesions. Decreases in MAP and increases in HR were only observed at the high dose of BI 703704. These results are the first demonstration of renal protection with sGC activation in a nephropathy model induced by type 2 diabetes. Importantly, beneficial effects were observed at doses that did not significantly alter MAP and HR.

Identification of (R)-6-(1-(4-cyano-3-methylphenyl)-5-cyclopentyl-4,5-dihydro-1H-pyrazol-3-yl)-2-methoxynicotinic acid, a highly potent and selective nonsteroidal mineralocorticoid receptor antagonist.

A novel series of nonsteroidal mineralocorticoid receptor (MR) antagonists identified as part of our strategy to follow up on the clinical candidate PF-03882845 (2) is reported. Optimization departed from the previously described pyrazoline 3a and focused on improving the selectivity for MR versus the progesterone receptor (PR) as an approach to avoid potential sex-hormone-related adverse effects and improving biopharmaceutical properties. From this effort, (R)-14c was identified as a potent nonsteroidal MR antagonist (IC50 = 4.5 nM) with higher than 500-fold selectivity versus PR and other related nuclear hormone receptors, with improved solubility as compared to 2 and pharmacokinetic properties suitable for oral administration. (R)-14c was evaluated in vivo using the increase of urinary Na(+)/K(+) ratio in rat as a mechanism biomarker of MR antagonism. Treatment with (R)-14c by oral administration resulted in significant increases in urinary Na(+)/K(+) ratio and demonstrated this novel compound acts as an MR antagonist.

PF-03882845, a non-steroidal mineralocorticoid receptor antagonist, prevents renal injury with reduced risk of hyperkalemia in an animal model of nephropathy.

The mineralocorticoid receptor (MR) antagonists PF-03882845 and eplerenone were evaluated for renal protection against aldosterone-mediated renal disease in uninephrectomized Sprague-Dawley (SD) rats maintained on a high salt diet and receiving aldosterone by osmotic mini-pump for 27 days. Serum K(+) and the urinary albumin to creatinine ratio (UACR) were assessed following 14 and 27 days of treatment. Aldosterone induced renal fibrosis as evidenced by increases in UACR, collagen IV staining in kidney cortex, and expression of pro-fibrotic genes relative to sham-operated controls not receiving aldosterone. While both PF-03882845 and eplerenone elevated serum K(+) levels with similar potencies, PF-03882845 was more potent than eplerenone in suppressing the rise in UACR. PF-03882845 prevented the increase in collagen IV staining at 5, 15 and 50 mg/kg BID while eplerenone was effective only at the highest dose tested (450 mg/kg BID). All doses of PF-03882845 suppressed aldosterone-induced increases in collagen IV, transforming growth factor-ß 1 (Tgf-ß 1), interleukin-6 (Il-6), intermolecular adhesion molecule-1 (Icam-1) and osteopontin gene expression in kidney while eplerenone was only effective at the highest dose. The therapeutic index (TI), calculated as the ratio of the EC50 for increasing serum K(+) to the EC50 for UACR lowering, was 83.8 for PF-03882845 and 1.47 for eplerenone. Thus, the TI of PF-03882845 against hyperkalemia was 57-fold superior to that of eplerenone indicating that PF-03882845 may present significantly less risk for hyperkalemia compared to eplerenone.

Angiotensin converting enzyme 2 contributes to sex differences in the development of obesity hypertension in C57BL/6 mice.

OBJECTIVE: Obesity promotes hypertension, but it is unclear if sex differences exist in obesity-related hypertension. Angiotensin converting enzyme 2 (ACE2) converts angiotensin II (AngII) to angiotensin-(1-7) (Ang-[1-7]), controlling peptide balance. We hypothesized that tissue-specific regulation of ACE2 by high-fat (HF) feeding and sex hormones contributes to sex differences in obesity-hypertension. METHODS AND RESULTS: HF-fed females gained more body weight and fat mass than males. HF-fed males exhibiting reduced kidney ACE2 activity had increased plasma angiotensin II levels and decreased plasma Ang-(1-7) levels. In contrast, HF-fed females exhibiting elevated adipose ACE2 activity had increased plasma Ang-(1-7) levels. HF-fed males had elevated systolic and diastolic blood pressure that were abolished by losartan. In contrast, HF-fed females did not exhibit increased systolic blood pressure until females were administered the Ang-(1-7) receptor antagonist, D-Ala-Ang-(1-7). Deficiency of ACE2 increased systolic blood pressure in HF-fed males and females, which was abolished by losartan. Ovariectomy of HF-fed female mice reduced adipose ACE2 activity and plasma Ang-(1-7) levels, and promoted obesity-hypertension. Finally, estrogen, but not other sex hormones, increased adipocyte ACE2 mRNA abundance. CONCLUSIONS: These results demonstrate that tissue-specific regulation of ACE2 by diet and sex hormones contributes to sex differences in obesity-hypertension.

The use of plasma aldosterone and urinary sodium to potassium ratio as translatable quantitative biomarkers of mineralocorticoid receptor antagonism.

BACKGROUND: Accumulating evidence supports the role of the mineralocorticoid receptor (MR) in the pathogenesis of diabetic nephropathy. These findings have generated renewed interest in novel MR antagonists with improved selectivity against other nuclear hormone receptors and a potentially reduced risk of hyperkalemia. Characterization of novel MR antagonists warrants establishing translatable biomarkers of activity at the MR receptor. We assessed the translatability of urinary sodium to potassium ratio (Na+/K+) and plasma aldosterone as biomarkers of MR antagonism using eplerenone (Inspra®), a commercially available MR antagonist. Further we utilized these biomarkers to demonstrate antagonism of MR by PF-03882845, a novel compound. METHODS: The effect of eplerenone and PF-03882845 on urinary Na+/K+ and plasma aldosterone were characterized in Sprague-Dawley rats and spontaneously hypertensive rats (SHR). Additionally, the effect of eplerenone on these biomarkers was determined in healthy volunteers. Drug exposure-response data were modeled to evaluate the translatability of these biomarkers from rats to humans. RESULTS: In Sprague-Dawley rats, eplerenone elicited a rapid effect on urinary Na+/K+ yielding an EC50 that was within 5-fold of the functional in vitro IC50. More importantly, the effect of eplerenone on urinary Na+/K+ in healthy volunteers yielded an EC50 that was within 2-fold of the EC50 generated in Sprague-Dawley rats. Similarly, the potency of PF-03882845 in elevating urinary Na+/K+ in Sprague-Dawley rats was within 3-fold of its in vitro functional potency. The effect of MR antagonism on urinary Na+/K+ was not sustained chronically; thus we studied the effect of the compounds on plasma aldosterone following chronic dosing in SHR. Modeling of drug exposure-response data for both eplerenone and PF-03882845 yielded EC50 values that were within 2-fold of that estimated from modeling of drug exposure with changes in urinary sodium and potassium excretion. Importantly, similar unbound concentrations of eplerenone in humans and SHR rats yielded the same magnitude of elevations in aldosterone, indicating a good translatability from rat to human. CONCLUSIONS: Urinary Na+/K+ and plasma aldosterone appear to be translatable biomarkers of MR antagonism following administration of single or multiple doses of compound, respectively. TRIAL REGISTRATION: For clinical study reference EE3-96-02-004, this study was completed in 1996 and falls out scope for disclosure requirements. Clinical study reference A6141115: http://clinicaltrials.gov, http://NIHclinicaltrails.gov; NCTID: NCT00990223.

Pharmacodynamic model of sodium-glucose transporter 2 (SGLT2) inhibition: implications for quantitative translational pharmacology.

Sodium-glucose co-transporter-2 (SGLT2) inhibitors are an emerging class of agents for use in the treatment of type 2 diabetes mellitus (T2DM). Inhibition of SGLT2 leads to improved glycemic control through increased urinary glucose excretion (UGE). In this study, a biologically based pharmacokinetic/pharmacodynamic (PK/PD) model of SGLT2 inhibitor-mediated UGE was developed. The derived model was used to characterize the acute PK/PD relationship of the SGLT2 inhibitor, dapagliflozin, in rats. The quantitative translational pharmacology of dapagliflozin was examined through both prospective simulation and direct modeling of mean literature data obtained for dapagliflozin in healthy subjects. Prospective simulations provided time courses of UGE that were of consistent shape to clinical observations, but were modestly biased toward under prediction. Direct modeling provided an improved characterization of the data and precise parameter estimates which were reasonably consistent with those predicted from preclinical data. Overall, these results indicate that the acute clinical pharmacology of SGLT2 inhibitors in healthy subjects can be reasonably well predicted from preclinical data through rational accounting of species differences in pharmacokinetics, physiology, and SGLT2 pharmacology. Because these data can be generated at the earliest stages of drug discovery, the proposed model is useful in the design and development of novel SGLT2 inhibitors. In addition, this model is expected to serve as a useful foundation for future efforts to understand and predict the effects of SGLT2 inhibition under chronic administration and in other patient populations.

Discovery of a clinical candidate from the structurally unique dioxa-bicyclo[3.2.1]octane class of sodium-dependent glucose cotransporter 2 inhibitors.

Compound 4 (PF-04971729) belongs to a new class of potent and selective sodium-dependent glucose cotransporter 2 inhibitors incorporating a unique dioxa-bicyclo[3.2.1]octane (bridged ketal) ring system. In this paper we present the design, synthesis, preclinical evaluation, and human dose predictions related to 4. This compound demonstrated robust urinary glucose excretion in rats and an excellent preclinical safety profile. It is currently in phase 2 clinical trials and is being evaluated for the treatment of type 2 diabetes.

Leptin potentiates the anti-obesity effects of rimonabant.

We hypothesized that a combination of low doses of rimonabant and leptin would markedly reduce body weight through the modulation of neuronal activity within the hypothalamus. To this end, high fat diet-induced obese rats were randomized to receive either leptin (0.5mg/kg subcutaneously), rimonabant (3mg/kg), the combination of both, or vehicle, daily for a duration of 2 weeks. A subset of rats was pair-fed to the combination-treated animals and received either vehicle or leptin. At the end of the weight loss phase, leptin treatment was maintained for 7 days while rimonabant was discontinued to assess changes in body weight during the rebound phase. The combination of rimonabant and leptin resulted in a marked inhibition of food intake and a profound reduction in body weight that was greater than achieved with either leptin or rimonabant alone. Treatment with leptin during the rebound phase inhibited compensatory increases in body weight associated with restitution of ad libitum feeding in previously pair-fed rats. Moreover, leptin partially blunted the rebound in food intake and body weight associated with cessation of rimonabant therapy.To investigate the effect of the combination on neuronal firing in the rat hypothalamus, single unit activity was recorded from brain slices containing the ventromedial and arcuate nuclei. The combination of rimonabant and leptin synergistically increased and decreased neuronal firing in the ventromedial and arcuate nuclei, respectively. Overall, these data demonstrate profound anti-obesity effects of combining cannabinoid type 1 receptor antagonists and leptin.