Ian Bytheway

PG545, a dual heparanase and angiogenesis inhibitor, induces potent anti-tumour and anti-metastatic efficacy in preclinical models

Background:PG545 is a heparan sulfate (HS) mimetic that inhibits tumour angiogenesis by sequestering angiogenic growth factors in the extracellular matrix (ECM), thus limiting subsequent binding to receptors. Importantly, PG545 also inhibits heparanase, the only endoglycosidase which cleaves HS chains in the ECM. The aim of the study was to assess PG545 in various solid tumour and metastasis models.Methods:The anti-angiogenic, anti-tumour and anti-metastatic properties of PG545 were assessed using in vivo angiogenesis, solid tumour and metastasis models. Pharmacokinetic (PK) data were also generated in tumour-bearing mice to gain an understanding of optimal dosing schedules and regimens.Results:PG545 was shown to inhibit angiogenesis in vivo and induce anti-tumour or anti-metastatic effects in murine models of breast, prostate, liver, lung, colon, head and neck cancers and melanoma. Enhanced anti-tumour activity was also noted when used in combination with sorafenib in a liver cancer model. PK data revealed that the half-life of PG545 was relatively long, with pharmacologically relevant concentrations of radiolabeled PG545 observed in liver tumours.Conclusion:PG545 is a new anti-angiogenic clinical candidate for cancer therapy. The anti-metastatic property of PG545, likely due to the inhibition of heparanase, may prove to be a critical attribute as the compound enters phase I clinical trials.

Discovery of PG545: A Highly Potent and Simultaneous Inhibitor of Angiogenesis, Tumor Growth, and Metastasis

Increasing the aglycone lipophilicity of a series of polysulfated oligosaccharide glycoside heparan sulfate (HS) mimetics via attachment of a steroid or long chain alkyl group resulted in compounds with significantly improved in vitro and ex vivo antiangiogenic activity. The compounds potently inhibited heparanase and HS-binding angiogenic growth factors and displayed improved antitumor and antimetastatic activity in vivo compared with the earlier series. Preliminary pharmacokinetic analyses also revealed significant increases in half-life following iv dosing, ultimately supporting less frequent dosing regimens in preclinical tumor models compared with other HS mimetics. The compounds also displayed only mild anticoagulant activity, a common side effect usually associated with HS mimetics. These efforts led to the identification of 3β-cholestanyl 2,3,4,6-tetra-O-sulfo-α-d-glucopyranosyl-(1→4)-2,3,6-tri-O-sulfo-α-d-glucopyranosyl-(1→4)-2,3,6-tri-O-sulfo-α-d-glucopyranosyl-(1→4)-2,3,6-tri-O-sulfo-β-d-glucopyranoside, tridecasodium salt (PG545, 18) as a clinical candidate. Compound 18 was recently evaluated in a phase I clinical trial in cancer patients.

The PG500 series: novel heparan sulfate mimetics as potent angiogenesis and heparanase inhibitors for cancer therapy

SummaryHeparan sulfate mimetics, which we have called the PG500 series, have been developed to target the inhibition of both angiogenesis and heparanase activity. This series extends the technology underpinning PI-88, a mixture of highly sulfated oligosaccharides which reached Phase III clinical development for hepatocellular carcinoma. Advances in the chemistry of the PG500 series provide numerous advantages over PI-88. These new compounds are fully sulfated, single entity oligosaccharides attached to a lipophilic moiety, which have been optimized for drug development. The rational design of these compounds has led to vast improvements in potency compared to PI-88, based on in vitro angiogenesis assays and in vivo tumor models. Based on these and other data, PG545 has been selected as the lead clinical candidate for oncology and is currently undergoing formal preclinical development as a novel treatment for advanced cancer.

Synthesis and biological evaluation of polysulfated oligosaccharide glycosides as inhibitors of angiogenesis and tumor growth

A series of polysulfated penta- and tetrasaccharide glycosides containing alpha(1-->3)/alpha(1-->2)-linked mannose residues were synthesized as heparan sulfate (HS) mimetics and evaluated for their ability to inhibit angiogenesis. The compounds bound tightly to angiogenic growth factors (FGF-1, FGF-2, and VEGF) and strongly inhibited heparanase activity. In addition, the compounds exhibited potent activity in cell-based and ex vivo assays indicative of angiogenesis, with tetrasaccharides exhibiting activity comparable to that of pentasaccharides. Selected compounds also showed good antitumor activity in vivo in a mouse melanoma (solid tumor) model resistant to the phase III HS mimetic 1 (muparfostat, formerly known as PI-88). The lipophilic modifications also resulted in reduced anticoagulant activity, a common side effect of HS mimetics, and conferred a reasonable pharmacokinetic profile in the rat, as exemplified by the sulfated octyl tetrasaccharide 5. The data support the further investigation of this class of compounds as potential antiangiogenic, anticancer therapeutics.

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.

An Experimental and Molecular‐Modeling Study of the Binding of Linked Sulfated Tetracyclitols to FGF‐1 and FGF‐2

The experimental binding affinities of a series of linked sulfated tetracyclitols [Cyc2N‐R‐NCyc2, where Cyc=C6H6(OSO3Na)3 and R=(CH2)n (n=2–10), p‐xylyl or (C2H4)2‐Ncyc] for the fibroblast growth factors FGF‐1 and FGF‐2 have been measured by using a surface plasmon resonance assay. The KD values range from 7.0 nM to 1.1 μM for the alkyl‐linked ligands. The binding affinity is independent of the flexibility of the linker, as replacement of the alkyl linker with a rigid p‐xylyl group did not affect the KD. Calculations suggest that binding modes for the p‐xylyl‐linked ligand are similar to those calculated for the flexible alkyl‐linked tetracyclitols. The possible formation of cross‐linked FGF:cyclitol complexes was examined by determining KD values at increasing protein concentrations. No changes in KD were observed; this suggesting that only 1:1 complexes are formed under these assay conditions. Monte Carlo multiple‐minima calculations of low‐energy conformers of the FGF‐bound ligands showed that all of the sulfated tetracyclitol ligands can bind effectively in the heparan sulfate‐binding sites of FGF‐1 and FGF‐2. Binding affinities of these complexes were estimated by the Linear Interaction Energy (LIE) method to within a root‐mean‐square deviation of 1 kcal mol−1 of the observed values. The effect of incorporating cations to balance the overall charge of the complexes during the LIE calculations was also explored.

Synthesis of a heparan sulfate mimetic disaccharide with a conformationally locked residue from a common intermediate.

A simple mimetic of a heparan sulfate disaccharide sequence that binds to the growth factors FGF-1 and FGF-2 was synthesized by coupling a 2-azido-2-deoxy-D-glucopyranosyl trichloroacetimidate donor with a 1,6-anhydro-2-azido-2-deoxy-beta-D-glucopyranose acceptor. Both the donor and acceptor were obtained from a common intermediate readily obtained from D-glucal. Molecular docking calculations showed that the predicted locations of the disaccharide sulfo groups in the binding site of FGF-1 and FGF-2 are similar to the positions observed for co-crystallized heparin-derived oligosaccharides obtained from published crystal structures.

Abstract A18: The dual angiogenesis/heparanase inhibitor PG545, but not the tyrosine kinase inhibitor sorafenib, inhibits spontaneous metastasis in models of breast and lung cancer

PG545 is a fully synthetic heparan sulfate (HS) mimetic selected as the lead oncology candidate for formal preclinical development. PG545 inhibits key processes in tumor progression (a) neovascularization ‐ by interfering with growth factor binding and (b) metastasis ‐ presumably by inhibition of heparanase activity. PG545 was assessed for anti‐angiogenic activity in vivo and for antitumor and anti‐metastatic activity in the T41 breast cancer and the Lewis Lung Carcinoma (LL/2) models using the tyrosine kinase inhibitor sorafenib as an anti‐angiogenic reference compound. Both PG545 (daily or twice weekly) and sorafenib (daily) significantly reduced CD31 staining in the AngioSponge™ model. Twice weekly treatment with 25 mg/kg PG545, but not daily treatment with 60mg/kg sorafenib, significantly inhibited solid tumor growth in the T41 model. Moreover, PG545 significantly inhibited spontaneous lung metastases in a dose‐dependent manner, whereas sorafenib significantly increased the number of metastases. In the LL/2 model, once weekly treatment with PG545 at 20 mg/kg significantly reduced solid tumor growth to a similar extent as sorafenib while the 40mg/kg dose appeared more efficacious than sorafenib. A significant reduction in spontaneous lung metastases was again associated with PG545 following either a single injection or once weekly treatment while sorafenib had no appreciable effect on metastases. PG545 was well tolerated with no significant loss in bodyweight noted. Taken together, the data demonstrate that PG545 is an effective angiogenesis inhibitor with anti‐metastatic activity ‐ an envious property for an anti‐cancer compound, allaying recently publicized concerns that angiogenesis inhibitors and even chemotherapeutic agents may have the potential to aggravate metastatic development. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A18.

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.

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

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 TKI9s 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