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Clinical Cancer Research 14, 2145, April 1, 2008. doi: 10.1158/1078-0432.CCR-07-4580
© 2008 American Association for Cancer Research
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Cancer Therapy: Preclinical

DTS-108, A Novel Peptidic Prodrug of SN38: In vivo Efficacy and Toxicokinetic Studies

Florence Meyer-Losic, Céline Nicolazzi, Jérôme Quinonero, Fabien Ribes, Matthieu Michel, Vincent Dubois, Catherine de Coupade, Matthieu Boukaissi, Anne-Sophie Chéné, Isabelle Tranchant, Valérie Arranz, Imane Zoubaa, Jean-Sébastien Fruchart, Denis Ravel and Jonathan Kearsey

Authors' Affiliation: Diatos S.A., Paris, 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.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Irinotecan is a prodrug converted to the active cytotoxic molecule SN38 predominantly by the action of liver carboxylesterases. The efficacy of irinotecan is limited by this hepatic activation that results in a low conversion rate, high interpatient variability, and dose-limiting gastrointestinal toxicity. The purpose of this study was to evaluate a novel peptidic prodrug of SN38 (DTS-108) developed to bypass this hepatic activation and thus reduce the gastrointestinal toxicity and interpatient variability compared with irinotecan.

Experimental Design: SN38 was conjugated to a cationic peptide (Vectocell) via an esterase cleavable linker. The preclinical development plan consisted of toxicity and efficacy evaluation in a number of different models and species.

Results: The conjugate (DTS-108) is highly soluble, with a human plasma half-life of 400 minutes in vitro. Studies in the dog showed that DTS-108 liberates significantly higher levels of free SN38 than irinotecan without causing gastrointestinal toxicity. In addition, the ratio of the inactive SN38-glucuronide metabolite compared with the active SN38 metabolite is significantly lower following DTS-108 administration, compared with irinotecan, which is consistent with reduced hepatic metabolism. In vivo efficacy studies showed that DTS-108 has improved activity compared with irinotecan. A significant dose-dependent antitumoral efficacy was observed in all models tested and DTS-108 showed synergistic effects in combination with other clinically relevant therapeutic agents.

Conclusions: DTS-108 is able to deliver significantly higher levels of SN38 than irinotecan, without the associated toxicity of irinotecan, resulting in an increased therapeutic window for DTS-108 in preclinical models. These encouraging data merit further preclinical and clinical investigation.

Irinotecan is an effective chemotherapeutic agent that is widely prescribed for advanced colorectal cancer as a first- or second-line treatment. Irinotecan has also been shown to be active in gastric cancer, non–small cell lung cancer, and small-cell lung cancer, alone or in combination with other cytotoxic agents (1). Currently, irinotecan is used in combination with 5-fluorouracil (5-FU) in first-line treatment for metastatic colorectal cancer (2). Irinotecan is also used with other agents, including the anti–vascular endothelial growth factor antibody bevacizumab (Avastin; refs. 3, 4). Following administration of irinotecan, the active metabolite SN38 (7-ethyl-10-hydroxycamptothecin) is formed by the action of carboxylesterases that are predominantly present in the liver (5, 6). SN38 is a topoisomerase I inhibitor with an activity a thousand times greater than irinotecan, but that cannot be administered directly as it is highly insoluble (1).

Several limitations to the clinical use of irinotecan arise due to its mechanism of activation, metabolism, and elimination. The first limitation is caused by the complexity of irinotecan metabolism, which results in high interpatient variability in both efficacy and toxicity, with only 2% to 8% of the administered dose of irinotecan converted into SN38 in man (7). Three potential irinotecan-activating carboxylesterases have been identified (CES1, CES2, and CES3) of which CES2 shows the greatest affinity for irinotecan (8). Studies have shown that genetic and environmental factors influence carboxylesterase enzyme activity by up to 10-fold (9). The detoxification of SN38 to the inactive SN38-glucuronide (SN38-G) by hepatic UDP-glucuronosyltransferases is also complex and variable. Seventeen human UDP-glucuronosyltransferases have been identified, the majority of which exhibits polymorphisms that could affect their activity (10). In addition, cytochrome CYP3A4–dependent irinotecan metabolism results in the formation of the inactive oxidation products 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxy camptothecin and 7-ethyl-10-[4-amino-1-piperidino]-carbonyloxy camptothecin (NPC), from which NPC can be further converted to SN38 by carboxylesterase (6).

The second limitation of irinotecan is the emergence of delayed, severe, and potentially life-threatening diarrhea, due to an accumulation of SN38 in the intestine resulting from bile excretion of the parent molecule and its metabolites. Once in the intestine, SN38 is formed either from irinotecan by intestinal carboxylesterase or from SN38-G by β-glucuronidase produced by intestinal bacteria (11, 12). Such late-onset grade 3 to 4 diarrhea is observed in 30% to 40% of treated patients (1, 13, 14). It is also a major limitation because the dose has to be reduced for further treatment cycles, and dose intensification of irinotecan in colon cancer patients is known to improve the antitumoral response (15–17).

A significant clinical advantage could therefore be gained from the direct administration of the active metabolite, SN38, using a drug delivery technology that is not reliant on hepatic activation and metabolism. This would allow higher plasma and tissular levels of SN38 with a consequential increase in antitumoral efficacy while limiting the gastrointestinal toxicity and interpatient variability. Based on this rationale, a novel water-soluble conjugate (DTS-108) was generated by linking SN38 to a highly charged oligopeptide of human origin (Vectocell Technology; refs. 18, 19). This technology has already been successfully used to enhance both the in vitro and in vivo efficacy of doxorubicin (20) by altering its physicochemical, pharmacokinetic, and cellular delivery properties, with no evidence of additional toxicity.

This report describes the application of the Vectocell technology to SN38. Solubility, stability, and in vitro cytotoxicity assays have been undertaken to evaluate this novel SN38-peptide (DTS-108) conjugate. Toxicokinetic studies in the dog were done to quantify the potential benefit of DTS-108 administration, whereas studies in both mice and rats were used to evaluate the antitumoral efficacy of this novel conjugate. Results show that conjugation of SN38 to a cationic peptide renders it soluble for i.v. administration in purely aqueous vehicles at high dose levels. DTS-108 permits significantly higher plasma levels of the active drug SN38 to be reached with consequential improved efficacy without increasing gastrointestinal toxicity compared with irinotecan administration.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Drugs. Irinotecan (677 g/mol) was obtained as a ready-to-use clinical formulation at 20 mg/mL (Campto, Pfizer). Clinical grade 5-FU (Fluorouracil, Teva, Pharma, 130 g/mol), at 50 mg/mL, and bevacizumab (Avastin, Roche, 149 kDa at 25 mg/mL) were used in in vivo combination studies. These reference drugs as well as DTS-108 (3170 g/mol) were diluted in sterile 0.9% NaCl (w/v) for in vivo administration. SN38 (392.3 g/mol), purchased from Abatra Technology, was first dissolved in DMSO and then diluted in culture medium for in vitro cytotoxicity assays. SN38-G was synthesized from SN38 using the UDP-glucuronosyltransferase UGT1A1 (Sigma) and the method described by Jinno et al. (21). In Results, doses and concentrations are given in milligrams or nanograms. To determine the amount of equivalent SN38 between DTS-108 and irinotecan, the molar ratio DTS-108/irinotecan (4.7) should be taken into account.

Synthesis of DTS-108. The 20-amino-acid DPV1047 Vectocell peptide (CVKRGLKLRHVRPRVTRMDV) was synthesized as a trifluoroacetate salt by NeoMPS.

The 4-{4-[(N-maleimydomethyl)cyclohexanecarboxamido]methyl}cyclohexane-1-carboxylic acid linker (BCH) was synthesized as follows: succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (11 g, SMCC; Pierce Biotechnology) was dissolved in 220 mL of acetonitrile and 55 mL of water. Trans-4-(aminomethyl)cyclohexane-1-carboxylic acid (16.7 g, Sigma-Aldrich) was dissolved in 125 mL of water and 20 mL of N,N'-diisopropylethylamine (DIPEA; Sigma-Aldrich) and added to the SMCC solution. The reaction mixture was stirred at ambient temperature for 1 h before the addition of dichloromethane (170 mL, SDS) and methanol (50 mL, SDS). The organic layer containing BCH was then collected and dried by rotary evaporation. The crude BCH linker was dissolved in 60 mL of dichloromethane and 30 mL of methanol and added slowly to methyl tert-butyl ether (400 mL, SDS) at 0°C. The resulting white precipitate (BCH) was filtered and dried under vacuum.

Five grams of SN38 (Abatra Technology) were added to BCH (5.8 g) and O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium (3.1 g, SDS), previously dissolved in N-methyl-2-pyrrolydone (50 mL, Sigma-Aldrich), followed by DIPEA (5.4 mL). The mixture was stirred for 4 h at 0°C and was then taken up in 125 mL of dichloromethane. The organic layer was washed thrice with 130 mL of 1 mol/L NaCl and thrice with 130 mL of 5% (w/v) aqueous citric acid and was then added slowly to 300 mL of methyl tert-butyl ether at 0°C. The yellow precipitate (10-O-BCH-SN38) was recovered by filtration and dried under vacuum.

Five grams of 10-O-BCH-SN38 were added to a solution containing DPV1047 (21.5 g) in 200 mL of N,N-dimethylformamide (Sigma-Aldrich) and stirred for 3 h at room temperature. Water (200 mL) was added and the aqueous layer was extracted thrice with 200 mL of dichloromethane and freeze dried.

DTS-108 hydrochloride (DTS-108·HCl) was generated by ion exchange chromatography of DTS-108·trifluoroacetate using Amberlite IRA-410 resin (Sigma-Aldrich) conditioned with 2 N HCl. Product-containing fractions were pooled, frozen, and lyophilized (typical purity/potency {approx}95%).

Solubility studies. Solubility studies were performed as previously described in the Organisation for Economic Cooperation and Development Guidelines for testing chemicals (22). The solubility of irinotecan, SN38, and DTS-108 was measured in water at 20°C. Solubility was initially evaluated by visual assessment at 50% (w/v); the different molecules were allowed to dissolve for 5 min with agitation. The quantity of water was then increased in increments of 5% (v/v) until the compound was completely dissolved.

Stability studies. The stability of the conjugates was tested in human, male beagle dog, and male CD1 mouse plasma. Mouse, dog, and human plasma were purchased from Charles River Laboratories and from EFS, respectively. DTS-108 (2.55 µmol/L) or irinotecan (2.55 µmol/L) were incubated at 37°C in citrated plasma and samples were harvested at 0, 3, 6, 20, 60, 180, and 360 min and at 24 and 48 h followed by analysis using the high-performance liquid chromatography fluorescence method described below (see Chromatographic equipment and conditions) following trifluoroacetate/methanol deproteination.

In vitro cytotoxicity. Viability assays were performed with DTS-108 or irinotecan on five human cancer cell lines treated with drugs for 48 h using the WST-1 assay (Roche). Three colon adenocarcinomas, HCT 116, HT-29, LS 174T; a lung adenocarcinoma, NCI-H460; and a mammary adenocarcinoma, MDA-MB-231 (all from American Type Culture Collection) were used in this study. Molar concentrations that inhibit 50% of cell viability (IC50 values) were calculated from sigmoid regressions, using the Graph Pad Prism 3.02 software.

Preliminary toxicity and toxicokinetic studies in the dog. Adult beagle dogs (1 dog for each drug and dose) were infused i.v. through the cephalic or saphenous vein. DTS-108 was administered at 5, 10, or 20 mg/kg as a 45-min infusion (0.4 mL/min). Irinotecan was injected at its maximum tolerated dose (MTD), that is, 30 mg/kg (23) as a 20-min infusion (0.4 mL/min). Clinical signs and hematologic toxicities were monitored for 15 days after infusion.

The body weight of each animal was recorded on days –1, 4, 10, and 13. Peripheral blood was sampled using EDTA tubes (Sarstedt) on days –1, 5, 11, and 14 for hematologic analyses. In addition, peripheral blood samples were taken at different times (up to 8 h) following infusion for pharmacokinetic analysis using 5 mL trisodium citrate tubes (Sarstedt S-Monovette 9NC) containing 2.5 mL of 5% trifluoroacetate (v/v). The area under the blood concentraion versus time curve (AUC) of the different compounds and their metabolites was measured up to the limit of quantification (7.8 ng/mL equivalent SN38). For irinotecan, blood was sampled at time 0; at the end of the infusion (20 min); and 10, 20, 40, 220, and 460 min after infusion. For DTS-108, blood was sampled at time 0; at the end of the infusion (45 min); and 10, 20, 40, 120, and 220 min after infusion. AUC values were calculated using the trapezoid summation rule from zero until the last sampling time point or until the limit of quantification was reached.

In vivo efficacy studies in human tumor xenograft models. All in vivo studies were performed in accordance with the guidelines set out by the European Economic Union and the United Kingdom's Co-ordinating Committee on Cancer Research (2nd edition; ref. 24). Human colon, lung, and mammary tumors were established by an intradermal or s.c. implantation of cells (1 x 107 HCT 116, 1 x 107 HT-29, 2 x 107 LS 174T, 3 x 106 NCI-H460, and 3 x 106 MDA-MB-231 cells injected) in the right flank of 7-wk-old female NMRI nude mice, or, for the LS 174T model, in Rh rnu/rnu nude rats (Harlan). Treatments were initiated when the tumors reached a size of ~100 mm3 in mice or 1,000 mm3 in rats [calculated using the following formula: (length x width2) / 2]. The day of the first injection animals were randomized into different groups (6 or 8 animals per group) and were treated by bolus i.v. injection (in the lateral tail vein) or i.p. injection (only for bevacizumab) of the different drugs dissolved in saline in a volume of 10 µL/g following the indicated administration schedule. During and following treatment, clinical signs, body weight, and tumor size were recorded regularly to evaluate the efficacy and toxicity of the injected drugs. The ratio (T/C, %) of the mean tumor volumes of treated (T) versus control (C) groups was used to evaluate treatment efficacy. The minimal T/C reflects the maximal tumor growth inhibition achieved.

For combination studies, suboptimal doses of DTS-108, 5-FU, and bevacizumab were administered (25, 26). The T/C ratio was calculated for each individual compound (bevacizumab, 5-FU, or DTS-108). The combination index of each pair of drugs was calculated by dividing the expected T/C (T/C of DTS-108 x T/C of bevacizumab or 5-FU) by the observed T/C. An index >1 shows a synergistic effect; an index of ~1 indicates an additive effect; and an index <1 indicates an antagonistic effect (27).

Chromatographic equipment and conditions. Blood samples (500 µL) were immediately diluted using an equal volume of trifluoroacetate 2.5% (v/v) to stabilize DTS-108 and prevent further cleavage of the conjugate. Each sample was placed in a 2 mL microcentrifuge tube and centrifuged at 16,000 x g for 3 min at +4°C. Twenty µL of a 1.12 µg/mL freshly prepared camptothecin (Sigma) solution (internal standard, +4°C) were added to 100 µL of the supernatant. Methanol (100 µL) was then immediately added and the mixture was vortexed for 5 s. Samples were then centrifuged at 16,000 x g, for 3 min at room temperature. The supernatant (150 µL) was recovered for high-performance liquid chromatography analysis. Drugs and metabolites were separated by high-performance liquid chromatography (Agilent 1100 series equipped with a fluorescence detector) using a 4.6 x 100 mm (3 µm particle size) Luna C18(2) column (Phenomenex ref. 00D-4251-E0). The aqueous component of the mobile phase (A) was 0.1% (v/v) trifluoroacetate in water. The organic modifier (B) was acetonitrile containing 0.1% (v/v) trifluoroacetate. For DTS-108 and its metabolites, an elution gradient was applied with a constant flow rate of 1.2 mL/min, increasing linearly the proportion of B from 15% to 37% in 2 min, from 37% to 47% in 6 min, followed by 2 min at 90%. The proportion of B was then returned to the initial condition for 3 min. For irinotecan and its metabolites, an elution gradient was applied with a constant flow rate at 1.2 mL/min, increasing linearly the proportion of B from 15% to 50% in 10 min, from 50% to 90% in 0.5 min, followed by 2 min at 90%. The proportion of B was then returned to the initial condition for 3 min. Fluorescence detection ({lambda}exc., 375 nm; {lambda}em., 560 nm) was used, and drugs and metabolites were identified according to their relative retention times as determined from a set of standards (RtDTS-108 = 4.7 min; RtSN38 = 5.3 min; Rtinternal standard camptothecin = 5.7 min).

The method was validated in terms of specificity, linearity, imprecision/inaccuracy, carryover, recovery, dilution effect, and stability (with a lower limit of quantification of 7.8 ng/mL equivalent SN38 and an upper limit of quantification of 1,000 ng/mL equivalent SN38). Extraction recoveries of 67 ± 10% were obtained for DTS-108 and SN38.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Peptide-SN38 conjugation, solubility and in vitro stability. SN38 is an efficient antitumoral agent. However, it is highly insoluble and therefore cannot be administered in pharmaceutically acceptable vehicles. The DPV1047 peptide was chemically conjugated to the 10-hydroxyl group of SN38 via a heterobifunctional cross-linker (BCH) with an ester bond (Fig. 1A ). The solubility of the SN38 conjugate DTS-108 in water (>500 mg/mL; 317.4 mmol/L) was found to be much greater than that of either irinotecan (<2.5 mg/mL; 3.7 mmol/L) or SN38 (<5 µg/mL; 0.013 mmol/L).


Figure 1
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  		Fig. 1. The structure of DTS-108 in comparison with irinotecan. DTS-108 (A) is a Vectocell peptide–SN38 prodrug generated by chemical coupling of the 10-hydroxyl group of SN38 via a heterobifunctional cross-linker (BCH) to the Vectocell peptide DPV1047 (CVKRGLKLRHVRPRVTRMDV). This linker results in the generation of an ester bond between the peptide and SN38. Irinotecan (B) is also an SN38 prodrug with a dipiperidine moiety coupled to the 10-hydroxyl group of SN38. For irinotecan, SN38 is linked via a carbamate bond to the dipiperidine moiety.

 
The BCH cross-linker was specifically designed for optimal stability of the conjugate in plasma, allowing SN38 release upon cleavage of the ester bond (28). This is contrary to irinotecan activation that requires hepatic carboxylesterase-mediated hydrolysis of a carbamate bond (5, 6). The in vitro stability of DTS-108 is species dependent, being superior in human and dog plasma (with half lives of 400 and 290 minutes, respectively) than in mouse plasma (where the half life is <3 minutes). In contrast, irinotecan is highly stable in human and dog plasma, showing only minimal release of SN38 (with a half life of >4,300 minutes in both human and dog plasma compared with 20 minutes in mouse plasma).

In vitro cytotoxicity of DTS-108. In vitro cytotoxicity experiments were performed to test whether the intact DTS-108 or its metabolites showed cytotoxic activity that was independent of the liver-specific activation of irinotecan. DTS-108 was shown to be cytotoxic in vitro in a panel of colon (HCT 116, HT-29, and LS 174T), lung (NCI-H460), and breast (MDA-MB-231) cancer cell lines after a 48-hour incubation. In these cell lines, the DTS-108 IC50 was 24, 94, 2, 40, and 834 nmol/L, respectively. In contrast, the prodrug irinotecan showed a very low cytotoxic activity (IC50 of 6, 50, 35, 11, and 85 µmol/L, respectively) as previously documented (29), with a 2 to 4 log decrease in activity compared with DTS-108.

Preliminary toxicity and toxicokinetic studies in the dog. The dog was selected as the most appropriate model for preliminary toxicity and toxicokinetic studies for DTS-108 because DTS-108 stability in dog plasma is similar to that observed in human plasma, and because, contrary to rodents, the dog is a relevant model that mirrors the human pharmacokinetics of irinotecan and the typical early- and late-stage diarrhea observed in man (30).

DTS-108 was administered at three doses (5, 10, and 20 mg/kg). No clinical signs of toxicity were observed at either 5 or 10 mg/kg, except for a moderate but non–dose-dependent decrease in WBC counts (Table 1 ). Administration of DTS-108 at 20 mg/kg induced toxicity, including a significant decrease in WBC counts (–93%), together with a decrease in food intake and late-stage diarrhea, suggesting that the MTD had been reached (Table 1). No early-stage diarrhea was observed following DTS-108 administration. Irinotecan was administered at its MTD (30 mg/kg), which resulted in the classic clinical signs (23), including early-stage (day 1) and late-stage (days 4-6) diarrhea, body weight loss, emesis, and a severe but reversible leucopenia on day 5 (Table 1).


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  		Table 1. Clinical and toxicokinetic comparison of DTS-108 and irinotecan following i.v. infusion in the dog

 
Typical blood concentration versus time curves following DTS-108 and irinotecan administration in the dog are shown in Fig. 2 . In both cases, the parent compound, SN38 and the glucuronoconjugate SN38-G were observed. The Cmax of DTS-108 is observed, as for irinotecan, at the end of the infusion. DTS-108 concentration then decreases much more rapidly than that of irinotecan. SN38 concentrations were significantly lower after irinotecan administration than after DTS-108. After treatment with DTS-108, the AUC of DTS-108 and SN38 increased in a dose-dependent manner in the tested range (5-20 mg/kg of DTS-108; Table 1). At the lowest dose of DTS-108 tested (5 mg/kg), the SN38 AUC was 920 ng x h/mL, whereas at its MTD (20 mg/kg) the SN38 AUC reached 4,756 ng x h/mL. Following irinotecan administration at its MTD, the AUC of SN38 was 18 ng x h/mL. This result is consistent with the low level of irinotecan reactivation observed previously in the dog (30). At all three doses of DTS-108, a significantly higher SN38 AUC than that observed following administration of irinotecan at its MTD was therefore achieved.


Figure 2
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  		Fig. 2. Blood concentration versus time curves following DTS-108 (A, 20 mg/kg) or irinotecan (B, 30 mg/kg) administration in the dog. The concentration of the parent compounds and the major metabolites, SN38 and SN38-G, were evaluated. DTS-108 ({lozenge}), irinotecan ({square}), SN38 (bullet), and SN38-G ({bigtriangleup}).

 
The AUC of the inactive metabolite SN38-G was also determined. Following DTS-108 (20 mg/kg) or irinotecan (30 mg/kg) administration, it was 1083 and 432 ng x h/mL, respectively. The ratio of the AUCs of SN38-G versus SN38 were therefore 0.23 for DTS-108 and 24 for irinotecan, suggesting a dramatic reduction in glucuronidation with the DTS-108 peptidic prodrug.

In vivo efficacy of DTS-108 in human tumor xenograft models. The in vivo antitumoral efficacy of DTS-108 was first characterized in mice bearing HCT 116 human colorectal tumors (Fig. 3A ). DTS-108 showed a significant dose-dependent effect in this model with transient tumor regressions and a minimal T/C ratio of 10% at the highest, well-tolerated dose level tested (95 mg/kg). Comparative studies between DTS-108 and irinotecan were also done, despite the fact that the activity of the latter is significantly overestimated in rodents compared with man due to its much higher conversion rate and the resulting increased exposure to SN38 (31, 32). Compared with irinotecan (Fig. 3B), an equimolar dose of DTS-108 results in improved antitumoral activity on HCT 116 tumors with a mean tumor volume of 813 ± 405 mm3 for the group treated with DTS-108 compared with 1,228 ± 247 mm3 for the group treated with irinotecan 28 days after treatment termination, although statistical significance was not reached.


Figure 3
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  		Fig. 3. Comparative in vivo efficacy study of DTS-108 in the human HCT 116 colorectal carcinoma model implanted intradermally into nude mice. Treatments were administered by the i.v. injection route on days 3, 7, and 11 (arrowheads). A, mice received either saline (—) or DTS-108 at 32 mg/kg (Figure 3), 62 mg/kg (Figure 3), or 95 mg/kg (Figure 3). B, mice received either saline (—), DTS-108 (Figure 3) at 95 mg/kg or irinotecan (Figure 3) at 20 mg/kg per injection (an equimolar dose of 30 µmol/kg). Points, mean tumor volume evolution; bars, SEM.

 
DTS-108 was then evaluated in a panel of human tumor models including colon (HCT 116), lung (NCI-H460), and breast (MDA-MB-231) carcinomas implanted into mice, using another injection schedule. These experiments confirmed that DTS-108 is extremely active with minimal T/C ratios of 3%, 23%, and 29% in the HCT 116, NCI-H460, and MDA-MB-231 tumor models, respectively (Fig. 4A-C ). Significant antitumoral efficacy (with a minimal T/C ratio of 44%) was also observed in rats bearing LS 174T colon tumors (Fig. 4D).


Figure 4
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  		Fig. 4. In vivo efficacy study of DTS-108 in human HCT 116 colorectal (A), NCI-H460 lung (B), MDA-MB-231 mammary (C), and LS 174T colorectal (D) carcinomas implanted into nude mice (A-C) or rats (D). Treatments were by i.v. injection on days 3, 5, 7, 10, 12, 14, 17, 19, and 21 (arrowheads) in mice or on days 14, 18, 21, 25, and 29 (arrowheads) in rats. Animals received either saline (—) or DTS-108 (Figure 4) at 80 mg/kg in mice or 63 mg/kg in the rat. Points, mean tumor volume; bars, SEM.

 
In preparation for future preclinical and clinical development, the trifluoroacetate salt was exchanged for HCl and no difference in efficacy or solubility was observed between the two salt forms of DTS-108. In vivo efficacy studies using the NCI-H460 lung adenocarcinoma model confirmed that DTS-108·HCl has a similar activity with an in vivo minimal T/C of 18% compared with 23% for DTS-108·trifluoroacetate.

In vivo studies were performed using mice bearing human HT-29 colon carcinomas to evaluate whether synergistic or additive effects are observed when DTS-108 is combined with the cytotoxic agent 5-FU or the anti–vascular endothelial growth factor antibody bevacizumab. The results (Fig. 5 ) show that DTS-108 alone is active in this model (even at the suboptimal dose tested) and that increased antitumoral efficacy is observed in combination with either 5-FU or bevacizumab. For 5-FU, the combination effect was synergistic (combination index >1), whereas for bevacizumab the combination effect was additive (combination index is ~1).


Figure 5
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  		Fig. 5. In vivo efficacy study of DTS-108 alone or in combination with either 5-FU or bevacizumab in the HT-29 human colorectal carcinoma tumor model. A, DTS-108 was administered by i.v. injections on days 3, 6, 10, 13, 17, and 20 (closed arrowheads) and 5-FU was administered on days 4, 11, and 18 (open arrowheads). The mice received either saline (—), DTS-108 at 40 mg/kg (Figure 5), 5-FU at 40 mg/kg (Figure 5), or DTS-108 at 40 mg/kg + 5-FU at 40 mg/kg (Figure 5). B, DTS-108 was administered by i.v. injections on days 3, 7, 11, 17, 21, and 25 (closed arrowheads) and bevacizumab was administered by i.p. injections on days 4, 7, 11, 14, 18, 21, 25, and 28 (open arrowheads). The mice received either saline (—), DTS-108 at 40 mg/kg (Figure 5), bevacizumab at 2 mg/kg (Figure 5), or DTS-108 at 40 mg/kg + bevacizumab at 2 mg/kg (Figure 5). Points, mean tumor volume; bars, SEM.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Irinotecan is a widely prescribed and effective antitumoral agent for the treatment of colorectal cancer. It is a prodrug of SN38 that was designed to improve the pharmaceutical properties, particularly water solubility, of this potent topoisomerase I inhibitor. However, the irinotecan therapeutic index in humans is much lower than expected based on preclinical rodent studies and is subject to significant interpatient variability. This is mainly due to significant interspecies differences in its metabolism.

In humans, the metabolism of irinotecan is extremely complex and predominantly mediated by hepatic specific enzymes, active transport proteins regulating intestinal absorption, and hepatobiliary secretion mechanisms. Irinotecan is converted to SN38 by hepatic carboxylesterase-specific cleavage of a carbamate bond between the 10-hydroxyl group of SN38 and the dipiperidine promoiety (7). This is in contrast to what is observed in rodents in which high levels of tissue and circulating esterases other than carboxylesterase convert irinotecan into SN38 to a much greater extent (32). Furthermore, the activity of carboxylesterases from animal species has been compared with the human enzymes and the latter show consistently lower activity than those of the other species (33). According to pharmacokinetic data, only 2% to 8% of irinotecan is converted into SN38 in humans with significant interpatient variability, whereas a 50% conversion rate is observed in rodents (7, 32).

The generation of SN38 in the liver favors its metabolism to the inactive glucurono-conjugate SN38-G by the enzyme UGT1A1. As a consequence, the plasma concentration of this glucuronyl metabolite is around 7-fold higher than that of SN38 (34). This also results in the accumulation of high levels of SN38 in the intestine through biliary excretion, which is the root cause of the late-onset diarrheas affecting 30% to 40% of treated patients (1, 15, 16). In addition, considerable variation in the conversion of SN38 into SN38-G in humans has been observed (35, 36) and genetic polymorphism of the gene encoding UGT1A1 has been reported in cancer patients. Recently, the labeling information for irinotecan was modified to indicate the role of UGT1A1*28 polymorphism in the metabolism of irinotecan and its effect on safety, and a lower dose is recommended for homozygous UGT1A1*28 patients. In August 2005, the Food and Drug Administration approved the marketing of a UGT1A1 molecular assay for use as an in vitro diagnostic test for detecting the *1 (wild-type) and *28 (variant) alleles of the gene (37, 38).

Several approaches have been considered to solubilize SN38, improve its pharmacokinetic properties, and increase its therapeutic efficacy, including liposomal or nanoparticle formulations, water-soluble polymer conjugates, vitamin E conjugates, or other derivatives (39–43). Very little data is, however, available to show that these approaches improve the therapeutic index of irinotecan. The present work was therefore undertaken to design a novel SN38 prodrug that would be water soluble and allow a rapid and efficient, non–hepatic release of the active drug. This, by avoiding the intervention of liver carboxylesterases and UDP-glucuronosyltransferases, should result in a higher and more reproducible exposure to SN38 compared with irinotecan, with only minimal gastrointestinal toxicity. Solubilization of SN38 was achieved through chemical conjugation with a cationic peptide via an optimized linker and an ester bond, which allows i.v. administration of the selected peptide-SN38 conjugate (DTS-108) using a purely aqueous vehicle.

As already discussed, rodents are not a good model for irinotecan pharmacokinetics or toxicity. DTS-108 and irinotecan pharmacokinetics and toxicity were therefore compared in the dog. Dogs and humans have similar rates of conversion of irinotecan into SN38 and both species develop the same gastrointestinal toxicity (23, 30). Furthermore, the kinetics of the in vitro release of SN38 from DTS-108 is also very close in dog and human plasma. In preliminary studies in the dog, the MTD of DTS-108 was found to be 20 mg/kg (6.3 µmol/kg) compared with 30 mg/kg (45 µmol/kg) for irinotecan (23). The dose-limiting toxicities were, as expected, hemotoxicity and late-onset diarrhea for both drugs. Irinotecan, contrary to DTS-108, also caused early-onset diarrhea. This was expected because such early-onset diarrhea is part of a cholinergic syndrome caused by the direct interaction of irinotecan with acetylcholine esterase and is often associated with lacrimation, hypersalivation, bradycardia, and abdominal cramps (44). Importantly, the SN38 plasma AUC values observed after DTS-108 treatment at 5, 10, and 20 mg/kg were 50-, 80-, and 270-fold higher than that observed with irinotecan at its MTD. Clearly, with DTS-108, much higher circulating levels of SN38 can be achieved without the intestinal toxicity associated with irinotecan administration. These improved pharmacokinetic and pharmacodynamic properties of DTS-108 are consistent with its anticipated non–hepatic mode of activation, the nature and the distribution of the esterases involved being different from that of the hepatic carboxylesterases that activate irinotecan (31). Also consistent with a reduction of the hepatic metabolism of DTS-108 compared with that of irinotecan is the observation of a significantly lower ratio of the AUCs of SN38-G versus SN38 (0.05 and 5.83, respectively).

Interestingly, optimization of both the peptide structure and linker stability was required to obtain an optimal therapeutic index (28), confirming that the benefit of the lead compound DTS-108 results from other specific properties, possibly tissue distribution characteristics and optimal stability of the conjugate, in addition to the ability of the peptide to solubilize SN38.

It is important to note that dose intensification with irinotecan in patients with metastatic colorectal cancer results in improved therapeutic responses (15–17), suggesting that increasing the circulating concentration of SN38 further, without the gastrointestinal toxicity, would be beneficial from an efficacy perspective. The results of the in vivo efficacy studies presented here confirm that DTS-108 has a significant, dose-dependent antitumoral activity in human colon, lung, and breast carcinoma models. DTS-108 was also shown to be more active than an equimolar dose of irinotecan, although rodent models overestimate irinotecan activity, as its conversion rate to SN38 in these species is much greater than in humans (32). Finally, as for irinotecan, DTS-108 has been shown to have synergistic or additive antitumoral effects in combination with either 5-FU or bevacizumab, which suggests that DTS-108 can be used in similar clinical settings.

Taken together, these results show that the i.v. injection of a new peptidic conjugate of SN38 improves antitumoral efficacy and safety compared with irinotecan as a result of a non–hepatic and efficient mode of activation. As the role of the two hepatic enzymes responsible for the variability of irinotecan metabolism (carboxylesterases and UDP-glucuronosyltransferases) is significantly reduced, the interpatient variability following DTS-108 administration is expected to be much lower. These results are highly encouraging and support the therapeutic potential of DTS-108. A phase I clinical study in cancer patients is planned to allow DTS-108 to be characterized further.


   Acknowledgments
 
We thank John Tchélingérian for his continued support and encouragement and Bertrand Alluis, Elise Assouly, and Audrey Darmon for their support on this project.


   Footnotes
 
Grant support: OSEO/ANVAR.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: F. Meyer-Losic and C. Nicolazzi contributed equally to this work.

Received 10/ 8/07; revised 11/30/07; accepted 12/26/07.


   References
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 Abstract
 Materials and Methods
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 References
 

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