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Cancer Therapy: Preclinical

Preclinical Toxicity, Toxicokinetics, and Antitumoral Efficacy Studies ofDTS-201,aTumor-SelectivePeptidicProdrugofDoxorubicin Denis Ravel,1Vincent Dubois,1Je¤ro“ me Quinonero,1Florence Meyer-Losic,1JeanPierre Delord,2 Philippe Rochaix,2 Ce¤line Nicolazzi,1Fabien Ribes,1Catherine Mazerolles,3 Elise Assouly,1 Karine Vialatte,1Ine'sHor,1Jonathan Kearsey,1and Andre¤Tr o u e t 1

Abstract Purpose: There is a clear clinical need for cytotoxic drugs with a lower systemic toxicity. DTS- 201 (CPI-0004Na) is a peptidic prodrug of doxorubicin that shows an improved therapeutic index in experimental models. The purpose of the current study was to complete its preclinical characterization before initiation of phase I clinical trials. Experimental Design:The preclinical development program consisted of a detailed assessment of the general and cardiac toxicity profiles of DTS-201in mice, rats, and dogs, together with mass balance and antitumoral efficacy studies in rodents. Neprilysin and thimet expres- sion, two enzymatic activators of DTS-201, was also characterized in human breast and prostate tumor biopsies. Results: The target organs of DTS-201toxicity in rodents and dogs are typically those of doxo- rubicin, albeit at much higher doses. Importantly, chronic treatment with DTS-201proved to be significantly less cardiotoxic than with doxorubicin at doses up to 8-fold higher in rats. The mass balance study showed that [14C] DTS-201does not accumulate in the body after intravenous administration. The improved therapeutic index of DTS-201compared with free doxorubicin was confirmed in three tumor xenograft models of prostate, breast, and . Neprilysin and/or are expressed in all experimental human tumor types thus far tested as well as in a large majority of human breast and prostate tumor biopsies. Conclusion: DTS-201gave promising results in terms of general toxicity, cardiovascular toler- ance, and in vivo efficacy in xenograft mouse models compared with free doxorubicin. Taken together, these results and the confirmation of the presence of activating in human tumor biopsies provide a strong rationale for a phase I clinical study in cancer patients.

Doxorubicin, an anthracycline antibiotic, remains among the Even if new targeted approaches, such as antibodies most widely used cytotoxic agents for the treatment of a broad (including trastuzumab; Herceptin; Roche) or tyrosine kinases spectrum of , including breast, stomach, non–Hodgkin inhibitors (e.g., lapatinib; GlaxoSmithKline), are significantly , and (1, 2). As with many cytotoxic less toxic than conventional cytotoxic agents, they often have to drugs, the use of doxorubicin is restricted due to severe dose- be used in combination with cytotoxic molecules to achieve limiting side effects resulting from its lack of tumor targeting. significant efficacy in the clinic (4, 5). There is therefore still a The major dose-limiting toxicities of free doxorubicin are real clinical need for a new generation of cytotoxic molecules neutropenia as an acute toxicity and cardiomyopathy, which is with increased efficacy and reduced systemic toxicity. One such cumulative and significantly increases the risk of congestive novel cytotoxic molecule is DTS-201 (CPI-0004Na), a peptidic as the total dose administered to a patient reaches prodrug of doxorubicin. DTS-201 (N-succinyl-h-alanyl-L- 2 values of 500 to 600 mg/m . As a result, treatment with leucyl-L-alanyl-L-leucyl-doxorubicin) is stable and inactive in doxorubicin or other anthracyclines frequently has to be its cell-impermeable prodrug form. The cell-impermeable discontinued, although patients are still responsive (1–3). nature of DTS-201 has been confirmed both in vitro and in vivo, using quantitative cell uptake assays, biodistribution studies, and pharmacokinetic studies that measure distribution Authors’ Affiliations: 1Diatos S.A., Paris, France; 2EA 3035, Institut Claudius- volumes (6, 7). In the vicinity of a tumor, the tetrapeptide Regaud, Universite Paul-Sabatier, Toulouse, France; and 3Service d’Anatomie et portion of the DTS-201 prodrug is cleaved by Cytologie Pathologiques, Ho“ pital de Rangueil,Toulouse, France that are released extracellularly in the tumor environment. This Received 5/11/07; revised 9/27/07; accepted 11/27/07. N N The costs of publication of this article were defrayed in part by the payment of page yields the metabolites -L-alanyl-L-leucyl-doxorubicin and -L- charges. This article must therefore be hereby marked advertisement in accordance leucyl-doxorubicin, which can enter cells and are converted to with 18 U.S.C. Section 1734 solely to indicate this fact. the active drug, doxorubicin. This results in an increased Requests for reprints: Jonathan Kearsey, Diatos S.A., 11rue Watt, 75013 concentration of doxorubicin in the tumor and reduced Paris, France. Phone: 33-1-53-80-93-49; Fax: 33-1-53-80-93-89; E-mail: [email protected]. doxorubicin levels in normal tissues (6, 7). F 2008 American Association for Cancer Research. Two tumor-specific endopeptidases have been identified that doi:10.1158/1078-0432.CCR-07-1165 cleave DTS-201: neprilysin (CD10, EC3.4.24.11; ref. 8) and

Clin Cancer Res 2008;14(4) February 15, 2008 1258 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2008 American Association for Cancer Research. DTS-201Preclinical Evaluation thimet oligopeptidase (TOP, EC3.4.24.15; ref. 9). These cinoma; ATCC# CL-188), HCT 116 (colon ; ATCC# CCL- endopeptidases are released in the extracellular space of solid 247), and Calu6 (lung anaplastic carcinoma; ATCC# HTB-26) were tumors by stromal, tumor, and neoangiogenic endothelial cells used for immunohistochemical analysis of enzymes expression in or expressed at their cell surface (10, 11). The extracellular tumor xenografts. Acute toxicity studies in rodents. Groups of 5 male and 5 female localization of these enzymes allows them to cleave and young adult Swiss CD-1 mice (Charles River Laboratories France) activate DTS-201 (8, 9). With the exception of some classes of received a single 15-minute intravenous infusion of vehicle (NaCl 0.9% and the brush borders of some epithelia, CD10 w/v), or 40, 60, 80, or 100 mg/kg of DTS-201. Clinical signs, body expression in normal tissues is limited, whereas it is over- weight, local tolerance, and mortality were monitored up to day 29, at expressed in a wide range of tumor types (12). TOP expression which point, the surviving animals were sacrificed for necropsy. Organs is less well-characterized. The normal tissue distribution of TOP were weighed immediately after dissection. has not yet been determined. Significant expression has been A similar study was done with groups of 6 male and 6 female young described in the central nervous system, testes, and some adult Sprague-Dawley rats (Charles River Laboratories France) using classes of lymphocytes. doses of DTS-201 of 30, 47, 60, and 73 mg/kg. Additional monitoring A significant therapeutic advantage of using the DTS-201 included hematologic and blood biochemistry investigations done on days 5, 11, 16, 22, and 29. –activated prodrug compared with free doxoru- Acute toxicity and toxicokinetics in the dog. Groups of 2 male and 2 bicin has already been shown in a number of different tumor female young adult Beagle dogs (Marshall Farms) received a single 15 models including breast, colon, prostate, and lung cancers minute intravenous infusion of vehicle or of DTS-201 at dose levels of (6–8, 13, 14). Pharmacokinetic and tissue distribution studies 4, 8, 16, or 32 mg/kg. The animals were monitored for clinical signs, on normal and tumor-bearing mice have confirmed that the local tolerance, body weight, rectal temperature, and mortality up to improved therapeutic index of the prodrug results from a day 29. In addition, electrocardiogram and blood pressure were tumor-selective release of doxorubicin, together with a signif- recorded before dosing, 4 and 24 h postdosing, and at the end of the icant decrease in doxorubicin levels in all normal tissues tested study. Hematologic and blood biochemistry investigations, as well as (6). In particular, a 10-fold decrease in doxorubicin levels in urine analysis, were also done predose and on days 5, 11, 16, 22, and cardiac tissue is observed after DTS-201 treatment, compared 29. Upon sacrifice, a complete macroscopic examination was done and organs were dissected and weighed. with an equimolar dose of free doxorubicin. Increased tumor Blood samples were collected on sodium citrate up to 48 h postdose exposure together with reduced cardiac exposure to doxorubi- for a toxicokinetic evaluation of DTS-201 in all animals, using a fully cin in animals after treatment with DTS-201 therefore provides validated method. DTS-201 and its metabolites (doxorubicin, N-L- a strong rationale for this prodrug approach. leucyl-doxorubicin and N-L-alanyl-L-leucyl-doxorubicin) were extracted This paper describes the preclinical development work that from plasma samples using solid-phase extraction and quantified by further supports the evaluation of this prodrug in a phase I high-performance liquid chromatography with tandem mass spectro- clinical study in patients with solid tumors. General toxicity metric detection analysis using a triple quadrupole mass spectrometer studies in rodents and dogs show that the target organs of DTS- (API 2000; PE Sciex) with a TurboIonSPray source. Briefly, plasma A A 201 toxicity are the same as for doxorubicin, but that this samples were supplemented with 40 L of methanol, 25 Lofa solution of flurbiprofen in methanol (internal standard), and 1 mL of a toxicity occurs at much higher dose levels. Reduced cardiac 0.2% formic acid solution. The mixtures were then loaded on Oasis toxicity was observed in a rat model that has been shown to be HLB cartridges (Waters) previously conditioned with 2 mL of methanol predictive for the human cumulative cardiotoxicity induced by followed by 2 mL of water. After washing with 1 mL of water, DTS-201 anthracyclines (15, 16). DTS-201 also showed an improved and its metabolites were eluted with 500 AL of methanol. Thirty efficacy in prostate, breast, and lung tumor models. The microliters of the eluates were then injected onto a 20 Â 3.9 mm, 5-Am potential utility of this prodrug approach in prostate and Oasis HLB column (Waters) and separated using a 15% to 60% has also been further explored through the acetonitrile gradient (in 5 Amol/L sodium trifluoroacetate-0.1% formic immunohistochemical analysis of the expression of the two acid) over 5 min at a flow rate of 0.8 mL/min. The three metabolites endopeptidases that cleave DTS-201 (TOP and CD10) in >200 were detected in the positive mode (first 3.5 min) and DTS-201 and flurbiprofen in the negative mode with multiple reaction monitoring human tumor biopsies. Expression of either one or both for transition of the parent ions to the product ions [Parent ion (m/z)/ endopeptidases was observed in almost all biopsies, suggesting Product ion (m/z): DTS-201, 1010/614; flurbiprofen, 243/199; AL-Dox, that both prostate and breast cancers are indications for which 728/314; L-Dox, 657/243; doxorubicin, 544/397]. The plasma concen- DTS-201 may show clinical value. tration of each analyte was determined by interpolation with the appropriate calibration curve. Subchronic toxicity studies in rodents. Groups of 10 to 15 male and Materials and Methods 10 to 15 female young adult Swiss CD-1 mice (Charles River Laboratories France) received one 15-minute intravenous infusion of Drugs and cell lines. DoxorubicinÁHCl (580 MW) was purchased vehicle or DTS-201 at 50, 100, or 150 mg/kg on day 1 and on day 22. In from Meiji Seika Pharma Kaisha, Ltd. DTS-201 (1034 MW) was addition to the monitoring described for the single-dose studies that synthesized in a similar manner as described elsewhere (13). The data was continued for 28 days after the second treatment, ophthalmologic in the results section are provided in mg/kg; to determine the quantity examinations, hematologic, and blood biochemistry investigations of doxorubicin present in the DTS-201 conjugate, the molecular weight were done. After sacrifice of all animals in the control and high-dose ratio DTS-201/doxorubicin (1.78) should therefore be taken into groups, a microscopic examination of the tissues defined in appendix A account. [14C-Doxorubicin] DTS-201 was prepared by Amersham of the European Medicines Evaluation Agency ‘‘Note for Guidance on Biosciences UK Limited. Saline was used as the vehicle for all drugs. Repeated Dose Toxicity’’ was done (17). The NCI-H1299 (non–small cell lung carcinoma; ATCC# CRL- A similar study was conducted with groups of 10 to 16 male and 10 5803), MDA-MB-231 (mammary adenocarcinoma; ATCC#HTB-26), to 16 female young adult Sprague-Dawley rats (Charles River and PC-3 (prostate carcinoma; ATCC# CRL-1435) human cell lines Laboratories France) treated with vehicle or 40, 80, or 100 mg/kg of were used for antitumoral activity studies. LS 174T (colon adenocar- DTS-201 on days 1 and 22.

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Acute cardiotoxicity study. Two male and two female adult Beagle maximum tolerated dose (MTD) was not reached in these dogs (CEDS) instrumented with telemetric transmitters (Transoma single-dose studies. In the mouse, treatment-related toxic effects Medical) for electrocardiogram, blood pressure, and heart rate were observed exclusively in males. At the highest dose level monitoring received successive intravenous infusions of DTS-201 at tested (100 mg/kg), a 6% body weight loss was observed on 3.2, 12.8, and 25.6 mg/kg with minimum washout periods of 7 days. average as well as a progressive paralysis of hind limbs starting Telemetric measurements started 24 h before dosing and continued for 24 h after treatment. 20 days after treatment. Degeneration of the testes was observed Chronic (cumulative) cardiotoxicity study. Groups of 10 male adult in all animals with a 60% reduction of the organ weight Sprague-Dawley rats (Charles River Laboratories France) received seven compared with controls whatever the dose level. In rats, besides consecutive weekly intravenous bolus injections of vehicle, doxorubi- a 4% to 16% dose-dependent decrease in body weight gain and cin, at 1.25 mg/kg, or DTS-201 at 2.23, 7.9, or 17.8 mg/kg. After a 9- a 20% reduction of testes weight in all male groups, a transient week treatment-free period, cardiotoxicity was evaluated semiquantita- decrease in WBC count was observed in both genders at doses tively by a histologic assessment of semi-thin sections of heart tissue of 47 mg/kg or higher. WBC counts were back to normal values using a scoring system specifically devised for anthracyclines and taking by day 16 after treatment. into account the severity (0-2) and extent (0-5) of the myocardial In the dog, GLP studies showed that DTS-201 induced no lesions as described elsewhere (15, 18). The mean total score (mean adverse effects at the doses of 4 and 8 mg/kg. Dose-dependent, total score = severity  extent, 0 to 10) was calculated for each group. Mass balance study. Three male and three female adult Sprague- transient, and moderate decreases in WBC counts (mainly Dawley rats (Harlan UK Limited) received a single intravenous dose of neutrophils and lymphocytes) were observed at doses of 16 and 50 mg/kg of [14C-Doxorubicin] DTS-201, corresponding to 3.7 MBq/kg 32 mg/kg (up to -38 to -41%). One male (of four) dosed at (14C is in position carbon 13 of doxorubicin). Urine, feces, and expired 32 mg/kg, experienced diarrhea, vomiting, and a 10% body air samples were quantitatively collected before treatment and at weight loss, and had to be prematurely sacrificed 7 days after 8 (urine only), 24, 48, 72, 96, and 120 h posttreatment. Urine and treatment. The same clinical signs were observed in the other expired air duplicate samples were mixed with 10 mL of Gold Star dogs treated at this dose level but proved to be transient. liquid scintillator (Meridian). Duplicate aliquots of feces were com- Consequently, the MTD of DTS-201 after a single intravenous busted using a sample oxidizer [PerkinElmer LAS (UK) Ltd.]. Carbosorb infusion to Beagle dogs was considered to be between 16 and and Permafluor [PerkinElmer LAS (UK) Ltd.] were used as absorbent 32 mg/kg. and scintillator, respectively. All samples were counted in a liquid scintillation counter [Model 2000TR; PerkinElmer LAS (UK) Ltd.]. A toxicokinetic evaluation of DTS-201 was conducted at the Antitumoral activity. Seven-week-old male or female NMRI nu/nu four dose levels tested (4 to 32 mg/kg). DTS-201 and three mice (Janvier) were respectively injected with a PC-3 cell suspension metabolites (N-L-alanyl-L-leucyl-doxorubicin, N-L-leucyl-doxo- (1  107 cells/mL) or a MDA-MB-231 suspension (6  107 cells/mL) in rubicin, and doxorubicin) were detected in the plasma of the ventral skin or implanted with a NCI-H1299 tumor fragment. all animals (Fig. 1A shows the pharmacokinetic profile at a Treatment began 14 (PC-3 and MDA-MB-231) or 12 (NCI-H1299) days representative dose of 8 mg/kg). The DTS-201 Cmax value was 3 after the graft, when tumors were at least 70 mm . Drugs were observed at the end of the 15-min infusion and ranged on A administered intravenously (bolus; 10 L per gram of body weight) average from 14.4 F 4.5 Amol/L (4 mg/kg) to 209 F 26 Amol/L weekly for 4 (NCI-H1299) or 5 (PC-3) consecutive weeks. In the MDA- (32 mg/kg), increasing almost linearly with dose MB-231 study, two cycles of one weekly administration for 3 weeks (Table 1). The C values observed for the metabolites were were done. Doses were experimentally determined based on the max injection schedule, together with the strain and sex of mice used. also reached at the end of the infusion or within the following F Tumor volumes were determined using the following formula: 25 to 120 min but were much lower, ranging from 1.9 1.30 (length  width2)/2. Results are presented as the evolution of mean to 29.6 F 3.4 nmol/L in the case of doxorubicin. A linear tumor volume as a function of time. Minimal treated versus control relationship was also observed between the doxorubicin Cmax (ratio of mean tumor volume of treated versus control mice) values were values and the DTS-201 dose level administered. Plasma used as a measure of treatment efficacy. Error bars represent SE. concentrations of DTS-201 and its metabolites then decreased studies. Xenograft samples and human rapidly and in a regular manner until the last quantifiable time tumors were fixed in formalin and embedded in paraffin. The human point (4 to 8 h for DTS-201, N-l-alanyl-l-leucyl-doxorubicin, tumor samples were then grouped in Tissue Micro Arrays. The N-l-leucyl-doxorubicin depending on the dose level, and 24 immunostaining was carried out on 5-Am thick sections using the to 48 h for doxorubicin). The area under the plasma con- anti-CD10 mouse monoclonal antibody 56C6 (Novocastra Laborato- ries) diluted to 1/50 or the anti-TOP rabbit polyclonal antibodies EP24- centration versus time curves (AUC) of DTS-201, as well as 15 (Proteimax) diluted to 1/500 after a microwave treatment in a citrate of free doxorubicin, also increased in a dose-dependent buffer [10 mmol/L (pH 6)]. Labeling was carried out using the two-step manner. The DTS-201 AUC (calculated from 0 up to the last enVision (Dako) according to the manufacturer’s instructions. The quantifiable time point) ranged from 5.1 F 1.5 (4 mg/kg) to percentage of labeled cells was evaluated for stromal, tumor, and 127 F 13 nmolÁh/L (32 mg/kg), whereas the free doxoru- neoangiogenic endothelial cells. The immunostaining results were bicin AUC was several hundred-fold smaller, ranging from reported for each of these cellular groups using a two-point scale: 6.06 F 5.23 to 771 F 186 nmolÁh/L. positive staining consisted in at least 10% of labeled cells, whereas Subchronic toxicity in rodents. In the mouse, two successive negative staining consisted in <10% of labeled cells. doses of 100 or 150 mg/kg of DTS-201 3 weeks apart proved to be toxic with progressive hind limb paralysis, a transient Results decrease of WBC counts, and a reduced body weight gain or weight losses. Laboratory investigations and the full histopath- Acute toxicity in rodents and in the dog. Good Laboratory ologic examination conducted 4 weeks after the second Practice (GLP) studies showed that DTS-201 was well-tolerated injection identified the thymus (lymphoid depletion), the in mice and rats, up to a maximum dose tested of 100 mg/kg in genital tract (degeneration of the testes and atrophied mice and 73 mg/kg in rats. There was no mortality and the endometrial cells), and the peripheral nervous system (myelin

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Fig. 1. DTS-201pharmacokinetics and elimination. A, pharmacokinetics after intravenous infusion in the dog: DTS-201 was administered as a 15-minute infusion at 8 mg/kg, and blood samples were taken 0.25, 0.66, 1, 2, 4, and 8 h after the beginning of the infusion. DTS-201 (5) and its metabolites, AL-Dox (o), L-Dox (Â), and Dox (.)weremeasured.B, mass balance in rats. Cumulative recovery of radioactivity in rats after intravenous injection of [14C-doxorubicin] DTS-201. Recovery in feces (E), urines (.), expired air (5), and total recovery (^).

degeneration and/or inflammation of the sciatic nerve and 17.8 mg/kg per week). A weekly dose of 1.25 mg/kg of roots of the spinal nerves) as target organs of DTS-201 toxicity. doxorubicin (i.e., 8-fold less than the highest dose of DTS-201 The dose of 50 mg/kg was well-tolerated and considered tested on a molar basis) induced severe and significant cardiac the MTD. lesions (mean total score = 6.8) in all animals, characterized by In rats treated similarly, two injections of 40 mg/kg were swelling of muscle fibers, vacuolation of myocytes, loss of well-tolerated, whereas two injections of 80 mg/kg were clearly myofilaments, and interstitial fibrosis (Fig. 2). The doxorubi- toxic. Their poor clinical condition (hind limb paralysis and cin-induced lesions proved to be significantly more severe in tongue abscesses, and body weight loss) prompted the the doxorubicin-treated rats compared with the animals treated premature sacrifice of all males and of 3 of 10 females in that with an 8-fold higher molar dose of DTS-201. Statistical group. For the same reasons, all animals in the 100 mg/kg dose analysis of the cardiotoxicity scores obtained after treatment group had to be sacrificed. Dose-related, transient decreases in with each dose of DTS-201 compared with vehicle-treated WBC counts were also observed 5 days after each treatment. As animals did not reveal any significant difference. in the mouse, the target organs of toxicity were the thymus, the With regard to acute cardiac toxicity, electrocardiographic genital tract, and the peripheral nervous system. In addition, monitoring was done after a single injection of DTS-201 in the the spleen also showed decreased follicular size and hypo- dog. The treatment did not affect the blood pressure, cellularity, as did the bone marrow. The tongue was also electrocardiography variables, heart rate, or cardiac conduction affected with inflammatory and ulcerative alterations in times (PR, PQ, QT, intervals duration and QRS complex) of animals treated at 80 and 100 mg/kg. Forty milligrams per dogs whatever the dose level used (up to a maximal dose tested kilogram was set as the MTD. of 25.6 mg/kg of DTS-201). Cardiac toxicity. Delayed, cumulative toxicity was evaluated Mass balance. A mass balance study was undertaken in the according to a standard protocol (15, 18) characterized by rat using [14C-doxorubicin] DTS-201 to characterize the kinetics seven consecutive weekly injections, followed by a free and route of elimination of the compound after a single treatment period of 9 weeks. In this study, DTS-201 was intravenous injection (50 mg/kg). As shown in Fig. 1B, 80% of compared with free doxorubicin at equimolar and at up to 8- the administered dose is eliminated within 24 h, and >95% is fold higher doses. The animals were sacrificed, and semiquan- eliminated after 5 days. Fecal excretion represents 51% of this titative evaluation of myocardial injury was done by histologic elimination and urinary excretion 36%. examination of heart tissue (Table 2). DTS-201–induced Antitumoral efficacy of DTS-201. DTS-201 and doxorubicin cardiac toxicity was found in a maximum of one animal per were compared at equitoxic doses in three human tumor group and, in the affected animals, was minimal with mean xenograft models. The PC-3 prostate tumor cells were total score values of V0.3 whatever the dose level (2.2, 8.9 or implanted into male mice, which are more sensitive to

Table 1. Toxicokinetic variables of DTS-201 and doxorubicin in Beagle dogs after a 15-min intravenous infusion of DTS-201 at increasing dose levels

Dose DTS-201 Doxorubicin

(mg/kg) (Mmol/kg) C max (Mmol/L) T max (min) AUC(0-t)*(MmolÁh/L) C max (nmol/L) T max (min) AUC(0-t)* (nmolÁh/L) 4 3.9 14.4 F 4.5 15 5.1 F 1.5 1.90 F 1.30 65 6.06 F 5.23 8 7.8 42.6 F 15.2 15 15.7 F 6.1 4.81 F 1.30 54 72.8 F 4.3 16 15.5 83.6 F 16.5 15 38.2 F 9.9 18.2 F 12.7 44 347 F 88 32 31.0 209 F 26 15 127 F 13 29.6 F 3.4 120 771 F 186

*AUC values calculated from 0 to the last quantifiable time point using the trapezoidal rule.

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tively, whereas doxorubicin was inactive under these experi- Table 2. Frequency and score of cardiac lesions mental conditions. Á after chronic injection of DTS-201 or doxorubicin HCl Analyses of these in vivo efficacy experiments confirmed a to Sprague-Dawley rats statistical difference between doxorubicin and DTS-201 in the NCI-H1299 model for both doses of DTS-201 (at 60 mg/kg, Dose Dose Wk 16 P P mg/kg* mg eq. Dox/kg < 0.05; at 80 mg/kg, < 0.001; Dunnett). For the MDA- D/E MTS MB-231 xenograft model, a statistical difference between P Control 0 0 0/5 0.0 DTS-201 and the control was observed ( < 0.05; Dunnett), DTS-201 2.2 1.25 1/5 0.2c whereas no statistical difference was observed between the 8.9 5 0/5 0.0 control group and free doxorubicin. For the PC-3 model, a b 17.8 10 1/4 0.3 statistical difference between the control group and the two Á c Doxorubicin HCl 1.25 1.25 5/5 6.8 treatment groups was observed (P > 0.05; Dunnett). An improved antitumor kill was observed for DTS-201 compared Abbreviations: D/E, damaged hearts/examined hearts; MTS, with doxorubicin, but this did not reach significance due to mean total score. the heterogeneity of this model. *Once a week for 7 consecutive week. cP < 0.01 versus control group (Kruskall Wallis test followed by Immunohistochemical analysis of NCI-H1299 tumors Dunn’s test). showed expression of both CD10 and TOP enzymes in a majority bP < 0.01 versus doxorubicinÁHCl group (Kruskall Wallis test of the cells (100% for CD10 and 90% for TOP). The stromal cells followed by Dunn’s test). in these tumor samples did not express the enzymes. Immunohistochemical analyses have shown that CD10 and TOP were also present in other in vivo tumor models including colorectal (LS 174T and HCT 116), lung (Calu 6), anthracycline toxicity than females. In this model, the MTD for ovary (SK-OV-3), and prostate (LnCap) . CD10 was DTS-201 and doxorubicin were defined as 40 and 4.5 mg/kg, predominantly expressed in carcinoma cells in LnCap tumors, respectively. DTS-201 at 40 mg/kg was more active than whereas CD10 expression was observed predominantly in doxorubicin at the end of the study (Fig. 3A). The PC-3 tumors stromal cells in Calu 6 tumors. expressed CD10 in only 5% of tumor cells but in 100% of CD10 and TOP expression in human breast and prostate tumor-infiltrated stromal cells. TOP was expressed in 70% of carcinoma biopsies. CD10 and TOP immunostaining was the cancer cells. carried out on 117 breast carcinomas and 98 prostate In the MDA-MB-231 breast tumor model, DTS-201 and carcinomas. CD10 was expressed in 105, and TOP was doxorubicin were administered at equitoxic doses (60 and 6 expressed in 113 of 117 breast carcinoma biopsies. Only one mg/kg, respectively). DTS-201 showed a higher activity in this biopsy showed no expression of either DTS-201 activating model compared with doxorubicin (Fig. 3B), with treated enzymes. Differences were observed in the expression of the versus control values of 25% and 43%, respectively. Interest- two ; CD10 was predominantly expressed by the ingly, only TOP expression was observed in these tumors. stromal cell population, whereas TOP was predominantly In the NCI-H1299 model, DTS-201 was expressed by both tumor and stromal cells (Fig. 4). administered at 60 mg/kg and at its MTD of 80 mg/kg In prostate carcinoma biopsies, CD10 and TOP were (Fig. 3C), and compared with doxorubicin at its MTD, i.e., expressed in 88 of 98 biopsies. Expression of CD10 was 6 mg/kg. DTS-201 showed significant dose-dependent efficacy, predominant in the prostate carcinoma cells, whereas TOP was with treated versus control values of 38% and 17%, respec- expressed in both carcinoma and stromal cell populations.

Fig. 2. Semi-thin sections of rat heart after chronic injection of DTS-201or doxorubicinÁHCl. Rats were sacrificed 9 wk after 7 weekly intravenous injections with vehicle (A), B, DTS-201 (17.8 mg/kg), and C, doxorubicinÁHCl (1.25 mg/kg).Toluidine blue staining; magnification, Â20.

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than free doxorubicin for mice and was shown to be more active at equitoxic dose levels in a number of human tumor xenograft models (6–8, 13, 14). These results were strongly supported by pharmacokinetic and tissue distribution studies that showed that normal tissues were much less exposed to doxorubicin after equimolar administration of DTS-201 com- pared with free doxorubicin, whereas the exposure of the xenografted tumors was almost doubled (6). Two peptidases, neprilysin (CD10) and TOP, had also been identified as candidates for the selective cleavage of DTS-201 into N-L-leucyl- doxorubicin or N-L-alanyl-L-leucyl-doxorubicin, respectively, in the vicinity of tumor cells (8, 9). However, other as yet unidentified enzymes may also result in tumor-specific DTS- 201 activation. DTS-201 was well-tolerated after a single administration to mice, rats, and dogs. Its MTD is >100 mg/kg (f300 mg/m2) and 73 mg/kg (f440 mg/m2) in mice and rats, respectively, and is between 16 and 32 mg/kg (f300 and f600 mg/m2)in the dog. After two consecutive injections, the MTD was found to be 50 mg/kg (f150 mg/m2) and 40 mg/kg (f240 mg/m2) in mice and rats, respectively. Comparing these values to those of doxorubicin documented in the literature, DTS-201 seems to be at least three to four times less toxic in rodents (6, 19) and up to 8 to 9-fold less toxic in the dog (16, 20). No evidence of toxicities unrelated to doxorubicin was found in DTS-201–treated animals. All the observed toxic effects after treatment with DTS-201 are typical of cytotoxic agents and are the same as those reported for free doxo- rubicin, but they occur at higher dose levels than with free doxorubicin (19). The main target organs of DTS-201 toxicity are bone marrow and the lymphoid tissues, the gastrointes- tinal tract and oral cavity, and the genital tract, as is the case for most cytotoxic anticancer agents and doxorubicin in particular (19). In rodents, the peripheral nervous system was also a key target, the animals systematically developing hind limb paralysis at toxic dose levels. This is a well-known toxicity of doxorubicin, described as rodent-specific and never reported in human patients (16, 21, 22). In agreement with this, no neurotoxic effects were observed with DTS- 201 in dogs. The observation of a dose-dependent plasma exposure to DTS-201 and doxorubicin after treatment with DTS-201 is also in good agreement with a dose-dependent toxicity, and the low plasma exposure to doxorubicin supports the improved tolerance. These toxicology studies were the basis for the European regulatory filing for a phase I clinical trial. DTS-201 did not induce any changes in the electrical activity of the heart after a single injection in the dog but more important was the demonstration of a much lower Fig. 3. In vivo antitumoral efficacy of DTS-201compared with free doxorubicin in PC-3 (A), MDA-MB-231 (B), and NCI-H1299 (C) human tumor models. cardiac toxicity for DTS-201 compared with free doxorubicin Mice were treated with DTS-201at 40 ( ), 60 ( ), or 80 ( )mg/kg. after chronic treatment in rats. This is consistent with the Doxorubicin was administered at 4.5 ( )or6( ) mg/kg.Treatments were done prodrug concept and with the results of tissue distribution weekly for 4 (NCI-H1299), 5 (PC-3), or 6 (MDA-MB-231) wk (arrowheads). Tumor volume was evaluated. studies that have shown a 10-fold reduction of heart expo- sure to doxorubicin after treatment of mice with DTS-201 compared with an equimolar dose of free doxorubicin (6). Discussion The fact that DTS-201 induces less severe lesions of heart tissue than doxorubicin at molar doses up to eight times DTS-201, a prodrug of doxorubicin, was selected as a higher highlights one of the major potential advantages of candidate for preclinical development on the basis of in vivo the prodrug. As already discussed, doxorubicin treatment of efficacy and tissue-distribution studies that showed its tumor- patients is frequently stopped because of an increased risk specific activation. DTS-201 proved to be significantly less toxic of cardiotoxicity, although tumors are still responsive to

www.aacrjournals.org 12 6 3 Clin Cancer Res 2008;14(4) February 15, 2008 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2008 American Association for Cancer Research. Cancer Therapy: Preclinical treatment (1, 3). If the reduced cardiac toxicity of DTS-201 is who are not potentially amenable to classic and/or anth- transposable to humans, significantly better clinical outcomes racycline chemotherapy, could also benefit from a treatment could be achieved in patients with doxorubicin-responsive with DTS-201. tumors. The tumor-targeting nature of the DTS-201 prodrug shown A good example of the need for a less cardiotoxic anthra- here and in previous studies (6, 7) has also the potential to cycline is breast cancer. The standard of care for most open new indications compared with the current use of breast cancer patients currently includes anthracyclines. For doxorubicin. The tumor-specific reactivation of DTS-201 results patients whose tumors express HER2/neu, the humanized from extracellular endopeptidase cleavage of the peptidic antibody trastuzumab is also an effective treatment, alone or moiety of the prodrug. The expression of two of the in combination with chemotherapy. It has been shown that endopeptidases (CD10 and TOP) that may be clinically combining trastuzumab with chemotherapy agents can im- relevant in the reactivation of DTS-201 has been characterized. prove efficacy (23, 24), but the use of trastuzumab with A total of 215 human tumor biopsies, 117 breast carcinomas, anthracyclines has been found to cause serious cardiac side and 98 prostate carcinomas were analyzed for CD10 and TOP effects (25). For this reason, combination of trastuzumab with expression. The simultaneous expression of both CD10 and doxorubicin is avoided. The reduced cardiotoxity of DTS- TOP by the carcinoma cells is very frequent in prostate 201 would provide a significant advantage in this setting. carcinoma (88 of 98) but infrequent in breast carcinoma Finally, patients at higher risk of developing a cardiomyopathy, (15 of 117). However, in the breast tumor samples, CD10 is

Fig. 4. Immunostaining of CD10 (left)and TOP (right) in human breast and prostate carcinomas. In breast carcinomas: CD10 immunostaining (A)wasmainly observed in fibroblasts and/or endothelial cells alone (101of 117); (B)itwasless frequently observed in tumor cells plus fibroblasts and/or endothelial cells (14 of 117); (C) TOP immunostaining was mainly observed in tumor cells, fibroblasts, and/or endothelial cells (88 of 117); (D) and it was less frequently observed in tumor cells alone (25 of 117) magnification, Â200 (for A, B,andD)andÂ100 (for C). In prostate carcinomas: (E) strong and diffuse CD10 staining of tumor cells, whereas normal epithelial cells are negative (88of98)and(F) weak immunostaining of TOP in tumor cells and in smooth muscular cells (88 of 98). Magnification, Â40.

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also expressed by stromal cells, and CD10 or TOP expression nonspecific reactivation of the prodrug should be expected. was observed in all but one of the breast tumor biopsies Toxicology studies were not able to identify new target organs, (tumoral and/or stromal cells). The expression of CD10 by the suggesting that no one normal organ expresses or releases stromal cells in the tumor environment is equally important sufficiently high levels of endopeptidases to induce localized because it would lead to a local activation of DTS-201, resulting release of doxorubicin, but detectable levels of free doxorubicin in a bystander effect and both stromal and tumoral cell killing. in the circulation suggests that some level of nonspecific In the MDA-MB-231 experimental tumor model, only reactivation occurs. This is, however, also true for the vast TOP expression was observed, suggesting that TOP alone, or a majority of targeted approaches, including all immunotoxins, yet unidentified endopeptidase, may be sufficient to reacti- HSP90, and HDAC inhibitors. vate DTS-201. In the PC-3 human prostate tumor model, Taken together, the efficacy, pharmacokinetics, and toxicity CD10 is expressed in only a low percentage of carcinoma cells of DTS-201 confirm the tumor-selective prodrug concept. The but a high percentage of stromal cells. Efficacy data in this preferential activation of DTS-201 in human tumors in mice model confirmed the benefit of DTS-201, which is suppor- supports the better therapeutic index compared with doxoru- tive of the aforementioned bystander effect. These efficacy bicin. On the basis of these preclinical data and of the broad data are consistent with that already observed for other breast clinical experience accumulated with doxorubicin, a starting and prostate tumor models after DTS-201 administration dose of DTS-201 of 80 mg/m2 (equivalent to 45 mg/m2 of (6–8). doxorubicin on a molar basis) was used for the clinical phase I Although it is clear from this and other publications that study that is under way in patients with advanced or metastatic the DTS-201 prodrug is able to deliver higher quantities of solid tumors (26). doxorubicin to the tumor, while sparing normal tissues (6–8), a potential limitation of this prodrug technology is that its activation relies on the expression of endogenous genes. These Acknowledgments endogenous genes are expressed in a number of different tissues, albeit at low levels, whereas tumor target is achieved by WethankDr.GuyMazue¤for his guidance concerning the toxicology program and Dr. Paola DellaTorre for her support with the cardiotoxicity studies in rats; Matthieu virtue of enhanced tumor expression and/or extracellular Michel, Nathalie Heylen, Fabienne Cournarie, Xavier Ranc¸on, and Vale¤rie Arranz for release of their products. As normal tissues express providing practical advice and suggestions; and Dr. JohnTchelingerian for his con- these genes, tumor targeting cannot be 100% specific, and some stant support and invaluable suggestions.

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Denis Ravel, Vincent Dubois, Jérôme Quinonero, et al.

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