Development of Albumin-Binding Camptothecin Prodrugs Using a

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Development of Albumin-Binding Camptothecin Prodrugs Using a Peptide Positional Scanning Library Björn Schmid,† André Warnecke,† Iduna Fichtner,‡ Manfred Jung,§ and Felix Kratz*,† Tumor Biology Center, Breisacher Straße 117, 79106 Freiburg, Germany, Max-Delbrück Centrum, Robert-Rössle-Straße 10, 13122 Berlin, Germany, and University of Freiburg, Institute of Pharmaceutical Sciences, Pharmaceutical and Medicinal Chemistry, Albertstraße 25, 79104, Freiburg, Germany. Received March 13, 2007; Revised Manuscript Received July 11, 2007

Designing truly tumor-specific prodrugs remains a challenge in the field of cancer chemotherapy. As a new strategy, we incubated homogenates of a spectrum of human colon tumor xenografts with a fluorogenic positional scanning tetrapeptide library in order to identify peptide sequences that are preferentially cleaved by colon tumors. Our screening experiments at pH 7.4 revealed that Met, Leu, and Lys were preferred amino acids in the position P1 and Tyr, Phe, and Met in P2, whereas in P3 and P4, the cleavage profiles were less characteristic. However, similar results were obtained when testing breast tumor material and homogenates from healthy murine organs. On the basis of these results, we developed albumin-binding camptothecin (CPT) prodrugs of the general formula EMC-Arg-P4-P3-P2-P1-Ala-CPT (EMC ) 6-maleimidocaproic acid) that incorporated two peptide linkers: H-ArgAla-Phe-Met-OH and H-Arg-Phe-Tyr-Met-OH (P4-P3-P2-P1). The incorporation of two arginine residues rendered the prodrugs water-soluble (>7 mg/mL), while the use of alanine as an amino acid spacer proved to be beneficial for the release of the active agent. Incubation studies with homogenates of HT-29 colon tumor tissue and murine spleen, liver, and kidneys demonstrated cleavage of the peptide linker with CPT–peptide derivatives and CPT being the major cleavage products. Although the peptide sequence is not selectively cleaved in colon tumors, an in ViVo study in a HT-29 xenograft model showed that the prodrug EMC-Arg-Arg-Ala-Phe-Met-Ala-CPT demonstrated enhanced antitumor efficacy when compared to CPT [(T/C max: 17% for the prodrug (2 × 12.5 mg/kg CPT equivalents) and 40% for CPT (3 × 12.5 mg/kg)].

INTRODUCTION One of the major limitations of clinically established anticancer drugs is their lack of tumor selectivity. Cytostatic drugs are evenly distributed in the organism, thus damaging healthy tissue and producing side effects. In order to overcome these drawbacks, a major goal of modern drug development is to provide efficient strategies that enable site-specific delivery of anticancer drugs. In the past, various low- and high-molecular-weight prodrugs have been evaluated with the aim of improving cancer therapy. Since it has been shown that macromolecules with a molecular weight of >20 kDa accumulate in tumor tissue due to passive targeting almost regardless of their chemical nature (1), conjugation of drugs with macromolecules has proved to be a promising and versatile platform technology for tumor targeting (2). Typically, the general design of such prodrugs can be formulated as carrier(-spacer-drug)n wherein n describes the number of drugs bound to one carrier molecule. In the past years, we have developed a macromolecular prodrug concept in which an anticancer agent is functionalized with a thiol-binding maleimide group. After intravenous administration, the prodrug binds rapidly and selectively to the cysteine-34 position of circulating serum albumin, thereby generating a macromolecular transport form of the drug in * To whom correspondence should be addressed. Dr. Felix Kratz, Tumor Biology Center, Department of Medical Oncology, Clinical Research, Breisacher Straße 117, D-79106 Freiburg, Federal Republic of Germany. Tel.: +49-761-2062930. Fax: +49-761-2062905. E-mail: [email protected]. † Tumor Biology Center. ‡ Max-Delbrück Centrum. § University of Freiburg.

situ (3–5). This strategy turned out to be applicable to a number of anticancer drugs such as doxorubicin (5–8), carboplatin (9), and camptothecin (10). These prodrugs demonstrated a lower toxicity combined with a superior in ViVo efficacy in mice models compared to the respective parent compounds. Our efforts led to the development of DOXO-EMCH, a 6-maleimidocaproic-hydrazone derivative of doxorubicin, which is the first albumin-binding prodrug to enter clinical trials. In animal experiments, DOXO-EMCH was well-tolerated and proved to be superior over doxorubicin in several preclinical tumor models (3, 4). In a clinical phase I study, DOXO-EMCH showed promising results (11), and a phase II study (with the compound renamed INNO-206) will be initiated at the beginning of 2007. Although inconspicuously named spacer or linker, the molecule realizing the link between the drug and the carrier plays a crucial role for the prodrug to exert antitumor efficacy. The spacer incorporates a predetermined breaking point and is thus responsible for a site-specific release of the drug after entering tumor cells or tumor tissue, on one hand. On the other hand, the spacer has to ensure the prodrug’s stability in the bloodstream and—ideally—healthy tissue. Apparently, a prodrug strategy can only be as effective as its underlying drug release mechanism. It is therefore of special interest to find new release strategies for enhanced tumor specificity and to customize appropriate spacer molecules. Whereas DOXO-EMCH incorporates an acid-sensitive hydrazone linker that is stable in plasma but is rapidly cleaved after cellular uptake in the acidic environment of endosomes in tumor cells, other strategies for achieving tumor-specific release of drugs are based on the overexpression of certain proteases in tumor tissue. Proteolytic enzymes play a decisive role in metastasis and tumor progression (12–14), and elevated levels were reported for cathepsins (15), matrix metalloproteases (16),

10.1021/bc0700842 CCC: $37.00  2007 American Chemical Society Published on Web 10/05/2007

Development of Albumin-Binding Camptothecin Prodrugs

plasmin (17), urokinase-type plasminogen activator (uPA) (17), and prostate-specific antigen (PSA) (18). In our previous work, we developed a number of albumin-binding prodrugs of doxorubicin that were cleaved by uPA (8), PSA (7), and the matrix metalloproteases 2 and 9 (6, 19). Disadvantages of targeting individual enzymes for cleaving the prodrug are their heterogeneous expression in the tumor and their variability among individual tumors, as well as their expression in healthy tissue. In the present work, we therefore decided not to focus on individual proteases but instead followed a pragmatic approach at attempting to identify those peptide sequences that are most efficiently cleaved by native tumor material irrespective of whether one or more than one enzyme were involved in the cleavage process. For this purpose, we screened tissue homogenates from colon and breast tumor xenografts as well as healthy murine organs with a positional scanning combinatorial tetrapeptide library. On the basis of these results, we selected two tetrapeptide sequences for the development of albuminbinding prodrugs with the anticancer agent camptothecin (CPT), an alkaloid from the chinese tree Camptotheca acuminata first isolated by Wall and co-workers, that shows strong cytotoxic activity (20), based on the inhibition of DNA-topoisomerase I (21). Due to its poor water-solubility (2.5 µg/mL) (22), CPT did not gain any clinical importance, whereas the water-soluble derivatives topotecan and irinotecan have meanwhile been approved for the therapy of colorectal, ovarian, and lung cancer (23, 24). By applying this novel approach, we hoped to obtain selective prodrugs for the treatment of colorectal tumors. Herein, we report on the screening experiments using a positional scanning library, the synthesis of the CPT prodrugs, and their cleavage properties and antitumor activity.

MATERIALS AND METHODS General. Chemicals and solvents were purchased from Sigma-Aldrich, Fluka, Merck, KMF, LGC Promochem and Roth, and were used without further purification. The Fmocprotected amino acids were purchased from Bachem AG (Switzerland). For the synthesis of the peptide libraries, polystyrol rink-amide AM-resin [Fmoc-RAM-AM, 100–200 mesh, loading (by Fmoc-determination): 0.65 mmol/g] from RAPP Polymere GmbH (Tübingen, FRG) was used. Camptothecin was purchased from Hande Tech Development, Inc. (USA). H-AlaCPT was synthesized according to a published procedure (25). EMC-Arg-Arg-Ala-Phe-Met-OH was custom-made by JPT Peptide Technologies GmbH (Berlin, Germany). Human serum albumin (5% solution) was purchased from Octapharma GmbH that contained approximately 60% free thiol groups as assessed with the Ellmann’s test. The buffers used were vacuum-filtered through a 0.2 µm membrane (Sartorius, Germany) and thoroughly degassed with ultrasound or by purging with nitrogen prior to use. Chemical reactions were carried out under a nitrogen atmosphere. Peptide substances were lyophilized with an Alpha 2–4 lyophilizer (Christ, Germany). Spectral Analyses. Mass spectra were obtained on a Thermoelectron LCQ Advantage with associated MAT SS 200 data system using electron spray ionization. UV/vis spectrophotometry was carried out with a double-beam spectrophotometer U-2000 from Hitachi. LC-MS-pos ESI spectrometry was carried out on a Finnigan LCQ Advantage mass spectrometer (capillary temperature 300 °C; source-induced dissociation voltage 25 V, Thermo Electron) coupled to a Finnigan Surveyor HPLC system (Thermo Electron, Dreieich, FRG) with a Jupiter C4 column (300–5, 150 × 2 mm; Phenomenex, Aschaffenburg, FRG). Chromatographic conditions are as follows: flow, 300 µL/min; mobile phase A, 1% MeCN, 99% demin H2O, 0.06% TFA;

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mobile phase B, 100% MeCN, 0.06% TFA; gradient 15–20 min increase from 10% mobile phase B to 50% mobile phase B; injection volume, 15 µL. Chromatography. See Supporting Information for chromatography. Synthesis of the Peptide Library. The peptides were built up on Rink amide resin (RAM-AM). The synthesis of ACC and P1-loaded ACC resin (H-P1-ACC-RAM-AM) was carried out according to a published procedure (26–28). We used 18 of the 20 standard amino acids (tryptophane and cysteine were omitted), while the published method used 19 amino acids (cysteine was omitted and norleucine was substituted for methionine), and in contrast to the published procedure, we did not use a fixed P1-position. The capacity of the resin (0.65 mmol/ g) was determined by a spectrophotometric Fmoc quantification assay (29). For preparation of the P1-position of the P1-library, the H-ACC-RAM-AM-resin was divided into 18 reaction columns (1.30 mmol/column) and was suspended in DMF. Consecutively, each of the 18 Fmoc-amino acids (5 equiv), 1,6collidine (5 equiv), and HATU (5 equiv) were added to the 18 reaction columns. The mixtures were stirred for 16 h and afterwards thoroughly washed with DMF. The yield of the reaction and the loading of the resin was determined via Fmoc quantitative assay (29). If the reaction afforded a yield of 95%. Synthesis of the EMC-Arg-Arg-Phe-Tyr-Met-OH Linker. The synthesis of the linker was performed on solid phase using methionine-preloaded Wang resin as the solid support. The Fmoc-amino acids were sequentially coupled following a standard Fmoc protocol (29). In a final step, 6-maleimidocaproic acid (EMC: 3.01 g, 14.25 mmol) was dissolved in DMF, and 10 equiv of HOBt and 10 equiv of DIPC were added. After preincubation for 2 min, the solution was added to the reaction

Schmid et al.

vessel containing the resin (total volume of DMF: 25 mL). Afterwards, the resin was treated with a mixture of TFA/water 95:5 (v/v) for 2 h. The resin was separated by filtration, and the filtrate was evaporated three times after addition of 10 mL of ethanol before the residue was dried in high vacuum. 1.624 g of a crude product was obtained. The crude product was dissolved in 15 mL of anhydrous methanol and precipitated with 200 mL anhydrous diethylether, then centrifuged and washed with anhydrous diethyl ether. This purification step yielded 600 mg of an off-white solid. Further purification was achieved by preparative C18-RP-HPLC chromatography. Preparative chromatography was carried out on a Nucleosil C18 column (100–7, 250 × 21 mm) with precolumn (100–7, 50 × 21 mm) from Macherey-Nagel. Chromatographic conditions are as follows: flow, 10.0 mL/min (0–26 min) and 15.0 mL/min (26–57 min); mobile phase A, 10% MeCN, 90% demin H2O, 0.1% TFA; mobile phase B, 40% MeCN, 60% demin H2O, 0.1% TFA; gradient, 0–26 min, 100% mobile phase A isocrat.; 26–36 min, 0%–100% mobile phase B; 36–46 min, 100% mobile phase B isocrat.; 46–55 min, 0%–100% mobile phase A; 55–57 min, 100% mobile phase A isocrat.; injection volume, 5 mL. After lyophilization, 243 mg of the parent compound was obtained as a white solid. ESI-MS dir-pos. (2.5 kV, MeOH): m/z (%) 965.3 ([M + H]+, 100); HPLC purity (λ ) 220 nm): ∼98%. Synthesis of EMC-Arg-Arg-Phe-Tyr-Met-Ala-CPT × 2 TFA (2). HATU (15.5 mg, 0.054 mmol) was added under a nitrogen atmosphere to a solution of camptothecin-20-O-(Lalaninate) × TFA (19.8 mg, 0.049 mmol), EMC-Arg-Arg-PheTyr-Met-OH × 2 TFA (65 mg, 0.054 mmol) and DIEA (25.5 µL, 0.198 mmol) in 1 mL DMF. The solution was stirred for 1 h at room temperature. Subsequently, the product was precipitated with diethyl ether, and the crude product was purified by preparative chromatography. Preparative chromatography was carried out in analogy to the isolation of 1. After lyophilization, 26.3 mg (33.7%) of 2 was obtained as a bright yellow solid. ESI-MS dir-pos (5.0 kV, 30% MeOH): m/z (%) 1366.4 ([M]+, 100), 1367.4 ([M + H]+, 70) (mass spectrum is included as Supporting Information); HPLC purity (λ ) 370 nm): >95%. Synthesis of the Albumin Conjugates of 1 (HSA-1) and 2 (HSA-2). 1.5 mg of the albumin-binding prodrug was dissolved in 2 mL of a 5% solution of human serum albumin (HSA) (∼100 mg of albumin) and incubated under stirring at 37 °C for 1 h after which no free 1 or 2 was detectable. The HPLC method to determine that the reaction was completed is available as Supporting Information. The sample was kept frozen at -20 °C and thawed prior to use. Incubation Studies of 1 and 2 with Mouse Blood Plasma. 0.75 mg of the albumin-binding prodrugs were dissolved in 100 µL of sterile-filtered glucose–phosphate buffer (10 mM sodium phosphate/5% D-glucose buffer, pH 5.8). 30 µL aliquots of these solutions were added immediately to 570 µL of mouse plasma (gratefully received from Dr. N. Esser, Tumor Biology Center, Freiburg) and incubated at 37 °C. Samples were collected after 5 min, 4 h, 8 h, and 24 h and were analyzed by HPLC. The decrease in the peak area of the conjugates at 370 nm over time was used to determine the halflives. Incubation Studies of HSA-1 and HSA-2 with Tissue Homogenates. 300 µL aliquots of the HSA-1 and HSA-2 stock solutions (500 µM) were mixed with 150 µL of TEAA buffer (20 mM, pH 7.4 and pH 5.0) and 150 µL of tissue homogenate (pH 7.4 and pH 5.0). The samples were incubated at 37 °C for 5 min, 4 h, and 24 h and were analyzed by HPLC. 150 µL of the 4 h and 24 h HT-29 samples were kept frozen at -20 °C and thawed prior to analysis by LC-MS spectrometry.


In ViWo Efficacy of 1. For the in ViVo testing of 1 in comparison with its parent compound, female NMRI: nu/nu mice (inhouse breeding) were used. The mice were held in individually ventilaged cages under sterile and standardized environmental conditions (25 ( 2 °C room temperature, 50 ( 10% relative humidity, 12 h light–dark rhythm). They received autoclaved food and bedding (ssniff, Soest, Germany) and acidified (pH 4.0) drinking water ad libitum. All animal experiments were performed under the auspices of the German Animal Protection Law. 107 cells of HT-29 were transplanted subcutaneously (s.c.) into the left flank region of anaesthetized (40 mg/kg i.p. Radenarkon, Asta Medica, Frankfurt, Germany) mice on day zero. Mice were randomly distributed to the experimental groups (6 mice per group). When the tumors were grown to a palpable size (90–130 mm3), treatment was initiated. HT-29 xenografted mice were treated intravenously at day 6, 13, and 20 with 10 mM sodium phosphate/5% D-glucose buffer pH 5.8, camptothecin, or 1 (compound was administered as a solution in 10 mM sodium phosphate/5% D-glucose buffer pH 5.8); for doses, see corresponding tables and figures. The volume of administration was 0.20 mL/20 g body weight. Tumor size was measured twice weekly with a caliper-like instrument in two dimensions. Individual tumor volumes (V) were calculated by the formula V ) (length + [width]2)/2 and related to the values on the first day of treatment (relative tumor volume, RTV). At each measurement day, treated/control values (T/C) were calculated as percentage for each experimental group; the optimum (lowest) values obtained within four weeks after treatment were used for evaluating the efficacy of the compounds, and optimum T/C values are presented in the respective tables. Statistical analysis was performed with the U-test (Mann and Whitney) with p < 0.05. The body weights of mice were determined every 3 to 4 days.

RESULTS Positional Scanning Library. The first aim of our work was to assess the proteolytic activity and specificity of selected native tumor tissue homogenates. Generally, for analyzing the substrate specificity of proteases, two different combinatorial strategies have been followed in the past: 1. Fluorogenic libraries of resin-bound peptides (one bead—one peptide) are screened with enzymes. Subsequent Edman degradation or mass spectrometry of selected hits provides exact information about the peptide sequence (33). 2. Fluorogenic libraries consist of a defined number of nonimmobilized sublibraries, each having one position fixed with one amino acid while all other positions are randomized (positional scanning (PS) libraries). Incubation of sublibraries with proteases and subsequent measuring of fluorescence provides information on the extent to which a single amino acid in a defined position influences the cleavage rate. Although screening proteases using the first strategy results in exact information, i.e., concrete peptide sequences which are cleaved by the target enzymes, the selection and analysis of hits is extremely time-consuming when assessing combinatorial peptides consisting of more than two amino acids and becomes nearly impossible when the number of amino acids exceeds four. In contrast, positional scanning—once the library is established— can be easily performed with high throughput and low cost. In addition, positional scanning has the advantage that cleavage studies can be carried out in solution, which allows a kinetic determination of proteolytic processes. Previously, fluorogenic PS-libraries have been successfully employed to explore the substrate specificity of a variety of proteases (26, 27, 34, 35). In our work, we synthesized a tetrapeptide PS-library based on 7-amino-4-carbamoylmethyl-


coumarin (ACC) as the fluorophor in accordance with a published procedure (27). Peptides were built up on solid phase (Rink amide resin) in five subsequent coupling steps: first the dye and then four amino acids were bound to the resin following straightforward Fmoc chemistry. For the randomized positions, an isokinetic mixture of Fmoc-protected amino acids was employed to ensure an even distribution (30). In order to provide sufficient structural diversity, we employed all standard amino acids except for tryptophane and cysteine. Hence, after cleaving the peptides from the resin, we obtained 4 × 18 sublibraries each having one position fixed. For instance, 18 sublibraries were obtained comprising the general formula Xxx-Yyy-Zzz-P1ACC (P1-library) with P1 fixed and positions Xxx, Yyy, and Zzz randomized. Once the bond between the first amino acid (in position P1) and the ACC dye is cleaved, a significant increase in fluorescence is observed. Therefore, ACC peptides can serve as model compounds for prodrugs in which peptides are bound to drugs via an amide bond. Incubation Studies of Different Tumor Homogenates and Tissue Homogenates of Healthy Organs with the PS Library. In a first step, the PS-library was used to study the protease specificity of native tumor tissue. For this purpose, tissue material of four different colon tumors (xenografts of HT29, 5735, 5854, and 5679) was homogenized according to two different protocols (31, 32): while homogenization at pH 5.0 was assumed to preserve activity of lysosomal proteases, a workup at pH 7.4 was performed to target extracellular enzymes. Figure 1A,B shows the relative increase in fluorescence (F1h/ F0h) of the peptide libraries P1–P4 after a 1 h incubation with the colon tumor homogenates at pH 7.4 and pH 5.0, respectively. On first inspection, all tissue samples exhibited significant proteolytic activity with the homogenates at pH 7.4 being more active than at pH 5.0. Furthermore, the selectivity of the libraries decreased from P1 to P4, and for the positions P3 and P4 at pH 5.0, it was not possible to identify specific amino acids that have a positive influence on the cleavage rate. Among the tested colon tumor samples, no significant differences in the overall cleavage profiles were noted, but there were differences in the absolute fluorescence values; e.g., HT-29 and 5735 showed a notably stronger increase in fluorescence than the other tested colon tumor samples within the P3 and P4 libraries at pH 7.4. For P1 and P2, a clear preference for the following amino acids can be stated at both pH values: pH 7.4. P1: methionine, lysine, and leucine. P2: tyrosine, methionine, and phenylalanine. pH 5.0. P1: methionine, leucine, and tyrosine. P2: methionine. This selection is based on an increase in the relative fluorescence value of >3 for P1 and P2 at pH 7.4 (Figure 1A) and an increase in the relative fluorescence value of >1.5 for P1 and P2 at pH 5.0 (Figure 1B) that was defined as to be able to score amino acids that promote cleavage. For P3 and P4, however, the cleavage profiles are less characteristic. While no preferred amino acid can be identified in these positions at pH 5.0, a marginal positive influence on the cleavage rate for tyrosine, phenylalanine, methionine, and alanine for the P3-position and for arginine, leucine, methionine, tyrosine, and lysine for P4 is noted at pH 7.4. In order to discover whether the cleavage profiles are colon tumor specific, the PS-library was additionally incubated with four different breast carcinoma samples (MCF-7, MDA-MB 435, Breast 6661, Breast 3366) at pH 7.4. The results of this experiment are depicted in Figure 2. On the whole, the cleavage profiles obtained from incubation with these homogenates revealed similar amino acid preferences compared to those of colon tumor homogenates.

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Figure 1. (A) Charts of the P1–P4 -libraries incubated with different colon tumor tissue homogenates at pH 7.4. The histograms show the relative increase in fluorescence (F1h/F0h) of the peptide libraries P1–P4 after a 1 h incubation with four different colon tumor homogenates at pH 7.4. (number of measurements: n ) 4). (B) Charts of the P1–P4 -libraries incubated with different colon tumor tissue homogenates at pH 5.0. The histograms show the relative increase in fluorescence (F1h/F0h) of the peptide libraries P1–P4 after a 1 h incubation with four different colon tumor homogenates at pH 5.0. (number of measurements: n ) 4.)

To elucidate the ACC–peptides’ stability in blood plasma, analogous incubation experiments with the PS libraries P1–P4 were carried out using human blood plasma at pH 7.4. The charts in Figure 3 show that no noteworthy increase in fluorescence with human plasma is detected. Hence, in contrast to tumor tissue, the proteolytic activity of plasma seems to be negligible after a 1 h incubation with the library.

Finally, we investigated the proteolytic activity of healthy organs (liver, kidney, and spleen from nude mice). The PSlibrary was incubated with tissue homogenate samples at pH 5.0 and pH 7.4 (Figures 4 and 5). At pH 7.4, the cleavage profile was quite similar to that of tumor homogenates with a preference for methionine, lysine, leucine, and additionally alanine in P1, tyrosine in P2, and a



Figure 2. Charts of the P1–P4 -libraries incubated with different breast tumor tissue homogenates at pH 7.4. The histograms show the relative increase in fluorescence (F1h/F0h) of the peptide libraries P1–P4 after 1 h incubation with four different breast tumor homogenates at pH 7.4. (number of measurements: n ) 4.)

Figure 3. Charts of the P1–P4 -library incubated with human blood plasma at pH 7.4. The histograms show the relative increase in fluorescence (F1h/F0h) of the peptide libraries P1–P4 after 1 h incubation with human blood plasma at pH 7.4. (number of measurements: n ) 4.)

less clear picture for P3 and P4 (Figure 4). As an exception, the cleavage pattern obtained with kidney homogenate differed markedly from those of liver and spleen. A pronounced cleavage was detected for peptides having arginine, lysine, or glycine in P1, whereas no clear preference could be seen in P2–P4. The

overall increase in fluorescence for the P3- and P4-libraries was higher in the healthy organs compared to the colon tumor samples. At pH 5.0, the overall increase in fluorescence was higher in the healthy organs compared to the tumor samples; incubation


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Figure 4. Charts of the P1–P4 -library incubated with healthy organs (liver, kidney, and spleen) from nude mice at pH 7.4. The histograms show the relative increase in fluorescence (F1h/F0h) of the peptide libraries P1–P4 after 1 h incubation with homogenates of murine healthy organs at pH 7.4. (number of measurements: n ) 4.)

Figure 5. Charts of the P1–P4 -library cleaved by healthy organs (liver, kidney, and spleen) from nude mice at pH 5.0. The histograms show the relative increase in fluorescence (F1h/F0h) of the peptide libraries P1–P4 after 1 h incubation with homogenates of murine healthy organs at pH 5.0. (number of measurements: n ) 4.)

studies with spleen homogenate showed the largest increase in fluorescence. For spleen and liver homogenates, a preference for methionine, lysine, leucine, and tyrosine in P1 and for methionine, valine, alanine, tyrosine, and phenylalanaine in P2

can be discerned. Incubation studies with kidney homogenates showed a less characteristic picture. In summary, the cleavage profiles of homogenates from human breast tumors, human colon tumors, and murine organs

Development of Albumin-Binding Camptothecin Prodrugs Scheme 1. Release of Camptothecin from CPT–Amino Acid Esters

were generally quite similar; albeit, a noteworthy exception was found after incubation with kidney homogenate at pH 7.4. When selecting a peptide sequence that would act as an efficient protease substrate in the prodrugs, we opted to choose and combine those amino acids with a high increase in fluorescence. This was methionine in the P1-position, either tyrosine or the related phenylalanine in the P2-position, alanine or phenylalanine the P3-position, and arginine in the P4-position, with the latter being chosen to enhance the water solubility of the prodrugs. The syntheses of two prodrugs with camptothecin incorporating Scheme 2. Synthesis of the Albumin-Binding CPT Prodrugs 1and 2


the peptide sequences Arg-Ala-Phe-Met or Arg-Phe-Tyr-Met are described below. Synthesis of the Camptothecin Prodrugs 1 and 2. Camptothecin (CPT), a pentacyclic alkaloid isolated from a Chinese tree, was chosen for the development of albumin-binding prodrugs incorporating the two aforementioned peptide sequences as enzymatically cleavable spacers. Although highly cytotoxic, CPT has not gained any clinical relevance due to its poor water solubility (2.5 µg/mL) (22), whereas the hydrophilic derivatives Topotecan and Irinotecan are meanwhile approved for the treatment of colorectal, lung, and ovarian carcinomas. To improve the water solubility and efficacy of CPT, a poly(ethylene glycol) conjugate of CPT (Prothecan) was developed that is currently undergoing phase II clinical trials (36). In this macromolecular prodrug, CPT is bound through an alanine spacer to the polymer. Generally, it could be shown that incorporating amino acids between the carrier and CPT turned out to be beneficial for the following reasons (37): (a) The ester bond between the amino acid and CPT is relatively stable under physiological conditions as long as the amino acid remains acylated (Scheme 1) (38); (b) once the amide bond between the carrier and the amino acid spacer is cleaved, the ester bond between the amino acid and CPT hydrolyzes spontaneously, liberating CPT in its active and less toxic lactone form. However, this in theory simple reaction proved to be quite complex, as previous mechanistic studies on the hydrolysis of H-Gly CPT revealed (39, 40). In our work, we chose to incorporate an alanine spacer between the peptide linker and the drug. Hence, C-terminal cleavage of the peptides results in the release of H-Ala-CPT, which further decomposes to alanine and the free drug. For the synthesis of the albumin-binding prodrugs 1 and 2, the maleimide-bearing peptides EMC-Arg-Arg-Ala-Phe-Met-OH and EMC-Arg-Arg-Phe-Tyr-Met-OH were reacted with camptothecin-20-O-(L-alaninate) (Scheme 2). The latter was readily prepared by a published two-step procedure, in which the first


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Figure 6. (A,B) Chromatograms of incubation studies of mouse blood plasma after 5 min, 4 h, 8 h, and 26 h with prodrugs 1 (A) and 2 (B) at 37 °C. The chromatograms show the stability of the albumin conjugate of prodrugs 1 and 2 over time, to determine the respective plasma half-lives. In addition, the chromatogram of the HSA-conjugate of 1 is displayed. For chromatographic conditions, see Supporting Information.

step was a stereoselective acylation of 20-(S)-camptothecin (25). Condensation of camptothecin-20-O-(L-alaninate) with the maleimide-bearing peptides was carried out using HATU as the coupling agent. The two prodrugs EMC-Arg-Arg-Ala-Phe-MetAla-CPT (1) and EMC-Arg-Arg-Phe-Tyr-Met-Ala-CPT (2) were purified by preparative HPLC and characterized by mass spectrometry. The incorporation of two arginine moieties in the peptide chain of the linkers of 1 and 2 resulted in excellent solubility in buffer solution (>7 mg/mL), while the parent compound is almost insoluble (CPT: 2.5 µg/mL). Albumin-Binding Properties and Stability of 1 and 2. In order to show that the prodrugs 1 and 2 bind rapidly and selectively to endogenous serum albumin, incubation with mouse plasma at 37 °C was performed for 5 min and HPLC profiles recorded at λ ) 370 nm, the maximum absorbance of CPT derivatives in the near-UV region. Figures S1 and S2 in the Supporting Information show the chromatographic profiles of 1 and 2 incubated with mouse blood plasma after 5 min (λ ) 370 nm). While the free prodrugs elute at ∼9 min (1) and ∼11 min (2), the signals disappeared almost completely after incubation with mouse plasma, and a single peak eluting at the retention time of mouse serum albumin (MSA) (33–37 min) was observed. In order to prove that the fast binding results from a Michael addition of the sulfhydryl group of MSA with the maleimide group of the prodrugs, analogous HPLC experiments were carried out in which the cysteine-34 position of MSA was blocked with an excess of -maleimidocaproic acid (EMC) prior

to incubation with 1 and 2 (see Figures S1 and S2 in the Supporting Information). In this case, marginal binding to MSA was observed after incubating plasma with 1 and 2 for 10 min. This observation is in accordance with our previous work on albumin-binding prodrugs with doxorubicin, camptothecin, and platinum derivatives where we have shown that albumin binding results from a Michael addition between the maleimide group and sulfhydryl group of the cysteine-34 position of serum albumin (3, 4, 8–10, 41, 42). In order to determine the plasma stability of the compounds 1 and 2 bound to albumin, plasma samples with 1 and 2 were analyzed at further time points (4 h, 8 h, and 26 h) by HPLC (Figure 6A,B) in order to obtain a first estimate of their plasma stability. From the decrease of the peak areas of the albumin conjugates of 1 and 2, the half-lives were approximately t1/2 ∼ 26 h (1) and t1/2 ∼ 25 h (2). Cleavage of HSA-1 and HSA-2 after Incubation with HT-29 Colon Tumor Homogenate and Tissue Homogenates of Healthy Murine Organs (Liver, Kidney, and Spleen). The cleavage properties of the albumin-bound form of 1 and 2 (HSA-1 and HSA-2) were studied in HT-29 colon tumor homogenates at pH 7.4 and 5.0 (Figure 7A,B and Figure 8A,B). Additionally, buffer stabilities (TEAA, 20 mM) of the albumin conjugates at pH 7.4 and pH 5.0 over 24 h were assessed, demonstrating that a certain degree of unspecific hydrolysis takes place: degradation at pH 7.4 for HSA-1 50%, pH 5.0 ∼75%. Interestingly, the cleavage profiles of both HSA-1 and HSA-2 at pH 7.4 showed H-Met-Ala-CPT, as well as H-Met(O)-AlaCPT in which the thiol group of methionine is oxidized.



Table 1. Antitumor Activity of Prodrug 1 against Human Colorectal Xenografts (HT-29) in ViWo

compound

dosea [mg/kg]

total mortality

body weight change [%] (days 6–9)

T/C [%] maximal

CPT 1

3 × 12.5 2 × 12.5

0/6 1/6 (day 13)

-2 -1

40 (day 22) 17b,c (day 22)

c

a Dose refers to CPT equivalents. Significant to camptothecin.

b

Significant to buffer solution.

Additional cleavage studies were carried out with HSA-1 and tissue homogenates of healthy, murine organs (liver, kidney, and spleen) at pH 5.0. The cleavage profiles (see Supporting Information Figures S3, S4, and S5) were compared to those of the incubation study with HSA-1 and HT-29 colon tumor homogenate at pH 5.0 (Figure 7A). The extent of the cleavage is comparable to the tumor homogenate study (overall degradation over 24 h ∼75%). The strongest cleavage was obtained with spleen homogenate. Overall, the cleavage profiles are very similar to those of the tumor homogenate study, with the exception of a lower generation of CPT in the carboxylate form. In ViWo Activity of 1. The in ViVo efficacy of 1 and CPT was assessed in xenografted nude mice using a human HT-29 colorectal cell line. CPT was evaluated at a dose of 3 × 12.5 mg/kg (i.v.) which was reported to be the maximum tolerated dose (MTD) for camptothecin in nude mice (43) and compared to prodrug 1 at the same dose of 3 × 12.5 mg/kg (i.v.) CPT equivalents. As a result of one toxic death that occurred after the second dose (day 13), a third dose of 1 was not administered. Table 1 and Figure 9 summarize the results of this experiment. Besides an acute toxicity, therapy with 1 produced no significant body weight loss (Figure S6 in Supporting Information). Whereas CPT (3 × 12.5 mg/kg) was moderately active (T/C max 40%), 1 exhibited superior antitumor activity at a lower dose of 2 × 12.5 mg/kg CPT equivalents (T/C max 17%).

DISCUSSION In this work, we set out to discover new peptide sequences for the design of prodrugs that are effectively cleaved within the tumor and, if possible, cleaved tumor-selectively. For this purpose, we screened homogenates of native human tumors and healthy murine organs with a positional scanning library. Positional scanning libraries have previously been used to determine the substrate-specificity of proteases such as plasmin or thrombin (27) and more recently of cathepsins (44). Although the number of studied biological samples was limited, a first and somewhat disappointing finding was the fact

that the cleavage profiles of samples from human breast carcinoma, human colon carcinoma, and murine spleen, kidney, and liver were quite similar. A noteworthy observation is the preference of methionine in P1, since this amino acid is mostly omitted in the development of peptide libraries and is not commonly found incorporated as an amino acid in protease substrates for ubiquitous enzymes. Using positional scanning, we were not able to identify amino acids in P3 and P4 that had a significant impact on the cleavage rate. Possible explanations are that these positions are variable or the sensitivity of our assay was too low. In theory, a low sensitivity for P3 and P4 can result from a high selectivity for P1 and P2 due to the small number of cleavable combinations (percentage of all tetrapeptides with the appropriate P1 and P2 combinations). Hence, sensitivity for P3 and P4 might be improved by developing a new peptide library with fixed P1 and P2. On the basis of the screening results, the selection of amino acids for developing camptothecin prodrugs was explorative in nature, with preference being given to those amino acids which had a significant impact on the cleavage rate at their respective positions. The resulting prodrugs EMC-Arg-Arg-Ala-Phe-Met-Ala-CPT (1) and EMC-Arg-Arg-Phe-Tyr-Met-Ala-CPT (2) showed fast albumin binding, acceptable stability in mouse plasma, and a distinct cleavage profile at pH 5.0 and 7.4. Surprisingly, cleavage of the albumin conjugates of 1 and 2 in HT-29 tumor homogenates was far more pronounced at pH 5.0 than at pH 7.4, considering that the reverse order would have been expected on the basis of the results from the screening experiments with our fluorogenic library. Our incubation studies with tumor homogenate revealed that both peptide sequences were primarily cleaved at the scissile bonds in P1 and P2. Results of in vivo studies in the HT-29 xenograft model were encouraging, since EMC-Arg-Arg-Ala-Phe-Met-Ala-CPT (1) showed significantly better antitumor activity than CPT at a lower dose. The incorporated peptide sequence in 1 cannot, however, be regarded as a colon tumor selective protease substrate, because a similar cleavage profile was observed with healthy murine organs. Reasons for the lack of tumor selectivity are manifold: The most obvious explanation is that there are no relevant differences in the expression pattern of proteases between individual tumors and healthy organs that can be detected with any degree of sensitivity using a positional scanning library. A further reason for the lack of tumor selectivity could be that the overall cleavage profile is determined by ubiquitous proteases that

Figure 9. Curves depicting tumor growth inhibition of subcutaneous HT-29 xenografts under therapy with camptothecin and prodrug 1. The dose of prodrug 1 is given as CPT equivalents. Camptothecin was adminstered at day 6, 13, and 20, whereas prodrug 1 was only administered at day 6 and 13.


overlay finer differences in the expression of proteases in healthy and malignant tissue. Another explanation could be that tumorassociated proteases are deactivated by inhibitors or during homogenate preparation. An alternative strategy for the evaluation of tumor-selective protease activity would be to consider the cellular pathways for the uptake of macromolecular prodrugs in more detail. In contrast to low-molecular-weight drugs, macromolecular prodrugs are taken up by internalization via the endosomal/ lysosomal pathway. As a consequence, assessing the proteolytic activity of pure lysosomal fractions of selected tumors and healthy organs could be an appropriate strategy for future trials in identifying truly tumor-specific peptide sequences. In summary, we have shown that prodrugs incorporating the peptide sequences Arg-Ala-Phe-Met and Arg-Phe-Tyr-Met that were selected from screening a positional scanning library with colon tumor homogenates were efficiently cleaved in their albumin-bound form after exposure to the initially used colon tumor homogenate. In addition, the antitumor efficacy of prodrug 1 with the Arg-Ala-Phe-Met linker was superior to free camptothecin. However, this sequence is not cleaved selectively in HT-29 colon tumors. It cannot be ruled out that esterases in the acidic milieu of the tumor tissue might be responsible for the cleavage of the ester bond between CPT and the peptide linker and lead to the release of the active compound at the tumor site. The improved activity of the prodrug in comparison to free CPT is presumably based on the much longer circulation of the macromolecular prodrug in the bloodstream due to reduced renal excretion associated with accumulation in the tumor mediated by the EPR effect. Subsequently, the albumin conjugate enters the tumor cell via the endosomal/lysosomal pathway and is presumably enzymatically cleaved to the active lactone form of CPT in the acidic environment of the lysosomes. In contrast, under physiological conditions the major part of free CPT is available in its inactive hydroxycarboxylate form and is rapidly excreted via the kidney.

ACKNOWLEDGMENT The support of the Wilhelm Sander-Stiftung, Germany, is gratefully acknowledged. We wish to thank Peter Lazar for carrying out the peptide syntheses and Christoph Warth for performing the LC-MS experiments. Supporting Information Available: Additional experimental data and spectra as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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Development of Albumin-Binding Camptothecin Prodrugs Using a

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