Treatment with the novel anti-angiogenic agent PI-88 is associated with immune-mediated thrombocytopenia

M. A. Rosenthal1,+, D. Rischin2, G. McArthur2, K. Ribbons3, B. Chong4, J. Fareed5, G. Toner2, M. D. Green1 and R. L. Basser1

From the Center for Developmental Cancer Therapeutics, Parkville, Victoria, Australia affiliates: 1Department of Medical Oncology and Clinical Hematology, Royal Melbourne Hospital, Melbourne; 2Department of Medical Oncology, Peter MacCallum Cancer Institute, East Melbourne, Victoria; 3Progen Industries, Brisbane, Queensland; 4Department of Hematology, Prince of Wales Hospital, NSW, Australia; 5Hemostasis and Thrombosis Research Laboratories, Loyola University Medical Center, IL, USA

Received 19 June 2001; revised 1 October 2001; accepted 23 October 2001.


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Conclusions
 References
 
Background

The novel molecule PI-88 is a highly sulfonated oligosaccharide which inhibits heparanase activity and competes with heparan sulfate binding of growth factors such as FGF and VEGF. Preclinical data demonstrates that PI-88 inhibits angiogenesis and has anti-metastatic effects. The aim of this phase I study was to determine the recommended dose and toxicity profile of PI-88.

Patients and methods

PI-88 was given intravenously in increasing duration of administration (0.57 mg/kg for 2 h, 0.57 mg/kg/day for 1 day, 4, 7 and 14 consecutive days) and then increasing dose for 14 consecutive days (1.14 mg/kg/day and 2.28 mg/kg/day) in patients with advanced malignancies until dose-limiting toxicity (DLT) was observed. Fourteen assessable patients with advanced malignancies received PI-88 intravenously.

Results

DLT was thrombocytopenia. The thrombocytopenia appeared to be immunologically mediated with the development of anti-heparin platelet factor 4 complex antibodies. There were no other significant toxicities. At the final dose and schedule (2.28 mg/kg/day for 14 days), there was limited evidence of biological activity as measured by the surrogate marker activated partial thromboplastin time (APTT), although two patients had stabilisation of disease.

Conclusions

In conclusion, PI-88 at a dose of 2.28 mg/kg/day for 14 days resulted in dose-limiting thrombocytopenia which appeared to be immune related. Limited evidence of biological activity was noted. Alternate scheduling and routes of administration are now being explored.

Key words: anti-angiogenic, PI-88, thrombocytopenia


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Conclusions
 References
 
Anti-angiogenic and anti-metastatic agents have an evolving role in the treatment of malignancy. Such agents act through a variety of mechanisms including tyrosine kinase inhibition, apoptotic pathways and cell adhesion [1, 2]. One selective approach is to interfere with tumor-derived heparanases that appear to be involved in the degradation of extracellular matrix [36]. Selective inhibition of the heparanase enzyme offers the opportunity to influence a range of biological processes including cellular extravasation through basement membranes and tissue remodeling during tumor growth. In addition, heparan sulfate, the substrate for heparanase can act as an essential cell surface receptor for angiogenic growth factors such as bFGF, aFGF and VEGF [7, 8].

PI-88 is a highly sulfonated oligosaccharide obtained as a randomly, but reproducibly, sulfated mixture with an average sulfation of three sulfates per mannose residue and a molecular weight of 2100–2585 Da [9]. PI-88 inhibits heparanase activity and competes with heparan sulfate binding of peptide growth factors involved in angiogenesis, such as FGF and VEGF [9]. PI-88 consistently prolongs the activated partial thromboplastin time (APTT) through activation of endogenous heparin cofactor II.

In vitro experiments demonstrate that PI-88 inhibits angiogenesis [9]. Similarly, in vivo models of rat- and human-derived tumors document that PI-88 has potent anti-angiogenic and anti-metastatic effects [911]. Apart from the anticipated anticoagulant effects, PI-88 was well tolerated in animal studies [12]. In a study of 24 normal volunteers, PI-88 was safe as a 2-h infusion and caused the anticipated biological effect of prolonged APTT. A two-fold increase in APTT was observed in patients receiving between 40 and 80 mg of PI-88. There were no other significant clinical, biological or hematological changes [12].

Limited data in rats using 14C labeled PI-88 suggests that the plasma half-life of PI-88 is 1–2 h following intravenous administration [12]. The drug appears to have low clearance and a low volume of distribution. There is currently no direct pharmacokinetic modeling assay available for PI-88. APTT had been previously used as a surrogate marker of PI-88 exposure in animal models and we elected to use APTT as a surrogate biological endpoint in this study. This phase Ib dose duration and dose escalation study represents the first use of the novel heparanase inhibitor, PI-88, in patients with malignant disease.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Conclusions
 References
 
Patient eligibility
Eligible patients had advanced malignancy where other effective therapy was either not available or had failed. Patients may have received chemotherapy or radiotherapy more than 30 days prior to enrollment. Other eligibility criteria included the following: Eastern Cooperative Oncology Group (ECOG) performance status of zero to two; >=18 years of age; neutrophil count >1.5 x 109/l; platelet count >100 x 109/l; bilirubin level <1.5-fold the upper limit of normal (ULN) and liver enzyme levels <2.5 x ULN; calculated creatinine clearance >60 ml/min; prothrombin time <1.5 x {Upsilon}ULN; normal APTT; geographically accessible and no other major coexisting medical problems. In addition, patients must have had no history of allergy/hypersensitivity to anticoagulants/thrombolytic agents, nor a history of acute or chronic gastrointestinal bleeding or any abnormal bleeding tendency. Patients with a history of heparin-induced thrombocytopenia, immune-mediated thrombocytopenia, thrombotic thrombocytopenic purpura or other platelet disease were ineligible. Patients receiving concomitant aspirin, non-steroidal anti-inflammatory drugs, heparin, low molecular weight heparin or warfarin were ineligible. The respective institutional ethics committees approved the protocol and written informed consent was obtained from all patients.

Study design
The study was performed at two centers as an open label non-comparative, non-randomized two-phase dose duration and dose escalation study. The study was in two parts (Table 1). First, the daily dose of PI-88 was kept constant but the duration of treatment extended. The dose was then escalated once the duration of infusion had reached 14 days. Dose escalation continued until dose-limiting toxicity (DLT) was identified. The study objective was to define the maximum tolerated dose (MTD) of PI-88 through the delineation of DLT and to study the biological activity of PI-88 using APTT as a surrogate biological marker. Single patient cohorts were used with increasing duration and dose of PI-88 administration starting at a dose of 0.57 mg/kg/day as a 2-h infusion. The infusional time was increased as follows: 2 h, 24 h, 4 days, 7 days and 14 days. At 14 days, the dose was subsequently escalated as follows: 0.57, 1.14 and 2.28 mg/kg/day (Table 1). All patients were monitored for 7 days after ceasing treatment before initiation of the next cohort accrual.


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Table 1. Duration and dose escalation schema
 
Drug-related toxicity (DRT) was defined as National Cancer Institute/Common Toxicity Criteria (NCI-CTC), version 2 grade 2 toxicity or APTT >=1.67 x ULN. DLT was defined as NCI-CTC grade 3 or 4 non-hematological toxicities, grade 4 neutropenia, grade 3 or 4 thrombocytopenia and APTT >2.34 x ULN. Once DRT was observed, the number of patients in that cohort and all subsequent cohorts was increased to three patients. If a DLT occurred, then the cohort was expanded to six patients and if two or more patients had a DLT then the cohort prior to this was deemed the MTD. A minimum of six patients were to be accrued at the MTD. If none of the additional patients experienced DLT then the study could proceed to the next cohort. Patients remained on study for 3 weeks (one cycle). At the completion of the study, two additional cycles could be administered to patients in the absence of intolerable toxicity or progressive disease. Further cycles of therapy were at the discretion of the investigator.

Treatment
PI-88 as a mixture of sulfonated oligosaccharides was supplied by Progen Industries (Brisbane, Australia) (Figure 1). Each 20 ml vial contained 400 mg of lyophilized PI-88 or a 10 mg/ml PI-88 solution that was reconstituted or diluted with normal saline. The solution was made up to 100 ml of normal saline in a 100 ml capacity CADD reservoir (Deltec, St Paul, MN, USA) under aseptic conditions. The dose was administered by way of a constant rate intravenous infusion such that 40 ml of solution was to be administered every 24 h by a CADD-1 ambulatory infusion pump. Four, 7 and 14 day infusions were given centrally via a PICC line or port-a-cath. Supportive treatments such as anti-emetics, analgesics and blood products could be given at the investigators discretion.



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Figure 1. Chemical structure of PI-88.

 
Pharmacokinetic and laboratory assays
Samples were collected for the assessment of PI-88 concentration and to carry out pharmacokinetic studies. Serum was frozen and samples are to be tested when a validated pharmacokinetic assay is established. Samples for APTT determination were taken before treatment and at 2, 4, 6, 8, 12 and 24 h after initiation of treatment, daily for the duration of treatment and 24 h, 3 and 7 days after the completion of treatment. Samples were processed centrally according to standard methods.

Available blood samples were retrospectively tested for evaluation of anti-heparin platelet factor 4 complex (AHPF4) antibodies. Two methods were used to identify immune-mediated effects. First, the serotonin-release assay (SRA) was performed according to standard methods in two separate laboratories to identify immune-mediated effects. Functional antibodies resulted in platelet aggregation [13]. AHPF4 complex antibodies (IgG, IgA and IgM subclasses) were also assayed by enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (GTI, Brookfield, WI, USA).


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Conclusions
 References
 
Patient characteristics and dose escalation
Fourteen patients with advanced malignancies were enrolled on the study. Patient characteristics are shown in Table 2. DLT was seen at 2.28 mg/kg/day given as a 14 day infusion. Twelve patients received one cycle of therapy and the remaining two patients received four and 12 cycles, respectively. Treatment with PI-88 was ceased for the following reasons: progressive disease in eight patients (58%), toxicity in three patients (21%), completion of the study in two patients (14%) and a catheter complication in the remaining patient (7%).


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Table 2. Patient characteristics
 
Hematological toxicity
Thrombocytopenia was the DLT at 2.28 mg/kg/day given as a 14 day infusion. Figure 2 shows platelet counts for five of the six patients who received this schedule and dose of PI-88. In one of the five patients (patient 11) in Figure 2 infusion was ceased on day 10 due to rapid disease progression. Two of six patients (patients 13 and 9) at this dose level developed grade 3 thrombocytopenia on days 8 and 14, respectively.



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Figure 2. Cohort seven platelet counts and anti-heparin antibody testing.

 
One further patient (patient 14) developed grade 1 thrombocytopenia at a dose of 2.28 mg/kg/day given as a 14 day infusion. On day 6 the platelet count was observed to have fallen from 195 x 109 to 110 x 109/l over a 24 h period. This represented a >40% fall in platelet count and no further PI-88 was administered. The platelet count continued to fall to a nadir of 87 x 109/l 2 days after ceasing therapy. One patient at the dose level of 0.57 mg/kg/day for 24 h developed grade 2 thrombocytopenia. This patient with metastatic lung cancer also had an underlying chronic myelomonocytic leukemia and developed a chest infection on day 4. A bone marrow biopsy and trephine demonstrated marked infiltration with carcinoma. This patient was deemed ineligible for toxicity evaluation and was replaced by an additional patient in cohort two. No patient suffered clinical complications of thrombocytopenia such as hemorrhage or purpura or evidence of thrombotic complications.

Other toxicities
There were no other toxicities attributable to PI-88 administration. In particular, there were no other hematological or biochemical abnormalities. However, two patients developed complications related to their intravenous access: one patient fractured his PICC line requiring replacement but continued on study, while another patient developed localized cellulitis at the PICC line entry site. This complication was successfully treated with intravenous antibiotics but the patient was taken off study after receiving 48 h of PI-88 treatment.

Platelet aggregation (SRA)
Functional AHPF4 antibodies (as indicated by platelet aggregation) were assayed using SRA in all 14 patients (Table 3). No patient had functional antibodies prior to the commencement of the study. Three patients had a positive result for functional antibodies at day 8, 17 and 90, respectively. Two of these three patients received 2.28 mg/kg/day for 8 and 14 days, respectively, and both patients developed grade 3 thrombocytopenia (Figure 2). Another patient who had a positive result for functional antibodies had received 0.57 mg/kg/day for 14 days for four cycles of therapy. This patient did not develop thrombocytopenia. The patient who developed grade 1 thrombocytopenia and ceased PI-88 on day 6 of treatment had an indeterminate SRA result due to heparin-dependent serotonin release detected at low and high heparin dosages suggesting non-specific effects.


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Table 3. Anti-heparin platelet factor 4 complex antibody studies
 
Anti-heparin platelet factor 4 complex antibodies (ELISA)
AHPF4 antibodies were assayed using ELISA in seven patients (Table 3). One patient had antibodies prior to the commencement of the study but these were absent at the cessation of the first cycle of treatment (0.57 mg/kg/day) in this patient. This patient received four cycles of treatment and did not develop thrombocytopenia. Four patients receiving 2.28 mg/kg/day developed AHPF4 antibodies on days 7, 7, 7 and 10, respectively (Figure 2). A positive AHPF4 titer was correlated with a positive SRA result in two patients with thrombocytopenia.

Antitumor activity
No objective tumor responses were documented. Two patients with documented progression of their tumor prior to commencing PI-88 had stabilisation of their disease. One patient (patient 5) with renal cell cancer developed stable disease and received 12 cycles of therapy (36 weeks) at which time his disease progressed and treatment was ceased. One patient (patient 7) with metastatic melanoma also achieved stable disease but developed disease progression after four cycles (12 weeks) of therapy and ceased treatment as a consequence.

Anticoagulant studies
Serial APTT estimations were documented in all 14 patients. Two patients developed prolongation of their APTT above baseline (30% and 80%, respectively). These patients were in cohorts one and seven, respectively. In both patients, APTT fell to within normal limits rapidly following the cessation of PI-88. Neither of these patients achieved a prolongation of APTT that met the protocol definition of DRT. No other patient demonstrated significant APTT prolongation. Neither of the patients who achieved stable disease developed prolongation of their APTT.

Other clotting parameters were also evaluated including prothrombin time (PT), fibrin degradation products, D-dimers and fibrinogen. There was no significant change in the values of these parameters during or at the completion of PI-88 treatment.


    Conclusions
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Conclusions
 References
 
PI-88 is a novel heparanase inhibitor with in vitro and in vivo anti-angiogenic and anti-metastatic activity [912]. It also has anticoagulant effects as indicated by prolongation of APTT, although it is less potent than either heparin or low molecular weight heparins [912]. This phase 1b study examined PI-88 in patients with advanced malignancies, using a single patient cohort, dose duration and dose escalation strategy. The study used APTT as a surrogate endpoint for biological activity and as a major consideration in identifying DLTs.

Treatment was complicated by thrombocytopenia that was unexpected and dose limiting at 2.28 mg/kg/day over a 14 day infusion period. It appears to have developed as an immune-mediated response with the formation of AHPF4 complex antibodies resulting in a syndrome similar to heparin-induced thrombocytopenic syndrome (HITS) [13]. Of the 14 patients treated, two (14%) developed a positive SRA suggesting the formation of functional AHPF4 complex antibodies and developed significant thrombocytopenia. Four of seven patients (57%) tested developed AHPF4 antibodies as detected by ELISA, with two of these patients developing thrombocytopenia. One additional patient ceased treatment at the discretion of the investigators due to concerns that they were developing a significant thrombocytopenia. This patient did not have antibodies detected by ELISA and had an indeterminate SRA result.

HITS is a well-characterized syndrome in which 0.5–5% of patients receiving full dose heparin develop thrombocytopenia [14]. In general, thrombocytopenia occurs 5–22 days after the commencement of heparin and may be severe [14]. Thrombocytopenia resolves within three to seven days in most cases. Bleeding complications are uncommon and the major sequelae are thrombotic events [15]. HITS may arise after any dose of heparin although it is more common following full dose therapy. Similarly, HITS may develop following administration of heparin via both the intravenous and subcutaneous routes, although it appears to be less common following subcutaneous administration [14].

A HITS-like phenomenon may occur with the administration of highly sulfated and highly negatively-charged oligosaccharides such as heparin, low molecular weight heparins, pentosan polysulfate and related molecules [14, 16]. AHPF4 antibodies form following administration of these compounds and result in platelet activation, aggregation and subsequent platelet and endothelial cell destruction [14].

Preclinical studies of PI-88 undertaken in monkeys and rats had not demonstrated any evidence of thrombocytopenia nor suggested that AHPF4 antibodies may be a clinical consequence of administration. Indeed, there was an expectation that antibody formation may have been less likely given the smaller molecular size of PI-88 compared with heparin. Furthermore, PI-88 had no effect on platelet function when administered at higher concentrations in pre-clinical studies. Nor was there evidence of thrombocytopenia in these studies or in the normal volunteer study. Finally, although pre-clinical studies demonstrated that PI-88 cross-reacted with anti-heparin antibodies, no in vivo preclinical models have been developed to examine HITS development.

The immune response following the administration of PI-88 in this small group of patients was heterogeneous. It is clear that PI-88 did result in the development or up-regulation of AHPF4 antibodies. However, the generation of AHPF4 antibodies did not necessarily result in a positive SRA or in thrombocytopenia itself. These inconsistent results may simply reflect the limitations regarding the specificity and sensitivity of both SRA and ELISA or the heterogeneity associated with HITS itself [17].

The possibility of pre-exposure to heparin or low molecular weight heparin in these patients has been considered as this may be partly responsible in potentiating the generation of AHPF4 antibodies after treatment with PI-88. One patient who developed thrombocytopenia had documented exposure to subcutaneous heparin (5000 U, b.d.) as surgical prophylaxis for a procedure performed 18 months prior to the current study. No other patient who developed a positive SRA, ELISA or thrombocytopenia had a documented prior exposure to heparin.

The early cessation of the study severely hampered interpretation of the biological activity of PI-88 and establishing the true incidence of immune-related thrombocytopenia. In this study, PI-88 had limited biological activity in the doses and schedules administered with only two patients demonstrating any evidence of APTT prolongation at the doses given prior to cessation of the study.

The true incidence of PI-88-induced immune-related thrombocytopenia cannot be deduced from this study. Single patient cohorts may erroneously give the impression of a DRT. Consideration was given to expanding cohort six in order to define the MTD according to protocol requirements. The investigators decided against this, taking into consideration the relative lack of biological activity according to the surrogate marker APTT, as well as the concern regarding the immunological nature of the thrombocytopenia that was unlikely to be dose-related [15]. Thus the study was closed and a recommended dose of PI-88 for future trials has not been established using this schedule of administration.

In conclusion, this study examined the tolerability and biological activity of PI-88, a novel heparanase inhibitor in the treatment of advanced malignancies. Thrombocytopenia was dose limiting at 2.28 mg/kg/day over a 14 day infusion period. Thrombocytopenia was related to the development of a HITS-like immune phenomenon. At this dose there was limited evidence of biological activity as measured by the surrogate of APTT although two patients achieved disease stabilisation. Further preclinical and clinical studies are required to examine alternate scheduling and routes of administration including shorter infusion times, bolus administration and subcutaneous delivery.


    Acknowledgements
 
We thank Heike Raunow and Rosetta Hart for their care of the patients.


    Footnotes
 
+ Correspondence to: Dr M. A. Rosenthal, Department of Medical Oncology and Clinical Oncology, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia. Tel: +61-3-9342-7560; Fax: +61-3-9347-7508; E-mail: mark.rosenthal@mh.org.au Back


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Conclusions
 References
 
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