Anti-CTGF Antibody Therapy With FG-3019 Prevents Diabetes-induced Increase in Vascular Complications in Streptozotocin (STZ) Treated Rats
Cardiovascular disease is the leading cause of morbidity and mortality in diabetic patients with renal disease. Both atherosclerosis and increased vascular permeability represent significant pathologies in this context. CTGF has been shown to be required to induce all forms of persistent fibrosis, and also has been implicated as a major factor involved in the pathogenesis of cardiovascular complications of diabetes. Diabetic patients undergo arterial stiffening, which has been implicated as an independent risk factor in cardiovascular mortality. To investigate the effects of FG-3019 (a fully human mAb reactive to human and rodent CTGF currently in clinical trials) on diabetic vascular complications, diabetes was induced in rats by injection of STZ. Animals were treated with control IgG, FG-3019 or captopril. Diabetic rats treated with IgG or captopril demonstrated increases in arterial stiffness compared to non-diabetic control animals. Specifically, force-pressure curves from carotid arteries of diabetic animals demonstrated increased axial stiffness of the arteries in IgG and captopril treated animals. Pressure-diameter curves demonstrated increased circumferential stiffness of the carotid arteries in diabetic animals treated with IgG or captopril. In contrast, both axial and circumferential stiffness of the carotid arteries of diabetic animals treated with FG-3019 remained similar to that of healthy control animals. Morphologic evaluations of arterial sections demonstrated an increase in adventitial thickening in diabetic rats, which was prevented by treatment with FG-3019 but not captopril. In addition, FG-3019 prevented diabetes-induced increases in skin vascular permeability. In summary, treatment with FG-3019 prevented the increase in biaxial stiffness of the carotid artery induced by diabetes and also reduced diabetes-induced capillary leakage. Since these benefits were not seen with captopril, FG-3019 may address pathways not affected by ACEi and may provide a novel therapeutic approach to treating micro and macrovascular complications associated with diabetes in a clinical setting.
© 2007 ASN
FG-3019, A Human Monoclonal Antibody to CTGF, With Gemcitabine/Erlotinib in Patients With Locally Advanced or Metastatic Pancreatic Ductal Adenocarcinoma
Background: Connective tissue growth factor (CTGF) is overexpressed in PDAC and facilitates local desmoplasia, tumor survival and metastasis. FG-3019 is a CTGF-specific monoclonal antibody that decreases tumor growth and metastases and prolongs survival in orthotopic and KPC mouse models. This study evaluates safety and efficacy of FG-3019 with gemcitabine/erlotinib in patients with PDAC. Methods: In an ongoing open-label, dose-escalation study, FG-3019 was used in combination with gemcitabine and erlotinib in patients with previously untreated, measurable, locally advanced or metastatic PDAC. Cohorts 1−6 received FG-3019 Q2W at 3, 10, 15, 25, 35 or 45 mg/kg. Cohort 7 received 35 mg/kg on Day 1 and then 17.5 mg/kg QW. Cohort 8 received 45 mg/kg on Day 1 and then 22.5 mg/kg QW. Results: 75 patients were enrolled at 7 centers. Baseline data: Stage III= 15, Stage IV=60; ECOG=0 (n=32), ECOG=1 (N=43). No SAEs or DLTs related to FG-3019 occurred at any dose. In per protocol population (n=68) median PFS and OS were 4.3 and 9.4 months respectively. Baseline plasma CTGF levels correlated inversely with PFS and OS (p=0.0029) (ITT population). Median FG-3019 Cmax and Cmin increased linearly with dose. Because of considerable overlap between subjects across cohorts, outcomes are correlated with drug exposure. OS, but not PFS, correlated with exposure after the first FG-3019 dose (Day 1 Cmax, p=0.009; Day 15 Cmin, p=0.005; ITT population). 47% of evaluable subjects had >50% decrease in CA19.9.The magnitude of reduction correlated with Day 1 Cmax (p=0.04) and inversely with baseline plasma CTGF (p=0.01). As FG-3019 accumulated over time, minimum FG-3019 exposure (Cmin) of 150 ug/mL on Day 43 appeared to be a threshold. Median OS was 7.7 and >8.6 months for subjects < (n=15) or ≥ (n=43) 150 ug/mL respectively on Day 43 (p=0.0034). Day 43 Cmin < 150, none alive at 12 months: Day 43 Cmin >150, 8 alive at 12 months, 17 alive 6.7-12 months. Conclusion: FG-3019 combined with gemcitabine/erlotonib is well tolerated. Interim results in this open-label study suggest OS improves with increasing exposure to FG-3019.
Preclinical and Clinical Proof of Concept (POC) for Treatment of IPF with Anti-CTGF Antibody FG-3019
Introduction: The matricellular protein, connective tissue growth factor (CTGF), is a central mediator of tissue remodeling and is elevated in patients with IPF, suggesting a key role for CTGF in lung remodeling diseases. FG-3019, a monoclonal antibody to CTGF, reverses tissue remodeling in animal models. A radiation-induced fibrosis model strengthened preclinical POC and prompted a Phase 2 trial of FG-3019 in IPF patients. Methods: Mouse lungs were injured with a single, full thorax irradiation. FG-3019 treatment began 16 weeks after irradiation, when significant increases in lung density were detectable by high-resolution computed tomography (HRCT). Gene expression and histological analyses were performed 18 weeks after irradiation. In an ongoing, open-label, Phase 2 clinical trial, the initial cohort of patients with moderate to severe IPF of ≤5 years duration, FVC 45−85% predicted, DLCO ≥30% predicted, and 10−50% parenchymal fibrosis by HRCT were infused with 15 mg/kg FG-3019 every 3 weeks. Pulmonary function tests and HRCT were performed every 3 and 6 months, respectively. Results: In the preclinical model, FG-3019 treatment modified gene expression patterns in remodeling lungs, reversed lung density increases within 2 weeks, and exhibited durable effects after 8 weeks of administration. Preliminary clinical data show stable or improved FVC in many patients and improved quantitative fibrosis score in 6 of the first 12 enrolled. These effects appear to be related to baseline disease severity. FG-3019 was well tolerated, with no drug-related SAEs reported. Conclusions: Preclinical and clinical experience with FG-3019 to date supports expansion of the Phase 2 IPF trial.
Phase 2 Trial of FG-3019, Anti-CTGF Monoclonal Antibody, In Idiopathic Pulmonary Fibrosis (IPF): Preliminary Safety and Efficacy Results
Introduction: Connective tissue growth factor (CTGF) is implicated in the pathogenesis of IPF and is a potential novel therapeutic target. Objectives: To evaluate the safety, tolerability, and efficacy of FG-3019 in subjects with IPF. Methods: Phase 2 prospective, open label study of FG-3019 (15 mg/kg IV every 3 weeks for 45 weeks) in subjects with well-defined IPF (duration ≤5 years, evidence of disease progression during the preceding year, FVC 45−85% predicted, DLCO ≥ 30% predicted, and 10−50% parenchymal fibrosis by HRCT). Treatment response was assessed by changes in extent of parenchymal disease (HRCT and FVC). Results: 54 subjects (males 83%, mean age 67 years, median FVC % predicted 63.2%) were enrolled. Quantified HRCT scores of whole lung fibrosis (QLF) and all abnormal interstitial lung disease (QILD) at week 24 showed decreases from baseline greater than analytical variability (±2%) in 6 (24%) and 8 (32%) of 25 subjects, respectively. Changes in both QLF and QILD score were significantly correlated with changes in FVC % predicted (for QILD r=-0.55, p=0.004). Mean decreases in FVC % predicted were less than in historical controls. Safety findings to date include 13 SAEs (none drug-related), 1 acute exacerbation, 9 respiratory-related hospitalizations, and 3 deaths (all related to IPF). Conclusions: FG-3019, a novel anti-fibrotic agent, is well tolerated by subjects with IPF. No drug-related SAEs have been reported to date. Promising results of measurement of quantified lung fibrosis scores and FVC warrant pursuing the clinical trial with a higher dose of FG-3019 to further assess efficacy and safety in subjects with IPF.
A Phase I Trial of the Monoclonal Antibody FG-3019 to Connective Tissue Growth Factor (CTGF) in Locally Advanced or Metastatic Pancreatic Cancer
Background: CTGF is highly expressed in pancreatic tumors and is thought to mediate local desmoplasia. FG-3019 is a fully human monoclonal antibody against CTGF. Studies using FG-3019 in murine xenograft models have shown reduced tumor growth and metastasis. Methods: This open-label, dose escalation study assessed the safety and pharmacokinetics of FG-3019 (3, 10, 15, and 25 mg/kg q14D). FG-3019 was initiated on D1 to assess single-agent toxicity. Standard gemcitabine and erlotinib were added on D15. Chemotherapy-naive patients with locally advanced or metastatic adenocarcinoma were eligible. Seventeen subjects (median age 66 yrs) were enrolled: n=4, 3, and 10 at 3, 10, and 15 mg/kg respectively. Enrollment is ongoing in the 25 mg/kg cohort. 7 subjects were female; 4 were Stage 3 and 13 were Stage 4. Results: No safety signals were detected with single-agent FG-3019. After beginning chemotherapy, 4 subjects experienced 7 SAEs, which were deemed unrelated to FG-3019 including 3 deaths: sepsis, suicide, and disease progression. Nine subjects experienced Grade 3 AEs, all of which were expected in patients with pancreatic cancer. There were no Grade 4 hematological abnormalities. AEs related to gemcitabine (hematological, abnormal LFTs) and erlotinib (rash) occurred at a rate and severity consistent with the prescribing information (preliminary data). Steady state Cmax (median 428, range 236 - 455 µg/mL), and T1/2 (median 6.6, range 6.3 - 6.7 days) at the 10 mg/kg dose level were comparable to PK data from subjects in non-oncological trials who received FG-3019 at the same dose level. One subject had a partial response by RECIST criteria for 9.7+ months. Another subject had a minor response for 7.7 months. Three of five subjects with PET scans at baseline and D15 experienced stable to reduced PET activity before starting chemotherapy. The median TTP across all cohorts was 3.7 months (95% CI 1.9-6.2), and the median OS was 9.4 months (95% CI 1.9-11.58). Conclusions: FG-3019 is well tolerated and dose escalation continues. Reduced PET activity after treatment with single-agent FG-3019 may indicate a biological effect of the agent.
The CTGF antibody FG-3019 blocks CTGF-stimulated migration of ovarian cancer cells
Ovarian tumors generally arise from the surface epithelium of the ovary, and require interactions with host stroma to promote tumor cell proliferation and expansion. The signaling mechanisms between tumor-associated stroma and adjacent ovarian epithelial tumor cells remain unclear. We have previously analyzed gene expression profiles of laser-capture microdissected stromal cells from normal ovary and ovarian tumors and have demonstrated that normal ovarian stroma undergoes significant gene expression changes in response to the epithelial tumor microenvironment. Connective Tissue Growth Factor (CTGF), a TGF-b-regulated gene, was identified as specifically up-regulated in tumor-associated versus normal stroma. Our study was the first to examine and identify gene expression changes in microdissected ovarian tumor stroma, including over-expression of CTGF. CTGF is a secreted protein that has been demonstrated to be involved in cellular proliferation and motility and has been studied in breast, prostate, lung and pancreatic cancers. FG-3019, a human monoclonal antibody against CTGF, has been demonstrated to inhibit pancreatic tumor growth and metastasis. The aim of this current study was to further elucidate the role of CTGF in ovarian cancer and examine its potential as a therapeutic target. We assessed the paracrine effect of CTGF on three different ovarian cancer cell lines, A224, OVCAR3, and SKOV3. Expression of endogenous CTGF is low in these cells which is consistent with the level found in primary ovarian tumor cells. Addition of recombinant CTGF for 6-hr stimulated migration of A224 (613 ± 13.4 vs. 349 ± 6.4 cells), OVCAR3 (88 ± 2.1 vs. 51 ± 3.5 cells) and SKOV3 (495 ± 32.5 vs. 185 ± 17.0 cells) in a transwell migration assay. Addition of the CTGF-blocking antibody FG-3019 decreased transwell migration in the presence of recombinant CTGF in all three cell lines (613 ± 13.4 vs. 187 ± 20.5 cells in A224, 88 ± 2.1 vs. 37 ± 1.4 cells in OVCAR3 and 495 ± 32.5 vs. 170 ± 18.4 cells in SKOV3). Additionally, CTGF-transfected OVCAR3 clones demonstrated higher anchorage-independent growth (114 ± 25.6 vs. 4 ± 1.8 colonies). However, recombinant CTGF did not promote proliferation of these cell lines. We demonstrate that CTGF may contribute to ovarian tumor biology and that its function can be blocked by FG-3019, thus determining CTGF as a potential therapeutic target in ovarian tumors. Clinical evaluation of FG-3019 is underway in pancreatic cancer patients and other tumor types are being considered for clinical testing.
Reversal of Established Fibrosis by Treatment with the Anti-CTGF Monoclonal Antibody FG-3019 in a Murine Model of Radiation-Induced Pulmonary Fibrosis
RATIONALE: Connective tissue growth factor (CTGF) is a matricellular protein that is a central mediator of tissue remodeling. CTGF expression is elevated in patients with idiopathic pulmonary fibrosis, and in mice administered bleomycin to induced pulmonary fibrosis. Several studies in different organs have suggested that CTGF is essential for sustained fibrosis. Therefore, inhibition of CTGF may be therapeutically beneficial to fibrosis patients. METHODS: Pulmonary fibrosis was initiated with a single, full thorax irradiation (20 Gy) to 6 groups of 25 mice. One group of mice remained untreated, while the other 5 groups received 8 weeks of control human IgG or FG-3019 administration (3 doses/week, 10 mg/kg). Administration of FG-3019 began 2 days before or after irradiation, or 20 days, or 16 weeks after irradiation. Two additional groups were not irradiated, but received 8 weeks of human IgG or FG-3019. Lung density of all surviving mice was monitored weekly by computed tomography (CT). 30 weeks after irradiation, oxygen saturation was determined by blood gas analysis of surviving mice. Mice sacrificed for histological examination were excluded from the survival analysis. RESULTS: All groups receiving FG-3019 exhibited better survival than irradiated, untreated groups, with the 20-day group demonstrating the best survival. Histological examination demonstrated that significant lung remodeling occurred between 13 and 19 weeks after irradiation. FG-3019 attenuated this remodeling in a schedule-dependent manner, with the 20-day group exhibiting the least remodeling. Increased lung densities observed between 22 and 31 weeks after irradiation were attenuated by FG-3019 beginning 2 days before or after irradiation. Administration of FG-3019 beginning 20 days or 16 weeks after irradiation had a more dramatic effect on lung densities, such that they became indistinguishable from those of unirradiated mice as early as 22 weeks after irradiation, with a sustained effect after cessation of FG-3019 administration. Since the lung density in the 16 week group had already increased at the time that FG-3019 administration began, achieving normal lung density required reversal of the radiation-induced changes. Comparison of oxygen saturation with lung density at 30 weeks demonstrated a good correlation and suggested that reduction in lung density is a good surrogate for improvement in lung function. CONCLUSIONS: FG-3019 reversed established fibrosis, improved lung function, and increased the survival of mice with radiation-induced pulmonary fibrosis. The data strongly support further clinical evaluation of FG-3019 (which is currently in Phase II clinical development) for treatment of pulmonary fibrosis.
A Randomized, Double-blind, Placebo-controlled, Phase 1 Study of Safety, Pharmacokinetics and Pharmacodynamics of FG-3019 in Subjects with Type 1 or Type 2 Diabetes Mellitus and Diabetic Kidney Disease (DKD) on Background ACEi and/or ARB Therapy
FG-3019 is a recombinant human IgG1 kappa monoclonal antibody that binds to domain 2 of connective tissue growth factor (CTGF), which plays a critical role in progressive fibrosis including DKD. In this phase 1 trial, 38 subjects with type 1/2 DM and DKD were enrolled. The study purpose was to characterize safety, tolerability, PK and PD of FG-3019 administered IV over 10 weeks in subjects with overt nephropathy on a stable regimen of angiotensin blockade. Eligible subjects were required to have 2 first morning ACR values of >0.2 g/g at least 48 hours apart, eGFR (by MDRD) of 20 to <90 mL/min/1.73 m2, and taking stable regimens of antihypertensive medications including at least one ACEi or ARB, and stable hypoglycemic and cholesterol-lowering medications. Subjects were randomized (1:1:1) to receive either a) placebo every 2 weeks, b) FG-3019 at 5 mg/kg every 2 weeks or c) FG-3019 at 10 mg/kg every 4 weeks (with one additional dose given at 2 weeks to reach steady state). Treatment assignment was stratified by CKD Stage 3 and 4. Measured PD effects were change from baseline to Day 85 in a) plasma CTGF-Whole and urine and plasma CTGF-N fragment +Whole levels, b) first morning urinary albumin/Cr and other analyte/Cr ratios, and c) plasma HbA1c, high sensitivity CRP, cystatin-C and BNP. At the time of this abstract, treatment assignment has not yet been unblinded. In a preliminary assessment of safety, 13 patients reported treatment emergent adverse events (AEs). These were primarily part of the AE spectrum of the underlying disease. 6 serious AEs were observed in 3 subjects. One SAE, stroke, was considered 'unlikely' related to study drug. All other SAEs were considered unrelated. No infusion-related AEs were reported. Overall, FG-3019 was shown to be well tolerated after 10 weeks of treatment in this patient population.
A Putative Role for Connective Tissue Growth Factor (CTGF) in Loss of Podocyte Slit Diaphragm Integrity and Actin Rearrangement
Diabetic Nephropathy (DN) is a serious and common complication of type I and type II diabetes, the leading cause of end-stage renal disease worldwide. Recent years have seen significant advances in understanding the biology of the glomerular podocyte and its role in the initiation and progression of DN. Studies have found that increased expression of CTGF is initially restricted to podocytes in the early disease process, consistent with a pathogenic role in the development of diabetic nephropathy, while over expression of CTGF in podocytes has been associated with worsening proteinuria and renal failure in STZ-induced diabetic mice. Here we describe, for the first time, the effects of CTGF on the immortalised human podocyte cell line, AB 8/13. Fully differentiated AB 8/13 cells exhibit many characteristics of primary podocytes including appropriate expression of components of the slit diaphragm. Cells exposed to rhCTGF underwent significant re-polarisation and exhibited loss of epithelial characteristics. rhCTGF stimulated reorganisation of the actin cytoskeleton accompanied by polarised redistribution of the acto-myosin contractile apparatus. These events are linked to activation of known signalling pathways including the p42/44 MAPK, PI3-kinase and integrin-linked paxillin phosphorylation. Changes in these polarity-inducing profiles in podocytes in vivo would have profound consequences for the integrity of the slit diaphragm and may mediate loss of podocin/nephrin and consequent foot process effacement. Understanding the importance of CTGF for integrity of the slit diaphragm and actin binding/regulatory programmes is crucially significant, as podocyte targeted anti-CTGF therapies may present a rational approach for future treatment of microalbuminaria, proteinuria and diabetic nephropathy.
© 2008 ASN
Connective Tissue Growth Factor (CCN2/CTGF) is Increased in High Peritoneal Solute Transport Rate in Peritoneal Dialysis Patients
Peritoneal fibrosis/sclerosis (PF) is an important complication of CAPD therapy that often occurs in association with high transport rate and ultrafiltration failure (UFF). As little is known about mechanism, we studied roles of CTGF, a fibrogenic cytokine downstream of TGF-β, in the relationship of PF and UFF. CTGF mRNA expression in peritoneal biopsy samples (n=54) was assessed by real-time PCR. We measured CTGF concentration by ELISA in peritoneal dialysis effluent of 155 patients and evaluated relationships with peritoneal transport rate and duration of treatment. Human mesothelial cells (HPMC) were isolated from the spent peritoneal dialysis effluent of 22 PD patients with variable peritoneal transport rate. CTGF mRNA expression in both HPMC and Met-5A mesothelial cell-line was studied under basal condition and 3, 12 and 24 hours post TGF-β1 stimulation (5ng/ml). CTGF mRNA expression was 13.8-fold higher in peritoneal membranes with UFF and 3.1-fold elevated in peritonitis tissues vs. biopsy samples at insertion of PD catheter (pre-PD renal failure peritoneum). There was a positive correlation between dialysate CTGF concentration and peritoneal creatinine ratio, in index of transport at constant dwell time and volume (D/P Cr) (Y=5.77X-12.4, R= 0.57), despite a weak correlation with duration of PD treatment (R=0.264). CTGF mRNA expression was increased and peaked at 12 hours post TGF-β1 exposure in both Met-5A and HPMC. CTGF mRNA induction at 12 and 24 hours in HPMC was correlated with D/P Cr in PD patients treated less than 2 yrs (Y = 6.09X -1.29, R=0.64; Y = 3.19X +0.23, R=0.53, respectively). No significant correlation of D/P Cr and CTGF basal expression was found. Our results suggest that high peritoneal transport state correlates with high peritoneal CTGF expression and higher CTGF induction by TGF-β. Functional alteration of mesothelial cells may be involved in progression of peritoneal fibrosis in high transport state.
© 2008 ASN
CTGF Inhibits BMP-7 Signaling in Diabetic Nephropathy
In diabetic nephropathy, connective tissue growth factor (CTGF) is upregulated and bone morphogenetic protein 7 (BMP-7) is downregulated. CTGF is known to inhibit BMP-4, but similar cross-talk between BMP-7 and CTGF has not been studied. In this study, it was hypothesized that CTGF acts as an inhibitor of BMP-7 signaling activity in diabetic nephropathy. Compared with diabetic wild-type CTGF(+/+) mice, diabetic CTGF(+/-) mice had approximately 50% lower CTGF mRNA and protein, less severe albuminuria, no thickening of the glomerular basement membrane, and preserved matrix metalloproteinase (MMP) activity. Although the amount of BMP-7 mRNA was similar in the kidneys of diabetic CTGF(+/+) and CTGF(+/-) mice, phosphorylation of the BMP signal transduction protein Smad1/5 and expression of the BMP target gene Id1 were lower in diabetic CTGF(+/+) mice. Moreover, renal Id1 mRNA expression correlated with albuminuria (R = -0.86) and MMP activity (R = 0.76). In normoglycemic mice, intraperitoneal injection of CTGF led to a decrease of pSmad1/5 in the renal cortex. In cultured renal glomerular and tubulointerstitial cells, CTGF diminished BMP-7 signaling activity, evidenced by lower levels of pSmad1/5, Id1 mRNA, and BMP-responsive element-luciferase activity. Co-immunoprecipitation, solid-phase binding assay, and surface plasmon resonance analysis showed that CTGF binds BMP-7 with high affinity (Kd approximately 14 nM). In conclusion, upregulation of CTGF inhibits BMP-7 signal transduction in the diabetic kidney and contributes to altered gene transcription, reduced MMP activity, glomerular basement membrane thickening, and albuminuria, all of which are hallmarks of diabetic nephropathy.
© 2008 ASN
Modular Signaling Activities of the CTGF/CCN2 Domain Structure: Implications for Therapeutic Intervention
Connective Tissue Growth Factor [CTGF]/CCN2, a member of the CCN family of secreted, cysteine-rich factors, important in development/progression of diabetic nephropathy. Over a decade ago CTGF was shown to contain four distinct modular domains that mediate interactions with growth factors, integrins, and extracellular components. Recently, studies have begun to assess how the CTGF domains orchestrate signals and control key biological processes. Here, we describe how the CTGF modules contribute to different cellular responses of human mesangial cells. We are also investigating the role of CTGF in regulating transforming growth factor beta1 (TGFβ-1)-induced cell signalling and its modulation by FG-3019; a human neutralizing CTGF mAb that binds to the VWC domain of CTGF. Preincubating mesangial cells with FG-3019 diminished CTGF-induced p42/44 MAPK, p38 MAPK, paxillin and Akt phosphorylation when compared to control IgG antibody. Antibodies directed against other domains of CTGF had no effect on CTGF-induced cell signalling. The N-terminal half fragment of CTGF was also capable of activating MAPK, paxillin and Akt phosphorylation. Consistent with these effects being VWC domain-dependent, addition of FG-3019 abrogated N-terminal CTGF-mediated p42/44 MAPK phosphorylation while other domain-specific antibodies did not. CTGF can also regulate TGF-β-induced cell signalling. Co-treatment with CTGF and TGF-β diminished TGF-β -induced Smad2 phosphorylation. Preincubation with FG-3019 reversed CTGF-mediated inhibition of TGF- -induced Smad2 phosphorylation. These results indicate that CTGF-mediated activation of MAPK, paxillin and Akt acts, at least in part, through the VWC domain. The role of CTGF in regulating TGF-β signalling responses and the ability of FG-3019 to reverse these effects emphasizes the potential utility of FG-3019 as an anti-CTGF directed therapy.
© 2008 ASN
BMP-Signaling and Podocyte Markers are Decreased in Human Diabetic Nephropathy in Association with CTGF Overexpression
Diabetic nephropathy is characterized by decreased expression of bone morphogenetic protein-7 (BMP-7), and decreased podocyte number and differentiation. However, BMP signalling activity in human diabetic nephropathy has not been studied. In addition to ligand availability, extracellular antagonists such as sclerostin domain-containing-1 (SOSTDC1; USAG-1) and connective tissue growth factor (CTGF; CCN-2) can strongly affect BMP signalling activity in the kidney.
We studied kidney tissue from five diabetic nephropathy patients and five non-diabetic individuals, and from diabetic CTGF+/+ and CTGF+/- mice. BMP signalling activity in glomeruli was visualized by pSmad1/5/8 immunostaining, and related to expression of CTGF, SOSTDC1, and the podocyte differentiation markers WT1, nephrin, and synaptopodin.
Phosphorylated Smad1/5/8 was mainly localized in podocytes and decreased in diabetic nephropathy. The decrease of pSmad1/5/8 correlated with, but exceeded loss of podocytes. Also for synaptopodin and nephrin, positive area and staining intensity were decreased. SOSTDC1 and CTGF were expressed exclusively in podocytes, as was shown by colocalization with podocyte markers. In diabetic glomeruli, SOSTDC1 decreased in parallel with podocyte number. CTGF, however, was strongly increased in diabetic glomeruli. Furthermore, in diabetic CTGF+/- mice pSmad1/5/8 was preserved compared to diabetic CTGF+/+ mice.
In human diabetic nephropathy BMP signalling activity is diminished, together with reduction of podocyte markers. This is associated with overexpression of CTGF but not SOSTDC1.
© 2008 ASN
A Targeted cDNA Microarray Identifies Cytoskeletal Regulatory Proteins as Transcriptional Targets of Connective Tissue Growth Factor (CTGF)/CCN2: Implications for Diabetic Nephropathy
Abstract: Hyperglycaemia-induced increases in glomerular mesangial extracellular matrix production and actin cytoskeleton rearrangement are key pathological hallmarks of diabetic nephropathy. Previously, we have described the use of a Suppressive Subtractive Hybridisation (SSH) screen to identify mRNA transcripts, which are differentially expressed in human glomerular mesangial cells (HMCs) propagated in vitro under conditions of either normal (5mM) or high (30mM) physiological glucose. In this study, we have used focused cDNA microarray technology to rapidly characterise the expression profile of genes derived from this SSH in HMCs in response to a key, profibrotic mediator of diabetic nephropathy, Connective Tissue Growth Factor (CTGF/CCN2). From the SSH screen, 171 distinct clones were amplified via PCR and arrayed onto glass slides. Analysis of the focused microarrays investigating HMCs stimulated with CTGF revealed induction of 10 distinct transcripts. CTGF was observed to induce the expression of the actin/myosin-binding protein caldesmon, the myosin regulatory chain and T-plastin alongside Arp-3 and fibronectin. In addition, CTGF caused a down-regulation of tubulin alpha-3 and the F-actin capping protein. These data suggest that CTGF activates an actin binding and regulatory protein cluster, representing a previously undescribed genetic programme which likely contributes to mesangial cell dysfunction in DN. When HMCs were stimulated with CTGF and stained for tubulin and F-actin, widespread microtubular and actin rearrangement was apparent. This was accompanied by a polarized redistribution of myosin, suggesting activation of the machinery of cell migration. Redistribution of myosin was facilitated by dephosphorylation of the myosin light and heavy chains in response to CTGF, which was inhibited by the addition of the myosin inhibitor 2,3 butanedionemonoxime. Increased expression of Arp-3 in response to CTGF was associated with cdc42 dependent PAK-1 phosphorylation. This data indicates that CCN2-mediated actin rearrangement likely contributes to the pathophysiology of the glomerular mesangium in diabetic nephropathy.
© 2007 ASN
Plasma Connective Tissue Growth Factor (CTGF; CCN-2) Predicts End-Stage Renal Disease and Mortality in Type 1 Diabetic Nephropathy
1 Pathology, UMCU, Utrecht, Netherlands; 2 Steno Diabetes Center, Gentofte, Denmark; 3 FibroGen, Inc., South San Francisco, CA, United States and 4 Nephrology, University School of Medicine, Nagoya, Japan. Background: Levels of CTGF in plasma are increased in experimental and human diabetic nephropathy. However, it is not known whether plasma CTGF might also be useful as a prognostic marker. Methods: We evaluated the predictive value of baseline plasma CTGF for outcome and disease progression in a large prospective study. Subjects were 198 type 1 diabetic patients with overt diabetic nephropathy, and 188 type 1 diabetic patients with persistent normoalbuminuria. Follow-up time was 12.8 years. Plasma CTGF was measured at baseline by sandwich ELISA. Results: In patients with nephropathy, addition of plasma CTGF content increased the correlation of urinary albumin excretion (UAE) with the rate of decline in GFR (cumulative R=0.46). Area under the ROC curve for prediction of end-stage renal disease (ESRD) by plasma CTGF was 0.74. A cutoff value of 394 pmol/L predicted ESRD with a sensitivity of 76% and a specificity of 61%. In addition, a standardized (1-SD) increase in plasma CTGF independently predicted ESRD [covariate-adjusted hazard ratio 1.6 (95% CI 1.1-2.5), P=0.03]. This was most prominent for patients in the highest UAE quartile [>2050 mg/day; covariate-adjusted hazard ratio 2.4 (1.3-4.2), P=0.003]. Of note, difference in UAE among patients within this quartile no longer predicted outcome. This points to unique potential application of plasma CTGF as a prognosticator of renal function in patients with more severe proteinuria. Furthermore, plasma CTGF content was an independent predictor of overall mortality in patients with diabetic nephropathy [covariate-adjusted hazard ratio 1.4 (1.1-1.8), P=0.003]. In contrast, in normoalbuminuric patients, plasma CTGF did not predict outcome nor correlate with clinical parameters. Conclusion: Addition of plasma CTGF content to conventional risk factor assessment significantly improves prediction of ESRD and mortality in patients with overt type 1 diabetic nephropathy.
© 2007 ASN
Urinary Connective Tissue Growth Factor (CTGF) Excretion in Patients with Renal Allograft
Abstract: Connective tissue growth factor (CTGF) up-regulation has been demonstrated in many fibrotic diseases as well as in experimental chronic allograft rejection. CTGF expression is enhanced by many pathogenic factors and processes that are relevant to chronic allograft nephropathy (CAN) and CTGF is considered the final effector of TGF-beta induced fibrogenesis. In the recent literature, urinary excretion of CTGF has been measured in patients with diabetic nephropathy and was found to be related to renal sclerosis. The aim of this study was evaluation of urinary CTGF excretion and correlation with clinical data in a cohort of kidney transplant recipients who received the graft in pediatric age. 67 fresh morning urine samples were obtained from 48 subjects (19F/29M) transplanted 6.75 ±3.4 years before (0.3-14) at a mean age of 13.3 ±7 years for nephropathies originating in early pediatric age. Mean serum Creatinine was 1.45 ±0.6 (range 0.6-3.5 mg/dL) and mean proteinuria 0.14 ±0.28 g/24h (range 0-2 g/24h). 7 patients received a biopsy for deterioration of renal functional and CAN was diagnosed. Fresh urines were obtained also from 46 healthy, age matched controls. CTGF was assayed by specific ELISA to detect both full length CTGF and N-terminal half fragments which are the predominant form in urine. Urinary CTGF (uCTGF) was significantly increased in transplanted patients vs controls: median 6.0 ng/mgCr (interquartile range 2.6-4.7 ng/mgCr) in transplanted vs 3.7 ng/mgCr (interquartile range 3.6-9.6 ng/mgCr) in controls (p<0.001). uCTGF did not correlate with creatinine clearance nor with proteinuria. A trend of correlation just below statistical significance was found between uCTGF and length of time occurred since transplant (P=0.06). uCTGF was not different in transplanted adults vs children. "Low" and "high" uCTGF excretors were subdivided choosing as a cut-off the value higher than the 99th percentile for normal controls (6 ng/mgCr): children with uCTGF >6 ng/mgCr were found to have an older graft, with a time since transplant significantly longer than those with uCTGF <6 ng/mgCr (7.15 years vs 4.3, p 0.02). This first report linking uCTGF with time since grafting appears to represent an encouraging useful marker of sclerosis which could be easily followed-up and monitored during the natural history of the transplant, particularly in children, potentially allowing a better screen for biopsy candidates and early alarm for sclerotic processes occurrence.
Dose-Escalation Phase I Study of FG-3019, Anti-CTGF Monoclonal Antibody, in Patients with Type 1/2 Diabetes Mellitus and Microalbuminuria
Abstract: Blood, urine and glomerular CTGF levels correlate with progression of diabetic nephropathy (DN). FG-3019 is a fully human IgG1 kappa neutralizing CTGF mAb effective in animal DN models. Subjects were ≥ 21 yrs with type 1 or 2 DM, BMI ≤ 32 kg/m2, SCr ≤ 1.1 (women) or ≤1.5 mg/dL (men), with microalbuminuria (MalbU) by first AM urine albumin/Cr ratio (ACR) of 30-300 mg/g. Ten subjects each received 3 or 10 mg/kg on Days 0, 14, 28 and 42, with 1 yr follow-up. The 3 mg/kg cohort is completed. The 10 mg/kg cohort is in progress; only demographic/adverse event (AE) data are included. Enrolled subjects (N=20; 60% male; 20% type I; 50% on insulin; 95% on ACEi and/or ARB) had a mean (±SD) age of 58 (±11), 17 (±7) yrs DM, SCr 0.9 (±0.2) mg/dL, eGFR (MDRD) 84 (±21) mL/min/1.73m2, and average MAP 114 (±11) mm Hg. One 3 mg/kg subject withdrew after 1 dose. 8/12 subjects with AE data reported an infusion day AE (flushing, dizzy, headache (HA), anxiety). 5/12 reported a non-infusion day possibly drug-related AE (HA, ↓Na, ↑LFT, paronychia, anemia). There was 1 unrelated SAE (day 343; gastroenteritis) and no severe AEs. For 3 mg/kg cohort: PK on Days 1/42 showed Tmax=2.25/4.12 hrs; Cmax=73/76 ug/mL; clearance=0.42/0.38 mL/hr/kg; T1/2=3.0/4.3 days. 0/9 pts developed anti-human antibodies. Day 0 ACR and albumin excretion rate (AER) were 48 (±27) mg/g and 80 (±77) mg/d. Day 56 mean ACR=29 (±15), a Δ of -19 (±25) mg/g, and AER was 41 (±22), a Δ of -36 (±68) mg/day. ACR decreased ≥ 50% in 3/9 pts (57-91 to 23-36 mg/g) and 3/9 pts (1 unique) decreased AER ≥ 50% (53-205 to 22-83 mg/day). Mean eGFR was stable (Δ= 3.6 ±15 mL/min/1.73m2); mean ΔMAP was -5 (±14) mm Hg. FG-3019 at 3 or 10 mg/kg was safely dosed over 42 days in DN pts with MalbU. PK showed 1 compartment distribution and saturable clearance. Urine ACR improved ≥ 50% in 33% of 3mg/kg pts. Anti-CTGF mAb may synergize with ACEi/ARBs.
© 2006 ASN
Connective Tissue Growth Factor-Specific Antibody Attenuates Tumor Growth, Metastasis, and Angiogenesis in an Orthotopic Mouse Model of Pancreatic Cancer
Connective tissue growth factor (CTGF) plays an important role in fibrosis by modulating cell migration and cell growth but may also modify tumor growth and metastasis. Because CTGF is overexpressed in pancreatic ductal adenocarcinoma, we investigated the in vitro effects of CTGF on the proliferation and invasiveness of PANC-1 pancreatic cancer cells and examined the consequences of its in vivo inhibition on the growth and metastasis of these cells using a fully human CTGF-specific monoclonal antibody (FG-3019) in an orthotopic nude mouse model. Although PANC-1 cells expressed relatively high levels of endogenous CTGF mRNA, the addition of CTGF to conditioned medium increased the proliferation and invasiveness of PANC-1 cells. Moreover, transforming growth factor-beta1 caused a further increase in CTGF expression in these cells. In vivo, the twice weekly i.p. administration of FG-3019 decreased tumor growth and metastasis and attenuated tumor angiogenesis and cancer cell proliferation. FG-3019 did not enhance apoptosis and did not attenuate the inhibitory effects of gemcitabine on tumor growth and metastasis. These findings suggest that CTGF may contribute to aberrant autocrine and paracrine pathways that promote pancreatic cancer cell growth, invasion, metastasis, and angiogenesis. Therefore, blocking CTGF actions with FG-3019 may represent a novel therapeutic approach in pancreatic ductal adenocarcinoma.
Connective Tissue Growth Factor Specific mAb Therapy Inhibits Pancreatic Tumor Growth and Metastasis
Pancreatic cancer is highly aggressive and refractory to most existing therapies. Past studies have shown that connective tissue growth factor (CTGF) expression is elevated in human pancreatic adenocarcinomas and some pancreatic cancer cell lines. To address whether and how CTGF influences tumor growth, we generated pancreatic tumor cell lines that overexpress different levels of human CTGF. The effect of CTGF overexpression on cell proliferation was measured in vitro in monolayer culture, suspension culture, or soft agar, and in vivo in tumor xenografts. Although there was no effect of CTGF expression on proliferation in two-dimensional cultures, anchorage-independent growth (AIG) was enhanced. The capacity of CTGF to enhance AIG in vitro was linked to enhanced pancreatic tumor growth in vivo when these cells were implanted s.c. in nude mice. Administration of a neutralizing CTGF-specific monoclonal antibody, FG-3019, had no effect on monolayer cell proliferation, but blocked AIG in soft agar. Consistent with this observation, anti-CTGF treatment of mice bearing established CTGF-expressing tumors abrogated CTGF-dependent tumor growth and inhibited lymph node metastases without any toxicity observed in normal tissue. Together, these studies implicate CTGF as a new target in pancreatic cancer and suggest that inhibition of CTGF with a human monoclonal antibody may control primary and metastatic tumor growth.
Anti-CTGF Human Antibody FG-3019 Prevents and Reverses Diabetes-Induced Cardiovascular Complications in Streptozotocin (STZ) Treated Rats
Cardiovascular disease is a leading cause of morbidity and mortality in diabetic patients. CTGF has been shown to be required to induce all forms of persistent fibrosis, and also has been implicated as a major factor involved in the pathogenesis of cardiovascular complications of diabetes. To investigate the effects of FG-3019 (a fully human mAb, reactive to human and rodent CTGF, in clinical trials for incipient diabetic nephropathy) on diabetic cardiovascular complications, diabetes was induced in rats by injection of STZ. Carotid arteries from diabetic rats treated with IgG or captopril for 6 weeks demonstrated increases in arterial stiffness (measured by force-pressure and pressure-diameter curves) compared to non-diabetic control animals. In contrast, carotid arterial force-pressure and pressure-diameter curves of diabetic animals treated with FG-3019 remained similar to that of healthy control animals. In addition, FG-3019 prevented diabetes-induced increases in skin vascular permeability and edema. In a follow-up study, diabetes and associated vascular changes were allowed to progress for six weeks prior to randomization for treatment. FG-3019 partially reversed diabetes-induced increases in carotid arterial stiffness, whereas IgG and captopril had no effect. In addition, animals in the FG-3019 treated group had improved cardiovascular function (LVEDP, EF, dP/dt) compared to IgG and captopril treated animals. FG-3019 also reversed diabetes-induced increases in skin vascular permeability. In summary, treatment with FG-3019 prevented and partially reversed diabetes-induced arterial stiffness, improved cardiovascular function and reduced diabetes-induced microvascular leakage. Since these benefits were not seen with captopril, FG-3019 may address pathways not affected by ACEi, and may provide a novel therapeutic approach to treating micro and macrovascular complications associated with diabetes in a clinical setting.
© 2006 American Diabetes Association From Diabetes®, Vol. 55, Suppl.1, 2006; A122 Reprinted with permission from The American Diabetes Association.
FG-3019, a Neutralizing CTGF Monoclonal Antibody, Provides Renal, Metabolic and Cardio-Protective Effects in Obese Type 2 Diabetic Mice
Connective tissue growth factor (CTGF) is implicated in multiple pathogenic processes that underlie the development and progression of diabetic comorbidities affecting the kidneys, heart, and eyes. CTGF is also a necessary factor in all forms of persistent or chronic fibrosis and has been implicated in proteinuria seen in diabetics. FG-3019, a human mAb reactive to human and rodent CTGF and currently in clinical trials for DN, has been shown to reduce albuminuria and to improve kidney function in hyperfiltering db/db mice, a model of obese Type 2 DN. Here, we further explored the potential of FG-3019 as a novel therapy for DN and cardiovascular disease using a model in db/db mice reported to be hypofiltering, i.e. reduced CrCl. Diabetic db/db mice were treated for two months with either FG-3019 or control human IgG (cIgG). Compared to non-diabetic mice, db/db mice exhibited elevated AER, enlarged kidneys and hearts, and increased urine volume. Diabetic mice treated with FG-3019 exhibited a significant and dose-dependent reduction of AER, had significantly reduced polyuria, and had completely normalized kidney size compared to cIgG-treated diabetic mice. These findings with FG-3019 contrast the lack of an effect of anti-TGFβ on albuminuria and polyuria in these mice (Ziyadeh, F., et al., PNAS, 2000). FG-3019 treatment also caused dose-dependent reductions in heart weight, blood LDL and HbA1c. Elevated levels of LDL and HbA1c are important surrogate markers and risk factors for diabetic complications and metabolic syndrome. Transcript analysis of heart tissue showed an increase in extracellular matrix gene expression in db/db mice that was reduced by antibody treatment. These data demonstrate a direct and causative role for CTGF in cardiovascular pathology. Blocking the activity of CTGF with FG-3019 represents a potential therapeutic approach to the prevention and treatment of DN and diabetes-associated metabolic and cardiovascular disorders.
© 2005 ASN
Long-Term Renal Effects of a Neutralizing Connective Tissue Growth Factor (CTGF)- Antibody in Obese Type 2 Diabetic Mice
CTGF is strongly implicated as a pathogenic factor in diabetic kidney disease. Elevated levels of CTGF in the urine, plasma and kidney correlate with the severity and progression of nephropathy in diabetes. FG-3019, a fully human IgG1 kappa monoclonal antibody (mAb) reactive to human CTGF (200 nM Kd), has been shown to significantly reduce collagen deposition in preclinical rodent models of fibrosis. These models may underestimate the effects of FG-3019 in humans, as the affinity of FG-3019 for rodent CTGF is 3-fold lower than for human CTGF. To explore a specific role for CTGF in renal pathology in type 2 diabetes, we examined the effects of FG-3019 in db/db mice, a model of obese type 2 diabetes. Diabetic db/db mice and nondiabetic db/+ mice were treated for two months with either FG-3019 or control human IgG (cIgG). FG-3019 treatment did not affect overall weight gain, blood glucose levels, urinary CTGF levels, or food intake in either strain. Diabetic control mice exhibited elevated levels of urinary CTGF and urinary albumin excretion (UAE), hyperfiltration as demonstrated by elevated creatinine clearance (CrCl), enlarged kidneys and increased urine volume. Diabetic mice treated with FG-3019 had normalized kidney filtration as evidenced by CrCl levels reduced by 82% (p<0.01) and exhibited a 69% reduction in UAE (p<0.01). FG-3019 treatment also significantly reduced kidney weight (p<0.05, not the result of reduced glomerular volume) and urine volume (p<0.01), compared to cIgG-treated diabetic mice. In conclusion, CTGF mediates early and critical features of renal pathology, including morphological and functional changes, in an animal model of type 2 diabetes. Blocking the activity of CTGF with FG-3019 represents a novel therapeutic approach to the prevention and treatment of diabetic nephropathy.
© 2004 ASN
Connective Tissue Growth Factor (CTGF) Activates Nuclear Factor-kB (NF-kB) in Tubulo Epithelial Cells
The connective tissue growth factor (CTGF) has been described as a novel fibrotic mediator. CTGF is overexpressed in several kidney diseases and it is induced by different factors involved in renal injury. There are few investigations about the intracellular mechanisms involved in CTGF response. In kidney diseases elevated tissue activity of the nuclear factor-kB (NF-kB) has been observed. This transcription factor regulates many proinflammatory genes and products involved in proliferation/apoptosis. Our aim was to investigate whether CTGF could activate NF-kB pathway in tubulo epithelial cells (MCT cell line). In growth-arrested cells, recombinant CTGF (100 to 1 ng/mL) increased NF-kB DNA binding activity, as early as 15 minutes, decreasing after 2 hours. This response was dose-dependent, and maximal after 1 hour with 10 ng/mL CTGF (3-fold over control, n=6, p<0.05, gel mobility shift assays). This effect was similar to that observed with proinflammatory cytokines (TNF-alpha and AngII). By immunofluorescence we have located NF-kB subunits. In control cells a diffuse cytoplasmic fluorescence was seen with p65 antibody. Treatment with CTGF for 1 hour caused nuclear staining of p65, showing translocation of NF-kB to the nuclei. We have also investigated whether CTGF regulates NF-kB-mediated gene expression, by using a luciferase reporter plasmid (NF-kB/luc) that contains five copies of the recognition site for the NF-kB sequence. MCT cells were transiently transfected with NF-kB/luc and renilla as internal control. CTGF potently and significantly increased NF-kB promoter activity. This activation was similar to that observed with the cytokines IL-1-beta and TNF-alpha. Our data demonstrate that CTGF activates NF-kB pathway in tubuloepithelial cells. Future studies are needed to evaluate the biological significance of the activation of this transcription factor. © 2004 ASN
Safety and Tolerability of Human Monoclonal Antibody FG-3019, Anti-Connective Tissue Growth Factor, in Patients with Idiopathic Pulmonary Fibrosis
PURPOSE: Idiopathic Pulmonary Fibrosis (IPF) is a progressive lung disease with a median survival of 2.5 to 3.5 years. No therapy has been shown to prolong survival. This is a Phase 1 study of FG-3019, a human monoclonal antibody against Connective Tissue Growth Factor (CTGF), in patients with mild to moderate IPF. CTGF is implicated in the pathogenesis of IPF and is considered the final common pathway of various profibrotic processes. Preclinical models of lung fibrosis demonstrate that FG-3019 reduces scarring associated with CTGF and excess deposition of matrix components (e.g., fibronectin and collagen). METHODS: This open-label, single dose, sequential-group, dose-escalation study is designed to evaluate safety, tolerability, pharmacokinetics, and immunogenicity of FG-3019 in patients with a well established diagnosis of IPF as defined by ATS criteria. FG-3019 (1 mg/kg, 3 mg/kg, or 10 mg/kg) is administered by intravenous infusion over 2 hours. RESULTS: Six patients have been treated with 1 mg/kg of FG-3019, and 9 patients with 3 mg/kg of FG-3019. No dose limiting toxicities have been reported. The mean plasma levels of FG-3019 varied from patient to patient but were above the predicted minimum effective concentration based on animal models of fibrosis for approximately 5 days and 13 days for the 1 and 3 mg/kg dose levels, respectively. CONCLUSION: These preliminary data suggest that a single 2-hour infusion of FG-3019 (1 mg/kg or 3 mg/kg) is safe and well tolerated. CLINICAL IMPLICATIONS: Further studies of FG-3019 are warranted to evaluate its utility in treating IPF.
© 2004 CHEST
Connective Tissue Growth Factor Differentially Mediates Transforming Growth Factorbeta1 Induced Fibrogenesis and Immunomodulation
Transforming growth factor-beta1 (TGF-beta1) functions as a pro-fibrogenic and immunomodulatory cytokine in the kidney. Emerging evidence suggests that CTGF is a downstream mediator of the profibrotic effects of TGF-beta1. However, the role of CTGF in the TGF-beta1 induced immunomodulation is still unknown. The present study was undertaken to determine whether CTGF is differentially involved in mediating TGF-beta1 induced fibrogenesis and immunomodulation in human kidney-2 (HK-2) cells. Fibronectin was used as a marker of fibrogenesis, and interleukin-8 (IL-8) and macrophage chemotactic protein-1 (MCP-1) were measured as immunomodulatory cytokines. CTGF gene was effectively silenced using small interfering RNA (siRNA) in HK-2 cells. RT-PCR confirmed a 95% reduction in CTGF mRNA levels. Wild-type, CTGF gene silenced and wild-type transfected with non-specific siRNA cells were then exposed to 2 ng/ml TGF-beta1 for 48 hrs to assess the fibrogenic response. Results were expressed relative to control values (wild-type cells in the absence of TGF-beta1). TGF-beta1 induced a 589+/- 215% increase in fibronectin secretion in wild-type cells (P<0.05). In CTGF gene silenced cells, TGF-beta1 induced a 244+/-63% increase in fibronectin secretion, significantly less than that observed in wild-type cells treated with TGF-beta1 (P<0.05). Exposure of wild type cells to TGF-beta1 induced IL-8 and MCP-1 to 356±88% (P<0.05) and 138±9% (P<0.05) respectively compared to control values. This stimulatory response was similar in CTGF gene silenced cells. Additional studies were performed to assess the direct effect of CTGF (20, 200 and 400 ng/ml) on IL-8 and MCP-1 at different time points (24, 48 and 72 hr). No stimulation was observed in wild type cells. These data suggest that TGF-beta1 mediates both fibrosis and immunomodulatory pathways, at least in part through a CTGF-dependent mechanism in HK-2 cells. However, TGF-beta1 induced IL-8 and MCP-1 is independent of CTGF.
© 2004 ASN
Connective Tissue Growth Factor (CTGF) Expression Level in Podocytes Relates to Glomerular Basement Membrane (GBM) Thickening in STZ-Induced Diabetes Mellitus
CTGF is a 36-38 kDa secreted protein which is strongly upregulated in fibrotic disorders, and has been suggested as a pathogenic factor in the development of diabetic nephropathy (DN). The objective of the present study was to investigate localization and expression levels of CTGF in relation to ECM accumulation in STZ-induced diabetic CTGF +/- and wildtype (CTGF +/+) mice. CTGF +/- mice (BalbC/129SV) were crossbred with wildtype C57Bl6/J mice. Type 1 diabetes (DM) was induced by STZ (i.p. 200 mg/kg) in the adult female F1 generation. Female littermates served as controls. After 9 weeks of DM, all mice were uninephrectomized to accelerate progression of DN. Mice were sacrificed after 17 weeks of DM. Localization of CTGF was assessed by ISH. CTGF and ECM protein mRNA levels in renal cortex were determined by Q-PCR. GBM thickness was measured by transmission EM. Also, mesangial matrix accumulation was quantified (PAS staining). Q-PCR showed that CTGF gene expression levels were 3-fold upregulated in renal cortex of DM +/+ mice as compared to controls. In DM +/- mice, CTGF mRNA was also increased, but remained 2-fold lower than in DM +/+ mice. In contrast to DM +/+ mice (17% increase), GBM thickness in DM +/- mice was not increased as compared to normoglycemic controls. ISH revealed that CTGF expression is mainly localized to podocytes. In addition, in vitro studies showed that fibronectin expression in human podocytes was induced dose-dependently by CTGF. However, albuminuria, mesangial matrix and mRNA levels of fibronectin and collagen IV in total renal cortex were all equally increased in both DM +/+ and +/- mice as compared to normoglycemic controls. These results indicate that the CTGF expression level in podocytes is involved in DM-related GBM thickening, but does not correlate with albuminuria.
© 2004 ASN
Connective Tissue Growth Factor (CTGF) Plasma Levels Correlate with Plasma Creatinine Levels in Patients with Type I Diabetes Mellitus (DM)
CTGF is a 36-38 kDa protein strongly upregulated in fibrotic disorders including diabetic nephropathy (DN). Plasma CTGF level and urinary CTGF excretion is increased in both human and experimental DN. Furthermore, plasma CTGF levels of type 1 diabetic patients were correlated with albuminuria and creatinine clearance. The aim of the present study was to investigate the possible association of plasma CTGF levels with markers of DN in a larger cross sectional study of patients with type 1 DM. 387 patients with type 1 DM were included in the study. 199 patients had DN, which was clinically defined as urinary albumin excretion rate (UAER) > 300 mg/24h in at least two of three consecutive 24h urine collections, presence of retinopathy and no evidence of other kidney disease. 188 DM patients with normoalbuminuria (NA), who were matched for sex, age and duration of DM, were included as controls. Plasma CTGF levels were determined by sandwich ELISA. Stepwise regression analysis was used to analyse the possible correlation between plasma CTGF levels and relevant patient characteristics. Plasma CTGF levels were significantly higher in DN patients (20±17 ng/ml) as compared to NA patients (10±6 ng/ml). When all 387 patients were included, stepwise regression analysis revealed a correlation between log plasma CTGF and log plasma creatinine levels (R=0.60, p<0.001). In addition, systolic blood pressure, log UAER, age and BMI also contributed as predictors of CTGF plasma level (cumulative R=0.64). When subgroups of patients were analysed separately, the correlation between log plasma CTGF and log plasma creatinine levels was present in the DN group (R=0.65, p<0.001) but absent in NA patients (R=0.018, p=0.81). CTGF plasma levels are increased in type 1 DM patients with DN. Furthermore, a correlation between plasma CTGF and plasma creatinine levels was found. The relative contributions of decreased filtration and increased production of CTGF in patients with DN remain to be established. Disclosure - Grant/Research Support: Fibrogen Inc.
© 2004 ASN
Upregulation of Connective Tissue Growth Factor (CTGF) in Glomerular Epithelial Cells of Mice with STZ-Induced Diabetes Mellitus
CTGF is a 36-38 kDa secreted protein which is strongly upregulated in fibrotic disorders and has been suggested as a pathogenic factor in the development of diabetic nephropathy (DN). The objective of the present study was to determine the relation between plasma, urinary and renal CTGF levels and development of DN in mice. In addition we investigated the localization of renal CTGF overexpression. Diabetes (DM) was induced in C57Bl6/J mice by STZ (200 mg/kg i.p.). DM mice as well as age-matched controls were sacrificed 9 weeks later. CTGF levels in plasma and urine as well as urinary albumin were determined by ELISA. CTGF gene expression levels in renal cortex were determined by Q-PCR. CTGF IHC was performed and glomerular CTGF protein expression as well as mesangial matrix (MM) and glomerular tuft area were quantified using morphometry (Optimas). DM mice were proteinuric, and showed increased glomerular tuft area (27%, p=0.01) and MM score (p=0.02). Plasma CTGF levels were increased 3-fold in DM mice (p=0.01) and urinary CTGF levels ranged from 12-186 mg/g creatinine, while urinary CTGF levels in control mice were below 2 mg/g creatinine. Moreover, in DM mice, albuminuria correlated with urinary CTGF excretion (r=0.82, p<0.001). Q-PCR showed that CTGF gene expression levels were upregulated in renal cortex of DM mice as compared to controls. Morphometry showed a 5-fold increase of CTGF positive surface area in glomeruli of DM mice (p=0.038). CTGF positive staining was mainly localized in visceral and parietal epithelial cells of the glomeruli, and less prominent in mesangial cells. In STZ-induced DM mice, CTGF levels were increased in plasma, urine and renal tissue. Urinary CTGF excretion correlated with albuminuria, the main characteristic of DN. Overexpression of CTGF was localized in the kidney, in particular in glomerular epithelial cells. In the context of the known profibrotic activity of CTGF, these findings suggest a role for CTGF in the development of DN.
© 2004 ASN
Beneficial Effect of Dual Blockade of the Renin-Angiotensin System (RAS) on Urinary Connective Tissue Growth Factor (CTGF) in Type 2 Diabetic Patients with Nephropathy
CTGF is a profibrotic growth factor implicated in the pathogenesis of diabetic nephropathy (DN) and urinary CTGF (U-CTGF) is significantly increased in patients with DN. We evaluated short-term changes in U-CTGF of dual blockade of the RAS by adding an angiotensin II receptor blocker (ARB) to treatment with maximal recommended doses of ACE-inhibitor (ACEI) in patients with type 2 diabetes (T2D) and DN. Twenty T2D patients with hypertension and DN were enrolled in this double-blinded randomized two-period crossover trial. Patients received eight weeks therapy with the ARB candesartan 16 mg daily and placebo, added in random order to existing treatment with lisinopril/enalapril 40 mg or captopril 150 mg daily. At the end of each treatment period we evaluated: U-CTGF (ELISA), albuminuria (turbidimetry), 24-h ambulatory blood pressure measurement (ABPM), and glomerular filtration rate (GFR). During dual blockade of the RAS by addition of candesartan, there was an overall mean reduction (95% CI) in U-CTGF of 18 (0 to 33) %, as compared to ACEI alone (p=.05). Albuminuria was reduced by 28 (17 to 38) % (p<0.001) and there was a modest reduction in systolic/diastolic 24-h ABPM and in GFR (NS). Interestingly, there was a significant carry-over effect by dual blockade on U-CTGF as reflected by a 36 (17 to 51) % (p<0.001) reduction in those 10 patients who received ACEI alone in the first period and dual blockade in the second period, whereas there was an insignificant change in U-CTGF of -5 (-38 to 20) % (p=0.71) in patients who received dual blockade in the first period and mono blockade with ACEI in the second period. A significant carry-over effect was not observed for albuminuria, SBP or GFR. Our short-term study demonstrates that ARB in combination with maximum recommended doses of ACEI significantly reduced U-CTGF and albuminuria as compared to monotherapy with ACEI. A significant carry-over effect on U-CTGF may suggest a prolonged effect of candesartan. Scientific Advisor: FibroGen, Inc.
© 2004 ASN
Amelioration of Diabetic Nephropathy (DN) Induced by Renal Ischemia-Reperfusion (IR) in Rats with Diabetes Mellitus (DM) by Treatment with FG-3019, a Monoclonal Antibody Against Connective Tissue Growth Factor (CTGF)
We tested effects of FG-3019 on DN in an animal model of combined hyperglycemia and ischemia mimicking human DN pathology - end-stage renal disease. Diabetes mellitus (DM) was induced in male Sprague Dawley rats by streptozotocin (STZ). Unilateral renal IR was achieved by clamping the left renal artery for 30 minutes. Treatment with FG-3019 (i.p. 5mg/kg) was started 1 day before IR and continued 3 times per week for 10 weeks. Blood chemistry was analyzed at weeks 0, 4, 8 and 10. Total 24-h urinary protein was determined at weeks 5 and 9. At the end, glomerular filtration rate (GFR) was estimated from inulin clearance, and kidneys were removed for biochemical and histopathological evaluation. In DM animals, there were significant increases in total 24-h urine volume and protein and BUN, indicating glomerular hyperfiltration and renal failure. Treatment with FG-3019 in these rats resulted in a significant reduction of proteinuria and transient reduction in urine volume and BUN. GFR was drastically reduced in the ischemic kidney of DM animals at 10 weeks. Treatment with FG-3019 significantly improved the GFR in ischemic kidneys. Biochemical analysis of the ischemic kidney tissue of DM rats revealed extensive fibrosis as determined by a significant increase in hydroxyproline content, a hallmark of collagen accumulation in fibrosis, while there was no fibrotic change in non-ischemic kidneys of DM animals. Furthermore, the ischemic kidney of DM rats exhibited prominent tubular atrophy and tubulointerstitial fibrosis as evaluated by histopathology. Treatment with FG-3019 resulted in a 30% decrease in the elevated collagen accumulation and a moderate reduction in fibrotic lesions in the ischemic kidneys of DM rats. This study shows that FG-3019 treatment ameliorated DN as indicated by a significant reduction in proteinuria, improvement in kidney function and a moderate decrease in renal fibrosis in the current rat DN model.
© 2004 ASN
Human Urinary CTGF (CCN2) as a Predictor of Progression of Chronic Renal Diseases
We have previously shown that connective tissue growth factor (CTGF/CCN2), a potent prosclerotic growth factor that mediates downstream effects of TGF, is upregulated in human and rat progressive renal diseases. We examined production and excretion of CTGF in the urine as a predictor of progression of renal dysfunction in diabetic nephropathy (DMN) and non-diabetic renal diseases (non-DMN). CTGF levels were measured in urine (n=312) of patients with type 2 diabetes, minimal change nephrotic syndrome (MCNS), membranous nephropathy (MN), IgA nephropathy (IgAN), focal glomerular sclerosis (FGS), nephrosclerosis (NS), hypertension, and healthy subjects using a sandwich ELISA that is specific for the N-terminal portion of CTGF. DMN was classified into five stages: normoalbuminuria, microalbuminuria, macroalbuminuria, renal insufficiency (RI), and renal failure (RF). CTGF values were standardized by urinary creatinine content.
Urinary CTGF (U-CTGF) content was significantly higher in RI and RF of DMN, NS, IgAN, and FGS than in healthy control. In both DMN and in non-DMN, the level of U-CTGF was inversely proportional to renal function. A positive and significant correlation between U-CTGF content and proteinuria was observed in DMN, however U-CTGF was not elevated in glomerular diseases characterized by non-inflammatory lesions and heavy proteinuria, ie. MCNS and MN. U-CTGF content was relatively higher in DMN than in non-DMN. We additionally measured U-CTGF levels every 3 - 4 months for 18 months in 12 patients with DMN and 2 patients with IgAN. Increased U-CTGF was correlated with extent of renal deterioration in all 7 diabetic patients whose renal function became worse. Furthermore, U-CTGF decreased in 2 diabetic patients whose renal function gradually improved during treatment with ACE-inhibitor or Angiotensin II receptor antagonist. These findings suggest that urinary CTGF is a predictor of renal disease progression and response to therapy.
© 2003 ASN
Connective Tissue Growth Factor (CTGF): A Biomarker of Chronic Allograft Nephropathy (CAN)
CAN continues to be one of the leading causes of late graft failure despite improvements in immunosuppressive strategies. Recently, we demonstrated that CTGF, a pleiotropic cytokine and downstream effector of TGF, is upregulated in mouse kidney allografts with CAN but not in nonrejecting isografts. We hypothesize that CTGF is upregulated in human transplant recipients and may be a valuable marker of disease. To this end, we prospectively measured CTGF protein by ELISA in the serum and urine of kidney transplant recipients (N=26) and obtained protocol kidney biopsy tissue at reperfusion and at serial intervals post-transplant for gene expression analysis.
In transplant recipients, serum (49.13.5 ng/ml) and urine (32.26.0 ng/mg creatinine) CTGF levels were significantly elevated compared to nontransplanted, healthy individuals (n=10; 3.91.3 ng/ml and 0.80.2 ng/mg creatinine respectively; p<0.001). While neither serum nor urinary levels correlated with recipient demographics, graft factors, or immunosuppression, mean urinary CTGF levels were highest in recipients with biopsy proven CAN (54.716.0 ng/mg creat), and lowest in recipients with normal histology (22.24.0 ng/mg creat; p=0.02). We also measured CTGF mRNA expression in protocol kidney biopsies by real-time PCR. In 5 patients with grade 2 or 3 CAN by Banff criteria, CTGF mRNA was 3-8-fold higher compared to normal kidney tissue, detected as early as day 19 post-transplant. While there were no significant increases in CTGF mRNA expression in recipients with acute cellular rejection, subclinical cellular rejection or normal histology, there was a 5-fold increase in CTGF expression in biopsies following reperfusion (p=0.01). Thus, CTGF is highly expressed in human transplant recipients with CAN. Furthermore, marked elevation in expression of this fibrogenic cytokine is seen following graft ischemia and reperfusion. These studies suggest that the induction of CTGF following transplantation may regulate the fibrogenic process of CAN. Understanding the regulation of this molecule may allow for earlier diagnosis and treatment of this disorder.
© 2003 ASN
Control of CTGF Expression by Fibroblasts: Role of IFNgamma, TNF-alpha and the Proteasome
Leask A., et al., (2000) 6th International Workshop on Scleroderma Research
Objective: In scleroderma connective tissue fibrosis results in the disruption of the normal architecture of affected organs and ultimately leads to their dysfunction and failure. The TGFbeta/Smad pathway and the activation of CTGF are key effector pathways likely to be responsible for the persistent activation of the genes encoding extracellular matrix proteins such as type I collagen, that underlie the fibrotic process. Understanding of the mode of action of these growth factors in particular the pathways they activate will allow the development of modulatory therapeutic strategies. We have studied the influence of the anti-fibrotic cytokines TNF-alpha and IFNgamma and inhibition of 26S proteasome function on TGFbeta induction of CTGF and type I collagen.
Methods: We have used primary dermal fibroblast cell cultures from normal individuals, ELISA, western blotting and transient transfection using reporter gene constructs to examine the influence of TNF-alpha and IFNgamma on the induction of fibroblast CTGF and type I collagen by TGFbeta, and the effect of inhibiting 26S proteasome on matrix gene expression.
Results: We found that TNF-alpha potently repressed TGFbeta induction of CTGF and type I collagen expression. A role for the transcription factor NFkappaB in this down-regulation was suggested by the ability of an inhibitor of the NFkappaB pathway to block the effects of TNF-alpha, and this was further supported by the ability of a dominant-negative mutant of IkappaBalpha to blocked the inhibition by TNF-alpha. The presence of IFNgamma a known inducer of the inhibitory Smad, Smad7, was also shown to cause marked down-regulation of TGFbeta-dependent type I collagen expression. Furthermore, we showed that addition of two inhibitors of the 26S proteasome, MG-132 or lactacystin, which inhibit degradation of ubiquitinated proteins prevented TNF-alpha inhibition of TGFbeta-dependent CTGF and type I collagen expression.
Conclusion: TNF-alpha appears to be able to regulate CTGF and type I collagen expression via the NFkappaB pathway, whereas IFNgamma may modulate TGFbeta-dependent type I collagen expression via the induction of Smad7. In addition, the ubiquitin-dependent degradation of signalling molecules relevant to the TGFbeta/Smad and the NFkappaB pathway is also likely to be key regulatory events in controlling the expression of pro-fibrotic growth factors and matrix production.
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Expression and Characterization of Recombinant Human Gelatin Fragments
Olsen D., et al. (2000) American Association of Pharmaceutical Scientists (AAPS) Annual Meeting and ExpositionPurpose. Gelatin is a common excipient in numerous pharmaceutical preparations including vaccines, capsules, and plasma substitutes and is used in surgical sponges and other medical devices. Gelatin is typically derived from animal tissue and therefore poses potential problems due to immunologic reactions and contaminants which cause transmissible spongiform encephalopathy (TSE). The purpose of our study was to develop a recombinant expression system that could produce homogenous, well-defined human gelatins of different sizes to test as vaccine stabilizers.
Methods. The host used to express recombinant gelatin was a Pichia pastoris strain which contains a single copy of the alpha and beta subunits of prolyl hydroxylase (P4H), the enzyme that catalyzes the post-translational conversion of peptide bound proline to hydroxyproline. Co-expression of P4H and gelatin fragments results in the expression of gelatin with the native human sequence. The expression plasmids used in our studies contain alpha1 (I) cDNA sequences of different sizes fused to the mating factor alpha prepro sequence and protein expression is regulated by the methanol-inducible alcohol oxidase promoter (PAOX1). Transformants were selected using zeocin or by complementation of a his4 auxotrophy. Gelatin producing strains were identified by SDS-PAGE analysis of conditioned media and P4H activity in extracts from shake flask cultures. Strains were then grown in 10 liter fermentors to generate media for large scale purification. Gelatin was purified from the media by ion-exchange chromatography and characterized by amino acid sequence and composition and tested in bioassays.
Results. Gelatin fragments of discrete sizes, ranging from 5-50 kd, were expressed and secreted into the media. Expression level in the 10 liter fermentor exceeded 3 grams/liter. High purity gelatin was obtained by a combination of anion and cation exchange chromatography. N-terminal sequence analysis demonstrated correct signal sequence processing. Some of the gelatin fragments demonstrated in vitro cell attachment activity.
Conclusions. These gelatin fragments represent a novel biomaterial which can be used in a variety of applications, such as vaccine stabilization, were animal derived gelatins are currently used. These new materials provide a safe, well-defined, non-immunogenic replacement for currently used gelatins.
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Anemia Correction with Roxadustat Lowers Hepcidin in Chronic Kidney Disease (CKD) Patients
Szczech L., et al (2015) J Am Soc Nephrol 26:237A
Background: The hypoxia-inducible factor prolyl hydroxylase inhibitor roxadustat (FG-4592) is being developed for treatment of CKD anemia. Hepcidin regulates iron metabolism and higher levels are associated with greater mortality. This analysis of phase 2 studies was undertaken to explore the consistency of the suppressive effect of roxadustat on hepcidin.
Methods: Among CKD-NDD (017 and 041) & CKD-DD (040 and 053) studies, roxadustat doses, study duration, and comparator (placebo or epoetin) varied. Studies restricted IV iron in general but allowed oral iron. Baseline (BL) hepcidin and change from BL (CFB) are summarized (mean±SD) overall and by BL tertile. Significant differences (p<0.05 vs BL) based on within-group comparisons (^).
Results: Mean BL hepcidin in CKD-NDD roxadustat subjects was 292.8±179.8 and 120.3±107.0 ng/mL (studies 017 & 041). Hepcidin fell with roxadustat treatment by 158.4±179.2 & 45.6±87.7. Mean BL hepcidin in CKD-DD Roxadustat subjects was 303.9±172.9 & 91.1±90.0 (040 and 053). Hepcidin fell with roxadustat treatment by 26.7±192.0 & 57.4 ±68.5. For both groups, the greatest declines were in the highest BL tertiles.
Hepcidin at BL
CFB during treatment
(4 wks, 8 wks for 041)
CFB: highest tertile
CFB middle tertile BL hepcidin
CFB: lowest tertile BL hepcidin
Epoetin alfa (N=34)
Conclusions: Roxadustat consistently lowered hepcidin in phase 2 studies. The decrement in hepcidin is greatest among those with the highest BL levels. The roxadustat phase 3 trials will include measurements of hepcidin to further define this effect.
© 2015 ASN
Randomized placebo-controlled dose-ranging and pharmacodynamics study of roxadustat (FG-4592) to treat anemia in nondialysis-dependent chronic kidney disease (NDD-CKD) patients
Anemia Correction with Roxadustat Improves Health Related Quality of Life (HRQOL) in Chronic Kidney Disease (CKD) PatientsBackground: The hypoxia-inducible factor prolyl hydroxylase inhibitor roxadustat is being developed for treatment of CKD anemia.This analysis of two phase 2 trials was undertaken to assess the effect of roxadustat on HRQOL in non-dialysis (NDD) and dialysis dependent (DD) CKD. Methods: HRQOL was assessed by SF-36 and FACT-An questionnaires in efficacy-evaluable populations in 2 open-label studies, CKD-NDD (041) and ESA-naïve incident CKD-DD (053). HRQOL was assessed at baseline (BL), 8 and 16 wks [end of treatment (EOT)] in 041 and at BL, 8 and 12 wks (EOT) in 053 [mean±SE changefrom BL (Δ)]. Missing data were imputed by last observation carried forward. Results: Data from 141 subjects with CKD-NDD and 55 subjects with CKD-DD were available. In both populations, SF-36 physical component summary and FACT-An scores improved compared to BL (p=0.005 & 0.001, CKD-NDD; p=0.01 & 0.02, CKD-DD). SF-36 vitality norm-based domain scores (NBDS) and FACT-An anemia score increased by an average of >4 points. Benefits were seen particularly among those with low BL scores. In Study 053, subjects with BL SF-36 Physical Functioning NBDS <35 experienced a mean increase of 8.7 (p=0.005) and those with BL Vitality NBDS <50 increased by 6.7 (p<0.0001). Subjects with BL FACT-An Anemia score<55 increased 10.3 (p=0.0001), and those with BL FACT-An Total Score<135 increased 16.0 (p=0.0005).
a p <0. 01 from other 2 gps; b not different from zero; C no difference between pair TIBC increased in all groups. Largest changes occurred in no Fe group. IV iron blocked hepcidin suppression & Ftn reduction. EoT TSAT (R2 = 0.40), Ftn (R2 = 0.41), & TIBC (R2 = 0.31) correlated with EoT hepcidin in the no Fe group. EoT Ftn (R2 = 0.73) & TIBC (R2 = 0.40) correlated with EoT hepcidin in the po Fe group. No correlations in IV iron group. BL and EoT Ftn levels correlated (R2 = 0.43 BL & 0.73 EOT) with hepcidin. Hepcidin correlated weakly with CRP [R2=0.11, p=0.015] at BL but not at EOT. Conclusions: Roxadustat consistently improved mean HRQOL sub- and summary scores. Improvements were greatest in subjects with low BL scores. Roxadustat is currently being evaluated in phase 3 trials in which HRQOL is further explored.
Population (Study) (n)
Δ at EOT
Physical Functioning NBDS
Physical Functioning NBDS
© 2015 ASN
Anemia Correction with Roxadustat Lowers Cholesterol in Chronic Kidney Disease (CKD) PatientsBackground: Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor roxadustat is being developed for CKD anemia.The HIF pathway affects cholesterol metabolism & ascension to altitude reduces total cholesterol (TC). This analysis of phase 2 studies explores roxadustat's effect on TC in non-dialysis (NDD) and dialysis (DD) CKD. Methods: In Phase 2 studies 41&47 in NDD & 40,48&53 in DD, roxadustat dose, study duration and comparator (placebo, epoetin) varied. Studies restricted IV iron but allowed oral iron. Baseline (BL) TC and change from BL (Δ) were summarized overall and by BL tertile. Data are mean±SD. Results: Among roxadustat NDD subjects (n=206), mean BL TC was 170.9±45.2 and 166.8±39.1 mg/dL (studies 41&47). TC fell with roxadustat by 25.8±29.7 and 33.7±31.8. Among roxadustat DD subjects (n=238), mean BL TC was 171.1±35.1, 174.3±57.3, and 171.0±56.7 (studies 48, 53&40). TC fell with roxadustat treatment by 14.2 ±36.4, 44.4±45.9 and 36.7±37.0. The greatest declines were among the highest BL tertile in both populations.
Conclusions: Roxadustat consistently lowered TC in phase 2 studies. The decrement in TC is greatest among those with highest BL levels. Roxadustat phase 3 trials will include tests to further define this effect and significance.
CFB during treatment
CFB in highest tertile of BL TC
CFB in middle tertile of BL TC
CFB in lowest tertile of BL TC
Epoetin alfa (N=22)
Epoetin alfa (N=36)
© 2015 ASN
Anemia Correction with Roxadustat Increases Soluble Transferrin Receptor (sTfR) in Chronic Kidney Disease (CKD) PatientsBackground: The hypoxia-inducible factor prolyl hydroxylase inhibitor roxadustat corrected anemia in phase 2 trials without IV iron.This analysis of 4 phase 2 studies explores roxadustat's effect on sTfR levels. Methods: In phase 2 studies CKD-NDD 017 & 047 and CKD-DD 040 & 048, subjects were randomized to roxadustat at different doses with comparator arms of placebo or epoetin-alfa. Studies restricted IV iron use in general but allowed oral iron. sTfR at baseline (BL) and % change from BL were summarized by treatment. Results: Among CKD-NDD subjects (n=125), BL sTfR mean±SD was 1.35±0.56 and 3.58±1.69 mg/L (studies 017 & 047 respectively) in roxadustat groups. sTfR rose with roxadustat treatment in both studies by 86.5% and 101.5%. These increases were greater than the placebo arm (15.3% & 2.6%, p=0.0081 & <0.0001 respectively). Among subjects with CKD-DD (n=199), BL sTfR mean±SD was 3.57±1.41 and 3.33±1.47 mg/L (studies 048 & 040 respectively) in the roxadustat group. In 040, the increase in the roxadustat group was greater than in the epoetin arm (31.1% vs - 0.7% respectively, p=0.0013). However, in 048, the changes in sTFR were comparable to epoetin arm (32.1 vs 39.9% respectively).
Conclusions: Roxadustat consistently increased sTfR in phase 2 studies with oral iron supplementation. Placebo had little effect on sTfr, and epoetin results varied. The consistent increase in sTfR supports sufficient iron delivery for erythropoiesis during the time periods studied. The coordinated erythropoiesis induced by roxadustat is being evaluated in phase 3 trials.
Study Population (N)
Baseline sTfR mg/L
% Change from BL
017 CKD-NDD (24)
047 CKD-NDD (61)
040 CKD-DD (78)
048 CKD-DD (73)
© 2015 ASN
Impact of Iron Regimen on Iron Indices and Hepcidin during Roxadustat Anemia Correction in Incident Dialysis PatientsBackground: Roxadustat (FG-4592) corrects anemia in incident ESA-naïve HD/PD patients assigned to no Fe, oral Fe, or IV Fe over a 12 wk period. Initial 6 wk ΔHb response was equal but maximal ΔHb & Hb at 12 weeks (EoT) was blunted with no Fe, while Hb response with oral iron was comparable to IV iron. This study evaluates the effect of each Fe regimen. Methods: Post hoc study. Results: Results are mean±SEM. At baseline (BL), iron parameters among iron regimens were similar reflecting iron depletion (TSAT/Ferritin (Ftn), but mean CHr 30.6pg; CRP 2 to 55 ng/ml; entry Hb 8.3 g/dL. CRP did not change; ΔHb was independent of BL CRP. Changes in iron parameters from BL to EoT are shown
TIBC increased in all groups. Largest changes occurred in no Fe group. IV iron blocked hepcidin suppression & Ftn reduction. EoT TSAT (R2 = 0.40), Ftn (R2 = 0.41), & TIBC (R2 = 0.31) correlated with EoT hepcidin in the no Fe group. EoT Ftn (R2 = 0.73) & TIBC (R2 = 0.40) correlated with EoT hepcidin in the po Fe group. No correlations in IV iron group. BL and EoT Ftn levels correlated (R2 = 0.43 BL & 0.73 EOT) with hepcidin. Hepcidin correlated weakly with CRP [R2=0.11, p=0.015] at BL but not at EOT. Conclusions: FG-4592 therapy is not impacted by inflammation and prevents iron-deficient erythropoiesis, removing parenteral iron-induced risks associated with IV iron. Even with IV iron, iron overload is avoided, likely attributable to HIF homeostasis. With FG-4592, po Fe regimen was optimal and physiologic. The combination of FG4592 and po Fe offers short term and perhaps long term benefits for many intermediate iron markers and other benchmarks of anemia care.Iron regimen (N)ΔHb (g/dL)Δ TSAT (%)ΔCHr (pg)ΔHepcidin (µM)ΔFtn (µM)No Iron (23)1.98 ± 0.28a-7.8 ± 1.43 a-2.91 ± 0.63 a-63.4 ± 13.3 c-121.5 ± 12.2 aOral Iron (22)2.68 ± 0.33 c1.07 ±1 .87 b,c-0.91 ± 0.13 c-54.1 ± 18.4 c- 51.9 ± 18.6IV Iron (10)3.25 ± 0.36 c0.69 ± 2.89 b,c-1.02 ± 0.14 c-12.6 ± 31.6 a,b-25.2 ± 33.5 b
© 2014 ASN
Hypoxia Inducing Factor Prolyl Hydroxylase Inhibitor FG-4592 Corrects Anemia In Peritoneal Dialysis
Background: FG-4592 inhibits prolyl hydroxylase and transiently stabilizes hypoxia-inducible factor being developed for CKD anemia treatment. Endogenous erythropoietin levels are transiently normalized to physiologic levels. The safety and efficacy of FG-4592 using various iron regimens was tested in a phase 2 study FGCL4592053 in hemo (HDP) and peritoneal (PDP) dialysis patients and reported. In that ~10% of patients with end stage renal disease (ESRD) globally receive PD, it is important to characterize the efficacy of FG-4592 in this group. Methods: 48 ESA-naïve HDP were randomized to FG-4592 and no, oral or IV iron. All PDP (12) received FG-4592 with oral iron. The cohorts of PDP and HDP receiving oral iron were compared (n=24). Hb target was 11-13 g/dL. Results: There were no clinically or statistically significant differences in demographics, medical history or baseline labs btw PDP and HDP. Among PDP, median age was 51.0 y (range 30-68). At baseline, Hb was 8.8 g/dL (0.7), ferritin level was 144.3 ng/mL (79.2) and TSAT was 19.4% (5.8) in the PD cohort. All PDP had hypertension while none had DM. PDP had a median baseline: peak change in Hb of 3.1 g/dL (range 2.4-4.7). All PDP and 11/12 HDP on oral iron responded (a change in Hb>1 g/dL from wks 3-13). Comparing PDP to HDP controlling for baseline Hb and iron stores, no difference in Hb change from baseline or in time to Hb response was detected btw groups (P=0.90 and 0.42, respectively). Median time to response was 4 wks (range 3-8). Adverse Event rates were similar between HDP and PDP and were generally mild-moderate and typical for ESRD patients. Conclusions: While the power afforded by the number of patients receiving PD is limited, these data do not suggest a difference in efficacy or safety for FG-4592 among patients with ESRD based on modality. Given these data and safety signals demonstrated in trials of ESAs, FG-4592 could represent a safer and more convenient option for anemia treatment in ESRD.
© 2013 ASN
A Randomized, Double-Blind, Placebo Controlled Trial of FG-4592 for Correction of Anemia in Subjects with Chronic Kidney Disease in ChinaFG-4592 is an oral inhibitor of hypoxia-inducible factor degradation being developed for treatment of anemia. This Phase 2b study tested the efficacy and safety of FG 4592 in anemic chronic kidney disease (CKD) subjects. CKD subjects not on dialysis with hemoglobin (Hb) <10 g/dL were randomized 1:1:1 to FG-4592 low or high dose or placebo orally 3 times a week (wk) for 8 wks. The primary efficacy endpoint was maximum change of Hb (ΔHbmax) from baseline (BL) by Wk 9. 91 subjects enrolled, 90 completed 8 wks dosing; 88 were evaluable for efficacy. BL characteristics for FG-4592 and placebo groups are comparable. In FG-4592-treated subjects, Hb increased from BL in a dose-related fashion. The proportion of Hb responders (ΔHb >1 g/dL from BL) and subjects achieving Hb ≥11.0 g/dL was significantly higher in FG-4592 vs placebo subjects (Table). Significant decreases in total and low-density lipoprotein cholesterol (ΔTC & ΔLDL-C) occurred in FG-4592 subjects. The incidences of treatment emergent adverse events (AEs) and serious AEs (SAEs) were nearly identical for FG-4592-treated and placebo subjects. AEs were generally mild or moderate and typical for CKD patients.
^p<0.01, #p < 0.0001Cohort12FG-4592PlaceboN30316130Mean (SD) dose, mg/kg/Wk4.16 (0.52)5.03 (0.67)4.60 (0.74)-BL Hb, g/dL8.828.848.838.93Hb @9 wks, g/dL10.74#11.38#11.07#9.4ΔHbmax , g/dL1.75#2.69#2.22#0.57Hb responders (%)24 (80)#27 (93.1)#51 (86.4)#7 (24.2)Subjects with Wk 9 Hb ≥11.0 g/dL (%)15 (50)^22 (75.9)#37 (62.7)#2 (6.9)ΔTC, mg/dL (%)-32 (-19.7)#-35 (-17.5)#-33.9 (-18.5)#+8 (+5.5)ΔLDL-Cmg/dL, (%)-23.7(-23.2)#-32(-23.2)#-27.9( -23.2)#+4Subjects w/ AEs (%)17 (56.7)19 (61.3)36 (59.0)19 (63.3)Subjects w/ SAEs (%)4 (13.3)5 (16.1)9 (14.8)4 (13.3)Subjects w/ cardiovascular SAE (%)0001 (3.3)
This study suggests that FG-4592 effectively corrects CKD anemia and is well-tolerated in CKD patients. FG-4592 may also lower cholesterol. P3 trials of FG-4592 in CKD patients are underway.
© 2013 ASN
FG-4592, an Oral Hypoxia-Inducible Factor Prolyl Hydroxylase-Inhibitor, Corrects Anemia Without Iron Supplementation in Incident Dialysis Patients
Background: Optimal iron management strategy in treating anemia is controversial. We evaluated whether FG-4592, with or without iron supplementation, can correct hemoglobin (Hb) levels in incident dialysis patients.
Methods: In an ongoing, open-label, phase 2 study, erythropoiesis-stimulating-agent-naïve patients on hemodialysis (HD) for 2 wks to 4 mos, with baseline (BL) Hb ≤ 10.0 g/dL, were randomized to 3 arms (N=12/arm) to receive FG-4592 thrice weekly for 12 wks without iron, with oral iron, or with intravenous (IV) iron. Another arm evaluated FG-4592 with oral iron in peritoneal dialysis patients (N=12). A confirmatory arm further assessed FG-4592 without iron in HD patients (N=12). Initial FG-4592 doses were tiered by weight; dose adjustments were allowed every 4 wks.
Results: Results are reported for 36 HD patients completing ≥8 weeks of treatment (Table); a majority were not iron replete at BL. Mean Hb changes from BL at Wk 8 were similar across arms. FG-4592 was well tolerated, with an adverse event profile consistent with the patient population.FG‑4592Arm(n=12/arm)N (%)Not Iron Replete at Baseline (BL)Mean ±SE Ferritin(ng/mL), BLMean ±SE TSAT (%), BLMean ± SE Hb (g/dL)Mean±SE
Change in Hb
In Hb (g/dL)Iron9 (75)150±1920±27.8±0.32.1±0.42.8±1.0Oral Iron6 (50)184±6321±28.5±0.32.2±0.43.3±1.9IV Iron9 (75)158±2523±58.3±0.32.3±0.73.2±1.7
Conclusions: Preliminary data indicated that treatment with oral FG-4592 TIW increased mean Hb in incident dialysis patients, regardless of BL iron status or iron supplementation. FG-4592 may prevent iron-deficient erythropoiesis, thus avoiding potential risks associated with parenteral iron.
© 2012 ASN
FG-4592, a Novel Oral Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor (HIF-PHI), Maintains Hemoglobin Levels and Lowers Cholesterol in Hemodialysis (HD) Patients: Phase 2 Comparison with Epoetin AlfaBackground: Dyslipidemia is a major factor associated with increased cardiovascular risk in advanced CKD. We evaluated effects of FG-4592 on hemoglobin (Hb) and total cholesterol (TC) in hemodialysis (HD) patients switched from epoetin alfa (rhEPO). Methods: In an ongoing open-label phase 2 study, 159 HD patients taking rhEPO 3X weekly (TIW) were randomized to FG-4592 TIW or continued rhEPO for 6 or 19 wks. FG-4592 doses were adjusted every 4 wks to maintain Hb 11-13 g/dL. Intravenous (IV) iron supplementation was disallowed. There were no protocol-specified restrictions on lipid medication use or thresholds for medication changes. Results: Hb data for patients treated 6 wks have been reported (Provenzano 2011). Mean±SD baseline (BL) Hb levels for patients treated 19 wks with FG-4592 (N=54) and rhEPO (N=19) were 11.3±0.7 and 11.4±0.9 g/dL, respectively. The corresponding mean±SD Hb changes from BL in the last 4 treatment wks were -0.2±1.4 and -0.6±1.3 g/dL, respectively; changes were not significantly different between the 2 groups. FG-4592 patients (N=91) had a Week 6 mean±SD 20%±15% TC reduction from BL, and rhEPO patients (N=32) had a mean±SD 4%±16% TC increase from BL (p<0.0001); the difference remained significant at Week 19 (p<0.0001). A substudy showed lower plasma erythropoietin levels with FG-4592 treatment compared with rhEPO treatment at screening. FG-4592 was well tolerated, with an adverse event profile consistent with the patient population. Conclusions: Oral FG-4592 TIW for 19 weeks maintained corrected Hb levels in HD patients without IV iron supplementation. The HIF-PHI mechanism of action appeared to reduce TC and plasma erythropoietin in patients given FG-4592 vs rhEPO.
© 2012 ASN
FG-4592 Oral Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor Corrects Anemia in Nondialysis CKD Patients without IV Iron
Anemia is largely undertreated in nondialysis CKD. We report data from an ongoing phase 2b, open-label study of FG-4592 to correct anemia in adult nondialysis CKD patients (pts). Ninety-six pts (Hb ≤10.5 g/dL at baseline [BL]) who had not received ESAs for the prior 12 weeks (wks) were randomized to 4 equal cohorts. Cohorts A and B: 16 wks initial tiered, weight-adjusted doses of 60−140 mg FG-4592 thrice weekly (TIW). Cohorts C and D: 24 wks initial fixed doses of 50 and 100 mg FG-4592 TIW, respectively. Cohort-dependent dose adjustment was allowed every 4 wks. IV iron was not permitted. BL demographics were similar across cohorts. For 95 efficacy-evaluable pts, overall mean±SD BL Hb was 9.7±0.7 g/dL. FG-4592 treatment led to increases from BL Hb in all cohorts (Table). After 16 wks of treatment, all A and B pts treated >5 wks (n=46) had a Hb response (>11 g/dL and ≥1 g/dL from BL), except 3 pts with BL Hb 8.5 g/dL who achieved mean Hb 10.4 g/dL. At wk 16, mean doses were 91 and 113 mg in A and B, respectively. B pts who had a Hb response were converted to twice weekly dosing. Responses were observed whether BL ferritin or TSAT was normal or subnormal. After 8 wks of treatment, there was a dose-dependent increase from BL Hb; and for 33 pts with data available, mean hepcidin decreased 36% from BL (p<0.0001). FG-4592 was well tolerated. No treatment-related serious adverse events were reported to date. Treatment with initial weight-adjusted or fixed oral FG-4592 doses corrected anemia in CKD pts in the absence of IV iron repletion.Mean±SD ΔHb (g/dL)CohortStarting Dose (mg)TIWWks of Treatmentn4816A60-140241.6±1.12.4±1.22.3±1.2B241.1±0.91.8±1.02.3±1.0C50230.6±0.71.0±0.9NAD100241.5±1.02.1±1.1NALOCF method used to impute missing data. NA=data not yet available.
© 2011 ASN
Inhibition of Prolyl Hydroxylases Increases Erythropoietin Production in ESRD
The reasons for inadequate production of erythropoietin (EPO) in patients with ESRD are poorly understood. A better understanding of EPO regulation, namely oxygen-dependent hydroxylation of the hypoxia-inducible transcription factor (HIF), may enable targeted pharmacological intervention. Here, we tested the ability of fibrotic kidneys and extrarenal tissues to produce EPO. In this phase 1 study, we used an orally active prolyl-hydroxylase inhibitor, FG-2216, to stabilize HIF independent of oxygen availability in 12 hemodialysis (HD) patients, six of whom were anephric, and in six healthy volunteers.FG-2216 increased plasma EPO levels 30.8-fold in HD patients with kidneys, 14.5-fold in anephric HDpatients, and 12.7-fold in healthy volunteers. These data demonstrate that pharmacologic manipulation of the HIF system can stimulate endogenous EPO production. Furthermore, the data indicate that deranged oxygen sensing—not a loss of EPO production capacity—causes renal anemia.
© 2011 ASN
FG-4592, A Novel Oral HIF Prolyl Hydroxylase Inhibitor, Elevates Hemoglobin in Anemic Stage 3/4 CKD Patients
Given heightened concerns about clinical outcomes arising from use of ESAs that cause supraphysiologic circulating EPO levels, use of agents producing transient, more physiologic increases in endogenous EPO (eEPO) may have significant benefit in CKD anemia. In this randomized, single-blind, placebo-controlled phase 2 study, 117 subjects were randomized with 116 (108 unique) treated: 88 to FG-4592 and 28 to placebo, in 4 dose cohorts (0.7, 1.0, 1.5, and 2.0 mg/kg FG-4592) administered 2 [BIW] or 3 [TIW] times weekly for 4 wks. 97 subjects who were anemic at baseline (BL Hb<11 g/dL) and treated for at least 2.5 wks were eligible for efficacy evaluation. BL Hb was 10.0 ± 0.7 g/dL (mean ± SD). Maximal Hb change from BL by wk 6 and Hb responder rate (% subjects with Hb increase >1 g/dL from BL at any time from wk 3-6) are shown in table. For responders, median time to Hb response was 22-43 and 15-22 days for BIW and TIW groups, respectively, generally faster than observed with ESA. FG-4592 administered orally for 4 wks was well-tolerated and led to dose-dependent Hb correction with observed peak eEPO levels 1-2 orders of magnitude lower than values reported for ESA. During treatment, one AE of increased BP was reported in the 0.7 mg/kg group; no significant BP change was observed across cohorts despite rapid rate of Hb rise with higher doses. There were no reports of thrombosis, sustained liver enzyme abnormality, or study drug related SAE. These results support a potentially unique mechanism of action permitting safer Hb increase and maintenance while avoiding thrombotic or hypertensive responses commonly observed with ESA.
Mean ± SD Maximal DHb g/dL (%Hb Responders)
Cohort BIW [N] TIW [N] 0.7 mg/kg 0.9±0.8 (33)  1.0±0.9 (62)  1.0 mg/kg 0.9±0.8 (60)  1.0±0.9 (60)  1.5 mg/kg 1.7±1.0 (80)  2.0±0.9 (91)  2.0 mg/kg 1.9±0.6 (100)  2.2±0.8(100)  placebo 0.4±0.5 (8) 
© 2010 ASN
Induction of Erythropoiesis in Rodents by Novel and Distinct Families of Orally Active HIF Prolyl Hydroxylase Inhibitors
FibroGen is developing a series of novel Hypoxia Inducible Factor (HIF) prolyl hydroxylase inhibitors (PHI) that stimulate HIF-dependent erythropoietin (Epo) secretion, and that with repeated oral dosing, selectively stimulate erythropoiesis in preclinical and clinical studies. Specific chemotypes were further optimized to enhance the potency and selectivity of novel HIF-PHI in vitro and in vivo, focusing on their capacity to enhance iron utilization and to work optimally under conditions of anemia and hyporesponsiveness to erythropoietic stimulating agents (ESA).
Optimized, next generation HIF-PHI were validated to selectively stimulate HIF-dependent Epo secretion in vitro, and with intravenous or oral administration in vivo, to elevate circulating endogenous Epo levels that were up to 1000-fold above levels in animals administered vehicle control. Multiple dosing regimens were explored to determine the optimal paradigm for HIF-dependent erythropoiesis. Repeated oral administration of HIF-PHI led to increased red blood cell number, hemoglobin (Hb) and hematocrit at doses as low as 0.5 mg/kg, with maximal Hb increases of ~ 5 g/dL within a 1-week period, and with no adverse effects at doses as high as 200 mg/kg, which was the highest dose tested. Depending on individual pharmacokinetic profiles, specific HIF-PHI were found to induce erythropoiesis at doses as low as 0.25 mg/kg.
Representative chemotypes from distinct structural classes were tested in preclinical models of anemia, including the rat remnant kidney model (5/6th nephrectomy) and a model of anemia of chronic disease (ACD) caused by inflammation. Oral dosing in the 5/6th model increased Hb levels as much as 5 g/dL over a 2 week period with no increase in serum creatinine or BUN and no elevation of systolic blood pressure. Oral administration in the ACD model increased Hb and concomitantly multiple parameters of iron utilization, including mean cell volume and mean cell Hb, under conditions where ESA were not efficacious.
These studies demonstrate that HIF-PHI promote pharmacologic stimulation of HIF-dependent erythropoiesis in a selective and well-tolerated manner, maintain efficacy with reduced renal mass and function, and work under conditions of ESA hyporesponsiveness. Thus, orally active HIF-PHI represent a novel therapeutic approach to treat anemia associated with chronic kidney disease and inflammation.
Correction of Anemia without Exacerbation of Hypertension in a Rat Model of Chronic Kidney Disease: Comparison of FG-2216 to Recombinant Erythropoietin
Cardiovascular disease is the leading cause of mortality among CKD patients. Uncontrolled blood pressure both increases progression of CKD and increases cardiovascular mortality risk. Hypertension has been regarded as one of the most significant complications from treatment with rhEPO in CKD patients; treatment with rhEPO is contraindicated in patients with uncontrolled hypertension. Hypertension associated with rhEPO has been attributed to many factors including rate of increase and absolute Hb rise. Here, we compare equipotent doses of FG-2216, a novel HIF-PH inhibitor (HIF-PHI), and rhEPO in a rat 5/6th remnant kidney model of CKD to evaluate effects on hypertension and erythropoiesis. Five wks after 5/6th nephrectomy (Nx), rats with demonstrated uremia, anemia and hypertension were randomized to receive vehicle, FG-2216, or rhEPO. Intermittent dosing with vehicle, FG-2216, or rhEPO continued 3 more wks. Hb values in both FG-2216-treated and rhEPO-treated groups increased to the same degree over the course of the study (>3 g/dL in 3 wks). Creatinine levels were not significantly different between treatment groups at the beginning or end of treatment. SBP was significantly elevated from sham-controls (50 mm Hg increase) and not different between groups at the beginning of the treatment period. Over the course of the 3-wk treatment, SBP increased significantly in the rhEPO group while decreasing significantly in the FG-2216 group. Treatment with either FG-2216 or rhEPO alleviated the anemia associated with 5/6th Nx; however, only FG-2216 was able to increase Hb without increasing SBP. Similar results were obtained in the same model using another HIF-PHI, FG-4592. The differential response to therapy may be related to the ability of FG-2216 and HIF-PHI in general to stimulate erythropoiesis with relatively low circulating levels of EPO (compared to levels required with recombinant therapies). These results indicate that HIF-PHI may provide clinical benefit over current therapies by allowing for correction of anemia without exacerbation of underlying hypertension and uremia.
© 2008 ASN
Beneficial Pharmacodynamic Effects Resulting from 'Complete Erythropoiesis' Induced by Novel HIF Prolyl Hydroxylase Inhibitors FG-2216 and FG-4592
FibroGen is developing a series of novel orally bioavailable HIF prolyl hydroxylase inhibitors (HIF-PHI) that selectively induce complete erythropoiesis, including FG-2216 and FG-4592, which are in phase 2 clinical development to treat CKD anemia. Complete erythropoiesis encompasses modulation of genes that promote RBC maturation, including increased expression of EPO/EPO-R, and genes that increase iron absorption/utilization (e.g., elevated Dcytb, DMT1, transferrin and its receptor, and decreased hepcidin). Coordinated regulation of genes mediating erythropoiesis is a novel approach to treat anemia of chronic inflammation, and makes HIF-PHI ideal to mitigate functional iron deficiency and hypo-responsiveness to erythropoietic stimulating agents (ESA). Additionally, current ESA therapies rely on supra-physiologic levels of recombinant EPO variants to induce erythropoiesis, leading to peak or sustained ESA levels greatly exceeding normal physiological levels of endogenous EPO (eEPO) that elevate Hb in response to hypoxia or phlebotomy, or in response to pharmacological activation of HIF. Available clinical data from phase 2 studies show modest and intermittent increases in eEPO induced by HIF-PHI (10- to 40-fold lower than ESA levels) are sufficient to mediate erythropoiesis in non-dialysis patients with CKD, without increased incidence of hypertension or thrombosis. Recent analysis of clinical studies and US renal databases shows that high doses of ESA are associated with an increased incidence of hypertension, thrombosis, and cardiovascular events, and an overall increased mortality, irrespective of Hb levels. Induction of complete erythropoiesis by HIF-PHI, and the relatively low levels of eEPO associated with HIF-PHI, may reduce potential adverse effects associated with treatment of anemia with supra-physiologic doses of ESA, including thrombosis and cardiovascular events, and mortality. HIF-PHI therapy may ultimately lead to anemia correction with more physiological levels of induced eEPO, and provide a novel approach to treat anemia associated with inflammation, functional iron deficiency, and ESA hypo-responsiveness.
© 2008 ASN
FG-2216, A Novel Oral HIF-PHI, Stimulates Erythropoiesis and Increases Hemoglobin Concentration in Patients With Non-Dialysis CKD
In this phase 2 single blind, placebo-controlled, dose-ranging study, we examined the ability of FG-2216 to correct anemia in ESA-naïve patients with CKD. 142 patients with CKD Stages 3 & 4 and Hb < 10.8 g/dL were randomized and treated for up to 15 weeks. Initial doses included 375mg (N=26), 625mg (N=52), or 1250mg (N=50), dosed twice weekly and placebo (N=14). After 4 weeks of stable dosing, elective titration was employed to achieve a target Hb response. The proportion of treated subjects with Hb response, defined as 1 g/dL increase, was 13 (50.0%), 31 (59.6%), 38 (76.0%) for the FG-2216 dose groups and none for placebo. Analysis of the completer per protocol population (N=96) excluding treatment non-completers due to early study termination by the sponsor reveals responder rates of 58.8%, 60.5%, and 90.6% for the FG-2216 dose arms. Mean baseline Hb levels for the FG-2216 starting doses and placebo were 10.2, 10.1, 9.9 and 10.1 g/dL, respectively; following up to 15 weeks of therapy, maximum mean Hb changes from baseline were 1.1, 1.4, 2.4 and 0.2 g/dL, respectively. Assessment of endogenous EPO and VEGF 12 hours after the first dose revealed a mean increase from baseline in EPO of 11.8, 28.9, 141.6 for the FG-2216 groups and 1.1 mIU/mL for placebo; mean change from baseline in VEGF was -7.8, -5.4, -3.9 for the FG-2216 groups and 5.2 pg/mL for placebo. There were 45 SAEs reported in 25 subjects, 2 assessed as possibly related to FG-2216 including 1 death due to fulminant hepatitis. There were no increases in BP observed with any dose level or correlated with increasing Hb levels; there was no increased use of BP medications over time. Oral administration of FG-2216 in ESA-naïve patients with CKD not on dialysis was well tolerated and may effectively treat anemia as evidenced by increasing erythropoietic effect with modest EPO elevation, no VEGF increase and no adverse BP changes.
© 2008 NKF
HIF Prolyl Hydroxylase Inhibitors Increase Erythropoiesis Without Promotion of Tumor Progression in the Presence or Absence of Concomitant Chemotherapy
Anemia is a frequent complication of cancer, in patients experiencing anemia of chronic disease (ACD; also Anemia of Cancer, AoC), and anemia associated with cancer chemotherapy (Chemotherapy Induced Anemia, CIA). FG-4592 and FG-2216, novel orally active hypoxia-inducible factor prolyl hydroxylase (HIF-PH) inhibitors, stabilize HIF and stimulate transcriptional activation of beneficial erythropoietic genes resulting in dose-dependent increases in EPO secretion, enhanced mobilization and utilization of free iron, and suppression of inflammatory interference on erythropoiesis. FG-4592 or FG-2216 treatment effects were monitored in the MDA-MB-435 and 786-O xenograft tumor models. FG-4592 and FG-2216 treatments resulted in increases in circulating EPO, RBC, Hb and Hct, indicating that these compounds stimulated erythropoiesis. No stimulation of tumor progression by either FG-4592 or FG-2216 treatment was observed. FG-4592 and FG-2216 did not affect circulating VEGF or tumor vascular density. In the Caki1 renal xenograft model, FG-2216 treatment was combined with chemotherapy treatment. Chemotherapy efficacy was not adversely affected by co-administration of FG-2216. Together, these data support the use of HIF prolyl hydroxylase inhibitors in treatment of anemia in cancer patients.
The Prolylhydroxylase Inhibitor FG2216 Stimulates EPO Production in Nephric and Anephric Dialysis Patients - Evidence for an Underutilized Production Capacity in Liver and Kidneys
Inadequate serum EPO levels are considered the main cause of renal anemia, and the reasons are poorly understood. It is assumed that the EPO-producing peritubular fibroblasts lose endocrine function during the course of kidney injury and that the liver does not sufficiently compensate. Progress in understanding the molecular control of oxygen-dependent EPO production revealed an important role of hypoxia-inducible transcription factors (HIF) in the oxygen-dependent regulation of EPO. HIF degradation occurs through oxygen-dependent hydroxylation of prolyl residues by prolyl hydroxylases (PHD). To test the ability of fibrotic kidneys to produce EPO and the ability of other organs to make EPO in anephric patients, we used FG-2216, a novel PHD inhibitor, which stabilizes HIF in an oxygen-independent fashion. A single dose Phase 1 study was performed in 12 hemodialysis (HD) patients, 6 of whom were anephric, and in 6 non-anemic volunteers. Recombinant EPO therapy was discontinued one week before dosing in all HD patients. Day 1 hemoglobin levels were 13.8 ±1.9, 14.2 ±1.2, and 15.5 ±0.6 g/dL for nephrics, anephrics and controls, respectively. FG-2216 (20mg/kg p.o.) resulted in a rise in EPO levels in all study subjects (Tmax was comparable for all groups). Median EPO levels increased from 17.4 to 240.6 (nephric), from 5.7 to 42.55 (anephric), and from 6.7 to 47.1 mU/ml in controls. Mean plasma half life of FG-2216 was similar in nephric and anephric patients, but twice as long as in controls. Dialysis clearance of FG-2216 was less than 2%, suggesting that timing of the dose of FG-2216 could be independent from HD. In conclusion, endogenous EPO production can be stimulated by pharmacological manipulation of the HIF system. The response in nephric and anephric patients demonstrates that diseased kidneys retain a significant production capacity for EPO and that liver production of EPO may also be useful in a clinical setting.
© 2007 ASN
Preliminary Results from a Randomized, Single-Blind, Placebo-Controlled Trial of FG-4592, a Novel Hypoxia Inducible Factor Prolyl Hydroxylase Inhibitor, in Subjects with CKD Anemia
Background: FG-4592 is an oral inhibitor of hypoxia inducible factor (HIF) prolyl hydroxylase in clinical development for the treatment of anemia. Stabilization of HIF, a cytosolic transcription factor, by FG-4592 leads to activation of the genes associated with erythropoiesis, including EPO and enzymes involved in iron metabolism.
Study Design: A randomized, single-blind, placebo-controlled, 4-week study of oral doses of FG-4592 (1 to 4 mg/kg) administered 2 or 3 times weekly. The objectives of this study were to characterize the safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of FG-4592 in subjects with CKD anemia. Thirty subjects were to be enrolled into 2 cohorts at each dose: Pharmacokinetic (PK, n=12, Hb<13 g/dL) and Treatment (TX, n=18, Hb<11 g/dL). All subjects were monitored for safety, including vital signs, clinical labs, and adverse events during treatment and for 4 weeks thereafter.
Results: Data from the 1 mg/kg cohort (21 active- and 8 placebo-treated) demonstrate that FG-4592 doses up to 120 mg are considered generally well-tolerated, with treatment emergent AEs attributed to study drug reported from only 2 subjects (1 active and 1 placebo). No clinically significant changes in serum chemistry or vital signs were observed in any subjects. Multiple samples from the PK subjects were collected and used to measure FG-4592 and circulating endogenous EPO levels. FG-4592 was rapidly absorbed with a mean Tmax= 1.8 hrs, mean Cmax= 5.9 µg/mL, and mean half-life= 11 hrs, which were comparable after the first and last dose, and similar to those previously observed in healthy subjects. Median peak plasma EPO levels of 115 mIU/mL occurred 8-12 hrs post-dose, and were comparable for first and last dose. In the TX group, 5 of 13 subjects (38%) treated with FG-4592 had increases in Hb=1 g/dL during the 4-week treatment period, and maintained that increase for 2-4 wks after stopping treatment. None of 4 placebo subjects had a similar Hb increase. In conclusion, 4 weeks of treatment with FG-4592 was well-tolerated and produced significant Hb increases in some subjects with CKD anemia.
© 2007 ASN
HIF-PH Inhibitor, FG-4592, Treats Anemia and Prevents Elevation of SBP in Uremic Rats
Inhibitors of hypoxia-inducible factor (HIF) prolyl hydroxylase (PH) stabilize HIF and increase transcription of endogenous erythropoietin and other critical genes in the erythropoietic pathway. FG-4592 is a novel and orally bioavailable HIF-PH inhibitor in clinical development to treat anemia associated with chronic kidney disease (CKD).
Oral administration of FG-4592 significantly increased reticulocytes, hematocrit, and hemoglobin (Hb) in normal rats. The effects of FG-4592 on anemia and hypertension associated with CKD were tested in a rat remnant kidney model in which one whole kidney and 2/3 of the other were surgically removed. Rats were prospectively randomized to receive vehicle or FG-4592 for 2 wks starting 5 wks after the 5/6th nephrectomy (Nx) or sham surgery at which time severe uremia and anemia was evident.
Treatment with FG-4592 significantly improved anemia in a dose-dependent fashion, without significant effects on body weight and uremia as reflected by similarly elevated serum creatinine levels. In addition, treatment of Nx rats with FG-4592 prevented the concomitant increase in systolic blood pressure (SBP) observed in vehicle-treated Nx rats, which exhibited significantly higher mean SBP (133.7 ± 3.2 mmHg) compared to all other groups (p<0.001). Mean SBP in Nx rats treated with 20 mg/kg FG-4592 (95.6 ± 9.8 mmHg) and with 40 mg/kg FG-4592 (84.1 ± 7.0 mmHg) was not significantly different from sham-operated controls (90.2 ± 5.5 mmHg).
In conclusion, FG-4592 increased reticulocyte counts and raised hematocrit and Hb in a dose-dependent and schedule-related fashion in normal rats and was effective in stimulating red blood cell production in animals with anemia due to severely reduced kidney mass and uremia. In addition, because hypertension generally develops from 3 - 6 wks in this model, the observation that mean SBP was lower in Nx rats treated with FG-4592 compared to vehicle-treated controls suggests that FG-4592 ameliorated hypertension. These non-clinical results provide a strong basis for clinical application of this HIF-PH inhibitor to treating anemia associated with CKD.
© 2007 ASN
HIF-Prolyl Hydroxylase Inhibition Results in Endogenous Erythropoietin Induction, Erythrocytosis, and Modest Fetal Hemoglobin Expression in Rhesus Macaques
Abstract: The hypoxia-inducible factor (HIF) pathway is crucial in mitigating the deleterious effects of oxygen deprivation. HIF-a is an essential component of the oxygen-sensing mechanisms and under normoxic conditions is targeted for degradation via hydroxylation by HIF–prolyl hydroxylases. Several HIF–prolyl hydroxylase inhibitors (PHIs) induced erythropoietin (epo) expression in vitro and in mice, with peak epo expression ranging from 5.6- to 207-fold above control animals. Furthermore, several PHIsinduced fetal hemoglobin (HbF) expression in primary human erythroid cells in vitro, as determined by flow cytometry. One PHI, FG-2216, was further tested in a nonhuman primate model without and withchronic phlebotomy. FG-2216 was orally bioavailable and induced significant and reversible Epo induction in vivo (82- to 309-fold at 60 mg/kg). Chronic oral dosing in male rhesus macaques was well tolerated, significantly increased erythropoiesis, and prevented anemia induced by weekly phlebotomy. Furthermore, modest increases in HbF-containing red cells and reticulocytes were demonstrated by flow cytometry, though significant increases in HbF were not demonstrated by high-pressure liquid chromatography (HPLC). HIF PHIs represent a novel class of molecules with broad potential clinical application for congenital and acquired anemias.
Introduction: Injectable recombinant erythropoietin (rHuEPO) is used in many clinical disorders of anemia. Prior attempts at EPO induction by gene transfer strategies were problematic rendering them less attractive. Hypoxia inducible factor (HIF) is important in mediating EPO expression and erythropoiesis. Under normoxic conditions, HIF-alpha protein is usually undetectable due to rapid inactivation by oxygen-dependent HIF prolyl hydroxylases (HIF-PH). In hypoxic conditions or with HIF-PH inhibitors, HIF-alpha protein levels are increased and maintained due to reduced activity of HIF-PH. Upon stabilization HIF-alpha is transported to the nucleus to increase the expression of EPO, aminolevulinic acid synthase (ALAS), iron-transport proteins, nitric oxide synthase, and others. FG-2216 is an orally active HIF-PH inhibitor that induces EPO expression and erythropoiesis in vitro and in vivo, and improves iron bioavaliability by increasing iron mobilization from intestinal and macrophage stores and by decreasing hepcidin expression. Here we report on the pharmacokinetics and pharmacodynamics of FG-2216 to induce EPO expression and erythropoiesis in non-human primates with chronic intermittent oral dosing.
Methods: FG-2216 was obtained from FibroGen, Inc. FG-2216 was given to rhesus macaques (4-7 yrs old) by oral gavage. Drug and EPO levels were measured at 0,2,4,8,10,24,48, and 72 hours following a single dose of 40 or 60mg/kg. 40 or 60mg/kg of drug was then given twice a week to 5 male animals for 3-6 months. After 6-8 weeks of twice weekly dosing at 60mg/kg, weekly 15-20% weight per volume phlebotomy was introduced for 6-8 weeks to simulate chronic anemia while drug dosing was continued. Weekly weights, complete blood counts, and metabolic profiles were obtained to assess efficacy and safety. Two male animals received vehicle control.
Results: FG-2216 is rapidly absorbed and has excellent oral bioavailability, with peak plasma drug concentration between 1-2 hours and a half-life of 7-8 hours. The pharmacokinetic curves were dose-proportional and predictable. After 6-8 weeks of 2 doses per week, all animals tolerated the drug without significant side effects, and animal weights were stable with no changes in serum electrolytes, hepatic, or renal parameters. Peak plasma EPO increased from a baseline of <20 to 132 and 1950 mIU/ml after 40 and 60 mg/kg doses, respectively. There were transient 2-3 fold increases in reticulocytes. All treated animals demonstrated an increase in total hemoglobin: 11.95 +/- 0.33 g/dL (baseline), 13.17 +/- 0.32 (40mg/kg), and 14.58 +/- 0.64 (60mg/kg). Absolute hemoglobin increases ranged from 0.66 to 1.8 (40mg/kg) and 0.97 to 4.04 (60mg/kg) g/dL. When phlebotomy was introduced with continued FG-2216 dosing, treated animals maintained their hemoglobin, 14.03 +/- 0.06 g/dL, while control animals had their hemoglobin reduced to 11.54 +/- 0.09g/dL.
Conclusion: FG-2216 is an orally bioavailable HIF-PH inhibitor that is well tolerated and a potent inducer of EPO, with greater than 50-fold increases in EPO above baseline with a 60mg/kg dose. When administered orally twice a week, FG-2216 significantly increases total hemoglobin in normal rhesus macaques. Under the stress of weekly phlebotomy, FG-2216 maintained the hemoglobin of treated animals in the normal range, averaging 2.5 g/dL higher than animals receiving placebo. These results have broad clinical implications.
This research was originally published in Blood. Hsieh et al. Blood. 2007;110:2140-7. © The American Society of Hematology.
HIF Prolyl Hydroxylase Inhibitors Potentiate EPO Signaling, Enhance Erythropoiesis and Overcome Inhibitory Effects of Pro-Inflammatory Cytokines in Anemia of Chronic Disease
Anemia of chronic disease (ACD) caused by chronic inflammation leads to blunted EPO responsiveness and reduced EPO production, as well as iron sequestration resulting in limited iron bioavailability for hemoglobin (Hb) synthesis. FibroGen has developed a series of selective HIF prolyl hydroxylase inhibitors (PHI) to treat anemia, including the clinical candidates FG-2216 and FG-4592, which induce EPO production and elevate Hb in preclinical models of ACD. Both FG-2216 and FG-4592 overcome suppression of EPO production by TNFα and IL-1β and enhance expression of genes that increase intestinal absorption and recycling to erythroid progenitors, thus enhancing iron bioavailability. To assess whether PHI have direct effects on EPO production and responsiveness in the bone marrow, FG-4592 was examined for its capacity to potentiate EPO signaling and drive erythroid maturation ex vivo in the absence and presence of TNFα and IL-1β. FG-4592 directly stimulated EPO and EPO receptor expression and enhanced maturation of erythroid progenitors in the absence and presence of exogenously added rHuEPO. Furthermore, FG-4592 mitigates the suppression of erythroid maturation in the presence of either TNFα or IL-1β. The results suggest PHI induce erythropoiesis by increasing EPO secretion from both renal and hepatic sources and also by autocrine and paracrine EPO production within the marrow microenvironment. Coupled with increased EPO responsiveness of the erythroid progenitors, the findings support our data whereby PHI induce erythropoiesis with only modest increases in circulating endogenous EPO, and are consistent with the enhanced EPO sensitivity of erythroid progenitors harboring the Chuvash mutation. Thus, PHI possesses therapeutic potential to treat ACD and other anemias associated with acute or chronic inflammation.
FG-2216 Increases Hemoglobin Concentration in Anemic Patients with Chronic Kidney Disease
Two Phase 2a dose escalation studies are evaluating safety and efficacy of FG-2216, an oral drug that stabilizes hypoxia-inducible factor (HIF) and induces the HIF2 mediated natural erythropoietic cascade. One study includes rHuEPO naïve CKD patients and the other CKD patients pretreated with rHuEPO (8+ weeks of continuous therapy). FG-2216 is dosed TIW for 4 weeks with 2 weeks of followup. Dose escalation was modeled from chronic intermittent hypoxia studies in animal and man. The low dose (6 mg/kg) was expected to be non-efficacious, and the higher doses, 15-20 mg/kg, to produce significant Hb responses. By day 42, 6 mg/kg of FG-2216 produced a statistically significant mean increase in Hb of 1.3 g/dL in EPO-naïve patients (n=5). Placebo patients (n=3) experienced a mean decrease in Hb from baseline of 0.35 g/dL. In rHuEPO pretreated patients, 6 mg/kg FG-2216 given after rHuEPO withdrawal increased Hb by 1.5 g/dL in one patient while another maintained baseline levels. Four patients showed Hb decreases to varying degrees, but the mean decrease in the FG-2216 group (-0.9 g/dL, n=6) was less than in the placebo group (-1.5 g/dL, n=3). The first patient (GFR=8) receiving 20 mg/kg FG-2216 after rHuEPO withdrawal experienced an increase of 1.8 g/dL Hb within 14 days, resulting in a decision to lower the dose in this cohort. The results show that CKD patients can produce EPO despite fibrosis, dialysis stage patients respond, and FG-2216 provides clinical benefit in anemia of CKD. The rapid Hb response induced by FG-2216 cannot be attributed to EPO alone and is likely due to HIF mediated iron mobilization. Moreover, lower circulating EPO levels required to induce erythropoiesis following FG-2216 (<10% of rHuEPO) are expected to reduce the known thrombotic risk associated with high EPO concentrations and may permit correction of Hb to normal levels in CKD and a more rapid amelioration of anemia in cancer.
© 2005 ASN
Induction of Erythropoiesis and Iron Utilization by the HIF Prolyl Hydroxylase Inhibitor FG-4592
FibroGen is developing HIF prolyl hydroxylase inhibitors (PHI) that selectively induce the expression of genes that mediate erythropoiesis, including erythropoietin (EPO) and genes that increase iron utilization by erythroid progenitors, and also overcome the suppression of erythropoiesis by chronic inflammation. FG-2216 is the first therapeutic PHI that has been shown to elevate circulating endogenous EPO (eEPO) and to increase hemoglobin (Hb) in healthy subjects and anemic patients with chronic kidney disease (CKD). FG-4592 represents one of several next generation PHI that has been optimized for multiple pharmacokinetic and pharmacodynamic parameters related to erythropoiesis, including selective inhibition of HIF prolyl and asparaginyl hydroxylases, potency, iron utilization, and ADME. FG-4592 stabilizes HIF-2 and induces EPO production in Hep3B cells, and increases circulating eEPO after a single i.v. or oral dose in rodents. FG-4592 elevates Hb in rodents when administered on an intermittent or daily dosing regimen, leading to significant dose-dependent elevation in Hb in less than 7 days. The erythropoietic potency of FG-4592 in rodents translates to efficacy in non-human primates with no adverse effects, with dose-dependent increases in circulating eEPO that exceed 1000 mIU/mL. Repeated daily dosing with FG-4592 in non-human primates for up to 28 days led to dramatic increases in circulating reticulocytes and RBC, with associated elevation of Hb of greater than 5 g/dl and no dose-limiting toxicities or adverse effects. The dose dependent increase in Hb was accompanied by increases in mean cell volume and mean cell hemoglobin, demonstrating that FG-4592 induces complete erythropoiesis by coordinately elevating eEPO and increasing iron utilization for newly emerging erythrocytes. Other hematopoietic lineages were not affected by repeated oral dosing with FG-4592. Together the data show that FG-4592 is a selective and potent oral erythropoietic agent that should be efficacious for treating anemia associated with CKD and chronic inflammation, with clinical studies scheduled to begin in 2005.
© 2005 ASN
FG-2216 Corrects Anemia and Improves Iron Utilization in a Rat Model of Anemia of Chronic Disease: Comparison to Darbepoetin
Anemia of Chronic Disease (ACD) is characterized by a blunted erythropoietin response, decreased iron utilization and impaired response of the bone marrow. Patients with impaired iron utilization are hyporesponsive to rHuEPO therapies. FG-2216 stimulates eEPO production, improves iron absorption and utilization and overcomes the inflammatory suppression of erythropoiesis. The objective of this study was to compare FG-2216 to darbepoetin in a rat model of ACD. Administration of peptidoglycan-polysaccharide polymers (PGPS) to female Lewis rats causes inflammation and disrupted iron utilization leading to a prolonged microcytic hypochromic anemia. Four weeks prior to treatment, rats were injected with PGPS to induce arthritis and anemia (Hb < 10 g/dL). Normal and anemic animals were treated with vehicle, FG-2216, darbepoetin or IV iron. After four weeks of treatment, FG-2216 and darbepoetin produced dramatic and equivalent increases in both Hb and Hct in normal animals. However, in PGPS challenged animals, FG-2216 increased Hb and Hct to levels comparable to normal controls, while darbepoetin had no effect. Intravenous iron had no effect in either normal or anemic animals. In addition, FG-2216 improved the microcytosis and increased MCH, demonstrating improved iron utilization. Darbepoetin did not improve MCV or MCH in anemic animals and reduced MCV and MCH in the normal group. Transcript analysis of duodenum and liver demonstrated increased expression of iron transport proteins and decreased expression of hepcidin (a hormone involved in negative regulation of circulating iron levels) in the FG-2216 treated group compared to all other groups. FG-2216 reversed the anemia associated with PGPS-induced arthritis in rats, while a functionally equivalent dose of darbepoetin was unable to stimulate erythropoiesis. By coordinating iron availability with erythropoietic stimulation, FG-2216 may be better able to overcome inflammatory suppression of erythropoiesis and thereby provide an important benefit to patients needing treatment for erythropoietin-resistant anemia and ACD.
© 2005 ASN
Novel and Beneficial Pharmacodynamic Properties of Endogenous EPO and 'Complete Erythropoiesis' Induced by Selective HIF Prolyl Hydroxylase Inhibitors
FibroGen is developing hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors (PHI) that stabilize HIF and stimulate 'complete erythropoiesis', as shown in preclinical studies where PHI induce endogenous EPO (eEPO), improve iron absorption and utilization, overcome the inflammatory suppression of erythropoiesis, and potently drive RBC maturation. Recent human data show that oral doses of the PHI FG-2216 are erythropoietic in patients with chronic kidney disease, at doses that induce only modest increases of eEPO in healthy subjects. The small increases in eEPO induced by erythropoietic doses of FG-2216 are consistent with preclinical studies in rodents and monkeys, where PHI elevate Hb at doses that increase eEPO by only 2-fold, and previous published studies in humans showing that repeated intermittent hypoxia, high altitude conditioning or standard phlebotomy are erythropoietic but lead to only modest increases in eEPO. Thus, stimulation of HIF-dependent erythropoiesis by physiological stimuli or PHI in several species requires only small increases in eEPO, which are 30 to 300-fold less than peak or sustained levels of rHuEPO associated with therapeutic doses of current recombinant therapeutics. The enhanced potency of PHI-induced erythropoiesis as compared to rHuEPO entails several novel and beneficial mechanisms, including coordinate regulation of eEPO and genes that improve iron utilization, eEPO signaling and responsiveness, and potential autocrine and paracrine EPO production within the marrow. Erythropoietic doses of PHI that modestly elevate eEPO could reduce the thrombotic risk caused by therapeutic doses of rHuEPO where supra-physiologic levels of rHuEPO lead to direct activation of the pro-thrombotic activities of platelets and the endothelium. PHI therapy may ultimately lead to anemia correction to target Hb levels that exceed current clinical practice guidelines and are closer to normal levels.
© 2005 ASN
FG-2216: Tumor Progression Studies and Correction of Anemia of Chronic Disease in Xenograft Models
Anemia is a frequent complication of cancer in patients experiencing anemia of chronic disease (also, Anemia of Cancer, AoC) and anemia associated with cancer chemotherapy (Chemotherapy Induced Anemia, CIA). Patient response rates to rHuEpo therapy have been reported in the 50% range in AoC and the 60% range in CIA. Factors adversely affecting erythropoietic response in AoC are common to anemia of chronic disease (ACD) and include inflammatory suppression of endogenous EPO production and erythroid progenitors, increased iron retention, decreased iron absorption, and decreased iron bioavailability in the bone marrow. FG-2216, a novel orally active inhibitor of HIF prolyl hydroxylases designed to activate HIF2, up-regulates EPO and iron mobilization genes, down-regulates hepcidin, and overcomes inflammatory suppression of erythropoiesis. FG-2216 has also been shown to be efficacious in treating anemic CKD patients. Here, FG-2216 was administered to animals for periods of 14 to 70 days in xenograft models of NSCLC. Key hematology parameters (Hct, RBC, Hb) were suppressed in vehicle treated tumor-bearing animals vs. controls, indicating the induction of ACD by xenograft tumors. In tumor-bearing animals, FG-2216 treatment resulted in increases in circulating EPO, RBC, Hb, and Hct, indicating that FG-2216 stimulated erythropoiesis by overcoming functional iron deficiency and inflammatory suppression. As measured by final excised tumor weights, no stimulation of tumor progression by FG-2216 treatment was observed. Similar conclusions were obtained by caliper measurement of tumor volumes in subcutaneous tumor studies. In these studies, FG-2216 treatment was associated with a small to moderate inhibition of tumor development. In an orthotopic model of lung tumor metastasis, FG-2216 treatment groups had lowered incidence of tumor-induced morbidity and metastasis. FG-2216 treatment was well tolerated, with no reduction in mean body weights or increase in frequency of animal deaths. Together, this data supports use of FG-2216 in AoC, CIA, and various other forms of EPO-hyporesponsive ACD, such as uremic CKD patients.
© 2005 ASN
Induction of Renal and Extra-Renal Erythropoietin Production by Orally Bioavailable HIF Prolyl Hydroxylase Inhibitors
FibroGen has developed a series of HIF prolyl hydroxylase inhibitors (PHI) that selectively induce the expression of genes that mediate erythropoiesis, including erythropoietin (EPO). Although EPO production in adults occurs primarily within the kidney, EPO is also produced constitutively and inducibly at extra-renal sites, particularly liver and brain. We determined the contribution of kidney and liver to total PHI-induced EPO levels in vivo by measurement of circulating endogenous EPO in sham operated mice and in mice receiving bilateral (total) nephrectomy (BNx). EPO mRNA expression was determined by qPCR analysis of kidney and liver derived EPO mRNA, and circulating endogenous EPO was determined in plasma by ELISA after oral administration of PHI. The results show that distinct PHI exhibited differential capacity to induce EPO after BNx in mice, suggesting that PHI-induced elevation of circulating endogenous EPO is due to differential production from renal and extra-renal sources, with different contributions from kidney and liver depending on the PHI employed. The relative proportions of EPO induced by PHI from renal and hepatic sources were generally independent of PHI dose employed. EPO expression was also examined in neural tissues by qPCR after intravenous administration of PHI, with PHI inducing both circulating and neural EPO or selective induction only in the circulation or brain. Compounds that selectively induce EPO expression in the brain may advance the development of novel neuroprotective therapeutics. The capacity of distinct PHI to differentially induce EPO from renal and hepatic tissue provides optimal flexibility in the development of therapeutic PHI to treat patients with anemia. Localized renal EPO production may maximally exploit the renoprotective properties of EPO that could slow progression to ESRD and provide cytoprotection against acute renal failure.
© 2005 ASN
Novel Prolyl Hydroxylase Inhibitors Overcome Inflammatory Cytokine-Mediated Suppression of EPO Secretion
Anemia of chronic diseases (ACD) such as rheumatoid arthritis and certain cancers is caused in part by elevated levels of circulating pro-inflammatory cytokines that suppress erythropoiesis. Pro-inflammatory cytokines such as TNF-alpha, IL-1beta and IL-6 directly inhibit erythropoietin (EPO) secretion, and further exacerbate anemia by reducing iron absorption and utilization that is necessary for hemoglobin synthesis. Hypoxia inducible factor-a (HIF-alpha) is a transcription factor that acts as the body's main sensor to low oxygen tension, and HIF responds to anemia (i.e. hypoxia) to increase erythropoiesis by elevating transcription of EPO and iron processing genes. HIF-alpha function is regulated by a family of conserved HIF prolyl hydroxylases (HIF-PH), which hydroxylate HIF-alpha and induce its degradation under normoxic conditions, but which are inhibited under hypoxia leading to HIF-alpha stabilization and activation. We developed a novel series of HIF prolyl hydroxylase inhibitors (PHI) that reversibly block HIF-PH function under normoxic conditions and which induce HIF-alpha activation and elevated levels of EPO secretion in PHI-treated cells. As expected, TNF-alpha and IL-1beta suppressed EPO secretion in a dose dependent fashion, but simultaneous or posttreatment of cells with PHI in the presence of either TNF-alpha or IL-1beta overcomes cytokine-mediated suppression of EPO secretion, with EPO induced to even higher levels than in the absence of TNF-alpha and IL-1beta. While IL-6 alone had little effect on EPO secretion, cells co-stimulated with IL-6 and PHI exhibit a synergistic increase in EPO secretion, to much higher levels than seen with PHI alone. The data indicate that PHI-mediated stabilization of HIF prevents the suppressive effects of TNF-alpha and IL-1beta on EPO production, and synergizes with IL-6 to induce even higher levels of EPO secretion. This suggests that prolyl hydroxylase inhibition may provide novel benefits in the treatment of anemias associated with chronic disease, for which available anemia therapeutics are not approved.
© 2004 ASN
Effect of FG-2216 on Anemia and Iron Transport in a Rat Model of Anemia of Chronic Disease
Anemia of chronic disease (ACD) is associated with various inflammatory conditions, including arthritis and neoplastic disease. This anemia is characterized by a relative reduction in erythropoietin production in response to decreased hematocrit, alterations in iron metabolism, reduced red cell lifespan and impaired response of the bone marrow. The objective of this study series was to determine the effect of a novel prolyl hydroxylase inhibitor, FG-2216 in a rat model of anemia of chronic disease. Female Lewis rats (approx. 160 gm) were injected IP with peptidoglycan-polysaccharide polymers (PGPS) @ 15 micrograms rhamnose/gm body weight to induce arthritis and anemia. Arthritis and anemia were allowed to develop prior to treatment with FG-2216 or vehicle. Each study contained one non-challenged control group. In one study, an additional non-challenged group received FG-2216. Studies ran from 2-6 weeks and included various intermittent dosing strategies and doses. Blood samples were taken for CBC analysis, serum iron and TIBC. Tissue samples were taken for genomic analysis. FG-2216 induced dose-dependent increases in hematocrit, hemoglobin and red cell count. After two weeks of intermittent FG-2216 treatment at the maximum dose tested, HCT, Hgb and RBC levels in PGPS challenged animals were comparable to those of non-challenged control animals. In addition, FG-2216 alleviated the microcytosis associated with the anemia and increased the mean cell hemoglobin. Iron status was improved in animals that were treated with FG-2216. Serum iron and transferrin saturation were increased in PGPS challenged animals following FG-2216 treatment. Genomic analysis of duodenal samples revealed treatment with FG-2216 induced transcription of Nramp2 and dcytb, genes involved in iron transport in the gut. Treatment with FG-2216 successfully reversed the anemia associated with PGPS-induced arthritis in rats. In addition, treatment with FG-2216 improved microcytosis and iron status. By increasing iron transport, FG-2216 facilitates complete erythropoiesis and may be useful in treating erythropoietin-resistant anemia and ACD.
© 2004 ASN
Upregulation of Endogenous EPO in Healthy Subjects by Inhibition of HIF-PH
High-altitude hypoxia stimulates erythropoiesis in anemic hemodialysis patients. Similarly, intermittent exposure to hypobaric hypoxia elevates EPO and stimulates erythropoiesis in normal subjects. HIF is a transcription factor that mediates the body's response to hypoxia. The stability and activity of HIF are regulated by HIF Prolyl Hydroxylase (HIF-PH). Inhibition of HIF-PH leads to rapid HIF stabilization and upregulation of erythropoietic genes. A FibroGen HIF-PH inhibitor, FG-2216, is an orally active small molecule with appropriate pharmacokinetic and pharmacodynamic activity for testing in humans. In animal studies, inhibitors of HIF-PH induce erythropoietic changes qualitatively similar to the effects of intermittent exposure to high altitude.
Phase 1 studies were initiated in healthy male subjects to determine the safety, tolerability, pharmacokinetics, and biologic activity of FG-2216 dosed orally (0.3 to 20 mg/kg). Serum levels of FG-2216 increased in a dose-dependent fashion; its elimination was characterized by a half-life of ~14 hr. Doses of 6 mg/kg and higher were associated with dose-dependent elevation in serum EPO levels. FG-2216 was subsequently administered using a repeated intermittent schedule, 2 or 3 times weekly. Doses of 10 and 20 mg/kg or placebo were given up to three weeks. Increased EPO was observed after each dose of FG-2216. There was no decrement in EPO response to subsequent doses, indicating a resetting of the EPO response during the dosing interval. Elevation in reticulocytes and soluble transferrin receptors was accompanied by a modest increase in hematocrit and hemoglobin above normal values at baseline. A total of 54 subjects received FG-2216, which was well tolerated. There were no serious adverse events or dose-limiting toxicities. Adverse events possibly related to FG-2216 were mild and subsided with continued administration. The data provide first proof of concept for upregulation of endogenous EPO by a specific HIF-PH inhibitor resulting in erythropoietic responses in humans.
© 2004 ASN
Stimulation of Erythropoiesis and Treatment of Anemia in Rodents by Oral Administration of FG-2216, a Novel HIF-Prolyl Hydroxylase Inhibitor
Hypoxia has long been known to induce endogenous erythropoietin (EPO), which stimulates red blood cell production, resulting in enhanced oxygen delivery to tissues. Hypoxia-inducible factor (HIF) is a master transcriptional switch in regulating the hypoxic response in animals. Inhibitors of HIF prolyl hydroxylase (HIF-PH) stabilize HIF and mimic certain aspects of the hypoxic response, activating the transcription of a number of erythropoietic genes. FG-2216, a novel HIF-PH inhibitor that stabilizes HIF protein and stimulates EPO production is highly bioavailable in vivo after oral administration. Several studies in normal mice and rats show that FG-2216 elevates serum concentration of endogenous EPO, increases reticulocyte counts and raises hematocrit and hemoglobin in a dose-dependent and schedule-related fashion. Studies also demonstrate that FG-2216 prevents anemia associated with acute renal failure in a rat model of renal ischemia-reperfusion injury; corrects anemia associated with end-stage renal failure in a rat remnant kidney model; improves anemia induced in rats by the chemotherapeutic agent cisplatin; and accelerates recovery from phlebotomy-induced anemia in mice. Our results suggest a number of important clinical applications for this HIF-PH inhibitor to induce full erythropoiesis in treating anemic conditions ranging from kidney failure to anemia of chronic disease to cancer chemotherapy to surgical blood loss.
© 2004 ASN
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Donor Pre-treatment with a HIF Prolyl Hydroxylase Inhibitor Improves Function and Increases Long-Term Graft Survival in an Allogenic Rat Transplant Model
Besides immunological aspects, the long-term survival of a renal allograft depends on the initial injury caused by the sequence of cold ischemia, warm ischemia and reperfusion and is thus already determined at the time of transplantation. Hypoxia-inducible transcription factors (HIF) are essential for adaptation to low oxygen. Normoxic inactivation of HIF is regulated by oxygen-dependent hydroxylation of specific prolyl-residues of HIF by prolyl-hydroxylases (PHD). Pharmacological inhibition of the PHD results in HIF accumulation with subsequent activation of a number of nephroprotective genes.
We examined the effect of donor treatment with a specific inhibitor of the PHD (FG-4497) on graft-function in a rat model of allogenic kidney transplantation (KTx). The Fisher-Lewis rat model of KTx was used. Isogenic transplantations served as controls. Orthotopic transplantation of the left donor-kidney was performed after 24h of cold storage. The right kidney was removed at the time of KTx (acute) or at day 10 (chronic). 6h prior to kidney explantation, donor animals were treated with a single dose of FG-4497 (40mg/kg i.v.) or vehicle (Veh) Recipients were followed up for 10 days (acute, n=6-8) or 24 weeks (chronic, n=13-14).
Donor-preconditioning with FG-4497 resulted in HIF accumulation and induction of HIF target genes, which persisted beyond the period of cold storage. It reduced acute renal injury (Serum creatinine FG-4497: 0.66±0.20 vs Veh 1.49±1.36; p<0.05) and improved histomorphology at 10 days. Donor-preconditioning improved long term survival of recipient animals (mortality after 24 wks: 7/13 Veh vs. 3/14 FG-4497 and 0/13 in the isogenic control group; p<0.05 FG-4497 vs. Veh).
In conclusion, pretreatment of organ donors with FG-4497 improves short- and long-term outcome after prolonged cold storage and subsequent allogeneic KTx. These findings may have significant clinical implications.
© 2007 ASN
Preconditional Activation of HIF Ameliorates Ischemic Acute Renal Failure
Activation of hypoxia-inducible factor(HIF),an α/β heterodimer,is essential in adaptation to low oxygen. Normoxic inactivation of HIFα is regulated by oxygen dependent hydroxylation of proline residues by prolyl hydroxylases (PHDs).Hypoxia or pharmacological inhibition of PHDs results in HIF accumulation with subsequent activation of renoprotective genes (EPO,HO-1).Therefore,preconditional inhibition of the PHDs may protect the kidney against hypoxic injury.To test this hypothesis we induced ischemic acute renal failure (iARF) in rats (right nephrectomy,clamping of the left renal artery for 40min). To stabilize HIF animals were pretreated with carbon monoxide 0.1%(CO) or a specific PHD-inhibitor (PHD-I). Vehicle treated(Veh),untreated(UnT) and sham operated(sham) rats served as controls. Serum creatinine (S-crea)was determined at 0h,24h and 72h.After 72h left kidney was processed for examination. Renal damage was quantified in a blinded analysis. Accumulation of HIFαwas examined by immunohistochemistry. Expression of HIF targets (e.g.EPO, HO-1) was analyzed by RNAse protection assay/ immunohistochemistry. CO and PHD-I led to renal HIF-accumulation and upregulation of EPO and HO-1.At 24h and 72h S-crea was significantly lower in the CO and PHD-I group (Table). Morphological changes were less severe in the CO and PHD-I group. These data provide a proof of principle that preconditional activation of the HIF pathway by CO or PHD-I protects against iARF. Inhibiting the HIF-hydroxylases to improve outcomes under ischemic conditions has considerable clinical perspectives.
S-creatinine (mg/dl) Group 0h 24h 72h Sham 0.3±0.02 0.49±0.05 0.48±0.04 UnT 0.37±0.16 3.58±0.62 2.28±0.7 CO 0.32±0.1 1.85±1.01# 1.08±0.5# Veh 0.39±0.1 3.76±1.05 2.57±1 PHD-I 0.41±0.2 2.43±0.54# 1.02±0.3# #p<0.05 vs. UnT /Veh
© 2005 ASN
Inhibition of Collagen Synthesis with Prolyl 4-Hydroxylase Inhibitor Improves Left Ventricular Function and Alters the Pattern of Left Ventricular Dilatation After Myocardial Infarction
Background- Left ventricular (LV) remodeling after myocardial infarction (MI) is associated with fibrosis, dilatation, and dysfunction. We postulated that prevention of fibrosis after MI with a prolyl 4-hydroxylase inhibitor (P4HI) would preserve LV function and attenuate LV enlargement. Methods and Results- Adult female rats (200 to 250 g) had experimental MI and were then randomized to treatment with P4HI (MI-FG041, n=29) or vehicle (MI-control, n=29) 48 hours after MI for 4 weeks in 2 phases. Echocardiograms were performed weekly with a 15-MHz linear transducer, and at 4 weeks, collagen isoform determinations and in vivo hemodynamics were performed. At randomization, the infarct size and LV function and size were similar in MI-FG041 and MI-control but significantly different from shams (n=9). At week 4, the LV function in MI-FG041 was significantly better than in MI-controls (fractional shortening 21% versus 16%, P=0.01; fractional area change 30% versus 19%, P=0.002; ejection fraction 35% versus 23%, P=0.001). In the FG041 group, LV area in systole was less (P<0.05), the dP/dt(max) after isoproterenol was higher (P<0.05), and types I and III collagen in noninfarcted LV were less than in MI-control. The hydroxyproline/proline ratio was increased by 64% in MI-control and reduced to the sham value in MI-FG041 rats. In the scar tissue, it was reduced by 24% in MI-FG041. Conclusions- This study demonstrates that prevention of interstitial fibrosis with a P4H inhibitor alters the pattern of LV enlargement and produces partial recovery of LV function after MI.
© 2001 Circulation
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