Mimetic PG545 Research Articles

Abstract 367: The heparan sulfate mimetic PG545 inhibits tumor growth in both male and female mouse model of pancreatic ductal adenocarcinoma (PDAC) with sex difference in the effect on cachexia

Abstract BACKGROUND: Cachexia occurs in up to 80% of PDAC patients and is characterized by weight loss mainly due to muscle and fat wasting. Cachexia leads to functional impairment, reduced anticancer therapy tolerance and increased mortality. PG545, a heparan sulfate (HS) mimetic, shows anticancer activity; however, it is unknown whether sex affects the anticancer activity and what effect PG545 has on cancer associated muscle and fat wasting. Given that many cytokines induced in patients with cancer cachexia can bind HS, the aim of this study was to assess PG545 effects on PDAC cachexia with emphasis on sex difference. METHODS: PDAC cells were injected into the pancreas of 10-week C57BL/6 male and female mice. The orthotopic tumor-bearing mice were treated with PG545 (10 mg/kg; i.p.). Body weight and body composition were monitored, and tumor and organs were collected. Skeletal muscles were cryosectioned for evaluation of cross-sectional area (CSA). C2C12 myotubes, the in vitro model of muscle wasting, were treated with PDAC cell-derived conditioned medium (CM) without or with PG545 and analyzed for the changes in size. PDAC cell proliferation was assessed by MTT assay. RESULTS: PG545 treatment markedly reduced tumor mass in both males and females compared to the vehicle controls. PG545 also reduced body weight but the difference gradually dissipated over time despite repeated dosing. However, male mice recovered from the negative effects more slowly than female mice. Similar sex difference was observed in the changes in the PG545-induced lean and fat mass loss. Of note, fat mass in PG545-treated females at the end of experiment was higher than in the vehicle controls. Furthermore, males had more reductions in muscle and fat weights than females in response to PG545. Grip strength in males was weakened by PG545 but returned to the level like that in the vehicle controls at the end, while PG545 did not weaken grip strength in females at the same time points. In addition, males, but not females, had smaller muscle fiber CSA after PG545 treatment versus their vehicle controls. In in vitro assay, PG545 inhibited PDAC cell proliferation, indicating a direct effect on cancer cells. Lastly, opposed to the in vivo effect, PG545 partly prevented myotube wasting induced by PDAC CM. CONCLUSION: PG545 has strong anti-PDAC activity in both male and female mice, at least partly through inhibition of cancer cell proliferation. Its initial negative effects on cachexia are gradually reversed with male tumor-bearing mice reversing more slowly than females. Because of the protective effect on C2C12 myotubes treated with PDAC CM, PG545 might induce some counteracting factors in vivo in the initial treatment and identification of these factors may offer molecular targets for combination therapy to counteract the negative effects while take advantage of the strong anti-cancer potential. Citation Format: Xiaoling Zhong, Keith Dredge, John M. Chirgwin, Michael G. House, Harikrishna Nakshatri, Teresa A. Zimmers. The heparan sulfate mimetic PG545 inhibits tumor growth in both male and female mouse model of pancreatic ductal adenocarcinoma (PDAC) with sex difference in the effect on cachexia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 367.

The Heparan Sulfate Mimetic PG545 Modulates T Cell Responses and Prevents Delayed-Type Hypersensitivity.

The heparan sulfate mimetic PG545 (pixatimod) is under evaluation as an inhibitor of angiogenesis and metastasis including in human clinical trials. We have examined the effects of PG545 on lymphocyte phenotypes and function. We report that PG545 treatment suppresses effector T cell activation and polarizes T cells away from Th17 and Th1 and toward Foxp3+ regulatory T cell subsets in vitro and in vivo. Mechanistically, PG545 inhibits Erk1/2 signaling, a pathway known to affect both T cell activation and subset polarization. Interestingly, these effects are also observed in heparanase-deficient T cells, indicating that PG545 has effects that are independent of its role in heparanase inhibition. Consistent with these findings, administration of PG545 in a Th1/Th17-dependent mouse model of a delayed-type hypersensitivity led to reduced footpad inflammation, reduced Th17 memory cells, and an increase in FoxP3+ Treg proliferation. PG545 also promoted Foxp3+ Treg induction by human T cells. Finally, we examined the effects of other heparan sulfate mimetics PI-88 and PG562 on lymphocyte polarization and found that these likewise induced Foxp3+ Treg in vitro but did not reduce Th17 numbers or improve delayed-type hypersensitivity in this model. Together, these data indicate that PG545 is a potent inhibitor of Th1/Th17 effector functions and inducer of FoxP3+ Treg. These findings may inform the adaptation of PG545 for clinical applications including in inflammatory pathologies associated with type IV hypersensitivity responses.

Immunomodulatory Activities of the Heparan Sulfate Mimetic PG545.

Heparanase regulates multiple biological activities that enhance tumor growth and metastatic spread. Heparanase cleaves and degrades heparan sulfate (HS), a key structural component of the extracellular matrix that serves as a barrier to cell invasion and also as a reservoir for cytokines and growth factors critical for tumor growth and metastasis. For this reason, heparanase is an attractive target for the development of novel anti-cancer therapies. Pixatimod (PG545), a heparanase inhibitor, has shown promising results in the treatment of multiple tumor types. PG545 offers a diversity of mechanisms of action in tumor therapy that include angiogenic inhibition, inhibition of growth factor release, inhibition of tumor cell migration, tumorcell apoptosis, activation of ER stress response, dysregulation of autophagy, and NK cell activation. Further investigation into the role that heparanase and its inhibitors play in tumor therapy can lead to the development of effective tumor therapies.

PG545 treatment reduces RRV-induced elevations of AST, ALT with secondary lymphoid organ alterations in C57BL/6 mice.

Recently the anti-viral effects of prophylactic treatment with the low-molecular-weight heparan sulfate mimetic PG545 in Ross River virus (RRV) infected mice were reported. We further investigated the related, transient pathophysiology of PG545 drug treatment in RRV-infected and mock-infected PG545-treated mice. PG545 treatment resulted in mild lethargy and piloerection, on days after the drug administration. Mice were treated with two or three doses of PG545 within a ten-day period and were subsequently culled at peak disease or at disease resolution. The treatment responses of the spleen and liver were assessed through histology, flow cytometry, gene arrays and serum biochemistry. Microscopy showed an expanded red pulp in the spleen following either two or three treatments with PG545. The red pulp expansion was further demonstrated by the proliferation of megakaryocytes and erythrocyte precursors within the spleen. In addition, flow cytometry and gene array analyses revealed a reduction of lymphocytes within the spleens of PG545-treated mice. Previously unreported, RRV-induced elevations of aspartate aminotransferase (AST) and alanine transaminase (ALT) enzymes and creatinine were also noted in the RRV-infected mice. However, PG545 only reduced AST and ALT levels but not the creatinine levels in infected mice during treatment. Mice treated with three doses of PG545 also showed hepatosplenomegaly and anaemia, which were reversed upon discontinuation of the treatment. In summary, this study demonstrates that dose and frequency related haemopoietic pathophysiology such as hepatosplenomegaly and anaemia, occurred in C57BL/6 mice treated with PG545. However, this effect was reversible once drug administration is terminated.

Dual targeting of dengue virus virions and NS1 protein with the heparan sulfate mimic PG545

Dengue virus (DENV) is the most prevalent mosquito-borne flavivirus that infects humans. At present, there are no specific antiviral drugs to treat DENV infection and vaccine development has met with challenges. DENV encodes two glycosaminoglycan (GAG) binding proteins; Envelope (E) and non-structural protein 1 (NS1). While previous work has validated the use of GAG analogues as inhibitors of E mediated virus-cell attachment, their potential for antiviral intervention in NS1 protein toxicity has not yet been explored. Here, we investigate the potential of the heparan sulfate mimetic PG545 as a dual purpose compound to target both DENV virion infectivity and NS1 function. In comparison to a non-sulfated analogue, we show that PG545 potently inhibits DENV infectivity with no cytotoxic effect. Against NS1, PG545 completely blocks the induction of cellular activation and abolishes NS1-mediated disruption of endothelial monolayer integrity. Furthermore, PG545 treatment moderately improves survival from lethal DENV challenge in a murine model. At peak disease, PG545-treated mice have lower viremia, circulating NS1 and serum TNF-α. Consistent with anti-NS1 activity, PG545 treatment also reduces systemic vascular leakage caused by DENV infection in vivo. Taken together, these findings demonstrate that the dual targeting of DENV virions and NS1 using GAG analogues offers a new avenue for DENV drug development.

Abstract 1135: Loss of HSulf-1 promotes defective autophagy and increased lipid droplet biogenesis in ovarian cancer

Abstract Dysregulation of autophagy and altered metabolic pathways are frequently observed in cancer. Due to these alterations, pharmacological targeting of these two pathways simultaneously could provide a viable therapeutic option. Although the association between these two pathways is well characterized in metabolic disorders, it is not well defined in ovarian cancer (OVCA). In this regard, we found that loss of endosulfatase HSulf-1, a known putative tumor suppressor, suppresses LC3-GFP foci formation and promotes increased lipid droplet (LD) biogenesis suggesting that absence of HSulf-1 in OVCA affects both autophagy and lipid metabolism. While isogenic cells with genetic ablation of HSulf-1 (OV202Sh1/2 and TOV2223Sh1 cells) displayed LDs, the nontargeted control transduced (NTC) OV202 and TOV2223 cells had significantly less LDs. In contrast, Transmission Electron Micrographs (TEMs) showed that OV202 and TOV2223 NTC cells had significantly more autophagic vacuoles (AVs) compared to their isogenic ShRNA targeted cells. Conversely, ectopic expression of HSulf-1 in SKOV3 cells decreased the number of LDs and increased the number of AVs compared to vector transfected controls. Here we report that OV202Sh1 cells and HSulf-1 deficient OV2008 cells have increased p-cPLA2α(ser505) levels that are associated with biogenesis of large number of LDs with reduced AVs. Interestingly, pharmacological inhibition of cPLA2α with AACOCF3 in OV202Sh1 cells resulted in reduced LD biogenesis, inhibited colony formation and reduced tumorigenesis in vivo. More importantly, treatment of HSulf-1 deficient cells with HS mimetic PG545 which can compensate for loss of HSulf-1, reduced LD biogenesis, promoted autophagy and inhibited tumor growth in vivo. Collectively, these results show a critical role of HSulf-1 in regulating both autophagy and LD biogenesis in ovarian cancer. Citation Format: Debarshi Roy, Susmita Mondal, Ashwani Khurana, Xiaoping He, Edward Hammond, Keith Dredge, Viji Shridhar. Loss of HSulf-1 promotes defective autophagy and increased lipid droplet biogenesis in ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1135. doi:10.1158/1538-7445.AM2015-1135

The heparan sulfate mimetic PG545 interferes with Wnt/β-catenin signaling and significantly suppresses pancreatic tumorigenesis alone and in combination with gemcitabine

The heparan sulfate mimetic PG545 has been shown to exert anti-angiogenic and anti-metastatic activity in vitro and in vivo cancer models. Although much of this activity has been attributed to inhibition of heparanase and heparan sulfate-binding growth factors, it was hypothesized that PG545 may additionally disrupt Wnt signaling, an important pathway underlying the malignancy of pancreatic cancer. We show that PG545, by directly interacting with Wnt3a and Wnt7a, inhibits Wnt/β-catenin signaling leading to inhibition of proliferation in pancreatic tumor cell lines. Additionally, we demonstrate for the first time that the combination of PG545 with gemcitabine has strong synergistic effects on viability, motility and apoptosis induction in several pancreatic cell lines. In an orthotopic xenograft mouse model, combination of PG545 with gemcitabine efficiently inhibited tumor growth and metastasis compared to single treatment alone. Also, PG545 treatment alone decreased the levels of β-catenin and its downstream targets, cyclin D1, MMP-7 and VEGF which is consistent with our in vitro data. Collectively, our findings suggest that PG545 exerts anti-tumor activity by disrupting Wnt/β-catenin signaling and combination with gemcitabine should be considered as a novel therapeutic strategy for pancreatic cancer treatment.

Abstract 3695: The utility of plasma vascular endothelial growth factor and heparanase as pharmacodynamic markers following treatment with PG545, a heparan sulfate mimetic: preliminary evidence from preclinical and clinical settings

Abstract Background: The heparan sulfate mimetic PG545 has been demonstrated to inhibit the function of vascular endothelial growth factor (VEGF) and heparanase (HPSE) in vitro whilst downregulating HPSE expression in tumor and metastatic tissue in vivo. Although there are reports of consistent drug-induced increases in plasma VEGF with VEGF inhibitors such as bevacizumab, similar data are lacking for HS mimetics. Given PG545's mechanism of action which involves inhibition of both angiogenesis and metastasis, the evaluation of plasma VEGF and HPSE may represent pharmacodynamic (PD) biomarkers for each of these key processes. Materials and Methods: The levels of plasma VEGF and HPSE were measured using ELISA in samples from preclinical models of ovarian cancer (syngeneic ID8 and xenogeneic A2780 models) and a small cohort of patients (dosed with PG545 by the subcutaneous route) with advanced solid tumors from a Phase I clinical study. In tumor models, PG545 was administered once weekly by intravenous or intraperitoneal routes and efficacy was determined by quantifying tumor burden using the following methods: measurement of palpable solid tumors, abdominal circumference, ascites volume and/or by tracking cells using bioluminescent reporter imaging (depending on the model). In the A2780 model and the clinical samples, the pharmacokinetics of PG545 was also evaluated using LC-MS/MS, which ensured multiple samples to facilitate the measurement of VEGF and HPSE in plasma over the duration of the dosing interval following treatment with PG545. Results: PG545 demonstrated potent antitumor and antimetastatic activity when administered once weekly via intravenous or intraperitoneal routes in preclinical studies. These studies also indicated that PG545 treatment led to increases in plasma VEGF and HPSE. Importantly, plasma samples from advanced cancer patients treated with PG545 also revealed increases in these PD markers with initial peaks often correlating with Cmax of PG545 but longer-term gradual increases also noted in some instances. We consider that these increases in plasma levels result from PG545 binding to, thereby inhibiting, VEGF and HPSE in the tumor microenvironment. Conclusion: Given the questionable utility of single time point measures of plasma PD markers (including VEGF), these preliminary data suggest that kinetic profiling can reveal the previously undetermined dynamics of circulating VEGF and HPSE following treatment with PG545. Thus, the measurement of VEGF and HPSE appears to represent potential markers of pharmacodynamic activity. A new Phase I trial with intravenously-administered PG545 has recently commenced and should further clarify the potential utility of these basic PD biomarker responses in advanced cancer patients. Citation Format: Keith Dredge, Edward Hammond, Boris Winterhoff, Shailendra Giri, Attila Teoman, Darryn Bampton, Michael Millward, Viji Shridhar. The utility of plasma vascular endothelial growth factor and heparanase as pharmacodynamic markers following treatment with PG545, a heparan sulfate mimetic: preliminary evidence from preclinical and clinical settings. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3695. doi:10.1158/1538-7445.AM2014-3695

Abstract 1: The novel heparin sulfate (HS) mimetic PG545 shows strong synergism when combined with cisplatin and paclitaxel in preclinical ovarian cancer models

Objectives: The majority of patients with ovarian cancer have recurrence of disease despite surgical debulking and adjuvant chemotherapy. The dual angiogenesis/metastasis inhibitor PG545 acts through interference with heparin-binding growth factors and through the inhibition of heparanase activity which leads to anti-tumor activity. Here we tested PG545 as a maintenance therapy as single agent or in addition to standard chemotherapy in ovarian cancer in vivo and in vitro.

Mechanisms of heparanase inhibition by the heparan sulfate mimetic PG545 and three structural analogues

The tetrasaccharide heparan sulfate (HS) mimetic PG545, a clinical anti-cancer candidate, is an inhibitor of the HS-degrading enzyme heparanase. The kinetics of heparanase inhibition by PG545 and three structural analogues were investigated to understand their modes of inhibition. The cholestanol aglycon of PG545 significantly increased affinity for heparanase and also modified the inhibition mode. For the tetrasaccharides, competitive inhibition was modified to parabolic competition by the addition of the cholestanol aglycon. For the trisaccharides, partial competitive inhibition was modified to parabolic competition. A schematic model to explain these findings is presented.

Abstract LB-309: The heparan sulfate mimetic PG545 increases plasma VEGF and FGF-2 in advanced cancer patients, and significantly inhibits tumor growth in preclinical models of ovarian cancer - implications for future therapy

Abstract PG545 is a synthetic heparan sulfate (HS) mimetic purported to inhibit angiogenesis via the blockade of VEGF and FGF-2 signaling, and block the enzymatic activity of heparanase - a molecule closely associated with metastatic dissemination. The attenuation of VEGF and FGF-2 signaling is supported by recent clinical data from the first four advanced cancer patients with multiple tumor types treated with PG545 which exhibited increases in plasma VEGF and FGF-2 levels, often in close association with the drug treatments/exposure profile during the first treatment cycle over 4 weeks. Unfortunately, due to local injection site reactions associated with subcutaneous (SC) administration, the trial was recently terminated. However, to support a re-entry to the clinic using the intravenous route, we tested PG545 in a preclinical model of ovarian cancer.The rationale to investigate PG545 in ovarian cancer is derived from recently generated data which found PG545 (5 µM) almost completely inhibited migration of SKOV3 ovarian carcinoma cells stimulated by each of the following HS binding growth factors: VEGF, HGF, FGF-2, SDF-1 and HB-EGF. Because HB-EGF is thought to play a major role in the spread of this cancer, PG545 was also tested against HB-EGF dependant SKOV3 cell invasion and was found to be a potent inhibitor of this process. The effect of PG545 on HB-EGF signaling pathways in these experiments were investigated and the results indicated that PG545 reduces activation of the HB-EGF receptor (EGFR) and, consequently, both AKTand MAPK signaling are also reduced. Given the strong mechanism of action data for ovarian carcinoma, PG545 was next tested in the A2780 ovarian xenograft model. The signficant antitumor activity using PG545 at 7.5 mg/kg twice weekly intravenous (IV) and 15 mg/kg once weekly IV was similar to a 20 mg/kg once weekly SC dose. Pharmacokinetic analysis confirmed that drug exposure (AUC) over the course of a week was consistent between groups. PG545 significantly reduced tumor burden in a cisplatin resistant ovarian tumor model (C200) when given intraperitoneally at the dose of 20mg/kg, once a week until the end of the study. Most importantly, PG545 completely reduced tumor mass and reduced ascites accumulation in a syngeneic immunocompetent mouse model of ovarian cancer (ID8). Given the known effects of PG545 on heparanase and in metastatic models, these emerging data on growth factors in the clinic and HB-EGF in preclinical models provide a strong rationale to re-enter the clinic using the IV route and to focus on ovarian cancer as the clinical indication. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-309. doi:1538-7445.AM2012-LB-309

Abstract A7: The heparan sulfate mimetic PG545 as a monotherapy or in combination with the standard-of-care agents paclitaxel or carboplatin inhibits solid tumor progression in two murine models of ovarian cancer.

Abstract PG545 is a fully synthetic heparan sulfate (HS) mimetic currently being assessed by subcutaneous administration in Phase I clinical trials for advanced cancer patients. PG545 inhibits the enzymatic activity of heparanase, angiogenesis, solid tumor growth and spontaneous metastases in different tumor types. As a role for tumor angiogenesis emerged in the development of ovarian cancer, the number the clinical investigations using avascular therapies have increased in recent years. Moreover, given the fact that ovarian cancers can metastasize by multiple routes, the dual antiangiogenic and antimetastatic activity of PG545 may offer a promising approach to treat ovarian cancer patients. Standard treatment with PG545 involved a loading and maintenance dosing regimen of 20 mg/kg followed by weekly injections of 10 mg/kg as previously reported. In these studies the antitumor effect of weekly dosing with PG545, both as a monotherapy and in combination with standard-of-care agents, was investigated. In the first study, mice bearing A2780 tumors were treated with PG545 alone, paclitaxel alone (15 mg/kg, qw) or in combination of both compounds. Treatment with PG545 reduced tumor growth (TGI = 42%) compared to vehicle control and was more effective than paclitaxel alone (TGI = 11%). In combination, PG545 and paclitaxel significantly reduced tumor volume (TGI = 61%) compared to vehicle control. In a second model, mice bearing SKOV3 tumors received PG545, carboplatin (60 mg/kg) or in combination. Treatment with PG545 significantly inhibited tumor growth (TGI = 40%) compared to vehicle control, almost equivalent to the effect of carboplatin monotherapy (TGI = 41%). The combination regimen further impacted tumor growth, showing a significant effect by day 19 of treatment (TGI = 71% versus the vehicle control and the monotherapy groups). In conclusion, PG545 demonstrates antitumor activity as a monotherapy in ovarian cancer models which can be substantially enhanced using combination regimens with current standard-of-care agents such as paclitaxel and carboplatin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A7.

Abstract 1461: The heparan sulfate mimetic PG545 potently inhibits spontaneous lung metastasis and significantly enhances overall survival in the 4T1 breast carcinoma model

Abstract PG545 is a fully synthetic heparan sulfate (HS) mimetic entering Phase I clinical trials for advanced cancer. PG545 inhibits key processes in tumor progression (a) angiogenesis – by interfering with growth factor binding and (b) metastasis – by inhibition of heparanase activity. The syngeneic 4T1 breast carcinoma murine model is a well known model of spontaneous metastasis and PG545 was assessed for effects on primary tumour growth, metastatic development in lung and overall survival. PG545 and the tyrosine kinase inhibitor (TKI) sorafenib (used as a comparative antiangiogenic agent) were investigated for their effect on primary tumour growth and the number of lung metastases after treatment for two weeks. PG545 significantly inhibited solid tumour growth and potently inhibited the formation of lung metastases when administered on a twice-weekly dosing schedule (2xqw). Although sorafenib inhibited primary tumour growth to a similar extent as PG545, sorafenib significantly increased the number of lung metastases. In a separate study to investigate overall survival, mice had their primary tumours resected (mastectomy) on day 11 post-inoculation and treatment with PG545 (1xqw), sorafenib (1xqd) or cisplatin (1xqw) was administered for 55 days. Interim data on day 30 using satellite animals confirmed the potent antimetastatic activity of PG545 by enumeration of lung metastases. This reduction in metastasis correlated with overall survival in the PG545-treated mice in the main study which was significantly enhanced compared to vehicle control or sorafenib (which had a similar survival profile as control). PG545 also outperformed cisplatin. Mice culled due to clinical signs associated with disease progression correlated with histological assessment of metastatic burden in lung tissue. The antimetastatic effects of PG545 were confirmed by histological assessment at the end of the study on day 55, with very few metastases observed in PG545-treated mice. In conclusion, PG545 has potent anti-metastatic activity that would appear to differentiate it from other antiangiogenic therapies such as TKI's which have also been recently reported by other groups to aggravate metastatic development under certain circumstances. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1461. doi:10.1158/1538-7445.AM2011-1461