Marked therapeutic efficacy of a novel polyethylene glycol-SN38 conjugate, EZN-2208, in xenograft models of B-cell non-Hodgkin's lymphoma

Examination of the clinical utility of SN38 (10-hydroxy-7-ethyl-camptothecin),the active metabolite of CPT-11, has not been possible to datedue to poor solubility of SN38. Here we evaluated the activityof EZN-2208, a water-soluble polyethyleneglycol-SN38 conjugate,in pre-clinical models of Burkitt’s non-Hodgkin’slymphoma (NHL) (Raji and Daudi), and follicular NHL (DoHH2).In vitro, the IC₅₀ of EZN-2208 ranged from 3–24 nM, whichwas 30- to 45-fold lower than CPT-11 or 2.5- to 3.5-fold higherthan SN38. In both an early-disease Raji model and an advanced-diseaseDaudi model, treatment with multiple doses of EZN-2208 resultedin 90% and 100% cures of animals, respectively (cure definedas no sign of tumors by gross observations at the terminationof study). The activity of EZN-2208 was dramatically superiorto that of CPT-11 in all three models. The excellent therapeuticefficacy of EZN-2208 in several B-cell NHL xenograft modelsmerits its evaluation in the clinic for lymphoid malignancies.

Key words: SN38, polyethylene glycol, drug conjugate, B-cell non-Hodgkin’s lymphoma, CPT-11, in vivo.

Introduction

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Introduction

Despite recent advances in the treatment of B-cell non-Hodgkin’slymphoma (NHL), novel therapeutics with improved efficacy andsafety are needed. Although a wide variety of chemotherapeuticoptions are available for patients with B-cell NHL, these drugs,even when combined with radiation therapy, have not resultedin long-term disease-free survival in the majority of the patients.^1,2Over the last decade, rituximab treatment has revolutionizedthe therapy of NHL. However, as these patients progress afterreceiving multiple rituximab-based regimens, alternative treatmentoptions are needed for this group of patients as well.³

CPT-11 (Camptosar®, irinotecan), a DNA topoisomerase I inhibitor,is widely used for the treatment of patients with advanced colorectalcancer and some other solid tumors. Although in some Phase Ior II trials a response rate of 25–62% has been reportedfor patients with relapsed or refractory aggressive lymphomaswho received CPT-11,^4–8 the small study sizes, the widespectrum of histological types of tumors, and the diverse dosageand schedule options have made it difficult to assess the efficacyof CPT-11 in this patient population. In addition, the highunpredictable gastrointestinal toxicity can compromise the clinicalutility of CPT-11. Furthermore, CPT-11 as a drug has severallimitations, including the requirement of an endogenous enzymeto convert the pro-drug (CPT-11) to the active form (SN38),leading to inter-individual variability in drug safety profileand antitumor acitivity. Therefore, novel topoisomerase I inhibitorsthat can mitigate these limitations are sought. SN38 (10-hydroxy-7-ethyl-camptothecin)is the active moiety of CPT-11. Hydrolysis of the water-solublepro-drug, CPT-11, to SN38 by carboxylesterase 2 is associatedwith a 100- to 1,000-fold increase in cytotoxicity.⁹ Despitepromising anti-cancer potential in the laboratory, currentlySN38 itself has not been used as an anticancer drug in humansdue to its poor solubility in any pharmaceutically acceptableexcipient. The solubility of SN38 can be vastly improved byPEGylation. EZN-2208 (polyethylene glycol [PEG]-SN38) is a water-solublePEGylated conjugate with approximately 3.5 to 4.0 SN38 moleculesattached to the multi-arm PEG backbone.¹⁰ We have previouslyshown in pre-clinical models using multiple solid tumors (includinga model of in vivo CPT-11 resistance), that EZN-2208 has a significantlyenhanced therapeutic index compared with CPT-11.¹¹ In thesestudies, EZN-2208 provides higher exposure of tumors to SN38via the preferential accumulation of EZN-2208 in the solid tumors(enhanced permeability and retention [EPR] effect) comparedwith CPT-11.¹¹

In this paper, we demonstrate that treatment with EZN-2208 cancure animals in pre-clinical models of B-cell NHL (Raji, Daudi,DoHH2). The high anti-tumor activity may be attributed, in part,to improved pharmacokinetics as reported by us previously.¹¹

Design and Methods

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Design and Methods

Materials, cell lines and animals
EZN-2208 was prepared as described previously.^10,11 Irinotecan(CPT-11, Camptosar®), was obtained from Bell Medical Services,NJ, USA. The human B-cell lymphoma cancer cells, Raji (Burkitt’s)and Daudi (Burkitt’s), were obtained from American TypeCulture Collection (Manassas, VA, USA), and the DoHH2 (follicular)cell line was obtained from Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH (DSMZ, Germany). Seven- to eight-weekold female BALB/c or CB17 SCID mice were purchased from Harlan,Indianapolis, IN, USA. All animal studies were approved by theUniversity of Medicine and Dentistry New Jersey InstitutionalAnimal Care and Use Committee.

Antiproliferation assay and in vitro apoptosis analysis
The antiproliferative activity of EZN-2208 was determined usingthe Cell Titer 96 Aqueous One Solution (MTS) (Promega, Madison,WI, USA) as described previously.¹¹ Apoptosis analysis was performedusing the annexin V-FITC/propidium iodide assay. (Invitrogen/Molecular Probes, Eugene, OR, USA) (further details are includedin the Online Supplementary Appendix).

In vivo therapeutic efficacy in B-cell NHL xenografts
The therapeutic efficacy of EZN-2208 was evaluated in threehuman xenograft models of B-cell NHL. Female CB17 SCID (n=6–10/group)mice were injected i.v. with Raji, DoHH-2 and Daudi cells at2.5x10⁶ cells/mouse, 10⁷ cells/mouse, and 1.5x10⁷ cells/mouse,respectively. For early-disease models, the mice received treatmentsof CPT-11 or EZN-2208 either at the single or multiple (q2dx5)MTD doses on day 1 (Raji and Daudi) or day 2 (DoHH2) post-administrationof cells. For the advanced-disease models, mice received EZN-2208or CPT-11 on day 6 (Daudi) or day 11 (DoHH2) post-administrationof cells. The single MTD of EZN-2208 and CPT-11 in SCID miceis 30 mg/kg and 60 mg/kg, respectively. The multiple (q2dx5)MTD of EZN-2208 and CPT-11 in SCID mice is 10 mg/kg and 40 mg/kg,respectively. In these models, tumors grow systemically andmice become paralyzed when tumor cells infiltrate the spinalcord, resulting in hind-leg paralysis. Mice were sacrificedat onset of paralysis or when they lost 20% of their pre-treatmentbody weight. The observation period lasted five to seven timesthe median survival time (MST) of control animals, at whichtime all mice were sacrificed and a gross necropsy was performed.In vivo survival studies were analyzed using Kaplan-Meier plots,with GraphPad Prism software. Cure of animals was defined asno evidence of tumor by gross observation at the terminationof the study. It is important to note that the doses or concentrationsof EZN-2208 stated in this paper refer to SN38 equivalents.For example, a dose of 30 mg/kg of EZN-2208 is equivalent toa dose containing 30 mg/kg of SN38, or 829 mg/kg (28-fold higher)of whole conjugate, when loading SN38 in the whole EZN-2208conjugate is 3.62%.

Results and Discussion

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Results and Discussion

We show here excellent therapeutic efficacy of EZN-2208 in threemodels of pre-clinical B-cell NHL. In vitro, EZN-2208, as wellas SN38, demonstrated potent cytotoxicity compared to CPT-11in a panel of human lymphoma cells lines. The IC₅₀ of EZN-2208ranged from 3–24 nM, which was 30- to 45-fold more potentthan CPT-11 (Table 1). The cytotoxicity of EZN-2208 was 2.5-to 3.5-fold less than that of SN38 (1.2 to 5 nM). The impressivelow nM IC₅₀ of SN38 or EZN-2208 in various lymphoma lines suggestthe utility of this agent in the clinic for patients with lymphoidmalignancies. In terms of apoptosis assays, no significant differencein apoptosis-inducing effects of EZN-2208 versus SN38 was observed(Online Supplementary Appendix). The major mechanism of cellkilling at 24 h was rapid progression to late apoptosis andcell necrosis. From 24 to 48 h, any residual viable cells underwenta slower progression to early apoptosis before progressing tonecrosis.

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Table 1. In vitro cytotoxicity.^a

Since experiments performed in vitro typically do not capturethe advantages of PEGylating SN38, such as improved pharmacokinetics,they may underestimate the efficacy of EZN-2208. In fact, excellentin vivo therapeutic efficacy of EZN-2208 was obtained in allthree models of B-cell NHL (Raji, Daudi and DoHH2). The firststudy was performed in the Raji model (Figure 1). Whereas controlanimals treated with saline had a median survival time (MST)of 16 days, treatment with a single MTD of EZN-2208 resultedin cure for 50% of animals (Figure 1A). In contrast, CPT-11dosed at its single MTD resulted in only a 25% increase in lifespan (ILS) (Figure 1A). In the same model, treatment of animalswith multiple doses of EZN-2208 resulted in cures of 90% ofanimals in contrast to no cures and 169% ILS observed for multipledose CPT-11 group (Figure 1B).

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Figure 1. Therapeutic efficacy of EZN-2208 in a xenograft model of NHL (Raji). CB17 SCID mice (6–10 mice per group) were inoculated i.v. with 2.5x10⁶ Raji cells per mouse. After one day, mice were treated with either a single (qdx1) or multiple (q2dx5) injections of EZN-2208 or CPT-11 at MTD. (A) Single MTD doses: Control ( ${blacksquare}$ ), EZN-2208 at 30 mg/kg ( ${blacktriangleup}$ ), CPT-11 at 60 mg/kg ( ${circ}$ ) (B) Multiple MTD doses (q2dx5): Control ( ${blacksquare}$ ), EZN-2208 at 10 mg/kg ( ${blacktriangleup}$ ), CPT-11 at 40 mg/kg ( ${circ}$ ).

We then evaluated the therapeutic efficacy of EZN-2208 in relationto tumor burden in two other B-cell NHL xenograft models, Daudiand DoHH2. For the Daudi model, treatments were given eitherone day or six days post-administration of cells, thus establishingan early-disease model or a late-disease model, respectively(Figure 2A). In the early-disease model, where control animalshad an MST of 35 days, a single MTD of EZN-2208 resulted incures of 6 of 9 animals in contrast to only a 66% ILS and nocures observed for the group that received a single MTD of CPT-11.In this setting, multiple injections of EZN-2208 cured 100%of animals. Significant cures (5 of 9) were also obtained whenCPT-11 was given as multiple injections in an early-diseasesetting. However, in the advanced-disease setting, CPT-11 efficacywas significantly diminished. A single MTD of CPT-11 was completelyineffective, and multiple injections of CPT-11 only caused 137%ILS with no cures. Conversely, EZN-2208 had a dramatic effectin the advanced-disease model. A single MTD of EZN-2208 cured5 of 9 animals, and multiple injections of EZN-2208 cured 100%of animals. Another experiment was performed in DoHH2 modelwhere the efficacy of EZN-2208 was begun two days after tumorinjection or with an even more advanced-disease setting comparedto the Daudi model above (treatment was initiated 11 days aftercell injection). Where control animals had MST of 19 days, multiple-doseEZN-2208 treatment started two days after cell injection resultedin cures of 72% of animals; however, CPT-11 treatment resultedin a MST of 66 days and 42% cures. When treatment was initiated11 days post-administration of cells, CPT-11 or EZN-2208 treatmentresulted in MST of 43 days or 77 days, respectively. We do realizethat these models are representative of Burkitt’s (Rajiand Daudi) or follicular (DoHH2) B-cell NHL and hence generalclaim of utility of EZN-2208 in all settings of lymphoid malignanciesmay not be applicable. However, the impressive pre-clinicalefficacy shown here in these models does serve as proof of conceptand merits the investigation of this agent in patients withlymphoid cancers. The excellent therapeutic efficacy of EZN-2208is attributed, in part, to the favorable pharmacokinetic profileachieved by the drug. We have shown previously in solid tumorxenograft models that EZN-2208 provides longer circulation timesto the active moiety (SN38) compared with CPT-11.¹¹

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Figure 2. (A) Therapeutic efficacy of EZN-2208 in an early- (first graph) versus advanced- (second graph) disease xenograft model of NHL (Daudi). CB17 SCID mice (6–10 mice per group) were inoculated i.v. with 1.5x10⁷ Daudi cells per mouse. EZN-2208 or CPT-11 was given either as single (qdx1) or multiple (q2dx5) MTD doses. Early-disease model treatment started on day 1, and advanced-disease model treatment started on day 6. Control ( ${blacksquare}$ ), EZN-2208 at 30 mg/kg qdx1 ( ${blacktriangleup}$ ), CPT-11 at 60 mg/kg qdx1 ( ${triangleup}$ ), EZN-2208 at 10 mg/kg q2dx5 ( $bullet$ ), CPT-11 at 40 mg/kg q2dx5 ( ${circ}$ ). (B) Therapeutic efficacy of EZN-2208 in an early-versus advanced-disease xenograft models of NHL (DoHH2). CB17 SCID mice (6–10 mice per group) were inoculated i.v. with 1.0x10⁷ DoHH2 cells per mouse. EZN-2208 or CPT-11 was given as multiple (q2dx5) MTD doses starting on day 2 or on day 11 post-administration of cells. Control ( ${blacksquare}$ ), EZN-2208 at 10 mg/kg q2dx5 starting on day 2 ( ${blacktriangleup}$ ), CPT-11 at 40 mg/kg q2dx5 starting on day 2 ( ${triangleup}$ ), EZN-2208 at 10 mg/kg q2dx5 starting on day 11 ( $bullet$ ), or CPT-11 at 40 mg/kg q2dx5 starting on day 11 ( ${circ}$ ).

Besides giving an improved pharmacokinetic advantage, EZN-2208may also have a novel mechanism of action. We have preliminaryevidence using a hypoxia-inducible factor-1 (HIF-1) reportercell line that treatment with a single dose of EZN-2208 resultsin potent, sustained downmodulation of HIF-1 ${alpha}$ (~80% by day 5),whereas CPT-11 has no effect; CPT-11 has minor effects onlywhen the drug is given in multiple doses.¹² This observationis in agreement with studies published by Rapisarda et al. inwhich topotecan, another topoisomerase I inhibitor, administeredon a daily but not an intermittent schedule, resulted in down-regulationof HIF-1 ${alpha}$ and tumor growth inhibition.^13,14 Thus, a prolongedexposure of SN38 by EZN-2208 can down-regulate HIF-1 ${alpha}$ in a morepotent and sustained manner compared to CPT-11. Besides beingassociated with poor prognosis in many types of solid tumors,the literature also suggests that HIF-1 is frequently activatedin lymphoma and may contribute to disease progression.¹⁵ Inone study, 44% of diffuse large B-cell lymphoma versus 11% offollicular lymphoma biopsies had moderate-to-high expressionof both HIF-1 ${alpha}$ and HIF-2 ${alpha}$ .¹⁶

A significant limitation of CPT-11 in the clinic is frequentand unpredictable gastrointestinal toxicity. In an ongoing PhaseI program in solid tumors and lymphoid malignancies, where EZN-2208was administered as a one hour intravenous infusion every threeweeks, no early late gastrointestinal toxicities were observed,and neutropenic fever was the dose-limiting toxicity.¹⁷

In conclusion, we have developed a novel PEGylated SN-38 drugconjugate which demonstrates significant therapeutic efficacyin several models of B-cell NHL and may have a distinct mechanismof action (via potent and sustained inhibition of HIF-1 ${alpha}$ ) comparedto CPT-11. Excellent pre-clinical data and a favorable safetyprofile of EZN-2208 in Phase I studies support further evaluationof this compound in patients with B-cell NHL.

Footnotes

Authorship and Disclosures

PS conceived and designed the experiments, analyzed and interpretedthe data and wrote the manuscript; PK, MM and JM performed thein vitro and in vivo experiments and analyzed the data; HZ developedthe chemistry of EZN-2208; LG and IH contributed in the designof experiments, interpretation of the data and writing of themanuscript; IH conceived the concept of developing EZN-2208.

All authors are full time employees of Enzon Pharmaceuticalsand own company stock options and/or units.

Received for publication March 5, 2009. Revision received April 23, 2009. Accepted for publication May 11, 2009.

References

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