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Cancer Therapy: Preclinical |
Authors' Affiliations: 1 Diatos S.A., Paris, France; 2 EA 3035, Institut Claudius-Regaud, Universite Paul-Sabatier, Toulouse, France; and 3 Service d'Anatomie et Cytologie Pathologiques, Hôpital de Rangueil, Toulouse, France
Requests for reprints: Jonathan Kearsey, Diatos S.A., 11 rue Watt, 75013 Paris, France. Phone: 33-1-53-80-93-49; Fax: 33-1-53-80-93-89; E-mail: jkearsey{at}diatos.com.
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Abstract |
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Experimental Design: The preclinical development program consisted of a detailed assessment of the general and cardiac toxicity profiles of DTS-201 in mice, rats, and dogs, together with mass balance and antitumoral efficacy studies in rodents. Neprilysin and thimet oligopeptidase expression, two enzymatic activators of DTS-201, was also characterized in human breast and prostate tumor biopsies.
Results: The target organs of DTS-201 toxicity in rodents and dogs are typically those of doxorubicin, albeit at much higher doses. Importantly, chronic treatment with DTS-201 proved 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-201 does not accumulate in the body after intravenous administration. The improved therapeutic index of DTS-201 compared with free doxorubicin was confirmed in three tumor xenograft models of prostate, breast, and lung cancer. Neprilysin and/or thimet oligopeptidase 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-201 gave promising results in terms of general toxicity, cardiovascular tolerance, and in vivo efficacy in xenograft mouse models compared with free doxorubicin. Taken together, these results and the confirmation of the presence of activating enzymes in human tumor biopsies provide a strong rationale for a phase I clinical study in cancer patients.
Even if new targeted approaches, such as antibodies (including trastuzumab; Herceptin; Roche) or tyrosine kinases inhibitors (e.g., lapatinib; GlaxoSmithKline), are significantly less toxic than conventional cytotoxic agents, they often have to be used in combination with cytotoxic molecules to achieve significant efficacy in the clinic (4, 5). There is therefore still a real clinical need for a new generation of cytotoxic molecules with increased efficacy and reduced systemic toxicity. One such novel cytotoxic molecule is DTS-201 (CPI-0004Na), a peptidic prodrug of doxorubicin. DTS-201 (N-succinyl-β-alanyl-L-leucyl-L-alanyl-L-leucyl-doxorubicin) is stable and inactive in its cell-impermeable prodrug form. The cell-impermeable 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 volumes (6, 7). In the vicinity of a tumor, the tetrapeptide portion of the DTS-201 prodrug is cleaved by endopeptidases that are released extracellularly in the tumor environment. This yields the metabolites N-L-alanyl-L-leucyl-doxorubicin and N-L-leucyl-doxorubicin, which can enter cells and are converted to the active drug, doxorubicin. This results in an increased concentration of doxorubicin in the tumor and reduced doxorubicin levels in normal tissues (6, 7).
Two tumor-specific endopeptidases have been identified that cleave DTS-201: neprilysin (CD10, EC3.4.24.11; ref. 8) and thimet oligopeptidase (TOP, EC3.4.24.15; ref. 9). These endopeptidases are released in the extracellular space of solid tumors by stromal, tumor, and neoangiogenic endothelial cells or expressed at their cell surface (10, 11). The extracellular localization of these enzymes allows them to cleave and activate DTS-201 (8, 9). With the exception of some classes of lymphocytes and the brush borders of some epithelia, CD10 expression in normal tissues is limited, whereas it is over- expressed in a wide range of tumor types (12). TOP expression is less well-characterized. The normal tissue distribution of TOP has not yet been determined. Significant expression has been described in the central nervous system, testes, and some classes of lymphocytes.
A significant therapeutic advantage of using the DTS-201 endopeptidase–activated prodrug compared with free doxorubicin has already been shown in a number of different tumor models including breast, colon, prostate, and lung cancers (6–8, 13, 14). Pharmacokinetic and tissue distribution studies on normal and tumor-bearing mice have confirmed that the improved therapeutic index of the prodrug results from a tumor-selective release of doxorubicin, together with a significant decrease in doxorubicin levels in all normal tissues tested (6). In particular, a 10-fold decrease in doxorubicin levels in cardiac tissue is observed after DTS-201 treatment, compared with an equimolar dose of free doxorubicin. Increased tumor exposure together with reduced cardiac exposure to doxorubicin in animals after treatment with DTS-201 therefore provides a strong rationale for this prodrug approach.
This paper describes the preclinical development work that further supports the evaluation of this prodrug in a phase I clinical study in patients with solid tumors. General toxicity studies in rodents and dogs show that the target organs of DTS-201 toxicity are the same as for doxorubicin, but that this toxicity occurs at much higher dose levels. Reduced cardiac toxicity was observed in a rat model that has been shown to be predictive for the human cumulative cardiotoxicity induced by anthracyclines (15, 16). DTS-201 also showed an improved efficacy in prostate, breast, and lung tumor models. The potential utility of this prodrug approach in prostate and breast cancer has also been further explored through the immunohistochemical analysis of the expression of the two endopeptidases that cleave DTS-201 (TOP and CD10) in >200 human tumor biopsies. Expression of either one or both endopeptidases was observed in almost all biopsies, suggesting that both prostate and breast cancers are indications for which DTS-201 may show clinical value.
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Materials and Methods |
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The NCI-H1299 (non–small cell lung carcinoma; ATCC# CRL-5803), MDA-MB-231 (mammary adenocarcinoma; ATCC#HTB-26), and PC-3 (prostate carcinoma; ATCC# CRL-1435) human cell lines were used for antitumoral activity studies. LS 174T (colon adenocarcinoma; ATCC# CL-188), HCT 116 (colon carcinoma; ATCC# CCL-247), and Calu6 (lung anaplastic carcinoma; ATCC# HTB-26) were used for immunohistochemical analysis of enzymes expression in tumor xenografts.
Acute toxicity studies in rodents. Groups of 5 male and 5 female young adult Swiss CD-1 mice (Charles River Laboratories France) received a single 15-minute intravenous infusion of vehicle (NaCl 0.9% w/v), or 40, 60, 80, or 100 mg/kg of DTS-201. Clinical signs, body weight, local tolerance, and mortality were monitored up to day 29, at which point, the surviving animals were sacrificed for necropsy. Organs were weighed immediately after dissection.
A similar study was done with groups of 6 male and 6 female young adult Sprague-Dawley rats (Charles River Laboratories France) using doses of DTS-201 of 30, 47, 60, and 73 mg/kg. Additional monitoring included hematologic and blood biochemistry investigations done on days 5, 11, 16, 22, and 29.
Acute toxicity and toxicokinetics in the dog. Groups of 2 male and 2 female young adult Beagle dogs (Marshall Farms) received a single 15 minute intravenous infusion of vehicle or of DTS-201 at dose levels of 4, 8, 16, or 32 mg/kg. The animals were monitored for clinical signs, local tolerance, body weight, rectal temperature, and mortality up to day 29. In addition, electrocardiogram and blood pressure were recorded before dosing, 4 and 24 h postdosing, and at the end of the study. Hematologic and blood biochemistry investigations, as well as urine analysis, were also done predose and on days 5, 11, 16, 22, and 29. Upon sacrifice, a complete macroscopic examination was done and organs were dissected and weighed.
Blood samples were collected on sodium citrate up to 48 h postdose for a toxicokinetic evaluation of DTS-201 in all animals, using a fully validated method. DTS-201 and its metabolites (doxorubicin, N-L-leucyl-doxorubicin and N-L-alanyl-L-leucyl-doxorubicin) were extracted from plasma samples using solid-phase extraction and quantified by high-performance liquid chromatography with tandem mass spectrometric detection analysis using a triple quadrupole mass spectrometer (API 2000; PE Sciex) with a TurboIonSPray source. Briefly, plasma samples were supplemented with 40 µL of methanol, 25 µL of a solution of flurbiprofen in methanol (internal standard), and 1 mL of a 0.2% formic acid solution. The mixtures were then loaded on Oasis HLB cartridges (Waters) previously conditioned with 2 mL of methanol followed by 2 mL of water. After washing with 1 mL of water, DTS-201 and its metabolites were eluted with 500 µL of methanol. Thirty microliters of the eluates were then injected onto a 20 x 3.9 mm, 5-µm Oasis HLB column (Waters) and separated using a 15% to 60% acetonitrile gradient (in 5 µmol/L sodium trifluoroacetate-0.1% formic acid) over 5 min at a flow rate of 0.8 mL/min. The three metabolites were detected in the positive mode (first 3.5 min) and DTS-201 and flurbiprofen in the negative mode with multiple reaction monitoring for transition of the parent ions to the product ions [Parent ion (m/z)/Product ion (m/z): DTS-201, 1010/614; flurbiprofen, 243/199; AL-Dox, 728/314; L-Dox, 657/243; doxorubicin, 544/397]. The plasma concentration of each analyte was determined by interpolation with the appropriate calibration curve.
Subchronic toxicity studies in rodents. Groups of 10 to 15 male and 10 to 15 female young adult Swiss CD-1 mice (Charles River Laboratories France) received one 15-minute intravenous infusion of vehicle or DTS-201 at 50, 100, or 150 mg/kg on day 1 and on day 22. In addition to the monitoring described for the single-dose studies that was continued for 28 days after the second treatment, ophthalmologic examinations, hematologic, and blood biochemistry investigations were done. After sacrifice of all animals in the control and high-dose groups, a microscopic examination of the tissues defined in appendix A of the European Medicines Evaluation Agency "Note for Guidance on Repeated Dose Toxicity" was done (17).
A similar study was conducted with groups of 10 to 16 male and 10 to 16 female young adult Sprague-Dawley rats (Charles River Laboratories France) treated with vehicle or 40, 80, or 100 mg/kg of DTS-201 on days 1 and 22.
Acute cardiotoxicity study. Two male and two female adult Beagle dogs (CEDS) instrumented with telemetric transmitters (Transoma Medical) for electrocardiogram, blood pressure, and heart rate monitoring received successive intravenous infusions of DTS-201 at 3.2, 12.8, and 25.6 mg/kg with minimum washout periods of 7 days. Telemetric measurements started 24 h before dosing and continued for 24 h after treatment.
Chronic (cumulative) cardiotoxicity study. Groups of 10 male adult Sprague-Dawley rats (Charles River Laboratories France) received seven consecutive weekly intravenous bolus injections of vehicle, doxorubicin, at 1.25 mg/kg, or DTS-201 at 2.23, 7.9, or 17.8 mg/kg. After a 9-week treatment-free period, cardiotoxicity was evaluated semiquantitatively by a histologic assessment of semi-thin sections of heart tissue using a scoring system specifically devised for anthracyclines and taking into account the severity (0-2) and extent (0-5) of the myocardial lesions as described elsewhere (15, 18). The mean total score (mean total score = severity x extent, 0 to 10) was calculated for each group.
Mass balance study. Three male and three female adult Sprague-Dawley rats (Harlan UK Limited) received a single intravenous dose of 50 mg/kg of [14C-Doxorubicin] DTS-201, corresponding to 3.7 MBq/kg (14C is in position carbon 13 of doxorubicin). Urine, feces, and expired air samples were quantitatively collected before treatment and at 8 (urine only), 24, 48, 72, 96, and 120 h posttreatment. Urine and expired air duplicate samples were mixed with 10 mL of Gold Star liquid scintillator (Meridian). Duplicate aliquots of feces were combusted using a sample oxidizer [PerkinElmer LAS (UK) Ltd.]. Carbosorb and Permafluor [PerkinElmer LAS (UK) Ltd.] were used as absorbent and scintillator, respectively. All samples were counted in a liquid scintillation counter [Model 2000TR; PerkinElmer LAS (UK) Ltd.].
Antitumoral activity. Seven-week-old male or female NMRI nu/nu mice (Janvier) were respectively injected with a PC-3 cell suspension (1 x 107 cells/mL) or a MDA-MB-231 suspension (6 x 107 cells/mL) in the ventral skin or implanted with a NCI-H1299 tumor fragment. Treatment began 14 (PC-3 and MDA-MB-231) or 12 (NCI-H1299) days after the graft, when tumors were at least 70 mm3. Drugs were administered intravenously (bolus; 10 µL per gram of body weight) weekly for 4 (NCI-H1299) or 5 (PC-3) consecutive weeks. In the MDA-MB-231 study, two cycles of one weekly administration for 3 weeks were done. Doses were experimentally determined based on the injection schedule, together with the strain and sex of mice used.
Tumor volumes were determined using the following formula: (length x width2)/2. Results are presented as the evolution of mean tumor volume as a function of time. Minimal treated versus control (ratio of mean tumor volume of treated versus control mice) values were used as a measure of treatment efficacy. Error bars represent SE.
Immunohistochemistry studies. Xenograft samples and human tumors were fixed in formalin and embedded in paraffin. The human tumor samples were then grouped in Tissue Micro Arrays. The immunostaining was carried out on 5-µm thick sections using the anti-CD10 mouse monoclonal antibody 56C6 (Novocastra Laboratories) diluted to 1/50 or the anti-TOP rabbit polyclonal antibodies EP24-15 (Proteimax) diluted to 1/500 after a microwave treatment in a citrate buffer [10 mmol/L (pH 6)]. Labeling was carried out using the two-step enVision kit (Dako) according to the manufacturer's instructions. The percentage of labeled cells was evaluated for stromal, tumor, and neoangiogenic endothelial cells. The immunostaining results were reported for each of these cellular groups using a two-point scale: positive staining consisted in at least 10% of labeled cells, whereas negative staining consisted in <10% of labeled cells.
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In the dog, GLP studies showed that DTS-201 induced no adverse effects at the doses of 4 and 8 mg/kg. Dose-dependent, transient, and moderate decreases in WBC counts (mainly neutrophils and lymphocytes) were observed at doses of 16 and 32 mg/kg (up to –38 to –41%). One male (of four) dosed at 32 mg/kg, experienced diarrhea, vomiting, and a 10% body weight loss, and had to be prematurely sacrificed 7 days after treatment. The same clinical signs were observed in the other dogs treated at this dose level but proved to be transient. Consequently, the MTD of DTS-201 after a single intravenous infusion to Beagle dogs was considered to be between 16 and 32 mg/kg.
A toxicokinetic evaluation of DTS-201 was conducted at the four dose levels tested (4 to 32 mg/kg). DTS-201 and three metabolites (N-L-alanyl-L-leucyl-doxorubicin, N-L-leucyl-doxorubicin, and doxorubicin) were detected in the plasma of all animals (Fig. 1A shows the pharmacokinetic profile at a representative dose of 8 mg/kg). The DTS-201 Cmax value was observed at the end of the 15-min infusion and ranged on average from 14.4 ± 4.5 µmol/L (4 mg/kg) to 209 ± 26 µmol/L (32 mg/kg), increasing almost linearly with dose (Table 1 ). The Cmax values observed for the metabolites were also reached at the end of the infusion or within the following 25 to 120 min but were much lower, ranging from 1.9 ± 1.30 to 29.6 ± 3.4 nmol/L in the case of doxorubicin. A linear relationship was also observed between the doxorubicin Cmax values and the DTS-201 dose level administered. Plasma concentrations of DTS-201 and its metabolites then decreased rapidly and in a regular manner until the last quantifiable time point (4 to 8 h for DTS-201, N-l-alanyl-l-leucyl-doxorubicin, N-l-leucyl-doxorubicin depending on the dose level, and 24 to 48 h for doxorubicin). The area under the plasma concentration versus time curves (AUC) of DTS-201, as well as of free doxorubicin, also increased in a dose-dependent manner. The DTS-201 AUC (calculated from 0 up to the last quantifiable time point) ranged from 5.1 ± 1.5 (4 mg/kg) to 127 ± 13 nmol·h/L (32 mg/kg), whereas the free doxorubicin AUC was several hundred-fold smaller, ranging from 6.06 ± 5.23 to 771 ± 186 nmol·h/L.
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In rats treated similarly, two injections of 40 mg/kg were well-tolerated, whereas two injections of 80 mg/kg were clearly toxic. Their poor clinical condition (hind limb paralysis and tongue abscesses, and body weight loss) prompted the premature sacrifice of all males and of 3 of 10 females in that group. For the same reasons, all animals in the 100 mg/kg dose group had to be sacrificed. Dose-related, transient decreases in WBC counts were also observed 5 days after each treatment. As in the mouse, the target organs of toxicity were the thymus, the genital tract, and the peripheral nervous system. In addition, the spleen also showed decreased follicular size and hypocellularity, as did the bone marrow. The tongue was also affected with inflammatory and ulcerative alterations in animals treated at 80 and 100 mg/kg. Forty milligrams per kilogram was set as the MTD.
Cardiac toxicity. Delayed, cumulative toxicity was evaluated according to a standard protocol (15, 18) characterized by seven consecutive weekly injections, followed by a free treatment period of 9 weeks. In this study, DTS-201 was compared with free doxorubicin at equimolar and at up to 8-fold higher doses. The animals were sacrificed, and semiquantitative evaluation of myocardial injury was done by histologic examination of heart tissue (Table 2
). DTS-201–induced cardiac toxicity was found in a maximum of one animal per group and, in the affected animals, was minimal with mean total score values of 
0.3 whatever the dose level (2.2, 8.9 or 17.8 mg/kg per week). A weekly dose of 1.25 mg/kg of doxorubicin (i.e., 8-fold less than the highest dose of DTS-201 tested on a molar basis) induced severe and significant cardiac lesions (mean total score = 6.8) in all animals, characterized by swelling of muscle fibers, vacuolation of myocytes, loss of myofilaments, and interstitial fibrosis (Fig. 2
). The doxorubicin-induced lesions proved to be significantly more severe in the doxorubicin-treated rats compared with the animals treated with an 8-fold higher molar dose of DTS-201. Statistical analysis of the cardiotoxicity scores obtained after treatment with each dose of DTS-201 compared with vehicle-treated animals did not reveal any significant difference.
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Mass balance. A mass balance study was undertaken in the rat using [14C-doxorubicin] DTS-201 to characterize the kinetics and route of elimination of the compound after a single intravenous injection (50 mg/kg). As shown in Fig. 1B, 80% of the administered dose is eliminated within 24 h, and >95% is eliminated after 5 days. Fecal excretion represents 51% of this elimination and urinary excretion 36%.
Antitumoral efficacy of DTS-201. DTS-201 and doxorubicin were compared at equitoxic doses in three human tumor xenograft models. The PC-3 prostate tumor cells were implanted into male mice, which are more sensitive to anthracycline toxicity than females. In this model, the MTD for DTS-201 and doxorubicin were defined as 40 and 4.5 mg/kg, respectively. DTS-201 at 40 mg/kg was more active than doxorubicin at the end of the study (Fig. 3A ). The PC-3 tumors expressed CD10 in only 5% of tumor cells but in 100% of tumor-infiltrated stromal cells. TOP was expressed in 70% of the cancer cells.
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In the NCI-H1299 lung cancer model, DTS-201 was administered at 60 mg/kg and at its MTD of 80 mg/kg (Fig. 3C), and compared with doxorubicin at its MTD, i.e., 6 mg/kg. DTS-201 showed significant dose-dependent efficacy, with treated versus control values of 38% and 17%, respectively, whereas doxorubicin was inactive under these experimental conditions.
Analyses of these in vivo efficacy experiments confirmed a statistical difference between doxorubicin and DTS-201 in the NCI-H1299 model for both doses of DTS-201 (at 60 mg/kg, P < 0.05; at 80 mg/kg, P < 0.001; Dunnett). For the MDA-MB-231 xenograft model, a statistical difference between DTS-201 and the control was observed (P < 0.05; Dunnett), whereas no statistical difference was observed between the control group and free doxorubicin. For the PC-3 model, a statistical difference between the control group and the two treatment groups was observed (P > 0.05; Dunnett). An improved antitumor kill was observed for DTS-201 compared with doxorubicin, but this did not reach significance due to the heterogeneity of this model.
Immunohistochemical analysis of NCI-H1299 tumors showed expression of both CD10 and TOP enzymes in a majority of the cells (100% for CD10 and 90% for TOP). The stromal cells 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), ovary (SK-OV-3), and prostate (LnCap) carcinomas. CD10 was predominantly expressed in carcinoma cells in LnCap tumors, whereas CD10 expression was observed predominantly in stromal cells in Calu 6 tumors.
CD10 and TOP expression in human breast and prostate carcinoma biopsies. CD10 and TOP immunostaining was carried out on 117 breast carcinomas and 98 prostate carcinomas. CD10 was expressed in 105, and TOP was expressed in 113 of 117 breast carcinoma biopsies. Only one biopsy showed no expression of either DTS-201 activating enzymes. Differences were observed in the expression of the two proteins; CD10 was predominantly expressed by the stromal cell population, whereas TOP was predominantly expressed by both tumor and stromal cells (Fig. 4 ).
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DTS-201 was well-tolerated after a single administration to mice, rats, and dogs. Its MTD is >100 mg/kg (
300 mg/m2) and 73 mg/kg (440 mg/m2) in mice and rats, respectively, and is between 16 and 32 mg/kg (300 and 600 mg/m2) in the dog. After two consecutive injections, the MTD was found to be 50 mg/kg (150 mg/m2) and 40 mg/kg (240 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 doxorubicin, 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 gastrointestinal 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 cardiac toxicity for DTS-201 compared with free doxorubicin after chronic treatment in rats. This is consistent with the prodrug concept and with the results of tissue distribution studies that have shown a 10-fold reduction of heart exposure to doxorubicin after treatment of mice with DTS-201 compared with an equimolar dose of free doxorubicin (6). The fact that DTS-201 induces less severe lesions of heart tissue than doxorubicin at molar doses up to eight times higher highlights one of the major potential advantages of the prodrug. As already discussed, doxorubicin treatment of patients is frequently stopped because of an increased risk of cardiotoxicity, although tumors are still responsive to treatment (1, 3). If the reduced cardiac toxicity of DTS-201 is transposable to humans, significantly better clinical outcomes could be achieved in patients with doxorubicin-responsive tumors.
A good example of the need for a less cardiotoxic anthracycline is breast cancer. The standard of care for most breast cancer patients currently includes anthracyclines. For patients whose tumors express HER2/neu, the humanized antibody trastuzumab is also an effective treatment, alone or in combination with chemotherapy. It has been shown that combining trastuzumab with chemotherapy agents can improve efficacy (23, 24), but the use of trastuzumab with anthracyclines has been found to cause serious cardiac side effects (25). For this reason, combination of trastuzumab with doxorubicin is avoided. The reduced cardiotoxity of DTS-201 would provide a significant advantage in this setting. Finally, patients at higher risk of developing a cardiomyopathy, who are not potentially amenable to classic and/or anthracycline chemotherapy, could also benefit from a treatment with DTS-201.
The tumor-targeting nature of the DTS-201 prodrug shown here and in previous studies (6, 7) has also the potential to open new indications compared with the current use of doxorubicin. The tumor-specific reactivation of DTS-201 results from extracellular endopeptidase cleavage of the peptidic moiety of the prodrug. The expression of two of the endopeptidases (CD10 and TOP) that may be clinically relevant in the reactivation of DTS-201 has been characterized. A total of 215 human tumor biopsies, 117 breast carcinomas, and 98 prostate carcinomas were analyzed for CD10 and TOP expression. The simultaneous expression of both CD10 and TOP by the carcinoma cells is very frequent in prostate carcinoma (88 of 98) but infrequent in breast carcinoma (15 of 117). However, in the breast tumor samples, CD10 is also expressed by stromal cells, and CD10 or TOP expression was observed in all but one of the breast tumor biopsies (tumoral and/or stromal cells). The expression of CD10 by the stromal cells in the tumor environment is equally important because it would lead to a local activation of DTS-201, resulting in a bystander effect and both stromal and tumoral cell killing.
In the MDA-MB-231 experimental tumor model, only TOP expression was observed, suggesting that TOP alone, or a yet unidentified endopeptidase, may be sufficient to reactivate DTS-201. In the PC-3 human prostate tumor model, CD10 is expressed in only a low percentage of carcinoma cells but a high percentage of stromal cells. Efficacy data in this model confirmed the benefit of DTS-201, which is supportive of the aforementioned bystander effect. These efficacy data are consistent with that already observed for other breast and prostate tumor models after DTS-201 administration (6–8).
Although it is clear from this and other publications that the DTS-201 prodrug is able to deliver higher quantities of 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 endogenous genes are expressed in a number of different tissues, albeit at low levels, whereas tumor target is achieved by virtue of enhanced tumor expression and/or extracellular release of their protein products. As normal tissues express these genes, tumor targeting cannot be 100% specific, and some nonspecific reactivation of the prodrug should be expected. Toxicology studies were not able to identify new target organs, suggesting that no one normal organ expresses or releases sufficiently high levels of endopeptidases to induce localized release of doxorubicin, but detectable levels of free doxorubicin in the circulation suggests that some level of nonspecific reactivation occurs. This is, however, also true for the vast majority of targeted approaches, including all immunotoxins, HSP90, and HDAC inhibitors.
Taken together, the efficacy, pharmacokinetics, and toxicity of DTS-201 confirm the tumor-selective prodrug concept. The preferential activation of DTS-201 in human tumors in mice supports the better therapeutic index compared with doxorubicin. On the basis of these preclinical data and of the broad clinical experience accumulated with doxorubicin, a starting dose of DTS-201 of 80 mg/m2 (equivalent to 45 mg/m2 of doxorubicin on a molar basis) was used for the clinical phase I study that is under way in patients with advanced or metastatic solid tumors (26).
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Acknowledgments |
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Received 5/11/07; revised 9/27/07; accepted 11/27/07.
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) and its metabolites, AL-Dox (
), L-Dox (x), and Dox (
) were measured. B, mass balance in rats. Cumulative recovery of radioactivity in rats after intravenous injection of [14C-doxorubicin] DTS-201. Recovery in feces (
), urines (
), 60 (
), or 80 (
) mg/kg. Doxorubicin was administered at 4.5 (
) or 6 (
) mg/kg. Treatments were done weekly for 4 (NCI-H1299), 5 (PC-3), or 6 (MDA-MB-231) wk (arrowheads). Tumor volume was evaluated.